Advances in Microbial Physiology, Volume 57.pdf
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<strong>Advances</strong> <strong>in</strong><br />
MICROBIAL<br />
PHYSIOLOGY<br />
VOLUME 57
This page <strong>in</strong>tentionally left blank
<strong>Advances</strong> <strong>in</strong><br />
MICROBIAL<br />
PHYSIOLOGY<br />
Edited by<br />
ROBERT K. POOLE<br />
West Rid<strong>in</strong>g Professor of Microbiology<br />
Department of Molecular Biology and Biotechnology<br />
The University of Sheffield<br />
Firth Court, Western Bank<br />
Sheffield, UK<br />
VOLUME 57<br />
Amsterdam Boston Heidelberg London New York Oxford<br />
Paris San Diego San Francisco S<strong>in</strong>gapore Sydney Tokyo<br />
Academic Press is an impr<strong>in</strong>t of Elsevier<br />
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Contents<br />
CONTRIBUTORS TO VOLUME 57 ............................................... vii<br />
Ammonia-Oxidis<strong>in</strong>g Archaea – <strong>Physiology</strong>, Ecology and Evolution<br />
Christa Schleper and Graeme W. Nicol<br />
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3<br />
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3<br />
2. Discovery of Archaea <strong>in</strong> Moderate Aerobic Habitats . . . . . . . . . . . . . . . . 4<br />
3. First Insights <strong>in</strong>to the <strong>Physiology</strong> of Ammonia-oxidis<strong>in</strong>g Archaea . . . 6<br />
4. Model Organisms of Ammonia-Oxidis<strong>in</strong>g Archaea . . . . . . . . . . . . . . . . . . 9<br />
5. Membrane Lipids of Ammonia-Oxidis<strong>in</strong>g Archaea . . . . . . . . . . . . . . . . . 13<br />
6. Genomes and Metagenomes of Ammonia-Oxidis<strong>in</strong>g Archaea . . . . . . 15<br />
7. Diversity, Distribution and Activity of Ammonia-oxidis<strong>in</strong>g<br />
Archaea <strong>in</strong> the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22<br />
8. Conclud<strong>in</strong>g Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33<br />
Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34<br />
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34<br />
Reductive Stress <strong>in</strong> Microbes: Implications for Understand<strong>in</strong>g<br />
Mycobacterium tuberculosis Disease and Persistence<br />
Aisha Farhana, Loni Guidry, Anup Srivastava, Amit S<strong>in</strong>gh,<br />
Mary K. Hondalus and Adrie J.C. Steyn<br />
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44<br />
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45<br />
2. Scope ............................................................... 46<br />
3. The Concept of Reductive Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47<br />
4. Overview: General Physiological Characteristics of<br />
Mycobacterium Tuberculosis ........................................ 49
vi CONTENTS<br />
5. Reductive S<strong>in</strong>ks <strong>in</strong> Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59<br />
6. Redox S<strong>in</strong>ks <strong>in</strong> Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76<br />
7. Conclud<strong>in</strong>g Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95<br />
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98<br />
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98<br />
Regulation of CtsR Activity <strong>in</strong> Low GC, Gram+ Bacteria<br />
Alexander K.W. Elsholz, Ulf Gerth and Michael Hecker<br />
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120<br />
1. Prote<strong>in</strong> Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120<br />
2. CtsR-Regulated Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122<br />
3. Cellular Functions of Genes Regulated by CtsR . . . . . . . . . . . . . . . . . . . 125<br />
4. Mechanisms for the Inactivation of the CtsR Repressor . . . . . . . . . . . 129<br />
5. Control of CtsR Degradation by the Regulated Adaptor McsB . . . . 136<br />
6. Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136<br />
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137<br />
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137<br />
AUTHOR INDEX ......................................................... 145<br />
SUBJECT INDEX ......................................................... 167
Contributors to <strong>Volume</strong> 57<br />
ALEXANDER K.W. ELSHOLZ, Ernst-Moritz-Arndt-University Greifswald,<br />
Institute of Microbiology, Greifswald, Germany<br />
AISHA FARHANA, Department of Microbiology, University of Alabama at<br />
Birm<strong>in</strong>gham, AL, USA<br />
ULF GERTH, Ernst-Moritz-Arndt-University Greifswald, Institute of<br />
Microbiology, Greifswald, Germany<br />
LONI GUIDRY, Department of Microbiology, University of Alabama at<br />
Birm<strong>in</strong>gham, AL, USA<br />
MICHAEL HECKER, Ernst-Moritz-Arndt-University Greifswald, Institute of<br />
Microbiology, Greifswald, Germany<br />
MARY K. HONDALUS, Department of Infectious Diseases, University of<br />
Georgia, Athens, GA, USA<br />
GRAEME W. NICOL, Institute of Biological & Environmental Sciences,<br />
Cruickshank Build<strong>in</strong>g, University of Aberdeen, Aberdeen, UK<br />
CHRISTA SCHLEPER, Department of Genetics <strong>in</strong> Ecology, University of<br />
Vienna, Vienna, Austria<br />
AMIT SINGH, International Center for Genetic Eng<strong>in</strong>eer<strong>in</strong>g and<br />
Biotechnology, Aruna Asaf Ali Marg, New Delhi, India<br />
ANUP SRIVASTAVA, Department of Microbiology, University of Alabama at<br />
Birm<strong>in</strong>gham, AL, USA<br />
ADRIE J.C. STEYN, Department of Microbiology, University of Alabama at<br />
Birm<strong>in</strong>gham, AL, USA
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Ammonia-Oxidis<strong>in</strong>g Archaea – <strong>Physiology</strong>,<br />
Ecology and Evolution<br />
Christa Schleper 1 and Graeme W. Nicol 2<br />
1 Department of Genetics <strong>in</strong> Ecology, University of Vienna, Vienna, Austria<br />
2 Institute of Biological & Environmental Sciences, Cruickshank Build<strong>in</strong>g,<br />
University of Aberdeen, Aberdeen, UK<br />
ABSTRACT<br />
Nitrification is a microbially mediated process that plays a central role <strong>in</strong><br />
the global cycl<strong>in</strong>g of nitrogen and is also of economic importance <strong>in</strong><br />
agriculture and wastewater treatment. The first step <strong>in</strong> nitrification is<br />
performed by ammonia-oxidis<strong>in</strong>g microorganisms, which convert<br />
ammonia <strong>in</strong>to nitrite ions. Ammonia-oxidis<strong>in</strong>g bacteria (AOB) have been<br />
known for more than 100 years. However, metagenomic studies and<br />
subsequent cultivation efforts have recently demonstrated that<br />
microorganisms of the doma<strong>in</strong> archaea are also capable of perform<strong>in</strong>g this<br />
process. Astonish<strong>in</strong>gly, members of this group of ammonia-oxidis<strong>in</strong>g<br />
archaea (AOA), which was overlooked for so long, are present <strong>in</strong> almost<br />
every environment on Earth and typically outnumber the known bacterial<br />
ammonia oxidisers by orders of magnitudes <strong>in</strong> common environments such<br />
as the mar<strong>in</strong>e plankton, soils, sediments and estuaries. Molecular studies<br />
<strong>in</strong>dicate that AOA are amongst the most abundant organisms on this<br />
planet, adapted to the most common environments, but are also present <strong>in</strong><br />
those considered extreme, such as hot spr<strong>in</strong>gs. The ecological distribution<br />
and community dynamics of these archaea are currently the subject of<br />
<strong>in</strong>tensive study by many research groups who are attempt<strong>in</strong>g to<br />
understand the physiological diversity and the ecosystem function of these<br />
ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 57 Copyright Ó 2010 by Elsevier Ltd.<br />
ISSN: 0065-2911 All rights reserved<br />
DOI:10.1016/B978-0-12-381045-8.00001-1
2 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
organisms. The cultivation of a s<strong>in</strong>gle mar<strong>in</strong>eisolateandtwoenrichments<br />
from hot terrestrial environments has demonstrated a<br />
chemolithoautotrophic mode of growth. Both pure culture-based and<br />
environmental studies <strong>in</strong>dicate that at least some AOA have a high<br />
substrate aff<strong>in</strong>ity for ammonia and are able to grow under extremely<br />
oligotrophic conditions. Information from the first available genomes of<br />
AOA <strong>in</strong>dicate that their metabolism is fundamentally different from that<br />
of their bacterial counterparts, <strong>in</strong>volv<strong>in</strong>g a highly copper-dependent<br />
system for ammonia oxidation and electron transport, as well as a novel<br />
carbon fixation pathway that has recently been discovered <strong>in</strong><br />
hyperthermophilic archaea. A dist<strong>in</strong>ct set of <strong>in</strong>formational process<strong>in</strong>g<br />
genes of AOA <strong>in</strong>dicates that they are members of a dist<strong>in</strong>ct and novel<br />
phylum with<strong>in</strong> the archaea, the ‘Thaumarchaeota’, which may even be a<br />
more ancient l<strong>in</strong>eage than the established Cren- and Euryarchaeota<br />
l<strong>in</strong>eages, rais<strong>in</strong>g questions about the evolutionary orig<strong>in</strong>s of archaea and<br />
the orig<strong>in</strong>s of ammonia-oxidis<strong>in</strong>g metabolism.<br />
Abbreviations . . . ............................................ 3<br />
1. Introduction ................................................ 3<br />
2. Discovery of archaea <strong>in</strong> moderate aerobic habitats. . ................ 4<br />
3. First <strong>in</strong>sights <strong>in</strong>to the physiology of ammonia-oxidis<strong>in</strong>g archaea ........ 6<br />
4. Model organisms of ammonia-oxidis<strong>in</strong>g archaea. ................... 9<br />
5. Membrane lipids of ammonia-oxidis<strong>in</strong>g archaea .................... 13<br />
6. Genomes and metagenomes of ammonia-oxidis<strong>in</strong>g archaea .......... 15<br />
6.1. Metagenomic Studies of Uncultivated Ammonia Oxidisers. ....... 15<br />
6.2. Predictions from Complete Genome Sequences of Two Mar<strong>in</strong>e<br />
Archaea . . . ............................................<br />
16<br />
6.3. Ammonia Oxidisers: A Dist<strong>in</strong>ct Phylum with<strong>in</strong> the Archaea . . . .... 19<br />
7. Diversity, distribution and activity of ammonia-oxidis<strong>in</strong>g archaea <strong>in</strong> the 22<br />
environment ................................................<br />
7.1. AOA <strong>in</strong> the Soil Environment ............................... 23<br />
7.2. AOA Activity <strong>in</strong> the Soil Environment. . . ...................... 25<br />
7.3. AOA <strong>in</strong> the Mar<strong>in</strong>e Environment . ........................... 28<br />
7.4. AOA Activity <strong>in</strong> the Mar<strong>in</strong>e Environment ...................... 29<br />
7.5. AOA <strong>in</strong> Sediments ....................................... 30<br />
7.6. AOA <strong>in</strong> Geothermal Environments .......................... 32<br />
7.7. AOA Associated with Mar<strong>in</strong>e Invertebrates. ................... 33<br />
8. Conclud<strong>in</strong>g remarks . . . ....................................... 33<br />
Acknowledgement ........................................... 34<br />
References. ................................................ 34<br />
22
AMMONIA-OXIDISING ARCHAEA 3<br />
ABBREVIATIONS<br />
AOA ammonia-oxidis<strong>in</strong>g archaea<br />
AOB ammonia-oxidis<strong>in</strong>g bacteria<br />
GDGT glycerol dialkyl glycerol tetraether<br />
16S rRNA 16S ribosomal ribonucleic acid<br />
1. INTRODUCTION<br />
In many ecosystems and <strong>in</strong> the biosphere as a whole, microorganisms are<br />
considered to constitute the largest component, <strong>in</strong> terms of both biomass and<br />
biological activity (Whitman et al., 1998). They are major players <strong>in</strong> regulat<strong>in</strong>g<br />
the biosphere through their participation <strong>in</strong> global biogeochemical<br />
cycles. Until recently, the role of archaea <strong>in</strong> these global cycles, as well as<br />
their phylogenetic and physiological diversity, had been largely underestimated.<br />
Only the distribution of methanogenic archaea <strong>in</strong> many different<br />
anaerobic habitats worldwide, as well as their role <strong>in</strong> the global carbon cycle,<br />
has been well described (Garcia et al., 2000). All other archaea were considered<br />
extremophiles, with specific adaptations allow<strong>in</strong>g them to <strong>in</strong>habit<br />
environments considered <strong>in</strong>hospitable for most other organisms, such as<br />
salt-saturated lakes, high-temperature terrestrial spr<strong>in</strong>gs and deep-sea vents<br />
(Woese, 1987). However, with the help of culture-<strong>in</strong>dependent molecular<br />
techniques, <strong>in</strong>volv<strong>in</strong>g the amplification of 16S rRNA genes directly from<br />
environmental samples, it has been shown over the past two decades that<br />
archaea are not conf<strong>in</strong>ed to extreme habitats. By contrast, they have a<br />
ubiquitous distribution on this planet and occur <strong>in</strong> significant numbers <strong>in</strong><br />
common environments such as soils, mar<strong>in</strong>e plankton and sediments as well<br />
as <strong>in</strong> the deep subsurface (DeLong, 1998; Schleper et al., 2005). However,<br />
s<strong>in</strong>ce their <strong>in</strong>itial discovery, it took over a decade before any aspect of the<br />
physiology or ecological role of moderate, aerobic archaea could be determ<strong>in</strong>ed.<br />
Only very recently, metagenomic and cultivation studies have<br />
provided evidence that moderate archaea of terrestrial and mar<strong>in</strong>e environments<br />
are capable of ammonia oxidation and thus potentially represent<br />
important players <strong>in</strong> the global nitrogen cycle.<br />
Nitrification, the biological conversion of ammonia to nitrate via nitrite, is<br />
a central component of the natural nitrogen cycle (Prosser, 1989; Kowalchuk<br />
and Stephen, 2001). It is a two-step, aerobic, microbially mediated process,<br />
with ammonia first oxidised to nitrite by ammonia-oxidis<strong>in</strong>g microorganisms,
4 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
and nitrite subsequently oxidised to nitrate by nitrite-oxidis<strong>in</strong>g microorganisms.<br />
The first step is considered to be rate limit<strong>in</strong>g for this process<br />
(Kowalchuk and Stephen, 2001). Nitrification ensures the conversion of<br />
ammonia (derived from organic nitrogen dur<strong>in</strong>g decomposition and m<strong>in</strong>eralisation<br />
of biomass) <strong>in</strong>to the oxidised and more soluble form of nitrate, and<br />
provides the substrate for denitrification, which returns nitrogen back to the<br />
atmosphere. Nitrate is the preferred substrate of plants and aerobic microorganisms.<br />
However, the consequences of nitrification for human activities<br />
are also considerable. The process is used <strong>in</strong> wastewater treatment plants to<br />
remove urea and ammonia from sewage. In agricultural soils, the oxidation<br />
of ammonia to nitrate <strong>in</strong>creases the availability of nitrogen for plants, but it<br />
also has negative consequences, because this results <strong>in</strong> loss of vast amounts of<br />
nitrogen fertiliser from agricultural land by leach<strong>in</strong>g of the more soluble<br />
nitrate, result<strong>in</strong>g <strong>in</strong> groundwater pollution. Ammonia oxidisers have a further<br />
substantial environmental impact as contributors to greenhouse gas emissions<br />
via both ammonia oxidation directly as well as nitrifier-denitrification<br />
mechanisms (Wrage et al., 2001).<br />
S<strong>in</strong>ce the recognition of ammonia and nitrite-oxidis<strong>in</strong>g bacteria by Percy<br />
Faraday Frankland and Sergei W<strong>in</strong>ogradsky and others over 100 years ago,<br />
only proteobacteria of the beta- and gamma-subdivisions were considered as<br />
capable of perform<strong>in</strong>g aerobic ammonia oxidation (Purkhold et al., 2000).<br />
Here, we summarise the current knowledge of the recently discovered<br />
ammonia-oxidis<strong>in</strong>g archaea (AOA), their physiology, genomic potential<br />
and distribution and activity <strong>in</strong> various environments.<br />
2. DISCOVERY OF ARCHAEA IN MODERATE AEROBIC<br />
HABITATS<br />
Archaea <strong>in</strong> moderate aerobic habitats were first recognised by Fuhrman et al.,<br />
1992Fuhrman and colleagues (1992) and DeLong (1992), based on 16S rRNA<br />
gene surveys of mar<strong>in</strong>e environments. They were <strong>in</strong>itially grouped <strong>in</strong>to three<br />
previously undetected l<strong>in</strong>eages that were termed Group I, affiliated to the<br />
k<strong>in</strong>gdom of Crenarchaeota, and Groups II and III with<strong>in</strong> the k<strong>in</strong>gdom<br />
Euryarchaeota (DeLong, 1992) (Fig. 1a). In particular, organisms with<strong>in</strong><br />
Group I (a l<strong>in</strong>eage dist<strong>in</strong>ct from, but specifically associated with, cultured<br />
hyperthermophilic organisms) were found <strong>in</strong> many moderate habitats <strong>in</strong>clud<strong>in</strong>g<br />
soils (B<strong>in</strong>trim et al., 1997; Buckley et al., 1998; Sandaa et al., 1999; Jurgens<br />
et al., 2000; Simon et al., 2000; Ochsenreiter et al.,2003), the ocean’s plankton<br />
(DeLong, 1992; Fuhrman et al., 1992), estuaries (Crump and Baross, 2000),
[(Figure_1)TD$FIG]<br />
Figure 1 (a) Phylogenetic relationship between various archaeal 16S rRNA gene-def<strong>in</strong>ed l<strong>in</strong>eages, <strong>in</strong>clud<strong>in</strong>g sequences from<br />
cultivated organisms and environmental samples. Groups 1, 2 and 3 represent l<strong>in</strong>eages which were orig<strong>in</strong>ally discovered <strong>in</strong><br />
planktonic mar<strong>in</strong>e habitats, with Group 1 sequences now recovered <strong>in</strong> nearly all terrestrial and aquatic habitats. (b) Phylogenetic<br />
relationships of Group I archaea. Black triangles represent groups <strong>in</strong> which amoA genes have been discovered (adapted from<br />
Prosser and Nicol, 2008).<br />
AMMONIA-OXIDISING ARCHAEA 5
6 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
mar<strong>in</strong>e and freshwater sediments (MacGregor et al., 1997;Schleperet al.,<br />
1997; Vetriani et al.,1998;Keoughet al.,2003), but also <strong>in</strong> the deep subsurface<br />
(Takai et al., 2001). Extensive surveys of 16S rRNA gene sequences from<br />
mar<strong>in</strong>e and soil samples revealed that Group I Crenarchaeota can be separated<br />
<strong>in</strong>to a number of dist<strong>in</strong>ct clades, with the majority of soil and mar<strong>in</strong>e<br />
sequences placed with<strong>in</strong> two of these, referred to as Group 1.1a and 1.1b<br />
l<strong>in</strong>eages, respectively (Fig. 1b). While aspects of the physiology and energy<br />
metabolism of these organisms rema<strong>in</strong>ed unknown for a long time, some<br />
<strong>in</strong>itial <strong>in</strong>direct <strong>in</strong>sights <strong>in</strong>to the carbon metabolism of mar<strong>in</strong>e archaea were<br />
obta<strong>in</strong>ed us<strong>in</strong>g stable isotope, microautoradiography or natural radiocarbon<br />
analyses. They <strong>in</strong>dicated that both modes of carbon assimilation occurred<br />
with<strong>in</strong> mar<strong>in</strong>e archaea, that is autotrophy (us<strong>in</strong>g <strong>in</strong>organic carbon as a nutrient<br />
source) (e.g. Kuypers et al., 2001; Pearson et al., 2001; Wuchter et al.,2003)<br />
and heterotrophy (us<strong>in</strong>g organic carbon compounds as nutrients) (Ouverney<br />
and Fuhrman, 2000; Herndl et al., 2005; Ingalls et al., 2006;Teiraet al., 2006).<br />
3. FIRST INSIGHTS INTO THE PHYSIOLOGY<br />
OF AMMONIA-OXIDISING ARCHAEA<br />
The first <strong>in</strong>sight <strong>in</strong>to a specific energy metabolism of Group I archaea<br />
stemmed from a fosmid clone derived from a soil metagenomic library<br />
(Treusch and Schleper, 2004; Treusch et al., 2004a,b, 2005). It conta<strong>in</strong>ed an<br />
<strong>in</strong>sert of about 43 kb. Based on 16S and 23S rRNA genes, clone ‘54d9’ was<br />
identified as belong<strong>in</strong>g to the Group 1.1b l<strong>in</strong>eage (Fig. 2, deposited <strong>in</strong><br />
GenBank, February 2004). In addition, it conta<strong>in</strong>ed homologues to bacterial<br />
genes <strong>in</strong>volved <strong>in</strong> nitrogen cycl<strong>in</strong>g. Specifically, it conta<strong>in</strong>ed two open<br />
read<strong>in</strong>g frames (ORFs) cod<strong>in</strong>g for putative alpha and beta subunits<br />
(AmoA and AmoB, respectively) of an ammonia monooxygenase<br />
(AMO) as well as a gene whose product was highly similar to copperdependent<br />
nitrite reductases (NirK) (Treusch et al., 2005). An <strong>in</strong> silico<br />
comparison to environmental sequences deposited <strong>in</strong> public databases<br />
showed that the soil-derived archaeal amoA andamoB genes were highly<br />
similar to archaea-associated scaffolds from the whole-genome shotgun<br />
(WGS) sequenc<strong>in</strong>g project of the Sargasso Sea (Venter et al., 2004;<br />
Schleper et al., 2005). Additionally, the genomic fragments from mar<strong>in</strong>e<br />
archaea assembled <strong>in</strong> the Sargasso Sea project conta<strong>in</strong>ed genes cod<strong>in</strong>g for<br />
the C-subunit of an AMO, apparently organised <strong>in</strong> a cluster together with<br />
amoA andamoB <strong>in</strong> a BCA gene order and contrasted with the CAB<br />
arrangement found <strong>in</strong> ammonia-oxidis<strong>in</strong>g bacteria (AOB) (Nicol and
[(Figure_2)TD$FIG]<br />
Figure 2 Schematic diagram show<strong>in</strong>g predicted ORFs on the 43 kb soil fosmid 54d9, some of which are potentially <strong>in</strong>volved <strong>in</strong><br />
nitrogen transformations (amoA and amoB: genes for potential subunits of ammonia monooxygenase; nirK for nitrite reductase gene,<br />
ORF38 conserved <strong>in</strong> amo clusters) (Treusch et al., 2005). Homologues present <strong>in</strong> scaffolds assembled from the Sargasso Sea sequenc<strong>in</strong>g<br />
project (Venter et al., 2004) presented underneath. (Adapted from Schleper et al. (2005), with permission.)<br />
AMMONIA-OXIDISING ARCHAEA 7
8 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
[(Figure_3)TD$FIG]<br />
Figure 3 Maximum-likelihood tree of derived am<strong>in</strong>o acid sequences show<strong>in</strong>g the<br />
phylogenetic relationship of ammonia monooxygenases and particulate methane<br />
monooxygenases (AMO and pMMO respectively) of bacteria and archaea. The<br />
phylogenetic tree is based on 156 unambiguously aligned positions.<br />
Schleper, 2006). Sequence comparison of the soil- and mar<strong>in</strong>e-derived<br />
archaeal AmoA sequences to the alpha subunits of the bacterial AMO<br />
and the particulate methane monooxygenase (pMMO) from bacterial methane<br />
oxidisers <strong>in</strong>dicated that they were quite dist<strong>in</strong>ct (Fig. 3), with only about<br />
40% similarity ( 25% identity) at the am<strong>in</strong>o acid level. In contrast, the<br />
similarity between the two related prote<strong>in</strong>s <strong>in</strong> AMO and pMMO <strong>in</strong> bacteria<br />
is much higher with up to 74% ( 50% identity). Furthermore, the putative<br />
amo/pmo genes of archaea were considerably shorter than those of their<br />
bacterial homologues. A comparison with structural data obta<strong>in</strong>ed of the<br />
pMMO of Methylococcus capsulatus (Lieberman and Rosenzweig, 2005)<br />
brought further evidence that the respective archaeal ORFs <strong>in</strong>deed coded<br />
for subunits of an AMO/pMMO-related prote<strong>in</strong>, as many am<strong>in</strong>o acid
AMMONIA-OXIDISING ARCHAEA 9<br />
[(Figure_4)TD$FIG]<br />
Figure 4 Aerobic ammonia oxidation dur<strong>in</strong>g nitrification. The oxidation of<br />
ammonia is performed by ammonia oxidisers (archaea and bacteria), and the nitrite<br />
produced is subsequently oxidised by nitrite-oxidis<strong>in</strong>g bacteria. In bacteria, ammonia<br />
is oxidised to nitrite via the <strong>in</strong>termediate hydroxylam<strong>in</strong>e and the enzyme hydroxylam<strong>in</strong>e<br />
oxidoreductase (HAO). No HAO homologue has yet been identified <strong>in</strong><br />
archaea, and oxidation of ammonia to nitrite may occur via a different biochemical<br />
pathway (see hypotheses Fig. 7).<br />
residues potentially <strong>in</strong>volved <strong>in</strong> copper b<strong>in</strong>d<strong>in</strong>g metal centres were also found<br />
to be conserved <strong>in</strong> the archaeal variants (Treusch et al., 2005). Moreover,<br />
microcosm experiments with soil slurries were conducted to study transcription<br />
of the archaeal amoA genes. Upon <strong>in</strong>cubation with NH4 + a significant<br />
<strong>in</strong>crease <strong>in</strong> transcriptional activity of the putative amoA gene was observed,<br />
suggest<strong>in</strong>g that the amo-like genes <strong>in</strong>deed coded for a monooxygenase<br />
<strong>in</strong>volved <strong>in</strong> the oxidation of ammonia (Treusch et al., 2005). The ultimate<br />
support for this hypothesis came from the cultivation of an autotrophic<br />
AOA, Nitrosopumilus maritimus, that conta<strong>in</strong>s amoA genes highly related<br />
to those found on the fosmid clone 54d9 (K€onneke et al., 2005). These<br />
f<strong>in</strong>d<strong>in</strong>gs <strong>in</strong>dicated that archaea could be <strong>in</strong>volved <strong>in</strong> ammonia oxidation<br />
(Fig. 4) <strong>in</strong> many terrestrial and mar<strong>in</strong>e environments because 16S rRNA<br />
and amoA gene sequences related to those of N. maritimus and 54d9 were<br />
found <strong>in</strong> many habitats around the planet.<br />
4. MODEL ORGANISMS OF AMMONIA-OXIDISING ARCHAEA<br />
Chemolithoautotrophic ammonia oxidisers, bacteria or archaea, are notoriously<br />
difficult to grow and ma<strong>in</strong>ta<strong>in</strong> <strong>in</strong> (pure) laboratory cultures. This is also<br />
illustrated by the fact that stra<strong>in</strong> collections of AOB are only kept <strong>in</strong> a few<br />
specialised laboratories (J. Prosser, University of Aberdeen, pers. comm.).<br />
The isolation of the first AOA, Candidatus N. maritimus [nitrosus (lat<strong>in</strong>):
10 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
nitrous; pumilus (Lat<strong>in</strong>): dwarf; maritimus (Lat<strong>in</strong>): of the sea] (K€onneke et al.,<br />
2005) has therefore represented a breakthrough for the characterisation of<br />
AOA, provid<strong>in</strong>g the first <strong>in</strong>sights <strong>in</strong>to the general physiology and specific<br />
metabolism of these organisms. N. maritimus stra<strong>in</strong> SCM1 was isolated from<br />
a tropical mar<strong>in</strong>e aquarium and grows with a near-stoichiometric conversion<br />
of ammonia <strong>in</strong>to nitrite under aerobic conditions. It grows to a maximum<br />
density of about 1.4 10 7 cells mL 1 at 28 C <strong>in</strong> def<strong>in</strong>ed m<strong>in</strong>eral medium<br />
supplemented with bicarbonate and 500 mM ammonium (K€onneke et al.,<br />
2005), and its growth is <strong>in</strong>hibited when organic compounds are added even <strong>in</strong><br />
low amounts. N. maritimus is a straight, relatively small rod with a diameter<br />
of 0.17–0.22 mm and a length of 0.5–0.9 mm (Fig. 5a). Based on 16S rRNA<br />
gene phylogeny, N. maritimus, like the earlier described sponge symbiont<br />
Cenarchaeum symbiosum (Preston et al., 1996), belongs to the Group 1.1a<br />
l<strong>in</strong>eage of mar<strong>in</strong>e archaea that is found <strong>in</strong> large numbers <strong>in</strong> the open ocean.<br />
Us<strong>in</strong>g the sequence <strong>in</strong>formation from metagenomic studies, K€onneke et al.<br />
(2005) designed primers aga<strong>in</strong>st amoA, amoB and amoC sequences and<br />
were able to amplify the respective homologous genes from N. maritimus.<br />
This analysis provided the direct l<strong>in</strong>k between the metagenomic predictions<br />
and physiological studies. In contrast to AOB, N. maritimus is apparently<br />
adapted to an extremely oligotrophic lifestyle (Martens-Habbena et al.,<br />
2009). It possesses a half-saturation constant (Km = 133 nM total ammonium)<br />
and a substrate threshold (
AMMONIA-OXIDISING ARCHAEA 11<br />
Two enrichments of thermophilic AOA have further demonstrated the<br />
capability of archaea to grow autotrophically with ammonia as a sole energy<br />
source. In an enrichment of ammonia-oxidis<strong>in</strong>g microorganisms from a<br />
microbial mat from the Siberian Garga hot spr<strong>in</strong>g (Lebedeva et al., 2005),<br />
cells of Candidatus Nitrososphaera gargensis [nitrosus (Lat<strong>in</strong>): nitrous;<br />
sphaera (Lat<strong>in</strong>): spherical; gargensis: from Garga spr<strong>in</strong>g] (Hatzenpichler<br />
et al., 2008) were visualised us<strong>in</strong>g CARD-FISH and simultaneous <strong>in</strong>corporation<br />
of radioactive labelled bicarbonate <strong>in</strong>dicat<strong>in</strong>g ammonia-dependent<br />
autotrophic growth at concentrations of 0.14 and 0.79 mM NH4 + .<br />
However, at higher concentrations, around 3 mM ammonium, or at lower<br />
concentrations but <strong>in</strong> the presence of allylthiourea, a potent <strong>in</strong>hibitor of<br />
ammonia oxidation, this <strong>in</strong>corporation was considerably reduced.<br />
Interest<strong>in</strong>gly, N. gargensis belongs to the l<strong>in</strong>eage of AOA dist<strong>in</strong>ct from<br />
mar<strong>in</strong>e AOA, the Group 1.1b l<strong>in</strong>eage, which is mostly represented by environmental<br />
sequences from soils and other terrestrial habitats (Ochsenreiter<br />
et al., 2003; Nicol et al., 2006). It is therefore the first representative of this<br />
second major l<strong>in</strong>eage which has been obta<strong>in</strong>ed <strong>in</strong> laboratory enrichments.<br />
The thermophilic AOA Candidatus Nitrosocaldus yellowstonii [nitrosus<br />
(Lat<strong>in</strong>): nitrous; caldus (Lat<strong>in</strong>): hot; yellowstonii: from Yellowstone] (de la<br />
Torre et al., 2008) obta<strong>in</strong>ed from a hot spr<strong>in</strong>g located <strong>in</strong> the Yellowstone<br />
National Park has an even higher optimal growth temperature between 65<br />
and 72 C, aga<strong>in</strong> grow<strong>in</strong>g with a stoichiometric conversion of ammonia to<br />
nitrite. This organism was particularly <strong>in</strong>terest<strong>in</strong>g as it not only extended the<br />
temperature limit for cultivated ammonia oxidisers, but broadened the<br />
known 16S rRNA and amoA gene diversity associated with ammonia oxidation<br />
(Table 1, Fig. 3).<br />
C. symbiosum [cen from Greek ka<strong>in</strong>os/ko<strong>in</strong>os: recent/common refers to<br />
the recent (derived from thermophiles) and widespread (common) occurrence<br />
of this group of archaea; (Preston et al., 1996)] was detected by 16S<br />
rRNA-based PCR surveys among the complex microbial communities<br />
with<strong>in</strong> the tissues of the mar<strong>in</strong>e sponge Ax<strong>in</strong>ella mexicana (Preston et al.,<br />
1996). S<strong>in</strong>ce it can be ‘quasi’-cultivated <strong>in</strong> the laboratory by ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g it <strong>in</strong><br />
stable association with its host under controlled conditions, it was the first<br />
described mesophilic crenarchaeon. The Cenarchaeum/Ax<strong>in</strong>ella association<br />
provided the first tractable system for the study of mar<strong>in</strong>e archaea and gave<br />
access to relatively large amounts of biomass of this species, allow<strong>in</strong>g the<br />
study of lipids, genomic DNA and cell structure by microscopy. As it is<br />
ma<strong>in</strong>ta<strong>in</strong>ed with<strong>in</strong> the tissues of the sponge and cannot be cultivated <strong>in</strong>dependently,<br />
ammonia oxidation by C. symbiosum has not been proven<br />
directly. However, the genome sequence of this organism, the presence of<br />
archaeal amoA genes and measured ammonia oxidation activity <strong>in</strong> other
Table 1 Ammonia-oxidis<strong>in</strong>g archaea <strong>in</strong> laboratory cultures or enrichments.<br />
Characteristics Nitrosopumilus<br />
maritimus<br />
Cenarchaeum<br />
symbiosum<br />
Nitrosocaldus<br />
yellowstonii<br />
Nitrososphaera<br />
gargensis<br />
Orig<strong>in</strong> Tropical mar<strong>in</strong>e Mar<strong>in</strong>e sponge symbiont Terrestrial hot<br />
Terrestrial warm spr<strong>in</strong>g<br />
aquarium<br />
spr<strong>in</strong>g<br />
Culture Pure laboratory Inside Ax<strong>in</strong>ella mexicana Laboratory<br />
Laboratory enrichment<br />
culture<br />
(aquarium)<br />
enrichment<br />
Affiliation Group 1.1a<br />
Group 1.1a<br />
HWCGIII Group 1.1b<br />
Thaumarchaeota Thaumarchaeota<br />
Thaumarchaeota<br />
Growth<br />
temperature<br />
28 C 10 C(8–18 C) 72 C (65–74 C) 46 C<br />
Shape Rod Curved rod Spherical Spherical, coccoid<br />
Genome 1.65 Mb 2.05 Mb n.d. >2.6 Mb, draft<br />
Reference K€onneke et al. (2005) Preston et al. (1996) de la Torre et al. (2008) Hatzenpichler et al. (2008),<br />
Spang et al. (2010)<br />
12 CHRISTA SCHLEPER AND GRAEME W. NICOL
AMMONIA-OXIDISING ARCHAEA 13<br />
mar<strong>in</strong>e sponges (Taylor et al., 2007; Bayer et al., 2008; Steger et al., 2008;<br />
Hoffmann et al., 2009) strongly support the hypothesis that C. symbiosum,<br />
like its close free-liv<strong>in</strong>g relatives <strong>in</strong> mar<strong>in</strong>e water, is capable of ammonia<br />
oxidation.<br />
5. MEMBRANE LIPIDS OF AMMONIA-OXIDISING ARCHAEA<br />
S<strong>in</strong>ce their discovery as a separate doma<strong>in</strong>, dist<strong>in</strong>ct from the eukaryotes (or<br />
Eukarya) and bacteria based on 16S rRNA gene phylogenies (Woese and<br />
Fox, 1977), the archaea have been found to possess a number of cellular and<br />
molecular features that clearly dist<strong>in</strong>guishes them from the other two<br />
doma<strong>in</strong>s. The best dist<strong>in</strong>guish<strong>in</strong>g feature is the membrane lipids of archaea,<br />
which are fundamentally different from all representatives <strong>in</strong> the other two<br />
doma<strong>in</strong>s (Brown and Doolittle, 1997). The phospholipids of archaea are<br />
glycerol–ether lipids <strong>in</strong> which isoprenoid side cha<strong>in</strong>s, not fatty acids like <strong>in</strong><br />
bacteria or Eukarya, are l<strong>in</strong>ked via an ether bond (<strong>in</strong>stead of an ester bond)<br />
to a glycerol moiety, and have a stereochemistry that is reverse of that <strong>in</strong><br />
bacteria and Eukarya. Furthermore, the C20-isoprenoid side cha<strong>in</strong>s are often<br />
l<strong>in</strong>ked to each other, which leads to the formation of a lipid monolayer with<br />
C 40 side cha<strong>in</strong>s, <strong>in</strong>stead of the typical membrane bilayer found <strong>in</strong> other<br />
organisms. Thus, the core, apolar component of archaeal cellular membrane<br />
lipids (<strong>in</strong> particular those of Crenarchaeota) are dom<strong>in</strong>ated by glycerol<br />
dialkyl glycerol tetraethers (GDGTs, see Fig. 6). The side cha<strong>in</strong>s may conta<strong>in</strong><br />
multiple cyclopentane moieties (see Fig. 5), whose numbers have been<br />
shown to <strong>in</strong>crease <strong>in</strong> response to an <strong>in</strong>crease <strong>in</strong> growth temperature of<br />
Archaeoglobus species (Lai et al., 2008). Interest<strong>in</strong>gly, a specific structure,<br />
a unique GDGT with a cyclohexane moiety <strong>in</strong> addition to the four cyclopentane<br />
r<strong>in</strong>gs (Fig. 5, bottom), was found <strong>in</strong> natural samples from moderate<br />
environments as well as <strong>in</strong> the mar<strong>in</strong>e sponge A. mexicana, which harbours<br />
C. symbiosum, a member of the mar<strong>in</strong>e Group 1.1a (Damste et al., 2002). It<br />
was thus termed ‘crenarchaeol’, although this lipid compound had never<br />
been found <strong>in</strong> cultivated hyperthermophilic Crenarchaeota and seems to<br />
be present <strong>in</strong> ‘mesophilic’ archaea only. In support of this, crenarchaeol<br />
and the other GDGT compounds have also been isolated from the laboratory<br />
cultures of N. maritimus (Schouten et al., 2008). In addition, with the<br />
f<strong>in</strong>d<strong>in</strong>g that the moderate thermophile N. gargensis (Pitcher et al., 2009) and<br />
the extremely thermophilic N. yellowstonii (de la Torre et al., 2008) also<br />
conta<strong>in</strong> crenarchaeol, and with the recovery of crenarchaeol from various<br />
hot spr<strong>in</strong>gs around the world (Pearson et al., 2004; Zhang et al., 2006;
14 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
[(Figure_6)TD$FIG]<br />
Figure 6 (a) Isoprenoidal glycerol dialkyl glycerol tetraethers (GDGTs) of<br />
archaea, <strong>in</strong>clud<strong>in</strong>g crenarchaeol that is so far exclusively found <strong>in</strong> cultured<br />
Thaumarchaeota and <strong>in</strong> many natural environments. (b) Correlation between<br />
GDGT abundance and AmoA gene copies (eight different soils types). (Adapted<br />
from data obta<strong>in</strong>ed by Le<strong>in</strong><strong>in</strong>ger et al. (2006), with permission.)<br />
Reigstad et al., 2008; Pitcher et al., 2009), it is now evident that this compound<br />
is not an <strong>in</strong>dicator for mesophilic archaea per se, but rather for certa<strong>in</strong><br />
l<strong>in</strong>eages of archaea to which ammonia oxidisers are affiliated (Pitcher<br />
et al., 2009). At least <strong>in</strong> some environments, crenarchaeol could be a suitable<br />
biomarker for AOA (Wuchter et al., 2004; Coolen et al., 2007; Schouten et al.,<br />
2007). For example, <strong>in</strong> 8 out of 12 <strong>in</strong>vestigated soils, the abundance of<br />
crenarchaeol (as well as the abundance of total GDGTs) correlated significantly<br />
with the abundance of amoA genes (Le<strong>in</strong><strong>in</strong>ger et al., 2006). Only the<br />
analysis of more isolates of AOA and of other more remotely related<br />
l<strong>in</strong>eages of archaea will help to clarify whether this lipid compound is<br />
restricted to ammonia oxidisers.<br />
It is <strong>in</strong>terest<strong>in</strong>g to note <strong>in</strong> this context that the relative abundance of<br />
crenarchaeol <strong>in</strong> the different laboratory cultures of AOA seems to vary quite<br />
considerably. The GDGTs of N. gargensis (grown at 46 C) consisted ma<strong>in</strong>ly<br />
of crenarchaeol, its regioisomer and a novel GDGT, while crenarchaeol was<br />
a m<strong>in</strong>or compound <strong>in</strong> the other organisms. Vary<strong>in</strong>g relative amounts of the<br />
crenarchaeol or its regioisomer <strong>in</strong> different l<strong>in</strong>eages of AOA could potentially<br />
confuse the TEX86 <strong>in</strong>dex that is used for paleothermometry, that is for<br />
reconstruct<strong>in</strong>g average temperatures experienced by plankton <strong>in</strong> earlier
AMMONIA-OXIDISING ARCHAEA 15<br />
times by measur<strong>in</strong>g the relative composition of sedimentary archaeal membrane<br />
lipids (Wuchter et al., 2004).<br />
6. GENOMES AND METAGENOMES<br />
OF AMMONIA-OXIDISING ARCHAEA<br />
Inspired by the rapid advances <strong>in</strong> genomic techniques applied to cultivated<br />
microorganisms, Ste<strong>in</strong> et al. (1996) used a BAC-derived fosmid vector to<br />
prepare a large-<strong>in</strong>sert library from mar<strong>in</strong>e water of the North-Eastern Pacific<br />
<strong>in</strong> order to characterise mar<strong>in</strong>e planktonic archaea. A 38.5-kb genomic<br />
fragment of an uncultivated mesophilic Crenarchaeota was identified with<strong>in</strong><br />
3552 clones us<strong>in</strong>g archaea-specific 16S rRNA gene probes. This study was<br />
the beg<strong>in</strong>n<strong>in</strong>g of a novel and now explod<strong>in</strong>g field of microbial environmental<br />
genomics or ‘metagenomics’. Large genome fragments are cloned <strong>in</strong>to bacterial<br />
artificial chromosomes (BACs) or, more commonly, BAC-derived<br />
fosmid vectors (a hybrid of a cosmid and BAC) which are archived <strong>in</strong><br />
Escherichia coli clone libraries (Handelsman, 2004; Treusch and Schleper,<br />
2005). Alternatively, large-scale sequenc<strong>in</strong>g us<strong>in</strong>g a whole-genome-shotgun<br />
approach allows the generation of small sequence reads from environmental<br />
samples that can be assembled <strong>in</strong>to larger fragments <strong>in</strong> silico (Venter et al.,<br />
2004). These techniques are now <strong>in</strong>creas<strong>in</strong>gly used for the characterisation of<br />
microbial communities, particularly s<strong>in</strong>ce novel deep sequenc<strong>in</strong>g technologies<br />
allow <strong>in</strong>creas<strong>in</strong>gly cheaper and high-throughput analysis. The first complete<br />
genome of a potential AOA (C. symbiosum) was also assembled from a<br />
metagenomic library, and now with cultivated or enriched organisms available,<br />
complete reference genomes of the first model organisms are easily<br />
obta<strong>in</strong>ed. While the complete genome sequences will be <strong>in</strong>valuable for the<br />
reconstruction of full metabolic pathways, the metagenomic datasets of<br />
AOA are of great importance to study the distribution and the genomic<br />
potential of the organisms <strong>in</strong> the various environments.<br />
6.1. Metagenomic Studies of Uncultivated Ammonia Oxidisers<br />
Several metagenomic libraries from microorganisms associated with a<br />
mar<strong>in</strong>e sponge, mar<strong>in</strong>e plankton or soil have been produced to characterise<br />
the genomic content of Group I archaea (Schleper et al., 1998; Beja et al.,<br />
2000, 2002; Quaiser et al., 2002; López-Garcıa et al., 2004; Treusch et al.,<br />
2004). Soil fosmid 54d9 was detected <strong>in</strong> such a library and led to the discovery<br />
of amo-related genes <strong>in</strong> archaea (Treusch et al., 2005). Comparative analyses
16 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
of metagenomic clones from the mar<strong>in</strong>e plankton <strong>in</strong>dicated the conservation<br />
of gene order around the 16S rRNA gene, thus confirm<strong>in</strong>g the close relationship<br />
of the planktonic archaea, even <strong>in</strong> stra<strong>in</strong>s from different oceanic<br />
prov<strong>in</strong>ces (Beja et al., 2002; López-Garcıa et al., 2004). Conversely, considerable<br />
genomic variation was dissected, <strong>in</strong>clud<strong>in</strong>g microheterogeneity, <strong>in</strong><br />
prote<strong>in</strong>-encod<strong>in</strong>g regions and <strong>in</strong>tergenic spacers, when genome fragments<br />
with otherwise identical or almost identical 16S rRNA genes were compared<br />
from the same DNA library (Schleper et al., 1998; Beja et al., 2002). Given the<br />
abundance and ubiquity of mar<strong>in</strong>e planktonic archaea, it is plausible that<br />
large numbers of archaeal genes would be detected <strong>in</strong> global random<br />
sequenc<strong>in</strong>g surveys of DNA obta<strong>in</strong>ed from filtered waters (Venter et al.,<br />
2004; Nealson and Venter, 2007). The huge databases from BAC and fosmid<br />
libraries (Treusch et al., 2004; Mart<strong>in</strong>-Cuadrado et al., 2008; Konstant<strong>in</strong>idis<br />
et al., 2009) as well as large-scale shotgun sequenc<strong>in</strong>g efforts are a valuable<br />
resource for dissect<strong>in</strong>g the diversity and distribution of AOA, particularly<br />
s<strong>in</strong>ce the first genomes of cultivated isolates are now available that allow us<br />
to test hypotheses on gene functions and metabolisms experimentally.<br />
6.2. Predictions from Complete Genome Sequences of Two<br />
Mar<strong>in</strong>e Archaea<br />
Although C. symbiosum has not been cultivated or completely physically<br />
separated from the tissues of its host (the mar<strong>in</strong>e sponge A. mexicana) and<br />
from the co-exist<strong>in</strong>g bacteria, cell fractions that were enriched with the<br />
archaeon were used for the construction of large-<strong>in</strong>sert genomic libraries<br />
(Schleper et al., 1997, 1998), facilitat<strong>in</strong>g the isolation of genome fragments<br />
and lead<strong>in</strong>g to a genome sequenc<strong>in</strong>g project that was completed <strong>in</strong> 2006<br />
(Hallam et al., 2006a, 2006bHallam et al., 2006a,b). The second genome from<br />
N. maritimus, the first cultivated isolate of mar<strong>in</strong>e AOA, has recently been<br />
completed (Walker et al., 2010).<br />
The C. symbiosum genome has a considerably higher G + C content<br />
(>55%) (Schleper et al., 1998; Hallam et al., 2006a) than its relatives <strong>in</strong> the<br />
mar<strong>in</strong>e plankton (approximately 34%), which might reflect adaptation to the<br />
lifestyle <strong>in</strong> the metazoan host, rather than a large evolutionary distance.<br />
Despite this difference, however, the two organisms show high overlap <strong>in</strong><br />
gene content with each other (approx. 1200 genes) and with mar<strong>in</strong>e metagenomes,<br />
<strong>in</strong>dicat<strong>in</strong>g that they can serve as suitable models to study the<br />
globally distributed and abundant mar<strong>in</strong>e planktonic archaea (Walker<br />
et al., 2010). With a few m<strong>in</strong>or exceptions, both genomes share similar gene<br />
content with respect to potential energy metabolism and carbon fixation
AMMONIA-OXIDISING ARCHAEA 17<br />
pathways, both of which seem to be clearly different from known bacterial<br />
ammonia oxidisers. AOA conta<strong>in</strong> amoA, B and C genes for the three subunits<br />
of a potential archaeal AMO, but no homologous genes of the bacterial<br />
hydroxylam<strong>in</strong>e oxidoreductase (HAO) complex that catalyses the second<br />
step of this process <strong>in</strong> bacteria, that is the oxidation of hydroxylam<strong>in</strong>e to<br />
nitrite (Hallam et al., 2006b; Walker et al., 2010). In AOB, this complex<br />
delivers electrons back to the AMO and to an electron transport cha<strong>in</strong> with<br />
cytochrome c prote<strong>in</strong>s (c 554 and c 552) required for electron flow to ubiqu<strong>in</strong>one<br />
(Fig. 6). These cytochromes are also not present <strong>in</strong> AOA, but they do<br />
possess numerous copper-conta<strong>in</strong><strong>in</strong>g prote<strong>in</strong>s such as multicopper oxidases,<br />
small blue copper-conta<strong>in</strong><strong>in</strong>g prote<strong>in</strong>s (Hallam et al., 2006a; Bartossek et al.,<br />
2010; Walker et al., 2010) as well as potential thiol-disulphide oxidoreductases,<br />
which may functionally replace cytochromes (Walker et al., 2010). In<br />
pr<strong>in</strong>ciple, it appears that the energy metabolism of AOA relies on copperrather<br />
than on iron-conta<strong>in</strong><strong>in</strong>g electron transfer systems (Hallam et al.,<br />
2006a; Walker et al., 2010).<br />
While C. symbiosum (like mar<strong>in</strong>e metagenomes) conta<strong>in</strong>s genes for urease,<br />
<strong>in</strong>dicat<strong>in</strong>g a potential broader substrate spectrum, N. maritimus does<br />
not. Both organisms also do not seem to conta<strong>in</strong> a homologue of nitric oxide<br />
reductase, that is part of the denitrification pathway of AOB, reduc<strong>in</strong>g nitric<br />
oxide (NO) to nitrous oxide (N 2O). N. maritimus (Walker et al., 2010), as well<br />
as soil archaea (Treusch et al., 2004a; Bartossek et al., 2010), does, however,<br />
conta<strong>in</strong> homologues of nitrite reductase, the first enzyme <strong>in</strong>volved <strong>in</strong> dentrification,<br />
that (<strong>in</strong> bacteria) reduces nitrite, the product of ammonia oxidation,<br />
to nitric oxide. In total, the genetic makeup of archaeal ammonia<br />
oxidisers <strong>in</strong>dicates that the biochemistry underly<strong>in</strong>g ammonia oxidation to<br />
nitrite could be fundamentally different from that of AOB. Even the key<br />
metabolic enzyme, AMO, shows only little sequence similarity to bacterial<br />
AMO and pMMOs. It is possible that AOA use the same pathway as AOB,<br />
that is oxidis<strong>in</strong>g ammonia via hydroxylam<strong>in</strong>e to nitrite, but with different<br />
enzymes. Alternatively, a fundamentally different pathway may be operat<strong>in</strong>g.<br />
Mart<strong>in</strong> Klotz (University of Louisville, Kentucky, USA) has proposed<br />
an alternative pathway for AOA that does not <strong>in</strong>volve hydroxylam<strong>in</strong>e as an<br />
<strong>in</strong>termediate, but rather nitroxyl (HNO) (see Walker et al., 2010)(Fig. 7). A<br />
nitroxyl oxidoreductase could then operate to oxidise nitroxyl to nitrite<br />
(Walker et al., 2010). In his extended model, Klotz proposes that the activation<br />
of AMO could be achieved by recycl<strong>in</strong>g NO, the product of nitrite<br />
reduction via nitrite reductase (see Fig. 7). The activation of O 2 by NO would<br />
then result <strong>in</strong> the production of N2. If this or a similar pathway for ammonia<br />
oxidation is <strong>in</strong>deed operat<strong>in</strong>g, it would <strong>in</strong>dicate most probably that AOA do<br />
not produce the greenhouse gas N2O, as do their bacterial counterparts. It is
18 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
[(Figure_7)TD$FIG]<br />
Figure 7 Proposed pathways of nitrogen, oxygen and electron flow <strong>in</strong> the qu<strong>in</strong>one-oxidis<strong>in</strong>g<br />
and -reduc<strong>in</strong>g branches of the electron transport cha<strong>in</strong>s (ETC) <strong>in</strong><br />
ammonia-oxidis<strong>in</strong>g bacteria (AOB) and archaea (AOA). (a) Basic <strong>in</strong>ventory encoded<br />
<strong>in</strong> all AOB. Ammonia monooxygenase (AMO); N-oxide-Ubiqu<strong>in</strong>one Redox Module<br />
(NURM) consist<strong>in</strong>g of hydroxylam<strong>in</strong>e oxidoreductase (HAO), cytochrome c554 and<br />
qu<strong>in</strong>one reductase cM552; cytochrome c552; cytochromes b and c1 (complex III), with<br />
several proton-pump<strong>in</strong>g oxygen-reduc<strong>in</strong>g heme-copper oxidases (complex IV) and<br />
several NO-reduc<strong>in</strong>g heme-copper oxidases (HCO-NOR) <strong>in</strong> the membrane-associated<br />
branch, and copper-conta<strong>in</strong><strong>in</strong>g nitrite reductase (NirK) and cytochrome c NOreductases<br />
<strong>in</strong> the soluble branch of the ETC, plus bacterial F0F 1-type ATP synthetase.<br />
The question mark <strong>in</strong>dicates that a direct qu<strong>in</strong>ol oxidase function of bacterial AMO/<br />
pMMO has not yet been demonstrated. (b) Basic <strong>in</strong>ventory predicted from genome<br />
sequences <strong>in</strong> AOA. AMO; NURM consist<strong>in</strong>g of nitroxyl oxidoreductase (NxOR) and<br />
plastocyan<strong>in</strong>-doma<strong>in</strong> conta<strong>in</strong><strong>in</strong>g membrane-associated putative qu<strong>in</strong>one reductase<br />
(Pcy); plastocyan<strong>in</strong>s (pcy); cytochrome b and associated plastocyan<strong>in</strong> (complex III)<br />
with several proton-pump<strong>in</strong>g oxygen-reduc<strong>in</strong>g plastocyan<strong>in</strong>-copper oxidases (complex<br />
IV) plus F0F 1-type ATP synthetase <strong>in</strong> the membrane-associated branch.<br />
Copper-conta<strong>in</strong><strong>in</strong>g nitrite reductase (NirK), active <strong>in</strong> the soluble branch of the<br />
ETC, has been identified <strong>in</strong> all AOA genomes with the exception of Cenarchaeum<br />
symbiosum (Bartossek et al., 2010). No <strong>in</strong>ventory encod<strong>in</strong>g cytochrome c and hemecopper<br />
NO-reductases has been identified. The asterisks <strong>in</strong>dicate the need for activation<br />
of oxygen <strong>in</strong> the monooxygenase reaction facilitated by AMO. Because a<br />
qu<strong>in</strong>ol oxidase function of archaeal AMO is not part of the model, it is predicted that<br />
oxygen activation <strong>in</strong> AOA is accomplished by utilis<strong>in</strong>g the NO radical produced <strong>in</strong> the<br />
soluble branch of the ETC. This would produce one-half molecule of d<strong>in</strong>itrogen gas<br />
per oxidised ammonia and <strong>in</strong>troduce AOA as non-classical aerobic denitrifiers. This<br />
chemistry needs to be experimentally tested as <strong>in</strong>dicated by ‘??’ (Figure and legend<br />
k<strong>in</strong>dly provided by Prof. Mart<strong>in</strong> G. Klotz, University of Louisville, USA).
AMMONIA-OXIDISING ARCHAEA 19<br />
<strong>in</strong>terest<strong>in</strong>g to note <strong>in</strong> this context that Bartossek et al. (2010) recently found<br />
variants of genes encod<strong>in</strong>g for copper-dependent nitrite reductases <strong>in</strong> soils<br />
and other environments, <strong>in</strong>dicat<strong>in</strong>g that this enzyme might <strong>in</strong>deed be widely<br />
distributed <strong>in</strong> AOA. Furthermore, transcription of the nitrite reductase<br />
homologue <strong>in</strong> soil archaea was observed even under aerobic (and potentially<br />
ammonia-oxidis<strong>in</strong>g) conditions, rather than under low-oxygen conditions<br />
that favour denitrification (Bartossek et al., 2010). Thus, the copperdependent<br />
nitrite reductase of archaea might <strong>in</strong>deed fulfill a different function<br />
<strong>in</strong> AOA metabolism than is assumed for the bacterial counterpart.<br />
From the genome sequences of the two mar<strong>in</strong>e organisms one can also<br />
deduce a possible pathway for carbon fixation. Whereas autotrophic AOB<br />
fix carbon with the ribulose bisphosphate carboxylase/oxygenase (RubisCO)<br />
<strong>in</strong> the Calv<strong>in</strong>–Bassham–Benson cycle, N. maritimus and C. symbiosum seem<br />
to conta<strong>in</strong> a different pathway, similar to that recently described for the<br />
hyperthermophilic archaeon Metallosphaera sedula (Berg et al., 2007). The<br />
pathway <strong>in</strong>volves the transformation of acetyl-CoA with two bicarbonate<br />
molecules via 3-hydroxyproprionate to succ<strong>in</strong>yl-CoA. This <strong>in</strong>termediate is<br />
reduced to 4-hydroxybutyrate and subsequently converted <strong>in</strong>to two acetyl-<br />
CoA molecules. Key enzymes for this pathway are found <strong>in</strong> both AOA<br />
organisms, such as the biot<strong>in</strong>ylated acetyl-CoA/propionyl-CoA carboxylase,<br />
methylmalonyl-CoA epimerase and mutase and 4-hydroxybutyrate dehydratase,<br />
but some prote<strong>in</strong>s of the M. sedula pathway are also miss<strong>in</strong>g,<br />
<strong>in</strong>dicat<strong>in</strong>g the AOA use a variant of this pathway or may possess nonorthologous<br />
gene replacements (Hallam et al., 2006b; Walker et al., 2010).<br />
While autotrophic growth of N. maritimus and its <strong>in</strong>hibition by organic<br />
compounds has been reported, both AOA conta<strong>in</strong> components of an oxidative<br />
TCA cycle that can potentially be utilised for the consumption of organic<br />
carbon or for the production of <strong>in</strong>termediates for am<strong>in</strong>o acid and cofactor<br />
biosynthesis. Mixotrophic growth has not been shown yet for any of the<br />
cultivated or enriched AOA, but it might well be possible that this growth<br />
mode will be found as more organisms are obta<strong>in</strong>ed <strong>in</strong> laboratory cultures.<br />
6.3. Ammonia Oxidisers: A Dist<strong>in</strong>ct Phylum with<strong>in</strong> the Archaea<br />
Based on 16S rRNA sequence phylogeny, AOA were orig<strong>in</strong>ally placed as a<br />
sister group of the Crenarchaeota (DeLong, 1992; Fuhrman et al., 1992),<br />
suggest<strong>in</strong>g that these archaea might have ancestors <strong>in</strong> hot spr<strong>in</strong>gs and only<br />
later radiated <strong>in</strong>to moderate environments. The AOA have s<strong>in</strong>ce been<br />
referred to as Crenarchaeota <strong>in</strong> all follow<strong>in</strong>g 16S rRNA-based diversity
20 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
studies. Us<strong>in</strong>g a concatenated dataset of 53 ribosomal prote<strong>in</strong>s from C.<br />
symbiosum which are shared by archaea and eukaryotes, Brochier-<br />
Armanet et al. (2008) calculated that ‘moderate Crenarchaeota’ constitute<br />
a separate phylum of the archaea that branches off before the separation of<br />
Crenarchaeota and Euryarchaeota. Includ<strong>in</strong>g the genomic <strong>in</strong>formation of N.<br />
maritimus and N. gargensis (draft genome) <strong>in</strong> this analysis confirmed the<br />
separation of AOA (Spang et al., 2010). The name Thaumarchaeota (from<br />
the Greek word ‘thaumas’ for wonder) was proposed for the new phylum<br />
(Brochier-Armanet et al., 2008).<br />
Several <strong>in</strong>formation process<strong>in</strong>g genes, whose presence or absence is characteristic<br />
for Euryarchaeota and/or Crenarchaeota, show a pattern <strong>in</strong> the<br />
three <strong>in</strong>vestigated Thaumarchaeota genomes that is dist<strong>in</strong>ctive from either of<br />
the two described phyla and this po<strong>in</strong>ts to fundamental differences <strong>in</strong> cellular<br />
processes. Thus, this gene content comparison strongly supports the<br />
Thaumarchaeota proposal (Table 2 and Brochier-Armanet et al., 2008;<br />
Spang et al., 2010). Most notably, Thaumarchaeota possess unsplit RNA<br />
Table 2 Distribution of core <strong>in</strong>formational process<strong>in</strong>g genes <strong>in</strong> the four different<br />
phyla of archaea.<br />
Thaumarchaeota<br />
Euryarchaeota<br />
Crenarchaeota<br />
Ribosomal prote<strong>in</strong>s<br />
r-prote<strong>in</strong> S25e + + +<br />
r-prote<strong>in</strong> S26e + + +<br />
r-prote<strong>in</strong> S30e + + +<br />
r-prote<strong>in</strong> L13e + +<br />
r-prote<strong>in</strong> L14e + (some) + +<br />
r-prote<strong>in</strong> L34e + (some) + +<br />
r-prote<strong>in</strong>s L38e W<br />
r-prote<strong>in</strong> L29p + + +<br />
r-prote<strong>in</strong> Lxa + (most) +<br />
Transcription/RNA polymerase<br />
rpoG (=rpo8) + +<br />
rpoA – s<strong>in</strong>gle ORF + split split +<br />
rpoB – s<strong>in</strong>gle ORF + W split + +<br />
Rpc34 + W +<br />
MBF 1 W + + +<br />
EIF 1 + +<br />
Korarchaeota<br />
(cont<strong>in</strong>ued)
AMMONIA-OXIDISING ARCHAEA 21<br />
Table 2 (cont<strong>in</strong>ued)<br />
Thaumarchaeota<br />
Euryarchaeota<br />
Crenarchaeota<br />
DNA polymerases/replication<br />
DNA pol D + + +<br />
RPA (Eury-like) + + +<br />
RPA/SSB + + (some) +/( ) +<br />
>one PCNA gene +<br />
Topoisomerases<br />
Topo IB +<br />
Topo IA W + + +<br />
Reverse gyrase + (HT) + +<br />
Topo IIA W<br />
Cell division<br />
ESCRT-III + +<br />
Vps4 (CdvC) + +<br />
CdvA + +<br />
Fts Z + + +<br />
Smc + + +<br />
ScpA + ScpB + + (many) +<br />
Histones (H3/H4)<br />
+ + (exc. 2) +<br />
Repair<br />
Hef-nuclease +<br />
ERCC4-type nuclease + + +<br />
ERRC4-type helicase +<br />
RadB +<br />
HSP70s, GrpE, Hsp40 + +<br />
UvrABC + W<br />
Korarchaeota<br />
polymerase A and B genes, both topoisomerases IA and B, and histones, but<br />
lack the archaea-specific r-prote<strong>in</strong> LXa. Thaumarchaeota, like all other<br />
archaea, conta<strong>in</strong> genes for central <strong>in</strong>formation process<strong>in</strong>g mach<strong>in</strong>ery (replication,<br />
transcription, etc.) that are shared with eukaryotes or are more<br />
closely related to eukaryotes than to bacteria (Bell and Jackson, 1998;<br />
Garrett and Klenk, 2007). In several phylogenetic analyses with <strong>in</strong>formation<br />
process<strong>in</strong>g factors, such as RNA polymerase subunits (see Fig. 8),<br />
Thaumarchaeota branch off early, <strong>in</strong>dicat<strong>in</strong>g that a more detailed characterisation<br />
of this previously enigmatic group might change our perception of the<br />
early evolution with<strong>in</strong> the archaeal and eukaryotic l<strong>in</strong>eage (Spang et al.,<br />
2010).
22 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
[(Figure_8)TD$FIG]<br />
Figure 8 Phylogenetic tree of RNA polymerase subunit A (rpoA) and schematic<br />
overview of gene arrangements <strong>in</strong> different archaeal l<strong>in</strong>eages and <strong>in</strong> bacteria and<br />
eukaryotes. Like the latter two, Thaumarchaeota have an unsplit rpoA gene. In l<strong>in</strong>e<br />
with this f<strong>in</strong>d<strong>in</strong>g, they form a separate and deeply branch<strong>in</strong>g l<strong>in</strong>eage with<strong>in</strong> the<br />
archaea <strong>in</strong> the phylogenetic analysis of the correspond<strong>in</strong>g prote<strong>in</strong> RpoA. (Modified<br />
from Spang et al. (2010), with permission.)<br />
7. DIVERSITY, DISTRIBUTION AND ACTIVITY OF AMMONIA-<br />
OXIDISING ARCHAEA IN THE ENVIRONMENT<br />
With<strong>in</strong> the archaeal doma<strong>in</strong>, the ability to oxidise ammonia appears thus far<br />
to be restricted to the Group 1 l<strong>in</strong>eage. After the <strong>in</strong>itial identification of<br />
AOA amo genes <strong>in</strong> soil and mar<strong>in</strong>e environments (Venter et al., 2004;<br />
Treusch et al., 2005), oligonucleotide primers were designed to target conserved<br />
regions of this gene and used <strong>in</strong> PCR, clon<strong>in</strong>g and sequenc<strong>in</strong>g<br />
approaches to exam<strong>in</strong>e the diversity and abundance of these organisms <strong>in</strong><br />
DNA extracted from environmental samples. It became clear that these<br />
organisms are globally distributed <strong>in</strong> most, if not all, environments (Fig. 9),<br />
similar to the f<strong>in</strong>d<strong>in</strong>gs for 16S rRNA gene surveys (Fig. 1). Current efforts are<br />
to determ<strong>in</strong>e the fundamental aspects of their ecophysiology and their ecological<br />
importance to nitrification processes globally by l<strong>in</strong>k<strong>in</strong>g environmental<br />
factors to AOA and AOB population dynamics.
AMMONIA-OXIDISING ARCHAEA 23<br />
[(Figure_9)TD$FIG]<br />
Figure 9 Phylogenetic tree describ<strong>in</strong>g major ammonia-oxidis<strong>in</strong>g archaeal amoA<br />
gene-def<strong>in</strong>ed l<strong>in</strong>eages and environments of orig<strong>in</strong>. Analyses were performed on an<br />
alignment of 486 positions from 188 sequences representative of known amoA diversity<br />
and the shape of triangular blocks are proportional to the number of sequences<br />
and maximum branch lengths with<strong>in</strong>. Multifurcat<strong>in</strong>g branches <strong>in</strong>dicate where the<br />
relative branch<strong>in</strong>g order of major l<strong>in</strong>eages could not be determ<strong>in</strong>ed <strong>in</strong> the majority<br />
of bootstrap replicates us<strong>in</strong>g various tree<strong>in</strong>g methods (distance, parsimony and maximum<br />
likelihood analyses). L<strong>in</strong>eages with cultivated representatives are highlighted<br />
together with soil fosmid clone 54d9.<br />
7.1. AOA <strong>in</strong> the Soil Environment<br />
Thaumarchaeota are the dom<strong>in</strong>ant archaea <strong>in</strong> most soil systems where they<br />
constitute up to 5% of all prokaryotes (e.g. Ochsenreiter et al., 2003;<br />
Lehtovirta et al., 2009). There are a number of thaumarchaeal l<strong>in</strong>eages found<br />
<strong>in</strong> high numbers <strong>in</strong> soil, and Group 1.1b (Fig. 1) is the dom<strong>in</strong>ant l<strong>in</strong>eage <strong>in</strong>
24 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
most soil systems (Auguet et al., 2010). This l<strong>in</strong>eage <strong>in</strong>cludes the archaeon<br />
from which soil fosmid 54d9 was derived, therefore <strong>in</strong>dicat<strong>in</strong>g that the most<br />
abundant archaea <strong>in</strong> soil may be capable of ammonia oxidation. Us<strong>in</strong>g<br />
quantitative PCR of amoA genes, Le<strong>in</strong><strong>in</strong>ger et al. (2006) exam<strong>in</strong>ed the<br />
abundance of AOA and AOB <strong>in</strong> 12 surface soils sampled across Europe.<br />
These soils represented a wide range of physicochemical properties and<br />
land-use types. Without exception, AOA represented the dom<strong>in</strong>ant group,<br />
with the ratio of AOA to AOB amoA genes rang<strong>in</strong>g from 1.5 to over 230<br />
(Fig. 10). This observation was confirmed for most soils exam<strong>in</strong>ed so far (e.g.<br />
He et al., 2007; Nicol et al., 2008; Jia and Conrad, 2009; Schauss et al., 2009; Di<br />
et al., 2010), although there are exceptions (Boyle-Yarwood et al., 2008).<br />
Another general trend observed is that the relative abundance of AOA to<br />
AOB changes with soil depth, with AOA numbers rema<strong>in</strong><strong>in</strong>g relatively<br />
constant but AOB numbers decreas<strong>in</strong>g dramatically (Le<strong>in</strong><strong>in</strong>ger et al., 2006;<br />
Jia and Conrad, 2009; Di et al., 2010), <strong>in</strong>dicat<strong>in</strong>g that AOA may be<br />
[(Figure_0)TD$FIG]<br />
Figure 10 AOA and AOB amoA gene abundance <strong>in</strong> 12 soils represent<strong>in</strong>g a wide<br />
geographical distribution and contrast<strong>in</strong>g physicochemical properties. Gene abundances<br />
(copies per dry weight soil gram) were calculated us<strong>in</strong>g two specific quantitative<br />
PCR assays. (From Le<strong>in</strong><strong>in</strong>ger et al. (2006), with permission.)
AMMONIA-OXIDISING ARCHAEA 25<br />
particularly well adapted to conditions with low levels of available nutrients<br />
and oxygen.<br />
7.2. AOA Activity <strong>in</strong> the Soil Environment<br />
With the co-occurrence of AOA and AOB <strong>in</strong> the soil environment, a major<br />
focus has been to determ<strong>in</strong>e the relative activities of both groups and<br />
what conditions determ<strong>in</strong>e their growth. Schauss and colleagues (2009) were<br />
the first to demonstrate growth of AOA <strong>in</strong> response to (organic) fertiliser<br />
additions. They demonstrated differences <strong>in</strong> the growth characteristics<br />
of AOA and AOB, but perhaps also provided some evidence for potential<br />
functional redundancy, with AOA and AOB both respond<strong>in</strong>g to the same<br />
source of ammonia: <strong>in</strong> microcosms amended with the antibiotic sulphadiaz<strong>in</strong>e,<br />
growth of AOB was <strong>in</strong>hibited while nitrification still occurred. Model<br />
calculations revealed that <strong>in</strong> such microcosms, a substantial contribution of<br />
ammonia oxidation must be attributed to AOA activity (Schauss et al.,<br />
2009).<br />
In some agricultural soils receiv<strong>in</strong>g significant N <strong>in</strong>puts, AOA have been<br />
shown to make a relatively small contribution to overall ammonia oxidation,<br />
with only the growth of AOB correlat<strong>in</strong>g with measured nitrification activity.<br />
Jia and Conrad (2009) demonstrated that <strong>in</strong> microcosms of agricultural soil<br />
receiv<strong>in</strong>g regular amendments of 7 mM <strong>in</strong>organic ammonium fertiliser,<br />
growth of AOB populations (only) correlated with ammonia oxidation activity,<br />
and growth of Group 1.1b AOA populations occurred even when all<br />
nitrification activity was <strong>in</strong>hibited by acetylene. In addition, grow<strong>in</strong>g AOA<br />
populations did not take up 13 C–CO 2, <strong>in</strong>dicat<strong>in</strong>g that they may possess<br />
heterotrophic metabolism. Di et al. (2009, 2010) reported similar f<strong>in</strong>d<strong>in</strong>gs<br />
<strong>in</strong> a number of experimental soils <strong>in</strong> New Zealand which were amended with<br />
high concentrations of urea–N (<strong>in</strong> the form of collected ur<strong>in</strong>e). Aga<strong>in</strong>, <strong>in</strong><br />
these field soil experiments, only AOB growth showed a positive relationship<br />
to nitrification activity. However, AOA growth (and not AOB) was<br />
observed <strong>in</strong> unamended (control) soils with low levels of nitrification fuelled<br />
by m<strong>in</strong>eralised organic nitrogen (Fig. 11), <strong>in</strong>dicat<strong>in</strong>g AOA growth associated<br />
with low levels of ammonia. Conclusive evidence of AOA growth <strong>in</strong> soil<br />
associated with nitrification activity was provided by studies of an agricultural<br />
soil, aga<strong>in</strong> receiv<strong>in</strong>g no fertiliser. Tourna et al. (2008) demonstrated that<br />
with different rates of nitrification (controlled as a function of temperature),<br />
changes were associated specifically with the transcript profiles of AOA<br />
amoA. These transcriptionally active populations grew dur<strong>in</strong>g nitrification<br />
(Offre et al., 2009), and their growth was completely <strong>in</strong>hibited when
[(Figure_1)TD$FIG]<br />
Figure 11 Contrast<strong>in</strong>g response of AOA and AOB communities to nitrogen deposition <strong>in</strong> a New Zealand agricultural soil. (a)<br />
Nitrification k<strong>in</strong>etics <strong>in</strong> soils receiv<strong>in</strong>g no amendment (control) and a high nitrogen load (collected dairy cow ur<strong>in</strong>e and added at an<br />
equivalent rate of 1000 kgN/ha). Growth dynamics of AOA (b) and AOB (c) communities <strong>in</strong> response to the different ammonia<br />
concentrations. (Adapted from data obta<strong>in</strong>ed by Di et al. (2010), with permission.)<br />
26 CHRISTA SCHLEPER AND GRAEME W. NICOL
AMMONIA-OXIDISING ARCHAEA 27<br />
[(Figure_2)TD$FIG]<br />
Figure 12 Growth of acetylene-sensitive ammonia-oxidis<strong>in</strong>g archaea <strong>in</strong> nitrify<strong>in</strong>g<br />
soil microcosms. (a) DGGE analysis of amoA-def<strong>in</strong>ed AOA communities. Arrow<br />
<strong>in</strong>dicates the growth of a specific population for which a specific qPCR assay was<br />
developed. (b) Inhibition of ammonia-oxidis<strong>in</strong>g activity <strong>in</strong> microcosms with a 10 Pa<br />
acetylene headspace partial pressure. (c) qPCR analysis demonstrat<strong>in</strong>g growth of<br />
AOA (group 1.1a archaea) only <strong>in</strong> microcosms with active nitrification. (Adapted<br />
from data obta<strong>in</strong>ed by Offre et al. (2009), with permission.)<br />
nitrification was <strong>in</strong>hibited with the addition of low concentrations of acetylene<br />
(Fig. 12), thus provid<strong>in</strong>g, for the first time, a direct l<strong>in</strong>k between soil<br />
nitrification and archaeal activity. However, one has to note that the active<br />
AOA phylotypes <strong>in</strong> these experiments were affiliated to Group 1.1a archaea<br />
typically found <strong>in</strong> the mar<strong>in</strong>e environment whereas Group 1.1b archaea<br />
(typically found <strong>in</strong> soils) did not exhibit activity <strong>in</strong> these experiments.<br />
Although based on a limited number of studies, published data describ<strong>in</strong>g<br />
the growth dynamics of AOA and AOB populations do h<strong>in</strong>t at fundamental<br />
differences <strong>in</strong> AOA and AOB physiology. From the relatively large number<br />
of cultivated AOB stra<strong>in</strong>s, it is known that there is a range of physiological<br />
diversity (adaptation to different ranges of ammonia concentrations,
28 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
temperature optima, contrast<strong>in</strong>g ureolytic capabilities, etc.) and it would<br />
seem likely that similar physiological diversity could be found with<strong>in</strong> the<br />
AOA. Therefore, it is probable that some populations of AOA and AOB do<br />
share or compete with<strong>in</strong> a similar dist<strong>in</strong>ct ecological niche present <strong>in</strong> soil.<br />
However, current evidence suggests the opposite may be the general rule,<br />
with populations of each l<strong>in</strong>eage resid<strong>in</strong>g <strong>in</strong> dist<strong>in</strong>ct ecological niches <strong>in</strong> the<br />
soil, and ammonia concentration (also pH) be<strong>in</strong>g the major driver for the<br />
relative activity of AOA and AOB. In addition, the source of ammonia may<br />
be a critical factor <strong>in</strong> determ<strong>in</strong><strong>in</strong>g relative growth. In all soil-based studies to<br />
date, where substantial AOA growth has been demonstrated, ammonium<br />
has been supplied to the system <strong>in</strong> the form of m<strong>in</strong>eralised organic N derived<br />
from composted manure (Schauss et al., 2009) or soil organic matter (Offre<br />
et al., 2009; Di et al., 2010) and AOB-dom<strong>in</strong>ated nitrification activity associated<br />
with ammonia from <strong>in</strong>organic fertiliser (Jia and Conrad, 2009) or<br />
(hydrolysed) urea (Di et al., 2010).<br />
7.3. AOA <strong>in</strong> the Mar<strong>in</strong>e Environment<br />
In the mar<strong>in</strong>e water column, nearly all AOA are placed with<strong>in</strong> a specific<br />
l<strong>in</strong>eage which is dist<strong>in</strong>ct from those associated with soil environments (Fig.<br />
3), and is congruent with the phylogenetic partition<strong>in</strong>g of 16S rRNA genes.<br />
Thaumarchaeota (formerly crenarchaeota) are found <strong>in</strong> very large numbers<br />
throughout the water column and they have been estimated to represent<br />
approximately 20% of prokaryotic cells <strong>in</strong> the water column (Karner et al.,<br />
2001). Indeed, the relative numbers of archaea decrease much less than<br />
bacteria and therefore generally represent a greater proportion of total<br />
prokaryotic numbers at depth. However, although thaumarchaeota are distributed<br />
throughout the water column, there is a clear phylogenetic separation<br />
of dist<strong>in</strong>ct AOA groups, with well-def<strong>in</strong>ed ‘shallow’ and ‘deep’ water<br />
l<strong>in</strong>eages (Francis et al., 2005; Hallam et al., 2006a; M<strong>in</strong>cer et al., 2007; Beman<br />
et al., 2008) and with only a small amount of overlap. Us<strong>in</strong>g specific qPCR<br />
assays for the ‘deep’ and ‘shallow’ l<strong>in</strong>eages, Beman et al. (2008) demonstrated<br />
that the shallow AOA l<strong>in</strong>eage was also found <strong>in</strong> deeper samples, but<br />
the deep l<strong>in</strong>eage demonstrated a more restricted distribution and did not<br />
occur <strong>in</strong> the shallower waters. This observation was also reflected <strong>in</strong> the<br />
detection of amoA mRNA transcripts of both these groups (Santoro et al.,<br />
2010).<br />
It is unclear whether all these thaumarchaeota are capable of ammonia<br />
oxidation and autotrophic growth. A recent study exam<strong>in</strong><strong>in</strong>g the ratio of<br />
thaumarchaeal 16S rRNA and AOA amoA genes <strong>in</strong>dicated that all shallow
AMMONIA-OXIDISING ARCHAEA 29<br />
archaeal populations may be capable of ammonia oxidation, with 16S rRNA:<br />
amoA gene ratios approach<strong>in</strong>g 1:1 (Agogue et al., 2008), and is consistent<br />
with the ratio found <strong>in</strong> the limited number of AOA genomes sequence thus<br />
far. However, with decreas<strong>in</strong>g depth this ratio <strong>in</strong>creases, with 16S rRNA:<br />
amoA gene ratio greater than 100:1 found at depths greater than 1000 m,<br />
thus <strong>in</strong>dicat<strong>in</strong>g that archaea <strong>in</strong> the deep ocean may not all be autotrophic<br />
ammonia oxidisers but heterotrophs (Agogue et al., 2008). However, analysis<br />
of radiocarbon data <strong>in</strong> archaeal lipids recovered from samples taken from the<br />
North Pacific Gyre have confirmed that the dom<strong>in</strong>ant thaumarchaeotal<br />
metabolism at depth does appear to be autotrophy (Ingalls et al., 2006),<br />
and potential discrepancies between amoA and 16 rRNA gene copy number<br />
may be due to a lack of coverage associated with certa<strong>in</strong> AOA amoA primer<br />
sets (Konstant<strong>in</strong>idis et al., 2009).<br />
There is strong correlative evidence that Group 1.1a archaea are not the<br />
only AOA l<strong>in</strong>eage present <strong>in</strong> the World’s oceans. Us<strong>in</strong>g quantitative PCR,<br />
M<strong>in</strong>cer et al. (2007) observed that there was a discrepancy <strong>in</strong> the abundance<br />
of AOA amoA and Group 1.1a 16S rRNA genes. However, when the<br />
abundance of a novel archaeal group related to the pSL12 clade [a l<strong>in</strong>eage<br />
orig<strong>in</strong>ally discovered <strong>in</strong> terrestrial hot spr<strong>in</strong>gs (Barns et al., 1996)] was taken<br />
<strong>in</strong>to account, a strong correlation was observed. Despite the relatively large<br />
sequence divergence between these two l<strong>in</strong>eages, the amoA genes of this<br />
l<strong>in</strong>eage appear to be <strong>in</strong>dist<strong>in</strong>guishable from Group 1.1a.<br />
7.4. AOA Activity <strong>in</strong> the Mar<strong>in</strong>e Environment<br />
It is perhaps <strong>in</strong> the mar<strong>in</strong>e environment that the clearest correlations<br />
between AOA abundance and nitrification activity are observed, and where<br />
AOA do appear to be both numerically dom<strong>in</strong>ant and functionally more<br />
active (relative to AOB). Abundances of AOA amoA and thaumarchaeal<br />
16S rRNA genes show a high correlation with nitrification rates, with up to<br />
10 4 –10 5 gene copies mL 1 <strong>in</strong> zones of high activity, and contrasts with the<br />
abundances of their bacterial counterparts which are frequently detected <strong>in</strong><br />
low numbers or are even undetectable (Ward, 2000; Wuchter et al., 2006;<br />
M<strong>in</strong>cer et al., 2007). AOA amoA abundance correlates with nitrite maxima<br />
<strong>in</strong> both oxygenated shallow waters and deeper waters <strong>in</strong> the oxygen m<strong>in</strong>imum<br />
zone (Coolen et al., 2007; Herfort et al., 2007; Beman et al., 2008).<br />
Nitrification activity (and AOA numbers) is greatest <strong>in</strong> the water column<br />
at the bottom of the euphotic zone. This may be due to competition for<br />
ammonia between nitrifiers and phytoplankton and/or light <strong>in</strong>hibition of<br />
the AMO enzyme (Ward, 2005). There is an <strong>in</strong>verse correlation between
30 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
thaumarchaeota and chlorophyll a (Murray et al., 1998), support<strong>in</strong>g the idea<br />
that phytoplankton have a negative effect on nitrifier communities (Ward,<br />
2005; Herfort et al., 2007). Additionally, previous studies have failed to f<strong>in</strong>d a<br />
correlation between bacterial ammonia-oxidis<strong>in</strong>g community structures and<br />
nitrification rates <strong>in</strong> ocean waters (O’Mullan and Ward, 2005).<br />
In an impressive time-series experiment over 11 months <strong>in</strong> the North Sea,<br />
Wuchter et al. (2006) quantified the abundance of <strong>in</strong>organic nitrogen concentrations<br />
together with bacterial and archaeal amoA gene copy numbers<br />
(Fig. 13). Ammonia concentrations were greatest <strong>in</strong> autumn and w<strong>in</strong>ter and<br />
decreased <strong>in</strong> the spr<strong>in</strong>g months. This decrease <strong>in</strong> ammonia concentration<br />
correlated with not only <strong>in</strong>creases <strong>in</strong> nitrite and nitrate concentrations (<strong>in</strong>dicative<br />
of aerobic ammonia oxidation), but also concomitant <strong>in</strong>creases <strong>in</strong><br />
archaea amoA and 16S rRNA gene copies and also thaumarchaeotal cell<br />
numbers (enumerated by fluorescent <strong>in</strong> situ hybridisation).<br />
Besides look<strong>in</strong>g at the correlation between <strong>in</strong>organic nitrogen concentrations<br />
and gene copies, Beman et al. (2008) also measured the oxidation of<br />
15 N-labelled ammonium pools <strong>in</strong> water samples taken from between the<br />
surface and 100 m depth <strong>in</strong> the Gulf of California. This study demonstrated<br />
that there was a correlation between AOA cell numbers and actual rates of<br />
ammonia oxidation.<br />
Correlations with the activity of other organisms <strong>in</strong>volved <strong>in</strong> the nitrogen<br />
cycle are also observed. For example, <strong>in</strong>creases <strong>in</strong> the abundance of AOA<br />
populations together with nitrite peaks <strong>in</strong> suboxic zones <strong>in</strong>dicate that they<br />
may supply the nitrite required for planctomycete bacteria perform<strong>in</strong>g the<br />
anammox processes (Coolen et al., 2007). In aerobic sub-surface waters at<br />
the bottom of the euphotic zone, correlations are observed between the<br />
abundance of AOA and nitrite-oxidis<strong>in</strong>g Nitrosp<strong>in</strong>a, suggest<strong>in</strong>g that the<br />
two groups are metabolically l<strong>in</strong>ked with AOA provid<strong>in</strong>g the nitrite substrate<br />
for Nitrosp<strong>in</strong>a populations and complet<strong>in</strong>g aerobic nitrification<br />
(M<strong>in</strong>cer et al., 2007; Santoro et al., 2010).<br />
7.5. AOA <strong>in</strong> Sediments<br />
AOA amoA sequences have been recovered from both freshwater<br />
(Herrmann et al., 2009) and estuar<strong>in</strong>e, coastal and deep-water mar<strong>in</strong>e sediments<br />
(e.g. Francis et al., 2005; Dang et al., 2009). There is a wide diversity of<br />
AOA <strong>in</strong> sediments, with sequences not only placed with<strong>in</strong> the mar<strong>in</strong>e water/<br />
sediment cluster where they are found <strong>in</strong> specific groups, but also affiliated to<br />
the major soil/sediment clade (Fig. 9). However, unlike <strong>in</strong> the mar<strong>in</strong>e column<br />
and most soils, the numerical dom<strong>in</strong>ance of AOA over AOB is not so
AMMONIA-OXIDISING ARCHAEA 31<br />
[(Figure_3)TD$FIG]<br />
Figure 13 Correlation of fluxes <strong>in</strong> <strong>in</strong>organic nitrogen concentrations and<br />
archaeal/AOA abundances dur<strong>in</strong>g an 11-month sampl<strong>in</strong>g time series. (a)<br />
Ammonia, nitrite and nitrate concentrations. (b) Enumeration of crenarchaeol (thaumarchaeal)<br />
abundance as determ<strong>in</strong>ed by qPCR and CARD-FISH with microscopy.<br />
(c) Abundance of AOA and AOB populations, as determ<strong>in</strong>ed by measur<strong>in</strong>g amoA<br />
gene copy numbers. (Adapted from data obta<strong>in</strong>ed by Wuchter et al. (2006), with<br />
permission.)
32 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
prevalent. Both communities show strong patterns of selection with different<br />
physicochemical properties (<strong>in</strong>clud<strong>in</strong>g sal<strong>in</strong>ity, ammonia and oxygen concentrations)<br />
(Mosier and Francis, 2008; Santoro et al., 2008), with AOA<br />
preferr<strong>in</strong>g lower sal<strong>in</strong>ities and lower ammonia concentrations. Estuaries<br />
are particularly important as they probably experience the highest concentrations<br />
of anthropogenic N <strong>in</strong>puts <strong>in</strong> the mar<strong>in</strong>e environment (particularly<br />
from agricultural run-off) and therefore represent an important area of<br />
nitrify<strong>in</strong>g transformations on a global scale.<br />
7.6. AOA <strong>in</strong> Geothermal Environments<br />
Although often referred to as ‘mesophilic’ or ‘nonthermophilic’ (cren)<br />
archaea, organisms associated with this l<strong>in</strong>eage were known to be present<br />
<strong>in</strong> terrestrial hot spr<strong>in</strong>gs, through the detection of 16S rRNA genes (Kvist<br />
et al., 2005, 2007) or archaeal-specific isoprenoid GDGTs lipids such as<br />
crenarchaeol (e.g. Pearson et al., 2004; Schouten et al., 2007). These f<strong>in</strong>d<strong>in</strong>gs<br />
therefore raised the possibility that AOA would also be found <strong>in</strong> such<br />
environments. This concept was particularly fasc<strong>in</strong>at<strong>in</strong>g as no known AOB<br />
had ever been found <strong>in</strong> such an environment, and it also raised questions<br />
about the potential orig<strong>in</strong>s of prokaryotic ammonia oxidation.<br />
There is now conclusive evidence that thaumarchaeota possess<strong>in</strong>g AMO<br />
are found <strong>in</strong> terrestrial environments of high temperature, with AOA amoA<br />
genes detected <strong>in</strong> a variety of habitats. These <strong>in</strong>clude speleothems (m<strong>in</strong>eral<br />
deposits), water and biofilms (Weidler et al., 2008) of geothermal caves<br />
and m<strong>in</strong>es, as well as terrestrial hot spr<strong>in</strong>gs. Thermal spr<strong>in</strong>gs (from where<br />
sulphur-dependent Crenarchaeota are typically cultured) which represent<br />
a wide range of temperatures and broad pH ranges located on the Russian<br />
Kamchatka pen<strong>in</strong>sula and on Iceland (Reigstad et al., 2008) as well as <strong>in</strong><br />
Yellowstone National Park (de la Torre et al., 2008) and further terrestrial<br />
hot spr<strong>in</strong>gs <strong>in</strong> the USA, Ch<strong>in</strong>a and Russia (Zhang et al., 2008) all harbour<br />
AOA gene markers. Reigstad et al. (2008) measured actual nitrification<br />
activity <strong>in</strong> an acidic hot muddy pool of 80 C under <strong>in</strong> situ conditions, demonstrat<strong>in</strong>g<br />
that this process is <strong>in</strong>deed found at considerable levels <strong>in</strong> terrestrial<br />
hot-spr<strong>in</strong>gs. A thermophilic AOA (N. yellowstonii) was grown <strong>in</strong> enrichment<br />
culture obta<strong>in</strong>ed from a hot spr<strong>in</strong>g located <strong>in</strong> the Yellowstone National<br />
Park with an optimal growth temperature between 65 and 72 C, grow<strong>in</strong>g<br />
with a stoichiometric conversion of ammonia to nitrite. Not only does this<br />
organism grow at the highest temperature for any known ammonia oxidiser,<br />
but it represents a separate l<strong>in</strong>eage outwith the ‘mar<strong>in</strong>e’ and ‘soil’ dom<strong>in</strong>ated<br />
groups (de la Torre et al., 2008).
AMMONIA-OXIDISING ARCHAEA 33<br />
7.7. AOA Associated with Mar<strong>in</strong>e Invertebrates<br />
Associations between AOA and a variety of mar<strong>in</strong>e <strong>in</strong>vertebrates are<br />
known, <strong>in</strong>clud<strong>in</strong>g mar<strong>in</strong>e sponges and corals, where they may play an important<br />
role <strong>in</strong> potentially complex nitrogen-cycl<strong>in</strong>g <strong>in</strong>teractions with<strong>in</strong> the host<br />
‘ecosystem’. The first Group 1 ‘model’ archaeon was the sponge symbiont C.<br />
symbiosum (Preston et al.,1996), which is found <strong>in</strong> the tissues of the mar<strong>in</strong>e<br />
sponge A. mexicana, and is closely related to those thaumarchaeota dom<strong>in</strong>at<strong>in</strong>g<br />
planktonic archaeal communities. The genome of this organism was<br />
the first to be sequenced with<strong>in</strong> the AOA l<strong>in</strong>eage (Hallam et al., 2006a,b) and<br />
possessed many <strong>in</strong>terest<strong>in</strong>g attributes, <strong>in</strong>clud<strong>in</strong>g the absence of some genes<br />
found <strong>in</strong> planktonic AOA (probably reflect<strong>in</strong>g the specific association with<br />
the sponge), and also had near-complete components of a 3-hydroxypropionate/4-hydroxybutyrate<br />
as well as TCA cycles, <strong>in</strong>dicative of both autotrophic<br />
and heterotrophic modes of growth, respectively. Recent studies of AOA<br />
amoA sequences <strong>in</strong> mar<strong>in</strong>e sponges (e.g. Meyer et al., 2008; Steger et al.,<br />
2008; Hoffmann et al., 2009) and corals (Beman et al., 2007; Siboni et al.,<br />
2008) have demonstrated that there are specific l<strong>in</strong>eages of AOA adapted<br />
to association with mar<strong>in</strong>e <strong>in</strong>vertebrates (Fig. 9), an observation<br />
previously found <strong>in</strong> 16S rRNA-based surveys. The association with AOA<br />
and mar<strong>in</strong>e sponges appears to be a cont<strong>in</strong>uous and stable one, with the<br />
transmission of AOA from adults to offspr<strong>in</strong>g <strong>in</strong> the larval stage observed<br />
<strong>in</strong> a number of different sponge species (Steger et al., 2008). A range<br />
of nitrogen transformative processes have been observed <strong>in</strong> sponges,<br />
with complex communities <strong>in</strong>clud<strong>in</strong>g anammox planctomycetes, nitrite<br />
oxidisers, denitrifiers, as well as AOA and AOB (Hoffmann et al., 2009;<br />
Mohamed et al., 2010). These sponge-associated communities therefore<br />
may represent a nitrogen cycl<strong>in</strong>g ecosystem which is dist<strong>in</strong>ct from that <strong>in</strong><br />
the surround<strong>in</strong>g water, and one which is essential for sponge health and<br />
cycl<strong>in</strong>g of waste.<br />
8. CONCLUDING REMARKS<br />
Based on the quantification of genes and cell numbers, Thaumarchaeota<br />
range among the most abundant microorganisms on this planet. Although<br />
their metabolic activity and versatility are still not entirely understood, there<br />
is no doubt that many of them are capable of ammonia oxidation and thus<br />
contribute significantly to global nitrogen and carbon cycl<strong>in</strong>g. Due to the<br />
extensive use of fertilisers <strong>in</strong> agriculture, the anthropogenic <strong>in</strong>put of fixed
34 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
nitrogen <strong>in</strong>to the World’s ecosystems is now estimated to be more than<br />
double that from natural processes (Rockstr€om et al., 2009). The major<br />
consequence of this shift <strong>in</strong> the equilibrium of the nitrogen cycle is an<br />
acceleration of nitrification, as well as eutrophication of freshwater and<br />
estuar<strong>in</strong>e environments. Another major consequence of accelerated rates<br />
of global nitrification is the <strong>in</strong>creased release of nitrogen oxides <strong>in</strong>to the<br />
atmosphere, which are produced by the denitrification activity of many<br />
bacteria, <strong>in</strong>clud<strong>in</strong>g ammonia oxidisers. However, it rema<strong>in</strong>s to be elucidated<br />
whether archaea also contribute to this process, with analyses of the first<br />
AOA genomes <strong>in</strong>dicat<strong>in</strong>g that ammonia oxidation is performed by a fundamentally<br />
different metabolic pathway. This new area of microbiology eagerly<br />
anticipates the results of current and future research which will compare the<br />
fundamental differences (or similarities) between bacterial and archaeal<br />
ammonia oxidation <strong>in</strong> various environments to understand whether these<br />
two groups of organisms have compet<strong>in</strong>g (or rather complement<strong>in</strong>g) roles <strong>in</strong><br />
various ecosystem processes.<br />
ACKNOWLEDGEMENT<br />
The authors would like to thank Mart<strong>in</strong> G. Klotz for discussions and for<br />
permission to <strong>in</strong>clude his hypotheses on the ammonia-oxidis<strong>in</strong>g metabolism<br />
of AOA, and provision of Fig. 7 used <strong>in</strong> this article. The authors also gratefully<br />
acknowledge the formatt<strong>in</strong>g work of Nathalia Jandl and support for<br />
Table 2 as well as Fig. 8 from Anja Spang.<br />
REFERENCES<br />
Agogue, H., Br<strong>in</strong>k, M., D<strong>in</strong>asquet, J. and Herndl, G.J. (2008) Major gradients <strong>in</strong><br />
putatively nitrify<strong>in</strong>g and non-nitrify<strong>in</strong>g Archaea <strong>in</strong> the deep North Atlantic.<br />
Nature 457, 550.<br />
Auguet, J., Barberan, A. and Casamayor, E.O. (2010) Global ecological patterns <strong>in</strong><br />
uncultured Archaea. ISME J. 4, 182–190.<br />
Barns, S.M., Delwiche, C.F., Palmer, J.D. and Pace, N.R. (1996) Perspectives on<br />
archaeal diversity, thermophily and monophyly from environmental rRNA<br />
sequences. Proc. Natl. Acad. Sci. U.S.A. 93, 9188–9193.<br />
Bartossek, R., Nicol, G., Lanzen, A., Klenk, H.-P. and Schleper, C. (2010)<br />
Homologues of nitrite reductases <strong>in</strong> ammonia-oxidiz<strong>in</strong>g Archaea: diversity and<br />
genomic context. Environ. Microbiol. 12, 1075–1088.<br />
Bayer, K., Schmitt, S. and Hentschel, U. (2008) <strong>Physiology</strong>, phylogeny and <strong>in</strong> situ<br />
evidence for bacterial and archaeal nitrifiers <strong>in</strong> the mar<strong>in</strong>e sponge Aplys<strong>in</strong>a aerophoba.<br />
Environ. Microbiol. 10, 2942–2955.
AMMONIA-OXIDISING ARCHAEA 35<br />
Beja, O., Koon<strong>in</strong>, E.V., Arav<strong>in</strong>d, L., Taylor, L.T., Seitz, H., Ste<strong>in</strong>, J.L., Bensen, D.C.,<br />
Feldman, R.A., Swanson, R.V. and DeLong, E.F. (2002) Comparative genomic<br />
analysis of archaeal genotypic variants <strong>in</strong> a s<strong>in</strong>gle population and <strong>in</strong> two different<br />
oceanic prov<strong>in</strong>ces. Appl. Environ. Microbiol. 68, 335–345.<br />
Beja, O., Suzuki, M.T., Koon<strong>in</strong>, E.V., Arav<strong>in</strong>d, L., Hadd, A., Nguyen, L.P.,<br />
Villacorta, R., Garrigues, C., Jovanovich, S.B., Feldman, R.A. and DeLong,<br />
E.F. (2000) Construction and analysis of bacterial artificial chromosome libraries<br />
from a mar<strong>in</strong>e microbial assemblage. Environ. Microbiol. 2, 516–529.<br />
Bell, S.D. and Jackson, S.P. (1998) Transcription <strong>in</strong> archaea. Cold Spr<strong>in</strong>g Harbor.<br />
Symp. Quant. Biol. 63, 41–51.<br />
Beman, J.M., Popp, B.N. and Francis, C.A. (2008) Molecular and biogeochemical<br />
evidence for ammonia oxidation by mar<strong>in</strong>e Crenarchaeota <strong>in</strong> the Gulf of<br />
California. ISME J. 2, 429–441.<br />
Beman, J.M., Roberts, K., Wegley, L., Rohwer, F. and Francis, C.A. (2007)<br />
Distribution and diversity of archaeal ammonia monooxygenase (amoA) genes<br />
associated with corals. Appl. Environ. Microbiol. 73, 5642–5647.<br />
Berg, I.A., Kockelkorn, D., Buckel, W. and Fuchs, G. (2007) A 3-hydroxypropionate/<br />
4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway <strong>in</strong> archaea.<br />
Science 318, 1782–1786.<br />
B<strong>in</strong>trim, S.B., Donohue, T.J., Handelsman, J., Roberts, G.P. and Goodman, R.M.<br />
(1997) Molecular phylogeny of Archaea from soil. Proc. Natl. Acad. Sci. USA 94,<br />
277–282.<br />
Boyle-Yarwood, S.A., Bottomley, P.J. and Myrold, D.D. (2008) Community composition<br />
of ammonia-oxidiz<strong>in</strong>g bacteria and archaea <strong>in</strong> soils under stands of red alder<br />
and Douglas fir <strong>in</strong> Oregon. Environ. Microbiol. 10, 2956–2965.<br />
Brochier-Armanet, C., Boussau, B., Gribaldo, S. and Forterre, P. (2008) Mesophilic<br />
Crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota. Nat.<br />
Rev. Microbiol. 6, 245–252.<br />
Brown, J.R. and Doolittle, W.F. (1997) Archaea and the prokaryote-to-eukaryote<br />
transition. Microbiol. Mol. Biol. Rev. 61, 456–502.<br />
Buckley, D.H., Graber, J.R. and Schmidt, T.M. (1998) Phylogenetic analysis of nonthermophilic<br />
members of the k<strong>in</strong>gdom crenarchaeota and their diversity and<br />
abundance <strong>in</strong> soils. Appl. Environ. Microbiol. 64, 4333–4339.<br />
Coolen, M.J., Abbas, B., van Bleijswijk, J., Hopmans, E.C., Kuypers, M.M., Wakeham,<br />
S.G. and Damste, J.S.S. (2007) Putative ammonia-oxidiz<strong>in</strong>g Crenarchaeota <strong>in</strong> suboxic<br />
waters of the Black Sea: a bas<strong>in</strong>-wide ecological study us<strong>in</strong>g 16S ribosomal and<br />
functional genes and membrane lipids. Environ. Microbiol. 9, 1001–1016.<br />
Crump, B.C. and Baross, J.A. (2000) Archaeaplankton <strong>in</strong> the Columbia River, its<br />
estuary and the adjacent coastal ocean, USA. FEMS Microbiol. Ecol. 31, 231–239.<br />
Damste, J.S.S., Schouten, S., Hopmans, E.C., van Du<strong>in</strong>, A.C. and Geenevasen, J.A.<br />
(2002) Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether<br />
membrane lipid of cosmopolitan pelagic crenarchaeota. J. Lipid Res. 43,<br />
1641–1651.<br />
Dang, H., Li, J., Zhang, X., Li, T., Tian, F. and J<strong>in</strong>, W. (2009) Diversity and spatial<br />
distribution of amoA-encod<strong>in</strong>g archaea <strong>in</strong> the deep-sea sediments of the tropical<br />
west pacific cont<strong>in</strong>ental marg<strong>in</strong>. J. Appl. Microbiol. 106, 1482–1493.<br />
de la Torre, J.R., Walker, C.B., Ingalls, A.E., K€onneke, M. and Stahl, D.A. (2008)<br />
Cultivation of a thermophilic ammonia-oxidiz<strong>in</strong>g archaeon synthesiz<strong>in</strong>g crenarchaeol.<br />
Environ. Microbiol. 10, 810–818.
36 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
DeLong, E.F. (1992) Archaea <strong>in</strong> coastal mar<strong>in</strong>e environments. Proc. Natl. Acad. Sci.<br />
USA 89, 5685–5689.<br />
DeLong, E.F. (1998) Everyth<strong>in</strong>g <strong>in</strong> moderation: archaea as ‘non-extremophiles’.<br />
Curr. Op<strong>in</strong>. Genet. Dev. 8, 649–654.<br />
Di, H.J., Cameron, K.C., Shen, J.P., W<strong>in</strong>efield, C.S., O’Callaghan, M., Bowatte, S.<br />
and He, J.Z. (2009) Nitrification driven by bacteria and not archaea <strong>in</strong> nitrogenrich<br />
grassland soils. Nat. Geosci. 2, 621–624.<br />
Di, H., Cameron, K., Shen, J., W<strong>in</strong>efield, C., O’Callaghan, M., Bowatte, S. and He, J.<br />
(2010) Ammonia-oxidiz<strong>in</strong>g bacteria and archaea grow under contrast<strong>in</strong>g soil<br />
nitrogen conditions. FEMS Microbiol. Ecol. 64, 167–174.<br />
Francis, C.A., Roberts, K.J., Beman, J.M., Santoro, A.E. and Oakley, B.B. (2005)<br />
Ubiquity and diversity of ammonia-oxidiz<strong>in</strong>g archaea <strong>in</strong> water columns and sediments<br />
of the ocean. Proc. Natl. Acad. Sci. USA 102, 14683–14688.<br />
Fuhrman, J.A., McCallum, K. and Davis, A.A. (1992) Novel major archaebacterial<br />
group from mar<strong>in</strong>e plankton. Nature 356, 148–149.<br />
Garcia, J.-L., Patel, B.K.C. and Ollivier, B. (2000) Taxonomic, phylogenetic, and<br />
ecological diversity of methanogenic Archaea. Anaerobe 6, 205–226.<br />
Garrett, R.A. and Klenk, H.P. (2007) Archaea: Evolution, <strong>Physiology</strong>, and Molecular<br />
Biology. Blackwell Publish<strong>in</strong>g, S<strong>in</strong>gapore.<br />
Hallam, S.J., Konstant<strong>in</strong>idis, K.T., Putnam, N., Schleper, C., Watanabe, Y., Sugahara,<br />
J., Preston, C., de la Torre, J., Richardson, P.M. and DeLong, E.F. (2006a)<br />
Genomic analysis of the uncultivated mar<strong>in</strong>e crenarchaeote Cenarchaeum symbiosum.<br />
Proc. Natl. Acad. Sci. USA 103, 18296–18301.<br />
Hallam, S.J., M<strong>in</strong>cer, T.J., Schleper, C., Preston, C.M., Roberts, K., Richardson, P.M.<br />
and DeLong, E.F. (2006b) Pathways of carbon assimilation and ammonia oxidation<br />
suggested by environmental genomic analyses of mar<strong>in</strong>e Crenarchaeota.<br />
PLoS Biol. 4, 520–536.<br />
Hatzenpichler, R., Lebedeva, E.V., Spieck, E., Stoecker, K., Richter, A., Daims, H.<br />
and Wagner, M. (2008) A moderately thermophilic ammonia-oxidiz<strong>in</strong>g crenarchaeote<br />
from a hot spr<strong>in</strong>g. Proc. Natl. Acad. Sci. USA 105, 2134–2139.<br />
He, J., Shen, J., Zhang, L., Zhu, Y., Zheng, Y., Xu, M. and Di, H. (2007) Quantitative<br />
analyses of the abundance and composition of ammonia-oxidiz<strong>in</strong>g bacteria and<br />
ammonia-oxidiz<strong>in</strong>g archaea of a Ch<strong>in</strong>ese upland red soil under long-term fertilization<br />
practices. Environ. Microbiol. 9, 2364–2374.<br />
Herfort, L., Schouten, S., Abbas, B., Veldhuis, M.J.W., Coolen, M.J.L., Wuchter, C.,<br />
Boon, J.P., Herndl, G.J. and Damste, J.S.S. (2007) Variations <strong>in</strong> spatial and<br />
temporal distribution of Archaea <strong>in</strong> the North Sea <strong>in</strong> relation to environmental<br />
variables. FEMS Microbiol. Ecol. 62, 242–257.<br />
Herndl, G.J., Re<strong>in</strong>thaler, T., Teira, E., van Aken, H., Veth, C., Pernthaler, A. and<br />
Pernthaler, J. (2005) Contribution of Archaea to total prokaryotic production <strong>in</strong><br />
the deep Atlantic Ocean. Appl. Environ. Microbiol. 71, 2303–2309.<br />
Herrmann, M., Saunders, A.M. and Schramm, A. (2009) Effect of lake trophic status<br />
and rooted macrophytes on community composition and abundance of ammoniaoxidiz<strong>in</strong>g<br />
prokaryotes <strong>in</strong> freshwater sediments. Appl. Environ. Microbiol. 75,<br />
3127–3136.<br />
Hoffmann, F., Radax, R., Woebken, D., Holtappels, M., Lavik, G., Rapp, H.T.,<br />
Schl€appy, M.-L., Schleper, C. and Kuypers, M.M.M. (2009) Complex nitrogen<br />
cycl<strong>in</strong>g <strong>in</strong> the sponge Geodia barretti. Environ. Microbiol. 11, 2228–2243.<br />
Ingalls, A.E., Shah, S.R., Hansman, R.L., Aluwihare, L.I., Santos, G.M., Druffel,<br />
E.R. and Pearson, A. (2006) Quantify<strong>in</strong>g archaeal community autotrophy <strong>in</strong> the
AMMONIA-OXIDISING ARCHAEA 37<br />
mesopelagic ocean us<strong>in</strong>g natural radiocarbon. Proc. Natl. Acad. Sci. USA 103,<br />
6442–6447.<br />
Jia, Z. and Conrad, R. (2009) Bacteria rather than Archaea dom<strong>in</strong>ate microbial<br />
ammonia oxidation <strong>in</strong> an agricultural soil. Environ. Microbiol. 11, 1658–1671.<br />
Jurgens, G., Glockner, F., Amann, R., Saano, A., Montonen, L., Likolammi, M. and<br />
Munster, U. (2000) Identification of novel Archaea <strong>in</strong> bacterioplankton of a boreal<br />
forest lake by phylogenetic analysis and fluorescent <strong>in</strong> situ hybridization. FEMS<br />
Microbiol. Ecol. 34, 45–56.<br />
Karner, M.B., DeLong, E.F. and Karl, D.M. (2001) Archaeal dom<strong>in</strong>ance <strong>in</strong> the<br />
mesopelagic zone of the Pacific Ocean. Nature 409, 507–510.<br />
Keough, B.P., Schmidt, T.M. and Hicks, R.E. (2003) Archaeal nucleic acids <strong>in</strong> picoplankton<br />
from great lakes on three cont<strong>in</strong>ents. Microb. Ecol. 46, 238–248.<br />
K€onneke, M., Bernhard, A.E., de la Torre, J.R., Walker, C.B., Waterbury, J.B. and<br />
Stahl, D.A. (2005) Isolation of an autotrophic ammonia-oxidiz<strong>in</strong>g mar<strong>in</strong>e<br />
archaeon. Nature 437, 543–546.<br />
Konstant<strong>in</strong>idis, K.T., Braff, J., Karl, D.M. and DeLong, E.F. (2009) Comparative<br />
metagenomic analysis of a microbial community resid<strong>in</strong>g at a depth of 4,000<br />
meters at station ALOHA <strong>in</strong> the North Pacific Subtropical Gyre. Appl.<br />
Environ. Microbiol. 75, 5345–5355.<br />
Kowalchuk, G.A. and Stephen, J.R. (2001) Ammonia-oxidiz<strong>in</strong>g bacteria: a model for<br />
molecular microbial ecology. Annu. Rev. Microbiol. 55, 485–529.<br />
Kuypers, M.M.M., Blokker, P., Erbacher, J., K<strong>in</strong>kel, H., Pancost, R.D., Schouten, S.<br />
and Damste, J.S.S. (2001) Massive expansion of mar<strong>in</strong>e archaea dur<strong>in</strong>g a mid-<br />
Cretaceous oceanic anoxic event. Science 293, 92–94.<br />
Kvist, T., Ahr<strong>in</strong>g, B.K. and Westermann, P. (2007) Archaeal diversity <strong>in</strong> Icelandic hot<br />
spr<strong>in</strong>gs. FEMS Microbiol. Ecol. 59, 71–80.<br />
Kvist, T., Mengewe<strong>in</strong>, A., Manzei, S., Ahr<strong>in</strong>g, B.K. and Westermann, P. (2005)<br />
Diversity of thermophilic and non-thermophilic Crenarchaeota at 80 degrees C.<br />
FEMS Microbiol. Lett. 244, 61–68.<br />
Lai, D., Spr<strong>in</strong>gstead, J.R. and Monbouquette, H.G. (2008) Effect of growth temperature<br />
on ether lipid biochemistry <strong>in</strong> Archaeoglobus fulgidus. Extremophiles 12,<br />
271–278.<br />
Lebedeva, E., Alawi, M., Fiencke, C., Namsaraev, B., Bock, E. and Spieck, E. (2005)<br />
Moderately thermophilic nitrify<strong>in</strong>g bacteria of a hot spr<strong>in</strong>g of the Baikal rift zone.<br />
FEMS Microbiol. Ecol. 54, 297–306.<br />
Lehtovirta, L.E., Prosser, J.I. and Nicol, G.W. (2009) Soil pH regulates the abundance<br />
and diversity of Group 1.1c Crenarchaeota. FEMS Microbiol. Ecol. 70,<br />
367–376.<br />
Le<strong>in</strong><strong>in</strong>ger, S., Urich, T., Schloter, M., Schwark, L., Qi, J., Nicol, G.W., Prosser, J.I.,<br />
Schuster, S.C. and Schleper, C. (2006) Archaea predom<strong>in</strong>ate among ammoniaoxidiz<strong>in</strong>g<br />
prokaryotes <strong>in</strong> soils. Nature 442, 806–809.<br />
Lieberman, R.L. and Rosenzweig, A.C. (2005) Crystal structure of a membranebound<br />
metalloenzyme that catalyses the biological oxidation of methane. Nature<br />
434, 177–182.<br />
López-Garcıa, P., Brochier, C., Moreira, D. and Rodrıguez-Valera, F. (2004)<br />
Comparative analysis of a genome fragment of an uncultivated mesopelagic crenarchaeote<br />
reveals multiple horizontal gene transfers. Environ. Microbiol. 6,<br />
19–34.<br />
MacGregor, B.J., Moser, D.P., Alm, E.W., Nealson, K.H. and Stahl, D.A. (1997)<br />
Crenarchaeota <strong>in</strong> Lake Michigan sediment. Appl. Environ. Microbiol. 63,<br />
1178–1181.
38 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
Martens-Habbena, W., Berube, P.M., Urakawa, H., de la Torre, J.R. and Stahl, D.A.<br />
(2009) Ammonia oxidation k<strong>in</strong>etics determ<strong>in</strong>e niche separation of nitrify<strong>in</strong>g<br />
Archaea and Bacteria. Nature 461, 976–979.<br />
Mart<strong>in</strong>-Cuadrado, A.B., Rodriguez-Valera, F., Moreira, D., Alba, J.C., Ivars-<br />
Martınez, E., Henn, M.R., Talla, E. and López-Garcıa, P. (2008) H<strong>in</strong>dsight <strong>in</strong><br />
the relative abundance, metabolic potential and genome dynamics of uncultivated<br />
mar<strong>in</strong>e archaea from comparative metagenomic analyses of bathypelagic plankton<br />
of different oceanic regions. ISME J. 2(8); 865–886.<br />
Meyer, M., Stenzel, U. and Hofreiter, M. (2008) Parallel tagged sequenc<strong>in</strong>g on the 454<br />
platform. Nat. Protoc. 3, 267–278.<br />
M<strong>in</strong>cer, T.J., Church, M.J., Taylor, L.T., Preston, C., Karl, D.M. and DeLong, E.F.<br />
(2007) Quantitative distribution of presumptive archaeal and bacterial nitrifiers <strong>in</strong><br />
Monterey Bay and the North Pacific Subtropical Gyre. Environ. Microbiol. 9,<br />
1162–1175.<br />
Mohamed, N.M., Saito, K., Tal, Y. and Hill, R.T. (2010) Diversity of aerobic and<br />
anaerobic ammonia-oxidiz<strong>in</strong>g bacteria <strong>in</strong> mar<strong>in</strong>e sponges. ISME J. 4, 38–48.<br />
Mosier, A.C. and Francis, C.A. (2008) Relative abundance and diversity of ammoniaoxidiz<strong>in</strong>g<br />
archaea and bacteria <strong>in</strong> the San Francisco Bay estuary. Environ.<br />
Microbiol. 10, 3002–3016.<br />
Murray, A.E., Preston, C.M., Massana, R., Taylor, L.T., Blakis, A., Wu, K. and<br />
DeLong, E.F. (1998) Seasonal and spatial variability of bacterial and archaeal<br />
assemblages <strong>in</strong> the coastal waters near Anvers Island, Antarctica. Appl. Environ.<br />
Microbiol. 64, 2585–2595.<br />
Nealson, K.H. and Venter, J.C. (2007) Metagenomics and the global ocean survey:<br />
what’s <strong>in</strong> it for us, and why should we care? ISME J. 1, 185–187.<br />
Nicol, G.W., Tscherko, D., Chang, L., Hammesfahr, U. and Prosser, J.I. (2006)<br />
Crenarchaeal community assembly and microdiversity <strong>in</strong> develop<strong>in</strong>g soils at two<br />
sites associated with deglaciation. Environ. Microbiol. 8, 1382–1393.<br />
Nicol, G.W., Le<strong>in</strong><strong>in</strong>ger, S., Schleper, C. and Prosser, J.I. (2008) The <strong>in</strong>fluence of soil<br />
pH on the diversity, abundance and transcriptional activity of ammonia-oxidiz<strong>in</strong>g<br />
archaea and bacteria. Environ. Microbiol.<br />
Nicol, G.W. and Schleper, C. (2006) Ammonia-oxidis<strong>in</strong>g Crenarchaeota: important<br />
players <strong>in</strong> the nitrogen cycle? Trends Microbiol. 14, 207–212.<br />
O’Mullan, G.D. and Ward, B.B. (2005) Relationship of temporal and spatial variabilities<br />
of ammonia-oxidiz<strong>in</strong>g bacteria to nitrification rates <strong>in</strong> Monterey Bay,<br />
California. Appl. Environ. Microbiol. 71, 697–705.<br />
Ochsenreiter, T., Selezi, D., Quaiser, A., Bonch-Osmolovskaya, L. and Schleper, C.<br />
(2003) Diversity and abundance of Crenarchaeota <strong>in</strong> terrestrial habitats studied by<br />
16S RNA surveys and real time PCR. Environ. Microbiol. 5, 787–797.<br />
Offre, P., Prosser, J.I. and Nicol, G. (2009) Growth of ammonia-oxidiz<strong>in</strong>g<br />
archaea <strong>in</strong> soil microcosms is <strong>in</strong>hibited by acetylene. FEMS Microbiol. Ecol.<br />
70, 99–108.<br />
Ouverney, C.C. and Fuhrman, J.A. (2000) Mar<strong>in</strong>e planktonic archaea take up am<strong>in</strong>o<br />
acids. Appl. Environ. Microbiol. 66, 4829–4833.<br />
Pearson, A., Huang, Z., Ingalls, A.E., Romanek, C.S., Wiegel, J., Freeman, K.H.,<br />
Smittenberg, R.H. and Zhang, C.L. (2004) Nonmar<strong>in</strong>e crenarchaeol <strong>in</strong> Nevada hot<br />
spr<strong>in</strong>gs. Appl. Environ. Microbiol. 70, 5229–5237.<br />
Pearson, A., McNichol, A.P., Benitez-Nelson, B.C., Hayes, J.M. and Egl<strong>in</strong>ton, T.I.<br />
(2001) Orig<strong>in</strong>s of lipid biomarkers <strong>in</strong> Santa Monica Bas<strong>in</strong> surface sediment: a case
AMMONIA-OXIDISING ARCHAEA 39<br />
study us<strong>in</strong>g compound-specific D14C analysis. Geochim. Cosmochim. Acta 65,<br />
3123–3137.<br />
Pitcher, A., Schouten, S. and Damste, J.S.S. (2009) In situ production of Crenarchaeol<br />
<strong>in</strong> two California hot spr<strong>in</strong>gs. Appl. Environ. Microbiol. 75, 4443–4451.<br />
Preston, C.M., Wu, K.Y., Mol<strong>in</strong>ski, T.F. and DeLong, E.F. (1996) A psychrophilic<br />
crenarchaeon <strong>in</strong>habits a mar<strong>in</strong>e sponge: Cenarchaeum symbiosum gen. nov., sp.<br />
nov. Proc. Natl. Acad. Sci. USA 93, 6241–6246.<br />
Prosser, J. (1989) Autotrophic nitrification <strong>in</strong> bacteria. Adv. Microbiol. Physiol. 30,<br />
125–181.<br />
Prosser, J.I. and Nicol, G.W. (2008) Relative contributions of archaea and bacteria to<br />
aerobic ammonia oxidation <strong>in</strong> the environment. Environ. Microbiol. 10,<br />
2931–2941.<br />
Purkhold, U., Pommeren<strong>in</strong>g-R€oser, A., Juretschko, S., Schmid, M.C., Koops, H.P.<br />
and Wagner, M. (2000) Phylogeny of all recognized species of ammonia oxidizers<br />
based on comparative 16S rRNA and amoA sequence analysis: implications for<br />
molecular diversity surveys. Appl. Environ. Microbiol. 66, 5368–5382.<br />
Quaiser, A., Ochsenreiter, T., Klenk, H.-P., Kletz<strong>in</strong>, A., Treusch, A.H., Meurer, G.b.,<br />
Eck, J., Sensen, C.W. and Schleper, C. (2002) First <strong>in</strong>sight <strong>in</strong>to the genome of an<br />
uncultivated crenarchaeote from soil. Environ. Microbiol. 4, 603–611.<br />
Reigstad, L.J., Richter, A., Daims, H., Urich, T., Schwark, L. and Schleper, C. (2008)<br />
Nitrification <strong>in</strong> terrestrial hot spr<strong>in</strong>gs of Iceland and Kamchatka. FEMS Microbiol.<br />
Ecol. 64, 167–174.<br />
Sandaa, R.A., Enger, O. and Torsvik, V. (1999) Abundance and diversity of Archaea<br />
<strong>in</strong> heavy-metal-contam<strong>in</strong>ated soils. Appl. Environ. Microbiol. 65, 3293–3297.<br />
Santoro, A.E., Francis, C.A., de Sieyes, N.R. and Boehm, A.B. (2008) Shifts <strong>in</strong> the<br />
relative abundance of ammonia-oxidiz<strong>in</strong>g bacteria and archaea across physicochemical<br />
gradients <strong>in</strong> a subterranean estuary. Environ. Microbiol. 10,<br />
1068–1079.<br />
Santoro, A.E., Casciotti, K.L. and Francis, C.A. (2010) Activity, abundance and<br />
diversity of nitrify<strong>in</strong>g archaea and bacteria <strong>in</strong> the central California Current.<br />
Environ. Microbiol. 12, 1989–2006.<br />
Schauss, K., Focks, A., Le<strong>in</strong><strong>in</strong>ger, S., Kotzerke, A., Heuer, H., Thiele-Bruhn, S.,<br />
Sharma, S., Wilke, B.M., Matthies, M., Smalla, K., Munch, J.C., Amelung, W.,<br />
Kaupenjohann, M., Schloter, M. and Schleper, C. (2009) Dynamics and functional<br />
relevance of ammonia-oxidiz<strong>in</strong>g archaea <strong>in</strong> two agricultural soils. Environ.<br />
Microbiol. 11, 446–456.<br />
Schleper, C., DeLong, E.F., Preston, C.M., Feldman, R.A., Wu, K.-Y. and Swanson,<br />
R.V. (1998) Genomic analysis reveals chromosomal variation <strong>in</strong> natural populations<br />
of the uncultured psychrophilic archaeon Cenarchaeum symbiosum.<br />
J. Bacteriol. 180, 5003–5009.<br />
Schleper, C., Holben, W. and Klenk, H.P. (1997) Recovery of crenarchaeotal ribosomal<br />
DNA sequences from freshwater-lake sediments. ApplEnviron Microbiol<br />
63, 321–323.<br />
Schleper, C., Jurgens, G. and Jonuscheit, M. (2005) Genomic studies of uncultivated<br />
archaea. Nat. Rev. Microbiol. 3, 479–488.<br />
Schouten, S., Hopmans, E.C., Baas, M., Boumann, H., Standfest, S., Konneke, M.,<br />
Stahl, D.A. and Damste, J.S.S. (2008) Intact membrane lipids of Candidatus<br />
‘Nitrosopumilus maritimus’, a cultivated representative of the cosmopolitan mesophilic<br />
Group I Crenarchaeota. Appl. Environ. Microbiol. 74, 2433–2440.
40 CHRISTA SCHLEPER AND GRAEME W. NICOL<br />
Schouten, S., van der Meer, M.T., Hopmans, E.C., Rijpstra, W.I., Reysenbach, A.L.,<br />
Ward, D.M. and Damste, J.S.S. (2007) Archaeal and bacterial glycerol dialkyl<br />
glycerol tetraether lipids <strong>in</strong> hot spr<strong>in</strong>gs of Yellowstone National Park (USA).<br />
Appl. Environ. Microbiol. 73, 6181–6191.<br />
Siboni, N., Ben-Dov, E., Sivan, A. and Kushmaro, A. (2008) Global distribution and<br />
diversity of coral-associated Archaea and their possible role <strong>in</strong> the coral holobiont<br />
nitrogen cycle. Environ. Microbiol. 10, 2979–2990.<br />
Simon, H.M., Dodsworth, J.A. and Goodman, R.M. (2000) Crenarchaeota colonize<br />
terrestrial plant roots. Environ. Microbiol. 2, 495–505.<br />
Spang, A., Hatzenpichler, R., Brochier-Armanet, C., Rattei, T., Tischler, P., Spieck,<br />
E., Streit, W., Stahl, D.A., Wagner, M. and Schleper, C. (2010) Dist<strong>in</strong>ct gene set <strong>in</strong><br />
two different l<strong>in</strong>eages of ammonia-oxidiz<strong>in</strong>g archaea supports the phylum<br />
Thaumarchaeota. Trends Microbiol. 18, 331–340.<br />
Steger, D., Ett<strong>in</strong>ger-Epste<strong>in</strong>, P., Whalan, S., Hentschel, U., de Nys, R., Wagner, M.<br />
and Taylor, M.W. (2008) Diversity and mode of transmission of ammonia-oxidiz<strong>in</strong>g<br />
archaea <strong>in</strong> mar<strong>in</strong>e sponges. Environ. Microbiol. 10, 1087–1094.<br />
Ste<strong>in</strong>, J.L., Marsh, T.L., Wu, K.Y., Shizuya, H. and DeLong, E.F. (1996)<br />
Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair<br />
genome fragment from a planktonic mar<strong>in</strong>e archaeon. J. Bacteriol. 178,<br />
591–599.<br />
Takai, K., Moser, D.P., DeFlaun, M., Onstott, T.C. and Fredrickson, J.K. (2001)<br />
Archaeal diversity <strong>in</strong> waters from deep South African gold m<strong>in</strong>es. Appl.<br />
Environ. Microbiol. 67, 5750–5760.<br />
Taylor, M.W., Radax, R., Steger, D. and Wagner, M. (2007) Sponge-associated<br />
microorganisms: evolution, ecology, and biotechnological potential. Microbiol.<br />
Mol. Biol. Rev. 71, 295–347.<br />
Teira, E., Van Aken, H., Veth, C. and Herndl, G.J. (2006) Archaeal uptake of<br />
enantiomeric am<strong>in</strong>o acids <strong>in</strong> the meso- and bathypelagic waters of the North<br />
Atlantic. Limnol. Oceanogr. 51, 60–69.<br />
Tourna, M., Freitag, T.E., Nicol, G.W. and Prosser, J.I. (2008) Growth, activity and<br />
temperature responses of ammonia-oxidiz<strong>in</strong>g archaea and bacteria <strong>in</strong> soil microcosms.<br />
Environ. Microbiol. 10, 1357–1364.<br />
Treusch, A.H., Kletz<strong>in</strong>, A., Raddatz, G., Ochsenreiter, T., Quaiser, A., Meurer, G.,<br />
Schuster, S.C. and Schleper, C. (2004a) Characterization of large-<strong>in</strong>sert DNA<br />
libraries from soil for environmental genomic studies of Archaea. Environ.<br />
Microbiol. 6, 970–980.<br />
Treusch, A.H. and Schleper, C. (2004b) The uncultivated crenarchaeota from soil:<br />
what can we learn from metagenomics? 10th International Symposium on<br />
<strong>Microbial</strong>Ecology (ISME). Cancun, Mexico.<br />
Treusch, A.H., Le<strong>in</strong><strong>in</strong>ger, S., Kletz<strong>in</strong>, A., Schuster, S.C., Klenk, H.P. and Schleper, C.<br />
(2005) Novel genes for nitrite reductase and Amo-related prote<strong>in</strong>s <strong>in</strong>dicate a role<br />
of uncultivated mesophilic crenarchaeota <strong>in</strong> nitrogen cycl<strong>in</strong>g. Environ. Microbiol.<br />
7, 1985–1995.<br />
Venter, J.C., Rem<strong>in</strong>gton, K., Heidelberg, J.F., Halpern, A.L., Rusch, D., Eisen, J.A.,<br />
Wu, D., Paulsen, I., Nelson, K.E., Nelson, W., Fouts, D.E., Levy, S., Knap, A.H.,<br />
Lomas, M.W., Nealson, K., White, O., Peterson, J., Hoffman, J., Parsons, R.,<br />
Baden-Tillson, H., Pfannkoch, C., Rogers, Y.H. and Smith, H.O. (2004)<br />
Environmental genome shotgun sequenc<strong>in</strong>g of the Sargasso Sea. Science 304,<br />
66–74.
AMMONIA-OXIDISING ARCHAEA 41<br />
Vetriani, C., Reysenbach, A. and Dore, J. (1998) Recovery and phylogenetic analysis<br />
of archaeal rRNA sequences from cont<strong>in</strong>ental shelf sediments. FEMS Microbiol.<br />
Lett. 161, 83–88.<br />
Walker, C.B., de la Torre, J., Klotz, M.G., Urakawa, H., P<strong>in</strong>el, N., Arp, D.J., Brochier-<br />
Armanet, C., Cha<strong>in</strong>, P.S.G., Chan, P.P., Gollabgir, A., Hemp, J., H€ugler, M., Karr,<br />
E.A., K€onneke, M., Sh<strong>in</strong>, M., Lawton, T.J., Lowe, T., Martens-Habbena, W.,<br />
Sayavedra-Soto, L.A., Lang, D., Sievert, S.M., Rosenzweig, A.C., Mann<strong>in</strong>g,<br />
G. and Stahl, D.A. (2010) Nitrosopumilus maritimus genome reveals unique<br />
mechanisms for nitrification and autotrophy <strong>in</strong> globally distributed mar<strong>in</strong>e<br />
crenarchaea. Proc. Natl. Acad. Sci. USA 107, 8818–8823.<br />
Ward, B.B. (2000) Nitrification and the mar<strong>in</strong>e nitrogen cycle. In: Kirchmann, D.L.<br />
(Ed.), <strong>Microbial</strong> Ecology of the Oceans. Wiley Series, New York, pp. 427–453.<br />
Ward, B.B. (2005) Temporal variability <strong>in</strong> nitrification rates and related biogeochemical<br />
factors <strong>in</strong> Monterey Bay, California, USA. Mar. Ecol. Prog. Ser. 292,97–109.<br />
Weidler, G.W., Gerbl, F.W. and Stan-Lotter, H. (2008) Crenarchaeota and their role<br />
<strong>in</strong> the nitrogen cycle <strong>in</strong> a subsurface radioactive thermal spr<strong>in</strong>g <strong>in</strong> the Austrian<br />
Central Alps. Appl. Environ. Microbiol. 74, 5934–5942.<br />
Whitman, W.B., Coleman, D.C. and Wiebe, W.J. (1998) Prokaryotes: the unseen<br />
majority. Proc. Natl. Acad. Sci. USA 95, 6578–6583.<br />
Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221–271.<br />
Woese, C.R. and Fox, G.E. (1977) Phylogenetic structure of the prokaryotic doma<strong>in</strong>:<br />
the primary k<strong>in</strong>gdoms. Proc. Natl. Acad. Sci. USA 74, 5088–5090.<br />
Wrage, N., Velthof, G.L., Van Beusichem, M.L. and Oenema, O. (2001) Role of<br />
nitrifier denitrification <strong>in</strong> the production of nitrous oxide. Soil Biol. Biochem. 33,<br />
1723–1732.<br />
Wuchter, C., Abbas, B., Coolen, M.J., Herfort, L., van Bleijswijk, J., Timmers, P.,<br />
Strous, M., Teira, E., Herndl, G.J., Middelburg, J.J., Schouten, S. and Damste, J.S.<br />
S. (2006) Archaeal nitrification <strong>in</strong> the ocean. Proc. Natl. Acad. Sci. USA 103,<br />
12317–12322.<br />
Wuchter, C., Schouten, S., Boschker, H.T. and Damste, J.S.S. (2003) Bicarbonate<br />
uptake by mar<strong>in</strong>e Crenarchaeota. FEMS Microbiol. Lett. 219, 203–207.<br />
Wuchter, C., Schouten, S., Coolen, M.J.L. and Damste, J.S.S. (2004) Temperaturedependent<br />
variation <strong>in</strong> the distribution of tetraether membrane lipids of mar<strong>in</strong>e<br />
Crenarchaeota: implications for TEX86 paleothermometry. Paleooceanography<br />
19, PA4028.<br />
Zhang, C.L., Pearson, A., Yi-Lian, L., Mills, G. and Wiegel, J. (2006) Thermophilic<br />
temperature optimum for crenarchaeol synthesis and its implication for archaeal<br />
evolution. Appl. Environ. Microbiol. 72, 4419–4422.<br />
Zhang, C.L., Ye, Q., Huang, Z.Y., Li, W.J., Chen, J.Q., Song, Z.Q., Zhao, W.D.,<br />
Bagwell, C., Inskeep, W.P., Ross, C., Gao, L., Wiegel, J., Romanek, C.S., Shock,<br />
E.L. and Hedlund, B.P. (2008) Global occurrence of archaeal amoA genes <strong>in</strong><br />
terrestrial hot spr<strong>in</strong>gs. Appl. Environ. Microbiol. 74, 6417–6426.
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Reductive Stress <strong>in</strong> Microbes: Implications for<br />
Understand<strong>in</strong>g Mycobacterium tuberculosis<br />
Disease and Persistence<br />
Aisha Farhana 1 , Loni Guidry 1 , Anup Srivastava 1 , Amit S<strong>in</strong>gh 2 ,<br />
Mary K. Hondalus 3 and Adrie J.C. Steyn 1<br />
1 Department of Microbiology, University of Alabama at Birm<strong>in</strong>gham, AL, USA<br />
2 International Center for Genetic Eng<strong>in</strong>eer<strong>in</strong>g and Biotechnology, Aruna Asaf Ali Marg,<br />
New Delhi, India<br />
3 Department of Infectious Diseases, University of Georgia, Athens, GA, USA<br />
ABSTRACT<br />
Mycobacterium tuberculosis (Mtb) is a remarkably successful pathogen that<br />
is capable of persist<strong>in</strong>g <strong>in</strong> host tissues for decades without caus<strong>in</strong>g disease.<br />
Years after <strong>in</strong>itial <strong>in</strong>fection, the bacilli may resume growth, the outcome of<br />
which is active tuberculosis (TB). In order to establish <strong>in</strong>fection, resist host<br />
defences and re-emerge, Mtb must coord<strong>in</strong>ate its metabolism with the<br />
<strong>in</strong> vivo environmental conditions and nutrient availability with<strong>in</strong> the primary<br />
site of <strong>in</strong>fection, the lung. Ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g metabolic homeostasis for an<br />
<strong>in</strong>tracellular pathogen such as Mtb requires a carefully orchestrated series of<br />
oxidation–reduction reactions, which, if unbalanced, generate oxidative or<br />
reductive stress. The importance of oxidative stress <strong>in</strong> microbial<br />
pathogenesis has been appreciated and well studied over the past several<br />
decades. However, the role of its counterpart, reductive stress, has been<br />
largely ignored. Reductive stress is def<strong>in</strong>ed as an aberrant <strong>in</strong>crease <strong>in</strong><br />
reduc<strong>in</strong>g equivalents, the magnitude and identity of which is determ<strong>in</strong>ed<br />
by host carbon source utilisation and <strong>in</strong>fluenced by the presence of<br />
host-generated gases (e.g. NO, CO, O2 and CO 2). This <strong>in</strong>creased reductive<br />
ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 57 Copyright Ó 2010 by Elsevier Ltd.<br />
ISSN: 0065-2911 All rights reserved<br />
DOI:10.1016/B978-0-12-381045-8.00002-3
44 AISHA FARHANA ET AL.<br />
power must be dissipated for bacterial survival. To recycle reduc<strong>in</strong>g<br />
equivalents, microbes have evolved unique electron ‘s<strong>in</strong>ks’ that are dist<strong>in</strong>ct<br />
for their particular environmental niche. In this review, we describe the<br />
specific mechanisms that some microbes have evolved to dispel reductive<br />
stress. The <strong>in</strong>tention of this review is to <strong>in</strong>troduce the concept of reductive<br />
stress, <strong>in</strong> tuberculosis research <strong>in</strong> particular, <strong>in</strong> the hope of stimulat<strong>in</strong>g new<br />
avenues of <strong>in</strong>vestigation.<br />
Abbreviations . . . ............................................ 44<br />
1. Introduction ................................................ 45<br />
2. Scope. . . .................................................. 46<br />
3. The Concept of Reductive Stress ............................... 47<br />
4. Overview: General Physiological Characteristics of Mycobacterium 49<br />
tuberculosis ................................................<br />
4.1. Historic Overview. ....................................... 49<br />
4.2. Environmental Factors that Influence Metabolism .............. 50<br />
5. Reductive S<strong>in</strong>ks <strong>in</strong> Microbes . . ................................. 59<br />
5.1. Fermentation ........................................... 59<br />
5.2. Polymer Deposition ...................................... 64<br />
5.3. Nitrate Reductase ....................................... 66<br />
5.4. Phenaz<strong>in</strong>e Production . . . ................................. 67<br />
5.5. Hydrogenases . . . ....................................... 70<br />
5.6. The Reverse TCA (rTCA) Cycle . ........................... 72<br />
5.7. Carbon Monoxide (CO) Dehydrogenase (CODH). .............. 74<br />
5.8. Other Mechanisms ...................................... 75<br />
6. Redox S<strong>in</strong>ks <strong>in</strong> Mycobacteria. . ................................. 76<br />
6.1. The Mycobacterial Intracellular Redox Environment. . . .......... 76<br />
6.2. The Mtb<br />
Dos Dormancy Regulon ........................... 79<br />
6.3. Mtb WhiB3 is an Intracellular Redox Sensor<br />
88<br />
that Counters Reductive Stress. . ...........................<br />
7. Conclud<strong>in</strong>g Remarks . . ....................................... 95<br />
Acknowledgements . . . ....................................... 98<br />
References. ................................................ 98<br />
ABBREVIATIONS<br />
GSH glutathione<br />
GSSG glutathione disulfide (oxidised glutathione)<br />
49<br />
88
REDUCTIVE STRESS IN MICROBES 45<br />
Sometimes scientific progress is not based on a discovery or the generation<br />
of new data but on a change of viewpo<strong>in</strong>t that allows one to see a set<br />
of already exist<strong>in</strong>g data <strong>in</strong> a new light’<br />
(Michael Reth)<br />
1. INTRODUCTION<br />
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a disease<br />
of great <strong>in</strong>ternational concern and the lead<strong>in</strong>g cause of death worldwide from<br />
a curable <strong>in</strong>fectious disease. Across the globe, one human life is lost to TB<br />
every 15 s (WHO Factsheet, 2009). The situation is further exacerbated by<br />
the coexistent HIV epidemic, and the emergence of multidrug resistant<br />
(MDR), extensively drug resistant (XDR) and Super-XDR (S-XDR) Mtb<br />
stra<strong>in</strong>s (Gandhi et al., 2006; Pillay and Sturm, 2007; Velayati et al., 2009).<br />
Despite the availability of ample genomic, proteomic and bio<strong>in</strong>formatic <strong>in</strong>formation<br />
on Mtb, it is estimated that <strong>in</strong> 2009 more TB-related deaths occurred<br />
than at any time <strong>in</strong> history (Fauci, 2008). The variably efficacious BCG<br />
(Bacille Calmette-Guer<strong>in</strong>) vacc<strong>in</strong>e rema<strong>in</strong>s the only available TB vacc<strong>in</strong>e<br />
and no new anti-mycobacterial drug has been deployed s<strong>in</strong>ce the discovery<br />
of rifampic<strong>in</strong> <strong>in</strong> 1963 (Duncan, 2004; Young et al., 2008; Kaufmann et al.,<br />
2010). The slow pace <strong>in</strong> the development of TB <strong>in</strong>tervention strategies compared<br />
to an overwhelm<strong>in</strong>g <strong>in</strong>crease <strong>in</strong> global TB <strong>in</strong>cidence compromises the<br />
achievements made <strong>in</strong> TB control. An important hurdle to the development<br />
of successful TB treatment regimes is the lack of knowledge concern<strong>in</strong>g the<br />
mechanisms by which Mtb is able to persist <strong>in</strong> a dormant state, unresponsive<br />
to anti-mycobacterial drugs (Gomez and McK<strong>in</strong>ney, 2004; Sacchett<strong>in</strong>i et al.,<br />
2008; Ma et al., 2010). We have yet to understand the physiological status of<br />
the persist<strong>in</strong>g mycobacterial organisms or the environmental cues which lead<br />
to reactivation of disease. Detailed knowledge of this persistent state of Mtb is<br />
crucial for the establishment of efficacious TB eradication schemes.<br />
Mtb displays a remarkable capacity to persist <strong>in</strong> latent form and switch<br />
between replicative and non-replicative (dormant) states <strong>in</strong> response to<br />
environmental signals generated by the host immune responses (Cosma<br />
et al., 2003; Warner and Mizrahi, 2007; Rustad et al., 2009). Mtb harbours<br />
the mach<strong>in</strong>ery necessary to synthesise almost all essential vitam<strong>in</strong>s, am<strong>in</strong>o<br />
acids and enzyme cofactors, provid<strong>in</strong>g the organism with the ability to alter<br />
its metabolic state enabl<strong>in</strong>g an aerobic (e.g. oxidative phosphorylation) and<br />
possibly an anaerobic mode of respiration (Wheeler and Ratledge, 1994;<br />
Muttucumaru et al., 2004). Importantly, this metabolic flexibility ensures
46 AISHA FARHANA ET AL.<br />
bacilli survival <strong>in</strong> the varied environments with<strong>in</strong> the human host rang<strong>in</strong>g<br />
from that of high oxygen tension <strong>in</strong> the lung alveolus to microaerophilic<br />
conditions with<strong>in</strong> the tuberculous granuloma (Ulrichs and Kaufmann, 2006).<br />
Most studies of the physiology and biochemistry of mycobacteria were<br />
carried out <strong>in</strong> the early 1910–1980s, and it was discovered that fundamental<br />
differences exist <strong>in</strong> the metabolism of Mtb cultured <strong>in</strong> vitro and that of bacilli<br />
grow<strong>in</strong>g <strong>in</strong> vivo. An important difference <strong>in</strong>cludes demonstrat<strong>in</strong>g that Mtb<br />
harvested from lungs showed <strong>in</strong>active respiratory responses to various carbohydrates<br />
and glycolytic <strong>in</strong>termediates, whereas positive responses were<br />
obta<strong>in</strong>ed to these same substrates by the same stra<strong>in</strong> when cultured <strong>in</strong> vitro<br />
(Dubos, 1953; Artman and Bekierkunst, 1961; Segal, 1965, 1984; Brez<strong>in</strong>a<br />
et al., 1967). Unfortunately, much of the data generated from these classical<br />
studies rema<strong>in</strong> hidden <strong>in</strong> the historical ‘archives’ not accessible through<br />
PubMed or similar literature database searches. In part, the goal of this<br />
review is to excavate some of this ‘buried’ <strong>in</strong>formation on Mtb and <strong>in</strong>tegrate<br />
it with the current understand<strong>in</strong>g of metabolic paradigms of prokaryotic and<br />
lower eukaryotic organisms.<br />
2. SCOPE<br />
In this review, we aim to <strong>in</strong>troduce the idea of ‘reductive stress’ <strong>in</strong> TB<br />
research. A strong emphasis is placed on the historical knowledge of Mtb<br />
physiology obta<strong>in</strong>ed by <strong>in</strong> vivo studies performed <strong>in</strong> the earlier half of the last<br />
century, because <strong>in</strong> some respects, these analyses are a lost art <strong>in</strong> the modern<br />
era of molecular techniques. This is then followed by a discussion of the<br />
<strong>in</strong> vivo factors that affect Mtb growth and metabolic mechanisms, such as<br />
redox s<strong>in</strong>ks, which microorganisms have evolved to ma<strong>in</strong>ta<strong>in</strong> redox homeostasis<br />
<strong>in</strong> response to oxido-reductive stress. Parallels between oxidoreductive<br />
pathways <strong>in</strong> mycobacteria versus other bacteria and yeast are<br />
highlighted. Metabolic eng<strong>in</strong>eer<strong>in</strong>g approaches that modulate reductive<br />
stress are also described. Next, the <strong>in</strong>tracellular redox environment of Mtb<br />
is discussed followed by a description of the best-known paradigm for signal<br />
transduction <strong>in</strong> Mtb: the Dos dormancy regulon, and its role <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g<br />
redox balance. Lastly, the role of the <strong>in</strong>tracellular redox sensor, Mtb WhiB3,<br />
<strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g redox homeostasis is discussed. This review does not cover<br />
oxidative stress per se, but it is considered when appropriate to the theme.<br />
Regard<strong>in</strong>g virulence and persistence, and general mycobacterial metabolism,<br />
we refer the reader to several articles that discuss these issues <strong>in</strong> detail<br />
(Ramakrishnan et al., 1972; Cosma et al., 2003; Boshoff and Barry, 2005; Hett
REDUCTIVE STRESS IN MICROBES 47<br />
and Rub<strong>in</strong>, 2008; Barry et al., 2009; Rustad et al., 2009; Meena and Rajni,<br />
2010; Paige and Bishai, 2010). In sum, we aim to present a clear analysis of<br />
the current knowledge of reductive stress <strong>in</strong> microorganisms <strong>in</strong> order to<br />
provide a better foundation for future <strong>in</strong>terpretation of the physiological<br />
events associated with Mtb <strong>in</strong>fection.<br />
3. THE CONCEPT OF REDUCTIVE STRESS<br />
The physiology and metabolism of Mtb are unique, allow<strong>in</strong>g it to survive<br />
under a wide range of <strong>in</strong> vitro and <strong>in</strong> vivo environmental conditions. This<br />
flexibility is evident from the fact that Mtb is exposed to a plethora of<br />
products <strong>in</strong>clud<strong>in</strong>g carbohydrates, organic acids, lipids, am<strong>in</strong>o acids, ions,<br />
etc., as well as gases such as nitric oxide (NO) (Voskuil et al., 2003, 2009)<br />
carbon monoxide (CO) (Kumar et al., 2007, 2008), carbon dioxide (CO 2)<br />
(Florczyk et al., 2003), oxygen (O 2)(Voskuil et al., 2003) and its correspond<strong>in</strong>g<br />
free radicals <strong>in</strong> vivo. These molecules subsequently <strong>in</strong>flict either oxidative<br />
or reductive stress with<strong>in</strong> the bacteria. Over the years, oxidative stress<br />
and the critical role it plays <strong>in</strong> a wide range of diseases has been well studied;<br />
however, the role of its counterpart, namely reductive stress, has largely been<br />
underappreciated. The likely reasons for this, primarily, <strong>in</strong>clude a lack of<br />
understand<strong>in</strong>g of the concept of reductive stress and the dearth of experimental<br />
techniques for exam<strong>in</strong><strong>in</strong>g it (Ghyczy and Boros, 2007).<br />
A crucial element <strong>in</strong> reductive stress is redox coupl<strong>in</strong>g, which entails<br />
electron transfer. More specifically, redox reactions <strong>in</strong>volve the transfer of<br />
electrons and hydrogen atoms from an electron donor (reductant or reduc<strong>in</strong>g<br />
agent) to an electron acceptor (oxidant or oxidis<strong>in</strong>g agent), which together<br />
function as a redox couple. These redox couples (e.g. NAD + /NADH,<br />
E 0’ = 315 mV; NADP + /NADPH, E 0’ = 320 mV; FAD/FADH2,<br />
E 0’ = 219 mV; 2GSH/GSSG, E hc = 250 mV [10 mM]) are vital to both<br />
anabolic and catabolic reactions. NADH functions as an energy-rich electron<br />
transfer coenzyme, which generates almost three ATPs for every NADH to<br />
NAD + oxidation event, whereas NAD + functions as a s<strong>in</strong>k for electrons. In<br />
contrast, NADPH is the primary source of electrons for reductive synthesis<br />
or anabolism of fatty acids (FAs) and reduction of the glutathione system,<br />
which is the key cellular antioxidant defence system. Thus, the NAD + /<br />
NADH coenzyme system required for catabolism contrasts with the<br />
NADPH/NADP + system that is required for anabolism (Voet et al., 2008).<br />
The above redox couples are often thermodynamically l<strong>in</strong>ked because<br />
elevated levels of either reductant or pro-oxidant are deleterious to the
48 AISHA FARHANA ET AL.<br />
microbial cell (Schafer and Buettner, 2001). Balanced rates of oxidation and<br />
reduction of these molecules are necessary for optimal metabolic function as<br />
redox imbalance <strong>in</strong> cells can lead to either oxidative or reductive stress, of<br />
which the latter has mostly escaped the attention of the scientific <strong>in</strong>vestigator.<br />
Reductive stress can be def<strong>in</strong>ed as an abnormal <strong>in</strong>crease <strong>in</strong> reductive<br />
equivalents (e.g. NADH, NADPH, GSH, etc.) or reduc<strong>in</strong>g power<br />
(Dimmeler and Zeiher, 2007; Ghyczy and Boros, 2007; Zhang et al., 2010).<br />
The central focus of this review is to exam<strong>in</strong>e the mechanisms used by<br />
microorganisms and Mtb <strong>in</strong> particular to recycle reduc<strong>in</strong>g equivalents <strong>in</strong><br />
order to ma<strong>in</strong>ta<strong>in</strong> redox balance.<br />
The formal concept of reductive stress emerged little over a decade ago.<br />
Us<strong>in</strong>g animal models for diabetes, several studies reported that the metabolic<br />
imbalance l<strong>in</strong>ked to an <strong>in</strong>creased blood flow to the ret<strong>in</strong>a, kidney and peripheral<br />
nerve is cytosolic reductive stress. This <strong>in</strong>creased NADH/NAD + ratio or<br />
hypoxia-like state is l<strong>in</strong>ked to the <strong>in</strong>creased oxidation of substrates such as<br />
sorbitol, glucoronic acid and non-esterified FAs, and to the reduction of<br />
NAD + to yield NADH (Ido et al., 1997; Tilton, 2002; Ido, 2007). S<strong>in</strong>ce an<br />
<strong>in</strong>crease <strong>in</strong> NADH was observed under hypoxic conditions, the observation<br />
was termed ‘pseudohypoxia’, orreferredtoasthe‘reductive stress hypothesis’<br />
(Ido, 2007). In a sem<strong>in</strong>al study, Rajasekaran et al. (2007) reported reductive<br />
stress <strong>in</strong> mice express<strong>in</strong>g the mutant human ab-crystall<strong>in</strong> gene. In this<br />
study, because of <strong>in</strong>creased activity of glucose-6-phosphate dehydrogenase<br />
(G6PD), enhanced levels of NADPH and GSH caused prote<strong>in</strong> aggregation,<br />
cardiomyopathy and <strong>in</strong>creased expression of heat shock prote<strong>in</strong>s (Hsp)<br />
<strong>in</strong>clud<strong>in</strong>g Hsp27. S<strong>in</strong>ce NADPH is a cofactor of NADPH oxidases and NO<br />
synthases, these f<strong>in</strong>d<strong>in</strong>gs established a l<strong>in</strong>k between reductive stress and<br />
oxidative or nitrosative stress signal<strong>in</strong>g pathways. Subsequently, it was shown<br />
that overexpression of Hsp27 <strong>in</strong>duces reductive stress <strong>in</strong> the heart<br />
(Zhang et al., 2010), which was evident by an <strong>in</strong>crease <strong>in</strong> 2GSH/GSSG, myocardial<br />
glutathione peroxidase activity and decreased levels of reactive oxygen<br />
species (ROS). Interest<strong>in</strong>gly, 2GSH levels rose but GSSG levels rema<strong>in</strong>ed<br />
unaltered. In another study, G6PD was overexpressed <strong>in</strong> Drosophila melanogaster,<br />
which resulted <strong>in</strong> <strong>in</strong>creased levels of NADH and NADPH, and<br />
<strong>in</strong>creased GSH/GSSG ratio. The presence of high amounts of these reduc<strong>in</strong>g<br />
equivalents enhanced resistance to oxidative stress and were associated with<br />
an extension of life span <strong>in</strong> the transgenic flies (Legan et al., 2008).<br />
On the other hand, <strong>in</strong>creased reductive stress may also lead to an<br />
<strong>in</strong>creased oxidative stress, as was demonstrated <strong>in</strong> hypoxic <strong>in</strong>jury studies<br />
(Gores et al., 1989; Khan and O’Brien, 1995). In those studies, it was proposed<br />
that reduction of electron carriers that are normally oxidised under<br />
aerobic conditions (reductive stress) promotes formation of toxic ROS upon
REDUCTIVE STRESS IN MICROBES 49<br />
O 2 availability. In an attempt to address the possible mode of action, it was<br />
shown that reduc<strong>in</strong>g equivalents can release redox-active iron lead<strong>in</strong>g to<br />
oxidative stress and cell <strong>in</strong>jury (Staubli and Boelsterli, 1998).<br />
In conclusion, it is clear that normal cellular functions essentially depend<br />
on ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g redox homeostasis. A redox imbalance can lead to either<br />
oxidative or reductive stress. Lastly, it is evident that reductive stress might<br />
be a common mechanism <strong>in</strong> many eukaryotic diseases but, unfortunately, its<br />
implications <strong>in</strong> microbial pathogenesis are poorly understood. The concept<br />
of reductive stress <strong>in</strong> bacteria, particularly as it applies to Mtb, is an example<br />
of a shift <strong>in</strong> perspective.<br />
4. OVERVIEW: GENERAL PHYSIOLOGICAL<br />
CHARACTERISTICS OF MYCOBACTERIUM<br />
TUBERCULOSIS<br />
4.1. Historic Overview<br />
Mtb is a prototrophic, obligate aerobe that is able to survive periods of<br />
extended anaerobiosis, although conditions are yet to be identified where<strong>in</strong><br />
the bacilli are capable of replication <strong>in</strong> the absence of O 2. In fact, studies<br />
reported <strong>in</strong> 1933 established that Mtb could survive for up to 12 years <strong>in</strong><br />
sealed tubes and rema<strong>in</strong> fully virulent (Corper and Cohn, 1933).<br />
Mycobacteria can utilise a wide range of carbon compounds for growth <strong>in</strong><br />
vitro <strong>in</strong>clud<strong>in</strong>g carbohydrates, lipids and prote<strong>in</strong>s, which suggests that the<br />
bacilli are able to assimilate a wide range of host substrates for growth<br />
<strong>in</strong> vivo. For example, micromolar quantities of organic acids (e.g. lactate,<br />
pyruvate, citrate, succ<strong>in</strong>ate, malate, acetoacetate, etc.), micro to millimolar<br />
quantities of carbohydrates (glucose, glycogen, fructose, etc.), micromolar<br />
quantities of virtually all am<strong>in</strong>o acids, nucleic acid precursors, nucleotides,<br />
and milligram to gram/litre quantities of lipids (<strong>in</strong>clud<strong>in</strong>g total and free FAs,<br />
triacylglycerol [TAG] and total cholesterol) are available as sources of metabolic<br />
energy (Wheeler and Ratledge, 1994). The degradative pathways of<br />
the above substrates converge on common <strong>in</strong>termediates, <strong>in</strong>clud<strong>in</strong>g <strong>in</strong> many<br />
cases acetyl-coenzyme A (acetyl-CoA) that eventually produce ATP.<br />
Decades ago, many of the scientific studies of Mtb metabolism and physiology<br />
were performed on <strong>in</strong> vivo-derived bacilli (Segal and Bloch, 1956;<br />
Artman and Bekierkunst, 1961; Segal, 1962, 1965, 1984). The complex and<br />
laborious approaches (e.g. isolat<strong>in</strong>g, purify<strong>in</strong>g and characteris<strong>in</strong>g bacilli from<br />
<strong>in</strong>fected mouse lungs) yielded a wealth of <strong>in</strong>formation regard<strong>in</strong>g the
50 AISHA FARHANA ET AL.<br />
differences between the <strong>in</strong> vivo and <strong>in</strong> vitro physiology of Mtb. In addition,<br />
differences between virulent and avirulent Mtb stra<strong>in</strong>s were noted. In recent<br />
years, a number of eloquent molecular and metabolic studies of Mtb have<br />
been performed. Although valuable <strong>in</strong>formation was obta<strong>in</strong>ed, an <strong>in</strong>herent<br />
weakness of much of that work was the use of <strong>in</strong> vitro cultured bacilli. The<br />
chemical makeup of artificial growth media critically <strong>in</strong>fluences the biochemical<br />
activity of the organism, and the applicability of data thus generated is<br />
limited to the conditions under which it was derived. Therefore, keep<strong>in</strong>g the<br />
complexity of the host environment <strong>in</strong> m<strong>in</strong>d, certa<strong>in</strong> aspects of Mtb physiology<br />
will undoubtedly have to be revisited when exam<strong>in</strong><strong>in</strong>g Mtb <strong>in</strong> vivo.<br />
Nonetheless, ample data exist to <strong>in</strong>dicate that Mtb adjusts its metabolism<br />
<strong>in</strong> response to the availability of nutrients and environmental gases dur<strong>in</strong>g<br />
different stages of <strong>in</strong>fection (Wheeler and Ratledge, 1994; McK<strong>in</strong>ney et al.,<br />
2000; Boshoff and Barry, 2005; Tian et al., 2005; Munoz-Elias et al., 2006;<br />
Ja<strong>in</strong> et al., 2007; Barry et al., 2009). It is therefore important to understand<br />
how this metabolic response permits the bacterium to persist long term <strong>in</strong> the<br />
human host.<br />
4.2. Environmental Factors that Influence Metabolism<br />
4.2.1. The TCA Cycle<br />
The global metabolic pathway of a microbial cell is an <strong>in</strong>terl<strong>in</strong>ked network of<br />
chemical reactions through which the cell breaks down substrate compounds<br />
<strong>in</strong>to smaller organic molecules, which then serve as precursors for the biosynthesis<br />
of diverse macromolecules. Microorganisms employ different metabolic<br />
strategies of which the ultimate goal is to generate a proton motive<br />
force and cellular energy, ATP. The TCA cycle is present <strong>in</strong> all aerobic<br />
organisms and serves as a means to oxidise carbohydrates such as glucose<br />
to CO2 and H2O and the energy released is efficiently harvested by the<br />
electron transport cha<strong>in</strong> (ETC). The TCA cycle is amphibolic because it<br />
can be used for both anabolic and catabolic processes, and yields much more<br />
energy per mole of glucose (38 moles of ATP) when completely oxidised<br />
than the 1–4 moles of ATP generated via anaerobic fermentation. The<br />
complete oxidation of 1 mole of glucose via glycolysis and the TCA cycle<br />
of Escherichia coli yields 10 moles of NAD(P)H and 2 moles of FADH 2<br />
(Vemuri et al., 2006) as depicted below:<br />
Glucose + 8NAD + + 2NADP + + 2FAD + 4ADP + 4Pi<br />
! 6CO2 + 8NADH + 2NADPH + 2FADH2 + 4ATP + 10H +
REDUCTIVE STRESS IN MICROBES 51<br />
Most of the TCA cycle enzymes are repressed by glucose and further<br />
repressed by anaerobiosis. Under aerobic conditions, succ<strong>in</strong>ate is formed<br />
through the oxidation of a-ketoglutarate (KG), whereas under anaerobic<br />
conditions bacteria form succ<strong>in</strong>ate through reduction of fumarate. Under<br />
anaerobic growth, the 2-oxoglutarate dehydrogenase complex (ODHC) and<br />
succ<strong>in</strong>ate dehydrogenase (SDH) are also repressed, caus<strong>in</strong>g the activity of<br />
the TCA cycle to virtually cease. The cycle is thus transformed <strong>in</strong>to its<br />
branched or non-cyclic form <strong>in</strong> which the carbon flows <strong>in</strong>to <strong>in</strong>dependent<br />
oxidative and reductive pathways lead<strong>in</strong>g to the formation of 2-oxoglutarate<br />
(glutamate), and succ<strong>in</strong>ate and succ<strong>in</strong>yl-CoA respectively (Amaras<strong>in</strong>gham<br />
and Davis, 1965; Spencer and Guest, 1987; Guest and Russell, 1992). In the<br />
branched or reductive pathway, SDH is replaced by fumarate reductase<br />
(FRD) which enables fumarate to be used as an electron acceptor <strong>in</strong> anaerobic<br />
respiration. Alternative anaerobic routes to succ<strong>in</strong>ate production occur<br />
either via aspartate <strong>in</strong>volv<strong>in</strong>g aspartate oxaloacetate am<strong>in</strong>otransferase, or<br />
via isocitrate catalysed by isocitrate lyase (Spencer and Guest, 1987; Guest<br />
and Russell, 1992).<br />
Environmental factors such as the availability of O2 and the nature (e.g.<br />
carbon oxidation state, COS; see Section 4.2.2) and quantity of the carbon<br />
source profoundly affect the status of the TCA cycle (Spencer and Guest,<br />
1987; Clark, 1989; Guest and Russell, 1992). O2 is a poisonous lethal gas,<br />
which allows aerobic microbes to survive as they have developed appropriate<br />
antioxidant defence mechanisms (Halliwell, 2008). TCA cycle enzymes<br />
*<br />
known to be <strong>in</strong>hibited by the O2 radical, superoxide anion (O2 ), and under<br />
high pO2 <strong>in</strong>clude aconitase, isocitrate dehydrogenase, a-ketoglutarate<br />
dehydrogenase (KDH) and fumarase (Halliwell, 2008). Several of these<br />
enzymes conta<strong>in</strong> 4Fe–4S clusters, which fall apart when targeted by O2 or<br />
*<br />
O2 . The <strong>in</strong>activation of these enzymes leads to the release of iron, which<br />
*<br />
can then promote the production of OH via the Fenton reaction. In addition,<br />
NO, a host-generated gas can effectively and irreversibly react with the<br />
Fe–S clusters of TCA cycle enzymes lead<strong>in</strong>g to the formation of a prote<strong>in</strong>bound<br />
DNIC complex, which affects specific enzymatic activity and overall<br />
metabolic activity of the cell (Imlay, 2006, 2008; Duan et al., 2009). In<br />
accordance with the critical role of the TCA enzymes <strong>in</strong> the production or<br />
*<br />
consumption of reduc<strong>in</strong>g equivalents, it is logical to believe that O2, O2 and NO also affect redox balance. Nonetheless, the impact of these diatomic<br />
gases and oxygen radicals on the components of the Mtb TCA cycle is an<br />
understudied area.<br />
The first evidence of TCA cycle activity dur<strong>in</strong>g <strong>in</strong> vivo growth of Mtb was<br />
the demonstration of SDH activity <strong>in</strong> <strong>in</strong> vivo-derived bacilli (Segal, 1962).<br />
Subsequently, the activity of all the TCA cycle enzymes with the exception of
52 AISHA FARHANA ET AL.<br />
KDH was established through the analysis of Mycobacterium lepraemurium<br />
harvested from mur<strong>in</strong>e lepromas (Mori et al., 1971; Tepper and Varma,<br />
1972). Another important study which characterised the enzymes of the<br />
Mtb TCA cycle noted that all the dehydrogenases, unlike those present <strong>in</strong><br />
other organisms, are NADP + -dependent with the exception of malate dehydrogenase<br />
which is NAD + -dependent (Murthy et al., 1962). Likely explanations<br />
for this f<strong>in</strong>d<strong>in</strong>g are: (i) the NADP + dependence of the Mtb dehydrogenases<br />
‘guarantees’ the presence of substantial quantities of NADPH, the<br />
reduc<strong>in</strong>g agent necessary for metabolic biosynthesis of essential lipids, and<br />
(ii) the presence of NADH oxidase ensures that adequate NAD + is cont<strong>in</strong>uously<br />
available as an oxidis<strong>in</strong>g agent dur<strong>in</strong>g these processes (Murthy et al.,<br />
1962). Present-day genomic analysis seems to support the above <strong>in</strong>terpretations<br />
<strong>in</strong> that a considerable portion of the Mtb genome is dedicated to lipid<br />
anabolism, which requires NADPH.<br />
The work of Wayne and others (Segal, 1984) identified a switch from<br />
aerobic to anaerobic metabolism dur<strong>in</strong>g <strong>in</strong> vivo growth and found this to<br />
be an important factor <strong>in</strong> virulence. Notably, the identification of hypoxia<br />
as an <strong>in</strong> vivo signal for metabolic transformation profoundly affected<br />
future scientific studies and led many years later to the widely used<br />
Wayne model of <strong>in</strong> vitro dormancy (Wayne and Hayes, 1996). This <strong>in</strong> turn<br />
facilitated the identification of the 48-member Mtb Dos dormancy regulon,<br />
a genetic response <strong>in</strong>duced by hypoxia, NO and CO (Sherman et al.,<br />
2001; Ohno et al., 2003; Voskuil et al., 2003; Kumar et al., 2008; Shiloh et al.,<br />
2008), which has become a paradigm for Mtb signal transduction <strong>in</strong><br />
response to host cues (see Section 6.2 for a complete discussion). The<br />
implication is that the metabolic adaptation or response of Mtb to the lack<br />
of O2 or the presence of NO and CO <strong>in</strong> vivo <strong>in</strong>duces the Dos regulon and<br />
allows establishment of a latent <strong>in</strong>fection. This raises several important<br />
questions such as which term<strong>in</strong>al electron acceptor, besides O 2,isused<br />
<strong>in</strong> vivo, and how are reduc<strong>in</strong>g equivalents re-oxidised to ma<strong>in</strong>ta<strong>in</strong> redox<br />
balance?<br />
4.2.2. The Carbon Oxidation State (COS)<br />
Experimental evidence <strong>in</strong> support of FAs as potential <strong>in</strong> vivo carbon<br />
sources for Mtb was provided several decades ago (Segal and Bloch,<br />
1956; Segal, 1984), and is supported by many recent studies (McK<strong>in</strong>ney<br />
et al., 2000; Munoz-Elias and McK<strong>in</strong>ney, 2005). In vivo grown Mtb and<br />
M. lepraemurium were shown to robustly oxidise long-cha<strong>in</strong> FA such as<br />
n-heptanoic, octanoic, oleic, palmitic, steric, l<strong>in</strong>oleic, l<strong>in</strong>olenic and luric
REDUCTIVE STRESS IN MICROBES 53<br />
acids, but failed to utilise carbohydrates (Segal and Bloch, 1956; Segal,<br />
1984). This disagrees with the impression that bacilli <strong>in</strong> <strong>in</strong>fected tissue exist<br />
<strong>in</strong> a reduced state of metabolic activity. These f<strong>in</strong>d<strong>in</strong>gs are further supported<br />
by recent Mtb genome data reveal<strong>in</strong>g the presence of 36 homologs<br />
of fadE and fadD genes catalys<strong>in</strong>g the first step of b-oxidation. Other<br />
bacteria such as E. coli and Salmonella enterica serovar Typhimurium,<br />
have only a s<strong>in</strong>gle fadE gene (Campbell and Cronan, 2002). Several studies<br />
strongly suggest that isocitrate lyase (icl), an enzyme of the glyoxylate<br />
cycle, enabl<strong>in</strong>g the recycl<strong>in</strong>g of acetyl-CoA (formed via b-oxidation), plays<br />
an important role <strong>in</strong> FA carbon source utilisation <strong>in</strong> vivo. Studies of an Mtb<br />
Dicl mutant <strong>in</strong> mice (McK<strong>in</strong>ney et al., 2000) showed that this mutant stra<strong>in</strong><br />
is attenuated at the onset of adaptive immunity (3 weeks post-<strong>in</strong>fection) <strong>in</strong><br />
immunocompetent animals, but rema<strong>in</strong>s virulent <strong>in</strong> g-IFN-deficient mice.<br />
Further research on icl (Munoz-Elias and McK<strong>in</strong>ney, 2005; Munoz-Elias<br />
et al., 2006) substantiates the importance of lipids as an important <strong>in</strong> vivo<br />
carbon source for Mtb.<br />
The effect of a carbon source (e.g. glucose vs. FA) on the ‘spontaneity’<br />
of a process, as def<strong>in</strong>ed by the Gibbs free energy (G) (Voet et al.,<br />
2008) is illustrated by the fact that the complete oxidation of glucose<br />
yields DG ’ = 2850 kJ/mol, whereas oxidation of a C 16 FA such as<br />
palmitate (C 16H 32O 2, a putative <strong>in</strong> vivo carbon substrate of Mtb) ismore<br />
exergonic and yields DG ’ = 9781 kJ/mol. Palmitate and oleate have<br />
highly reduced carbon oxidation states (COSs) of 28 and 30 respectively,<br />
compared to other FA precursors such as propionate (COS = 1),<br />
valerate (COS = 6) and carbohydrates such as glucose (COS = 0) and<br />
sorbitol (COS = 1). Subsequent b-oxidation of palmitate generates 106<br />
ATP, whereas oxidation of glucose produces 38 ATP. Importantly,<br />
b-oxidation of FA yields one NADH and one FADH2 molecule for every<br />
acetyl-CoA generated, a condition which has the potential to cause<br />
cellular redox imbalance lead<strong>in</strong>g to reductive stress if the consequent<br />
buildup of reduc<strong>in</strong>g equivalents is not dissipated. Thus, the oxidation<br />
state of the carbon source determ<strong>in</strong>es the amount of reduc<strong>in</strong>g equivalents<br />
[e.g. NAD(P)H] to be recycled and consequently also the excreted<br />
products (see Section 4.2.3).<br />
In E. coli, it has been shown that the COS, extracellular oxido-reduction<br />
potential and environmental pH (Kleman and Strohl, 1994) <strong>in</strong>fluence the<br />
composition of excreted fermentation products. For example, <strong>in</strong> E. coli,<br />
oxidation of glucose and sorbitol generates two and three reduc<strong>in</strong>g equivalents<br />
respectively, whereas utilisation of the highly oxidised sugar glucuronic<br />
acid (COS = +2) results <strong>in</strong> no NADH production. Thus, <strong>in</strong> order to recycle<br />
the NADH produced dur<strong>in</strong>g growth on the more reduced substrate, sorbitol,
54 AISHA FARHANA ET AL.<br />
E. coli excretes reduced ethanol (COS = 2). In contrast, cells grown on<br />
glucuronic acid are redox balanced and do not need to produce ethanol;<br />
rather, glucuronic acid is converted to acetate (COS = 0) (Wolfe, 2005).<br />
Other studies have shown that as the pH drops, E. coli produces lactate<br />
rather than acetate or formate (Bunch et al., 1997), and that the rate of<br />
glycolysis is dramatically reduced (Og<strong>in</strong>o et al., 1980). Clearly, the physicochemical<br />
properties of a particular carbon source (e.g. FA or glucose and<br />
therefore the COS), environmental factors and the metabolites produced<br />
dur<strong>in</strong>g substrate utilisation profoundly affect redox balance and thus overall<br />
microbial physiology.<br />
4.2.3. Excretion of Metabolites and Redox Balance<br />
E. coli regenerates NAD + under anaerobic conditions via the production and<br />
excretion of partially oxidised metabolic <strong>in</strong>termediates such as D-lactate,<br />
succ<strong>in</strong>ate, formate and ethanol. Similarly, acetate excretion by E. coli occurs<br />
anaerobically dur<strong>in</strong>g mixed acid fermentation <strong>in</strong> order to regenerate the<br />
NAD + consumed by glycolysis and to recycle Coenzyme A (CoASH) utilised<br />
dur<strong>in</strong>g the conversion of pyruvate to acetyl-CoA (Wolfe, 2005). Acetate can<br />
also be excreted dur<strong>in</strong>g aerobic growth on high concentrations of glucose<br />
(Crabtree effect), which <strong>in</strong>hibits respiration (Ko et al., 1993; Wolfe, 2005).<br />
Because NAD + is required by the glycolytic enzyme glyceraldehyde-3phosphate<br />
dehydrogenase (GAPDH), E. coli must re-oxidise NADH to<br />
ma<strong>in</strong>ta<strong>in</strong> a work<strong>in</strong>g glycolytic pathway. In the absence of a functional<br />
TCA cycle dur<strong>in</strong>g anaerobic growth, the reduc<strong>in</strong>g equivalents are recycled<br />
by the production of metabolic <strong>in</strong>termediates such as D-lactate, ethanol,<br />
succ<strong>in</strong>ate and formate, which are secreted along with acetate <strong>in</strong>to the culture<br />
medium. However, acetate excretion produces ATP, whereas the other<br />
metabolites are not used as energy harvest<strong>in</strong>g molecules, but rather consume<br />
reduc<strong>in</strong>g equivalents (Wolfe, 2005). Thus, under anaerobic conditions, bacteria<br />
excrete a range of products <strong>in</strong> order to regenerate NAD + and to<br />
ma<strong>in</strong>ta<strong>in</strong> redox balance.<br />
Other studies have yielded a few clues as to the <strong>in</strong>termediary metabolic<br />
changes mycobacteria undergo dur<strong>in</strong>g aerobic respiration and oxidation of<br />
diverse substrates. An <strong>in</strong>terest<strong>in</strong>g observation made <strong>in</strong> 1930 (Merrill, 1930)<br />
was that mycobacteria utilise carbohydrates without the production of acids,<br />
suggest<strong>in</strong>g that carbohydrates are completely oxidised, leav<strong>in</strong>g <strong>in</strong>significant<br />
amounts of partially oxidised products (e.g. acids) <strong>in</strong> the medium (Merrill,<br />
1930; Edson, 1951). Initially, precise manometer measurements of respiratory<br />
changes (the respiratory quotient) were determ<strong>in</strong>ed by measur<strong>in</strong>g O2 consumption<br />
and CO2 production of bacilli grow<strong>in</strong>g on carbon sources such as
REDUCTIVE STRESS IN MICROBES 55<br />
glucose or glycerol. However, it quickly became clear that <strong>in</strong> order to accurately<br />
<strong>in</strong>terpret the respiratory quotients, lipid and prote<strong>in</strong> content of the bacilli<br />
had to be determ<strong>in</strong>ed. Subsequently, ‘starved’ bacilli (achieved by float<strong>in</strong>g<br />
bacilli on phosphate buffered sal<strong>in</strong>e for several days) rather than ‘washed’ cell<br />
suspensions were used <strong>in</strong> manometer techniques. Notably, autorespiration was<br />
barely impaired after 1–4 days of starvation. It subsequently became clear that<br />
glycerol, acetate and FA enhanced respiration whereas glucose stimulation<br />
was weak, and arab<strong>in</strong>ose, fructose, mannose and <strong>in</strong>ositol showed no effect<br />
(Edson, 1951). However, an <strong>in</strong>trigu<strong>in</strong>g and important observation was that<br />
there did not seem to be a direct correlation between growth and the respir<strong>in</strong>g<br />
capacity of a substrate. In fact, a substrate that promotes respiration could<br />
<strong>in</strong>hibit or <strong>in</strong>duce bacterial growth, or may have no <strong>in</strong>fluence at all (Bloch et al.,<br />
1947). Carbon balance experiments have shown that when glucose was used as<br />
a carbon source, 34% of its carbon was recovered as CO 2, 63% was found <strong>in</strong><br />
the bacilli and 2–6% rema<strong>in</strong>ed <strong>in</strong> the media (Edson, 1951).<br />
Several studies demonstrated that human and bov<strong>in</strong>e tubercle bacilli<br />
grow<strong>in</strong>g <strong>in</strong> glycerol medium generated alkal<strong>in</strong>e culture supernatants<br />
(reviewed <strong>in</strong> Merrill, 1930). This contrasts with the vast majority of bacteria,<br />
which produce organic acids as cleavage products upon the utilisation of<br />
carbohydrates. Some researchers also argued that m<strong>in</strong>ute quantities of acids<br />
were formed from the oxidation of glycerol, whereas others believed that<br />
glycerol was completely utilised without the production of <strong>in</strong>termediates.<br />
However, unconfirmed studies (Fowler et al., 1960) claimed that virulent and<br />
avirulent mycobacterial stra<strong>in</strong>s accumulate acetic acid, succ<strong>in</strong>ic acid, malic<br />
acid, citric acid, oxalic acid and pyruvic acid <strong>in</strong> the culture filtrate. Further,<br />
Mycobacterium butyricum became a model organism for study<strong>in</strong>g acid formation,<br />
s<strong>in</strong>ce it was noted that M. butyricum acidifies its culture medium.<br />
Us<strong>in</strong>g this organism, several studies demonstrated the excretion of a-ketoglutaric<br />
acid (2-oxoglutaric acid), succ<strong>in</strong>ic acid, acetic acid and pyruvic acid<br />
<strong>in</strong>to the culture filtrate (Hunter, 1953; Wright, 1959). Succ<strong>in</strong>ic, acetic and<br />
fumaric acids and DL-5-carboxymethylhydanto<strong>in</strong> were also isolated as crystall<strong>in</strong>e<br />
products from Mtb and Mycobacterium ranae cultured <strong>in</strong> a def<strong>in</strong>ed<br />
medium conta<strong>in</strong><strong>in</strong>g asparag<strong>in</strong>e, glycerol and trace quantities of citrate<br />
(Fowler et al., 1960). Acetyl L-isoleuc<strong>in</strong>e and acetyl L-leuc<strong>in</strong>e were also<br />
identified <strong>in</strong> culture filtrates of M. ranae (Fowler et al., 1961). Although a<br />
recent mass spectrometry-based study identified small quantities of pyruvate<br />
(18 mM), succ<strong>in</strong>ate (15 mM) and lactate (15 mM) (Goodw<strong>in</strong> et al., 2006) <strong>in</strong> the<br />
culture supernatants of Mtb, the experimental conditions were limited,<br />
necessitat<strong>in</strong>g a more comprehensive <strong>in</strong>vestigations to exam<strong>in</strong>e excreted metabolic<br />
<strong>in</strong>termediates of Mtb under a range of environmental conditions.<br />
Thus, unlike E. coli, mycobacterial species <strong>in</strong> general appears not to excrete
56 AISHA FARHANA ET AL.<br />
large amounts of <strong>in</strong>termediary metabolites and therefore has a dist<strong>in</strong>ct metabolic<br />
mechanism to ma<strong>in</strong>ta<strong>in</strong> <strong>in</strong>tracellular redox balance.<br />
4.2.4. The Balanc<strong>in</strong>g Act In Vitro and In Vivo<br />
In agreement with prior f<strong>in</strong>d<strong>in</strong>gs (Segal and Bloch, 1956), many studies have<br />
confirmed that there are metabolic dist<strong>in</strong>ctions between <strong>in</strong> vivo and <strong>in</strong> vitro<br />
grown mycobacteria. For example, differences exist between phtiocol, tuberculostearic<br />
acid, phtioic acid, specific polysaccharides (Anderson et al., 1943)<br />
and the lipid content of <strong>in</strong> vitro cultured Mtb and that of bacilli <strong>in</strong> tuberculous<br />
lung tissue (Sheehan and Whitwell, 1949). The subsequent development of<br />
differential centrifugation techniques to purify Mtb from animal lung tissue<br />
(Segal and Bloch, 1956), and biochemical comparison with <strong>in</strong> vitro grown<br />
Mtb led to a profoundly new understand<strong>in</strong>g of the phenotypic and metabolic<br />
states of Mtb grown <strong>in</strong> vitro and <strong>in</strong> vivo. In these studies, separation of<br />
tubercle bacilli from <strong>in</strong>fected lungs <strong>in</strong>volved the use of isotonic sucrose<br />
and differential centrifugation at low temperatures to yield considerable<br />
quantities of highly purified bacilli. Us<strong>in</strong>g Warburg manometry (which measures<br />
O2 consumption) and test<strong>in</strong>g the hydrogen transfer capacity of bacilli <strong>in</strong><br />
the presence of a range of substrates and the electron acceptor 2,3,5-triphenyl<br />
tetrazolium chloride, it was shown that the metabolic activity of <strong>in</strong> vivo<br />
grown Mtb was very low compared to that of <strong>in</strong> vitro cultured Mtb, which was<br />
ma<strong>in</strong>ta<strong>in</strong>ed for at least 20 h post-purification. In addition, the respiratory<br />
response of <strong>in</strong> vivo grown Mtb to glucose, glycerol, sodium lactate, sodium<br />
acetate and sodium pyruvate was shown to be virtually absent. On the other<br />
hand, salicylate and the FA n-heptanoic acid, octanoic acid and oleic acid,<br />
stimulated respiration to the same degree <strong>in</strong> <strong>in</strong> vivo grown bacilli as that<br />
observed <strong>in</strong> <strong>in</strong> vitro cultured Mtb. The robust respiratory responses of the<br />
bacilli isolated from the lungs towards FAs suggest that <strong>in</strong> vivo bacilli do not<br />
exist <strong>in</strong> a reduced state of metabolic activity. Intrigu<strong>in</strong>gly, <strong>in</strong> vitro cultur<strong>in</strong>g of<br />
the lung-derived Mtb <strong>in</strong> standard culture medium rapidly reversed the <strong>in</strong> vivo<br />
phenotype to that of the <strong>in</strong> vitro cultured bacilli (Segal and Bloch, 1956).<br />
An additional study (Segal, 1962) raised concerns regard<strong>in</strong>g the validity of<br />
<strong>in</strong> vitro based experiments by demonstrat<strong>in</strong>g that untreated whole cells of<br />
<strong>in</strong> vivo grown Mtb exhibit active SDH activity, whereas <strong>in</strong> vitro cultured Mtb<br />
cells are negative for SDH activity (cell-free extracts of the latter were shown<br />
to be positive for SDH activity). In another study, the lack of respiratory<br />
responses of <strong>in</strong> vivo grown Mtb for succ<strong>in</strong>ate, fumarate, a-oxoglutarate,<br />
malate and glyoxylate was <strong>in</strong> fact due to the impermeability of the bacilli<br />
because cell free extracts, but not <strong>in</strong>tact cells, oxidised these substrates <strong>in</strong> the<br />
presence of an electron acceptor (Murthy et al., 1962). This metabolic
REDUCTIVE STRESS IN MICROBES 57<br />
disparity between <strong>in</strong> vitro and <strong>in</strong> vivo grown Mtb was hypothesised to be<br />
(i) <strong>in</strong>itial repression of the TCA cycle and/or repression of the ETC dur<strong>in</strong>g<br />
<strong>in</strong> vivo growth, (ii) host-<strong>in</strong>duced <strong>in</strong>hibition of Mtb oxidative enzymes dur<strong>in</strong>g<br />
<strong>in</strong> vivo growth or (iii) substrate impermeability dur<strong>in</strong>g <strong>in</strong> vivo growth (Segal,<br />
1984). Collectively, the metabolic dissimilarity between <strong>in</strong> vivo and <strong>in</strong> vitro<br />
grown bacilli po<strong>in</strong>ted to a metabolic shift away from the respiratory pathway<br />
towards anaerobic glycolysis (Segal, 1984).<br />
The gross morphological and biochemical differences between <strong>in</strong> vivo and<br />
<strong>in</strong> vitro cultured Mtb, as <strong>in</strong>dicated by the above-mentioned studies, are <strong>in</strong><br />
agreement with modern expression studies demonstrat<strong>in</strong>g differential<br />
expression of genes encod<strong>in</strong>g prote<strong>in</strong>s needed for cell wall synthesis, virulence<br />
lipid anabolism and energy production <strong>in</strong> macrophages, animal models<br />
and humans (Triccas et al., 1999; Talaat et al., 2004, 2007; Shi et al., 2005;<br />
Rachman et al., 2006; Srivastava et al., 2007, 2008; Fontan et al., 2008). In a<br />
sem<strong>in</strong>al study, transcriptional analysis of Mtb derived from <strong>in</strong>fected lung<br />
samples (Rachman et al., 2006) found dramatic changes <strong>in</strong> genes <strong>in</strong>volved<br />
<strong>in</strong> cell envelope, lipid biosynthesis, FA and mycolic acid biosynthesis, and<br />
anaerobic respiration. In addition, us<strong>in</strong>g the mouse model for TB, <strong>in</strong> vivo<br />
lipidomics studies have suggested a l<strong>in</strong>k between host lipid catabolism and<br />
<strong>in</strong>creased production of Mtb virulence lipid (PDIM, SL-1) (Ja<strong>in</strong> et al., 2007).<br />
In an elegant study exam<strong>in</strong><strong>in</strong>g the transcriptional profile of Mtb <strong>in</strong> sputum,<br />
genes <strong>in</strong>volved <strong>in</strong> anaerobic respiration and tgs1, which encodes for the<br />
enzyme responsible for produc<strong>in</strong>g TAG (Garton et al., 2008), were found<br />
to be overexpressed. Tgs1 is under the strict control of the Dos dormancy<br />
regulon that is <strong>in</strong>duced by hypoxia, NO and CO (Sherman et al., 2001; Ohno<br />
et al., 2003; Voskuil et al., 2004). TAG production <strong>in</strong> sputum contradicts the<br />
assumption that sputum conta<strong>in</strong>s aerobically replicat<strong>in</strong>g bacilli. It has been<br />
argued that hypoxic conditions do not exist <strong>in</strong> sputum and thus the Dos<br />
dormancy regulon would not be <strong>in</strong>duced (Barry et al., 2009). While the pO 2<br />
concentration of tuberculous sputum has not been established, Worlitzsch<br />
et al. (2002) reported an <strong>in</strong> situ pO2 concentration of 2.5 mm Hg <strong>in</strong> the mucus<br />
of cystic fibrosis (CF) patients, a measurement made via aClarkeelectrode<br />
attached to a fibre-optic bronchoscope. The latter f<strong>in</strong>d<strong>in</strong>g along with the<br />
severely restricted diffusion of O2 through mucopurulent lum<strong>in</strong>al material<br />
(Worlitzsch et al., 2002) provides good evidence that a hypoxic environment<br />
can <strong>in</strong>deed be generated <strong>in</strong> sputum.<br />
Us<strong>in</strong>g fluorescent dyes and cell surface antibodies to exam<strong>in</strong>e the ultrastructure<br />
of Mtb <strong>in</strong> mice and gu<strong>in</strong>ea pigs, it was shown that Mtb exists <strong>in</strong><br />
different subpopulations (Ryan et al., 2010). This suggests that Mtb <strong>in</strong> vivo<br />
exist as vary<strong>in</strong>g stochastic phenotypes, which may allow the bacilli to adapt to<br />
a chang<strong>in</strong>g host environment. Collectively, modern-day gene expression,
58 AISHA FARHANA ET AL.<br />
cellular and morphological data are <strong>in</strong> support of the classical biochemical<br />
studies (Anderson et al., 1943; Segal and Bloch, 1956; Segal, 1965, 1984),<br />
which reported profound differences <strong>in</strong> cell wall architecture, virulence, lipid<br />
production and energy metabolism between <strong>in</strong> vivo grown Mtb and <strong>in</strong> vitro<br />
cultured bacilli.<br />
4.2.5. The Gaseous Environment of the Lung<br />
An irrefutable f<strong>in</strong>d<strong>in</strong>g, based upon 100 years of study, ascerta<strong>in</strong>ed that Mtb<br />
cannot replicate <strong>in</strong> the absence of O2. The rate of Mtb multiplication<br />
decreases rapidly as the partial O2 pressure (pO2) falls below that of room<br />
air (Dubos, 1953; Wayne and Hayes, 1996). The pO2 of atmospheric O2 is<br />
150–160 mm Hg and drops substantially <strong>in</strong> the lung (60–150 mm Hg) and<br />
blood ( 104 mm Hg) (Aly et al., 2006; Brahimi-Horn and Pouyssegur, 2007),<br />
the rat spleen ( 16 mm Hg) and thymus (10 mm Hg) (Braun et al., 2001) and<br />
the TB granuloma (1.59 mm Hg) (Via et al., 2008).<br />
The total alveolar surface area encountered by O2 <strong>in</strong> <strong>in</strong>haled air is<br />
130 m 2 , which allows for optimum O2 exchange (Murray, 2010). Besides<br />
O2, another gas, NO, is encountered by Mtb dur<strong>in</strong>g <strong>in</strong>fection. NO is a small,<br />
highly diffusible free radical. Inducible nitric oxide synthase (iNOS) and<br />
therefore NO production are crucial for protection of mice aga<strong>in</strong>st Mtb<br />
(MacMick<strong>in</strong>g et al., 1997; Chan et al.,2001). Importantly, human macrophages<br />
present <strong>in</strong> Mtb-<strong>in</strong>fected tissues have been demonstrated to express iNOS<br />
(Nicholson et al., 1996). Similarly, <strong>in</strong>creased exhaled NO and NO3 levels<br />
<strong>in</strong> patients with active pulmonary TB were shown to be due to <strong>in</strong>creased<br />
iNOS production (Wang et al., 1998). In macrophages, iNOS uses NADPH<br />
and O2 as cofactors and produces NO and its oxidative products NO2 and<br />
NO3 . The diffusion distance of NO is 175 mm (Leone et al., 1996) and the<br />
concentrations <strong>in</strong> sk<strong>in</strong>, a s<strong>in</strong>gle endothelial cell, and rat lung ranges from 0.14<br />
to 0.95 mM (Clough et al., 1998; Brovkovych et al., 1999). Although it is<br />
<strong>in</strong>tuitively assumed that NO is present <strong>in</strong> TB lesions, the fact that iNOS<br />
requires O2 as cofactor for its enzymatic function (KmO2 ¼ 135 mM) (Dweik,<br />
2005) suggests that NO production would be severely <strong>in</strong>hibited with<strong>in</strong> these<br />
hypoxic granulomas [1.59 mm Hg (Via et al., 2008)].<br />
CO is a diatomic gas that is endogenously produced by heme oxygenase-1<br />
(HO-1) <strong>in</strong> the human lungs <strong>in</strong> response to oxidative stress. HO-1 enzymatic<br />
activity requires three moles of molecular O2 per heme molecule oxidised<br />
and NADPH or NADH (albeit only <strong>in</strong> vitro) as reduc<strong>in</strong>g equivalents (Chung<br />
et al., 2009; Ryter and Choi, 2009). A credible role for CO <strong>in</strong> Mtb persistence<br />
was first discovered dur<strong>in</strong>g the biochemical and biophysical characterisations<br />
of DosS and DosT (Kumar et al., 2007; Sousa et al., 2007). CO was
REDUCTIVE STRESS IN MICROBES 59<br />
subsequently shown to <strong>in</strong>duce the complete Mtb Dos dormancy regulon and<br />
was demonstrated to be produced <strong>in</strong> the lungs of Mtb-<strong>in</strong>fected mice (Kumar<br />
et al., 2008; Shiloh et al., 2008). It is known that hypoxia, NO (Voskuil et al.,<br />
2003) and CO (Davidge et al., 2009) each <strong>in</strong>dividually <strong>in</strong>hibits respiration and<br />
that comb<strong>in</strong>ations of these gases may even act synergistically to do the same.<br />
CO 2 has also been shown to be extremely unfavourable for <strong>in</strong> vitro survival<br />
(Dubos, 1953) and survival of Mtb <strong>in</strong> the cavities of TB patients<br />
(Haapanen et al., 1959). On the other hand, low concentrations of CO 2 have<br />
been shown to enhance survival of BCG under hypoxic conditions<br />
(Florczyk et al., 2003).<br />
Differences exist <strong>in</strong> ventilation and perfusion of various areas of the lung,<br />
as do the degree of blood oxygenation and pO2. In the upper lung regions,<br />
higher O 2 tension is present (Rich and Follis, 1942; Rasmussen, 1957; Riley,<br />
1957; West, 1977). As proposed earlier (Kumar et al., 2008), Mtb likely<br />
encounters gradients of gases (e.g. NO, CO, O2 or CO2) dur<strong>in</strong>g the course<br />
of <strong>in</strong>fection, which contribute towards produc<strong>in</strong>g vary<strong>in</strong>g microenvironments<br />
and dist<strong>in</strong>ct granulomatous populations. These microenvironments<br />
may <strong>in</strong>clude caseous, fibrotic and non-necrotis<strong>in</strong>g granulomas all occurr<strong>in</strong>g<br />
with<strong>in</strong> the same lung (Barry et al., 2009). Thus, it is reasonable to conclude<br />
that if anaerobic granulomas exist, Mtb will not be able to survive <strong>in</strong> them.<br />
However, hypoxic (as opposed to anaerobic) granulomas provide bacilli with<br />
the capacity to respire, albeit at a low metabolic state, allow<strong>in</strong>g survival<br />
and promot<strong>in</strong>g antimicrobial tolerance (see Section 6.2.4.1 on the role of<br />
NO3 /NO2 <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g Mtb viability under anaerobic conditions).<br />
Mtb has an extraord<strong>in</strong>ary capacity to persist for decades <strong>in</strong> the human lung<br />
despite conditions that should be detrimental to its survival. Low levels of O 2<br />
<strong>in</strong> TB granulomas and the presence of diatomic host gases <strong>in</strong>hibit respiration,<br />
effects which profoundly <strong>in</strong>fluence the metabolic state of the tubercle bacillus.<br />
Detailed knowledge of the metabolic state of Mtb with<strong>in</strong> the granulomatous<br />
host environment is lack<strong>in</strong>g and severely hampers our understand<strong>in</strong>g<br />
of the mechanism(s) of Mtb persistence.<br />
5. REDUCTIVE SINKS IN MICROBES<br />
5.1. Fermentation<br />
5.1.1. Saccharomyces cerevisiae<br />
A vast literature shows that mycobacterial species are <strong>in</strong>capable of fermentation.<br />
Nonetheless, s<strong>in</strong>ce Mtb is exposed to a hypoxic environment <strong>in</strong> vivo
60 AISHA FARHANA ET AL.<br />
and encounters host defence molecules such as NO and CO, which <strong>in</strong>hibit<br />
respiration, it is important to identify the metabolic pathways <strong>in</strong>volved <strong>in</strong> the<br />
reoxidation of reduc<strong>in</strong>g equivalents <strong>in</strong> order to ma<strong>in</strong>ta<strong>in</strong> redox balance. It<br />
therefore becomes imperative to look at examples provided by other model<br />
organisms to identify parallels of such metabolic events.<br />
The lower eukaryote Saccharomyces cerevisiae (S. cerevisiae) isaparticularly<br />
attractive model organism s<strong>in</strong>ce, as is the case for eukaryotes, its cytoplasm<br />
is highly reduced [2GSH/GSSG = 70–190:1 (Grant et al., 1998; Garrido<br />
and Grant, 2002)], whereas the endoplasmic reticulum (ER), where oxidative<br />
prote<strong>in</strong> fold<strong>in</strong>g occurs, is oxidised [2GSH/GSSG = 1–3:1 (Hwang et al.,1992)].<br />
Compartmentalisation of these oxido-reductive events is essential for proper<br />
cellular function<strong>in</strong>g of the organism, and more specifically, to protect <strong>in</strong>tracellular<br />
components from non-specific oxidation or reduction. In order to<br />
dissect the oxido-reductive events associated with these functionally dist<strong>in</strong>ct<br />
cellular compartments, the strong reduc<strong>in</strong>g agent dithiothreitol (DTT)<br />
was used as a molecular tool to promote reductive stress. Comprehensive<br />
microarray analysis showed that S. cerevisiae treated with DTT generates a<br />
transcriptional response that is dist<strong>in</strong>ct from other stresses such as hyperosmotic<br />
stress, starvation and heat shock (Gasch et al., 2000). DTT exposure<br />
also <strong>in</strong>duces the upregulation of prote<strong>in</strong> disulfide isomerase, prote<strong>in</strong> fold<strong>in</strong>g<br />
chaperones localised to the ER and other genes that respond to changes <strong>in</strong><br />
the cellular redox potential. Furthermore, the upregulation of genes <strong>in</strong>volved<br />
<strong>in</strong> cell wall synthesis and signal<strong>in</strong>g pathways responsive to cell wall damage<br />
was noted and led the <strong>in</strong>vestigators to conclude that cell wall defects eventually<br />
<strong>in</strong>itiate the environmental stress response (ESR). Further <strong>in</strong>vestigations<br />
us<strong>in</strong>g DTT exposure showed that the loss of S. cerevisiae genes encod<strong>in</strong>g two<br />
thioredox<strong>in</strong>s (trx1, trx2) causes sensitivity to DTT (Trotter and Grant, 2002).<br />
S<strong>in</strong>ce thioredox<strong>in</strong>s are small oxidoreductases that typically protect cells<br />
aga<strong>in</strong>st oxidative stress, the observed sensitivity to DTT was <strong>in</strong>trigu<strong>in</strong>g.<br />
Given that thioredox<strong>in</strong> loss was previously shown to cause an imbalance <strong>in</strong><br />
the 2GSH:GSSG ratio (Muller, 1996; Garrido and Grant, 2002), the f<strong>in</strong>d<strong>in</strong>gs<br />
suggest that the yeast cellular redox mach<strong>in</strong>ery requires precise regulation to<br />
protect aga<strong>in</strong>st oxidative and reductive stress, and that thioredox<strong>in</strong>s help<br />
ma<strong>in</strong>ta<strong>in</strong> redox homeostasis <strong>in</strong> response to both oxidative and reductive<br />
stress. The mode of action of the yeast redox mach<strong>in</strong>ery is functionally dist<strong>in</strong>ct<br />
from that of bacteria, s<strong>in</strong>ce loss of bacterial thioredox<strong>in</strong> results <strong>in</strong> sensitivity to<br />
the thiol-oxidis<strong>in</strong>g agent diamide (Ritz et al., 2000), whereas loss of trx1 and<br />
trx2 <strong>in</strong> S. cerevisiae produced diamide resistance (Trotter and Grant, 2002).<br />
Thioredox<strong>in</strong>s, glutaredox<strong>in</strong>s and GSH are thermodynamically l<strong>in</strong>ked, s<strong>in</strong>ce<br />
oxidised thioredox<strong>in</strong>s are reduced by NADPH and thioredox<strong>in</strong> reductase,<br />
whereas oxidised glutaredox<strong>in</strong>s are reduced by GSH and NADPH.
REDUCTIVE STRESS IN MICROBES 61<br />
In cont<strong>in</strong>uation of the above studies (Rand and Grant, 2006), S. cerevisiae<br />
was used <strong>in</strong> a screen to identify mutants sensitive to DTT. A large number of<br />
mutations were found that affected gene expression, metabolism and components<br />
of the secretory pathway, <strong>in</strong>clud<strong>in</strong>g a mutation that resulted <strong>in</strong> the<br />
loss of TSA1, encod<strong>in</strong>g a peroxiredox<strong>in</strong>. It was observed that TSA1 mutants<br />
accumulate aggregated ribosomal prote<strong>in</strong>s, thus impair<strong>in</strong>g translation<br />
<strong>in</strong>itiation (Rand and Grant, 2006). A complementary and physiologically<br />
relevant approach to study<strong>in</strong>g DTT exposure <strong>in</strong>cluded analysis of a glycerol-<br />
3-phosphate dehydrogenase (GPD2) mutant shown to have altered <strong>in</strong>tracellular<br />
NADH levels. Excessive NADH levels are thought to be the underly<strong>in</strong>g<br />
reason for the attenuated anaerobic growth of S. cerevisiae lack<strong>in</strong>g GPD2<br />
(Ansell et al., 1997).<br />
Dur<strong>in</strong>g S. cerevisiae fermentation, NADH/NAD + redox balance is ma<strong>in</strong>ta<strong>in</strong>ed<br />
when acetaldehyde is reduced to ethanol, which is redox neutral.<br />
Dur<strong>in</strong>g anaerobic growth, glycerol is follow<strong>in</strong>g ethanol and CO2, the most<br />
abundant byproduct, which is produced by the NADH-mediated reduction<br />
of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, followed<br />
by dephosphorylation (van Dijken and Scheffers, 1986). Nonetheless, the<br />
reduction of NAD + to NADH occurs via metabolite and biomass synthesis<br />
and NADH <strong>in</strong> turn must be re-oxidised to NAD + to ma<strong>in</strong>ta<strong>in</strong> redox balance.<br />
Thus, <strong>in</strong>tr<strong>in</strong>sically, glycerol functions as a redox s<strong>in</strong>k for anaerobic growth of<br />
S. cerevisiae, and as such, glycerol must be cont<strong>in</strong>uously produced to ma<strong>in</strong>ta<strong>in</strong><br />
redox balance (van Dijken and Scheffers, 1986). S. cerevisiae<br />
Dgpd1Dgpd2 cells are unable to synthesise glycerol under anaerobic conditions<br />
(Ansell et al., 1997) and consequently cannot re-oxidise NADH, which<br />
leads to NADH accumulation and growth arrest. However, provision of<br />
exogenous aceto<strong>in</strong> or acetaldehyde to the media (Ansell et al., 1997) as<br />
electron acceptors restored redox balance and growth. 2D-PAGE analyses<br />
of anaerobically grown S. cerevisiae Dgpd2 showed <strong>in</strong>creased expression of<br />
Tdh1p, the m<strong>in</strong>or isoform of G3PD, an effect which could be reversed by the<br />
addition of aceto<strong>in</strong>. S<strong>in</strong>ce deletion of TDH1 improved anaerobic growth of<br />
S. cerevisiae Dgpd2, it was speculated that TDH1 functions as a reporter for<br />
<strong>in</strong>tracellular NADH reductive stress (Valadi et al., 2004).<br />
The requirement of glycerol formation as a redox s<strong>in</strong>k for NADH<br />
<strong>in</strong> anaerobically cultured S. cerevisiae was abrogated by the NADHdependent<br />
reduction of acetic acid to ethanol (Med<strong>in</strong>a et al., 2010). In this<br />
study, the E. coli mhpF gene, encod<strong>in</strong>g the acetylat<strong>in</strong>g NAD + -dependent<br />
acetaldehyde dehydrogenase, was expressed <strong>in</strong> S. cerevisiae Dgpd1Dgpd2<br />
and was able to restore growth of the mutant under anaerobic conditions<br />
when the medium was supplemented with acetate as an electron acceptor<br />
(Med<strong>in</strong>a et al., 2010). An alternative approach for the reoxidation of
62 AISHA FARHANA ET AL.<br />
NADH, based upon the absence of transhydrogenase activity (NADH<br />
+NADP + $ NAD + +NADPH)<strong>in</strong>yeasts(van Dijken and Scheffers, 1986)<br />
was attempted, where<strong>in</strong> the ultimate goal was to heterologously <strong>in</strong>troduce<br />
a different pathway for reoxidation of NADH <strong>in</strong> yeast when glycerol<br />
synthesis was impaired. This would lead to an <strong>in</strong>creased ethanol production<br />
under aerobic and anaerobic conditions. Unfortunately, the system<br />
appeared to be more complex than anticipated, and attempts to <strong>in</strong>troduce<br />
an alternative NADH oxidation pathway by express<strong>in</strong>g the transhydrogenase<br />
of Azotobacter v<strong>in</strong>elandii <strong>in</strong> S. cerevisiae Dgpd1Dgpd2 was unsuccessful<br />
(Nissen et al., 2000).<br />
Formate is a particularly important NADH-generat<strong>in</strong>g substrate utilised<br />
by S. cerevisiae under aerobic conditions, s<strong>in</strong>ce CO2 [product of the formate<br />
dehydrogenase (FDH) reaction] does not accumulate <strong>in</strong> solution. In a metabolic<br />
eng<strong>in</strong>eer<strong>in</strong>g approach, formate, which cannot act as a carbon source<br />
for biomass formation, was used to <strong>in</strong>crease glycerol production under<br />
anaerobic conditions (Geertman et al., 2006). However, formate oxidation<br />
was shown to be <strong>in</strong>complete. S<strong>in</strong>ce low <strong>in</strong>tracellular NAD + concentrations<br />
negatively affect the <strong>in</strong> vivo Km of FDH for formate, GPD2 was overexpressed<br />
to re-oxidise NADH. The concurrent overexpression of FDH1 with<br />
GPD2 demonstrated a synergistic effect that resulted <strong>in</strong> consumption of 70%<br />
of the supplied formate (Geertman et al., 2006).<br />
In sum, overproduction of NAD(P)H has to be balanced by NAD(P)Hconsum<strong>in</strong>g<br />
pathways. In order to ma<strong>in</strong>ta<strong>in</strong> redox balance, yeast may secrete<br />
ethanol, polyalcohols, monocarboxylic acids and di- and tricarboxylic acids.<br />
5.1.2. Escherichia coli<br />
Bacteria have evolved a variety of mechanisms to balance the rate of oxidation<br />
and reduction. Reoxidation of NAD(P)H requires electron acceptors<br />
acquired from the environment (external acceptors) or they may be generated<br />
<strong>in</strong>tracellularly. When electron transfer occurs <strong>in</strong> a membrane-bound<br />
process, NADH oxidation may be l<strong>in</strong>ked to respiration (either aerobic or<br />
anaerobic) (de Graef et al., 1999), whereas electron transfer that occurs <strong>in</strong> the<br />
cytosol, aka fermentation, also re-oxidises NADH to generate NAD +<br />
(Wolfe, 2005).<br />
Pyruvate catabolism is the major switch po<strong>in</strong>t between the respiratory and<br />
fermentative responses. In the absence of O 2, energy must be supplied by<br />
either anaerobic respiration coupled to electron acceptors such as nitrate<br />
(NO 3 ) and fumarate, or by fermentation (Gray et al., 1966; Clark, 1989).<br />
For example, facultative anaerobes such as E. coli can use term<strong>in</strong>al electron<br />
acceptors such as fumarate, NO3 or DMSO <strong>in</strong> the process of anaerobic
REDUCTIVE STRESS IN MICROBES 63<br />
respiration. Alternatively, if no term<strong>in</strong>al electron acceptor is available,<br />
E. coli switches to fermentation, which makes use of endogenous organic<br />
compounds as electron acceptors to generate soluble products <strong>in</strong>clud<strong>in</strong>g<br />
acetate, ethanol, lactate, formate and succ<strong>in</strong>ate and gaseous products such<br />
as H 2 and CO 2 (Clark, 1989). Notably, because redox balance has to be<br />
ma<strong>in</strong>ta<strong>in</strong>ed, the ratio of these products is <strong>in</strong>fluenced by the number of<br />
reduc<strong>in</strong>g equivalents generated dur<strong>in</strong>g breakdown of the substrate. In the<br />
ethanolic fermentation pathway, ethanol, propionic acid and CO 2 are the<br />
end products whose formation is coupled with the conversion of NADH to<br />
NAD + . In the case of mixed acid fermentation carried out by E. coli and<br />
members of the genera Salmonella and Shigella, pyruvate is converted <strong>in</strong>to<br />
ethanol, acetate, succ<strong>in</strong>ate, formate, molecular H 2, lactate and CO2 (Gray<br />
et al., 1966; Wolfe, 2005). Dur<strong>in</strong>g synthesis of these products, NADH is<br />
re-oxidised to NAD + , and acetate production is accompanied by ATP formation<br />
via substrate-level phosphorylation. In addition to carbohydrates,<br />
am<strong>in</strong>o acids such as arg<strong>in</strong><strong>in</strong>e can be fermented by Clostridium, Streptococcus<br />
and Mycoplasma spp. to ornith<strong>in</strong>e, CO 2 and NH 3. Clostridia can<br />
ferment multiple am<strong>in</strong>o acids through the Stickland reaction <strong>in</strong> which one<br />
am<strong>in</strong>o acid functions as an electron donor and the other as an electron<br />
acceptor, allow<strong>in</strong>g regeneration of reduc<strong>in</strong>g equivalents (Atlas, 1996).<br />
The <strong>in</strong>tracellular redox state of E. coli as <strong>in</strong>dicated by the NADH/NAD +<br />
ratio is strongly <strong>in</strong>fluenced by the availability and nature of the external<br />
electron acceptors present <strong>in</strong> the extracellular environment (de Graef<br />
et al., 1999). Both fumarate and nitrate were shown to be electron acceptors<br />
capable of function<strong>in</strong>g as effective reductive (NADH) s<strong>in</strong>ks. The highest<br />
NADH/NAD + ratios occurred dur<strong>in</strong>g fermentation followed by fumarate<br />
respiration and nitrite respiration. Furthermore, <strong>in</strong> exam<strong>in</strong><strong>in</strong>g the relationship<br />
between dissolved O 2 tension (DOT) and the <strong>in</strong>tracellular levels of<br />
reduc<strong>in</strong>g equivalents, the NADH/NAD + redox ratio was found to <strong>in</strong>versely<br />
correlate with DOT; the lower the DOT, the higher the ratio, such that the<br />
most anaerobic condition exam<strong>in</strong>ed (DOT of 0.1%) was associated with the<br />
largest ratio (de Graef et al., 1999).<br />
As described earlier, acetogenesis, or the excretion of acetic acid <strong>in</strong>to the<br />
culture medium, can either occur as a result of growth <strong>in</strong>volv<strong>in</strong>g a high rate of<br />
glucose consumption <strong>in</strong> the presence of ample O2 (Crabtree effect), or<br />
dur<strong>in</strong>g growth under anaerobic conditions when the TCA cycle is not operat<strong>in</strong>g.<br />
Although acetic acid production can be viewed as a mechanism to<br />
reduce NAD(P)H accumulation, it subsequently leads to the production of<br />
ATP, whereas D-lactate, succ<strong>in</strong>ate, ethanol, formate and CO 2 are the<br />
excreted products that function as s<strong>in</strong>ks for accumulat<strong>in</strong>g reduc<strong>in</strong>g equivalents<br />
to ma<strong>in</strong>ta<strong>in</strong> redox balance (Wolfe, 2005).
64 AISHA FARHANA ET AL.<br />
To exam<strong>in</strong>e the role of the NADH/NAD + ratio <strong>in</strong> acetic acid overflow<br />
metabolism, the redox ratio <strong>in</strong> E. coli was modulated by overexpress<strong>in</strong>g<br />
the Streptococcus pneumoniae nox gene (encod<strong>in</strong>g water-form<strong>in</strong>g<br />
NADH oxidase), which decouples NADH oxidation from respiration<br />
(Vemuri et al., 2006). The data demonstrated that an <strong>in</strong>crease <strong>in</strong> oxidation<br />
of excess NADH led to decreased acetate formation and biomass yield, and<br />
<strong>in</strong>creased the glucose consumption rate by 50%. As expected, the redox<br />
ratio was always greater for nox bacteria than for the nox + stra<strong>in</strong>.<br />
However, acetate formation for both stra<strong>in</strong>s occurred at an identical<br />
NADH/NAD + ratio of 0.06, thereby establish<strong>in</strong>g a relationship between<br />
the redox state of the cell and overflow metabolism (Vemuri et al., 2006).<br />
Conversely, the effect of <strong>in</strong>creas<strong>in</strong>g <strong>in</strong>tracellular NADH was studied by<br />
substitut<strong>in</strong>g the native cofactor-<strong>in</strong>dependent FDH with an NAD + -dependent<br />
FDH from Candida boid<strong>in</strong>ii (Berrios-Rivera et al., 2002a,b,c).<br />
Overexpression of the yeast FDH <strong>in</strong> E. coli under anaerobic conditions<br />
caused an <strong>in</strong>crease <strong>in</strong> NADH and favoured the production of more reduced<br />
metabolites such as ethanol, which also generated a three- to fourfold<br />
<strong>in</strong>crease <strong>in</strong> the ethanol/acetate ratio (Berrios-Rivera et al., 2002b). In fact,<br />
the <strong>in</strong>creased availability of NADH <strong>in</strong>duced a shift towards fermentation <strong>in</strong><br />
the presence of O2 evident by the production of lactate, ethanol and succ<strong>in</strong>ate,<br />
all metabolites typically produced dur<strong>in</strong>g anaerobic fermentation<br />
(Berrios-Rivera et al., 2002a,b).<br />
5.2. Polymer Deposition<br />
5.2.1. Polyhydroxyalkonate (PHA), Poly-b-Hydroxybutyrate (PHB)<br />
and Triacylglycerol (TAG)<br />
Biosynthesis of polyketide or lipid-like molecules <strong>in</strong> response to a change <strong>in</strong><br />
<strong>in</strong>tracellular redox balance may be a general compensatory mechanism<br />
found <strong>in</strong> many bacteria. In fact, polyhydroxyalkonate (PHA) and polyb-hydroxybutyrate<br />
(PHB) are accumulated by diverse bacteria as carbon<br />
and reductive-power storage molecules (Encarnacion et al., 1995; Cevallos<br />
et al., 1996). As <strong>in</strong> the case of the TCA cycle, carbon flow through the<br />
pathways necessary for PHB or PHA accumulation is greatly <strong>in</strong>fluenced<br />
by growth and environmental conditions (e.g. O2 concentration) (Senior<br />
and Dawes, 1973).<br />
Biosynthesis of PHA, a storage lipid, <strong>in</strong> Azotobacter beijer<strong>in</strong>ckii was<br />
shown to be regulated by O2 concentration and carbon source (Senior and
REDUCTIVE STRESS IN MICROBES 65<br />
Dawes, 1973). The central regulator of PHA production is the flux of acetyl-<br />
CoA, which may be oxidised via the TCA cycle or can serve as a precursor<br />
for PHA synthesis depend<strong>in</strong>g upon oxygen concentration. Under oxygen<br />
limitation, when the NADH/NAD + ratio <strong>in</strong>creases, the activities of the TCA<br />
cycle enzymes, citrate synthase and isocitrate dehydrogenase are <strong>in</strong>hibited<br />
by NADH and as a consequence, acetyl-CoA could no longer enters the<br />
TCA cycle. Instead, acetyl-CoA is converted to acetoacetyl-CoA by 3ketothiolase,<br />
the first enzyme <strong>in</strong> the PHA biosynthesis pathway. Based on<br />
these f<strong>in</strong>d<strong>in</strong>gs, it was proposed that PHA serves not only as a reserve carbon<br />
and energy source, but also as a reductant s<strong>in</strong>k, similar to what was suggested<br />
for TAG (Senior and Dawes, 1973). Direct evidence <strong>in</strong> support of PHA <strong>in</strong><br />
regulat<strong>in</strong>g <strong>in</strong>tracellular redox balance was provided by Page and Knosp<br />
(1989), us<strong>in</strong>g an NADH oxidase-deficient stra<strong>in</strong> of A. v<strong>in</strong>elandii. This stra<strong>in</strong><br />
is unable to re-oxidise NADH via oxygen-dependent respiration and <strong>in</strong>stead<br />
accumulates large amounts of PHA, as a mechanism for the disposal of<br />
excess reductants (Page and Knosp, 1989).<br />
Species belong<strong>in</strong>g to the genera Rhizobium, Bradyrhizobium and<br />
Azorhizobium synthesise PHB dur<strong>in</strong>g symbiosis and <strong>in</strong> free-liv<strong>in</strong>g state<br />
(Encarnacion et al., 1995; Cevallos et al., 1996). Interest<strong>in</strong>gly, even though<br />
Rhizobium spp. are strict aerobes that are well adapted to survive microaerophilically,<br />
a fermentative response was also described. Besides serv<strong>in</strong>g<br />
as a s<strong>in</strong>k of reductive power, PHB is also a fermentative product, which is<br />
secreted like other organic acids and am<strong>in</strong>o acids (Encarnacion et al., 1995).<br />
Studies <strong>in</strong> Rhizobium etli have shown that a PHB mutant excreted significantly<br />
more pyruvate, fumarate, lactate, acetate and b-hydroxybutyrate<br />
compared to the wild-type (wt) stra<strong>in</strong> (Cevallos et al., 1996). This data,<br />
together with the observation that the NAD + /NADH ratio is much reduced,<br />
<strong>in</strong>dicates that the oxidative capacity of the organism is significantly<br />
decreased because of the absence of a s<strong>in</strong>k for reductive power<br />
(Cevallos et al., 1996).<br />
TAG is a water-<strong>in</strong>soluble triester of glycerol with FA and an excellent<br />
reserve substrate because of the reduced COS relative to carbohydrates or<br />
prote<strong>in</strong>s. Because of these properties, it yields significantly more energy<br />
when oxidised (Alvarez and Ste<strong>in</strong>buchel, 2002; Waltermann et al., 2007).<br />
b-Oxidation of the FA cha<strong>in</strong>s of TAG generates large quantities of reduc<strong>in</strong>g<br />
equivalents, which require subsequent oxidation. This requirement<br />
might be the reason why TAG-produc<strong>in</strong>g bacteria are all aerobes.<br />
Consistent with this notion, it has been suggested that, <strong>in</strong> act<strong>in</strong>omycetes,<br />
TAG could serve as a s<strong>in</strong>k for excess reductants accumulated <strong>in</strong> the<br />
absence of term<strong>in</strong>al electron acceptors (Alvarez and Ste<strong>in</strong>buchel, 2002).<br />
Rhodococcus ruber is capable of accumulat<strong>in</strong>g both TAG and PHA and
66 AISHA FARHANA ET AL.<br />
disruption of PHA biosynthesis leads to <strong>in</strong>creased accumulation of TAG,<br />
suggest<strong>in</strong>g a metabolic l<strong>in</strong>k between these two triesters (Alvarez et al.,<br />
2000; Alvarez and Ste<strong>in</strong>buchel, 2002). Studies <strong>in</strong> Rhodococcus opacus<br />
PD630 suggested that TAG production can be promoted by cultur<strong>in</strong>g<br />
bacteria under limited aeration (Alvarez and Ste<strong>in</strong>buchel, 2002).<br />
Similarly, <strong>in</strong>creased expression of Mtb tgs1 lead<strong>in</strong>g to TAG accumulation<br />
occurs under hypoxic conditions and dur<strong>in</strong>g exposure to NO and CO<br />
(Sherman et al., 2001; Ohno et al., 2003; Voskuil et al., 2003; Kumar<br />
et al., 2008). Consistent with the biological function of TAG and PHA, it<br />
appears that <strong>in</strong>hibition of respiration by NO or lack of O2 as term<strong>in</strong>al<br />
electron acceptor leads to an <strong>in</strong>creased amount of reduc<strong>in</strong>g equivalents,<br />
which can be dissipated via TAG, PHA or PHB anabolism.<br />
Recent reports provide important mechanistic l<strong>in</strong>ks between reductive<br />
stress, polyketide and TAG anabolism (see Section 6.2.4.2). It has been<br />
shown that dur<strong>in</strong>g persistence Mtb accumulates NAD(P)H, confirm<strong>in</strong>g the<br />
physiological presence of reductive stress <strong>in</strong> Mtb pathogenesis (Boshoff et al.,<br />
2008). Furthermore, <strong>in</strong> the mouse model for TB (Ja<strong>in</strong> et al., 2007) and <strong>in</strong> the<br />
<strong>in</strong> vitro model for dormancy (Daniel et al., 2004), Mtb <strong>in</strong>duces production of<br />
complex polyketides, such as PDIM and SL-1, and the storage lipid TAG<br />
respectively. S<strong>in</strong>ce accumulation of NADH can eventually lead to oxidative<br />
*<br />
stress by auto-oxidation and reduction of O2 to generate O2 , it has been<br />
proposed that polyketide and TAG anabolism could serve as efficient reductant<br />
disposal mechanisms utilised by Mtb to alleviate reductive stress for<br />
long-term persistence (S<strong>in</strong>gh et al., 2009). TAG is metabolised by Mtb upon<br />
reactivation from the Wayne model of <strong>in</strong> vitro dormancy (Deb et al., 2006),<br />
<strong>in</strong>dicat<strong>in</strong>g a possible role for TAG <strong>in</strong> emergence from a persistent state.<br />
5.3. Nitrate Reductase<br />
Under normal grow<strong>in</strong>g conditions, aerobic respiration <strong>in</strong> bacteria is primarily<br />
required to generate a proton motive force for ATP synthesis. Several<br />
studies suggest that <strong>in</strong>hibition of aerobic respiration, due to the lack of<br />
oxygen or exposure to NO, results <strong>in</strong> accumulation of reduc<strong>in</strong>g equivalents<br />
and depletion of ATP (de Graef et al., 1999; San et al., 2002; Berrios-Rivera<br />
et al., 2004; Sanchez et al., 2005; Vemuri et al., 2006; Husa<strong>in</strong> et al., 2008). It has<br />
been shown that <strong>in</strong> addition to hypoxia and NO, oxidative metabolism of<br />
highly reduced carbon substrates (e.g. palmitate, caproate, butyrate, oleate)<br />
also results <strong>in</strong> <strong>in</strong>tracellular reductive stress even <strong>in</strong> the presence of oxygen<br />
(Alam and Clark, 1989; Clark, 1989; Sears et al., 2000; Berrios-Rivera et al.,<br />
2004; L<strong>in</strong> et al., 2005; Sanchez et al., 2005). These studies suggest that under
REDUCTIVE STRESS IN MICROBES 67<br />
conditions of reductive stress, respiration is not coupled to proton translocation;<br />
rather, disposal of excess reductants from cells without ATP generation<br />
may be the dom<strong>in</strong>ant function of respiration (Sears et al., 2000). For<br />
example, <strong>in</strong> Paracoccus pantotrophus, electrons flow from ubiqu<strong>in</strong>ol to periplasmic<br />
nitrate reductase (Nap) without proton translocation dur<strong>in</strong>g growth<br />
on reduced carbon substrates such as acetate and butyrate (Ell<strong>in</strong>gton et al.,<br />
2002). P. pantotrophus is capable of both aerobic and anaerobic respiration<br />
on NO 3 <strong>in</strong> the presence of a variety of carbon sources with broad oxidation<br />
states. It has been shown that Nap is required to ma<strong>in</strong>ta<strong>in</strong> cellular redox<br />
homeostasis by provid<strong>in</strong>g an alternate route for the oxidation of excess<br />
reductants generated from the oxidative metabolism of highly reduced carbon<br />
substrates (Richardson, 2000). Furthermore, demonstrat<strong>in</strong>g that highly<br />
reduced carbon substrates (e.g. caproate and butyrate) <strong>in</strong>crease Nap activity<br />
whereas oxidised carbon substrates (e.g. succ<strong>in</strong>ate and malate) decrease Nap<br />
activity po<strong>in</strong>ts to a clear correlation between Nap activity and the COS of a<br />
carbon substrate (Sears et al., 2000). More importantly, transcription of the<br />
nap operon is shown to be dependent on carbon source, imply<strong>in</strong>g that nap<br />
transcription responds to a change <strong>in</strong> the cellular redox state due to the<br />
metabolism of the reduced carbon substrates (Sears et al., 2000). A strict<br />
hierarchical preference for carbon sources was exhibited by P. pantotrophus.<br />
The less reduced carbon source, succ<strong>in</strong>ate, is preferred over acetate, which <strong>in</strong><br />
turn is preferred over butyrate for growth. This hierarchical preference for<br />
carbon substrates is directly correlated with the expression of nap, such that<br />
nap is maximally <strong>in</strong>duced dur<strong>in</strong>g growth on butyrate followed by acetate and<br />
then succ<strong>in</strong>ate (Ell<strong>in</strong>gton et al., 2002). As was demonstrated by the low<br />
growth rate, P. pantotrophus cells grown on butyrate were ‘stra<strong>in</strong>ed’ as<br />
compared to succ<strong>in</strong>ate. This suggests a growth-restrict<strong>in</strong>g effect of reduced<br />
carbon source. Consistent with cellular energetics, butyrate as a carbon<br />
substrate is more reduced than most of the sugars and thus generates an<br />
excess of toxic reduc<strong>in</strong>g equivalents, which must be dissipated. The excess<br />
reductants can be disposed only if there is a mechanism for uncoupl<strong>in</strong>g the<br />
respiratory electron flow from ATP synthesis. In P. pantotrophus, the ubiqu<strong>in</strong>ol-Nap<br />
nitrate reductase pathway is one such mechanism (Richardson,<br />
2000; Sears et al., 2000). See Section 6.2.4.1 for a description of the role of<br />
Mtb nitrate reductase <strong>in</strong> reductive stress.<br />
5.4. Phenaz<strong>in</strong>e Production<br />
Phenaz<strong>in</strong>es are redox-active heterocyclic compounds produced naturally<br />
and modified at different positions on their r<strong>in</strong>gs by various phenaz<strong>in</strong>e-
68 AISHA FARHANA ET AL.<br />
generat<strong>in</strong>g bacterial species. To date, there are approximately 100 natural<br />
phenaz<strong>in</strong>e products that are almost exclusively produced <strong>in</strong> high levels (mg<br />
to g/L) by eubacteria (Mavrodi et al., 2006). Many members of the genus<br />
Streptomyces, which are high G + C content bacteria, also produce simple<br />
and complex phenaz<strong>in</strong>es (Mavrodi et al., 2006; Mentel et al., 2009;<br />
W<strong>in</strong>stanley and Fothergill, 2009). Pyocyan<strong>in</strong> (PYO; 5-N-methyl-1-hydroxyphenaz<strong>in</strong>e)<br />
was the first described phenaz<strong>in</strong>e and is produced by<br />
Pseudomonas aerug<strong>in</strong>osa. PYO naturally occurs as a zwitterion that has<br />
hydrophobic and hydrophilic regions, which can easily penetrate cytoplasmic<br />
membranes, and can undergo cellular redox cycl<strong>in</strong>g <strong>in</strong> the presence of<br />
*<br />
NADPH, NADH and O2 to generate O2 and H2O2 (Dietrich et al., 2008;<br />
Mentel et al., 2009). PYO and phenaz<strong>in</strong>e-1-carboxylic acid (another major<br />
phenaz<strong>in</strong>e produced by P. aerug<strong>in</strong>osa) have redox potentials of 34 mV<br />
(Friedheim and Michaelis, 1931) and 116 mV (Price-Whelan et al., 2007)<br />
respectively, and therefore can be reduced by NADH (E 0’ = 320 mV). Not<br />
surpris<strong>in</strong>gly, establish<strong>in</strong>g that NADH can react with PYO <strong>in</strong> vitro led to the<br />
conclusion that bacteria may use PYO as a mechanism to ma<strong>in</strong>ta<strong>in</strong> <strong>in</strong>tracellular<br />
redox homoeostasis (Friedheim, 1931; Price-Whelan et al., 2006).<br />
Consistent with this, recent studies have shown that PYO can directly activate<br />
SoxR, a Fe–S cluster and regulatory prote<strong>in</strong> that is typically upregulated<br />
<strong>in</strong> response to oxidative stress (Dietrich et al., 2006). It was also suggested<br />
that excreted phenaz<strong>in</strong>es reduce Fe 3+ to the more soluble Fe 2+ form that can<br />
be taken up by siderophores (Hernandez et al., 2004). Thus, it is clear that<br />
phenaz<strong>in</strong>es have the potential to generate substantial oxidative stress on<br />
cells.<br />
Because phenaz<strong>in</strong>es have low mid-po<strong>in</strong>t redox potentials, it has been<br />
suggested that they can be directly reduced by NADH or GSH<br />
(Hernandez and Newman, 2001). Several groups observed that both synthetic<br />
and natural phenaz<strong>in</strong>es are reduced by prokaryotes, but <strong>in</strong> most of<br />
these studies the physiological effect of this reduction was not exam<strong>in</strong>ed.<br />
However, Methanobacterium mazei Go1, an archeon produc<strong>in</strong>g methanophenaz<strong>in</strong>e<br />
(a phenaz<strong>in</strong>e derivative), has been shown to utilise phenaz<strong>in</strong>es<br />
<strong>in</strong>stead of qu<strong>in</strong>ones <strong>in</strong> the ETC <strong>in</strong> order to generate ATP (Deppenmeier,<br />
2002). Thus, <strong>in</strong> M. mazei, phenaz<strong>in</strong>e production is not only crucial to energy<br />
metabolism, but also for re-oxidis<strong>in</strong>g NADH (Deppenmeier, 2004). This<br />
raises the possibility that phenaz<strong>in</strong>es may be similarly <strong>in</strong>volved <strong>in</strong> dissipat<strong>in</strong>g<br />
reductive stress <strong>in</strong> pseudomonads.<br />
In an <strong>in</strong>terest<strong>in</strong>g report exam<strong>in</strong><strong>in</strong>g the NAD + and NADH concentrations<br />
<strong>in</strong> Clostridium welchii, Klebsiella aerogenes, E. coli, Staphylococcus albus<br />
and P. aerug<strong>in</strong>osa (Wimpenny and Firth, 1972), it was demonstrated that<br />
all the species, with the exception of P. aerug<strong>in</strong>osa, have NADH/NAD +
REDUCTIVE STRESS IN MICROBES 69<br />
ratios of
70 AISHA FARHANA ET AL.<br />
Phenaz<strong>in</strong>es have the potential to directly associate with the respiratory cha<strong>in</strong><br />
to either couple their reduction to ATP generation or dissipate reductive<br />
stress (Hernandez and Newman, 2001; Mavrodi et al., 2006; Price-Whelan<br />
et al., 2006, 2007).<br />
5.5. Hydrogenases<br />
Hydrogenases (H 2ases) are redox metalloenzymes found throughout the<br />
eukaryotic and prokaryotic genera, the majority of which conta<strong>in</strong> an iron–<br />
sulphur (Fe–S) cluster or alternatively two metal atoms (two Fe atoms or an<br />
Ni–Fe comb<strong>in</strong>ation) (Vignais et al., 2001; Jenney and Adams, 2008). H 2ases<br />
catalyse the reversible conversion/oxidation of hydrogen gas (Nandi and<br />
Sengupta, 1998; Meyer, 2007; Jenney and Adams, 2008):<br />
H2 $ 2H + +2e<br />
Membrane-bound hydrogenases split the protons from H2, thus creat<strong>in</strong>g a<br />
proton gradient across the cytoplasmic membrane. The electrons (e ) produced<br />
<strong>in</strong> the H2 oxidation process are transferred to electron carriers <strong>in</strong> the<br />
bacterial membrane and ultimately to term<strong>in</strong>al electron acceptors such as O2 (aerobic respiration) or fumarate, NO3 ,SO4 2 and CO2 (anaerobic respiration)<br />
(Vignais et al., 2001; Vignais and Colbeau, 2004). In addition, some<br />
bacteria can use the protons as oxidants to dispose of excess reduc<strong>in</strong>g power,<br />
thereby reoxidis<strong>in</strong>g their coenzymes <strong>in</strong> the process (Cammack et al., 2001;<br />
Vignais et al., 2001). S<strong>in</strong>ce the oxidation of H2 is reversible, the H2ase will<br />
produce H2 <strong>in</strong> the presence of an electron donor and will act as a H2 uptake<br />
enzyme <strong>in</strong> the presence of an electron acceptor. H2ase activity is modulated<br />
by numerous regulatory pathways and responds to changes <strong>in</strong> the valance<br />
state of the metal cofactors, O2 levels and pH (Vignais and Colbeau, 2004).<br />
H2 is a high-energy reductant that is present <strong>in</strong> the mucus layer of the mouse<br />
stomach at 43 mM, <strong>in</strong> the liver at 50 mM(Olson and Maier, 2002; Maier et al.,<br />
2003) and spreads throughout host tissues by diffusion, or alternatively is<br />
carried through the bloodstream to organs such as the lungs (Levitt, 1969).<br />
H2 has a likely role to play <strong>in</strong> microbial pathogenesis as it was recently<br />
*<br />
demonstrated that H2 is highly effective <strong>in</strong> reduc<strong>in</strong>g OH and alleviat<strong>in</strong>g<br />
*<br />
OH -<strong>in</strong>duced cytotoxicity without affect<strong>in</strong>g other ROS (Ohsawa et al., 2007).<br />
H2ases can be classified <strong>in</strong>to three categories based on the composition of<br />
their metal centres: (i) [NiFe]-hydrogenases, (ii) [FeFe]-hydrogenases and<br />
(iii) the Fe–S cluster-free hydrogenases (Nandi and Sengupta, 1998; Vignais<br />
and Colbeau, 2004; Burgdorf et al., 2005; Jenney and Adams, 2008). S<strong>in</strong>ce the<br />
enzymatic activity of uptake H2ases can <strong>in</strong>crease under anaerobic conditions
REDUCTIVE STRESS IN MICROBES 71<br />
(reductive activation) (Maier et al., 2003), it has implications for a wide range<br />
of pathogens. For example, <strong>in</strong> a sem<strong>in</strong>al study, a role for H2 <strong>in</strong> Helicobacter<br />
pathogenesis was reported (Olson and Maier, 2002). It was shown that H2<br />
produced by the gastric flora <strong>in</strong> mice functions as a respiratory substrate for<br />
Helicobacter pylori and substantially <strong>in</strong>creases its ability to colonise the<br />
stomach (Olson and Maier, 2002). On the other hand, Salmonella H 2ases<br />
(e.g. Hyd) may oxidise H2 with O2 as the term<strong>in</strong>al electron acceptor with the<br />
<strong>in</strong>tention of conserv<strong>in</strong>g energy dur<strong>in</strong>g different stages of host <strong>in</strong>fection (Zbell<br />
et al., 2008). Genetic studies <strong>in</strong> Salmonella have shown that H2 can be<br />
generated and oxidised by the same organism (Zbell and Maier, 2009).<br />
Us<strong>in</strong>g H 2 as an energy source <strong>in</strong> vivo by bacterial pathogens is not unique<br />
and has been reported previously (Olson and Maier, 2002; Zbell et al., 2007;<br />
da Silva et al., 2008).<br />
S<strong>in</strong>ce the TCA cycle is repressed under anaerobic conditions, fermentative<br />
grow<strong>in</strong>g bacteria must oxidise reduc<strong>in</strong>g equivalents such as NADH to<br />
ensure cont<strong>in</strong>uous conversion of the substrate. Klebsiella pneumoniae has<br />
evolved a clever mechanism by which the bacteria ga<strong>in</strong> limited quantities of<br />
the reduc<strong>in</strong>g equivalent NADPH from the oxidation of citrate (Pfenn<strong>in</strong>ger-<br />
Li and Dimroth, 1992), but because of the downregulation of the TCA cycle,<br />
sufficient quantities of NADH is lack<strong>in</strong>g. K. pneumoniae solves this problem<br />
by generat<strong>in</strong>g NADPH via the oxidation of H 2 dur<strong>in</strong>g citrate fermentation by<br />
aH 2:NAD(P) + oxidoreductase (hydrogenase). Thus, reduc<strong>in</strong>g equivalents<br />
are provided for the production of biomass (Steuber et al., 1999).<br />
Pyrococcus furiosus employs a different strategy. This organism can use a<br />
range of carbohydrates, convert<strong>in</strong>g them to acetate, CO2,H2 and, if elemental<br />
sulphur (S 0 ) is present, H 2S(Ma et al., 1993). An important characteristic<br />
is that ferredox<strong>in</strong> serves as electron acceptor dur<strong>in</strong>g glycolysis and that no<br />
NAD(P) + is generated. Subsequently, it was found that a H2-evolv<strong>in</strong>g membrane-bound<br />
H 2ase couples the oxidation of ferredox<strong>in</strong> and the reduction of<br />
2H + to H 2 production. Next, a membrane-bound oxidase oxidises ferredox<strong>in</strong><br />
and reduces NADP, which is used by a NAD(P)H S 0 oxidoreductase to<br />
reduce S 0 to H 2S. Thus, P. furiosus disposes of excess reductant us<strong>in</strong>g protons<br />
or S 0 as electron acceptors to yield H 2S(Ma et al., 1993; Schut et al., 2007;<br />
Jenney and Adams, 2008). The production of H2 via hydrogenases is a<br />
specific mechanism to dispose of surplus reduc<strong>in</strong>g equivalents (Nandi and<br />
Sengupta, 1998).<br />
A recent study suggested a feasible and physiologically relevant role for<br />
mycobacterial hydrogenases <strong>in</strong> scaveng<strong>in</strong>g H 2 as an energy source or as a<br />
redox s<strong>in</strong>k (Berney and Cook, 2010). Microarray analysis of hypoxic, carbonlimited<br />
cont<strong>in</strong>uous cultures of Mycobacterium smegmatis identified a number<br />
of hydrogenases that are differentially regulated under energy- or
72 AISHA FARHANA ET AL.<br />
oxygen-limit<strong>in</strong>g conditions. S<strong>in</strong>ce it rema<strong>in</strong>s unclear how mycobacteria recycle<br />
reduc<strong>in</strong>g equivalents under hypoxic conditions lack<strong>in</strong>g exogenous electron<br />
acceptors, it was hypothesised that (i) mycobacteria switch to NAD + /<br />
NADH-<strong>in</strong>dependent enzymes and use ferredox<strong>in</strong>s as electron carriers,<br />
which is typical for anaerobic bacteria, and that (ii) hydrogenases either<br />
oxidise H 2 or produce H 2, or perform both. The <strong>in</strong>vestigators cited the<br />
significant upregulation of many ferredox<strong>in</strong>-reduc<strong>in</strong>g and oxidis<strong>in</strong>g enzymes<br />
identified <strong>in</strong> their study, as well as the results of another study identify<strong>in</strong>g an<br />
anaerobic type a-ketoglutarate:ferredox<strong>in</strong> oxidoreductase (KOR), which<br />
upon disruption requires CO2 for growth (Baughn et al., 2009), to provide<br />
credence to their hypotheses (Berney and Cook, 2010). In order to <strong>in</strong>vestigate<br />
the role of H 2ases under conditions of energy limitation, a highly upregulated<br />
putative hydrogen:qu<strong>in</strong>one oxidoreductase of Msm was disrupted.<br />
The Msm mutant showed a 20% reduction <strong>in</strong> biomass, thus demonstrat<strong>in</strong>g<br />
the essential role of this particular H2ase <strong>in</strong> adapt<strong>in</strong>g to energy limitation.<br />
F<strong>in</strong>ally, based on the Msm studies, it was argued that acidic, hypoxic and<br />
lipid-rich environments (which contribute to a highly reductive <strong>in</strong>tracellular<br />
milieu) may require H2ases to function as reductive s<strong>in</strong>ks, or alternatively,<br />
that H2 could serve as an energy source for Mtb (Berney and Cook, 2010).<br />
The role of H 2ases <strong>in</strong> Mtb pathogenesis is a highly relevant, albeit unexplored<br />
area of <strong>in</strong>vestigation.<br />
5.6. The Reverse TCA (rTCA) Cycle<br />
The TCA cycle is widely distributed <strong>in</strong> aerobic microorganisms and is an<br />
energy acquisition pathway that is exergonic (spontaneous). Although this<br />
cycle only operates under aerobic conditions, it is also present <strong>in</strong> anaerobes<br />
(Sr<strong>in</strong>ivasan and Morowitz, 2006). The reverse TCA (rTCA) cycle is generally<br />
regarded as the phylogenetic orig<strong>in</strong> of the TCA cycle and uses CO2 as the<br />
key source for carbon fixation. The rTCA cycle is, <strong>in</strong> fact, a reversal of the<br />
oxidative TCA cycle and is an endergonic (unfavourable, non-spontaneous)<br />
anabolic pathway that requires reduc<strong>in</strong>g equivalents such as NADH,<br />
NADPH and FADH2 to complete the cycle (Hugler et al., 2005; Sr<strong>in</strong>ivasan<br />
and Morowitz, 2006; Aoshima, 2007; Ikeda et al., 2010). The end result is the<br />
fixation of two molecules of CO 2 and the production of one molecule of<br />
acetyl-CoA, which is reductively carboxylated to pyruvate from which many<br />
central metabolites can be formed. Three enzymes allow the TCA cycle to<br />
operate <strong>in</strong> the reverse direction and their activity is <strong>in</strong>dicative of a function<strong>in</strong>g<br />
rTCA cycle (Hugler et al., 2005; Sr<strong>in</strong>ivasan and Morowitz, 2006). These<br />
enzymes are ATP citrate lyase, 2-oxoglutarate:ferredox<strong>in</strong> oxidoreductase
REDUCTIVE STRESS IN MICROBES 73<br />
(OGFO) and FRD, which catalyse the ATP-dependent cleavage of citrate<br />
to acetyl-CoA and oxaloacetate, the carboxylation of succ<strong>in</strong>yl-CoA to 2oxoglutarate<br />
and the reduction of fumarate to form succ<strong>in</strong>ate, respectively.<br />
Hav<strong>in</strong>g these three enzymes to drive the rTCA cycle provides pathogens<br />
with the flexibility to effectively respond to diverse environments <strong>in</strong> order to<br />
survive <strong>in</strong> their physiological niche (Hugler et al., 2005; Sr<strong>in</strong>ivasan and<br />
Morowitz, 2006; Aoshima, 2007).<br />
S. cerevisiae conta<strong>in</strong>s two FRDs that use FADH 2,FMNH 2 or reduced<br />
riboflav<strong>in</strong> as electron donors to irreversibly reduce fumarate to succ<strong>in</strong>ate. It<br />
has been suggested that these FRDs are <strong>in</strong>volved <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g redox<br />
homeostasis dur<strong>in</strong>g anaerobiosis (Enomoto et al., 2002; Camarasa et al.,<br />
2007). The oxidative and reductive branches of the TCA cycle can also<br />
operate under anaerobic fermentation. For example, dur<strong>in</strong>g yeast fermentation,<br />
if aspartate is the nitrogen source, succ<strong>in</strong>ate can be generated via the<br />
reductive branch. On the other hand, if glutamate is the nitrogen source,<br />
succ<strong>in</strong>ate can be made via the oxidative branch (Sr<strong>in</strong>ivasan and Morowitz,<br />
2006). Thus, it is clear that depend<strong>in</strong>g on the environmental conditions such<br />
as O2 concentration and energy source availability, the rTCA cycle can<br />
operate <strong>in</strong> either a reductive or oxidative direction.<br />
S<strong>in</strong>ce Mtb resides <strong>in</strong> a hypoxic, presumably nutrient-starved environment,<br />
it was speculated that these environmental conditions may severely restrict<br />
the ability of the organism to re-oxidise reduc<strong>in</strong>g equivalents to ma<strong>in</strong>ta<strong>in</strong><br />
redox homeostasis (Boshoff and Barry, 2005; Sr<strong>in</strong>ivasan and Morowitz,<br />
2006; Leistikow et al., 2010), which would eventually lead to a redox imbalance<br />
and death. S<strong>in</strong>ce tubercle lesions generate substantial quantities of CO2<br />
(Haapanen et al., 1959), the rTCA cycle, with its capacity to fix CO 2, may be<br />
an effective mechanism to dissipate reduc<strong>in</strong>g equivalents generated by hypoxic<br />
or microaerophilic conditions and host gases (NO or CO) that <strong>in</strong>hibit<br />
Mtb respiration or by the catabolism of highly reduced <strong>in</strong> vivo carbon sources<br />
such as FA.<br />
Intrigu<strong>in</strong>gly, Mtb appears to lack a CoA-dependent KDH because the<br />
correspond<strong>in</strong>g enzymatic activity was absent <strong>in</strong> crude cellular extracts<br />
(Tian et al., 2005). This f<strong>in</strong>d<strong>in</strong>g suggests that Mtb might operate separate<br />
oxidative and reductive TCA half-cycles. Therefore, the study proposed that<br />
the oxidative branch (that produces a-ketoglutarate [KG] and glutamate)<br />
and reductive branch (that produces succ<strong>in</strong>ate) may be l<strong>in</strong>ked by a-ketoglutarate<br />
decarboxylase (KGD) and succ<strong>in</strong>ic semialdehyde dehydrogenase<br />
(SSADH) to generate succ<strong>in</strong>ate from KG via succ<strong>in</strong>ic semialdehyde (SSA)<br />
(Tian et al., 2005). In conflict with the above <strong>in</strong>terpretation are the results of<br />
an <strong>in</strong>dependent study where<strong>in</strong> an anaerobic type CoA-dependent KG dehydrogenase<br />
activity was assayed <strong>in</strong> Mtb (Baughn et al., 2009). An <strong>in</strong>terest<strong>in</strong>g
74 AISHA FARHANA ET AL.<br />
f<strong>in</strong>d<strong>in</strong>g was that this KOR is highly stable under aerobic conditions. KOR is<br />
expendable for growth when the glyoxylate shunt is non-functional whereas<br />
KGD is critical for this bypass. Collectively, these f<strong>in</strong>d<strong>in</strong>gs po<strong>in</strong>t to a<br />
pathway that operates concomitantly with b-oxidation (KOR-dependent)<br />
and another pathway that functions <strong>in</strong> the absence of b-oxidation (KGDdependent).<br />
Intrigu<strong>in</strong>gly, an Mtb korAB mutant stra<strong>in</strong> required substantial<br />
amounts of CO 2 for growth, suggest<strong>in</strong>g that the KOR-dependent decarboxylation<br />
of KG is a valuable source of CO2 <strong>in</strong> Mtb metabolism (Baughn et al.,<br />
2009). This f<strong>in</strong>d<strong>in</strong>g may have important physiological implications <strong>in</strong> vivo<br />
as alveolar air, blood and <strong>in</strong>terstitial fluids conta<strong>in</strong> 5–6% CO 2 (Florczyk<br />
et al., 2003). Furthermore, CO 2 preserves microaerophilic growth of<br />
Mycobacterium bovis BCG and prevents growth <strong>in</strong>hibition when O2 is<br />
rapidly removed (Florczyk et al., 2003), suggest<strong>in</strong>g a role of CO 2 <strong>in</strong> mycobacterial<br />
persistence. Despite differences <strong>in</strong> mechanisms proposed (Tian<br />
et al., 2005; Baughn et al., 2009), the production of succ<strong>in</strong>ate under lowoxygen<br />
conditions via the rTCA cycle is a feasible survival mechanism<br />
under this physiological condition. In agreement with this, FRD was<br />
recently speculated to be <strong>in</strong>volved <strong>in</strong> survival and ATP production dur<strong>in</strong>g<br />
hypoxia (Rao et al., 2008).<br />
5.7. Carbon Monoxide (CO) Dehydrogenase (CODH)<br />
Carboxydotrophic microbes are aerobic organisms that use CO as the sole<br />
source of carbon and energy dur<strong>in</strong>g chemolithoautotrophic growth. These<br />
CO-utilis<strong>in</strong>g microorganisms use the enzyme CODH to reversibly oxidise<br />
CO accord<strong>in</strong>g to the follow<strong>in</strong>g reaction (Ferry, 1995; Ragsdale, 2004;<br />
Seravalli and Ragsdale, 2008):<br />
CO + H 2O $ CO 2 +2H + +2e<br />
CODH enzymes are divided <strong>in</strong>to two classes accord<strong>in</strong>g to metal or cofactor<br />
content or metabolic role and catalytic activity. Aerobic carboxydotrophic<br />
bacteria uses a Cu-, Fe- and Mo-conta<strong>in</strong><strong>in</strong>g flavoenzyme, whereas anaerobic<br />
and archaebacteria use an oxygen-sensitive Ni- and Fe-conta<strong>in</strong><strong>in</strong>g CODH<br />
(Ragsdale, 2004; Jeoung and Dobbek, 2007; Oelgeschlager and Rother,<br />
2008). Genes encod<strong>in</strong>g aerobic CODH are usually denoted as carbon monoxide<br />
oxidases (cox). CODH oxidises CO to generate CO 2, which is then<br />
fixed <strong>in</strong>to biomass via the rTCA cycle, the Calv<strong>in</strong>–Benson–Bassham cycle,<br />
the 3-hydroxypropionate cycle or the Wood–Ljungdahl pathway (Ragsdale,<br />
2004; Seravalli and Ragsdale, 2008). When CODH is coupled with acetyl-<br />
CoA synthase (ACS) (CODH/ACS), acetyl-CoA is generated from CO2,
REDUCTIVE STRESS IN MICROBES 75<br />
CoA and a methyl group. In this scenario, CODH/ACS reduces CO 2 to CO,<br />
which supplies the carbonyl group of acetyl-CoA <strong>in</strong> the Wood–Ljungdahl<br />
pathway. Acetyl-CoA is then used <strong>in</strong> ATP production or for biomass synthesis<br />
(Oelgeschlager and Rother, 2008).<br />
Diverse respiratory events <strong>in</strong>clud<strong>in</strong>g oxygen respiration, hydrogenesis,<br />
desulphurication, acetogenesis and methanogenesis can all be coupled to<br />
CO oxidation (Oelgeschlager and Rother, 2008). These f<strong>in</strong>d<strong>in</strong>gs serve to<br />
demonstrate the range of microbes capable of us<strong>in</strong>g CO as an energy source.<br />
In a recent 13 C-metabolic flux analysis study, (McK<strong>in</strong>lay and Harwood, 2010)<br />
it was reported that the Calv<strong>in</strong>–Benson–Bassham (Calv<strong>in</strong>) cycle plays an<br />
important role <strong>in</strong> reoxidis<strong>in</strong>g approximately half of the reduced cofactors<br />
generated by conversion of acetate <strong>in</strong>to biomass. Thus, the Calv<strong>in</strong> cycle is an<br />
electron accept<strong>in</strong>g process and helps ma<strong>in</strong>ta<strong>in</strong> redox balance by fix<strong>in</strong>g CO 2<br />
and oxidis<strong>in</strong>g the reduced cofactor NADH to NAD + .<br />
Phylogenetic and growth analyses (K<strong>in</strong>g, 2003; Song et al., 2010), and<br />
exam<strong>in</strong>ation of crude extracts suggest the presence of CODH (Park et al.,<br />
2003) with NO dehydrogenase (NODH) activity (Park et al., 2007) <strong>in</strong><br />
Mtb. The presence of CODH subunit homologues (Rv0373c, Rv0374c,<br />
Rv0375c) <strong>in</strong> the Mtb genome are consistent with the above observation.<br />
Furthermore, CODH can jo<strong>in</strong> the Calv<strong>in</strong> cycle and the rTCA cycle (see<br />
above) as a means to fix CO 2 <strong>in</strong>to biomass (Meyer and Schlegel, 1983;<br />
Ferry, 1995). However, s<strong>in</strong>ce the Calv<strong>in</strong> cycle appears to be absent <strong>in</strong> Mtb<br />
(Park et al., 2003), the rTCA is a plausible mechanism for conversion of<br />
exogenous CO2 to biomass. A sub-lethal source of CO2 may be the cavities<br />
of lesions found <strong>in</strong> TB patients (Haapanen et al., 1959), which are essential<br />
for Mtb growth (Baughn et al., 2009). Thus, the rTCA cycle <strong>in</strong> Mtb may<br />
operate when encounter<strong>in</strong>g reductive stress generated by host FA catabolism,<br />
hypoxia, NO and CO (and other signals), <strong>in</strong> order to fix CO2 and to<br />
consume reduc<strong>in</strong>g equivalents. The presence of CODH and NODH activities<br />
<strong>in</strong> Mtb suggest that the tubercle bacillus may detoxify host-generated<br />
NO and/or CO <strong>in</strong> order to survive and persist <strong>in</strong> the host. The roles<br />
of CODH and NODH <strong>in</strong> Mtb pathogenesis represent an important area<br />
of research.<br />
5.8. Other Mechanisms<br />
Under aerobic culture conditions, Staphylococcus aureus primarily excretes<br />
acetate whereas under ferment<strong>in</strong>g growth conditions the organism produces<br />
L-lactate, ethanol and formate. In an elegant study, it was shown that upon<br />
NO exposure the cells exclusively produce L-lactate from both aerobically
76 AISHA FARHANA ET AL.<br />
respir<strong>in</strong>g and ferment<strong>in</strong>g cells (Richardson et al., 2008). Subsequently, it was<br />
demonstrated that S. aureus exposure to NO caused an <strong>in</strong>crease <strong>in</strong> reduc<strong>in</strong>g<br />
equivalents, which was metabolically balanced via L-lactate dehydrogenase<br />
activity to generate L-lactate. Thus, the NO-<strong>in</strong>ducible L-lactate dehydrogenase<br />
dissipates reductive stress by reoxidis<strong>in</strong>g NADH to generate NAD + and<br />
L-lactate (Richardson et al., 2008). In the case of Enterococcus faecalis, the<br />
regeneration of NAD + from excess NADH was accomplished by generat<strong>in</strong>g<br />
extracellular a-ketoisocaproic acid, 1,1-dihydroxy-4-methyl-2-pentanone<br />
(Ward et al., 2000). Other mechanisms the microorganisms have evolved<br />
to dissipate excess reduc<strong>in</strong>g power <strong>in</strong>clude the use of the Calv<strong>in</strong> cycle as a<br />
reductive s<strong>in</strong>k (Xanthobacter flavus)(Van Keulen et al., 2000), the synthesis<br />
of branched cha<strong>in</strong> am<strong>in</strong>o acids (Aspergillus nidulans) (Shimizu et al., 2010)<br />
and the overproduction of L-alan<strong>in</strong>e dehydrogenase by non-pathogenic<br />
mycobacteria (Hutter and Dick, 1998; Feng et al., 2002) and Arthrobacter<br />
oxydans (Hashimoto and Katsumata, 1999).<br />
6. REDOX SINKS IN MYCOBACTERIA<br />
6.1. The Mycobacterial Intracellular Redox Environment<br />
In aerobic microbes, the production of ROS is not perfectly balanced by<br />
antioxidants. The latter dampens the effects of the former rather than elim<strong>in</strong>at<strong>in</strong>g<br />
them (Halliwell, 2008). If the balance is severely disturbed, a state of<br />
either oxidative or reductive stress ensues. The presence of substantial quantities<br />
of redox buffers such as glutathione or mycothiol <strong>in</strong> the microbial<br />
cytoplasm generates a reduc<strong>in</strong>g environment. Nonetheless, dur<strong>in</strong>g aerobic<br />
respiration, redox couples such as NAD + /NADH exist predom<strong>in</strong>antly <strong>in</strong> the<br />
oxidised, NAD + state because of rapid electron flow to the ETC (Green and<br />
Paget, 2004).<br />
To study the redox environment of a microbial cell, it is impractical and<br />
impossible to measure the concentration of all l<strong>in</strong>ked redox couples. Rather,<br />
quantification of a representative redox couple can be used to <strong>in</strong>fer the<br />
overall redox environment of the cell. For example, <strong>in</strong> eukaryotes and many<br />
bacteria, the glutathione disulfide–glutathione couple (GSSG/2GSH) is the<br />
major thiol-disulfide redox buffer and the redox state of this couple can be<br />
used to <strong>in</strong>fer the status of the redox environment. As elegantly discussed<br />
(Schafer and Buettner, 2001), the reduction potential of GSSG/2GSH is<br />
dependent on the absolute concentration of GSH, as well as the GSSG/<br />
2GSH ratio. This circumstance differs from that of NAD + /NADH and
REDUCTIVE STRESS IN MICROBES 77<br />
NADP + /NADPH couples <strong>in</strong> which assessment of the absolute concentration<br />
of the <strong>in</strong>dividual components is not necessary and measurement of the ratio<br />
of the oxidised and reduced species of the redox couples is sufficient.<br />
Therefore, the redox state of a redox couple is def<strong>in</strong>ed by the half-cell<br />
reduction potential and the reduc<strong>in</strong>g capacity of that couple (Schafer and<br />
Buettner, 2001).<br />
As is the case for GSH, thioredox<strong>in</strong> acts as an antioxidant by facilitat<strong>in</strong>g<br />
the reduction of cyst<strong>in</strong>e residues <strong>in</strong> prote<strong>in</strong>s by cyste<strong>in</strong>e thiol-disulphide<br />
exchange. GSH is typically present <strong>in</strong> millimolar concentration <strong>in</strong> bacterial<br />
and mammalian cells, whereas the concentration of thioredox<strong>in</strong> [TrxSS/Trx<br />
(SH) 2] is <strong>in</strong> the micromolar range <strong>in</strong> bacteria (Halliwell, 2008). Notably, s<strong>in</strong>ce<br />
the Trx and GSH systems use NADPH as a reduc<strong>in</strong>g factor, the NADP + /<br />
NADPH, GSSG/2GSH and TrxSS/Trx(SH) 2 redox systems are not isolated<br />
systems and are thermodynamically l<strong>in</strong>ked to one another (Schafer and<br />
Buettner, 2001). Intrigu<strong>in</strong>gly, mycobacterial species do not produce glutathione;<br />
<strong>in</strong>stead, they produce millimolar quantities of mycothiol (MSH) as a<br />
redox buffer.<br />
6.1.1. Mycothiol: The Mycobacterial Redox Buffer<br />
In addition to act<strong>in</strong>g as the major redox buffer <strong>in</strong> mycobacteria, the lowmolecular-weight<br />
(LMW) thiol, mycothiol, is <strong>in</strong>volved <strong>in</strong> the removal of toxic<br />
compounds from the cell. Besides, another antioxidant, ergothione<strong>in</strong>e,<br />
(ERG; ERGox/ERGred), is also synthesised by mycobacteria (Genghof<br />
and Van Damme, 1964, 1968), although little is known so far about its role<br />
<strong>in</strong> these bacteria.<br />
MSH is produced <strong>in</strong> a five-step process and all but one of the enzymes<br />
responsible for its synthesis have been identified. The first step <strong>in</strong>volves<br />
l<strong>in</strong>k<strong>in</strong>g 1L-myo-<strong>in</strong>ositol-1-phosphate (derived from glucose-6-phosphate)<br />
to UDP-N-acetylglucosam<strong>in</strong>e to produce N-acetylglucosam<strong>in</strong>yl<strong>in</strong>ositol<br />
phosphate, catalysed by the glycosyltransferase encoded by mshA (Newton<br />
et al., 2003). MshA2, the MSH phosphatase whose gene is not yet identified,<br />
then dephosphorylates N-acetylglucosam<strong>in</strong>yl<strong>in</strong>ositol phosphate (Newton<br />
et al., 2006) followed by deacetylation by MshB, the MSH deacetylase, to<br />
produce glucosam<strong>in</strong>yl<strong>in</strong>ositol [1-O-(2-am<strong>in</strong>o-1-deoxy-a-D-glucopyranosyl)-<br />
D-myo-<strong>in</strong>ositol] (Newton et al., 2000a). Cyste<strong>in</strong>e is ligated to this compound<br />
via its carboxyl group <strong>in</strong> an ATP-dependent reaction catalysed by the MSH<br />
ligase, MshC (Sareen et al., 2002). The f<strong>in</strong>al step is the acetylation of the<br />
am<strong>in</strong>o group of cyste<strong>in</strong>e by MshD, the MSH acetyltransferase (Koled<strong>in</strong> et al.,<br />
2002), a reaction which serves to make mycothiol more resistant to autoxidation<br />
than free cyste<strong>in</strong>e (Newton et al., 1995, 2008).
78 AISHA FARHANA ET AL.<br />
Mutants <strong>in</strong> the mycothiol biosynthetic pathway have been isolated <strong>in</strong><br />
M. smegmatis (Msm) and Mtb. In Msm and the Mtb stra<strong>in</strong>s H37Rv,<br />
Erdman and CDC1551, the loss of MshA activity results <strong>in</strong> undetectable<br />
levels of MSH and its <strong>in</strong>termediates (Newton et al., 1999, 2003; Vilcheze<br />
et al., 2008). mshB mutants of Msm and Mtb display accumulation of<br />
pathway <strong>in</strong>termediate, N-acetylglucosam<strong>in</strong>yl<strong>in</strong>ositol, but are still able to<br />
produce low levels of MSH, which may be attributed to an uncharacterised<br />
enzyme with a complementary deacetylase function (Buchmeier et al.,<br />
2003; Rawat et al., 2003). An mshC mutant <strong>in</strong> Msm does not produce<br />
detectable levels of MSH but has <strong>in</strong>creased glucosam<strong>in</strong>yl<strong>in</strong>ositol levels<br />
(Rawat et al., 2002). Efforts to produce an mshC mutant of Mtb Erdman<br />
were unsuccessful and the researchers attributed this <strong>in</strong>ability to the essentiality<br />
of mycothiol for Mtb survival (Sareen et al., 2003). mshD mutants of<br />
Msm and Mtb have been shown to produce decreased MSH, <strong>in</strong>creased<br />
levels of its immediate precursor and two novel thiols (Newton et al.,2005;<br />
Buchmeier et al., 2006).<br />
Based on the phenotypes of various MSH mutants <strong>in</strong> response to redox<br />
stress, it is clear that MSH contributes greatly to the ma<strong>in</strong>tenance of redox<br />
homeostasis <strong>in</strong> mycobacteria. Increased sensitivity to oxidative stress is a<br />
common theme, and <strong>in</strong> agreement to this, the Msm mycothiol mutants have<br />
decreased survival compared to wild type after treatment with hydrogen<br />
peroxide and plumbag<strong>in</strong>, a superoxide generator (Newton et al., 1999;<br />
Rawat et al., 2002, 2007). In Mtb, the mshB and mshD mutants have<br />
<strong>in</strong>creased sensitivity to cumene hydroperoxide and hydrogen peroxide<br />
respectively (Buchmeier et al., 2003, 2006). Additionally, it has been shown<br />
that the MSH mutants of Msm are more sensitive than wild type to reduc<strong>in</strong>g<br />
stress based on disk assays <strong>in</strong> the presence of 10 mM DTT, a reductant<br />
(Rawat et al., 2007). Antibiotic resistance is also altered <strong>in</strong> the mutants;<br />
<strong>in</strong>creased resistance to ethionamide (ETH) has been reported for all Msm<br />
mutants except mshC (Rawat et al., 2003, 2007). However, the mshA and<br />
mshD Msm mutants are more resistant to isoniazid (INH) (Koled<strong>in</strong> et al.,<br />
2002; Newton et al., 2003). In Mtb, the mshA mutant has <strong>in</strong>creased resistance<br />
to INH and ETH (Vilcheze et al., 2008), and INH resistance has also been<br />
reported for the mshB mutant (Buchmeier et al., 2003).<br />
The regulation of MSH biosynthesis has not yet been fully elucidated.<br />
Transcriptional regulators are located upstream of mshA and mshD <strong>in</strong> Mtb,<br />
but not a s<strong>in</strong>gle study has been carried out to determ<strong>in</strong>e their role <strong>in</strong> MSH<br />
regulation (Newton et al., 2008). MSH levels are <strong>in</strong>fluenced by growth phase<br />
<strong>in</strong> Mtb and are <strong>in</strong>creased greater than threefold <strong>in</strong> stationary phase as compared<br />
to early exponential phase (Buchmeier et al., 2006). However, it is not<br />
known what causes this effect. No direct transcriptional analysis of the genes<br />
<strong>in</strong>volved <strong>in</strong> MSH biosynthesis at these different phases has been performed
REDUCTIVE STRESS IN MICROBES 79<br />
(Newton et al., 2008), but microarray analysis to identify genes whose expression<br />
levels differ <strong>in</strong> exponential and stationary phases did not identify the<br />
MSH-encod<strong>in</strong>g genes (Voskuil et al., 2004). MshB activity is <strong>in</strong>hibited <strong>in</strong> the<br />
presence of MSH; thus, MSH biosynthesis may be regulated <strong>in</strong> part by<br />
feedback <strong>in</strong>hibition (Newton and Fahey, 2002).<br />
Many MSH-dependent enzymes are present <strong>in</strong> mycobacteria such as MSH<br />
disulfide reductase, or Mtr. In the course of ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g the reduc<strong>in</strong>g environment<br />
of the cell, MSH becomes oxidised (MSSM) and must be re-reduced<br />
to ensure proper function<strong>in</strong>g of cellular processes. This crucial task is performed<br />
by Mtr, which uses FAD as a cofactor <strong>in</strong> a reaction that consumes<br />
NADPH (Rietveld et al., 1994; Patel and Blanchard, 1998, 1999). MSH has a<br />
role <strong>in</strong> the removal of toxic compounds such as antibiotics, via the action of<br />
mycothiol S-conjugate amidase (Mca). MSH fuses with the toxic compound<br />
and then Mca breaks down the complex <strong>in</strong>to glucosam<strong>in</strong>yl<strong>in</strong>ositol that is used<br />
to synthesise more MSH and a mercapturic acid, which is subsequently<br />
excreted from the cell (Newton et al., 2000b; Newton and Fahey, 2002).<br />
Similarly, the detoxification of formaldehyde is accomplished due to the<br />
action of formaldehyde dehydrogenase (MscR), an enzyme that also acts<br />
as a nitrosothiol reductase. MSH can react with formaldehyde, and <strong>in</strong> the<br />
presence of NAD + , the formaldehyde is oxidised by MscR to the less toxic<br />
formate (Vogt et al., 2003). The nitrosothiol reductase activity of MscR is<br />
greater than its formaldehyde dehydrogenase activity (Vogt et al., 2003).<br />
This activity protects the cell from NO, and <strong>in</strong> fact, it was shown that<br />
mycothiol plays a major part <strong>in</strong> protection of mycobacteria from NO<br />
(Miller et al., 2007). MSNO, an adduct of MSH and NO, is formed, which<br />
is subsequently broken down <strong>in</strong>to nitrate and MSH by the action of MscR <strong>in</strong><br />
the presence of NADH (Vogt et al., 2003).<br />
It has been established that MSH is the most abundant LMW thiol <strong>in</strong><br />
mycobacteria, which ma<strong>in</strong>ta<strong>in</strong>s redox homeostasis and is thermodynamically<br />
l<strong>in</strong>ked with a variety of enzymes to elim<strong>in</strong>ate toxic compounds.<br />
Unfortunately, little is known thus far of the mechanisms utilised by mycobacteria<br />
to regulate the production of MSH.<br />
6.2. The Mtb Dos Dormancy Regulon<br />
6.2.1. Biological Role and Function<br />
The Dos two-component system was first identified <strong>in</strong> a virulent stra<strong>in</strong> of<br />
Mtb (K<strong>in</strong>ger and Tyagi, 1993) where<strong>in</strong> DosR (Rv3133c) was demonstrated<br />
to possess homology to response regulator prote<strong>in</strong>s of the NarL/UhpA/<br />
FixJ subfamily and DosS (Rv3132c) to sensor histid<strong>in</strong>e k<strong>in</strong>ases. The
80 AISHA FARHANA ET AL.<br />
two-component pair genes, dosR and dosS, are cotranscribed and conserved<br />
<strong>in</strong> Mtb and M. bovis BCG (Dasgupta et al., 2000) and their expression is<br />
hypoxia-responsive <strong>in</strong> pathogenic and non-pathogenic mycobacteria <strong>in</strong>clud<strong>in</strong>g<br />
Mtb (Sherman et al., 2001), M. bovis BCG (Boon et al., 2001)andM. smegmatis<br />
(Mayuri et al.,2002). A series of studies revealed that significant overlap<br />
exists between the expression profiles of Mtb cells cultured under hypoxic<br />
conditions and those exposed to NO and that the transcriptional response is<br />
under direct control of DosR (Sherman et al., 2001; Ohno et al., 2003; Voskuil<br />
et al., 2003). Subsequently, DosT (Rv2027c), a homologue of DosS, was<br />
discovered and was also shown to phosphorylate DosR (Roberts et al.,<br />
2004; Sa<strong>in</strong>i et al., 2004), <strong>in</strong>dicat<strong>in</strong>g a crosstalk between DosS, DosT and<br />
DosR. The role of DosT rema<strong>in</strong>s enigmatic as it is an orphan sensor k<strong>in</strong>ase,<br />
has no cognate response regulator and is not upregulated under any known<br />
condition. Thus, DosS/T/R can be regarded as a ‘three’-component system<br />
believed to facilitate transition from active to the latent form of <strong>in</strong>fection.<br />
Detailed studies have shown that DosT is active early <strong>in</strong> the hypoxia response.<br />
As O 2 becomes more limit<strong>in</strong>g, DosT loses its activity and DosS cont<strong>in</strong>ues to<br />
<strong>in</strong>duce the regulon thereafter. Thus, it has been proposed that Mtb responds to<br />
hypoxia <strong>in</strong> a stepwise manner (Honaker et al.,2009). More recently, significant<br />
studies have demonstrated that <strong>in</strong> addition to hypoxia and NO, the complete<br />
Dos regulon is also <strong>in</strong>duced by CO (Kumar et al., 2007, 2008; Shiloh et al.,<br />
2008). Because of the acknowledged role of hypoxia and NO (and likely also<br />
CO) <strong>in</strong> Mtb persistence, the Dos regulon is a paradigm for Mtb signal transduction,<br />
illustrat<strong>in</strong>g that sens<strong>in</strong>g and relay<strong>in</strong>g of physiologically relevant host<br />
<strong>in</strong>formation <strong>in</strong>duces an adaptive bacterial transcriptional response.<br />
The response of the Dos regulon to O 2, NO and CO consists of the altered<br />
expression of 100 genes (Sherman et al., 2001; Voskuil et al., 2003, 2009;<br />
Kumar et al., 2007, 2008; Shiloh et al., 2008) that <strong>in</strong>clude the repression of<br />
well-characterised genes of prote<strong>in</strong> synthesis, DNA synthesis/cell division,<br />
lipid or am<strong>in</strong>o acid synthesis, aerobic metabolism and the <strong>in</strong>duction of 48<br />
genes which are mostly uncharacterised. Nearly all of the 48 genes <strong>in</strong>itially<br />
<strong>in</strong>duced by hypoxia, NO and CO require DosR for their <strong>in</strong>duction (Honaker<br />
et al., 2008, 2009). Among these 48 genes, with known or predicted function,<br />
many are speculated to play a role <strong>in</strong> adaptation to hypoxic stress, such as acr<br />
(rv2031c; chaperone function), narX (rv1736c; unknown function), nark2<br />
(rv1737c; nitrate/nitrite transport), fdxA (rv2007c; ferredox<strong>in</strong>), nrdZ<br />
(rv0570; ribonuclease reductase), tgs1 (rv3130; triglyceride synthase) and<br />
Mtb orthologues of the universal stress prote<strong>in</strong> family (rv1996, rv2005c,<br />
rv2028c, rv2623, rv2624c, rv3134c) (Voskuil et al., 2009).<br />
A particularly <strong>in</strong>terest<strong>in</strong>g f<strong>in</strong>d<strong>in</strong>g is that isolates of the W-Beij<strong>in</strong>g l<strong>in</strong>eage<br />
of Mtb constitutively overexpress the Dos regulon under <strong>in</strong> vitro conditions
REDUCTIVE STRESS IN MICROBES 81<br />
(Reed et al., 2007), suggest<strong>in</strong>g that the Dos regulon may confer an adaptive<br />
advantage for growth under conditions that <strong>in</strong>hibit aerobic respiration.<br />
Studies of a Mtb Ddos rv3134c/rv3133c/rv3132c mutant deficient <strong>in</strong> expression<br />
of the Dos regulon (Leistikow et al., 2010) demonstrate its essential role<br />
<strong>in</strong> Mtb survival <strong>in</strong> the Wayne model for <strong>in</strong> vitro dormancy where<strong>in</strong> O 2 is<br />
limit<strong>in</strong>g. Notably, the Dos regulon is important for resumption of growth<br />
once Mtb exits this anaerobic state (Leistikow et al., 2010). More recently, an<br />
additional transcriptional response, def<strong>in</strong>ed as the endur<strong>in</strong>g hypoxia<br />
response, was identified and comprised a set of 230 genes <strong>in</strong>duced subsequent<br />
to the Dos dormancy response and expressed long term (Rustad et al.,<br />
2008).<br />
Several l<strong>in</strong>es of evidence suggest that the diatomic gases O2, NO and CO<br />
likely play roles <strong>in</strong> mycobacterial persistence. First, it was demonstrated that<br />
tubercle bacilli require oxygen for growth, and s<strong>in</strong>ce reactivation disease<br />
occurs primarily <strong>in</strong> the oxygen-rich upper lung lobes (Medlar, 1948), the role<br />
of oxygen tension <strong>in</strong> TB has received wide attention (Dubos, 1953; Wayne<br />
and Hayes, 1996; Boon et al., 2001; Voskuil et al., 2009; Gengenbacher et al.,<br />
2010). Rapid withdrawal of oxygen is lethal to Mtb; however, gradual depletion<br />
allows time for adaptation and bacterial survival (Wayne and Hayes,<br />
1996). Furthermore, TB is associated with the most O2-rich site (upper<br />
region of the lung) with<strong>in</strong> the body (Rich and Follis, 1942; Rasmussen,<br />
1957; Riley, 1957), and significantly more bacilli are present <strong>in</strong> TB lung<br />
lesions connected to open airways versus lesions closed to airways<br />
(Medlar, 1948; Haapanen et al., 1959). Secondly, iNOS and therefore NO<br />
production is crucial for protection of mice aga<strong>in</strong>st Mtb (MacMick<strong>in</strong>g et al.,<br />
1997), and human macrophages <strong>in</strong> Mtb-<strong>in</strong>fected tissues are also shown to<br />
express iNOS (Nicholson et al., 1996; Nathan and Shiloh, 2000). Conv<strong>in</strong>c<strong>in</strong>g<br />
studies have demonstrated iNOS RNA and prote<strong>in</strong> <strong>in</strong> bronchoalveolar<br />
lavage specimens from active pulmonary TB patients (Nicholson et al.,<br />
1996). Studies have also shown that <strong>in</strong>creased exhaled NO and nitrite <strong>in</strong><br />
patients with active pulmonary TB is due to an <strong>in</strong>creased iNOS production<br />
(Wang et al., 1998) and that a polymorphism <strong>in</strong> iNOS <strong>in</strong>fluences susceptibility<br />
to TB (Gomez et al., 2007). Thirdly, significant overlap exists between the<br />
gene expression profiles of Mtb cells treated <strong>in</strong> vitro with NO or CO and that<br />
of bacilli cultured under hypoxic conditions (Voskuil et al., 2003, 2009).<br />
Fourthly, <strong>in</strong> recent studies it was shown that NO, O2 and CO (Ioanoviciu<br />
et al., 2007; Kumar et al., 2007, 2008; Sousa et al., 2007; Yukl et al., 2007) are<br />
modulatory ligands of the heme sensor k<strong>in</strong>ases DosT and DosS and that they<br />
either b<strong>in</strong>d directly to the heme-irons or oxidise the heme irons, suggest<strong>in</strong>g<br />
that the bacilli have the mach<strong>in</strong>ery to cont<strong>in</strong>uously monitor O2, NO and CO<br />
levels dur<strong>in</strong>g the course of <strong>in</strong>fection. Regardless of the aforementioned data,
82 AISHA FARHANA ET AL.<br />
the evidence l<strong>in</strong>k<strong>in</strong>g hypoxia, NO and CO to latent TB <strong>in</strong> humans rema<strong>in</strong>s<br />
circumstantial.<br />
Is there a role for CO <strong>in</strong> human TB? Intrigu<strong>in</strong>gly, a role for environmental<br />
CO <strong>in</strong> TB was first described as early as 1923 (Hazleton, 1923) when <strong>in</strong>dividuals<br />
exposed to coal gas (which conta<strong>in</strong>s large quantities of CO) contracted<br />
TB. In observ<strong>in</strong>g a large number of similar cases, the <strong>in</strong>vestigator concluded<br />
that the <strong>in</strong>halation of small quantities of coal gas for a considerable period of<br />
time might have been a predispos<strong>in</strong>g cause of TB. Also, more recently, <strong>in</strong><br />
exam<strong>in</strong><strong>in</strong>g the historical statistics on coal consumption and TB, an <strong>in</strong>terest<strong>in</strong>g<br />
hypothesis that l<strong>in</strong>ks TB and coal-generated CO (and thus air pollution) was<br />
developed (Tremblay, 2007). The newly discovered role of b<strong>in</strong>d<strong>in</strong>g of CO to<br />
DosS and DosT (Kumar et al., 2007; Sousa et al., 2007) and evidence suggest<strong>in</strong>g<br />
that mycobacteria can oxidise CO (K<strong>in</strong>g, 2003; Park et al., 2003, 2007)<br />
have implications for understand<strong>in</strong>g Mtb disease and persistence. Lastly, a<br />
sem<strong>in</strong>al study demonstrated a role for HO-1 generated CO <strong>in</strong> protection<br />
aga<strong>in</strong>st experimental cerebral malaria (Pamplona et al., 2007). Thus, hostgenerated<br />
CO production may be part of a generalised host response to a<br />
variety of pathogens.<br />
6.2.2. The Mtb Dos Dormancy Regulon and Virulence<br />
For many decades, it has been postulated that pO2 <strong>in</strong>fluences the progression<br />
of human TB (Rich and Follis, 1942; Medlar, 1948; Rasmussen, 1957).<br />
Present-day studies us<strong>in</strong>g the hypoxia marker pimonidazole hydrochloride<br />
yielded the surpris<strong>in</strong>g discovery that granulomatous tissue <strong>in</strong> the Mtb<strong>in</strong>fected<br />
mouse model is relatively aerobic ( 37 mm Hg) (Aly et al., 2006;<br />
Tsai et al., 2006), but that tuberculous pulmonary lesions <strong>in</strong> macaques, rabbits<br />
and gu<strong>in</strong>ea pigs are hypoxic (Via et al., 2008). In fact, a detailed analysis<br />
us<strong>in</strong>g a fibre-optic O 2 sensor showed that the pO 2 <strong>in</strong> rabbit pulmonary lesions<br />
was 1.59 mm Hg (Via et al., 2008), demonstrat<strong>in</strong>g that the TB granuloma is<br />
<strong>in</strong>deed hypoxic. In vivo virulence studies have shown that an Mtb dosR<br />
mutant is attenuated <strong>in</strong> the gu<strong>in</strong>ea pig TB <strong>in</strong>fection model (Malhotra et al.,<br />
2004; Converse et al., 2009). However, studies <strong>in</strong> mice (Parish et al., 2003;<br />
Rustad et al., 2008) have produced conflict<strong>in</strong>g results. The discrepancies <strong>in</strong><br />
data obta<strong>in</strong>ed <strong>in</strong> the various <strong>in</strong> vivo studies have been thoroughly discussed<br />
elsewhere (Converse et al., 2009).<br />
The pronounced expression of the Dos regulon and the presence of<br />
TAG <strong>in</strong> bacilli procured from the sputum of Mtb-<strong>in</strong>fected <strong>in</strong>dividuals<br />
(Garton et al., 2008) suggest that sputum conta<strong>in</strong>s non-replicat<strong>in</strong>g bacilli<br />
and has significant implications for understand<strong>in</strong>g the physiological makeup<br />
of the host environment that allows Mtb to persist. For example, because of
REDUCTIVE STRESS IN MICROBES 83<br />
the low O 2 diffusion capability <strong>in</strong> mucus (Worlitzsch et al., 2002), it is plausible<br />
that the sputum is depleted for O2, which leads to upregulation of the<br />
Dos regulon. Furthermore, the direct <strong>in</strong>duction of the Dos regulon by NO,<br />
CO or a comb<strong>in</strong>ation thereof cannot be excluded, s<strong>in</strong>ce TB patients (<strong>in</strong>clud<strong>in</strong>g<br />
healthy <strong>in</strong>dividuals) exhale NO (Gustafsson et al., 1991; Borland et al.,<br />
1993; Wang et al., 1998). Lastly, the diffusion of NO metabolites (NO 3 ,<br />
NO2 ) across the alveolar capillary membrane followed by aerosolisation of<br />
airway epithelial l<strong>in</strong><strong>in</strong>g fluid and conf<strong>in</strong>ement of NO2 /NO 3 to the oropharyngeal<br />
tract and trachea (Marteus et al., 2005; Br<strong>in</strong>dicci et al., 2009;<br />
Fitzpatrick et al., 2009), po<strong>in</strong>t to an environment <strong>in</strong> which access to NO 3<br />
may enable Mtb to oxidise excess NADH via NarG (see Section 6.2.4.1).<br />
6.2.3. The Dos Regulon and CO<br />
The biochemical and biophysical basis of the Mtb response to O2, NO and<br />
CO <strong>in</strong>volv<strong>in</strong>g the heme-irons of the sensor k<strong>in</strong>ases DosS and DosT has been<br />
previously reviewed (Voskuil et al., 2009). However, s<strong>in</strong>ce the role of CO <strong>in</strong><br />
<strong>in</strong>duction of the Dos regulon has only recently been discovered, a short<br />
overview will be given here on the biological relevance of this host gas.<br />
CO is a LMW gas that is endogenously produced by HO-1 <strong>in</strong> humans dur<strong>in</strong>g<br />
normal metabolism and it is <strong>in</strong>creased <strong>in</strong> response to oxidative stress (Ferry,<br />
1995; Chung et al., 2009). CO b<strong>in</strong>d<strong>in</strong>g to the heme-irons of DosS and DosT<br />
modulates their autok<strong>in</strong>ase activity lead<strong>in</strong>g to the upregulation of key members<br />
of the Dos regulon (e.g. dosR, hspX and fdxA)(Kumar et al., 2007). This<br />
f<strong>in</strong>d<strong>in</strong>g led the <strong>in</strong>vestigators to hypothesise that CO is capable of <strong>in</strong>duc<strong>in</strong>g the<br />
Dos regulon. Indeed, subsequent microarray studies performed by two <strong>in</strong>dependent<br />
groups have shown that exposure to CO <strong>in</strong>duces the 48-member<br />
Dos regulon (Kumar et al., 2008; Shiloh et al., 2008).<br />
HO-1 requires O 2 and NADPH as cofactors and utilises heme as a substrate,<br />
ultimately generat<strong>in</strong>g biliverd<strong>in</strong>, iron and CO. HO-1 confers protection<br />
aga<strong>in</strong>st oxidative stress via the anti-<strong>in</strong>flammatory and anti-apoptotic<br />
activities of its byproducts (Chung et al., 2009; Ryter and Choi, 2009).<br />
Infection of macrophages by Mtb <strong>in</strong>duces the expression of HO-1 mRNA,<br />
prote<strong>in</strong> levels and enzymatic activity (Kumar et al., 2008). The ability of HO-<br />
1 generated CO to <strong>in</strong>duce the Dos regulon was demonstrated by <strong>in</strong>fection of<br />
HO-1 +/+ and HO-1 / bone marrow-derived macrophages with Mtb, followed<br />
by expression analysis of the Dos regulon members (Kumar et al.,<br />
2008; Shiloh et al., 2008). Importantly, upregulation of HO-1 was found to be<br />
<strong>in</strong>dependent of the NO pathway (Kumar et al., 2008). In addition, HO-1 was<br />
found to be produced <strong>in</strong> the lungs of Mtb-<strong>in</strong>fected mice (Kumar et al., 2008;<br />
Shiloh et al., 2008). Thus, the comb<strong>in</strong>ed presence of NO and CO at the site of
84 AISHA FARHANA ET AL.<br />
Mtb <strong>in</strong>fection, which might also be hypoxic, provides new <strong>in</strong>sight <strong>in</strong>to how<br />
gaseous gradients may <strong>in</strong>fluence disease progression (Kumar et al., 2008).<br />
However, as mentioned earlier (Section 6.2.1), s<strong>in</strong>ce O2 is a cofactor for<br />
iNOS [KmO2<br />
¼ 135 mM; (Dweik et al., 1998; Dweik, 2005)], the hypoxic<br />
nature of the granuloma (Via et al., 2008) might prevent production of<br />
significant quantities of NO.<br />
Although CO typically <strong>in</strong>hibits respiration <strong>in</strong> other bacteria<br />
(Davidge et al., 2009), Mtb is unusual <strong>in</strong> the sense that it is capable of<br />
tolerat<strong>in</strong>g relatively high concentrations of CO [80 mM (Shiloh et al.,<br />
2008)]. An important functional difference between NO and CO is that<br />
CO can only react with ferrous iron (Fe 2+ ) whereas NO can react with both<br />
Fe 2+ and ferric iron (Fe 3+ )(Kumar et al., 2007). Recently, a protective role<br />
for CO <strong>in</strong> experimental cerebral malaria was demonstrated and it was proposed<br />
that CO locks cell free haemoglob<strong>in</strong> <strong>in</strong> the Fe 2+ state, thereby prevent<strong>in</strong>g<br />
oxidation to the Fe 3+ state, which upon reaction with ROS would<br />
lead to disruption of the blood–bra<strong>in</strong> barrier (Pamplona et al., 2007). A<br />
similar model termed the ‘sense-and-lock’ model was proposed for the<br />
mechanisms of DosS and DosT <strong>in</strong> sens<strong>in</strong>g O2, NO and CO (Kumar et al.,<br />
2007). However, a def<strong>in</strong>ite role for CO <strong>in</strong> Mtb pathogenesis has yet to be<br />
demonstrated, and represents an important but underexplored area of<br />
<strong>in</strong>vestigation.<br />
6.2.4. The Mtb Dos Regulon and Reductive Stress<br />
In physiological terms, it makes bioenergetic sense for an aerobe such as Mtb<br />
to express energy-dissipat<strong>in</strong>g systems under hypoxic conditions <strong>in</strong> order to<br />
ma<strong>in</strong>ta<strong>in</strong> redox balance. For example, when Mtb encounters highly reduced<br />
carbon sources <strong>in</strong> vivo, the carbon source must be oxidised to the po<strong>in</strong>t of<br />
assimilation. If oxidation releases more reduc<strong>in</strong>g equivalents, for example<br />
via b-oxidation, than is needed for ATP generation (reductive stress), the<br />
bacillus must <strong>in</strong>itiate mechanisms to dispose of excess reductants, otherwise<br />
the growth rate will be slowed to the rate at which NADH can be oxidised or<br />
ADP be phosphorylated.<br />
Us<strong>in</strong>g an <strong>in</strong> vitro dormancy model, studies have shown that <strong>in</strong> an Mtb Ddos<br />
rv3134c/rv3133c/rv3132c mutant, the ATP level decreased to 10% of that<br />
measured under aerobic growth. In contrast, the ATP level <strong>in</strong> the wt stra<strong>in</strong><br />
stabilised at 25% of the aerobic level (Leistikow et al., 2010). These f<strong>in</strong>d<strong>in</strong>gs<br />
are <strong>in</strong> agreement with an <strong>in</strong>dependent study demonstrat<strong>in</strong>g that, dur<strong>in</strong>g<br />
hypoxia, Mtb has a much reduced but cont<strong>in</strong>uous pool of ATP (Rao et al.,<br />
2008). In fact, it was shown that further reduction of ATP (three to fourfold)<br />
under hypoxic conditions <strong>in</strong>creases cell death to more than 90% whereas a
REDUCTIVE STRESS IN MICROBES 85<br />
depletion of more than 90% of ATP was required to see a kill<strong>in</strong>g effect under<br />
aerobic growth conditions. Consistent with these f<strong>in</strong>d<strong>in</strong>gs, it was demonstrated<br />
that the membrane of Mtb cultured under hypoxia is fully energised<br />
and is required for ATP production (Rao et al., 2008).<br />
NAD + and NADH concentrations decrease by 50% when wt Mtb is grown<br />
under hypoxic conditions, yet the overall redox couple ratio is ma<strong>in</strong>ta<strong>in</strong>ed.<br />
This is an <strong>in</strong>trigu<strong>in</strong>g observation as the NADH/NAD + ratio typically<br />
<strong>in</strong>creases <strong>in</strong> bacteria as O2 levels decrease (de Graef et al., 1999; Berrios-<br />
Rivera et al., 2002a). One explanation might be that <strong>in</strong>creased NADH generated<br />
under hypoxia is consumed dur<strong>in</strong>g TAG synthesis, thereby ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g<br />
an ‘optimal’ NADH/NAD + ratio. Regardless, <strong>in</strong> the Mtb Ddos mutant,<br />
the NAD + levels dropped to 10% of the wt level, and the NAD + /NADH<br />
ratio was sixfold less under hypoxia. Thus, the data clearly suggest that<br />
the Mtb Dos regulon plays a role <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g redox homeostasis<br />
(Leistikow et al., 2010). Furthermore, it appears that ndh-2 rather than<br />
ndh-1 contributes to the generation of the proton motive force for ATP<br />
production under hypoxic conditions and that Ndh-2 is responsible for<br />
replenish<strong>in</strong>g NAD + <strong>in</strong> Mtb grown under hypoxic conditions (Rao et al.,<br />
2008).<br />
Two reports provide direct evidence for the presence of reductive stress <strong>in</strong><br />
Mtb. First, the accumulation of extraord<strong>in</strong>arily high levels of NAD(P)H<br />
relative to NAD(P) + was found <strong>in</strong> bacilli derived from the lungs of <strong>in</strong>fected<br />
mice (Boshoff et al., 2008). Secondly, polyketide and TAG anabolism have<br />
been demonstrated to occur <strong>in</strong> vivo and the synthesis of such may serve as<br />
efficient reductant disposal mechanisms to alleviate reductive stress for longterm<br />
persistence (S<strong>in</strong>gh et al., 2009) (Fig. 1) (see also Section 6.2.4.2).<br />
In conclusion, a role for the Dos regulon <strong>in</strong> reductive stress has largely<br />
been underappreciated. Nonetheless, the DosR-controlled NarGHJI/NarK2<br />
and Tgs1 prote<strong>in</strong>s are likely candidates for dispos<strong>in</strong>g of excess reduc<strong>in</strong>g<br />
equivalents and thereby ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g redox balance.<br />
6.2.4.1. Nitrate reductase<br />
Several functions can be attributed to nitrate reduction <strong>in</strong> bacteria (Cole,<br />
1996; Richardson et al., 2001; Morozk<strong>in</strong>a and Zvyagilskaya, 2007) and<br />
<strong>in</strong>clude (i) NO3 assimilation (us<strong>in</strong>g NO3 as a nitrogen source), (ii) NO3 dissimilation (disposal of excess reduc<strong>in</strong>g equivalents to ma<strong>in</strong>ta<strong>in</strong> redox<br />
balance) and (iii) NO3 respiration (us<strong>in</strong>g NO3 as the term<strong>in</strong>al electron<br />
acceptor for the production of energy). Mtb conta<strong>in</strong>s two possible nitrate<br />
reductases, the NarGHJI (RV1161–1164) complex, which catalyses the<br />
reduction of NO3 to NO2 and NarX that has no known function to date.
86 AISHA FARHANA ET AL.<br />
[(Figure_1)TD$FIG]<br />
Figure 1 Model depict<strong>in</strong>g the dissipation of excess reductants through the Dos<br />
regulon to ma<strong>in</strong>ta<strong>in</strong> redox homeostasis. Hypoxia, NO and likely CO, which <strong>in</strong>hibit<br />
respiration, are sensed by DosS/T lead<strong>in</strong>g to activation of the Dos regulon via DosR.<br />
Included <strong>in</strong> the Dos regulon are genes encod<strong>in</strong>g for components of the rTCA cycle,<br />
and narGHJI-narK. These pathways serve as alternate electron s<strong>in</strong>ks for reductive<br />
stress dissipation. The excess NADH, NADPH and FADH2 that are produced upon<br />
<strong>in</strong>hibition of respiration or b-oxidation of host FA are utilised for the generation of<br />
metabolic <strong>in</strong>termediates. (a) The rTCA cycle <strong>in</strong>corporates one molecule of CO 2 <strong>in</strong> an<br />
energy-consum<strong>in</strong>g process, where<strong>in</strong> the energy is provided by the reduc<strong>in</strong>g equivalents.<br />
This allows the recycl<strong>in</strong>g of excess reductants for the production of essential<br />
metabolites, thereby lower<strong>in</strong>g their <strong>in</strong>tracellular concentrations. (b) The uptake of<br />
NO 3 by NarK2 and its correspond<strong>in</strong>g reduction to NO 2 by NarG consumes cellular<br />
reduc<strong>in</strong>g equivalents. As the concentration of cytosolic NO 2 <strong>in</strong>creases to reach toxic<br />
levels, it is pumped out via NarK. As extracellular levels of NO3 decrease, NO2 is<br />
transported back <strong>in</strong>to the cell and reduced via NirBD to NH3. (c) NADPH is a<br />
cofactor <strong>in</strong> the FASI pathway and is consumed dur<strong>in</strong>g anabolism of the storage<br />
lipid, TAG.
REDUCTIVE STRESS IN MICROBES 87<br />
Several studies have shown that nitrate reductase activity <strong>in</strong>creases dramatically<br />
dur<strong>in</strong>g cultur<strong>in</strong>g of Mtb under microaerophilic conditions (Sohaskey<br />
and Wayne, 2003; Sohaskey, 2005, 2008). The narGHJI operon is constitutively<br />
expressed <strong>in</strong> Mtb whereas narK2, encod<strong>in</strong>g for the NO 3 /NO 2 transporter,<br />
is under control of DosR and therefore a component of the<br />
Dos regulon that is highly <strong>in</strong>duced dur<strong>in</strong>g hypoxia and upon exposure to<br />
NO and CO (Sherman et al., 2001; Voskuil et al., 2003; Kumar et al.,<br />
2008). Depend<strong>in</strong>g on the organism, nitrate reductases require NADH<br />
or NADPH as cofactors (Richardson et al., 2001; Morozk<strong>in</strong>a and<br />
Zvyagilskaya, 2007). Genetic experiments suggest that nitrate reductase<br />
activity <strong>in</strong> Mtb is not coupled to proton translocation and ATP synthesis<br />
(Sohaskey and Wayne, 2003). Rather, it has been concluded that nitrate<br />
reductase may be <strong>in</strong>volved <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g redox balance dur<strong>in</strong>g microaerophilic<br />
growth by dissipat<strong>in</strong>g excess reduc<strong>in</strong>g equivalents (Sohaskey, 2008).<br />
The redox potential of NO3 /NO2 is +420 mV and the change <strong>in</strong> Gibbs free<br />
energy approximates 82 kJ/mol (Seifritz et al., 1993), which suggests that<br />
NO 3 is an effective electron s<strong>in</strong>k.<br />
Although Mtb can utilise NO3 as a term<strong>in</strong>al electron acceptor under<br />
anaerobic conditions to ma<strong>in</strong>ta<strong>in</strong> some degree of viability or to prolong<br />
survival, active replication does not occur <strong>in</strong> the absence of O 2 (Sohaskey<br />
and Wayne, 2003; Aly et al., 2006; Sohaskey, 2008; Gengenbacher et al.,<br />
2010). S<strong>in</strong>ce Mtb does not actively respire anaerobically, it is tempt<strong>in</strong>g to<br />
theorise that the release of Gibbs free energy from the reduction of NO 3 to<br />
NO2 contributes towards ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g viability of the bacilli when encounter<strong>in</strong>g<br />
hypoxia <strong>in</strong> vivo. Complementation of an E. coli nark narU mutant with<br />
Mtb narK2 strongly suggests that NarK2 transports NO 3 <strong>in</strong>to and NO 2 out<br />
of Mtb. Thus, under low-O2 conditions, NO3 is transported <strong>in</strong>to the cell,<br />
reduced to NO2 and, when toxic levels of NO2 are reached, exported out.<br />
An <strong>in</strong>terest<strong>in</strong>g parallel exists between Mtb and Staphylococcus carnosus. In<br />
case of the latter, the exported NO2 is transported back <strong>in</strong>to the cell and<br />
reduced to ammonia, which aga<strong>in</strong> accumulates <strong>in</strong> the medium (Neubauer<br />
and Gotz, 1996). This event is rem<strong>in</strong>iscent of the <strong>in</strong>creased alkal<strong>in</strong>ity (as<br />
opposed to acidity) detected <strong>in</strong> the culture supernatant of Mtb, which was<br />
shown to be due to buildup of ammonia (Merrill, 1930). It is tempt<strong>in</strong>g to<br />
speculate that NirB (Rv0252) and NirD (Rv0253) are Mtb nitrite reductase<br />
subunits that reduce NO2 to ammonia without liberat<strong>in</strong>g <strong>in</strong>termediate<br />
products and may expla<strong>in</strong> the observed alkal<strong>in</strong>ity by Merrill (1930).<br />
An obvious question is: does Mtb encounter NO 3 and/or NO 2 <strong>in</strong> vivo?<br />
Indeed, the presence of NO3 plus NO 2 <strong>in</strong> saliva (313 mM), NO 3 <strong>in</strong> trachael<br />
secretions (144–421 mM) (Grasemann et al., 1998; L<strong>in</strong>nane et al., 1998;<br />
Worlitzsch et al., 2002), NO3 (0.5–37 mM) and NO2 (1.4–15 mM) <strong>in</strong>
88 AISHA FARHANA ET AL.<br />
bronchoalveolar lavage fluid (Dweik et al., 2001; Fitzpatrick et al., 2009), and<br />
NO2 /NO3 <strong>in</strong> exhaled breath condensate (17 mmol) and sputum (449 mmol)<br />
(Br<strong>in</strong>dicci et al., 2009) provides evidence that Mtb is likely to encounter<br />
NO 2 and/or NO 3 dur<strong>in</strong>g the full spectrum of disease.<br />
6.2.4.2. TAG production<br />
An underappreciated consequence of Mtb utilisation of host lipids for energy<br />
is the production of large quantities of reduc<strong>in</strong>g equivalents that are essential<br />
cofactors for the production of storage lipids (TAG) and virulence lipids such<br />
as SL-1, PDIM and PAT/DAT. At least two widely known biological functions<br />
of TAG <strong>in</strong> prokaryotes <strong>in</strong>clude its use as an effective reserve carbon<br />
source because of its reduced COS, and its service as an electron s<strong>in</strong>k for<br />
excess reduc<strong>in</strong>g equivalents (Alvarez and Ste<strong>in</strong>buchel, 2002). Thus, it appears<br />
that TAG anabolism is a plausible mechanism for dissipation of reductive<br />
stress <strong>in</strong> Mtb (S<strong>in</strong>gh et al., 2009). TAG production is under the control of the<br />
DosR regulon and its generation is highly <strong>in</strong>creased when Mtb cells are<br />
exposed to hypoxia, NO or CO (Voskuil et al., 2009). Interest<strong>in</strong>gly, TAG is<br />
found <strong>in</strong> the sputum of TB patients (Garton et al., 2008). Studies us<strong>in</strong>g the <strong>in</strong><br />
vitro Wayne model for dormancy have shown that TAG is catabolised by Mtb<br />
upon reactivation (Deb et al., 2006) and thus may implicate a possible role for<br />
TAG <strong>in</strong> emergence of Mtb from a persistent state <strong>in</strong> vivo.<br />
6.3. Mtb WhiB3 is an Intracellular Redox Sensor<br />
that Counters Reductive Stress<br />
6.3.1. The WhiB Family of Prote<strong>in</strong>s<br />
The first WhiB-like prote<strong>in</strong> was characterised <strong>in</strong> Streptomyces <strong>in</strong> 1992 (Davis<br />
and Chater, 1992) and was predicted to have a putative helix-turn-helix (HTH)<br />
motif at its C-term<strong>in</strong>us. WhiB-like prote<strong>in</strong>s are LMW (81–122 am<strong>in</strong>o acids)<br />
and are restricted to act<strong>in</strong>omycetes (den Hengst and Buttner, 2008). WhiB<br />
members conta<strong>in</strong> four conserved Cys residues, with one exception (Cole et al.,<br />
1998). Mtb conta<strong>in</strong>s seven homologues of WhiB (WhiB1, Rv3219; WhiB2,<br />
Rv3260c; WhiB3, Rv3416; WhiB4, Rv3681c; WhiB5, Rv0022c; WhiB6,<br />
Rv3862c and WhiB7, Rv3197A) that show strong homology to regulatory<br />
prote<strong>in</strong>s of Streptomyces spp. critical for sporulation (Chater, 1972; Flardh<br />
et al., 1999). In one of the <strong>in</strong>itial studies on mycobacterial WhiB prote<strong>in</strong>s, it<br />
was shown that Msm WhmD is essential and is <strong>in</strong>volved <strong>in</strong> septum formation<br />
and fragmentation (Gomez and Bishai, 2000). Subsequently, other members
REDUCTIVE STRESS IN MICROBES 89<br />
of the WhiB family have been shown to play a role <strong>in</strong> combat<strong>in</strong>g oxidative<br />
stress <strong>in</strong> Corynebacterium glutamicum (Kim et al.,2005). An important f<strong>in</strong>d<strong>in</strong>g<br />
was the establishment of Mtb WhiB3 as a virulence factor (Steyn et al., 2002)<br />
(see Section 6.3.2 below for a complete description). Moreover, the observations<br />
that Mtb WhiB7 plays a role <strong>in</strong> antibiotic resistance and that it is highly<br />
upregulated <strong>in</strong> the presence of palmitic acid, a putative <strong>in</strong> vivo carbon source,<br />
are particularly <strong>in</strong>terest<strong>in</strong>g (Morris et al.,2005) and suggest that, dur<strong>in</strong>g <strong>in</strong>fection,<br />
the specific <strong>in</strong> vivo environment might allow Mtb to effectively resist<br />
chemotherapeutic <strong>in</strong>tervention strategies. Exam<strong>in</strong>ation of the differential<br />
expression of all seven Mtb whiB genes dur<strong>in</strong>g growth and upon exposure<br />
to a wide range of antibiotics or to a variety of <strong>in</strong> vitro stress conditions<br />
<strong>in</strong>dicates that the Mtb WhiB family is strongly reactive to a wide range of<br />
environmental stress conditions (Geiman et al., 2006).<br />
Consistent with the presence of conserved Cys residues <strong>in</strong> the WhiB<br />
family, it was demonstrated that WhiD, a Streptomyces member of the<br />
WhiB family, conta<strong>in</strong>s a 4Fe–4S cluster (Jakimowicz et al., 2005). Subsequently,<br />
it was demonstrated that Mtb WhiB3 also possesses a 4Fe–4S<br />
cluster (S<strong>in</strong>gh et al., 2007), suggest<strong>in</strong>g that this feature might be common<br />
to all seven Mtb homologues. Studies on Mtb apo-WhiB1 (Garg et al., 2007)<br />
and apo-WhiB4 (Alam et al., 2007) suggest that they function as disulphide<br />
reductases. Us<strong>in</strong>g the yeast two-hybrid system, the same group has shown<br />
that WhiB1 <strong>in</strong>teracts with GlgB (an a-1,4-glucan branch<strong>in</strong>g enzyme) and<br />
reduces the <strong>in</strong>tra-molecular disulfide bond of GlgB (Garg et al., 2009).<br />
Additionally, a biochemical study provides some evidence that all the Mtb<br />
WhiB’s conta<strong>in</strong> Fe–S clusters (Alam et al., 2009). However, s<strong>in</strong>ce the study<br />
predom<strong>in</strong>antly used UV-visible spectroscopy and not electron paramagnetic<br />
resonance spectroscopy to characterise the Fe–S clusters, the type of Fe–S<br />
cluster (4Fe–4S, 3Fe–4S or 2Fe–2S) of each homologue was not conclusively<br />
demonstrated.<br />
Lastly, an <strong>in</strong>terest<strong>in</strong>g study has shown that the WhiB-like prote<strong>in</strong> of<br />
mycobacteriophage TM4 leads to the downregulation of Mtb whiB2 with<br />
subsequent filamentation and growth <strong>in</strong>hibition (Rybniker et al., 2010). The<br />
likely redox-mediated regulation of a host mycobacterial prote<strong>in</strong> by a viral<br />
factor is an excit<strong>in</strong>g f<strong>in</strong>d<strong>in</strong>g and warrants further <strong>in</strong>vestigations.<br />
6.3.2. Mtb WhiB3 and Virulence<br />
In 1995, a s<strong>in</strong>gle po<strong>in</strong>t mutation (Arg515–His) <strong>in</strong> the 4.2 region of rpoV<br />
(encod<strong>in</strong>g the pr<strong>in</strong>cipal s-factor SigA) was shown to be responsible for the<br />
loss of virulence of wt M. bovis (Steyn et al., 2002). rpoV was the first gene<br />
identified <strong>in</strong> the Mtb complex required for virulence. The biological
90 AISHA FARHANA ET AL.<br />
mechanism of attenuation caused by this mutation rema<strong>in</strong>ed unknown until a<br />
yeast two-hybrid screen, which employed the wt rpoV 4.2 region allele as<br />
bait, identified a small regulatory prote<strong>in</strong>, WhiB3, as an <strong>in</strong>teract<strong>in</strong>g partner.<br />
Significantly, WhiB3 <strong>in</strong>teraction with the attenuated RpoV allele conta<strong>in</strong><strong>in</strong>g<br />
the s<strong>in</strong>gle po<strong>in</strong>t mutation described above was severely impaired<br />
(Steyn et al., 2002). Therefore, loss of <strong>in</strong>teraction with WhiB3 was the likely<br />
reason for the attenuat<strong>in</strong>g nature of the RpoV R-H mutation. Although<br />
whiB3 (Rv3416) deletion mutants of both Mtb H37Rv and M. bovis displayed<br />
no observable phenotypic changes dur<strong>in</strong>g <strong>in</strong> vitro growth, both were<br />
shown to be attenuated <strong>in</strong> vivo. Infection of gu<strong>in</strong>ea pigs with the M. bovis<br />
DwhiB3 mutant demonstrated that this stra<strong>in</strong> was unable to colonise the<br />
spleen. Perhaps a more strik<strong>in</strong>g f<strong>in</strong>d<strong>in</strong>g was that the Mtb DwhiB3 deletion<br />
mutant showed wt-like growth <strong>in</strong> all organs, yet the mean survival time of<br />
C57BL/6 mice <strong>in</strong>fected with the Mtb DwhiB3 deletion stra<strong>in</strong> (280 days) was<br />
virtually twice to that of mice similarly <strong>in</strong>fected with wt Mtb H37Rv (150<br />
days). Furthermore, despite identical organ burdens, a dramatic difference <strong>in</strong><br />
lung pathology <strong>in</strong>duced by mutant and wt was observed. The lungs of the Mtb<br />
DwhiB3-<strong>in</strong>fected mice showed less pathology and cellular <strong>in</strong>filtration compared<br />
to those of mice <strong>in</strong>fected with wt Mtb, an observation that suggested<br />
that <strong>in</strong>fection with the Mtb DwhiB3 mutant was not associated with a harmful<br />
<strong>in</strong>flammatory response (Steyn et al., 2002). Ow<strong>in</strong>g to the reduced immunopathology<br />
caused by this mutant <strong>in</strong> mice and because a Streptomyces whiD<br />
mutant has repressed expression of whiE that encodes a polyketide synthase<br />
(Kelemen et al., 1998), a connection between whiB3, the production of Mtb<br />
polyketides and detrimental immunopathology was proposed (Steyn et al.,<br />
2002). Consistent with the idea that WhiB3 is needed for the synthesis of cellsurface<br />
polyketides is the observation of differences <strong>in</strong> colony morphology<br />
displayed by the wt and the DwhiB3 mutant stra<strong>in</strong>s (S<strong>in</strong>gh et al., 2007).<br />
F<strong>in</strong>ally, whiB3 expression was found to <strong>in</strong>versely correlate with bacterial<br />
density <strong>in</strong> vivo <strong>in</strong> the mouse system, suggest<strong>in</strong>g a possible role for WhiB3 <strong>in</strong><br />
quorum sens<strong>in</strong>g (Banaiee et al., 2006).<br />
An important f<strong>in</strong>d<strong>in</strong>g was the demonstration that Mtb conta<strong>in</strong>s a cyste<strong>in</strong>e<br />
desulphurase (IscS; Rv3025c) capable of assembl<strong>in</strong>g the WhiB3 4Fe–4S<br />
cluster (S<strong>in</strong>gh et al., 2007). The four WhiB3 Cys residues were subsequently<br />
shown to be essential <strong>in</strong> coord<strong>in</strong>at<strong>in</strong>g a Fe–S cluster. Exposure of purified<br />
WhiB3 to air or NO led to cluster degradation or formation of a d<strong>in</strong>itrosyl<br />
iron–dithiol complex respectively. Therefore, WhiB3 reacts directly with O 2<br />
and NO and likely functions as an endogenous sensor of these two dormancy<br />
gases (S<strong>in</strong>gh et al., 2007).<br />
Aside from the identification of a functional IscS, not much is known<br />
about Fe–S cluster assembly systems <strong>in</strong> mycobacteria. A putative Mtb SUF
REDUCTIVE STRESS IN MICROBES 91<br />
(mobilisation of sulphur) system (SufBCD) was identified through prote<strong>in</strong>–<br />
prote<strong>in</strong> <strong>in</strong>teraction studies (Huet et al., 2005), and formation of this prote<strong>in</strong><br />
complex depends on the prote<strong>in</strong> splic<strong>in</strong>g (via an <strong>in</strong>te<strong>in</strong>) of SufB (Huet et al.,<br />
2006). Mtb conta<strong>in</strong>s more than one IscS homolog and the SufBCD system,<br />
which are virtually uncharacterised <strong>in</strong> terms of general Fe–S cluster<br />
assembly.<br />
It has now been demonstrated that WhiB3 is a regulator of cellular metabolism,<br />
where<strong>in</strong> it is important for growth <strong>in</strong> the presence of glucose, pyruvate,<br />
succ<strong>in</strong>ate and fumarate as sole carbon sources (S<strong>in</strong>gh et al., 2007). The<br />
defective growth exhibited by the Mtb DwhiB3 mutant on glucose or succ<strong>in</strong>ate<br />
as the sole carbon source can be rescued by complementation with wt<br />
WhiB3, but not with WhiB3 that has all four Cys residues mutated. Thus, the<br />
[4Fe–4S] + cluster is necessary for WhiB3 to function as a metabolic regulator.<br />
Intrigu<strong>in</strong>gly, the Mtb DwhiB3 mutant grows much better <strong>in</strong> media conta<strong>in</strong><strong>in</strong>g<br />
the short-cha<strong>in</strong> FA acetate, suggest<strong>in</strong>g that WhiB3 is a potential regulator of<br />
FA metabolism <strong>in</strong> Mtb. It has been proposed that WhiB3 senses or responds<br />
to cellular substrates via its [4Fe–4S] + cluster <strong>in</strong> order to affect a metabolic<br />
switchover to the use of FA as the preferred carbon source <strong>in</strong> vivo<br />
(S<strong>in</strong>gh et al., 2007). The role of WhiB3 <strong>in</strong> core <strong>in</strong>termediary metabolism is<br />
consistent with studies on FNR and ArcA, which regulate key enzymes <strong>in</strong> the<br />
glycolytic and TCA cycles <strong>in</strong> response to carbon source starvation (Levanon<br />
et al., 2005; Shalel-Levanon et al., 2005).<br />
In conclusion, the pathological defect <strong>in</strong>duced by Mtb DwhiB3 <strong>in</strong> the<br />
mouse model (Steyn et al., 2002) as well as the altered colony morphology<br />
and growth properties of Mtb DwhiB3 on carbon-limit<strong>in</strong>g media, <strong>in</strong> particular<br />
FAs (S<strong>in</strong>gh et al., 2007), suggests that WhiB3 is <strong>in</strong>volved <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g<br />
redox homeostasis by regulat<strong>in</strong>g catabolism and polyketide biosynthesis <strong>in</strong><br />
Mtb. The precise mechanism by which this occurs rema<strong>in</strong>s to be <strong>in</strong>vestigated.<br />
6.3.3. WhiB3 and Reductive Stress<br />
Mtb WhiB3 is essential for ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g bacterial cell shape and size, and<br />
modulates the biosynthesis of complex virulence lipids <strong>in</strong>clud<strong>in</strong>g PAT, DAT,<br />
SL-1 and PDIM (S<strong>in</strong>gh et al., 2009). An <strong>in</strong>terest<strong>in</strong>g f<strong>in</strong>d<strong>in</strong>g was that Mtb<br />
DwhiB3 displayed defective production of the methyl-branched polar lipids<br />
PAT, DAT and SL-1 but showed <strong>in</strong>creased synthesis of PDIM and, to a<br />
lesser extent, TAG. The f<strong>in</strong>d<strong>in</strong>g that Mtb DwhiB3 accumulates PDIM and<br />
TAG is unique and has not yet been reported for any Mtb mutant to date.<br />
Exam<strong>in</strong>ation of the mycolic acid profiles demonstrated a decrease <strong>in</strong><br />
a,a’-trehalose dimycolate (TDM) and a,a’-trehalose monomycolate<br />
(TMM) <strong>in</strong> Mtb DwhiB3 as compared to wt Mtb. Importantly, further studies
92 AISHA FARHANA ET AL.<br />
revealed that WhiB3 regulates the production of PAT, PDIM and TAG <strong>in</strong> a<br />
redox-dependent manner <strong>in</strong> vitro (S<strong>in</strong>gh et al., 2009). The discovery of a<br />
redox-switch<strong>in</strong>g mechanism of Mtb virulence lipid production is the first of its<br />
k<strong>in</strong>d and provides important <strong>in</strong>sight <strong>in</strong>to how oxido-reductive host factors<br />
could modulate the synthesis of lipids <strong>in</strong>volved <strong>in</strong> virulence. In these experiments,<br />
diamide and DTT were used as a thiol-specific oxidant and reductant<br />
respectively. An <strong>in</strong>terest<strong>in</strong>g observation was that under reductive stress<br />
conditions (treatment with DTT), a significant <strong>in</strong>crease <strong>in</strong> TAG production<br />
was observed <strong>in</strong> Mtb DwhiB3 compared to wt Mtb (S<strong>in</strong>gh et al., 2009). These<br />
f<strong>in</strong>d<strong>in</strong>gs have several important biological implications. First, they establish a<br />
l<strong>in</strong>k between the extracellular heme-based Dos dormancy signal<strong>in</strong>g pathway<br />
(TAG is under DosR control), the <strong>in</strong>tracellular 4Fe–4S cluster WhiB3<br />
signal<strong>in</strong>g pathway and reductive stress. Secondly, they suggest that <strong>in</strong>tracellular<br />
oxido-reductive stress modulates diverse polyketides, which are implicated<br />
<strong>in</strong> virulence (Cox et al., 1999; Trivedi et al., 2005), and TAG production<br />
that might be essential for emergence from a persistent state (Deb et al.,<br />
2006). Thirdly, s<strong>in</strong>ce it is well known that TAG can function as an effective<br />
electron s<strong>in</strong>k <strong>in</strong> bacteria (Alvarez and Ste<strong>in</strong>buchel, 2002), the data implicate<br />
WhiB3 <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g <strong>in</strong>tracellular redox homeostasis.<br />
As host FAs are degraded via b-oxidation, potentially toxic levels of<br />
propionate, a byproduct of odd cha<strong>in</strong> FA assimilation, are generated (Ja<strong>in</strong><br />
et al., 2007; Upton and McK<strong>in</strong>ney, 2007). The fact that Mtb DwhiB3 was able<br />
to grow on much higher concentrations of propionate compared to wt Mtb,<br />
and that it accumulated PDIM and TAG dur<strong>in</strong>g <strong>in</strong>tra-macrophage growth,<br />
suggests that the <strong>in</strong>creased resistance to propionate toxicity is because propionate<br />
is channelled <strong>in</strong>to PDIM via the methyl-malonyl CoA (MMCoA)<br />
pathway, and <strong>in</strong>to TAG (S<strong>in</strong>gh et al., 2009). Thus, WhiB3-mediated regulation<br />
of virulence lipid anabolism is a plausible mechanism for the detoxification<br />
of excess levels of propionate <strong>in</strong> vivo. In fact, it was shown that<br />
<strong>in</strong>creased flux of MMCoA augments the size and abundance of PDIM and<br />
SL-1. The <strong>in</strong>creased synthesis and mass of PDIM that occurred dur<strong>in</strong>g <strong>in</strong>fection<br />
of mice provides credence to the notion that propionate toxicity <strong>in</strong> vivo is<br />
relieved via PDIM anabolism (Ja<strong>in</strong> et al., 2007).<br />
Large quantities of NADH and NADPH are generated from b-oxidation<br />
of host FA (e.g. palmitate) (see Section 4.2.2), which, if not properly balanced,<br />
can lead to reductive stress. A central question is: How does Mtb<br />
WhiB3 modulate the disposal of, or dissipate excess reduc<strong>in</strong>g equivalents to<br />
ma<strong>in</strong>ta<strong>in</strong> redox balance? In light of the fact that Mtb does not use NO 3 as a<br />
term<strong>in</strong>al electron acceptor under anaerobic conditions to promote replication,<br />
does not ferment and, to the best of our knowledge, does not secrete<br />
redox active molecules or use electron bifurcation mechanisms (Thauer,
REDUCTIVE STRESS IN MICROBES 93<br />
1988; Darrouzet and Daldal, 2003; Xia et al., 2007; Thauer et al., 2008; Costa<br />
et al., 2010) for energy production; this raises the question as to what the<br />
primary electron s<strong>in</strong>k <strong>in</strong> Mtb is? Although this is an emerg<strong>in</strong>g area of <strong>in</strong>vestigation<br />
and not much is known at this po<strong>in</strong>t of time, several recent f<strong>in</strong>d<strong>in</strong>gs<br />
suggest that Mtb lipid anabolism, which requires NAD(P)H as cofactors,<br />
functions as an effective electron s<strong>in</strong>k and that WhiB3 plays an important<br />
regulatory role <strong>in</strong> this process. First, <strong>in</strong> vivo studies have shown that Mtb<br />
isolated from <strong>in</strong>fected mouse lungs have extraord<strong>in</strong>arily high levels of NAD<br />
(P)H, provid<strong>in</strong>g evidence that Mtb experiences severe reductive stress<br />
(Boshoff et al., 2008). Second, studies have shown that PDIM levels and<br />
bacterial mass <strong>in</strong>crease significantly <strong>in</strong> <strong>in</strong>fected mice (Ja<strong>in</strong> et al., 2007).<br />
Third, WhiB3 was shown to modulate the differential anabolism of mycobacterial<br />
lipids <strong>in</strong>clud<strong>in</strong>g methyl-branched FAs (PDIM, SL-1, PAT and<br />
DAT) and TAG dur<strong>in</strong>g growth <strong>in</strong> vitro and <strong>in</strong> macrophages (S<strong>in</strong>gh et al.,<br />
2009). Fourth, Mtb DwhiB3 treated with DTT <strong>in</strong>creased synthesis of TAG<br />
dramatically (S<strong>in</strong>gh et al., 2009). Fifth, us<strong>in</strong>g [ 14 C] nicot<strong>in</strong>amide <strong>in</strong>corporation<br />
and enzymatic cycl<strong>in</strong>g assays, it was shown that Mtb DwhiB3 accumulated<br />
large quantities of NADPH <strong>in</strong> macrophages and therefore experienced<br />
reductive stress (S<strong>in</strong>gh et al., 2009). Collectively, the data suggest that Mtb<br />
lipid anabolism <strong>in</strong> vitro and <strong>in</strong> vivo demand substantial quantities of NAD(P)<br />
H and therefore functions as an effective electron s<strong>in</strong>k to alleviate excess<br />
reduc<strong>in</strong>g equivalents and reductive stress.<br />
In order to expla<strong>in</strong> these events, a model (Fig. 2) was proposed to illustrate<br />
the catabolism of highly reduced host FAs, as well as exposure of the bacilli<br />
to hypoxic conditions or NO, thereby generat<strong>in</strong>g reductive stress. In order to<br />
dispose or dissipate the excess reduc<strong>in</strong>g equivalents [NAD(P)H], Mtb anabolises<br />
PAT/DAT, SL-1, PDIM and predom<strong>in</strong>antly TAG, which function as<br />
electron s<strong>in</strong>ks. In conclusion, the mode of action of Mtb WhiB3 represents an<br />
elegant avenue for exam<strong>in</strong><strong>in</strong>g the mechanistic basis of reductive stress dissipation<br />
<strong>in</strong> bacterial pathogens and should open new areas of <strong>in</strong>vestigation.<br />
6.3.4. WhiB3 and DNA B<strong>in</strong>d<strong>in</strong>g<br />
For more than 15 years, WhiB homologues <strong>in</strong> act<strong>in</strong>omycetes have been<br />
speculated to be putative DNA-b<strong>in</strong>d<strong>in</strong>g transcription factors (den Hengst<br />
and Buttner, 2008). However, a formal proof demonstrat<strong>in</strong>g their DNA<br />
b<strong>in</strong>d<strong>in</strong>g activity was lack<strong>in</strong>g until recently. S<strong>in</strong>gh et al. (2009) provided the<br />
first evidence that a member of the WhiB family can b<strong>in</strong>d to DNA. Gel<br />
retardation studies have shown that the redox state of the Mtb WhiB3 4Fe–<br />
4S cluster does not <strong>in</strong>fluence DNA b<strong>in</strong>d<strong>in</strong>g and that oxidised apo-WhiB3<br />
b<strong>in</strong>ds DNA tightly compared to holo-WhiB3. This is unusual because either
94 AISHA FARHANA ET AL.<br />
[(Figure_2)TD$FIG]<br />
Figure 2 Model depict<strong>in</strong>g the role of Mtb WhiB3 <strong>in</strong> sens<strong>in</strong>g and dissipat<strong>in</strong>g<br />
reductive stress to ma<strong>in</strong>ta<strong>in</strong> redox homeostasis. (a) Generation of reductants. b-oxidation<br />
of host FA and the <strong>in</strong>hibition of respiration by hypoxia, NO and possibly CO<br />
lead to the generation of NADH and NADPH that are major contributors to reductive<br />
stress <strong>in</strong> Mtb. (b) Regulation. WhiB3 ma<strong>in</strong>ta<strong>in</strong>s <strong>in</strong>tracellular redox homeostasis<br />
through its Fe–S cluster via an alteration <strong>in</strong> its apo and holo redox states. This<br />
modulates DNA b<strong>in</strong>d<strong>in</strong>g and regulates the expression of genes necessary for lipid<br />
anabolism. (c) Dissipat<strong>in</strong>g reductive stress. The <strong>in</strong>duction of lipid anabolism by WhiB3<br />
leads to the dissipation of excess reductants. Anabolism of a wide range of lipids<br />
implicated <strong>in</strong> virulence (PAT, DAT, PDIM and SL-1) via MMCoA, and the synthesis<br />
of storage lipid (TAG) by the FASI pathway functions as electron s<strong>in</strong>ks. Note that the<br />
Dos dormancy regulon and the WhiB3 signal<strong>in</strong>g pathway are l<strong>in</strong>ked via TAG anabolism<br />
(S<strong>in</strong>gh et al., 2009).<br />
classic bacterial Fe–S cluster prote<strong>in</strong>s such as FNR and SufR require an Fe–S<br />
cluster for DNA b<strong>in</strong>d<strong>in</strong>g (Green and Paget, 2004), or b<strong>in</strong>d<strong>in</strong>g is dictated by<br />
the redox state of the Fe–S cluster (Shen et al., 2007). In a recent study, apo-<br />
WhiB b<strong>in</strong>d<strong>in</strong>g was also demonstrated for Mtb WhiB2 and a mycobacteria<br />
phage TM4 WhiB homologue, WhiBTM4 (Rybniker et al., 2010), suggest<strong>in</strong>g
REDUCTIVE STRESS IN MICROBES 95<br />
that apo-WhiB b<strong>in</strong>d<strong>in</strong>g of DNA might be a common feature of the WhiB<br />
family.<br />
Although DNA b<strong>in</strong>d<strong>in</strong>g does not provide categorical proof for transcriptional<br />
regulation, evidence from a previous study demonstrat<strong>in</strong>g that WhiB3<br />
selectively <strong>in</strong>teracts with the 4.2 doma<strong>in</strong> of the Mtb pr<strong>in</strong>cipal sigma factor,<br />
RpoV (SigA) (Steyn et al., 2002), suggests a plausible role for WhiB3 <strong>in</strong><br />
transcriptional regulation. Consistent with this observation, WhiB3 directly<br />
regulates the transcription of various lipid/polyketide biosynthetic genes<br />
<strong>in</strong>clud<strong>in</strong>g pks2 (for SL-1 production), pks3 (PAT/DAT production), fbpA<br />
(TDM biosynthesis), mas, ppsA, fadD26 and fadD28 (PDIM production) by<br />
directly b<strong>in</strong>d<strong>in</strong>g to their promoter regions (S<strong>in</strong>gh et al., 2009). However, the<br />
role of the WhiB3 4Fe–4S cluster and the oxidation state thereof <strong>in</strong> transcriptional<br />
regulation of those genes rema<strong>in</strong>s to be determ<strong>in</strong>ed.<br />
7. CONCLUDING REMARKS<br />
Development of new <strong>in</strong>tervention strategies aga<strong>in</strong>st latent TB h<strong>in</strong>ges on<br />
ga<strong>in</strong><strong>in</strong>g a substantially better understand<strong>in</strong>g of the <strong>in</strong> vivo physiology of<br />
Mtb, <strong>in</strong>clud<strong>in</strong>g the critical anabolic and catabolic pathways which allow the<br />
bacillus to persist <strong>in</strong> spite of host immune responses and/or chemotherapy.<br />
The crucial role of redox couples (e.g. NADH/NAD + , NADPH/NADP + ,<br />
FADH2/FAD, MSH/MSSM, ERG ox/ERG red) <strong>in</strong> metabolic homeostasis<br />
clearly suggests that oxidation–reduction reactions play an <strong>in</strong>dispensible role<br />
<strong>in</strong> the ma<strong>in</strong>tenance of latency. Even though the balance of such reactions can<br />
sw<strong>in</strong>g either way, research has focused primarily on oxidative stress and its<br />
counterpart, reductive stress, has largely been ignored.<br />
An emerg<strong>in</strong>g hypothesis regard<strong>in</strong>g the trigger<strong>in</strong>g events result<strong>in</strong>g <strong>in</strong> the<br />
Mtb latency response might be that of ‘reductive stress’. As stated earlier,<br />
reductive stress is def<strong>in</strong>ed as <strong>in</strong>creased ‘reduc<strong>in</strong>g power’, usually caused by<br />
an excess of high-energy reductant <strong>in</strong>clud<strong>in</strong>g NADH or NADPH, or a failure<br />
of the mechanism(s) to counter those excesses, lead<strong>in</strong>g to a reductive<br />
cytosolic environment (Ido et al., 1997). Although very little is known about<br />
reductive stress <strong>in</strong> bacterial biology, it is possibly more prevalent than<br />
oxidative stress and may also be the ma<strong>in</strong> source of ROS (Ghyczy and<br />
Boros, 2007).<br />
The phenomenon of reductive stress is well established <strong>in</strong> eukaryotic<br />
biology and its role has been demonstrated <strong>in</strong> various human diseases <strong>in</strong>clud<strong>in</strong>g<br />
diabetes and cardiomyopathy (Boucher, 2007; Zhang et al., 2010).<br />
Although the role of reductive stress <strong>in</strong> microbial-<strong>in</strong>duced diseases is <strong>in</strong>vit<strong>in</strong>g
96 AISHA FARHANA ET AL.<br />
an <strong>in</strong>creas<strong>in</strong>g research <strong>in</strong>terest, very few such studies have been reported.<br />
Nonetheless, the concept of reductive stress has provided researchers with a<br />
plethora of new research avenues that <strong>in</strong>clude the potential for development<br />
of new diagnostic and therapeutic <strong>in</strong>tervention strategies.<br />
Numerous studies have demonstrated <strong>in</strong>creased sensitivity to both oxidative<br />
and reductive stress <strong>in</strong> MSH mutants, emphasis<strong>in</strong>g the importance of<br />
MSH as the major redox buffer <strong>in</strong> mycobacteria. However, ERG <strong>in</strong> mycobacteria<br />
has not been studied for the past 50 years and warrants further<br />
<strong>in</strong>vestigation. Also, the l<strong>in</strong>k between redox homeostasis and drug efficacy <strong>in</strong><br />
mycobacteria is important, s<strong>in</strong>ce many anti-mycobacterial drugs are prodrugs<br />
that are activated upon reduction <strong>in</strong> the mycobacterial cytoplasm.<br />
This leads to the logical conclusion that mechanisms <strong>in</strong>volved <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g<br />
the <strong>in</strong>tracellular redox homeostasis of Mtb are important factors <strong>in</strong> drug<br />
resistance studies. However, the genetically <strong>in</strong>tractable nature of this pathogen<br />
and the lack of novel tools allow<strong>in</strong>g measurement of the <strong>in</strong>tracellular<br />
redox environment of Mtb hamper novel drug discovery approaches <strong>in</strong> this<br />
regard.<br />
The wealth of anabolic and catabolic <strong>in</strong>formation gathered from a diverse<br />
spectrum of non-pathogenic and pathogenic bacteria, lower eukaryotes and<br />
other model microbes has revealed numerous electron s<strong>in</strong>ks (e.g. fermentation,<br />
<strong>in</strong>tracellular lipid accumulation, Calv<strong>in</strong> cycle, rTCA cycle, hydrogenase<br />
activity, lipid anabolism, etc.) for the recycl<strong>in</strong>g of reduc<strong>in</strong>g equivalents.<br />
These recycl<strong>in</strong>g s<strong>in</strong>ks are unique for the particular environmental niche<br />
the microbe occupies. Despite some recent progress (S<strong>in</strong>gh et al., 2009), their<br />
place <strong>in</strong> Mtb physiology is poorly def<strong>in</strong>ed at present. As more is learned<br />
about the role of electron s<strong>in</strong>ks <strong>in</strong> model microbes, Mtb researchers should<br />
be compelled to revisit the exist<strong>in</strong>g paradigms for bacilli persistence, and to<br />
seek new testable hypotheses.<br />
As is clear from many historical studies, Mtb utilises carbohydrates dur<strong>in</strong>g<br />
<strong>in</strong> vitro growth without the production of acids; rather, the culture media<br />
becomes alkal<strong>in</strong>e. The latter f<strong>in</strong>d<strong>in</strong>g begs the questions of how carbohydrate<br />
metabolism by Mtb is dist<strong>in</strong>ct from other bacteria that produce acids, and<br />
what are the generated end products, if any? Data <strong>in</strong>dicat<strong>in</strong>g that lipids are<br />
the preferred <strong>in</strong> vivo carbon sources are compell<strong>in</strong>g, but are based upon<br />
<strong>in</strong>direct gene knockout <strong>in</strong>ferences and genome sequence <strong>in</strong>formation.<br />
Detailed <strong>in</strong> vivo studies us<strong>in</strong>g radiolabeled FA precursors should be undertaken.<br />
The effect of exogenous host lipid catabolism on endogenous Mtb<br />
lipid anabolism must be explored. Insights <strong>in</strong>to how these metabolic events<br />
are coord<strong>in</strong>ated to recycle reduc<strong>in</strong>g equivalents are needed. Furthermore,<br />
direct measurement of the <strong>in</strong>tracellular redox environment of Mtb dur<strong>in</strong>g<br />
<strong>in</strong> vitro growth and dur<strong>in</strong>g <strong>in</strong>fection will prove <strong>in</strong>valuable.
REDUCTIVE STRESS IN MICROBES 97<br />
The majority of <strong>in</strong>vestigations address<strong>in</strong>g the fundamental aspects of<br />
<strong>in</strong> vivo Mtb physiology (mean<strong>in</strong>g the study of Mtb isolated from <strong>in</strong>fected<br />
lung tissue) are decades old. Although they rema<strong>in</strong> remarkably perceptive,<br />
there is an urgent need to revisit those studies us<strong>in</strong>g modern molecular<br />
biological tools. Mtb is an excellent candidate for <strong>in</strong> vivo study because<br />
answers to key questions regard<strong>in</strong>g the mechanisms of persistence have<br />
not yet been obta<strong>in</strong>ed us<strong>in</strong>g <strong>in</strong> vitro model systems. In pr<strong>in</strong>ciple, the need<br />
to study <strong>in</strong> vivo-derived organisms was recognised long ago, particularly<br />
when it was elegantly revealed that the respiratory responses to a spectrum<br />
of carbohydrates and FAs were vastly different between <strong>in</strong> vitro-cultured<br />
and <strong>in</strong> vivo-isolated bacilli (Segal and Bloch, 1956). The marked response of<br />
<strong>in</strong> vivo-derived bacilli to FAs and lack of utilisation of carbohydrates suggest<br />
physiological and biochemical adaptation to widely diverse environments,<br />
changes which should be analysed by modern-day molecular tools such as<br />
DNA microarray, mass spectrometry and metabolic profil<strong>in</strong>g. Us<strong>in</strong>g gene<br />
knock-out strategies and enzyme activity assays, a systematic <strong>in</strong> vitro and<br />
<strong>in</strong> vivo analysis of the Krebs cycle components and constituents of other<br />
pathways (e.g. glycolysis) should be undertaken. 13 C-metabolic flux analysis<br />
of <strong>in</strong> vivo-isolated Mtb versus <strong>in</strong> vitro-cultured bacilli should reveal important<br />
<strong>in</strong>sights <strong>in</strong>to pathways that are specific for <strong>in</strong>fection, and perhaps<br />
persistence.<br />
As discussed, the <strong>in</strong> vivo carbon source(s) used by Mtb is important from<br />
the perspective of energy generation and the choice almost certa<strong>in</strong>ly <strong>in</strong>fluences<br />
metabolic homeostasis. The comb<strong>in</strong>ed effect of <strong>in</strong> vivo carbon substrate<br />
utilisation (most likely lipids and cholesterol) and the presence of<br />
environmental gases (e.g. NO, CO2) that <strong>in</strong>hibit respiration results <strong>in</strong> the<br />
accumulation of reduc<strong>in</strong>g equivalents that require recycl<strong>in</strong>g to ma<strong>in</strong>ta<strong>in</strong><br />
redox balance. The admittedly limited biochemical and physiological data<br />
on reductive stress <strong>in</strong> Mtb po<strong>in</strong>ts to lipid anabolism as an electron s<strong>in</strong>k for<br />
recycl<strong>in</strong>g reduc<strong>in</strong>g equivalents (S<strong>in</strong>gh et al., 2009). The latter f<strong>in</strong>d<strong>in</strong>g raises<br />
hopes for the future unveil<strong>in</strong>g of novel features of the <strong>in</strong> vivo Mtb metabolome,<br />
lipodome and redoxome. Furthermore, the potential roles of hydrogenases,<br />
CODH and the rTCA cycle <strong>in</strong> dissipat<strong>in</strong>g reductive stress <strong>in</strong> Mtb are<br />
unexplored and therefore represent new research areas <strong>in</strong> need of <strong>in</strong>vestigations.<br />
Insight <strong>in</strong>to the role of WhiB3 <strong>in</strong> the ma<strong>in</strong>tenance of redox homeostasis<br />
by divert<strong>in</strong>g reductive stress <strong>in</strong> macrophages (S<strong>in</strong>gh et al., 2009) and the<br />
observation that an extraord<strong>in</strong>ary amount of reduc<strong>in</strong>g equivalents are produced<br />
<strong>in</strong> the lungs of Mtb-<strong>in</strong>fected mice (Boshoff et al., 2008) represent<br />
start<strong>in</strong>g po<strong>in</strong>ts for other <strong>in</strong>novative avenues of research.<br />
Studies on the microenvironment of the granuloma where<strong>in</strong> Mtb resides<br />
<strong>in</strong> vivo rema<strong>in</strong> at an early stage of <strong>in</strong>vestigation, and few reports are
98 AISHA FARHANA ET AL.<br />
available. The current paradigm, which suggests that Mtb resides <strong>in</strong> dist<strong>in</strong>ct<br />
and dynamic microenvironments with<strong>in</strong> the lung (Barry et al., 2009), represents<br />
a biological predicament for the mycobacteriologist, s<strong>in</strong>ce the metabolic<br />
state of the bacilli with<strong>in</strong> each <strong>in</strong>dependent granuloma may be dissimilar<br />
from that of another. For example, the microaerophilic or anaerobic state<br />
of the particular granuloma, presence of gases, and exposure of the bacilli to<br />
a vast range of host factors that vary throughout the course of <strong>in</strong>fection,<br />
illustrate the challenges of exam<strong>in</strong><strong>in</strong>g Mtb <strong>in</strong> vivo. Creatively exploit<strong>in</strong>g<br />
genome-wide technologies that exam<strong>in</strong>e the transcriptional state and metabolic<br />
profile of Mtb with<strong>in</strong> dist<strong>in</strong>ct granulomas is essential towards identify<strong>in</strong>g<br />
new pathways and metabolites, and establish<strong>in</strong>g whether exist<strong>in</strong>g pathways<br />
are ‘active’ dur<strong>in</strong>g <strong>in</strong>fection. As more is learned about the physiology<br />
and biochemistry of Mtb, a better understand<strong>in</strong>g of TB is obta<strong>in</strong>ed which is<br />
key to the development of effective anti-mycobacterial strategies.<br />
ACKNOWLEDGEMENTS<br />
We wish to thank members of the Steyn laboratory for critical read<strong>in</strong>g of this<br />
manuscript. Research <strong>in</strong> our laboratories is supported <strong>in</strong> whole or <strong>in</strong> part, by<br />
National Institutes of Health Grants AI058131, AI076389 (to A.J.C.S.) and<br />
AI060469 (to M.K.H.) This work was also supported by the University of<br />
Alabama at Birm<strong>in</strong>gham (UAB) Center for AIDS Research, UAB Center<br />
for Free Radical Biology and UAB Center for Emerg<strong>in</strong>g Infections and<br />
Emergency Preparedness (A.J.C.S.). A.J.C.S. is a Burroughs Wellcome<br />
Investigator <strong>in</strong> the Pathogenesis of Infectious Diseases.<br />
REFERENCES<br />
Alam, K.Y. and Clark, D.P. (1989) Anaerobic fermentation balance of Escherichia<br />
coli as observed by <strong>in</strong> vivo nuclear magnetic resonance spectroscopy. J. Bacteriol.<br />
171, 6213–6217.<br />
Alam, M.S., Garg, S.K. and Agrawal, P. (2007) Molecular function of WhiB4/<br />
Rv3681c of Mycobacterium tuberculosis H37Rv: a [4Fe–4S] cluster co-ord<strong>in</strong>at<strong>in</strong>g<br />
prote<strong>in</strong> disulphide reductase. Mol. Microbiol. 63, 1414–1431.<br />
Alam, M.S., Garg, S.K. and Agrawal, P. (2009) Studies on structural and functional<br />
divergence among seven WhiB prote<strong>in</strong>s of Mycobacterium tuberculosis H37Rv.<br />
FEBS J. 276, 76–93.<br />
Alvarez-Ortega, C. and Harwood, C.S. (2007) Responses of Pseudomonas aerug<strong>in</strong>osa<br />
to low oxygen <strong>in</strong>dicate that growth <strong>in</strong> the cystic fibrosis lung is by aerobic<br />
respiration. Mol. Microbiol. 65, 153–165.
REDUCTIVE STRESS IN MICROBES 99<br />
Alvarez, H.M., Kalscheuer, R. and Ste<strong>in</strong>buchel, A. (2000) Accumulation and mobilization<br />
of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber<br />
NCIMB 40126. Appl. Microbiol. Biotechnol. 54, 218–223.<br />
Alvarez, H.M. and Ste<strong>in</strong>buchel, A. (2002) Triacylglycerols <strong>in</strong> prokaryotic microorganisms.<br />
Appl. Microbiol. Biotechnol. 60, 367–376.<br />
Aly, S., Wagner, K., Keller, C., Malm, S., Malzan, A., Brandau, S., Bange, F.C. and<br />
Ehlers, S. (2006) Oxygen status of lung granulomas <strong>in</strong> Mycobacterium tuberculosis-<strong>in</strong>fected<br />
mice. J. Pathol. 210, 298–305.<br />
Amaras<strong>in</strong>gham, C.R. and Davis, B.D. (1965) Regulation of alpha-ketoglutarate<br />
dehydrogenase formation <strong>in</strong> Escherichia coli. J. Biol. Chem. 240, 3664–3668.<br />
Anderson, R.J., Reeves, R.E., Creighton, M.M. and Lothrop, W.C. (1943) The<br />
chemistry of the lipids of tubercle bacilli, LXV. An <strong>in</strong>vestigation of tuberculous<br />
lung tissue. Am. Rev. Tuberc. 48, 65–75.<br />
Ansell, R., Granath, K., Hohmann, S., Thevele<strong>in</strong>, J.M. and Adler, L. (1997) The two<br />
isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase<br />
encoded by GPD1 and GPD2 have dist<strong>in</strong>ct roles <strong>in</strong> osmoadaptation and redox<br />
regulation. EMBO J. 16, 2179–2187.<br />
Aoshima, M. (2007) Novel enzyme reactions related to the tricarboxylic acid cycle:<br />
phylogenetic/functional implications and biotechnological applications. Appl.<br />
Microbiol. Biotechnol. 75, 249–255.<br />
Artman, M. and Bekierkunst, A. (1961) Studies on Mycobacterium tuberculosis<br />
H37RV grown <strong>in</strong> vivo. Am. Rev. Respir. Dis. 83, 100–106.<br />
Atlas, R.L. (1996) Pr<strong>in</strong>ciples of Microbiology, 2nd edn. Wm. C. Brown Publishers<br />
pp. 117.<br />
Banaiee, N., Jacobs Jr., W.R. and Ernst, J.D. (2006) Regulation of Mycobacterium<br />
tuberculosis whiB3 <strong>in</strong> the mouse lung and macrophages. Infect. Immun. 74, 6449.<br />
Barry 3rd, C.E., Boshoff, H.I., Dartois, V., Dick, T., Ehrt, S., Flynn, J., Schnapp<strong>in</strong>ger,<br />
D., Wilk<strong>in</strong>son, R.J. and Young, D. (2009) The spectrum of latent tuberculosis:<br />
reth<strong>in</strong>k<strong>in</strong>g the biology and <strong>in</strong>tervention strategies. Nat. Rev. Microbiol. 7,<br />
845–855.<br />
Baughn, A.D., Garforth, S.J., Vilcheze, C. and Jacobs Jr., W.R. (2009) An<br />
anaerobic-type alpha-ketoglutarate ferredox<strong>in</strong> oxidoreductase completes the<br />
oxidative tricarboxylic acid cycle of Mycobacterium tuberculosis. PLoS<br />
Pathog. 5, e1000662.<br />
Berney, M. and Cook, G.M. (2010) Unique flexibility <strong>in</strong> energy metabolism allows<br />
mycobacteria to combat starvation and hypoxia. PLoS One 5, e8614.<br />
Berrios-Rivera, S.J., Bennett, G.N. and San, K.Y. (2002a) The effect of <strong>in</strong>creas<strong>in</strong>g<br />
NADH availability on the redistribution of metabolic fluxes <strong>in</strong> Escherichia coli<br />
chemostat cultures. Metab. Eng. 4, 230–237.<br />
Berrios-Rivera, S.J., Bennett, G.N. and San, K.Y. (2002b) Metabolic eng<strong>in</strong>eer<strong>in</strong>g of<br />
Escherichia coli: <strong>in</strong>crease of NADH availability by overexpress<strong>in</strong>g an NAD<br />
(+)-dependent formate dehydrogenase. Metab. Eng. 4, 217–229.<br />
Berrios-Rivera, S.J., San, K.Y. and Bennett, G.N. (2002c) The effect of NAPRTase<br />
overexpression on the total levels of NAD, the NADH/NAD+ ratio, and the<br />
distribution of metabolites <strong>in</strong> Escherichia coli. Metab. Eng. 4, 238–247.<br />
Berrios-Rivera, S.J., Sanchez, A.M., Bennett, G.N. and San, K.Y. (2004) Effect of<br />
different levels of NADH availability on metabolite distribution <strong>in</strong> Escherichia<br />
coli fermentation <strong>in</strong> m<strong>in</strong>imal and complex media. Appl. Microbiol. Biotechnol. 65,<br />
426–432.
100 AISHA FARHANA ET AL.<br />
Bloch, H., Matter, E. and Suter, E. (1947) The effect of glycerol and related substances<br />
on the growth and the oxygen uptake of the tubercle bacillus. Am. Rev.<br />
Tuberc. 55, 540–551.<br />
Boon, C., Li, R., Qi, R. and Dick, T. (2001) Prote<strong>in</strong>s of Mycobacterium bovis BCG<br />
<strong>in</strong>duced <strong>in</strong> the Wayne dormancy model. J. Bacteriol. 183, 2672–2676.<br />
Borland, C., Cox, Y. and Higenbottam, T. (1993) Measurement of exhaled nitric<br />
oxide <strong>in</strong> man. Thorax 48, 1160–1162.<br />
Boshoff, H.I. and Barry 3rd, C.E. (2005) Tuberculosis – metabolism and respiration <strong>in</strong><br />
the absence of growth. Nat. Rev. Microbiol. 3, 70–80.<br />
Boshoff, H.I., Xu, X., Tahlan, K., Dowd, C.S., Pethe, K., Camacho, L.R., Park, T.H.,<br />
Yun, C.S., Schnapp<strong>in</strong>ger, D., Ehrt, S., Williams, K.J. and Barry 3rd, C.E. (2008)<br />
Biosynthesis and recycl<strong>in</strong>g of nicot<strong>in</strong>amide cofactors <strong>in</strong> Mycobacterium tuberculosis.<br />
An essential role for NAD <strong>in</strong> nonreplicat<strong>in</strong>g bacilli. J. Biol. Chem. 283,<br />
19329–19341.<br />
Boucher, B.J. (2007) Nutrition Discussion Forum. Br. J. Nutr. 80, 115–117.<br />
Brahimi-Horn, M.C. and Pouyssegur, J. (2007) Oxygen, a source of life and stress.<br />
FEBS Lett. 581, 3582–3591.<br />
Braun, R.D., Lanzen, J.L., Snyder, S.A. and Dewhirst, M.W. (2001) Comparison<br />
of tumor and normal tissue oxygen tension measurements us<strong>in</strong>g OxyLite or<br />
microelectrodes <strong>in</strong> rodents. Am. J. Physiol. Heart Circ. Physiol. 280, H2533–<br />
H2544.<br />
Brez<strong>in</strong>a, O., Drobnicova, I. and Drobnica, L. (1967) Proliferation of Mycobacterium<br />
tuberculosis from mouse lung tissue on various carbon sources. Nature 214,<br />
1036–1037.<br />
Br<strong>in</strong>dicci, C., Ito, K., Torre, O., Barnes, P.J. and Kharitonov, S.A. (2009) Effects of<br />
am<strong>in</strong>oguanid<strong>in</strong>e, an <strong>in</strong>hibitor of <strong>in</strong>ducible nitric oxide synthase, on nitric oxide<br />
production and its metabolites <strong>in</strong> healthy control subjects, healthy smokers, and<br />
COPD patients. Chest 135, 353–367.<br />
Brovkovych, V., Stolarczyk, E., Oman, J., Tomboulian, P. and Mal<strong>in</strong>ski, T. (1999)<br />
Direct electrochemical measurement of nitric oxide <strong>in</strong> vascular endothelium.<br />
J. Pharm. Biomed. Anal. 19, 135–143.<br />
Buchmeier, N.A., Newton, G.L. and Fahey, R.C. (2006) A mycothiol synthase mutant<br />
of Mycobacterium tuberculosis has an altered thiol-disulfide content and limited<br />
tolerance to stress. J. Bacteriol. 188, 6245–6252.<br />
Buchmeier, N.A., Newton, G.L., Koled<strong>in</strong>, T. and Fahey, R.C. (2003) Association of<br />
mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and<br />
antibiotics. Mol. Microbiol. 47, 1723–1732.<br />
Bunch, P.K., Mat-Jan, F., Lee, N. and Clark, D.P. (1997) The ldhA gene encod<strong>in</strong>g the<br />
fermentative lactate dehydrogenase of Escherichia coli. Microbiology 143(Pt 1);<br />
187–195.<br />
Burgdorf, T., Lenz, O., Buhrke, T., Van der L<strong>in</strong>den, E., Jones, A.K., Albracht, S.P.J.<br />
and Friedrich, B. (2005) [NiFe]-Hydrogenases of Ralstonia eutropha H16: modular<br />
enzymes for oxygen-tolerant biological hydrogen oxidation. J. Molec.<br />
Microbiol. Biotechnol. 10, 181–196.<br />
Camarasa, C., Faucet, V. and Dequ<strong>in</strong>, S. (2007) Role <strong>in</strong> anaerobiosis of the isoenzymes<br />
for Saccharomyces cerevisiae fumarate reductase encoded by OSM1 and<br />
FRDS1. Yeast 24, 391–401.<br />
Cammack, R., Frey, M. and Robson, R. (2001) Hydrogen as Fuel. Learn<strong>in</strong>g from<br />
Nature. Taylor & Francis, London.
REDUCTIVE STRESS IN MICROBES 101<br />
Campbell, J.W. and Cronan Jr., J.E. (2002) The enigmatic Escherichia coli fadE gene<br />
is yafH. J. Bacteriol. 184, 3759–3764.<br />
Cevallos, M.A., Encarnacion, S., Leija, A., Mora, Y. and Mora, J. (1996) Genetic and<br />
physiological characterization of a Rhizobium etli mutant stra<strong>in</strong> unable to synthesize<br />
poly-beta-hydroxybutyrate. J. Bacteriol. 178, 1646–1654.<br />
Chan, E.D., Chan, J. and Schluger, N.W. (2001) What is the role of nitric oxide <strong>in</strong><br />
mur<strong>in</strong>e and human host defense aga<strong>in</strong>st tuberculosis? Current knowledge. Am. J.<br />
Respir. Cell Mol. Biol. 25, 606–612.<br />
Chater, K.F. (1972) A morphological and genetic mapp<strong>in</strong>g study of white colony<br />
mutants of Streptomyces coelicolor. J. Gen. Microbiol. 72, 9–28.<br />
Chung, S.W., Hall, S.R. and Perrella, M.A. (2009) Role of haem oxygenase-1 <strong>in</strong><br />
microbial host defence. Cell. Microbiol. 11, 199–207.<br />
Clark, D.P. (1989) The fermentation pathways of Escherichia coli. FEMS Microbiol.<br />
Rev. 5, 223–234.<br />
Clough, G.F., Bennett, A.R. and Church, M.K. (1998) Measurement of nitric oxide<br />
concentration <strong>in</strong> human sk<strong>in</strong> <strong>in</strong> vivo us<strong>in</strong>g dermal microdialysis. Exp. Physiol. 83,<br />
431–434.<br />
Cole, J. (1996) Nitrate reduction to ammonia by enteric bacteria: redundancy, or a<br />
strategy for survival dur<strong>in</strong>g oxygen starvation? FEMS Microbiol. Lett. 136, 1–11.<br />
Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.<br />
V., Eiglmeier, K., Gas, S., Barry 3rd, C.E., Tekaia, F., Badcock, K., Basham, D.,<br />
Brown, D., Chill<strong>in</strong>gworth, T., Connor, R., Davies, R., Devl<strong>in</strong>, K., Feltwell, T.,<br />
Gentles, S., Haml<strong>in</strong>, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean,<br />
J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M.A., Rajandream, M.A.,<br />
Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J.E.,<br />
Taylor, K., Whitehead, S. and Barrell, B.G. (1998) Decipher<strong>in</strong>g the biology of<br />
Mycobacterium tuberculosis from the complete genome sequence. Nature 393,<br />
537–544.<br />
Converse, P.J., Karakousis, P.C., Kl<strong>in</strong>kenberg, L.G., Kesavan, A.K., Ly, L.H., Allen,<br />
S.S., Grosset, J.H., Ja<strong>in</strong>, S.K., Lamichhane, G. and Manabe, Y.C. (2009) Role of<br />
the dosR-dosS two-component regulatory system <strong>in</strong> Mycobacterium tuberculosis<br />
virulence <strong>in</strong> three animal models. Infect. Immun. 77, 1230.<br />
Corper, H.J. and Cohn, M.L. (1933) The viability and virulence of old cultures of<br />
tubercle bacilli: studies on twelve-year broth cultures ma<strong>in</strong>ta<strong>in</strong>ed at <strong>in</strong>cubator<br />
temperature. Am. Rev. Tuberc. Pulm. Dis. 28, 856–874.<br />
Cosma, C.L., Sherman, D.R. and Ramakrishnan, L. (2003) The secret lives of the<br />
pathogenic mycobacteria. Annu. Rev. Microbiol. 57, 641–676.<br />
Costa, K.C., Wong, P.M., Wang, T., Lie, T.J., Dodsworth, J.A., Swanson, I., Burn,<br />
J.A., Hackett, M. and Leigh, J.A. (2010) Prote<strong>in</strong> complex<strong>in</strong>g <strong>in</strong> a methanogen<br />
suggests electron bifurcation and electron delivery from formate to heterodisulfide<br />
reductase. Proc. Natl. Acad. Sci. 107(24); 11050–11055.<br />
Cox, J.S., Chen, B., McNeil, M. and Jacobs Jr., W.R. (1999) Complex lipid determ<strong>in</strong>es<br />
tissue-specific replication of Mycobacterium tuberculosis <strong>in</strong> mice. Nature 402,<br />
79–83.<br />
da Silva, S.M., Venceslau, S.S., Fernandes, C.L.V., Valente, F.M.A. and Pereira, I.A.<br />
C. (2008) Hydrogen as an energy source for the human pathogen Bilophila wadsworthia.<br />
Antonie Leeuwenhoek 93, 381–390.<br />
Daniel, J., Deb, C., Dubey, V.S., Sirakova, T.D., Abomoelak, B., Morbidoni, H.R.<br />
and Kolattukudy, P.E. (2004) Induction of a novel class of diacylglycerol
102 AISHA FARHANA ET AL.<br />
acyltransferases and triacylglycerol accumulation <strong>in</strong> Mycobacterium tuberculosis<br />
as it goes <strong>in</strong>to a dormancy-like state <strong>in</strong> culture. J. Bacteriol. 186, 5017–5030.<br />
Darrouzet, E. and Daldal, F. (2003) Prote<strong>in</strong>-prote<strong>in</strong> <strong>in</strong>teractions between<br />
Cytochrome b and the Fe–S prote<strong>in</strong> subunits dur<strong>in</strong>g QH2 oxidation and largescale<br />
doma<strong>in</strong> movement <strong>in</strong> the bc1 complex. Biochemistry 42, 1499–1507.<br />
Dasgupta, N., Kapur, V., S<strong>in</strong>gh, K.K., Das, T.K., Sachdeva, S., Jyothisri, K. and<br />
Tyagi, J.S. (2000) Characterization of a two-component system, devR-devS,<br />
of Mycobacterium tuberculosis. Tuberc. Lung Dis. 80, 141–159.<br />
Davidge, K.S., Sangu<strong>in</strong>etti, G., Yee, C.H., Cox, A.G., McLeod, C.W., Monk, C.E.,<br />
Mann, B.E., Motterl<strong>in</strong>i, R. and Poole, R.K. (2009) Carbon monoxide-releas<strong>in</strong>g<br />
antibacterial molecules target respiration and global transcriptional regulators.<br />
J. Biol. Chem. 284, 4516–4524.<br />
Davis, N.K. and Chater, K.F. (1992) The Streptomyces coelicolor whiB gene encodes<br />
a small transcription factor-like prote<strong>in</strong> dispensable for growth but essential for<br />
sporulation. Mol. Gen. Genet. 232, 351–358.<br />
de Graef, M.R., Alexeeva, S., Snoep, J.L. and Teixeira de Mattos, M.J. (1999) The<br />
steady-state <strong>in</strong>ternal redox state (NADH/NAD) reflects the external redox state<br />
and is correlated with catabolic adaptation <strong>in</strong> Escherichia coli. J. Bacteriol. 181,<br />
2351–2357.<br />
Deb, C., Daniel, J., Sirakova, T.D., Abomoelak, B., Dubey, V.S. and Kolattukudy,<br />
P.E. (2006) A novel lipase belong<strong>in</strong>g to the hormone-sensitive lipase family<br />
<strong>in</strong>duced under starvation to utilize stored triacylglycerol <strong>in</strong> Mycobacterium tuberculosis.<br />
J. Biol. Chem. 281, 3866–3875.<br />
den Hengst, C.D. and Buttner, M.J. (2008) Redox control <strong>in</strong> act<strong>in</strong>obacteria. Biochim.<br />
Biophys. Acta 1780, 1201–1216.<br />
Deppenmeier, U. (2002) The unique biochemistry of methanogenesis. Prog. Nucleic<br />
Acid Res. Mol. Biol. 71, 223–283.<br />
Deppenmeier, U. (2004) The membrane-bound electron transport system of<br />
Methanosarc<strong>in</strong>a species. J. Bioenerg. Biomembr. 36, 55–64.<br />
Dietrich, L.E., Price-Whelan, A., Petersen, A., Whiteley, M. and Newman, D.K.<br />
(2006) The phenaz<strong>in</strong>e pyocyan<strong>in</strong> is a term<strong>in</strong>al signall<strong>in</strong>g factor <strong>in</strong> the quorum<br />
sens<strong>in</strong>g network of Pseudomonas aerug<strong>in</strong>osa. Mol. Microbiol. 61, 1308–1321.<br />
Dietrich, L.E., Teal, T.K., Price-Whelan, A. and Newman, D.K. (2008) Redox-active<br />
antibiotics control gene expression and community behavior <strong>in</strong> divergent bacteria.<br />
Science 321, 1203–1206.<br />
Dimmeler, S. and Zeiher, A.M. (2007) A ‘reductionist’ view of cardiomyopathy. Cell<br />
130, 401–402.<br />
Duan, X., Yang, J., Ren, B., Tan, G. and D<strong>in</strong>g, H. (2009) Reactivity of nitric oxide<br />
with the [4Fe–4S] cluster of dihydroxyacid dehydratase from Escherichia coli.<br />
Biochem. J. 417, 783–789.<br />
Dubos, R.J. (1953) Effect of the composition of the gaseous and aqueous environments<br />
on the survival of tubercle bacilli <strong>in</strong> vitro. J. Exp. Med. 97, 357.<br />
Duncan, K. (2004) Identification and validation of novel drug targets <strong>in</strong> tuberculosis.<br />
Curr. Pharm. Des. 10, 3185–3194.<br />
Dweik, R.A. (2005) Nitric oxide, hypoxia, and superoxide: the good, the bad, and the<br />
ugly! Thorax 60, 265–267.<br />
Dweik, R.A., Comhair, S.A., Gaston, B., Thunnissen, F.B., Farver, C., Thomassen,<br />
M.J., Kavuru, M., Hammel, J., Abu-Soud, H.M. and Erzurum, S.C. (2001) NO
REDUCTIVE STRESS IN MICROBES 103<br />
chemical events <strong>in</strong> the human airway dur<strong>in</strong>g the immediate and late antigen<strong>in</strong>duced<br />
asthmatic response. Proc. Natl. Acad. Sci. USA 98, 2622–2627.<br />
Dweik, R.A., Laskowski, D., Abu-Soud, H.M., Kaneko, F., Hutte, R., Stuehr, D.J.<br />
and Erzurum, S.C. (1998) Nitric oxide synthesis <strong>in</strong> the lung. Regulation by oxygen<br />
through a k<strong>in</strong>etic mechanism. J. Cl<strong>in</strong>. Invest. 101, 660–666.<br />
Edson, N.L. (1951) The <strong>in</strong>termediary metabolism of the mycobacteria. Microbiol.<br />
Molec. Biol. Rev. 15, 147.<br />
Ell<strong>in</strong>gton, M.J., Bhakoo, K.K., Sawers, G., Richardson, D.J. and Ferguson, S.J. (2002)<br />
Hierarchy of carbon source selection <strong>in</strong> Paracoccus pantotrophus: strict correlation<br />
between reduction state of the carbon substrate and aerobic expression of the<br />
nap operon. J. Bacteriol. 184, 4767–4774.<br />
Encarnacion, S., Dunn, M., Willms, K. and Mora, J. (1995) Fermentative and aerobic<br />
metabolism <strong>in</strong> Rhizobium etli. J. Bacteriol. 177, 3058–3066.<br />
Enomoto, K., Arikawa, Y. and Muratsubaki, H. (2002) Physiological role of soluble<br />
fumarate reductase <strong>in</strong> redox balanc<strong>in</strong>g dur<strong>in</strong>g anaerobiosis <strong>in</strong> Saccharomyces<br />
cerevisiae. FEMS Microbiol. Lett. 215, 103–108.<br />
Fauci, A.S. (2008) Multidrug-resistant and extensively drug-resistant tuberculosis:<br />
the National Institute of Allergy and Infectious Diseases Research agenda and<br />
recommendations for priority research. J. Infect. Dis. 197, 1493–1498.<br />
Feng, Z., Caceres, N.E., Sarath, G. and Barletta, R.G. (2002) Mycobacterium<br />
smegmatisL-alan<strong>in</strong>e dehydrogenase (Ald) is required for proficient utilization of<br />
alan<strong>in</strong>e as a sole nitrogen source and susta<strong>in</strong>ed anaerobic growth. J. Bacteriol. 184,<br />
5001–5010.<br />
Ferry, J.G. (1995) CO dehydrogenase. Annu. Rev. Microbiol. 49, 305–333.<br />
Fitzpatrick, A.M., Brown, L.A., Holgu<strong>in</strong>, F. and Teague, W.G. (2009) Levels of nitric<br />
oxide oxidation products are <strong>in</strong>creased <strong>in</strong> the epithelial l<strong>in</strong><strong>in</strong>g fluid of children with<br />
persistent asthma. J. Allergy Cl<strong>in</strong>. Immunol. 124, 990–996 e1-9.<br />
Flardh, K., F<strong>in</strong>dlay, K.C. and Chater, K.F. (1999) Association of early sporulation<br />
genes with suggested developmental decision po<strong>in</strong>ts <strong>in</strong> Streptomyces coelicolor A3<br />
(2). Microbiology 145, 2229.<br />
Florczyk, M.A., McCue, L.A., Purkayastha, A., Currenti, E., Wol<strong>in</strong>, M.J. and<br />
McDonough, K.A. (2003) A family of acr-coregulated Mycobacterium tuberculosis<br />
genes shares a common DNA motif and requires Rv3133c (dosR or devR) for<br />
expression. Infect. Immun. 71, 5332–5343.<br />
Fontan, P., Aris, V., Ghanny, S., Soteropoulos, P. and Smith, I. (2008) Global transcriptional<br />
profile of Mycobacterium tuberculosis dur<strong>in</strong>g THP-1 human macrophage<br />
<strong>in</strong>fection. Infect. Immun. 76, 717–725.<br />
Fowler, A.V., Camien, M.N. and Dunn, M.S. (1960) Extracellular acids produced by<br />
Mycobacterium ranae and Mycobacterium tuberculosis H37Rv. J. Biol. Chem. 235,<br />
1386–1389.<br />
Fowler, A.V., Camien, M.N. and Dunn, M.S. (1961) Acetyl L-isoleuc<strong>in</strong>e and acetyl<br />
L-leuc<strong>in</strong>e as extracellular products of Mycobacterium ranae. J. Bacteriol. 81, 163.<br />
Friedheim, E. (1931) Pyocyan<strong>in</strong>e, an accessory respiratory pigment. J. Exp. Med. 54,<br />
207–221.<br />
Friedheim, E. and Michaelis, L. (1931) Potentiometric study of the pyocyan<strong>in</strong>e.<br />
J. Biol. Chem. 91, 355–368.<br />
Gandhi, N.R., Moll, A., Sturm, A.W., Paw<strong>in</strong>ski, R., Govender, T., Lalloo, U., Zeller,<br />
K., Andrews, J. and Friedland, G. (2006) Extensively drug-resistant tuberculosis
104 AISHA FARHANA ET AL.<br />
as a cause of death <strong>in</strong> patients co-<strong>in</strong>fected with tuberculosis and HIV <strong>in</strong> a rural area<br />
of South Africa. Lancet 368, 1575–1580.<br />
Garg, S., Alam, M.S., Bajpai, R., Kishan, K.V. and Agrawal, P. (2009) Redox biology<br />
of Mycobacterium tuberculosis H 37 Rv: prote<strong>in</strong>-prote<strong>in</strong> <strong>in</strong>teraction between GlgB<br />
and WhiB 1 <strong>in</strong>volves exchange of thiol-disulfide. BMC Biochem. 10, 1.<br />
Garg, S.K., Suhail Alam, M., Soni, V., Radha Kishan, K.V. and Agrawal, P. (2007)<br />
Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a prote<strong>in</strong> disulfide<br />
reductase. Prote<strong>in</strong> Expr. Purif. 52, 422–432.<br />
Garrido, E.O. and Grant, C.M. (2002) Role of thioredox<strong>in</strong>s <strong>in</strong> the response of<br />
Saccharomyces cerevisiae to oxidative stress <strong>in</strong>duced by hydroperoxides. Mol.<br />
Microbiol. 43, 993–1003.<br />
Garton, N.J., Waddell, S.J., Sherratt, A.L., Lee, S.M., Smith, R.J., Senner, C., H<strong>in</strong>ds,<br />
J., Rajakumar, K., Adegbola, R.A. and Besra, G.S. (2008) Cytological and transcript<br />
analyses reveal fat and lazy persister-like bacilli <strong>in</strong> tuberculous sputum.<br />
PLoS Med. 5, e75.<br />
Gasch, A.P., Spellman, P.T., Kao, C.M., Carmel-Harel, O., Eisen, M.B., Storz, G.,<br />
Botste<strong>in</strong>, D. and Brown, P.O. (2000) Genomic expression programs <strong>in</strong> the<br />
response of yeast cells to environmental changes. Molec. Biol. Cell 11, 4241.<br />
Geertman, J.M., van Dijken, J.P. and Pronk, J.T. (2006) Eng<strong>in</strong>eer<strong>in</strong>g NADH metabolism<br />
<strong>in</strong> Saccharomyces cerevisiae: formate as an electron donor for glycerol<br />
production by anaerobic, glucose-limited chemostat cultures. FEMS Yeast Res.<br />
6, 1193–1203.<br />
Geiman, D.E., Raghunand, T.R., Agarwal, N. and Bishai, W.R. (2006) Differential<br />
gene expression <strong>in</strong> response to exposure to antimycobacterial agents and other<br />
stress conditions among seven Mycobacterium tuberculosis whiB-like genes.<br />
Antimicrob. Agents Chemother. 50, 2836.<br />
Gengenbacher, M., Rao, S.P., Pethe, K. and Dick, T. (2010) Nutrient-starved, nonreplicat<strong>in</strong>g<br />
Mycobacterium tuberculosis requires respiration, ATP synthase and<br />
isocitrate lyase for ma<strong>in</strong>tenance of ATP homeostasis and viability. Microbiology<br />
156, 81–87.<br />
Genghof, D.S. and Van Damme, O. (1964) Biosynthesis of ergothione<strong>in</strong>e and hercyn<strong>in</strong>e<br />
by Mycobacteria. J. Bacteriol. 87, 852–862.<br />
Genghof, D.S. and Van Damme, O. (1968) Biosynthesis of ergothione<strong>in</strong>e from<br />
endogenous hercyn<strong>in</strong>e <strong>in</strong> Mycobacterium smegmatis. J. Bacteriol. 95, 340–344.<br />
Ghyczy, M. and Boros, M. (2007) Electrophilic methyl groups present <strong>in</strong> the diet<br />
ameliorate pathological states <strong>in</strong>duced by reductive and oxidative stress: a hypothesis.<br />
Br. J. Nutr. 85, 409–414.<br />
Gomez, J.E. and Bishai, W.R. (2000) whmD is an essential mycobacterial gene<br />
required for proper septation and cell division. Proc. Natl. Acad. Sci. USA 97, 8554.<br />
Gomez, J.E. and McK<strong>in</strong>ney, J.D. (2004) M. tuberculosis persistence, latency, and<br />
drug tolerance. Tuberculosis 84, 29–44.<br />
Gomez, L.M., Anaya, J.M., Vilchez, J.R., Cadena, J., H<strong>in</strong>ojosa, R., Velez, L.,<br />
Lopez-Nevot, M.A. and Mart<strong>in</strong>, J. (2007) A polymorphism <strong>in</strong> the <strong>in</strong>ducible<br />
nitric oxide synthase gene is associated with tuberculosis. Tuberculosis<br />
(Ed<strong>in</strong>b.) 87, 288–294.<br />
Goodw<strong>in</strong>, M.B., Boshoff, H.I. and Barry, C.E. (2006) Quantification of small molecule<br />
organic acids from Mycobacterium tuberculosis culture supernatant us<strong>in</strong>g ion<br />
exclusion liquid chromatography/mass spectrometry. Rapid Commun. Mass<br />
Spectrom. 20, 3345–3350.
REDUCTIVE STRESS IN MICROBES 105<br />
Gores, G.J., Flarsheim, C.E., Dawson, T.L., Niem<strong>in</strong>en, A.L., Herman, B. and<br />
Lemasters, J.J. (1989) Swell<strong>in</strong>g, reductive stress, and cell death dur<strong>in</strong>g chemical<br />
hypoxia <strong>in</strong> hepatocytes. Am. J. Physiol. Cell Physiol. 257, C347.<br />
Grant, C.M., Perrone, G. and Dawes, I.W. (1998) Glutathione and catalase provide<br />
overlapp<strong>in</strong>g defenses for protection aga<strong>in</strong>st hydrogen peroxide <strong>in</strong> the yeast<br />
Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 253, 893–898.<br />
Grasemann, H., Ioannidis, I., Tomkiewicz, R.P., De Groot, H., Rub<strong>in</strong>, B.K. and<br />
Ratjen, F. (1998) Nitric oxide metabolites <strong>in</strong> cystic fibrosis lung disease. Arch.<br />
Dis. Childhood 78, 49.<br />
Gray, C.T., Wimpenny, J.W. and Mossman, M.R. (1966) Regulation of metabolism <strong>in</strong><br />
facultative bacteria. II. Effects of aerobiosis, anaerobiosis and nutrition on the<br />
formation of Krebs cycle enzymes <strong>in</strong> Escherichia coli. Biochim. Biophys. Acta 117,<br />
33–41.<br />
Green, J. and Paget, M.S. (2004) Bacterial redox sensors. Nat. Rev. Microbiol. 2,<br />
954–966.<br />
Guest, J.R. and Russell, G.C. (1992) Complexes and complexities of the citric acid<br />
cycle <strong>in</strong> Escherichia coli. Curr. Top Cell. Regul. 33, 231–247.<br />
Gustafsson, L.E., Leone, A.M., Persson, M.G., Wiklund, N.P. and Moncada, S. (1991)<br />
Endogenous nitric oxide is present <strong>in</strong> the exhaled air of rabbits, gu<strong>in</strong>ea pigs and<br />
humans. Biochem. Biophys. Res. Commun. 181, 852–857.<br />
Haapanen, J.H., Kass, I., Gens<strong>in</strong>i, G. and Middlebrook, G. (1959) Studies on the<br />
gaseous content of tuberculous cavities. Am. Rev. Respir. Dis. 80, 1.<br />
Halliwell, B. and Gutterridge, J.M.C. (2008) Free Radicals <strong>in</strong> Biology and Medic<strong>in</strong>e,<br />
4th edn. Oxford University Press, pp. 30-78.<br />
Hashimoto, S. and Katsumata, R. (1999) Mechanism of alan<strong>in</strong>e hyperproduction by<br />
Arthrobacter oxydans HAP-1: metabolic shift to fermentation under nongrowth<br />
aerobic conditions. Appl. Environ. Microbiol. 65, 2781.<br />
Hazleton, E.B. (1923) Carbon monoxide a predispos<strong>in</strong>g cause of pulmonary tuberculosis.<br />
Br. Med. J. ii, 763–764.<br />
Hernandez, M.E., Kappler, A. and Newman, D.K. (2004) Phenaz<strong>in</strong>es and other<br />
redox-active antibiotics promote microbial m<strong>in</strong>eral reduction. Appl. Environ.<br />
Microbiol. 70, 921–928.<br />
Hernandez, M.E. and Newman, D.K. (2001) Extracellular electron transfer. Cell Mol.<br />
Life Sci. 58, 1562–1571.<br />
Hett, E.C. and Rub<strong>in</strong>, E.J. (2008) Bacterial growth and cell division: a mycobacterial<br />
perspective. Microbiol. Mol. Biol. Rev. 72, 126–156 table of contents.<br />
Honaker, R.W., Leistikow, R.L., Bartek, I.L. and Voskuil, M.I. (2009) Unique roles<br />
of DosT and DosS <strong>in</strong> DosR regulon <strong>in</strong>duction and Mycobacterium tuberculosis<br />
dormancy. Infect. Immun. 77, 3258.<br />
Honaker, R.W., Stewart, A., Schittone, S., Izzo, A., Kle<strong>in</strong>, M.R. and Voskuil, M.I.<br />
(2008) Mycobacterium bovis BCG vacc<strong>in</strong>e stra<strong>in</strong>s lack narK2 and narX <strong>in</strong>duction<br />
and exhibit altered phenotypes dur<strong>in</strong>g dormancy. Infect. Immun. 76, 2587–2593.<br />
Huet, G., Casta<strong>in</strong>g, J.P., Fournier, D., Daffe, M. and Saves, I. (2006) Prote<strong>in</strong> splic<strong>in</strong>g<br />
of SufB is crucial for the functionality of the Mycobacterium tuberculosis SUF<br />
mach<strong>in</strong>ery. J. Bacteriol. 188, 3412.<br />
Huet, G., Daffe, M. and Saves, I. (2005) Identification of the Mycobacterium tuberculosis<br />
SUF mach<strong>in</strong>ery as the exclusive mycobacterial system of [Fe–S] cluster<br />
assembly: evidence for its implication <strong>in</strong> the pathogen’s survival. J. Bacteriol. 187,<br />
6137.
106 AISHA FARHANA ET AL.<br />
Hugler, M., Wirsen, C.O., Fuchs, G., Taylor, C.D. and Sievert, S.M. (2005)<br />
Evidence for autotrophic CO2 fixation via the reductive tricarboxylic acid cycle<br />
by members of the epsilon subdivision of proteobacteria. J. Bacteriol. 187,<br />
3020–3027.<br />
Hunter, G.J.E. (1953) Acid formation <strong>in</strong> cultures of Mycobacterium butyricum.<br />
J. Bacteriol. 66, 676.<br />
Husa<strong>in</strong>, M., Bourret, T.J., McCollister, B.D., Jones-Carson, J., Laughl<strong>in</strong>, J. and<br />
Vazquez-Torres, A. (2008) Nitric oxide evokes an adaptive response to oxidative<br />
stress by arrest<strong>in</strong>g respiration. J. Biol. Chem. 283, 7682–7689.<br />
Hutter, B. and Dick, T. (1998) Increased alan<strong>in</strong>e dehydrogenase activity dur<strong>in</strong>g<br />
dormancy <strong>in</strong> Mycobacterium smegmatis. FEMS Microbiol. Lett. 167, 7–11.<br />
Hwang, C., S<strong>in</strong>skey, A.J. and Lodish, H.F. (1992) Oxidized redox state of glutathione<br />
<strong>in</strong> the endoplasmic reticulum. Science 257, 1496–1502.<br />
Ido, Y. (2007) Pyrid<strong>in</strong>e nucleotide redox abnormalities <strong>in</strong> diabetes. Antioxid. Redox<br />
Signal. 9, 931–942.<br />
Ido, Y., Kilo, C. and Williamson, J.R. (1997) Cytosolic NADH/NAD+, free radicals,<br />
and vascular dysfunction <strong>in</strong> early diabetes mellitus. Diabetologia 40, 115–117.<br />
Ikeda, T., Yamamoto, M., Arai, H., Ohmori, D., Ishii, M. and Igarashi, Y. (2010)<br />
Enzymatic and electron paramagnetic resonance studies of anabolic pyruvate<br />
synthesis by pyruvate:ferredox<strong>in</strong> oxidoreductase from Hydrogenobacter thermophilus.<br />
FEBS J. 277, 501–510.<br />
Imlay, J.A. (2006) Iron-sulphur clusters and the problem with oxygen. Mol.<br />
Microbiol. 59, 1073–1082.<br />
Imlay, J.A. (2008) Cellular defenses aga<strong>in</strong>st superoxide and hydrogen peroxide.<br />
Annu. Rev. Biochem. 77, 755–776.<br />
Ioanoviciu, A., Yukl, E.T., Moenne-Loccoz, P. and de Montellano, P.R. (2007) DevS,<br />
a heme-conta<strong>in</strong><strong>in</strong>g two-component oxygen sensor of Mycobacterium tuberculosis.<br />
Biochemistry 46, 4250–4260.<br />
Ja<strong>in</strong>, M., Petzold, C.J., Schelle, M.W., Leavell, M.D., Mougous, J.D., Bertozzi, C.R.,<br />
Leary, J.A. and Cox, J.S. (2007) Lipidomics reveals control of Mycobacterium<br />
tuberculosis virulence lipids via metabolic coupl<strong>in</strong>g. Proc. Natl. Acad. Sci. USA<br />
104, 5133–5138.<br />
Jakimowicz, P., Cheesman, M.R., Bishai, W.R., Chater, K.F., Thomson, A.J. and<br />
Buttner, M.J. (2005) Evidence that the Streptomyces developmental prote<strong>in</strong><br />
WhiD, a member of the WhiB family, b<strong>in</strong>ds a [4Fe–4S] cluster. J. Biol. Chem.<br />
280, 8309–8315.<br />
Jenney Jr., F.E. and Adams, M.W.W. (2008) Hydrogenases of the model hyperthermophiles.<br />
Ann. NY Acad. Sci. 1125, 252–266.<br />
Jeoung, J.H. and Dobbek, H. (2007) Carbon dioxide activation at the Ni, Fe-cluster of<br />
anaerobic carbon monoxide dehydrogenase. Science 318, 1461–1464.<br />
Kaufmann, S.H., Hussey, G. and Lambert, P.H. (2010) New vacc<strong>in</strong>es for tuberculosis.<br />
Lancet 375, 2110–2119.<br />
Kelemen, G.H., Brian, P., Flardh, K., Chamberl<strong>in</strong>, L., Chater, K.F. and Buttner,<br />
M.J. (1998) Developmental regulation of transcription of whiE, a locus specify<strong>in</strong>g<br />
the polyketide spore pigment <strong>in</strong> Streptomyces coelicolor A3 (2).<br />
J. Bacteriol. 180, 2515.<br />
Khan, S. and O’Brien, P.J. (1995) Modulat<strong>in</strong>g hypoxia-<strong>in</strong>duced hepatocyte <strong>in</strong>jury by<br />
affect<strong>in</strong>g <strong>in</strong>tracellular redox state. Biochim. Biophys. Acta (BBA) Molec. Cell Res.<br />
1269, 153–161.
REDUCTIVE STRESS IN MICROBES 107<br />
Kim, T.H., Park, J.S., Kim, H.J., Kim, Y., Kim, P. and Lee, H.S. (2005) The whcE gene<br />
of Corynebacterium glutamicum is important for survival follow<strong>in</strong>g heat and oxidative<br />
stress. Biochem. Biophys. Res. Commun. 337, 757–764.<br />
K<strong>in</strong>g, G.M. (2003) Molecular and culture-based analyses of aerobic carbon monoxide<br />
oxidizer diversity. Appl. Environ. Microbiol. 69, 7257–7265.<br />
K<strong>in</strong>ger, A.K. and Tyagi, J.S. (1993) Identification and clon<strong>in</strong>g of genes differentially<br />
expressed <strong>in</strong> the virulent stra<strong>in</strong> of Mycobacterium tuberculosis. Gene 131, 113–117.<br />
Kleman, G.L. and Strohl, W.R. (1994) Acetate metabolism by Escherichia coli <strong>in</strong><br />
high-cell-density fermentation. Appl. Environ. Microbiol. 60, 3952–3958.<br />
Ko, Y.F., Bentley, W.E. and Weigand, W.A. (1993) An <strong>in</strong>tegrated metabolic model<strong>in</strong>g<br />
approach to describe the energy efficiency of Escherichia coli fermentations<br />
under oxygen-limited conditions: cellular energetics, carbon flux, and acetate<br />
production. Biotechnol. Bioeng. 42, 843–853.<br />
Koled<strong>in</strong>, T., Newton, G.L. and Fahey, R.C. (2002) Identification of the mycothiol<br />
synthase gene (mshD) encod<strong>in</strong>g the acetyltransferase produc<strong>in</strong>g mycothiol <strong>in</strong><br />
act<strong>in</strong>omycetes. Arch. Microbiol. 178, 331–337.<br />
Kumar, A., Deshane, J.S., Crossman, D.K., Bolisetty, S., Yan, B.S., Kramnik, I.,<br />
Agarwal, A. and Steyn, A.J. (2008) Heme oxygenase-1-derived carbon monoxide<br />
<strong>in</strong>duces the Mycobacterium tuberculosis dormancy regulon. J. Biol. Chem. 283,<br />
18032–18039.<br />
Kumar, A., Toledo, J.C., Patel, R.P., Lancaster Jr., J.R. and Steyn, A.J. (2007)<br />
Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor.<br />
Proc. Natl. Acad. Sci. USA 104, 11568–11573.<br />
Legan, S.K., Rebr<strong>in</strong>, I., Mockett, R.J., Radyuk, S.N., Klichko, V.I., Sohal, R.S. and<br />
Orr, W.C. (2008) Overexpression of glucose-6-phosphate dehydrogenase extends<br />
the life span of Drosophila melanogaster. J. Biol. Chem. 283, 32492.<br />
Leistikow, R.L., Morton, R.A., Bartek, I.L., Frimpong, I., Wagner, K. and Voskuil,<br />
M.I. (2010) The Mycobacterium tuberculosis DosR regulon assists <strong>in</strong> metabolic<br />
homeostasis and enables rapid recovery from nonrespir<strong>in</strong>g dormancy. J. Bacteriol.<br />
192, 1662.<br />
Leone, A.M., Furst, V.W., Foxwell, N.A., Cellek, S. and Moncada, S. (1996)<br />
Visualisation of nitric oxide generated by activated mur<strong>in</strong>e macrophages.<br />
Biochem. Biophys. Res. Commun. 221, 37–41.<br />
Levanon, S.S., San, K.Y. and Bennett, G.N. (2005) Effect of oxygen on the<br />
Escherichia coli ArcA and FNR regulation systems and metabolic responses.<br />
Biotechnol. Bioeng. 89, 556–564.<br />
Levitt, M.D. (1969) Production and excretion of hydrogen gas <strong>in</strong> man. N. Engl. J.<br />
Med. 281, 122–127.<br />
L<strong>in</strong>, H., Bennett, G.N. and San, K.Y. (2005) Effect of carbon sources differ<strong>in</strong>g <strong>in</strong><br />
oxidation state and transport route on succ<strong>in</strong>ate production <strong>in</strong> metabolically eng<strong>in</strong>eered<br />
Escherichia coli. J. Ind. Microbiol. Biotechnol. 32, 87–93.<br />
L<strong>in</strong>nane, S.J., Keat<strong>in</strong>gs, V.M., Costello, C.M., Moynihan, J.B., O’Connor, C.M.,<br />
Fitzgerald, M.X. and McLoughl<strong>in</strong>, P. (1998) Total sputum nitrate plus nitrite is<br />
raised dur<strong>in</strong>g acute pulmonary <strong>in</strong>fection <strong>in</strong> cystic fibrosis. Am. J. Respir. Crit. Care<br />
Med. 158, 207.<br />
Ma, K., Schicho, R.N., Kelly, R.M. and Adams, M.W. (1993) Hydrogenase of the<br />
hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase:<br />
evidence for a sulfur-reduc<strong>in</strong>g hydrogenase ancestor. Proc. Natl. Acad.<br />
Sci. USA 90, 5341.
108 AISHA FARHANA ET AL.<br />
Ma, Z., Lienhardt, C., McIlleron, H., Nunn, A.J. and Wang, X. (2010) Global tuberculosis<br />
drug development pipel<strong>in</strong>e: the need and the reality. Lancet 375,<br />
2100–2109.<br />
MacMick<strong>in</strong>g, J.D., North, R.J., LaCourse, R., Mudgett, J.S., Shah, S.K. and Nathan,<br />
C.F. (1997) Identification of nitric oxide synthase as a protective locus aga<strong>in</strong>st<br />
tuberculosis. Proc. Natl. Acad. Sci. USA 94, 5243–5248.<br />
Maier, R.J., Olson, J. and Olczak, A. (2003) Hydrogen-oxidiz<strong>in</strong>g capabilities of<br />
Helicobacter hepaticus and <strong>in</strong> vivo availability of the substrate. J. Bacteriol. 185,<br />
2680.<br />
Malhotra, V., Sharma, D., Ramanathan, V.D., Shakila, H., Sa<strong>in</strong>i, D.K., Chakravorty,<br />
S., Das, T.K., Li, Q., Silver, R.F., Narayanan, P.R. and Tyagi, J.S. (2004)<br />
Disruption of response regulator gene, devR, leads to attenuation <strong>in</strong> virulence<br />
of Mycobacterium tuberculosis. FEMS Microbiol. Lett. 231, 237–245.<br />
Marteus, H., T€ornberg, D.C., Weitzberg, E., Sched<strong>in</strong>, U. and Alv<strong>in</strong>g, K. (2005) Orig<strong>in</strong><br />
of nitrite and nitrate <strong>in</strong> nasal and exhaled breath condensate and relation to nitric<br />
oxide formation. Br. Med. J. 60, 219.<br />
Mavrodi, D.V., Blankenfeldt, W. and Thomashow, L.S. (2006) Phenaz<strong>in</strong>e compounds<br />
<strong>in</strong> fluorescent Pseudomonas spp. biosynthesis and regulation. Annu. Rev.<br />
Phytopathol. 44, 417–445.<br />
Mayuri, Bagchi, G., Das, T.K. and Tyagi, J.S. (2002) Molecular analysis of the<br />
dormancy response <strong>in</strong> Mycobacterium smegmatis: expression analysis of genes<br />
encod<strong>in</strong>g the DevR-DevS two-component system, Rv3134c and chaperone<br />
alpha-crystall<strong>in</strong> homologues. FEMS Microbiol. Lett. 211, 231–237.<br />
McK<strong>in</strong>lay, J.B. and Harwood, C.S. (2010) Carbon dioxide fixation as a central<br />
redox cofactor recycl<strong>in</strong>g mechanism <strong>in</strong> bacteria. Proc. Natl. Acad. Sci. 107,<br />
11669.<br />
McK<strong>in</strong>ney, J.D., Honer zu Bentrup, K., Munoz-Elias, E.J., Miczak, A., Chen, B.,<br />
Chan, W.T., Swenson, D., Sacchett<strong>in</strong>i, J.C., Jacobs Jr., W.R. and Russell,<br />
D.G. (2000) Persistence of Mycobacterium tuberculosis <strong>in</strong> macrophages<br />
and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406,<br />
735–738.<br />
Med<strong>in</strong>a, V.G., Almer<strong>in</strong>g, M.J.H., van Maris, A.J.A. and Pronk, J.T. (2010)<br />
Elim<strong>in</strong>ation of glycerol production <strong>in</strong> anaerobic cultures of a Saccharomyces<br />
cerevisiae stra<strong>in</strong> eng<strong>in</strong>eered to use acetic acid as an electron acceptor. Appl.<br />
Environ. Microbiol. 76, 190.<br />
Medlar, E.M. (1948) The pathogenesis of m<strong>in</strong>imal pulmonary tuberculosis: a study<br />
of 1,225 necropsies <strong>in</strong> cases of sudden and unexpected death. Am. Rev. Tuberc.<br />
58, 583.<br />
Meena, L.S and Rajni (2010) Survival mechanisms of pathogenic Mycobacterium<br />
tuberculosis H37Rv. FEBS J. 277, 2416–2427.<br />
Mentel, M., Ahuja, E.G., Mavrodi, D.V., Bre<strong>in</strong>bauer, R., Thomashow, L.S. and<br />
Blankenfeldt, W. (2009) Of two make one: the biosynthesis of phenaz<strong>in</strong>es.<br />
Chembiochem 10, 2295–2304.<br />
Merrill, M.H. (1930) Carbohydrate metabolism of organisms of the genus<br />
Mycobacterium. J. Bacteriol. 20, 235.<br />
Meyer, J. (2007) [FeFe] hydrogenases and their evolution: a genomic perspective.<br />
Cell. Molec. Life Sci. 64, 1063–1084.<br />
Meyer, O. and Schlegel, H.G. (1983) Biology of aerobic carbon monoxide-oxidiz<strong>in</strong>g<br />
bacteria. Annu. Rev. Microbiol. 37, 277–310.
REDUCTIVE STRESS IN MICROBES 109<br />
Miller, C.C., Rawat, M., Johnson, T. and Av-Gay, Y. (2007) Innate protection of<br />
Mycobacterium smegmatis aga<strong>in</strong>st the antimicrobial activity of nitric oxide is<br />
provided by mycothiol. Antimicrob. Agents Chemother. 51, 3364–3366.<br />
Mori, T., Kosaka, K. and Tanaka, Y. (1971) Tricarboxylic acid cycle <strong>in</strong> M. lepraemurium.<br />
Int. J. Lepr. Other Mycobact. Dis. 39, 796–812.<br />
Morozk<strong>in</strong>a, E.V. and Zvyagilskaya, R.A. (2007) Nitrate reductases: structure, functions,<br />
and effect of stress factors. Biochemistry (Moscow) 72, 1151–1160.<br />
Morris, R.P., Nguyen, L., Gatfield, J., Visconti, K., Nguyen, K., Schnapp<strong>in</strong>ger, D.,<br />
Ehrt, S., Liu, Y., Heifets, L. and Pieters, J. (2005) Ancestral antibiotic resistance <strong>in</strong><br />
Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 102, 12200.<br />
Muller, E.G. (1996) A glutathione reductase mutant of yeast accumulates high levels<br />
of oxidized glutathione and requires thioredox<strong>in</strong> for growth. Mol. Biol. Cell 7,<br />
1805–1813.<br />
Munoz-Elias, E.J. and McK<strong>in</strong>ney, J.D. (2005) Mycobacterium tuberculosis isocitrate<br />
lyases 1 and 2 are jo<strong>in</strong>tly required for <strong>in</strong> vivo growth and virulence. Nat. Med. 11,<br />
638–644.<br />
Munoz-Elias, E.J., Upton, A.M., Cherian, J. and McK<strong>in</strong>ney, J.D. (2006) Role of the<br />
methylcitrate cycle <strong>in</strong> Mycobacterium tuberculosis metabolism, <strong>in</strong>tracellular<br />
growth, and virulence. Mol. Microbiol. 60, 1109–1122.<br />
Murray, J.F. (2010) The structure and function of the lung. Int. J. Tuberc. Lung Dis.<br />
14, 391–396.<br />
Murthy, P.S., Sirsi, M. and Ramakrishnan, T. (1962) Tricarboxylic acid cycle and<br />
related enzymes <strong>in</strong> cell-free extracts of Mycobacterium tuberculosis H37Rv.<br />
Biochem. J. 84, 263–269.<br />
Muttucumaru, D.G., Roberts, G., H<strong>in</strong>ds, J., Stabler, R.A. and Parish, T. (2004) Gene<br />
expression profile of Mycobacterium tuberculosis <strong>in</strong> a non-replicat<strong>in</strong>g state.<br />
Tuberculosis (Ed<strong>in</strong>b.) 84, 239–246.<br />
Nandi, R. and Sengupta, S. (1998) <strong>Microbial</strong> production of hydrogen: an overview.<br />
Crit. Rev. Microbiol. 24, 61–84.<br />
Nathan, C. and Shiloh, M.U. (2000) Reactive oxygen and nitrogen <strong>in</strong>termediates <strong>in</strong><br />
the relationship between mammalian hosts and microbial pathogens. Proc. Natl.<br />
Acad. Sci. USA 97, 8841–8848.<br />
Neubauer, H. and Gotz, F. (1996) <strong>Physiology</strong> and <strong>in</strong>teraction of nitrate and nitrite<br />
reduction <strong>in</strong> Staphylococcus carnosus. J. Bacteriol. 178, 2005.<br />
Newton, G.L., Av-Gay, Y. and Fahey, R.C. (2000a) N-Acetyl-1-D-myo-<strong>in</strong>osityl-2am<strong>in</strong>o-2-deoxy-alpha-D-glucopyranoside<br />
deacetylase (MshB) is a key enzyme <strong>in</strong><br />
mycothiol biosynthesis. J. Bacteriol. 182, 6958–6963.<br />
Newton, G.L., Av-Gay, Y. and Fahey, R.C. (2000b) A novel mycothiol-dependent<br />
detoxification pathway <strong>in</strong> mycobacteria <strong>in</strong>volv<strong>in</strong>g mycothiol S-conjugate amidase.<br />
Biochemistry 39, 10739–10746.<br />
Newton, G.L., Bewley, C.A., Dwyer, T.J., Horn, R., Aharonowitz, Y., Cohen, G.,<br />
Davies, J., Faulkner, D.J. and Fahey, R.C. (1995) The structure of U17 isolated<br />
from Streptomyces clavuligerus and its properties as an antioxidant thiol. Eur. J.<br />
Biochem. 230, 821–825.<br />
Newton, G.L., Buchmeier, N. and Fahey, R.C. (2008) Biosynthesis and functions of<br />
mycothiol, the unique protective thiol of Act<strong>in</strong>obacteria. Microbiol. Mol. Biol.<br />
Rev. 72, 471–494.<br />
Newton, G.L. and Fahey, R.C. (2002) Mycothiol biochemistry. Arch. Microbiol. 178,<br />
388–394.
110 AISHA FARHANA ET AL.<br />
Newton, G.L., Koled<strong>in</strong>, T., Gorovitz, B., Rawat, M., Fahey, R.C. and Av-Gay, Y.<br />
(2003) The glycosyltransferase gene encod<strong>in</strong>g the enzyme catalyz<strong>in</strong>g the first step<br />
of mycothiol biosynthesis (mshA). J. Bacteriol. 185, 3476–3479.<br />
Newton, G.L., Ta, P., Bzymek, K.P. and Fahey, R.C. (2006) Biochemistry of the <strong>in</strong>itial<br />
steps of mycothiol biosynthesis. J. Biol. Chem. 281, 33910–33920.<br />
Newton, G.L., Ta, P. and Fahey, R.C. (2005) A mycothiol synthase mutant of<br />
Mycobacterium smegmatis produces novel thiols and has an altered thiol redox<br />
status. J. Bacteriol. 187, 7309–7316.<br />
Newton, G.L., Unson, M.D., Anderberg, S.J., Aguilera, J.A., Oh, N.N., delCardayre,<br />
S.B., Av-Gay, Y. and Fahey, R.C. (1999) Characterization of Mycobacterium<br />
smegmatis mutants defective <strong>in</strong> 1-D-myo-<strong>in</strong>osityl-2-am<strong>in</strong>o-2-deoxy-alpha-D-glucopyranoside<br />
and mycothiol biosynthesis. Biochem. Biophys. Res. Commun. 255,<br />
239–244.<br />
Nicholson, S., Bonec<strong>in</strong>i-Almeida Mda, G., Lapa e Silva, J.R., Nathan, C., Xie, Q.W.,<br />
Mumford, R., Weidner, J.R., Calaycay, J., Geng, J., Boechat, N., L<strong>in</strong>hares, C.,<br />
Rom, W. and Ho, J.L. (1996) Inducible nitric oxide synthase <strong>in</strong> pulmonary alveolar<br />
macrophages from patients with tuberculosis. J. Exp. Med. 183, 2293–2302.<br />
Nissen, T.L., Hamann, C.W., Kielland-Brandt, M.C., Nielsen, J. and Villadsen, J.<br />
(2000) Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae<br />
mutants impaired <strong>in</strong> glycerol synthesis. Yeast 16, 463–474.<br />
Oelgeschlager, E. and Rother, M. (2008) Carbon monoxide-dependent energy<br />
metabolism <strong>in</strong> anaerobic bacteria and archaea. Arch. Microbiol. 190, 257–269.<br />
Og<strong>in</strong>o, T., Arata, Y. and Fujiwara, S. (1980) Proton correlation nuclear magnetic<br />
resonance study of metabolic regulations and pyruvate transport <strong>in</strong> anaerobic<br />
Escherichia coli cells. Biochemistry 19, 3684–3691.<br />
Ohno, H., Zhu, G., Mohan, V.P., Chu, D., Kohno, S., Jacobs Jr., W.R. and Chan, J.<br />
(2003) The effects of reactive nitrogen <strong>in</strong>termediates on gene expression <strong>in</strong><br />
Mycobacterium tuberculosis. Cell. Microbiol. 5, 637–648.<br />
Ohsawa, I., Ishikawa, M., Takahashi, K., Watanabe, M., Nishimaki, K., Yamagata, K.,<br />
Katsura, K., Katayama, Y., Asoh, S. and Ohta, S. (2007) Hydrogen acts as a<br />
therapeutic antioxidant by selectively reduc<strong>in</strong>g cytotoxic oxygen radicals. Nat.<br />
Med. 13, 688–694.<br />
Olson, J.W. and Maier, R.J. (2002) Molecular hydrogen as an energy source for<br />
Helicobacter pylori. Science 298, 1788.<br />
Page, W.J. and Knosp, O. (1989) Hyperproduction of poly-beta-hydroxybutyrate<br />
dur<strong>in</strong>g exponential growth of Azotobacter v<strong>in</strong>elandii UWD. Appl. Environ.<br />
Microbiol. 55, 1334–1339.<br />
Paige, C. and Bishai, W.R. (2010) Penitentiary or penthouse condo: the tuberculous<br />
granuloma from the microbe’s po<strong>in</strong>t of view. Cell. Microbiol. 12, 301–309.<br />
Pamplona, A., Ferreira, A., Balla, J., Jeney, V., Balla, G., Epiphanio, S., Chora, A.,<br />
Rodrigues, C.D., Gregoire, I.P., Cunha-Rodrigues, M., Portugal, S., Soares, M.P.<br />
and Mota, M.M. (2007) Heme oxygenase-1 and carbon monoxide suppress the<br />
pathogenesis of experimental cerebral malaria. Nat. Med. 13, 703–710.<br />
Parish, T., Smith, D.A., Kendall, S., Casali, N., Bancroft, G.J. and Stoker, N.G. (2003)<br />
Deletion of two-component regulatory systems <strong>in</strong>creases the virulence of<br />
Mycobacterium tuberculosis. Infect. Immun. 71, 1134–1140.<br />
Park, S.W., Hwang, E.H., Park, H., Kim, J.A., Heo, J., Lee, K.H., Song, T., Kim, E.,<br />
Ro, Y.T., Kim, S.W. and Kim, Y.M. (2003) Growth of mycobacteria on carbon<br />
monoxide and methanol. J. Bacteriol. 185, 142–147.
REDUCTIVE STRESS IN MICROBES 111<br />
Park, S.W., Song, T., Kim, S.Y., Kim, E., Oh, J.I., Eom, C.Y. and Kim, Y.M. (2007)<br />
Carbon monoxide dehydrogenase <strong>in</strong> mycobacteria possesses a nitric oxide dehydrogenase<br />
activity. Biochem. Biophys. Res. Commun. 362, 449–453.<br />
Patel, M.P. and Blanchard, J.S. (1998) Synthesis of des-myo-<strong>in</strong>ositol mycothiol and<br />
demonstration of a mycobacterial specific reductase activity. J. Am. Chem. Soc.<br />
120, 11538–11539.<br />
Patel, M.P. and Blanchard, J.S. (1999) Expression, purification, and characterization<br />
of Mycobacterium tuberculosis mycothione reductase. Biochemistry 38,<br />
11827–11833.<br />
Pfenn<strong>in</strong>ger-Li, X.D. and Dimroth, P. (1992) NADH formation by Na(+)-coupled<br />
reversed electron transfer <strong>in</strong> Klebsiella pneumoniae. Mol. Microbiol. 6, 1943–1948.<br />
Pillay, M. and Sturm, A.W. (2007) Evolution of the extensively drug-resistant F15/<br />
LAM4/KZN stra<strong>in</strong> of Mycobacterium tuberculosis <strong>in</strong> KwaZulu-Natal, South<br />
Africa. Cl<strong>in</strong>. Infect. Dis. 45, 1409–1414.<br />
Price-Whelan, A., Dietrich, L.E. and Newman, D.K. (2006) Reth<strong>in</strong>k<strong>in</strong>g ‘secondary’<br />
metabolism: physiological roles for phenaz<strong>in</strong>e antibiotics. Nat. Chem. Biol. 2,<br />
71–78.<br />
Price-Whelan, A., Dietrich, L.E. and Newman, D.K. (2007) Pyocyan<strong>in</strong> alters redox<br />
homeostasis and carbon flux through central metabolic pathways <strong>in</strong> Pseudomonas<br />
aerug<strong>in</strong>osa PA14. J. Bacteriol. 189, 6372–6381.<br />
Rachman, H., Strong, M., Ulrichs, T., Grode, L., Schuchhardt, J., Mollenkopf, H.,<br />
Kosmiadi, G.A., Eisenberg, D. and Kaufmann, S.H.E. (2006) Unique transcriptome<br />
signature of Mycobacterium tuberculosis <strong>in</strong> pulmonary tuberculosis. Infect.<br />
Immun. 74, 1233.<br />
Ragsdale, S.W. (2004) Life with carbon monoxide. Crit. Rev. Biochem. Mol. Biol. 39,<br />
165–195.<br />
Rajasekaran, N.S., Connell, P., Christians, E.S., Yan, L.J., Taylor, R.P., Orosz, A.,<br />
Zhang, X.Q., Stevenson, T.J., Peshock, R.M., Leopold, J.A., Barry, W.H.,<br />
Loscalzo, J., Odelberg, S.J. and Benjam<strong>in</strong>, I.J. (2007) Human alpha B-crystall<strong>in</strong><br />
mutation causes oxido-reductive stress and prote<strong>in</strong> aggregation cardiomyopathy<br />
<strong>in</strong> mice. Cell 130, 427–439.<br />
Ramakrishnan, T., Murthy, P.S. and Gop<strong>in</strong>athan, K.P. (1972) Intermediary metabolism<br />
of mycobacteria. Bacteriol. Rev. 36, 65–108.<br />
Rand, J.D. and Grant, C.M. (2006) The thioredox<strong>in</strong> system protects ribosomes<br />
aga<strong>in</strong>st stress-<strong>in</strong>duced aggregation. Molec. Biol. Cell 17, 387.<br />
Rao, S.P., Alonso, S., Rand, L., Dick, T. and Pethe, K. (2008) The protonmotive<br />
force is required for ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g ATP homeostasis and viability of hypoxic,<br />
nonreplicat<strong>in</strong>g Mycobacterium tuberculosis. Proc.Natl.Acad.Sci.USA105,<br />
11945–11950.<br />
Rasmussen, K.N. (1957) The apical localization of pulmonary tuberculosis. Acta<br />
Tuberc. Scand. 34, 245.<br />
Rawat, M., Johnson, C., Cadiz, V. and Av-Gay, Y. (2007) Comparative analysis of<br />
mutants <strong>in</strong> the mycothiol biosynthesis pathway <strong>in</strong> Mycobacterium smegmatis.<br />
Biochem. Biophys. Res. Commun. 363, 71–76.<br />
Rawat, M., Kovacevic, S., Billman-Jacobe, H. and Av-Gay, Y. (2003) Inactivation of<br />
mshB, a key gene <strong>in</strong> the mycothiol biosynthesis pathway <strong>in</strong> Mycobacterium smegmatis.<br />
Microbiology 149, 1341–1349.<br />
Rawat, M., Newton, G.L., Ko, M., Mart<strong>in</strong>ez, G.J., Fahey, R.C. and Av-Gay, Y. (2002)<br />
Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to
112 AISHA FARHANA ET AL.<br />
alkylat<strong>in</strong>g agents, free radicals, and antibiotics. Antimicrob. Agents Chemother. 46,<br />
3348–3355.<br />
Reed, M.B., Gagneux, S., DeRiemer, K., Small, P.M. and Barry III, C.E. (2007) The<br />
W-Beij<strong>in</strong>g l<strong>in</strong>eage of Mycobacterium tuberculosis overproduces triglycerides and<br />
has the DosR dormancy regulon constitutively upregulated. J. Bacteriol. 189, 2583.<br />
Rich, A.R. and Follis Jr., R.H. (1942) The effect of low oxygen tension upon the<br />
development of experimental tuberculosis. Bull. Johns Hopk<strong>in</strong>s Hosp. 71,<br />
345–363.<br />
Richardson, A.R., Libby, S.J. and Fang, F.C. (2008) A nitric oxide-<strong>in</strong>ducible lactate<br />
dehydrogenase enables Staphylococcus aureus to resist <strong>in</strong>nate immunity. Science<br />
319, 1672–1676.<br />
Richardson, D.J. (2000) Bacterial respiration: a flexible process for a chang<strong>in</strong>g environment.<br />
Microbiology 146(Pt 3); 551–571.<br />
Richardson, D.J., Berks, B.C., Russell, D.A., Spiro, S. and Taylor, C.J. (2001)<br />
Functional, biochemical and genetic diversity of prokaryotic nitrate reductases.<br />
Cell. Molec. Life Sci. 58, 165–178.<br />
Rietveld, P., Arscott, L.D., Berry, A., Scrutton, N.S., Deonara<strong>in</strong>, M.P., Perham, R.N.<br />
and Williams Jr., C.H. (1994) Reductive and oxidative half-reactions of glutathione<br />
reductase from Escherichia coli. Biochemistry 33, 13888–13895.<br />
Riley, R.L. (1957) Aerial dissem<strong>in</strong>ation of pulmonary tuberculosis. Am. Rev. Tuberc.<br />
Pulm. Dis. 76, 931–941.<br />
Ritz, D., Patel, H., Doan, B., Zheng, M., Aslund, F., Storz, G. and Beckwith, J. (2000)<br />
Thioredox<strong>in</strong> 2 is <strong>in</strong>volved <strong>in</strong> the oxidative stress response <strong>in</strong> Escherichia coli.<br />
J. Biol. Chem. 275, 2505–2512.<br />
Roberts, D.M., Liao, R.P., Wisedchaisri, G., Hol, W.G. and Sherman, D.R. (2004)<br />
Two sensor k<strong>in</strong>ases contribute to the hypoxic response of Mycobacterium tuberculosis.<br />
J. Biol. Chem. 279, 23082–23087.<br />
Rustad, T.R., Harrell, M.I., Liao, R. and Sherman, D.R. (2008) The endur<strong>in</strong>g hypoxic<br />
response of Mycobacterium tuberculosis. PLoS One 3, e1502.<br />
Rustad, T.R., Sherrid, A.M., M<strong>in</strong>ch, K.J. and Sherman, D.R. (2009) Hypoxia: a<br />
w<strong>in</strong>dow <strong>in</strong>to Mycobacterium tuberculosis latency. Cell. Microbiol. 11, 1151–1159.<br />
Ryan, G.J., Hoff, D.R., Driver, E.R., Voskuil, M.I., Gonzalez-Juarrero, M., Basaraba,<br />
R.J., Crick, D.C., Spencer, J.S. and Lenaerts, A.J. (2010) Multiple M. tuberculosis<br />
phenotypes <strong>in</strong> mouse and gu<strong>in</strong>ea pig lung tissue revealed by a dual-sta<strong>in</strong><strong>in</strong>g<br />
approach. PLoS One 5, e11108.<br />
Rybniker, J., Nowag, A., van Gumpel, E., Nissen, N., Rob<strong>in</strong>son, N., Plum, G. and<br />
Hartmann, P. (2010) Insights <strong>in</strong>to the function of the WhiB-like prote<strong>in</strong> of mycobacteriophage<br />
TM4 – a transcriptional <strong>in</strong>hibitor of WhiB2. Mol. Microbiol. 77,<br />
642–657.<br />
Ryter, S.W. and Choi, A.M. (2009) Heme oxygenase-1/carbon monoxide: from<br />
metabolism to molecular therapy. Am. J. Respir. Cell Mol. Biol. 41, 251–260.<br />
Sacchett<strong>in</strong>i, J.C., Rub<strong>in</strong>, E.J. and Freundlich, J.S. (2008) Drugs versus bugs: <strong>in</strong> pursuit<br />
of the persistent predator Mycobacterium tuberculosis. Nat. Rev. Microbiol. 6,<br />
41–52.<br />
Sa<strong>in</strong>i, D.K., Malhotra, V. and Tyagi, J.S. (2004) Cross talk between DevS sensor<br />
k<strong>in</strong>ase homologue, Rv2027c, and DevR response regulator of Mycobacterium<br />
tuberculosis. FEBS Lett. 565, 75–80.<br />
San, K.Y., Bennett, G.N., Berrios-Rivera, S.J., Vadali, R.V., Yang, Y.T., Horton, E.,<br />
Rudolph, F.B., Sariyar, B. and Blackwood, K. (2002) Metabolic eng<strong>in</strong>eer<strong>in</strong>g
REDUCTIVE STRESS IN MICROBES 113<br />
through cofactor manipulation and its effects on metabolic flux redistribution <strong>in</strong><br />
Escherichia coli. Metab. Eng. 4, 182–192.<br />
Sanchez, A.M., Bennett, G.N. and San, K.Y. (2005) Effect of different levels of<br />
NADH availability on metabolic fluxes of Escherichia coli chemostat cultures <strong>in</strong><br />
def<strong>in</strong>ed medium. J. Biotechnol. 117, 395–405.<br />
Sareen, D., Newton, G.L., Fahey, R.C. and Buchmeier, N.A. (2003) Mycothiol is<br />
essential for growth of Mycobacterium tuberculosis Erdman. J. Bacteriol. 185,<br />
6736–6740.<br />
Sareen, D., Steffek, M., Newton, G.L. and Fahey, R.C. (2002) ATP-dependent<br />
L-cyste<strong>in</strong>e:1D-myo-<strong>in</strong>osityl 2-am<strong>in</strong>o-2-deoxy-alpha-D-glucopyranoside ligase,<br />
mycothiol biosynthesis enzyme MshC, is related to class I cyste<strong>in</strong>yl-tRNA synthetases.<br />
Biochemistry 41, 6885–6890.<br />
Schafer, F.Q. and Buettner, G.R. (2001) Redox environment of the cell as viewed<br />
through the redox state of the glutathione disulfide/glutathione couple. Free Radic.<br />
Biol. Med. 30, 1191–1212.<br />
Schut, G.J., Bridger, S.L. and Adams, M.W. (2007) Insights <strong>in</strong>to the metabolism of<br />
elemental sulfur by the hyperthermophilic archaeon Pyrococcus furiosus: characterization<br />
of a coenzyme A-dependent NAD(P)H sulfur oxidoreductase.<br />
J. Bacteriol. 189, 4431–4441.<br />
Sears, H.J., Sawers, G., Berks, B.C., Ferguson, S.J. and Richardson, D.J. (2000)<br />
Control of periplasmic nitrate reductase gene expression (napEDABC) from<br />
Paracoccus pantotrophus <strong>in</strong> response to oxygen and carbon substrates.<br />
Microbiology 146(Pt 11); 2977–2985.<br />
Segal, W. (1962) Differential succ<strong>in</strong>ic dehydrogenase activity of <strong>in</strong>tact cells of<br />
Mycobacterium tuberculosis grown <strong>in</strong> vitro and <strong>in</strong> vivo. Bacteriol. Proc. 62, 96.<br />
Segal, W. (1965) Comparative study of Mycobacterium grown <strong>in</strong> vivo and <strong>in</strong> vitro. V.<br />
Differences <strong>in</strong> sta<strong>in</strong><strong>in</strong>g properties. Am. Rev. Respir. Dis. 91, 285.<br />
Segal, W. (1984) Growth Dynamics of the In Vivo and In Vitro Grown Mycobacterial<br />
Pathogens. Marcel Dekker, Inc., New York.<br />
Segal, W. and Bloch, H. (1956) Biochemical differentiation of Mycobacterium tuberculosis<br />
grown <strong>in</strong> vivo and <strong>in</strong> vitro. J. Bacteriol. 72, 132.<br />
Seifritz, C., Daniel, S.L., Gossner, A. and Drake, H.L. (1993) Nitrate as a preferred<br />
electron s<strong>in</strong>k for the acetogen Clostridium thermoaceticum. J. Bacteriol. 175,<br />
8008.<br />
Senior, P.J. and Dawes, E.A. (1973) The regulation of poly-beta-hydroxybutyrate<br />
metabolism <strong>in</strong> Azotobacter beijer<strong>in</strong>ckii. Biochem. J. 134, 225–238.<br />
Seravalli, J. and Ragsdale, S.W. (2008) 13 C NMR characterization of an exchange<br />
reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase.<br />
Biochemistry 47, 6770–6781.<br />
Shalel-Levanon, S., San, K.Y. and Bennett, G.N. (2005) Effect of ArcA and FNR on<br />
the expression of genes related to the oxygen regulation and the glycolysis pathway<br />
<strong>in</strong> Escherichia coli under microaerobic growth conditions. Biotechnol. Bioeng.<br />
92, 147–159.<br />
Sheehan, H.L. and Whitwell, F. (1949) The sta<strong>in</strong><strong>in</strong>g of tubercle bacilli with Sudan<br />
black B. J. Pathol. Bacteriol. 61, 269–271 pl.<br />
Shen, G., Balasubramanian, R., Wang, T., Wu, Y., Hoffart, L.M., Krebs, C., Bryant,<br />
D.A. and Golbeck, J.H. (2007) SufR coord<strong>in</strong>ates two [4Fe–4S]2+, 1+ clusters and<br />
functions as a transcriptional repressor of the sufBCDS operon and an autoregulator<br />
of sufR <strong>in</strong> cyanobacteria. J. Biol. Chem. 282, 31909–31919.
114 AISHA FARHANA ET AL.<br />
Sherman, D.R., Voskuil, M., Schnapp<strong>in</strong>ger, D., Liao, R., Harrell, M.I. and Schoolnik,<br />
G.K. (2001) Regulation of the Mycobacterium tuberculosis hypoxic response gene<br />
encod<strong>in</strong>g -crystall<strong>in</strong>. Proc. Natl. Acad. Sci. 98, 7534.<br />
Shi, L., Sohaskey, C.D., Kana, B.D., Dawes, S., North, R.J., Mizrahi, V. and Gennaro,<br />
M.L. (2005) Changes <strong>in</strong> energy metabolism of Mycobacterium tuberculosis <strong>in</strong><br />
mouse lung and under <strong>in</strong> vitro conditions affect<strong>in</strong>g aerobic respiration. Proc.<br />
Natl. Acad. Sci. USA 102, 15629.<br />
Shiloh, M.U., Manzanillo, P. and Cox, J.S. (2008) Mycobacterium tuberculosis senses<br />
host-derived carbon monoxide dur<strong>in</strong>g macrophage <strong>in</strong>fection. Cell Host Microbe 3,<br />
323–330.<br />
Shimizu, M., Fujii, T., Masuo, S. and Takaya, N. (2010) Mechanism of de novo<br />
branched-cha<strong>in</strong> am<strong>in</strong>o acid synthesis as an alternative electron s<strong>in</strong>k <strong>in</strong> hypoxic<br />
Aspergillus nidulans cells. Appl. Environ. Microbiol. 76, 1507–1515.<br />
S<strong>in</strong>gh, A., Crossman, D.K., Mai, D., Guidry, L., Voskuil, M.I., Renfrow, M.B. and<br />
Steyn, A.J. (2009) Mycobacterium tuberculosis WhiB3 ma<strong>in</strong>ta<strong>in</strong>s redox homeostasis<br />
by regulat<strong>in</strong>g virulence lipid anabolism to modulate macrophage response.<br />
PLoS Pathog. 5, e1000545.<br />
S<strong>in</strong>gh, A., Guidry, L., Narasimhulu, K.V., Mai, D., Trombley, J., Redd<strong>in</strong>g, K.E.,<br />
Giles, G.I., Lancaster, J.R. and Steyn, A.J.C. (2007) Mycobacterium tuberculosis<br />
WhiB3 responds to O2 and nitric oxide via its [4Fe–4S] cluster and is essential for<br />
nutrient starvation survival. Proc. Natl. Acad. Sci. 104, 11562.<br />
Sohaskey, C.D. (2005) Regulation of nitrate reductase activity <strong>in</strong> Mycobacterium<br />
tuberculosis by oxygen and nitric oxide. Microbiology 151, 3803–3810.<br />
Sohaskey, C.D. (2008) Nitrate enhances the survival of Mycobacterium tuberculosis<br />
dur<strong>in</strong>g <strong>in</strong>hibition of respiration. J. Bacteriol. 190, 2981–2986.<br />
Sohaskey, C.D. and Wayne, L.G. (2003) Role of narK2X and narGHJI <strong>in</strong> hypoxic<br />
upregulation of nitrate reduction by Mycobacterium tuberculosis. J. Bacteriol. 185,<br />
7247–7256.<br />
Song, T., Park, S.W., Park, S.J., Kim, J.H., Yu, J.Y., Oh, J.I. and Kim, Y.M. (2010)<br />
Clon<strong>in</strong>g and expression analysis of the duplicated genes for carbon monoxide<br />
dehydrogenase of Mycobacterium sp. stra<strong>in</strong> JC1 DSM 3803. Microbiology 156,<br />
999–1008.<br />
Sousa, E.H., Tuckerman, J.R., Gonzalez, G. and Gilles-Gonzalez, M.A. (2007) DosT<br />
and DevS are oxygen-switched k<strong>in</strong>ases <strong>in</strong> Mycobacterium tuberculosis. Prote<strong>in</strong> Sci.<br />
16, 1708–1719.<br />
Spencer, M.E. and Guest, J.R. (1987) Regulation of citric acid cycle genes <strong>in</strong> facultative<br />
bacteria. Microbiol. Sci. 4, 164–168.<br />
Sr<strong>in</strong>ivasan, V. and Morowitz, H.J. (2006) Ancient genes <strong>in</strong> contemporary persistent<br />
microbial pathogens. Biol. Bull. 210, 1–9.<br />
Srivastava, V., Ja<strong>in</strong>, A., Srivastava, B.S. and Srivastava, R. (2008) Selection of genes<br />
of Mycobacterium tuberculosis upregulated dur<strong>in</strong>g residence <strong>in</strong> lungs of <strong>in</strong>fected<br />
mice. Tuberculosis (Ed<strong>in</strong>b.) 88, 171–177.<br />
Srivastava, V., Rouanet, C., Srivastava, R., Ramal<strong>in</strong>gam, B., Locht, C. and Srivastava,<br />
B.S. (2007) Macrophage-specific Mycobacterium tuberculosis genes: identification<br />
by green fluorescent prote<strong>in</strong> and kanamyc<strong>in</strong> resistance selection. Microbiology<br />
153, 659–666.<br />
Staubli, A. and Boelsterli, U.A. (1998) The labile iron pool <strong>in</strong> hepatocytes: prooxidant-<strong>in</strong>duced<br />
<strong>in</strong>crease <strong>in</strong> free iron precedes oxidative cell <strong>in</strong>jury. Am. J. Physiol.<br />
Gastro<strong>in</strong>test. Liver Physiol. 274, 1031.
REDUCTIVE STRESS IN MICROBES 115<br />
Steuber, J., Krebs, W., Bott, M. and Dimroth, P. (1999) A membrane-bound NAD (P)<br />
+-reduc<strong>in</strong>g hydrogenase provides reduced pyrid<strong>in</strong>e nucleotides dur<strong>in</strong>g citrate<br />
fermentation by Klebsiella pneumoniae. J. Bacteriol. 181, 241.<br />
Steyn, A.J., Coll<strong>in</strong>s, D.M., Hondalus, M.K., Jacobs Jr., W.R., Kawakami, R.P. and<br />
Bloom, B.R. (2002) Mycobacterium tuberculosis WhiB3 <strong>in</strong>teracts with RpoV to<br />
affect host survival but is dispensable for <strong>in</strong> vivo growth. Proc. Natl. Acad. Sci.<br />
USA 99, 3147–3152.<br />
Talaat, A.M., Lyons, R., Howard, S.T. and Johnston, S.A. (2004) The temporal<br />
expression profile of Mycobacterium tuberculosis <strong>in</strong>fection <strong>in</strong> mice. Proc. Natl.<br />
Acad. Sci. USA 101, 4602.<br />
Talaat, A.M., Ward, S.K., Wu, C.W., Rondon, E., Tavano, C., Bannant<strong>in</strong>e, J.P.,<br />
Lyons, R. and Johnston, S.A. (2007) Mycobacterial bacilli are metabolically active<br />
dur<strong>in</strong>g chronic tuberculosis <strong>in</strong> mur<strong>in</strong>e lungs: <strong>in</strong>sights from genome-wide transcriptional<br />
profil<strong>in</strong>g. J. Bacteriol. 189, 4265.<br />
Tepper, R. and Varma, K.G. (1972) Metabolic activity of purified suspensions of<br />
Mycobacterium lepraemurium. J. Gen. Microbiol. 73, 143–152.<br />
Thauer, R.K. (1988) Citric-acid cycle, 50 years on. Eur. J. Biochem. 176, 497–508.<br />
Thauer, R.K., Kaster, A.K., Seedorf, H., Buckel, W. and Hedderich, R. (2008)<br />
Methanogenic archaea: ecologically relevant differences <strong>in</strong> energy conservation.<br />
Nat. Rev. Microbiol. 6, 579–591.<br />
Tian, J., Bryk, R., Itoh, M., Suematsu, M. and Nathan, C. (2005) Variant tricarboxylic<br />
acid cycle <strong>in</strong> Mycobacterium tuberculosis: identification of alpha-ketoglutarate<br />
decarboxylase. Proc. Natl. Acad. Sci. USA 102, 10670–10675.<br />
Tilton, R.G. (2002) Diabetic vascular dysfunction: l<strong>in</strong>ks to glucose-<strong>in</strong>duced reductive<br />
stress and VEGF. Microsc. Res. Tech. 57, 390–407.<br />
Tremblay, G.A. (2007) Historical statistics support a hypothesis l<strong>in</strong>k<strong>in</strong>g tuberculosis<br />
and air pollution caused by coal. Int. J. Tuberc. Lung Dis. 11, 722–732.<br />
Triccas, J.A., Berthet, F.X., Pelicic, V. and Gicquel, B. (1999) Use of fluorescence<br />
<strong>in</strong>duction and sucrose counterselection to identify Mycobacterium tuberculosis<br />
genes expressed with<strong>in</strong> host cells. Microbiology 145(Pt 10); 2923–2930.<br />
Trivedi, O.A., Arora, P., Vats, A., Ansari, M.Z., Tickoo, R., Sridharan, V., Mohanty,<br />
D. and Gokhale, R.S. (2005) Dissect<strong>in</strong>g the mechanism and assembly of a complex<br />
virulence mycobacterial lipid. Mol. Cell 17, 631–643.<br />
Trotter, E.W. and Grant, C.M. (2002) Thioredox<strong>in</strong>s are required for protection<br />
aga<strong>in</strong>st a reductive stress <strong>in</strong> the yeast Saccharomyces cerevisiae. Molec.<br />
Microbiol. 46, 869–878.<br />
Tsai, M.C., Chakravarty, S., Zhu, G., Xu, J., Tanaka, K., Koch, C., Tufariello, J.A.,<br />
Flynn, J.A. and Chan, J. (2006) Characterization of the tuberculous granuloma <strong>in</strong><br />
mur<strong>in</strong>e and human lungs: cellular composition and relative tissue oxygen tension.<br />
Cell. Microbiol. 8, 218–232.<br />
Ulrichs, T. and Kaufmann, S.H. (2006) New <strong>in</strong>sights <strong>in</strong>to the function of granulomas<br />
<strong>in</strong> human tuberculosis. J. Pathol. 208, 261–269.<br />
Upton, A.M. and McK<strong>in</strong>ney, J.D. (2007) Role of the methylcitrate cycle <strong>in</strong> propionate<br />
metabolism and detoxification <strong>in</strong> Mycobacterium smegmatis. Microbiology 153,<br />
3973–3982.<br />
Valadi, H., Valadi, A., Ansell, R., Gustafsson, L., Adler, L., Norbeck, J. and<br />
Blomberg, A. (2004) NADH-reductive stress <strong>in</strong> Saccharomyces cerevisiae <strong>in</strong>duces<br />
the expression of the m<strong>in</strong>or isoform of glyceraldehyde-3-phosphate dehydrogenase<br />
(TDH1). Curr. Genet. 45, 90–95.
116 AISHA FARHANA ET AL.<br />
van Dijken, J.P. and Scheffers, W.A. (1986) Redox balances <strong>in</strong> the metabolism of<br />
sugars by yeasts. FEMS Microbiol. Lett. 32, 199–224.<br />
Van Keulen, G., Dijkhuizen, L. and Meijer, W.G. (2000) Effects of the Calv<strong>in</strong> cycle<br />
on nicot<strong>in</strong>amide aden<strong>in</strong>e d<strong>in</strong>ucleotide concentrations and redox balances of<br />
Xanthobacter flavus. J. Bacteriol. 182, 4637.<br />
Velayati, A.A., Masjedi, M.R., Farnia, P., Tabarsi, P., Ghanavi, J., Ziazarifi, A.H. and<br />
Hoffner, S.E. (2009) Emergence of new forms of totally drug-resistant tuberculosis<br />
bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant<br />
stra<strong>in</strong>s <strong>in</strong> Iran. Chest 136, 420–425.<br />
Vemuri, G.N., Altman, E., Sangurdekar, D.P., Khodursky, A.B. and Eiteman, M.A.<br />
(2006) Overflow metabolism <strong>in</strong> Escherichia coli dur<strong>in</strong>g steady-state growth: transcriptional<br />
regulation and effect of the redox ratio. Appl. Environ. Microbiol. 72,<br />
3653–3661.<br />
Via, L.E., L<strong>in</strong>, P.L., Ray, S.M., Carrillo, J., Allen, S.S., Eum, S.Y., Taylor, K., Kle<strong>in</strong>,<br />
E., Manjunatha, U. and Gonzales, J. (2008) Tuberculous granulomas are hypoxic<br />
<strong>in</strong> gu<strong>in</strong>ea pigs, rabbits, and nonhuman primates. Infect. Immun. 76, 2333.<br />
Vignais, P.M., Billoud, B. and Meyer, J. (2001) Classification and phylogeny of<br />
hydrogenases. FEMS Microbiol. Rev. 25, 455–501.<br />
Vignais, P.M. and Colbeau, A. (2004) Molecular biology of microbial hydrogenases.<br />
Curr. Issues Mol. Biol. 6, 159–188.<br />
Vilcheze, C., Av-Gay, Y., Attarian, R., Liu, Z., Hazbon, M.H., Colangeli, R., Chen,<br />
B., Liu, W., Alland, D., Sacchett<strong>in</strong>i, J.C. and Jacobs Jr., W.R. (2008) Mycothiol<br />
biosynthesis is essential for ethionamide susceptibility <strong>in</strong> Mycobacterium tuberculosis.<br />
Mol. Microbiol. 69, 1316–1329.<br />
Voet, D., Voet, J.G. and Pratt, C.W. (2008) Fundamental of Biochemistry: Life at a<br />
Molecualr Level. John Wiley and Sons.<br />
Vogt, R.N., Steenkamp, D.J., Zheng, R. and Blanchard, J.S. (2003) The metabolism<br />
of nitrosothiols <strong>in</strong> the Mycobacteria: identification and characterization of Snitrosomycothiol<br />
reductase. Biochem. J. 374, 657–666.<br />
Voskuil, M.I., Honaker, R.W. and Steyn, A.J.C. (2009) In: Parish, T., Brown, A.<br />
(Eds.), Mycobacterium Genomics and Molecualar Biology. Caister Academic<br />
Press, Norfolk, UK, pp. 119–147.<br />
Voskuil, M.I., Schnapp<strong>in</strong>ger, D., Visconti, K.C., Harrell, M.I., Dolganov, G.M.,<br />
Sherman, D.R. and Schoolnik, G.K. (2003) Inhibition of respiration by nitric oxide<br />
<strong>in</strong>duces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198,<br />
705–713.<br />
Voskuil, M.I., Visconti, K.C. and Schoolnik, G.K. (2004) Mycobacterium tuberculosis<br />
gene expression dur<strong>in</strong>g adaptation to stationary phase and low-oxygen dormancy.<br />
Tuberculosis (Ed<strong>in</strong>b.) 84, 218–227.<br />
Waltermann, M., Stoveken, T. and Ste<strong>in</strong>buchel, A. (2007) Key enzymes for biosynthesis<br />
of neutral lipid storage compounds <strong>in</strong> prokaryotes: properties, function and<br />
occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases.<br />
Biochimie 89, 230–242.<br />
Wang, C.H., Liu, C.Y., L<strong>in</strong>, H.C., Yu, C.T., Chung, K.F. and Kuo, H.P. (1998)<br />
Increased exhaled nitric oxide <strong>in</strong> active pulmonary tuberculosis due to <strong>in</strong>ducible<br />
NO synthase upregulation <strong>in</strong> alveolar macrophages. Eur. Respir. J. 11, 809–815.<br />
Ward, D.E., Van Der Weijden, C.C., Van Der Merwe, M.J., Westerhoff, H.V.,<br />
Claiborne, A. and Snoep, J.L. (2000) Branched-cha<strong>in</strong> alpha-keto acid catabolism
REDUCTIVE STRESS IN MICROBES 117<br />
via the gene products of the bkd operon <strong>in</strong> Enterococcus faecalis: a new, secreted<br />
metabolite serv<strong>in</strong>g as a temporary redox s<strong>in</strong>k. J. Bacteriol. 182, 3239.<br />
Warner, D.F. Mizrahi, V. (2007) The survival kit of Mycobacterium tuberculosis.<br />
Nat. Med. 13, 282–284.<br />
Wayne, L.G. and Hayes, L.G. (1996) An <strong>in</strong> vitro model for sequential study of<br />
shiftdown of Mycobacterium tuberculosis through two stages of nonreplicat<strong>in</strong>g<br />
persistence. Infect. Immun. 64, 2062–2069.<br />
West, J.B. (1977) Regional Differences <strong>in</strong> the Lung. Academic Press, New York.<br />
Wheeler, P.R. and Ratledge, C. (1994) In: Bloom, B.R. (Ed.), Tuberculosis:<br />
Pathogenesis, Protection, and Control. ASM Press, Wash<strong>in</strong>gton, pp. 353–385.<br />
Wimpenny, J.W.T. and Firth, A. (1972) Levels of nicot<strong>in</strong>amide aden<strong>in</strong>e d<strong>in</strong>ucleotide<br />
and reduced nicot<strong>in</strong>amide aden<strong>in</strong>e d<strong>in</strong>ucleotide <strong>in</strong> facultative bacteria and the<br />
effect of oxygen. J. Bacteriol. 111, 24.<br />
W<strong>in</strong>stanley, C. and Fothergill, J.L. (2009) The role of quorum sens<strong>in</strong>g <strong>in</strong> chronic cystic<br />
fibrosis Pseudomonas aerug<strong>in</strong>osa <strong>in</strong>fections. FEMS Microbiol. Lett. 290, 1–9.<br />
Wolfe, A.J. (2005) The acetate switch. Microbiol. Mol. Biol. Rev. 69, 12–50.<br />
Worlitzsch, D., Tarran, R., Ulrich, M., Schwab, U., Cekici, A., Meyer, K.C., Birrer, P.,<br />
Bellon, G., Berger, J., Weiss, T., Botzenhart, K., Yankaskas, J.R., Randell, S.,<br />
Boucher, R.C. and Dor<strong>in</strong>g, G. (2002) Effects of reduced mucus oxygen concentration<br />
<strong>in</strong> airway Pseudomonas <strong>in</strong>fections of cystic fibrosis patients. J. Cl<strong>in</strong>. Invest.<br />
109, 317–325.<br />
Wright, D.E. (1959) The formation of keto and am<strong>in</strong>o acids by Mycobacterium<br />
butyricum grow<strong>in</strong>g <strong>in</strong> a chemically def<strong>in</strong>ed medium. Microbiology 20, 554.<br />
Xia, D., Esser, L., Yu, L. and Yu, C.A. (2007) Structural basis for the mechanism of<br />
electron bifurcation at the qu<strong>in</strong>ol oxidation site of the cytochrome bc 1 complex.<br />
Photosyn. Res. 92, 17–34.<br />
Young, D.B., Perk<strong>in</strong>s, M.D., Duncan, K. and Barry 3rd, C.E. (2008) Confront<strong>in</strong>g the<br />
scientific obstacles to global control of tuberculosis. J. Cl<strong>in</strong>. Invest. 118, 1255–1265.<br />
Yukl, E.T., Ioanoviciu, A., de Montellano, P.R. and Moenne-Loccoz, P. (2007)<br />
Interdoma<strong>in</strong> <strong>in</strong>teractions with<strong>in</strong> the two-component heme-based sensor DevS<br />
from Mycobacterium tuberculosis. Biochemistry 46, 9728–9736.<br />
Zbell, A.L., Benoit, S.L. and Maier, R.J. (2007) Differential expression of NiFe<br />
uptake-type hydrogenase genes <strong>in</strong> Salmonella enterica serovar Typhimurium.<br />
Microbiology 153, 3508–3516.<br />
Zbell, A.L. and Maier, R.J. (2009) Role of the Hya hydrogenase <strong>in</strong> recycl<strong>in</strong>g of<br />
anaerobically produced H 2 <strong>in</strong> Salmonella enterica serovar Typhimurium. Appl.<br />
Environ. Microbiol. 75, 1456.<br />
Zbell, A.L., Maier, S.E. and Maier, R.J. (2008) Salmonella enterica serovar<br />
Typhimurium NiFe uptake-type hydrogenases are differentially expressed<br />
<strong>in</strong> vivo. Infect. Immun. 76, 4445.<br />
Zhang, X., M<strong>in</strong>, X., Li, C., Benjam<strong>in</strong>, I.J., Qian, B., D<strong>in</strong>g, Z., Gao, X., Yao, Y., Ma, Y.,<br />
Cheng, Y. and Liu, L. (2010) Involvement of reductive stress <strong>in</strong> the cardiomyopathy<br />
<strong>in</strong> transgenic mice with cardiac-specific overexpression of heat shock prote<strong>in</strong><br />
27. Hypertension 55, 1412–1417.
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Regulation of CtsR Activity <strong>in</strong> Low GC,<br />
Gram+ Bacteria<br />
Alexander K.W. Elsholz, Ulf Gerth and Michael Hecker<br />
Ernst-Moritz-Arndt-University Greifswald, Institute of Microbiology, Greifswald, Germany<br />
ABSTRACT<br />
CtsR is the global transcriptional regulator of the core prote<strong>in</strong> quality<br />
networks <strong>in</strong> low GC, Gram+ bacteria. Balanc<strong>in</strong>g these networks dur<strong>in</strong>g<br />
environmental stress is of considerable importance for moderate survival of<br />
the bacteria, and also for virulence of pathogenic species. Therefore,<br />
<strong>in</strong>activation of the CtsR repressor is one of the major cellular responses for<br />
fast and efficient adaptation to different prote<strong>in</strong> stress conditions.<br />
Historically, CtsR <strong>in</strong>activation was ma<strong>in</strong>ly studied for the heat stress<br />
response, and recently it has been shown that CtsR is an <strong>in</strong>tr<strong>in</strong>sic<br />
thermosensor. Moreover, it has been demonstrated that CtsR degradation is<br />
regulated by a two-step mechanism dur<strong>in</strong>g heat stress, dependent on the<br />
arg<strong>in</strong><strong>in</strong>e k<strong>in</strong>ase activity of McsB. Interest<strong>in</strong>gly, CtsR is also <strong>in</strong>activated<br />
dur<strong>in</strong>g oxidative stress, but by a thiol-dependent regulatory pathway. These<br />
observations suggest that dual activity control of CtsR activity has developed<br />
dur<strong>in</strong>g the course of evolution.<br />
Abbreviations . . . ........................................... 120<br />
1. Prote<strong>in</strong> Quality Control. . ..................................... 120<br />
2. Ctsr-Regulated Genes . . ..................................... 122<br />
3. Cellular Functions Of Genes Regulated by CtsR. .................. 125<br />
4. Mechanisms for the Inactivation of the CtsR Repressor ............. 129<br />
4.1. Heat Inactivation of CtsR. . ............................... 129<br />
4.2. CtsR Inactivation Dur<strong>in</strong>g Oxidative Stress ................... 132<br />
ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 57 Copyright Ó 2010 by Elsevier Ltd.<br />
ISSN: 0065-2911 All rights reserved<br />
DOI:10.1016/B978-0-12-381045-8.00003-5
120 ALEXANDER K.W. ELSHOLZ ET AL.<br />
4.3. CtsR Inactivation Dur<strong>in</strong>g Other Stress Conditions ............. 134<br />
5. Control of Ctsr Degradation by the Regulated Adaptor McsB . ........ 136<br />
6. Summary And Outlook. . ..................................... 136<br />
Acknowledgement .......................................... 137<br />
References. ............................................... 137<br />
ABBREVIATIONS<br />
HTH helix-turn-helix<br />
HHP high-hydrostatic pressure<br />
LMWPTP low-molecular-weight prote<strong>in</strong> tyros<strong>in</strong>e phosphatase<br />
1. PROTEIN QUALITY CONTROL<br />
The ma<strong>in</strong>tenance of proper prote<strong>in</strong> homeostasis is important for viability and<br />
growth of all liv<strong>in</strong>g organisms, and cells have evolved two major strategies.<br />
Prote<strong>in</strong> quality networks ensure the correct prote<strong>in</strong> function as molecular<br />
chaperones promote prote<strong>in</strong> fold<strong>in</strong>g and mediate refold<strong>in</strong>g while ATPdependent<br />
proteases degrade misfolded or aggregated prote<strong>in</strong>s to prevent<br />
cell <strong>in</strong>jury when refold<strong>in</strong>g by molecular chaperones has failed. Stress conditions<br />
such as heat, oxidative stress and extreme pH values result <strong>in</strong> damage of<br />
the prote<strong>in</strong> structure that may lead to aggregation of prote<strong>in</strong>s which fail to<br />
fulfill their physiological function and are often lethal for the cell. Hence,<br />
enhanced expression of these critical prote<strong>in</strong> classes is needed for moderate<br />
survival of the cell dur<strong>in</strong>g severe prote<strong>in</strong> stress conditions (Geth<strong>in</strong>g and<br />
Sambrook, 1992; Wickner et al., 1999; Sauer et al., 2004; Hartl and Hayer-<br />
Hartl, 2009).<br />
The cellular chaperone mach<strong>in</strong>ery assists <strong>in</strong> non-covalent fold<strong>in</strong>g or<br />
unfold<strong>in</strong>g of macromolecular complexes, but molecular chaperones are<br />
not <strong>in</strong>volved <strong>in</strong> the normal enzymatic reactions of these complexes. In<br />
all three doma<strong>in</strong>s of life, a plethora of structurally unrelated chaperones<br />
have evolved (Young et al., 2004), but general modes of operation are<br />
conserved for all molecular chaperones. In general, all chaperones recognise<br />
hydrophobic residues and/or unstructured backbone regions <strong>in</strong> prote<strong>in</strong>s,<br />
that is structural features normally buried upon completion of<br />
fold<strong>in</strong>g but typically exposed by non-native prote<strong>in</strong>s. This ensures that
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 121<br />
only <strong>in</strong>correctly folded prote<strong>in</strong>s become targets for molecular chaperones<br />
(Hartl and Hayer-Hartl, 2009).<br />
Energy-dependent prote<strong>in</strong> degradation is achieved by large cyl<strong>in</strong>drical<br />
assemblies with a common r<strong>in</strong>g-stack<strong>in</strong>g architecture of diverse complexity.<br />
This architecture is highly conserved <strong>in</strong> all liv<strong>in</strong>g organisms and operates with<br />
similar pr<strong>in</strong>ciples. It is composed of a chaperone r<strong>in</strong>g compris<strong>in</strong>g ATPase<br />
doma<strong>in</strong>s of the AAA+-type (ATPase associated with a variety of cellular<br />
activities) that caps both ends of self-compartmentalised proteases whereby<br />
the active sites are located <strong>in</strong> an <strong>in</strong>ternal chamber and are thereby separated<br />
from the cytosol (Wickner et al., 1999; Sauer et al., 2004). Hydrolysis of ATP<br />
by the AAA+ superfamily of prote<strong>in</strong>s is translated <strong>in</strong>to force that unfolds<br />
substrates and translocates them <strong>in</strong>to the proteolytic chamber of the protease<br />
subunit where the peptide bonds are hydrolysed (Neuwald et al., 1999; Baker<br />
and Sauer, 2006).<br />
Prote<strong>in</strong> degradation <strong>in</strong> eukaryotes is performed by the 26S proteasome<br />
comprised of a 19S cap particle with six different AAA+ prote<strong>in</strong>s at its base,<br />
and a 20S core particle which conta<strong>in</strong>s the proteolytic site (Pickart and<br />
Cohen, 2004). A 26S proteasome system is not present <strong>in</strong> eubacteria, except<br />
for some act<strong>in</strong>obacteria (Darw<strong>in</strong>, 2009). However, at least four ATPdependent<br />
proteases (Clp, HslUV, Lon and FtsH) operat<strong>in</strong>g on a similar<br />
pr<strong>in</strong>ciple have evolved <strong>in</strong> eubacteria (Gottesman, 2003).<br />
In low GC, Gram+ bacteria the Clp mach<strong>in</strong>ery seems to be the major<br />
system for general prote<strong>in</strong> turnover (Frees and Ingmer, 1999; Kr€uger et al.,<br />
2000; Kock et al., 2004b; Gerth et al., 2008). The Clp protease is constituted of<br />
Hsp 100/Clp prote<strong>in</strong>s of the AAA+ superfamily and an associated barrel-like<br />
protease complex formed by ClpP (Gottesman, 2003). The multimeric barrellike<br />
structure of ClpP is formed by two stacked heptameric r<strong>in</strong>gs of ClpP<br />
homomers which create a catalytic cavity where<strong>in</strong> the 14 proteolytic active<br />
ser<strong>in</strong>e residues are enclosed (Wang et al.,1997). ClpP, on its own, is only able<br />
to degrade small peptides and with very restricted efficiency (Jenn<strong>in</strong>gs et al.,<br />
2008). Therefore, a regulatory AAA+ prote<strong>in</strong> of the HSP 100/Clp family<br />
flanks both ends of the ClpP complex, display<strong>in</strong>g a gateway to the protease.<br />
These regulatory prote<strong>in</strong>s form hexameric r<strong>in</strong>gs with a narrow pore <strong>in</strong> the<br />
middle through which the substrate is translocated <strong>in</strong>to the <strong>in</strong>ternal chamber<br />
of the peptidase (Zolkiewski, 2006). Substrate unfold<strong>in</strong>g and thread<strong>in</strong>g<br />
through the narrow pore <strong>in</strong>to the proteolytic chamber by the Hsp 100/Clp<br />
prote<strong>in</strong>s is ATP dependent (Weber-Ban et al., 1999). Once the unfolded<br />
polypeptide enters the ClpP proteolytic chamber, it is rapidly degraded,<br />
without further utilisation of ATP, to short peptides of 7–10 am<strong>in</strong>o acids<br />
length (Thompson et al., 1994).
122 ALEXANDER K.W. ELSHOLZ ET AL.<br />
2. CtsR-REGULATED GENES<br />
In the Gram-negative model organism Escherichia coli the regulation of<br />
classical heat shock genes depends ma<strong>in</strong>ly on the level of the alternative<br />
transcription factor s 32 (Yura, 1996). However, <strong>in</strong> the low GC, Gram+ model<br />
organism Bacillus subtilis regulation of prote<strong>in</strong> quality control systems differs<br />
significantly. To date, at least six different classes have been dist<strong>in</strong>guished for<br />
<strong>in</strong>duced expression of heat shock prote<strong>in</strong>s (Schumann, 2004). Class one was<br />
def<strong>in</strong>ed as genes regulated by the global repressor HrcA, class two encompasses<br />
genes under the control of the alternative sigma factor SigB, class<br />
three genes belong to the CtsR regulon, class four conta<strong>in</strong>s only the htpG<br />
gene, class five is controlled by the CssRS two-component system and class<br />
six is reserved for genes whose expression is <strong>in</strong>duced dur<strong>in</strong>g heat stress but<br />
with no known regulatory mechanism (Schumann, 2004). The central core of<br />
the prote<strong>in</strong> quality control <strong>in</strong> all low GC, Gram+ bacteria is under the control<br />
of CtsR (class three stress gene repressor), as long as a ctsR gene is present <strong>in</strong><br />
the correspond<strong>in</strong>g genome. To date, the only bacilli lack<strong>in</strong>g a ctsR gene<br />
belong to the Lactobacillus acidophilus group (van de Guchte et al., 2006).<br />
Although these six different classes of heat shock prote<strong>in</strong>s are not present<br />
<strong>in</strong> all Gram+ bacteria, regulation of heat shock <strong>in</strong>duction rema<strong>in</strong>s complex<br />
and is also divided <strong>in</strong>to several groups <strong>in</strong> other low GC, Gram+ bacteria.<br />
Nevertheless, these regulons are not very committed <strong>in</strong> their specific contributions<br />
and thus partial or full overlap between different classes can be<br />
found <strong>in</strong> some species, whereas these classes are very dist<strong>in</strong>ct <strong>in</strong> others.<br />
The alternative sigma factor SigB is only conserved <strong>in</strong> the order Bacillales<br />
(Hecker et al., 2007) and is def<strong>in</strong>ed as class two <strong>in</strong> the heat shock response of<br />
B. subtilis. SigB overlaps with CtsR <strong>in</strong> the regulation of the clpC operon and<br />
clpP <strong>in</strong> B. subtilis (Kr€uger et al., 1996; Gerth et al., 1998) as well as for the<br />
clpC, clpP and clpB operons <strong>in</strong> Listeria monocytogenes (Hu et al., 2007).<br />
However, <strong>in</strong> Staphylococcus aureus no functional overlap between SigB and<br />
CtsR was found. The clpC, clpP and clpB operons are solely under CtsR<br />
control, whereas SigB alone is responsible for heat <strong>in</strong>duction of clpL (Gertz<br />
et al., 2000; Chastanet et al., 2003). Dual regulation of heat shock gene<br />
regulation by SigB and CtsR does not mean that a SigB stimulus is sufficient<br />
for an effective <strong>in</strong>duction of transcription while CtsR is still active as a<br />
transcriptional repressor. Such a scenario leads to only a slight <strong>in</strong>duction<br />
of the dually regulated clpC operon <strong>in</strong> B. subtilis, for example dur<strong>in</strong>g different<br />
SigB activat<strong>in</strong>g stress conditions such as glucose limitation or ethanol<br />
stress (Kr€uger et al., 1994). SigB activity alone does not lead to an appropriate<br />
<strong>in</strong>duction of the target genes because the impact of CtsR repression on<br />
transcription is super-ord<strong>in</strong>ated, and thus CtsR <strong>in</strong>activation is str<strong>in</strong>gent for
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 123<br />
adequate expression. The major task of SigB for dually regulated genes<br />
maybe is to enhance the <strong>in</strong>duction of transcription when CtsR is <strong>in</strong>activated.<br />
Heat shock class one <strong>in</strong> B. subtilis is regulated by the global repressor<br />
HrcA (heat shock regulation by CIRCE) (Zuber and Schumann, 1994; Yuan<br />
and Wong, 1995) and regulates only two operons cod<strong>in</strong>g for the classical<br />
molecular chaperones dnaK and groE (Homuth et al., 1997). In B. subtilis<br />
and close relatives, the HrcA and the CtsR regulon are dist<strong>in</strong>ct from each<br />
other. However, <strong>in</strong> other groups of low GC, Gram+ bacteria, CtsR is also<br />
able to regulate expression of HrcA-dependent genes (Chastanet et al.,<br />
2003). In most cases, HrcA-regulated genes are under dual control of<br />
both transcriptional regulators, and only <strong>in</strong> Streptococcus salivarius and<br />
Streptococcus thermophilus clpP is <strong>in</strong> addition to CtsR under HrcA control<br />
(Chastanet and Msadek, 2003). In S. aureus both HrcA-dependent operons<br />
also belong to the CtsR regulon, whereas <strong>in</strong> the order of Lactobacillales only<br />
the groE operon stands under dual regulation (Chastanet et al., 2003).<br />
Moreover, <strong>in</strong> species where one of the two global heat shock repressors is<br />
absent, the other has taken over the regulation of both regulons. For example,<br />
CtsR is the global stress repressor <strong>in</strong> Oenococcus oeni (Grandvalet et al.,<br />
2005), a lactic acid bacterium miss<strong>in</strong>g hrcA <strong>in</strong> its genome. In contrast, HrcA is<br />
the correspond<strong>in</strong>g regulator of clp expression <strong>in</strong> the L. acidophilus group,<br />
which lack ctsR <strong>in</strong> their genome (van de Guchte et al., 2006) (Fig. 1).<br />
In contrast to dual regulation by a transcriptional ‘activator’ such as SigB<br />
and a repressor such as CtsR the <strong>in</strong>terplay between two transcriptional<br />
repressors seems odd. The major effect of this crosstalk is that basal transcription<br />
under repressor active conditions is lower compared to expression<br />
when only one repressor is responsible for regulation (Chastanet et al., 2003).<br />
HrcA activity is directly dependent on prote<strong>in</strong> stress (Mogk et al., 1997),<br />
whereas the CtsR repressor senses different stress <strong>in</strong>puts (Elsholz et al., 2010,<br />
manuscript submitted) and is also <strong>in</strong>activated dur<strong>in</strong>g virulence-related stress<br />
conditions. This gives space for speculation that dual regulation of the HrcA<br />
regulon ensures that the molecular chaperones, which are also crucial for<br />
virulence (see Section 3), are strongly <strong>in</strong>duced dur<strong>in</strong>g both specific stress and<br />
virulence conditions (Chastanet et al., 2003).<br />
The ctsR gene itself stands under its own control and is auto-regulated<br />
(Derre et al., 1999b). The ctsR gene is co-transcribed with clpC <strong>in</strong> an operon<br />
for all low GC, Gram+ bacteria and the AAA+ prote<strong>in</strong> ClpC belongs to the<br />
CtsR regulon <strong>in</strong> all cases when ctsR gene is present <strong>in</strong> a genome. In the<br />
order Bacillales of low GC, Gram+ bacteria the clpC operon is tetra-cistronic<br />
and, <strong>in</strong> addition to ctsR and clpC, also encodes the two modulators of<br />
CtsR activity, mcsA and mcsB. However, genes for these two modulators<br />
are absent <strong>in</strong> the order of Lactobacillales (Varmanen et al., 2000;
[(Figure_1)TD$FIG]<br />
Figure 1 Graphical presentation of the distribution of CtsR-regulated prote<strong>in</strong>s for different low GC, Gram+ species and known<br />
overlaps with other regulators such as SigB or HrcA. An arrow <strong>in</strong>dicates specific <strong>in</strong>fluence of transcriptional regulator for the<br />
expression of the correspond<strong>in</strong>g gene. Prote<strong>in</strong>s whose expression is solely dependent on CtsR activity are depicted <strong>in</strong> red, whereas<br />
prote<strong>in</strong>s that are dually regulated by CtsR and SigB (green) are shown <strong>in</strong> blue. Prote<strong>in</strong>s controlled by CtsR as well as HrcA are<br />
presented <strong>in</strong> grey, and prote<strong>in</strong>s that are regulated only by HrcA are shown <strong>in</strong> purple (See Colour Plate Section).<br />
124 ALEXANDER K.W. ELSHOLZ ET AL.
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 125<br />
Chastanet et al., 2001). In these species, ctsR is co-transcribed only with clpC<br />
<strong>in</strong> a bi-cistronic operon.<br />
The proteolytic component of the ATP-dependent protease ClpP is also a<br />
permanent member of the CtsR regulon <strong>in</strong> all low GC, Gram+ bacteria, as<br />
long as the ctsR gene is present <strong>in</strong> the correspond<strong>in</strong>g genome. With the<br />
exception of S. salivarius and S. thermophilus, where dual regulation with<br />
CtsR and HrcA is present (Chastanet and Msadek, 2003), expression of the<br />
clpP operon stands solely under control of CtsR. In low GC Gram+ bacteria,<br />
clpP is mostly encoded by a mono-cistronic gene (Gerth et al., 1996), but <strong>in</strong><br />
O. oeni clpP can be also co-transcribed with clpL (Beltramo et al., 2004).<br />
The molecular chaperones ClpB and ClpL lack an IGF loop and thus are<br />
probably not able to <strong>in</strong>teract directly with the proteolytic component ClpP<br />
(Kim et al., 2001; Frees et al., 2004; Weibezahn et al., 2004). ClpB and ClpL<br />
are not present <strong>in</strong> the families Bacillaceae and Listeriaceae, but can be found<br />
<strong>in</strong> Staphylococcaceae and more often <strong>in</strong> Lactobacillaceae. Both genes stand<br />
under CtsR control with the exception of clpL <strong>in</strong> staphylococci (Gertz et al.,<br />
2000). ClpE, another Hsp 100/Clp prote<strong>in</strong>, always stands under CtsR control<br />
as far as both clpE and ctsR genes are present <strong>in</strong> the genome.<br />
Interest<strong>in</strong>gly, new studies have revealed so far unknown and not typical<br />
members of the CtsR regulon. In Lactobacillus plantarum the ATP-dependent<br />
membrane-bound AAA+ protease FtsH and the small heat shock<br />
prote<strong>in</strong> Hsp1 are under direct CtsR control (Fiocco et al., 2008, 2010). In<br />
addition, a second small heat shock prote<strong>in</strong>, Hsp18, was found regulated by<br />
CtsR <strong>in</strong> O. oeni (Grandvalet et al., 2005).<br />
In O. oeni, a CtsR consensus sequence was surpris<strong>in</strong>gly found <strong>in</strong> front of<br />
the clpX operon, but no <strong>in</strong>fluence of CtsR was detected for clpX transcription<br />
(Grandvalet et al., 2005). It was speculated that this b<strong>in</strong>d<strong>in</strong>g site was an<br />
evolutionary remnant. Nevertheless, regulation of clpX expression rema<strong>in</strong>s<br />
unclear for all low GC, Gram+ bacteria, and to date, no CtsR-dependent<br />
regulation has been shown for ClpX.<br />
3. CELLULAR FUNCTIONS OF GENES REGULATED BY CtsR<br />
The physiological function of the CtsR regulon members lies not only <strong>in</strong> their<br />
important role <strong>in</strong> prote<strong>in</strong> quality control and the result<strong>in</strong>g effect <strong>in</strong> stress<br />
adaptation and general cellular processes, but also <strong>in</strong> their specific role <strong>in</strong><br />
regulated degradation of key regulators for essential cellular and physiological<br />
programmes <strong>in</strong> response to temporal, spatial or environmental stimuli.<br />
Not only the specific regulated proteolysis of transcription factors is
126 ALEXANDER K.W. ELSHOLZ ET AL.<br />
important, but also the re-arrangement of the complete proteome after a<br />
specific stress <strong>in</strong>put to atta<strong>in</strong> a better adaptation of the cell (Sauer et al., 2004;<br />
Neher et al., 2006). However, specific adaptor prote<strong>in</strong>s are needed for Clpspecific<br />
degradation. To date, only a few adaptor prote<strong>in</strong>s are known for the<br />
low GC, Gram+ model organism B. subtilis, but additional adaptor prote<strong>in</strong>s<br />
must exist (Kirste<strong>in</strong> et al., 2009), suggest<strong>in</strong>g that <strong>in</strong>duction of the Clp proteolytic<br />
mach<strong>in</strong>ery by <strong>in</strong>activation of CtsR alone is not sufficient for regulated<br />
degradation, with the exception of McsB-dependent proteolysis. Thus, the<br />
CtsR regulon seems to be embedded <strong>in</strong>to stress-specific modulons, where<br />
additional regulatory mechanisms <strong>in</strong>duce expression of stress-specific adaptor<br />
prote<strong>in</strong>s allow<strong>in</strong>g a concatenated stress response. Consequently, regulated<br />
prote<strong>in</strong> degradation seems to be under multiple control.<br />
The peptidase ClpP is the proteolytic component of the Clp degradation<br />
mach<strong>in</strong>ery and is ma<strong>in</strong>ly <strong>in</strong>volved <strong>in</strong> (1) prote<strong>in</strong> quality control (degradation<br />
of irreversibly damaged or ssrA-tagged prote<strong>in</strong>s) (Kr€uger et al., 2000;<br />
Wiegert and Schumann, 2001); (2) specific degradation of regulatory prote<strong>in</strong>s<br />
such as CtsR, ComK, SpoIIAB, Spx, DegU-P or RsiW (Turgay et al.,<br />
1998; Kr€uger et al., 2001; Pan et al., 2001; Nakano et al., 2002; Zellmeier et al.,<br />
2006; Ogura and Tsukahara, 2010); and (3) unemployed prote<strong>in</strong>s such as<br />
biosynthetic enzymes, which are no longer needed <strong>in</strong> starv<strong>in</strong>g cells such as<br />
MurAA, IlvB, PurF or PyrB (Kock et al., 2004a; Gerth et al., 2008) and also<br />
for virulence of pathogenic low GC, Gram+ bacteria (Frees et al., 2007).<br />
Synthesis of clpP is <strong>in</strong>duced dur<strong>in</strong>g several environmental stress conditions<br />
that causes damage to the prote<strong>in</strong> structure (Gerth et al., 1998, 2004;<br />
Frees and Ingmer, 1999; Gaillot et al., 2000; Chastanet et al., 2001; Frees et al.,<br />
2003a). Consequently, clpP <strong>in</strong>activation <strong>in</strong> low GC, Gram+ bacteria leads to<br />
a pleiotropic mutant phenotype <strong>in</strong>dicat<strong>in</strong>g the extraord<strong>in</strong>ary role of the Clp<br />
mach<strong>in</strong>ery for prote<strong>in</strong> quality control <strong>in</strong> low GC, Gram+ bacteria (Gerth<br />
et al., 1998; Msadek et al., 1998).<br />
A B. subtilis clpP mutant failed to activate specific developmental programmes<br />
due to failure <strong>in</strong> regulated degradation of key regulators, result<strong>in</strong>g<br />
<strong>in</strong> a deficiency <strong>in</strong> competence development and sporulation (Msadek et al.,<br />
1998). Both deficiencies depend on ClpCP-dependent degradation of either<br />
the master regulator of competence development ComK (Turgay et al., 1998)<br />
or the anti-sigma factor SpoIIAB, which regulates the sporulation-specific<br />
sigma factor s F <strong>in</strong> the forespore (Pan et al., 2001).<br />
A clpP mutant is more sensitive aga<strong>in</strong>st heat, oxidative or low pH stress <strong>in</strong><br />
Lactococcus lactis (Frees and Ingmer, 1999), L. monocytogenes (Gaillot<br />
et al., 2000), Streptococcus pneumoniae (Chastanet et al., 2001),<br />
Streptococcus mutans (Lemos and Burne, 2002), S. aureus (Frees et al.,<br />
2003a) andO. oeni (Beltramo et al., 2004). ClpP is also responsible for the
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 127<br />
controlled degradation of the MazE antitox<strong>in</strong>s and probably all other antitox<strong>in</strong>s<br />
<strong>in</strong> S. aureus (Donegan et al., 2010). In L. lactis, activity of the transcriptional<br />
stress repressor HdiR (heat and DNA damage-<strong>in</strong>ducible regulator)<br />
is controlled by ClpP-dependent degradation (Savijoki et al., 2003).<br />
F<strong>in</strong>ally, ClpP is also <strong>in</strong>volved <strong>in</strong> the virulence of pathogenic low GC, Gram<br />
+ bacteria. ClpP is essential <strong>in</strong> a mouse <strong>in</strong>fection model and for <strong>in</strong>tracellular<br />
survival of L. monocytogenes (Gaillot et al., 2000), <strong>in</strong> a mur<strong>in</strong>e sk<strong>in</strong> abscess<br />
model for S. aureus (Frees et al., 2003a), for survival of S. pneumoniae <strong>in</strong> mice<br />
(Robertson et al., 2002) and for expression of virulence genes <strong>in</strong> S. mutans<br />
(Kajfasz et al., 2009). In the last few years, different roles for ClpP <strong>in</strong> the<br />
expression of virulence factors were described. In L. monocytogenes, ClpP<br />
affects expression of listeriolys<strong>in</strong> O (Gaillot et al., 2000) and SvpA (Borezee<br />
et al., 2001). In S. aureus, ClpP <strong>in</strong>fluences expression of extracellular virulence<br />
factors such as hla (Frees et al., 2003a). In S. pneumoniae (Robertson<br />
et al., 2002; Kwon et al., 2003; Ibrahim et al., 2005) andS. mutans (Kajfasz<br />
et al., 2009), it was demonstrated that ClpP <strong>in</strong>fluences virulence gene<br />
expression.<br />
Based on the important cellular and physiological function of ClpP for<br />
stress adaptation and virulence of pathogenic bacteria, ClpP seems to be a<br />
promis<strong>in</strong>g target for newly developed antibiotics (Br€otz-Oesterhelt et al.,<br />
2005; B€ottcher and Sieber, 2008).<br />
ClpP needs a Clp ATPase to perform efficient prote<strong>in</strong> degradation. In<br />
low GC, Gram+ bacteria only ClpX, ClpC and ClpE are able to perform<br />
this function, but only clpC and clpE are under CtsR control. Interest<strong>in</strong>gly,<br />
B. subtilis ClpC needs specific adaptor prote<strong>in</strong>s for oligomerisation and<br />
ATPase activity (Kirste<strong>in</strong> et al., 2006),whichareneededforanefficient<br />
ClpCP activity. Contrarily, B. subtilis ClpE possesses an <strong>in</strong>tr<strong>in</strong>sic ATPase<br />
activity, which depends on a putative z<strong>in</strong>c f<strong>in</strong>ger (Miethke et al., 2006). A<br />
clpC mutant exhibits thermosensitivity <strong>in</strong> B. subtilis (Msadek et al., 1994),<br />
S. aureus (Frees et al., 2003a) andL. monocytogenes (Rouquette et al.,<br />
1996), but a clpC deletion <strong>in</strong> L. lactis (Ingmer et al., 1999)andS. pneumoniae<br />
(Chastanet et al., 2001) did not affect growth at higher temperatures.<br />
Opposite observations were made for ClpE. A clpE mutant has no obvious<br />
phenotype <strong>in</strong> B. subtilis (Derre et al., 1999a), whereas a clpE deletion <strong>in</strong><br />
L. lactis (Ingmer et al., 1999)orS. pneumoniae (Chastanet et al., 2001)has<br />
a severe thermosensitive phenotype. One could speculate that this observation<br />
is l<strong>in</strong>ked to the occurrence of the specific heat-activated ClpC<br />
adaptor McsB, which is present <strong>in</strong> the order of Bacillales but is absent <strong>in</strong><br />
Lactobacillales.<br />
ClpC is the major ATPase for prote<strong>in</strong> turnover and regulated degradation<br />
<strong>in</strong> B. subtilis (Kr€uger et al., 2000; Gerth et al., 2008) and a correspond<strong>in</strong>g
128 ALEXANDER K.W. ELSHOLZ ET AL.<br />
mutant has a severe phenotype (Kr€uger et al., 1994; Msadek et al., 1994).<br />
Therefore, degradation of key regulators or ‘unemployed’ prote<strong>in</strong>s such as<br />
SpoIIAB, ComK, MurAA and GlmS are ma<strong>in</strong>ly performed by the ClpCP<br />
system (Turgay et al., 1998; Pan et al., 2001; Kock et al., 2004a; Gerth et al.,<br />
2008). ClpC is also important <strong>in</strong> other low GC, Gram+ bacteria and a clpC<br />
mutant thus has also a severe phenotype <strong>in</strong> L. monocytogenes (Rouquette<br />
et al., 1998), S. pneumoniae (Charpentier et al., 2000) and S. aureus (Frees<br />
et al., 2004; Chatterjee et al., 2005). ClpC is <strong>in</strong>volved <strong>in</strong> the virulence of<br />
pathogenic bacteria such as L. monocytogenes (Rouquette et al., 1996,<br />
1998; Nair et al., 2000b), S. aureus (Frees et al., 2004) and S. pneumoniae<br />
(Charpentier et al., 2000). Similar to ClpP, ClpC also affects expression of<br />
virulence factors such as SvpA (Borezee et al., 2001) and listeriolys<strong>in</strong> O<br />
(Rouquette et al., 1998)<strong>in</strong>L. monocytogenes or general virulence expression<br />
<strong>in</strong> S. pneumoniae (Ibrahim et al., 2005).<br />
In B. subtilis, clpE does not show a severe phenotype (Derre et al., 1999a)<br />
and a specific function for ClpE <strong>in</strong> B. subtilis could only be l<strong>in</strong>ked to CtsR<br />
(Miethke et al., 2006). Deletion of clpE affects the re-repression of CtsRdependent<br />
transcription, suggest<strong>in</strong>g a role for ClpE <strong>in</strong> the re-activation of<br />
CtsR <strong>in</strong> B. subtilis (Miethke et al., 2006)andL. lactis (Varmanen et al., 2003).<br />
ClpE also participates <strong>in</strong> the degradation of CtsR, partially <strong>in</strong> B. subtilis<br />
(Miethke et al., 2006) and fully <strong>in</strong> L. lactis (Elsholz et al., manuscript<br />
submitted). In other low GC, Gram+ bacteria a clpE mutant is deficient <strong>in</strong><br />
stress survival <strong>in</strong> L. monocytogenes (Nair et al., 1999), L. lactis (Ingmer et al.,<br />
1999) and S. pneumoniae (Chastanet et al., 2001). In addition, ClpE is also<br />
needed for virulence of L. monocytogenes (Nair et al., 1999) and S. pneumoniae<br />
(Zhang et al., 2009).<br />
Effects of clpL or clpB knock-outs <strong>in</strong> S. aureus can only depend on<br />
chaperone activity because ClpL and ClpB lack the IGF loop which is crucial<br />
for <strong>in</strong>teraction with ClpP (Kim et al., 2001). A clpL mutant displays a heatsensitive<br />
phenotype and decreased virulence for S. pneumoniae (Kwon et al.,<br />
2003) and S. mutans. ClpL is <strong>in</strong>volved <strong>in</strong> stress tolerance and viability<br />
(Kajfasz et al., 2009). ClpB <strong>in</strong> L. monocytogenes is needed for virulence<br />
and <strong>in</strong>duction of thermotolerance, but not for other stress conditions<br />
(Chastanet et al., 2004). In S. aureus a clpB mutant showed decreased virulence<br />
and thermotolerance (Frees et al., 2004).<br />
The molecular chaperones DnaK and GroEL are highly conserved among<br />
pro- and eukaryotes (Craig, 1985), and are important for stress resistance. In<br />
S. aureus, both prote<strong>in</strong>s are important for virulence (Qoronfleh et al., 1993,<br />
1998). Deletion of either dnaK or groEL leads to reduced virulence and<br />
stress sensitivity (S<strong>in</strong>gh et al., 2007). S. mutans dnaK and groEL are needed<br />
for general stress tolerance (Lemos et al., 2007).
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 129<br />
The two modulators McsA and McsB <strong>in</strong> Bacillales were shown to act as an<br />
adaptor complex for ClpC (Kirste<strong>in</strong> et al., 2007). Recently, it has also been<br />
demonstrated that the McsAB adaptor complex mediates the delocalisation<br />
of competence prote<strong>in</strong>s from cell poles <strong>in</strong> B. subtilis (Hahn et al., 2009).<br />
Inactivation of CtsR itself leads to an enhanced stress tolerance accord<strong>in</strong>g<br />
to the <strong>in</strong>creased expression of the prote<strong>in</strong> quality control prote<strong>in</strong>s <strong>in</strong><br />
L. monocytogenes (Nair et al., 2000a; Karatzas et al., 2003), L. lactis<br />
(Varmanen et al., 2000), S. thermophilus (Zotta et al., 2009) and<br />
Lactobacillus sakeii (H€ufner et al., 2007; H€ufner and Hertel, 2008), where<br />
the utilisation of a ctsR mutant represents an advantage for <strong>in</strong>dustrial<br />
production <strong>in</strong> a fermenter.<br />
4. MECHANISMS FOR THE INACTIVATION OF THE CtsR<br />
REPRESSOR<br />
4.1. Heat Inactivation of CtsR<br />
CtsR, the first gene of the clpC operon, was described to conta<strong>in</strong> a putative<br />
helix-turn-helix (HTH) DNA b<strong>in</strong>d<strong>in</strong>g motif <strong>in</strong> low GC, Gram+ bacteria<br />
(Kr€uger et al., 1997). Later, CtsR was identified as the correspond<strong>in</strong>g repressor<br />
for clpC and clpP expression <strong>in</strong> B. subtilis. CtsR directly b<strong>in</strong>ds to the<br />
promoters and recognises the direct heptanucleotide repeat sequence (A/<br />
GGTCA A ANANA/GGTCA A A) (Derre et al., 1999b). This consensus<br />
sequence is highly conserved among low GC, Gram+ bacteria. Moreover,<br />
the ctsR gene itself is also highly conserved. However, CtsR is very specific<br />
for low GC, Gram+ bacteria because no homologues were found <strong>in</strong> either<br />
Gram-negative bacteria or the act<strong>in</strong>obacteria branch.<br />
An <strong>in</strong>itial characterisation <strong>in</strong> B. subtilis revealed that CtsR is only active as<br />
a dimer (Derre et al., 2000). The 2nd and 3rd genes of the tetra-cistronic clpCoperon<br />
<strong>in</strong> B. subtilis were renamed McsA and McsB (modulators of CtsR<br />
activity) accord<strong>in</strong>g to their <strong>in</strong>fluence on CtsR activity. McsB shows homology<br />
to ATP:guanid<strong>in</strong>o phosphotransferases such as arg<strong>in</strong><strong>in</strong>e k<strong>in</strong>ases and <strong>in</strong>hibited<br />
CtsR DNA b<strong>in</strong>d<strong>in</strong>g <strong>in</strong> vitro. 2D Western analysis suggested that CtsR<br />
became phosphorylated by McsB dur<strong>in</strong>g heat stress (Kr€uger et al., 2001).<br />
McsA, a putative z<strong>in</strong>c f<strong>in</strong>ger prote<strong>in</strong>, was shown to be essential for CtsR<br />
activity <strong>in</strong> vivo because CtsR is no longer active <strong>in</strong> a mcsA mutant.<br />
Furthermore, it became clear that CtsR is degraded <strong>in</strong> ClpCP-dependent<br />
manner dur<strong>in</strong>g heat stress (Kr€uger et al., 2001).<br />
ClpE is needed for an efficient and timely re-repression of CtsRdependent<br />
transcription. In a L. lactis clpE mutant, re-repression was
130 ALEXANDER K.W. ELSHOLZ ET AL.<br />
delayed, but still occurred with a time lag. Therefore, ClpE does not seem<br />
solely responsible for the reconstitution of CtsR activity. In addition, the z<strong>in</strong>c<br />
f<strong>in</strong>ger of ClpE was identified to be crucial for re-repression (Varmanen et al.,<br />
2003). This observation was later also confirmed for B. subtilis. Moreover,<br />
the z<strong>in</strong>c f<strong>in</strong>ger of ClpE was l<strong>in</strong>ked to the autonomic ATPase function of ClpE<br />
(Miethke et al., 2006), as was also shown for ClpX <strong>in</strong> E. coli (Banecki et al.,<br />
2001).<br />
McsB was characterised as a tyros<strong>in</strong>e k<strong>in</strong>ase <strong>in</strong> vitro that specifically phosphorylates<br />
McsA, CtsR, ClpC as well as itself (Kirste<strong>in</strong> et al., 2005).<br />
However, McsB k<strong>in</strong>ase activity needs McsA as an activator for efficient<br />
phosphorylation. Specific phosphorylation sites for McsB, tyros<strong>in</strong>e residue<br />
155 and 210, as well as tyros<strong>in</strong>e residues for McsA were described. Inter-<br />
(McsB!McsA) and <strong>in</strong>tramolecular (McsB!McsB) phosphate transfer was<br />
observed. The low-molecular-weight prote<strong>in</strong> tyros<strong>in</strong>e phosphatase<br />
(LMWPTP) YwlE was identified as the cognate McsB phosphatase that<br />
dephosphorylates CtsR, McsA, McsB as well as ClpC <strong>in</strong> vitro. Furthermore,<br />
McsB is the only direct <strong>in</strong>teraction partner of CtsR and McsBdependent<br />
release of CtsR from the DNA <strong>in</strong> vitro is strongly enhanced when<br />
McsB is active as a k<strong>in</strong>ase. Additionally, ClpCP-dependent CtsR degradation<br />
dur<strong>in</strong>g heat stress is also dependent on McsA and McsB. Based on the<br />
observation that ClpC <strong>in</strong>hibits McsB activity and this repression is restricted<br />
when prote<strong>in</strong> aggregates are present, a titration model was postulated<br />
(Kirste<strong>in</strong> et al., 2005). Such a titration model is also known for other heat<br />
shockregulatorssuchass 32 (Bukau, 1993) orHrcA(Mogk et al., 1997), and<br />
seems to be an elegant model for regulation of CtsR activity as well (Fig. 2).<br />
A recent <strong>in</strong> vitro study with CtsR and McsB from Bacillus stearothermophilus<br />
revealed that McsB is an arg<strong>in</strong><strong>in</strong>e k<strong>in</strong>ase <strong>in</strong>stead of a tyros<strong>in</strong>e k<strong>in</strong>ase<br />
(Fuhrmann et al., 2009). Furthermore, the structure of CtsR b<strong>in</strong>d<strong>in</strong>g to its<br />
DNA operator was solved. It was shown that CtsR is a w<strong>in</strong>ged HTH prote<strong>in</strong><br />
where the HTH doma<strong>in</strong> b<strong>in</strong>ds <strong>in</strong> the major groove of the DNA whereas the<br />
b-hairp<strong>in</strong> w<strong>in</strong>g grabs <strong>in</strong>to the m<strong>in</strong>or groove. It was also demonstrated that<br />
McsB phosphorylates CtsR on specific arg<strong>in</strong><strong>in</strong>e residues with<strong>in</strong> the w<strong>in</strong>ged<br />
HTH region <strong>in</strong> vitro. Because the determ<strong>in</strong>ed phospho-sites are also<br />
<strong>in</strong>volved <strong>in</strong> DNA b<strong>in</strong>d<strong>in</strong>g of CtsR, it was thought that McsB is only able to<br />
b<strong>in</strong>d and phosphorylate free CtsR (Fuhrmann et al., 2009).<br />
Such a model supports only a transient <strong>in</strong>duction of gene expression, but<br />
not rapid and strong <strong>in</strong>duction with<strong>in</strong> 1–2 m<strong>in</strong>, as was observed for CtsRdependent<br />
transcription dur<strong>in</strong>g heat stress (Kr€uger et al., 1994). Exclusive<br />
phosphorylation of free CtsR would result <strong>in</strong> slow enhancement of transcription<br />
peak<strong>in</strong>g long after the stress <strong>in</strong>put because <strong>in</strong>activation of CtsR would<br />
depend on prior dissociation of DNA-bound CtsR. In addition, it has long
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 131<br />
[(Figure_2)TD$FIG]<br />
Figure 2 Graphical presentation of regulation and degradation of CtsR <strong>in</strong> the<br />
different low GC, Gram+ orders under specific stress conditions. (a) CtsR <strong>in</strong>activation<br />
and degradation dur<strong>in</strong>g heat stress <strong>in</strong> the order Bacillales. Under control conditions<br />
CtsR b<strong>in</strong>ds to its DNA operator and McsB k<strong>in</strong>ase is repressed by b<strong>in</strong>d<strong>in</strong>g to<br />
ClpC. Elevated temperatures lead to a loss of CtsR DNA b<strong>in</strong>d<strong>in</strong>g. In addition, McsB<br />
is released from ClpC and becomes activated as a k<strong>in</strong>ase by McsA. This activation<br />
results <strong>in</strong> target<strong>in</strong>g of free CtsR for a ClpCP-mediated proteolysis. (b) CtsR <strong>in</strong>activation<br />
and degradation dur<strong>in</strong>g heat stress <strong>in</strong> the order Lactobacillales that misses the<br />
two modulators McsA and McsB. Under control conditions CtsR b<strong>in</strong>ds to its DNA<br />
operator. Elevated temperatures lead to a loss of CtsR DNA b<strong>in</strong>d<strong>in</strong>g and ClpEPmediated<br />
degradation. (c) CtsR <strong>in</strong>activation dur<strong>in</strong>g oxidative stress conditions <strong>in</strong> the<br />
order Bacillales. Under control conditions CtsR b<strong>in</strong>ds to its DNA operator and McsB<br />
k<strong>in</strong>ase is repressed by b<strong>in</strong>d<strong>in</strong>g to ClpC. Dur<strong>in</strong>g oxidative stress critical thiols are<br />
oxidised with<strong>in</strong> the oxidative stress sensor prote<strong>in</strong> McsA. This oxidation disturbs<br />
<strong>in</strong>teraction of McsA/McsB and activates a specific McsB function. Consequently,<br />
McsB is now able to b<strong>in</strong>d and <strong>in</strong>activate DNA-bound CtsR, but CtsR cannot be<br />
degraded due to the <strong>in</strong>active McsB k<strong>in</strong>ase. (d) CtsR <strong>in</strong>activation dur<strong>in</strong>g oxidative<br />
stress <strong>in</strong> the order Lactobacillales. Under control conditions CtsR b<strong>in</strong>ds to its DNA<br />
operator. Oxidative stress leads to oxidation of critical thiols with<strong>in</strong> ClpE. This<br />
oxidation causes an activation of ClpE which is now able to target DNA-bound<br />
CtsR (See Colour Plate Section).
132 ALEXANDER K.W. ELSHOLZ ET AL.<br />
been known that the two modulators McsA and McsB are absent <strong>in</strong> the<br />
Lactobacillales order (Varmanen et al., 2000; Lemos and Burne, 2002).<br />
Recently, Elsholz and co-workers revealed that McsB is not <strong>in</strong>volved <strong>in</strong><br />
the regulation of CtsR activity dur<strong>in</strong>g heat stress <strong>in</strong> vivo, support<strong>in</strong>g a<br />
hypothesis that CtsR activity is identically regulated <strong>in</strong> all low GC, Gram+<br />
bacteria. It was suggested that CtsR is able to sense and respond to elevated<br />
temperatures by act<strong>in</strong>g as a prote<strong>in</strong> thermometer <strong>in</strong> all low GC, Gram+<br />
bacteria, which adjusts its activity <strong>in</strong>tr<strong>in</strong>sically to the surround<strong>in</strong>g temperatures.<br />
CtsR is able to b<strong>in</strong>d to its cognate DNA operator sequence with high<br />
aff<strong>in</strong>ity under control conditions, but DNA b<strong>in</strong>d<strong>in</strong>g under heat shock conditions<br />
is dramatically reduced. Furthermore, the CtsR prote<strong>in</strong> is adapted to<br />
the ecological niche of the specific low GC, Gram+ bacteria, and thus can<br />
respond to the very species-specific heat shock temperatures (Elsholz et al.,<br />
2010).<br />
The auto-<strong>in</strong>activation of CtsR is not an on/off decision, where CtsR activity<br />
is switched off above a specific temperature. CtsR is only partially <strong>in</strong>activated<br />
at modest heat temperatures and becomes fully <strong>in</strong>activated only at<br />
maximal heat stress temperatures, as observed for CtsR from B. subtilis<br />
(Kr€uger et al., 1994), S. aureus (Fleury et al., 2009) and L. lactis (Elsholz<br />
et al., 2010). This mechanism secures that only the needed amounts of heat<br />
shock prote<strong>in</strong>s become expressed under specific circumstances and excludes<br />
that an excess of prote<strong>in</strong> quality systems are expressed under a modest<br />
prote<strong>in</strong> stress which would waste important cellular resources.<br />
The b hairp<strong>in</strong> w<strong>in</strong>g with its tetra-glyc<strong>in</strong>e loop was identified as the region<br />
that is responsible for thermosens<strong>in</strong>g of CtsR <strong>in</strong> all low GC, Gram+ bacteria<br />
(Elsholz et al., 2010). This loop occupies an outstand<strong>in</strong>g position as it is<br />
directly located at the tip of the w<strong>in</strong>g implicat<strong>in</strong>g a potential regulatory role<br />
(Fuhrmann et al., 2009). It has long been known that glyc<strong>in</strong>e residues exhibit<br />
great conformational freedom and can adopt a conformation to a much wider<br />
range than the other am<strong>in</strong>o acid residues due to their free phi and psi angles<br />
(Matthews et al., 1987). As a result, glyc<strong>in</strong>e residues have more backbone<br />
conformational flexibility, which <strong>in</strong>creases the configurational entropy of the<br />
denatured state. Therefore, more free energy is needed to ma<strong>in</strong>ta<strong>in</strong> the<br />
structural <strong>in</strong>tegrity of the native state. This effect results <strong>in</strong> decreased thermostability<br />
of the prote<strong>in</strong> (Elsholz et al., 2010).<br />
4.2. CtsR Inactivation Dur<strong>in</strong>g Oxidative Stress<br />
It has long been known that, <strong>in</strong> addition to heat stress, CtsR is also <strong>in</strong>activated<br />
dur<strong>in</strong>g other stress conditions such as disulfide stress (Leichert et al.,
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 133<br />
2003), H 2O 2 (Mostertz et al., 2004), carbonyl electrophiles (Nguyen et al.,<br />
2009) and low pH (Frees et al., 2003b). As long as a titration model regard<strong>in</strong>g<br />
McsB was applicable, the <strong>in</strong>duction mechanism seemed to be clear and<br />
unique because all these stress conditions should result <strong>in</strong> damage of prote<strong>in</strong><br />
structure which may lead to prote<strong>in</strong> aggregation (Hartl and Hayer-Hartl,<br />
2009). However, s<strong>in</strong>ce the observation that CtsR is an <strong>in</strong>tr<strong>in</strong>sic heat sensor<br />
(Elsholz et al., 2010), the assumption that a common mechanism for CtsR<br />
<strong>in</strong>activation exists for all stress conditions turned out to be obsolete. Thus,<br />
dual activity control of CtsR activity must have evolved for CtsR <strong>in</strong>activation<br />
dur<strong>in</strong>g different stress conditions. This model is also underl<strong>in</strong>ed by the fact<br />
that CtsR-dependent transcription differs significantly dur<strong>in</strong>g oxidative<br />
stress and heat stress (Elsholz et al., manuscript submitted).<br />
A new mechanism for CtsR <strong>in</strong>activation dur<strong>in</strong>g oxidative stress was<br />
uncovered that depends solely on McsB, but not on its k<strong>in</strong>ase activity.<br />
McsB activity dur<strong>in</strong>g oxidative stress is regulated by McsA, which acts as a<br />
redox-sens<strong>in</strong>g prote<strong>in</strong> to adjust McsB function. Not only CtsR activity is<br />
dually regulated, but also McsA acts as a dual regulator of McsB. Under<br />
control and heat stress conditions, McsA and McsB are associated, and<br />
McsA can activate McsB k<strong>in</strong>ase as long as McsB is not <strong>in</strong>hibited by ClpC.<br />
On the other hand, McsA can also act as an <strong>in</strong>hibitor of McsB, and McsB<br />
ceases to <strong>in</strong>activate DNA-bound CtsR, as long as both prote<strong>in</strong>s are associated.<br />
Under oxidative stress conditions, the critical thiols of the second z<strong>in</strong>c<br />
f<strong>in</strong>ger of McsA become oxidised. As a result, these thiols act as a molecular<br />
redox switch to regulate the activity of McsB by decreas<strong>in</strong>g the <strong>in</strong>teraction of<br />
McsA with McsB. When McsB is free of McsA, it is able to b<strong>in</strong>d and<br />
<strong>in</strong>activate DNA-bound CtsR, result<strong>in</strong>g <strong>in</strong> <strong>in</strong>duced transcription of target<br />
genes. But due to the <strong>in</strong>active McsB k<strong>in</strong>ase, CtsR is not degraded. McsA is<br />
processed <strong>in</strong> a ClpCP-dependent manner, with the essential help of the<br />
adaptor prote<strong>in</strong> McsB, result<strong>in</strong>g <strong>in</strong> a cleaved McsA prote<strong>in</strong>, which may<br />
prevent b<strong>in</strong>d<strong>in</strong>g to McsB. Interest<strong>in</strong>gly, reversible oxidation of McsA seems<br />
to be required for correct cleavage of McsA (Elsholz et al., manuscript<br />
submitted).<br />
McsA, with its crucial cyste<strong>in</strong>e residues, is a redox-sens<strong>in</strong>g prote<strong>in</strong> which<br />
specifically regulates the activity of McsB and thus is responsible for a<br />
modest response to oxidative stress. Yet the oxidative stress sens<strong>in</strong>g complex<br />
McsA/B is not present <strong>in</strong> the order of Lactobacillales. The observation<br />
that CtsR-dependent transcription <strong>in</strong> L. lactis is strongly <strong>in</strong>duced<br />
dur<strong>in</strong>g oxidative stress suggests that another mechanism for CtsR <strong>in</strong>activation<br />
exists. It was found that ClpE is essential for CtsR <strong>in</strong>activation<br />
dur<strong>in</strong>g oxidative stress <strong>in</strong> L. lactis, reveal<strong>in</strong>g a new ClpE-mediated <strong>in</strong>activation<br />
mechanism for CtsR. Remarkably, L. lactis clpE expression is
134 ALEXANDER K.W. ELSHOLZ ET AL.<br />
relatively higher under control conditions when compared with B. subtilis<br />
to secure that ClpE is always present and can act as an redox-sensor<br />
prote<strong>in</strong> (Elsholz et al., manuscript submitted). This is due to the fact that<br />
the number of L. lactis CtsR b<strong>in</strong>d<strong>in</strong>g sites <strong>in</strong> front of clpE is lower compared<br />
with B. subtilis (Varmanen et al., 2000). It was demonstrated that<br />
the previously described thiol-dependent cleavage of ClpE by ClpP<br />
(Varmanen et al., 2003) depends on the reversible oxidation of the critical<br />
thiols. Interest<strong>in</strong>gly, this cleavage was not observed for B. subtilis ClpE,<br />
suggest<strong>in</strong>g that L. lactis ClpE specifically ga<strong>in</strong>ed this feature <strong>in</strong> the course<br />
of evolution (Elsholz et al., manuscript submitted).<br />
Signal transduction dur<strong>in</strong>g oxidative stress result<strong>in</strong>g <strong>in</strong> CtsR <strong>in</strong>activation is<br />
mediated by different signall<strong>in</strong>g pathways <strong>in</strong> low GC, Gram+ bacteria,<br />
dependent on the presence or evolutionary development of the redoxsens<strong>in</strong>g<br />
prote<strong>in</strong>s. Nevertheless, both aforementioned mechanisms depend<br />
on critical thiols, which act as nano-switches to adjust prote<strong>in</strong> activity <strong>in</strong> order<br />
to counteract oxidative stress (Elsholz et al., manuscript submitted). Both<br />
pathways are also completely different <strong>in</strong> comparison to heat <strong>in</strong>activation,<br />
demonstrat<strong>in</strong>g dual activity control of CtsR, which may be important for<br />
appropriate adaptation to different stress conditions.<br />
4.3. CtsR Inactivation Dur<strong>in</strong>g Other Stress Conditions<br />
Two dist<strong>in</strong>ct mechanisms are known for the <strong>in</strong>activation of CtsR, a temperature-dependent<br />
one for the response to elevated temperatures and a thioldependent<br />
one for sens<strong>in</strong>g of oxidative stress. Nevertheless, a few stress<br />
conditions which result <strong>in</strong> CtsR <strong>in</strong>activation cannot be expla<strong>in</strong>ed sufficiently<br />
by these two mechanisms. Inactivation of CtsR by prote<strong>in</strong> stress caused by<br />
specific antibiotics such as puromyc<strong>in</strong> (Kr€uger et al., 1996; Frees and Ingmer,<br />
1999) or low pH (Frees et al., 2003b) is not expla<strong>in</strong>able by the two identified<br />
mechanisms. Neither a heat-sens<strong>in</strong>g doma<strong>in</strong> nor critical thiols are known to<br />
respond to prote<strong>in</strong> aggregates. CtsR <strong>in</strong>activation dur<strong>in</strong>g these conditions<br />
may depend on activation of McsB k<strong>in</strong>ase by prote<strong>in</strong> aggregates. However,<br />
one could also speculate that an additional mechanism has become established<br />
for these conditions.<br />
Interest<strong>in</strong>gly, <strong>in</strong>duction of CtsR-dependent prote<strong>in</strong> quality control systems<br />
<strong>in</strong> L. monocytogenes under hydrostatic pressure was shown not to<br />
depend on a transcriptional signall<strong>in</strong>g cascade, but is regulated at the genomic<br />
level by genetic variation. Most of the isolated L. monocytogenes spontaneous<br />
high-hydrostatic pressure (HHP)-tolerant mutants bear a mutation<br />
<strong>in</strong> a specific region of CtsR which results <strong>in</strong> an <strong>in</strong>active CtsR prote<strong>in</strong>
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 135<br />
(Karatzas et al., 2003). The mutated region codes for the b hairp<strong>in</strong> w<strong>in</strong>g and<br />
the mutations are located <strong>in</strong> the tetra-glyc<strong>in</strong>e loop. This loop is encoded <strong>in</strong> L.<br />
monocytogenes by a short sequence repeat of GGT and all mutations were<br />
located with<strong>in</strong> this DNA repeat (Karatzas et al., 2005). Such hot spots of<br />
genetic variation, where Rec-<strong>in</strong>dependent mutations accumulate at high<br />
frequency, have been known for a long time. DNA repeats are very common<br />
<strong>in</strong> this group and most likely cause strand slippage of the DNA polymerase,<br />
thus promot<strong>in</strong>g genetic variability (Treangen et al., 2009). It was demonstrated<br />
for E. coli that such tandem repeats are very common <strong>in</strong> stress<br />
response genes to ensure a moderate stress survival (Rocha et al., 2002). In<br />
addition, such specific DNA repeats <strong>in</strong> virulence genes are of tremendous<br />
relevance for the adaptation of pathogenic bacteria to their specific host<br />
(Moxon et al., 2006).<br />
As noted above, a ctsR mutant displays an <strong>in</strong>creased stress tolerance due<br />
to the permanent <strong>in</strong>duction of the prote<strong>in</strong> quality control genes. Thus, transient<br />
gene silenc<strong>in</strong>g of ctsR would enhance fitness and stress tolerance and<br />
give the bacteria an advantage <strong>in</strong> the adaptation to a new ecological niche<br />
and their specific conditions. In fact, <strong>in</strong>activation of L. monocytogenes ctsR<br />
by a s<strong>in</strong>gle codon deletion enhances the stress tolerance and decreases the<br />
virulence (Karatzas et al., 2003), most probably to escape the immune system<br />
and persist with<strong>in</strong> the host.<br />
It seems likely that evolution has allowed an emergency exit for low GC,<br />
Gram+ bacteria to cope with conformational stress conditions when activation<br />
of prote<strong>in</strong> quality control genes through regular transcriptional<br />
regulation of CtsR is not applicable. As a consequence, a regulatory layer<br />
at the genomic level was <strong>in</strong>troduced to give the bacteria an opportunity to<br />
escape stress conditions by <strong>in</strong>creas<strong>in</strong>g the amount of prote<strong>in</strong> quality systems.<br />
Hence, specific mutations accumulate <strong>in</strong> a DNA repeat with<strong>in</strong> the<br />
ctsR gene dur<strong>in</strong>g evolutionary pressure to <strong>in</strong>activate CtsR and to give<br />
the bacteria a growth advantage, ensur<strong>in</strong>g that a subset of cells survive<br />
the stress.<br />
However, such a stress-specific genetic mutation is only shown for<br />
piezo-tolerant SCV of L. monocytogenes (Karatzas et al., 2003) and not<br />
for other low GC, Gram+ bacteria. In addition, <strong>in</strong> piezo-tolerant SCV of<br />
S. aureus no mutations for CtsR were detected (Karatzas et al., 2007), and<br />
the DNA repeats of the tetra-glyc<strong>in</strong>e doma<strong>in</strong> <strong>in</strong> ctsR are not so conserved<br />
<strong>in</strong> low GC, Gram+ bacteria as at the prote<strong>in</strong> level (Karatzas et al., 2005).<br />
To date, only these two studies have been reported, but this could be a<br />
widely used mechanism. Therefore, additional genetic studies must be<br />
performed to <strong>in</strong>vestigate more prote<strong>in</strong> stress conditions <strong>in</strong> different low<br />
GC, Gram+ bacteria.
136 ALEXANDER K.W. ELSHOLZ ET AL.<br />
5. CONTROL OF CTSR DEGRADATION BY THE REGULATED<br />
ADAPTOR McsB<br />
Controlled degradation of key transcriptional regulators plays a critical role<br />
<strong>in</strong> many bacterial regulatory circuits (Gottesman, 2003) However, CtsR<br />
degradation is not needed for <strong>in</strong>duction of ctsR-dependent genes. In vivo<br />
and <strong>in</strong> vitro experiments demonstrated that CtsR is a substrate for the ClpCP<br />
protease (Kr€uger et al., 2001), but CtsR is very stable under control conditions<br />
and only becomes degraded under stress conditions such as heat or<br />
puromyc<strong>in</strong> treatment (Kr€uger et al., 2001). It was revealed that the two<br />
modulators of CtsR activity, McsA and McsB, <strong>in</strong> B. subtilis are necessary<br />
for CtsR degradation dur<strong>in</strong>g heat stress (Kirste<strong>in</strong> et al., 2005). It was shown<br />
by <strong>in</strong> vitro experiments that specifically McsB k<strong>in</strong>ase is essential for CtsR<br />
degradation, but CtsR phosphorylation is not sufficient for CtsR degradation<br />
(Kirste<strong>in</strong> et al., 2007). Additionally, YwlE was identified as the cognate<br />
phosphatase of the McsB k<strong>in</strong>ase <strong>in</strong> vitro (Kirste<strong>in</strong> et al., 2005).<br />
Recently, it has been demonstrated that McsB k<strong>in</strong>ase activity is needed for<br />
an efficient CtsR degradation <strong>in</strong> vivo, underl<strong>in</strong><strong>in</strong>g the role of McsB as an<br />
adaptor for ClpC. CtsR degradation depends on the activation of McsB as an<br />
adaptor prote<strong>in</strong> by auto-phosphorylation and not on a direct CtsR phosphorylation.<br />
Only phosphorylated McsB is able to b<strong>in</strong>d CtsR. Consequently, a<br />
sophisticated regulatory two-step mechanism for CtsR degradation was<br />
uncovered as CtsR is only accessible for McsB when it is not bound to its<br />
DNA operator, and McsB can only target free CtsR when heat activated as<br />
an adaptor prote<strong>in</strong> (Elsholz et al., 2010).<br />
YwlE was shown to counteract McsB adaptor activity <strong>in</strong> vivo. When,<br />
dephosphorylation of McsB by YwlE failed, McsB adaptor activity is shutdown<br />
rapidly by degradation when phosphorylated. This regulatory switch<br />
prevents degradation of re-activated CtsR (Elsholz et al., 2010).<br />
CtsR was also shown to be rapidly degraded dur<strong>in</strong>g heat stress <strong>in</strong> the order<br />
of Lactobacillales, where mcsA and mcsB are not present. CtsR degradation<br />
<strong>in</strong> these bacteria was shown to depend on ClpE (Elsholz et al., 2010), but the<br />
precise mechanism of CtsR target<strong>in</strong>g and degradation, that is whether ClpE<br />
is heat activated or not, rema<strong>in</strong>s to be uncovered.<br />
6. SUMMARY AND OUTLOOK<br />
The regulation of CtsR activity and its controlled degradation has become<br />
one of the best-studied regulatory heat stress mechanisms <strong>in</strong> low GC, Gram+
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 137<br />
bacteria. In recent years, CtsR was established as a model system par excellence<br />
to study precise and f<strong>in</strong>e-tuned regulatory mechanisms <strong>in</strong> molecular<br />
detail. Generally, elucidation of CtsR activity provides deeper <strong>in</strong>sights <strong>in</strong>to a<br />
fundamental, highly conserved and global bacterial stress response system.<br />
Nevertheless, ‘solved’ problems tend to raise more questions. Thus, a further<br />
characterisation of the structural details of how CtsR gets <strong>in</strong>activated dur<strong>in</strong>g<br />
heat stress and how CtsR is re-activated as a DNA b<strong>in</strong>d<strong>in</strong>g repressor, as well<br />
as a molecular characterisation of CtsR degradation, rema<strong>in</strong>s an <strong>in</strong>terest<strong>in</strong>g<br />
subject for future studies.<br />
The identification of other prote<strong>in</strong> stress conditions that lead to CtsR<br />
<strong>in</strong>activation, as well as the underly<strong>in</strong>g molecular mechanisms, will provide<br />
a deeper understand<strong>in</strong>g of the function and role of CtsR <strong>in</strong> low GC, Gram+<br />
bacteria. In addition, new members of the CtsR regulon will probably also<br />
contribute to the physiological important function of CtsR. One of the major<br />
challenges is to connect the different mechanisms perta<strong>in</strong><strong>in</strong>g to the regulation<br />
of CtsR activity and stability, to <strong>in</strong>vestigate why different mechanisms<br />
have become established for the same physiological function <strong>in</strong> the different<br />
low GC, Gram+ orders and to place all the <strong>in</strong>formation <strong>in</strong>to a systems<br />
biology context.<br />
ACKNOWLEDGEMENT<br />
The authors are grateful to Volker Br€ozel (South Dakota State Univ., USA)<br />
for critical read<strong>in</strong>g of the manuscript and helpful comments.<br />
REFERENCES<br />
Baker, T.A. and Sauer, R.T. (2006) ATP-dependent proteases of bacteria: recognition<br />
logic and operat<strong>in</strong>g pr<strong>in</strong>ciples. Trend Biochem. Sci. 31, 647–653.<br />
Banecki, B., Wawrzynow, A., Puzewicz, J., Georgopoulos, C. and Zylicz, M. (2001)<br />
Structure–function analysis of the z<strong>in</strong>c-b<strong>in</strong>d<strong>in</strong>g region of the Clpx molecular chaperone.<br />
J. Biol. Chem. 276, 18843–18848.<br />
Beltramo, C., Grandvalet, C., Pierre, F. and Guzzo, J. (2004) Evidence for multiple<br />
levels of regulation of Oenococcus oeni clpP-clpL locus expression <strong>in</strong> response to<br />
stress. J. Bacteriol. 186, 2200–2205.<br />
Borezee, E., Pellegr<strong>in</strong>i, E., Beretti, J.L. and Berche, P. (2001) SvpA, a novel surface<br />
virulence-associated prote<strong>in</strong> required for <strong>in</strong>tracellular survival of Listeria monocytogenes.<br />
Microbiology (Read<strong>in</strong>g Engl.) 147, 2913–2923.<br />
B€ottcher, T. and Sieber, S.A. (2008) Beta-lactones as specific <strong>in</strong>hibitors of ClpP<br />
attenuate the production of extracellular virulence factors of Staphylococcus<br />
aureus. J. Am. Chem. Soc. 130, 14400–14401.
138 ALEXANDER K.W. ELSHOLZ ET AL.<br />
Br€otz-Oesterhelt, H., Beyer, D., Kroll, H.-P., Endermann, R., Ladel, C., Schroeder,<br />
W., H<strong>in</strong>zen, B., Raddatz, S., Paulsen, H., Henn<strong>in</strong>ger, K., Bandow, J.E., Sahl, H.-G.<br />
and Labisch<strong>in</strong>ski, H. (2005) Dysregulation of bacterial proteolytic mach<strong>in</strong>ery by a<br />
new class of antibiotics. Nat. Med. 11, 1082–1087.<br />
Bukau, B. (1993) Regulation of the Escherichia coli heat-shock response. Mol.<br />
Microbiol. 9, 671–680.<br />
Charpentier, E., Novak, R. and Tuomanen, E. (2000) Regulation of growth <strong>in</strong>hibition<br />
at high temperature, autolysis, transformation and adherence <strong>in</strong> Streptococcus<br />
pneumoniae by clpC. Mol. Microbiol. 37, 717–726.<br />
Chastanet, A., Derre, I., Nair, S. and Msadek, T. (2004) clpB, a novel member of the<br />
Listeria monocytogenes CtsR regulon, is <strong>in</strong>volved <strong>in</strong> virulence but not <strong>in</strong> general<br />
stress tolerance. J. Bacteriol. 186, 1165–1174.<br />
Chastanet, A., Fert, J. and Msadek, T. (2003) Comparative genomics reveal novel<br />
heat shock regulatory mechanisms <strong>in</strong> Staphylococcus aureus and other Grampositive<br />
bacteria. Mol. Microbiol. 47, 1061–1073.<br />
Chastanet, A. and Msadek, T. (2003) ClpP of Streptococcus salivarius is a novel<br />
member of the dually regulated class of stress response genes <strong>in</strong> gram-positive<br />
bacteria. J. Bacteriol. 185, 683–687.<br />
Chastanet, A., Prudhomme, M., Claverys, J.P. and Msadek, T. (2001) Regulation of<br />
Streptococcus pneumoniae clp genes and their role <strong>in</strong> competence development<br />
and stress survival. J. Bacteriol. 183, 7295–7307.<br />
Chatterjee, I., Becker, P., Grundmeier, M., Bischoff, M., Somerville, G.A., Peters, G.,<br />
S<strong>in</strong>ha, B., Harraghy, N., Proctor, R.A. and Herrmann, M. (2005) Staphylococcus<br />
aureus ClpC is required for stress resistance, aconitase activity, growth recovery,<br />
and death. J. Bacteriol. 187, 4488–4496.<br />
Craig, E.A. (1985) The heat shock response. CRC Crit. Rev. Biochem. 18, 239–280.<br />
Darw<strong>in</strong>, K.H. (2009) Prokaryotic ubiquit<strong>in</strong>-like prote<strong>in</strong> (Pup), proteasomes and<br />
pathogenesis. Nat. Rev. Micro. 7, 485–491.<br />
Derre, I., Rapoport, G., Dev<strong>in</strong>e, K., Rose, M. and Msadek, T. (1999a) ClpE, a novel<br />
type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis.<br />
Mol. Microbiol. 32, 581–593.<br />
Derre, I., Rapoport, G. and Msadek, T. (1999b) CtsR, a novel regulator of stress and<br />
heat shock response, controls clp and molecular chaperone gene expression <strong>in</strong><br />
gram positive bacteria. Mol. Microbiol. 31, 117–131.<br />
Derre, I., Rapoport, G. and Msadek, T. (2000) The CtsR regulator of stress response<br />
is active as a dimer and specifically degraded <strong>in</strong> vivo at 37 degrees C. Mol.<br />
Microbiol. 38, 335–347.<br />
Donegan, N.P., Thompson, E.T., Fu, Z. and Cheung, A.L. (2010) Proteolytic regulation<br />
of tox<strong>in</strong>-antitox<strong>in</strong> systems by ClpPC <strong>in</strong> Staphylococcus aureus. J. Bacteriol.<br />
192, 1416–1422.<br />
Elsholz, A.K.W., Hempel, K., P€other, D.C., Becher, D., Hecker, M. and Gerth, U. A<br />
new mechanism for CtsR <strong>in</strong>activation dur<strong>in</strong>g oxidative stress <strong>in</strong> low GC, Gram+<br />
bacteria. Manuscript Submitted.<br />
Elsholz, A.K.W., Michalik, S., Z€uhlke, D., Hecker, M. and Gerth, U. (2010) CtsR, the<br />
Gram positive master regulator of prote<strong>in</strong> quality control, feels the heat. EMBO J<br />
Fiocco, D., Capozzi, V., Coll<strong>in</strong>s, M., Gallone, A., Hols, P., Guzzo, J., Weidmann, S.,<br />
Rieu, A., Msadek, T. and Spano, G. (2010) Characterization of the CtsR stress<br />
response regulon <strong>in</strong> Lactobacillus plantarum. J. Bacteriol. 192, 896–900.
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 139<br />
Fiocco, D., Coll<strong>in</strong>s, M., Muscariello, L., Hols, P., Kleerebezem, M., Msadek, T. and<br />
Spano, G. (2008) The Lactobacillus plantarum ftsH gene is a novel member of the<br />
CtsR stress response regulon. J. Bacteriol. 191, 1688–1694.<br />
Fleury, B., Kelley, W.L., Lew, D., G€otz, F., Proctor, R.A. and Vaudaux, P. (2009)<br />
Transcriptomic and metabolic responses of Staphylococcus aureus exposed to<br />
supra-physiological temperatures. BMC Microbiol. 9, 76.<br />
Frees, D., Chastanet, A., Qazi, S., Sørensen, K., Hill, P., Msadek, T. and Ingmer, H. (2004)<br />
Clp ATPases are required for stress tolerance, <strong>in</strong>tracellular replication and biofilm formation<br />
<strong>in</strong> Staphylococcus aureus. Mol. Microbiol 54, 1445–1462.<br />
Frees, D. and Ingmer, H. (1999) ClpP participates <strong>in</strong> the degradation of misfolded<br />
prote<strong>in</strong> <strong>in</strong> Lactococcus lactis. Mol. Microbiol. 31, 79–87.<br />
Frees, D., Qazi, S.N.A., Hill, P.J. and Ingmer, H. (2003a) Alternative roles of ClpX<br />
and ClpP <strong>in</strong> Staphylococcus aureus stress tolerance and virulence. Mol. Microbiol.<br />
48, 1565–1578.<br />
Frees, D., Savijoki, K., Varmanen, P. and Ingmer, H. (2007) Clp ATPases and ClpP<br />
proteolytic complexes regulate vital biological processes <strong>in</strong> low GC, Grampositive<br />
bacteria. Mol. Microbiol. 63, 1285–1295.<br />
Frees, D., Vogensen, F.K. and Ingmer, H. (2003b) Identification of prote<strong>in</strong>s <strong>in</strong>duced<br />
at low pH <strong>in</strong> Lactococcus lactis. Int. J. Food Microbiol. 87, 293–300.<br />
Fuhrmann, J., Schmidt, A., Spiess, S., Lehner, A., Turgay, K., Mechtler, K.,<br />
Charpentier, E. and Clausen, T. (2009) McsB is a prote<strong>in</strong> arg<strong>in</strong><strong>in</strong>e k<strong>in</strong>ase that<br />
phosphorylates and <strong>in</strong>hibits the heat-shock regulator CtsR. Science 324,<br />
1323–1327.<br />
Gaillot, O., Pellegr<strong>in</strong>i, E., Bregenholt, S., Nair, S. and Berche, P. (2000) The ClpP<br />
ser<strong>in</strong>e protease is essential for the <strong>in</strong>tracellular parasitism and virulence of Listeria<br />
monocytogenes. Mol. Microbiol. 35, 1286–1294.<br />
Gerth, U., Kirste<strong>in</strong>, J., Mostertz, J., Waldm<strong>in</strong>ghaus, T., Miethke, M., Kock, H. and<br />
Hecker, M. (2004) F<strong>in</strong>e-tun<strong>in</strong>g <strong>in</strong> regulation of Clp prote<strong>in</strong> content <strong>in</strong> Bacillus<br />
subtilis. J. Bacteriol. 186, 179–191.<br />
Gerth, U., Kock, H., Kusters, I., Michalik, S., Switzer, R.L. and Hecker, M. (2008) Clp<br />
dependent proteolysis down-regulates central metabolic pathways <strong>in</strong> glucose<br />
starved Bacillus subtilis. J. Bacteriol. 190, 321–331.<br />
Gerth, U., Kr€uger, E., Derre, I., Msadek, T. and Hecker, M. (1998) Stress <strong>in</strong>duction of<br />
the Bacillus subtilis clpP gene encod<strong>in</strong>g a homologue of the proteolytic component<br />
of the Clp protease and the <strong>in</strong>volvement of ClpP and ClpX <strong>in</strong> stress tolerance. Mol.<br />
Microbiol. 28, 787–802.<br />
Gerth, U., Wipat, A., Harwood, C.R., Carter, N., Emmerson, P.T. and Hecker, M.<br />
(1996) Sequence and transcriptional analysis of clpX, a class-III heat-shock gene<br />
of Bacillus subtilis. Gene 181, 77–83.<br />
Gertz, S., Engelmann, S., Schmid, R., Ziebandt, A.K., Tischer, K., Scharf, C., Hacker,<br />
J. and Hecker, M. (2000) Characterization of the sigma(B) regulon <strong>in</strong><br />
Staphylococcus aureus. J. Bacteriol. 182, 6983–6991.<br />
Geth<strong>in</strong>g, M.J. and Sambrook, J. (1992) Prote<strong>in</strong> fold<strong>in</strong>g <strong>in</strong> the cell. Nature 355,33–45.<br />
Gottesman, S. (2003) Proteolysis <strong>in</strong> bacterial regulatory circuits. Annu. Rev. Cell Dev.<br />
Biol. 19, 565–587.<br />
Grandvalet, C., Coucheney, F., Beltramo, C. and Guzzo, J. (2005) CtsR is the master<br />
regulator of stress response gene expression <strong>in</strong> Oenococcus oeni. J. Bacteriol. 187,<br />
5614–5623.
140 ALEXANDER K.W. ELSHOLZ ET AL.<br />
Hahn, J., Kramer, N., Briley, K. and Dubnau, D. (2009) McsA and B mediate the<br />
delocalization of competence prote<strong>in</strong>s from the cell poles of Bacillus subtilis. Mol.<br />
Microbiol. 72, 202–215.<br />
Hartl, F.U. and Hayer-Hartl, M. (2009) Converg<strong>in</strong>g concepts of prote<strong>in</strong> fold<strong>in</strong>g <strong>in</strong><br />
vitro and <strong>in</strong> vivo. Nat. Struct. Mol. Biol. 16, 574–581.<br />
Hecker, M., Pane-Farre, J. and V€olker, U. (2007) SigB-dependent general stress<br />
response <strong>in</strong> Bacillus subtilis and related gram-positive bacteria. Annu. Rev.<br />
Microbiol. 61, 215–236.<br />
Homuth, G., Masuda, S., Mogk, A., Kobayashi, Y. and Schumann, W. (1997) The<br />
dnaK operon of Bacillus subtilis is heptacistronic. J. Bacteriol. 179, 1153–1164.<br />
Hu, Y., Raengpradub, S., Schwab, U., Loss, C., Orsi, R.H., Wiedmann, M. and Boor,<br />
K.J. (2007) Phenotypic and transcriptomic analyses demonstrate <strong>in</strong>teractions<br />
between the transcriptional regulators CtsR and Sigma B <strong>in</strong> Listeria monocytogenes.<br />
Appl. Environ. Microbiol. 73, 7967–7980.<br />
H€ufner, E. and Hertel, C. (2008) Improvement of raw sausage fermentation by stress<br />
condition<strong>in</strong>g of the starter organism Lactobacillus sakei. Curr. Microbiol. 57,<br />
490–496.<br />
H€ufner, E., Markieton, T., Chaillou, S., Crutz-Le Coq, A.-M., Zagorec, M. and<br />
Hertel, C. (2007) Identification of Lactobacillus sakei genes <strong>in</strong>duced dur<strong>in</strong>g meat<br />
fermentation and their role <strong>in</strong> survival and growth. Appl. Environ. Microbiol. 73,<br />
2522–2531.<br />
Ibrahim, Y.M., Kerr, A.R., Silva, N.A. and Mitchell, T.J. (2005) Contribution of the<br />
ATP dependent protease ClpCP to the autolysis and virulence of Streptococcus<br />
pneumoniae. Infect. Immun. 73, 730–740.<br />
Ingmer, H., Vogensen, F.K., Hammer, K. and Kilstrup, M. (1999) Disruption and<br />
analysis of the clpB, clpC, and clpE genes <strong>in</strong> Lactococcus lactis: ClpE, a new Clp<br />
family <strong>in</strong> gram-positive bacteria. J. Bacteriol. 181, 2075–2083.<br />
Jenn<strong>in</strong>gs, L.D., Lun, D.S., Medard, M. and Licht, S. (2008) ClpP hydrolyzes a prote<strong>in</strong><br />
substrate processively <strong>in</strong> the absence of the ClpA ATPase: mechanistic studies of<br />
ATP-<strong>in</strong>dependent proteolysis. Biochemistry 47, 11536–11546.<br />
Kajfasz, J.K., Mart<strong>in</strong>ez, A.R., Rivera-Ramos, I., Abranches, J., Koo, H., Quivey, R.G.<br />
and Lemos, J.A. (2009) Role of Clp prote<strong>in</strong>s <strong>in</strong> expression of virulence properties<br />
of Streptococcus mutans. J. Bacteriol. 191, 2060–2068.<br />
Karatzas, K.A.G., Valdramidis, V.P. and Wells-Bennik, M.H.J. (2005) Cont<strong>in</strong>gency<br />
locus <strong>in</strong> ctsR of Listeria monocytogenes Scott A: a strategy for occurrence of<br />
abundant piezo tolerant isolates with<strong>in</strong> clonal populations. Appl. Environ.<br />
Microbiol. 71, 8390–8396.<br />
Karatzas, K.A.G., Wouters, J.A., Gahan, C.G.M., Hill, C., Abee, T. and Bennik, M.<br />
H.J. (2003) The CtsR regulator of Listeria monocytogenes conta<strong>in</strong>s a variant<br />
glyc<strong>in</strong>e repeat region that affects piezo tolerance, stress resistance, motility and<br />
virulence. Mol. Microbiol. 49, 1227–1238.<br />
Karatzas, K.A.G., Zervos, A., Tassou, C.C., Mallidis, C.G. and Humphrey, T.J.<br />
(2007) Piezotolerant small-colony variants with <strong>in</strong>creased thermotolerance, antibiotic<br />
susceptibility, and low <strong>in</strong>vasiveness <strong>in</strong> a clonal Staphylococcus aureus population.<br />
Appl. Environ. Microbiol. 73, 1873–1881.<br />
Kim, Y.I., Levchenko, I., Fraczkowska, K., Woodruff, R.V., Sauer, R.T. and Baker, T.<br />
A. (2001) Molecular determ<strong>in</strong>ants of complex formation between Clp/Hsp100<br />
ATPases and the ClpP peptidase. Nat. Struct. Biol. 8, 230–233.
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 141<br />
Kirste<strong>in</strong>, J., Dougan, D.A., Gerth, U., Hecker, M. and Turgay, K. (2007) The tyros<strong>in</strong>e<br />
k<strong>in</strong>ase McsB is a regulated adaptor prote<strong>in</strong> for ClpCP. EMBO J. 26, 2061–2070.<br />
Kirste<strong>in</strong>, J., Moliere, N., Dougan, D.A. and Turgay, K. (2009) Adapt<strong>in</strong>g the mach<strong>in</strong>e:<br />
adaptor prote<strong>in</strong>s for Hsp100/Clp and AAA+ proteases. Nat. Rev. Micro. 7,589–599.<br />
Kirste<strong>in</strong>, J., Schlothauer, T., Dougan, D.A., Lilie, H., Tischendorf, G., Mogk, A.,<br />
Bukau, B. and Turgay, K. (2006) Adaptor prote<strong>in</strong> controlled oligomerization<br />
activates the AAA+ prote<strong>in</strong> ClpC. EMBO J. 25, 1481–1491.<br />
Kirste<strong>in</strong>, J., Z€uhlke, D., Gerth, U., Turgay, K. and Hecker, M. (2005) A tyros<strong>in</strong>e<br />
k<strong>in</strong>ase and its activator control the activity of the CtsR heat shock repressor <strong>in</strong> B.<br />
subtilis. EMBO J. 24, 3435–3445.<br />
Kock, H., Gerth, U. and Hecker, M. (2004a) MurAA, catalys<strong>in</strong>g the first committed<br />
step <strong>in</strong> peptidoglycan biosynthesis, is a target of Clp-dependent proteolysis <strong>in</strong><br />
Bacillus subtilis. Mol. Microbiol. 51, 1087–1102.<br />
Kock, H., Gerth, U. and Hecker, M. (2004b) The ClpP peptidase is the major determ<strong>in</strong>ant<br />
of bulk prote<strong>in</strong> turnover <strong>in</strong> Bacillus subtilis. J. Bacteriol. 186, 5856–5864.<br />
Kr€uger, E., Msadek, T. and Hecker, M. (1996) Alternate promoters direct stress<strong>in</strong>duced<br />
transcription of the Bacillus subtilis clpC operon. Mol. Microbiol. 20,<br />
713–723.<br />
Kr€uger, E., Msadek, T., Ohlmeier, S. and Hecker, M. (1997) The Bacillus subtilis clpC<br />
operon encodes DNA repair and competence prote<strong>in</strong>s. Microbiology (Read<strong>in</strong>g<br />
Engl.) 143(Pt 4); 1309–1316.<br />
Kr€uger, E., V€olker, U. and Hecker, M. (1994) Stress <strong>in</strong>duction of clpC <strong>in</strong> Bacillus<br />
subtilis and its <strong>in</strong>volvement <strong>in</strong> stress tolerance. J. Bacteriol. 176, 3360–3367.<br />
Kr€uger, E., Witt, E., Ohlmeier, S., Hanschke, R. and Hecker, M. (2000) The clp<br />
proteases of Bacillus subtilis are directly <strong>in</strong>volved <strong>in</strong> degradation of misfolded<br />
prote<strong>in</strong>s. J. Bacteriol. 182, 3259–3265.<br />
Kr€uger, E., Z€uhlke, D., Witt, E., Ludwig, H. and Hecker, M. (2001) Clp-mediated<br />
proteolysis <strong>in</strong> Gram-positive bacteria is autoregulated by the stability of a repressor.<br />
EMBO J. 20, 852–863.<br />
Kwon, H.-Y., Kim, S.-W., Choi, M.-H., Ogunniyi, A.D., Paton, J.C., Park, S.-H., Pyo,<br />
S.-N. and Rhee, D.-K. (2003) Effect of heat shock and mutations <strong>in</strong> ClpL and ClpP<br />
on virulence gene expression <strong>in</strong> Streptococcus pneumoniae. Infect. Immun. 71,<br />
3757–3765.<br />
Leichert, L.I.O., Scharf, C. and Hecker, M. (2003) Global characterization of disulfide<br />
stress <strong>in</strong> Bacillus subtilis. J. Bacteriol. 185, 1967–1975.<br />
Lemos, J.A., Luzardo, Y. and Burne, R.A. (2007) Physiologic effects of forced downregulation<br />
of dnaK and groEL expression <strong>in</strong> Streptococcus mutans. J. Bacteriol.<br />
189, 1582–1588.<br />
Lemos, J.A.C. and Burne, R.A. (2002) Regulation and physiological significance of<br />
ClpC and ClpP <strong>in</strong> Streptococcus mutans. J. Bacteriol. 184, 6357–6366.<br />
Matthews, B.W., Nicholson, H. and Becktel, W.J. (1987) Enhanced prote<strong>in</strong> thermostability<br />
from site-directed mutations that decrease the entropy of unfold<strong>in</strong>g. Proc.<br />
Natl. Acad. Sci. U.S.A. 84, 6663–6667.<br />
Miethke, M., Hecker, M. and Gerth, U. (2006) Involvement of Bacillus subtilis ClpE<br />
<strong>in</strong> CtsR degradation and prote<strong>in</strong> quality control. J. Bacteriol. 188, 4610–4619.<br />
Mogk, A., Homuth, G., Scholz, C., Kim, L., Schmid, F.X. and Schumann, W. (1997)<br />
The GroE chaperon<strong>in</strong> mach<strong>in</strong>e is a major modulator of the CIRCE heat shock<br />
regulon of Bacillus subtilis. EMBO J. 16, 4579–4590.
142 ALEXANDER K.W. ELSHOLZ ET AL.<br />
Mostertz, J., Scharf, C., Hecker, M. and Homuth, G. (2004) Transcriptome and<br />
proteome analysis of Bacillus subtilis gene expression <strong>in</strong> response to superoxide<br />
and peroxide stress. Microbiology (Read<strong>in</strong>g Engl.) 150, 497–512.<br />
Moxon, R., Bayliss, C. and Hood, D. (2006) Bacterial cont<strong>in</strong>gency loci: the role of<br />
simple sequence DNA repeats <strong>in</strong> bacterial adaptation. Annu. Rev. Genet. 40,<br />
307–333.<br />
Msadek, T., Dartois, V., Kunst, F., Herbaud, M.L., Denizot, F. and Rapoport, G.<br />
(1998) ClpP of Bacillus subtilis is required for competence development, motility,<br />
degradative enzyme synthesis, growth at high temperature and sporulation. Mol.<br />
Microbiol. 27, 899–914.<br />
Msadek, T., Kunst, F. and Rapoport, G. (1994) MecB of Bacillus subtilis, a member of<br />
the ClpC ATPase family, is a pleiotropic regulator controll<strong>in</strong>g competence gene<br />
expression and growth at high temperature. Proc. Natl. Acad. Sci. U.S.A. 91,<br />
5788–5792.<br />
Nair, S., Derre, I., Msadek, T., Gaillot, O. and Berche, P. (2000a) CtsR controls class<br />
III heat shock gene expression <strong>in</strong> the human pathogen Listeria monocytogenes.<br />
Mol. Microbiol. 35, 800–811.<br />
Nair, S., Frehel, C., Nguyen, L., Escuyer, V. and Berche, P. (1999) ClpE, a novel<br />
member of the HSP100 family, is <strong>in</strong>volved <strong>in</strong> cell division and virulence of Listeria<br />
monocytogenes. Mol. Microbiol. 31, 185–196.<br />
Nair, S., Milohanic, E. and Berche, P. (2000b) ClpC ATPase is required for cell<br />
adhesion and <strong>in</strong>vasion of Listeria monocytogenes. Infect. Immun. 68, 7061–7068.<br />
Nakano, S., Zheng, G., Nakano, M.M. and Zuber, P. (2002) Multiple pathways of Spx<br />
(YjbD) proteolysis <strong>in</strong> Bacillus subtilis. J. Bacteriol. 184, 3664–3670.<br />
Neher, S.B., Villen, J., Oakes, E.C., Bakalarski, C.E., Sauer, R.T., Gygi, S.P. and<br />
Baker, T.A. (2006) Proteomic profil<strong>in</strong>g of ClpXP substrates after DNA damage<br />
reveals extensive <strong>in</strong>stability with<strong>in</strong> SOS regulon. Mol. Cell 22, 193–204.<br />
Neuwald, A.F., Arav<strong>in</strong>d, L., Spouge, J.L. and Koon<strong>in</strong>, E.V. (1999) AAA+: A class of<br />
chaperone-like ATPases associated with the assembly, operation, and disassembly<br />
of prote<strong>in</strong> complexes. Genome Res. 9, 27–43.<br />
Nguyen, T.T.H., Eiamphungporn, W., M€ader, U., Liebeke, M., Lalk, M., Hecker, M.,<br />
Helmann, J.D. and Antelmann, H. (2009) Genome-wide responses to carbonyl<br />
electrophiles <strong>in</strong> Bacillus subtilis: control of the thiol-dependent formaldehyde<br />
dehydrogenase AdhA and cyste<strong>in</strong>e prote<strong>in</strong>ase YraA by the MerR-family regulator<br />
YraB (AdhR). Mol. Microbiol. 71, 876–894.<br />
Ogura, M. and Tsukahara, K. (2010) Autoregulation of the Bacillus subtilis response<br />
regulator gene degU is coupled with the proteolysis of DegU-P by ClpCP. Mol.<br />
Microbiol. 75, 1592–1596.<br />
Pan, Q., Gars<strong>in</strong>, D.A. and Losick, R. (2001) Self-re<strong>in</strong>forc<strong>in</strong>g activation of a cellspecific<br />
transcription factor by proteolysis of an anti-sigma factor <strong>in</strong> B. subtilis.<br />
Mol. Cell 8, 873–883.<br />
Pickart, C.M. and Cohen, R.E. (2004) Proteasomes and their k<strong>in</strong>: proteases <strong>in</strong> the<br />
mach<strong>in</strong>e age. Nat. Rev. Mol. Cell Biol. 5, 177–187.<br />
Qoronfleh, M.W., Bortner, C.A., Schwartzberg, P. and Wilk<strong>in</strong>son, B.J. (1998)<br />
Enhanced levels of Staphylococcus aureus stress prote<strong>in</strong> GroEL and DnaK homologs<br />
early <strong>in</strong> <strong>in</strong>fection of human epithelial cells. Infect. Immun. 66, 3024–3027.<br />
Qoronfleh, M.W., Weraarchakul, W. and Wilk<strong>in</strong>son, B.J. (1993) Antibodies to a<br />
range of Staphylococcus aureus and Escherichia coli heat shock prote<strong>in</strong>s <strong>in</strong> sera<br />
from patients with S. aureus endocarditis. Infect. Immun. 61, 1567–1570.
REGULATION OF CtsR ACTIVITY IN LOW GC, GRAM+ BACTERIA 143<br />
Robertson, G.T., Ng, W.-L., Foley, J., Gilmour, R. and W<strong>in</strong>kler, M.E. (2002) Global<br />
transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and<br />
their effects on physiology and virulence. J. Bacteriol. 184, 3508–3520.<br />
Rocha, E.P.C., Matic, I. and Taddei, F. (2002) Over-representation of repeats <strong>in</strong> stress<br />
response genes: a strategy to <strong>in</strong>crease versatility under stressful conditions?<br />
Nucleic Acids Res. 30, 1886–1894.<br />
Rouquette, C., de Chastellier, C., Nair, S. and Berche, P. (1998) The ClpC ATPase of<br />
Listeria monocytogenes is a general stress prote<strong>in</strong> required for virulence and<br />
promot<strong>in</strong>g early bacterial escape from the phagosome of macrophages. Mol.<br />
Microbiol. 27, 1235–1245.<br />
Rouquette, C., Ripio, M.T., Pellegr<strong>in</strong>i, E., Bolla, J.M., Tascon, R.I., Vazquez-Boland,<br />
J.A. and Berche, P. (1996) Identification of a ClpC ATPase required for stress<br />
tolerance and <strong>in</strong> vivo survival of Listeria monocytogenes. Mol. Microbiol. 21,<br />
977–987.<br />
Sauer, R.T., Bolon, D.N., Burton, B.M., Burton, R.E., Flynn, J.M., Grant, R.A.,<br />
Hersch, G.L., Joshi, S.A., Kenniston, J.A., Levchenko, I., Neher, S.B., Oakes,<br />
E.S.C., Siddiqui, S.M., Wah, D.A. and Baker, T.A. (2004) Sculpt<strong>in</strong>g the proteome<br />
with AAA(+) proteases and disassembly mach<strong>in</strong>es. Cell 119, 9–18.<br />
Savijoki, K., Ingmer, H., Frees, D., Vogensen, F.K., Palva, A. and Varmanen, P.<br />
(2003) Heat and DNA damage <strong>in</strong>duction of the LexA-like regulator HdiR<br />
from Lactococcus lactis is mediated by RecA and ClpP. Mol. Microbiol. 50,<br />
609–621.<br />
Schumann, W. (2004) The Bacillus subtilis heat shock stimulon. Cell Stress Chaperone<br />
8, 207–217.<br />
S<strong>in</strong>gh, V.K., Utaida, S., Jackson, L.S., Jayaswal, R.K., Wilk<strong>in</strong>son, B.J. and<br />
Chamberla<strong>in</strong>, N.R. (2007) Role for dnaK locus <strong>in</strong> tolerance of multiple stresses<br />
<strong>in</strong> Staphylococcus aureus. Microbiology (Read<strong>in</strong>g Engl.) 153, 3162–3173.<br />
Thompson, M.W., S<strong>in</strong>gh, S.K. and Maurizi, M.R. (1994) Processive degradation of<br />
prote<strong>in</strong>s by the ATP-dependent Clp protease from Escherichia coli. Requirement<br />
for the multiple array of active sites <strong>in</strong> ClpP but not ATP hydrolysis. J. Biol. Chem.<br />
269, 18209–18215.<br />
Treangen, T.J., Abraham, A.-L., Touchon, M. and Rocha, E.P.C. (2009) Genesis,<br />
effects and fates of repeats <strong>in</strong> prokaryotic genomes. FEMS Microbiol. Rev. 33,<br />
539–571.<br />
Turgay, K., Hahn, J., Burghoorn, J. and Dubnau, D. (1998) Competence <strong>in</strong> Bacillus<br />
subtilis is controlled by regulated proteolysis of a transcription factor. EMBO J. 17,<br />
6730–6738.<br />
van de Guchte, M., Penaud, S., Grimaldi, C., Barbe, V., Bryson, K., Nicolas, P.,<br />
Robert, C., Oztas, S., Mangenot, S., Couloux, A., Loux, V., Dervyn, R., Bossy,<br />
R., Bolot<strong>in</strong>, A., Batto, J.-M., Walunas, T., Gibrat, J.-F., Bessieres, P.,<br />
Weissenbach, J., Ehrlich, S.D. and Magu<strong>in</strong>, E. (2006) The complete genome<br />
sequence of Lactobacillus bulgaricus reveals extensive and ongo<strong>in</strong>g reductive<br />
evolution. Proc. Natl. Acad. Sci. U.S.A. 103, 9274–9279.<br />
Varmanen, P., Ingmer, H. and Vogensen, F.K. (2000) ctsR of Lactococcus lactis<br />
encodes a negative regulator of clp gene expression. Microbiology (Read<strong>in</strong>g<br />
Engl.) 146(Pt 6); 1447–1455.<br />
Varmanen, P., Vogensen, F.K., Hammer, K., Palva, A. and Ingmer, H. (2003) ClpE<br />
from Lactococcus lactis promotes repression of CtsR-dependent gene expression.<br />
J. Bacteriol. 185, 5117–5124.
144 ALEXANDER K.W. ELSHOLZ ET AL.<br />
Wang, J., Hartl<strong>in</strong>g, J.A. and Flanagan, J.M. (1997) The structure of ClpP at 2.3<br />
resolution suggests a model for ATP-dependent proteolysis. Cell 91, 447–456.<br />
Weber-Ban, E.U., Reid, B.G., Miranker, A.D. and Horwich, A.L. (1999) Global<br />
unfold<strong>in</strong>g of a substrate prote<strong>in</strong> by the Hsp100 chaperone ClpA. Nature 401,<br />
90–93.<br />
Weibezahn, J., Tessarz, P., Schlieker, C., Zahn, R., Maglica, Z., Lee, S., Zentgraf, H.,<br />
Weber-Ban, E.U., Dougan, D.A., Tsai, F.T.F., Mogk, A. and Bukau, B. (2004)<br />
Thermotolerance requires refold<strong>in</strong>g of aggregated prote<strong>in</strong>s by substrate translocation<br />
through the central pore of ClpB. Cell 119, 653–665.<br />
Wickner, S., Maurizi, M.R. and Gottesman, S. (1999) Posttranslational quality control:<br />
fold<strong>in</strong>g, refold<strong>in</strong>g, and degrad<strong>in</strong>g prote<strong>in</strong>s. Science 286, 1888–1893.<br />
Wiegert, T. and Schumann, W. (2001) SsrA-mediated tagg<strong>in</strong>g <strong>in</strong> Bacillus subtilis. J.<br />
Bacteriol. 183, 3885–3889.<br />
Young, J.C., Agashe, V.R., Siegers, K. and Hartl, F.U. (2004) Pathways of chaperone<br />
mediated prote<strong>in</strong> fold<strong>in</strong>g <strong>in</strong> the cytosol. Nat. Rev. Mol. Cell Biol. 5, 781–791.<br />
Yuan, G. and Wong, S.L. (1995) Isolation and characterization of Bacillus subtilis<br />
groE regulatory mutants: evidence for orf39 <strong>in</strong> the dnaK operon as a repressor<br />
gene <strong>in</strong> regulat<strong>in</strong>g the expression of both groE and dnaK. J. Bacteriol. 177,<br />
6462–6468.<br />
Yura, T. (1996) Regulation and conservation of the heat-shock transcription factor<br />
sigma32. Genes Cells 1, 277–284.<br />
Zellmeier, S., Schumann, W. and Wiegert, T. (2006) Involvement of Clp protease<br />
activity <strong>in</strong> modulat<strong>in</strong>g the Bacillus subtilis sigma-w stress response. Mol.<br />
Microbiol. 61, 1569–1582.<br />
Zhang, Q., Xu, S.-X., Wang, H., Xu, W.-C., Zhang, X.-M., Wu, K.-F., Liu, L. and Y<strong>in</strong>,<br />
Y.-B. (2009) Contribution of ClpE to virulence of Streptococcus pneumoniae. Can.<br />
J. Microbiol. 55, 1187–1194.<br />
Zolkiewski, M. (2006) A camel passes through the eye of a needle: prote<strong>in</strong> unfold<strong>in</strong>g<br />
activity of Clp ATPases. Mol. Microbiol. 61, 1094–1100.<br />
Zotta, T., Aster<strong>in</strong>ou, K., Rossano, R., Ricciardi, A., Varcamonti, M. and Parente, E.<br />
(2009) Effect of <strong>in</strong>activation of stress response regulators on the growth and<br />
survival of Streptococcus thermophilus Sfi39. Int. J. Food Microbiol. 129, 211–220.<br />
Zuber, U. and Schumann, W. (1994) CIRCE, a novel heat shock element <strong>in</strong>volved <strong>in</strong><br />
regulation of heat shock operon dnaK of Bacillus subtilis. J. Bacteriol. 176,<br />
1359–1363.
[(Plate_1)TD$FIG]<br />
Plate 1 Graphical presentation of the distribution of CtsR-regulated prote<strong>in</strong>s for different low GC, Gram+ species and known<br />
overlaps with other regulators such as SigB or HrcA. An arrow <strong>in</strong>dicates specific <strong>in</strong>fluence of transcriptional regulator for the expression<br />
of the correspond<strong>in</strong>g gene. Prote<strong>in</strong>s whose expression is solely dependent on CtsR activity are depicted <strong>in</strong> red, whereas prote<strong>in</strong>s that are<br />
dually regulated by CtsR and SigB (green) are shown <strong>in</strong> blue. Prote<strong>in</strong>s controlled by CtsR as well as HrcA are presented <strong>in</strong> grey, and<br />
prote<strong>in</strong>s that are regulated only by HrcA are shown <strong>in</strong> purple. (For b/w version, see page 126 <strong>in</strong> the volume.)
(a)<br />
(b)<br />
(c)<br />
(d)<br />
[(Plate_2)TD$FIG]<br />
Plate 2 Graphical presentation of regulation and degradation of CtsR <strong>in</strong> the<br />
different low GC, Gram+ orders under specific stress conditions. (a) CtsR <strong>in</strong>activation<br />
and degradation dur<strong>in</strong>g heat stress <strong>in</strong> the order Bacillales. Under control conditions<br />
CtsR b<strong>in</strong>ds to its DNA operator and McsB k<strong>in</strong>ase is repressed by b<strong>in</strong>d<strong>in</strong>g to<br />
ClpC. Elevated temperatures lead to a loss of CtsR DNA b<strong>in</strong>d<strong>in</strong>g. In addition, McsB<br />
is released from ClpC and becomes activated as a k<strong>in</strong>ase by McsA. This activation<br />
results <strong>in</strong> target<strong>in</strong>g of free CtsR for a ClpCP-mediated proteolysis. (b) CtsR <strong>in</strong>activation<br />
and degradation dur<strong>in</strong>g heat stress <strong>in</strong> the order Lactobacillales that misses the<br />
two modulators McsA and McsB. Under control conditions CtsR b<strong>in</strong>ds to its DNA<br />
operator. Elevated temperatures lead to a loss of CtsR DNA b<strong>in</strong>d<strong>in</strong>g and ClpEPmediated<br />
degradation. (c) CtsR <strong>in</strong>activation dur<strong>in</strong>g oxidative stress conditions <strong>in</strong> the<br />
order Bacillales. Under control conditions CtsR b<strong>in</strong>ds to its DNA operator and McsB<br />
k<strong>in</strong>ase is repressed by b<strong>in</strong>d<strong>in</strong>g to ClpC. Dur<strong>in</strong>g oxidative stress critical thiols are<br />
oxidised with<strong>in</strong> the oxidative stress sensor prote<strong>in</strong> McsA. This oxidation disturbs<br />
<strong>in</strong>teraction of McsA/McsB and activates a specific McsB function. Consequently,<br />
McsB is now able to b<strong>in</strong>d and <strong>in</strong>activate DNA-bound CtsR, but CtsR cannot be<br />
degraded due to the <strong>in</strong>active McsB k<strong>in</strong>ase. (d) CtsR <strong>in</strong>activation dur<strong>in</strong>g oxidative<br />
stress <strong>in</strong> the order Lactobacillales. Under control conditions CtsR b<strong>in</strong>ds to its DNA<br />
operator. Oxidative stress leads to oxidation of critical thiols with<strong>in</strong> ClpE. This<br />
oxidation causes an activation of ClpE which is now able to target DNA-bound<br />
CtsR. (For b/w version, see page 133 <strong>in</strong> the volume.)
Abbas, B., 14, 30, 32<br />
Abee, T., 131, 137<br />
Abomoelak, B., 68, 90, 94<br />
Abraham, A.-L., 137<br />
Abranches, J., 129, 130<br />
Abu-Soud, H.M., 86, 90<br />
Adams, M.W., 73<br />
Adams, M.W.W., 72, 73<br />
Adegbola, R.A., 59, 84, 90<br />
Adler, L., 63<br />
Agarwal, A., 49, 54, 61, 68, 85, 86, 89<br />
Agarwal, N., 91<br />
Agashe, V.R., 122<br />
Agogue, H., 29<br />
Agrawal, P., 91<br />
Aguilera, J.A., 80<br />
Aharonowitz, Y., 79<br />
Ahr<strong>in</strong>g, B.K., 32<br />
Ahuja, E.G., 70<br />
Alam, K.Y., 68<br />
Alam, M.S., 91<br />
Alawi, M., 11<br />
Albracht, S.P.J., 72<br />
Alexeeva, S., 64, 65, 68, 87<br />
Alland, D., 80<br />
Allen, S.S., 60, 84, 86<br />
Alm, E.W., 6<br />
Almer<strong>in</strong>g, M.J.H., 63<br />
Alonso, S., 76, 86, 87<br />
Altman, E., 52, 66, 68<br />
Aluwihare, L.I., 6, 29<br />
Alvarez, H.M., 67, 68, 90, 94<br />
Alvarez-Ortega, C., 71<br />
Alv<strong>in</strong>g, K., 85<br />
Aly, S., 60, 84, 89<br />
Amann, R., 4<br />
Amaras<strong>in</strong>gham, C.R., 53<br />
Amelung, W., 23, 25, 28<br />
Anaya, J.M., 83<br />
Author Index<br />
Anderberg, S.J., 80<br />
Anderson, R.J., 58, 60<br />
Andrews, J., 47<br />
Andrews-Pfannkoch, C., 34<br />
Ansari, M.Z., 94<br />
Ansell, R., 63<br />
Antelmann, H., 135<br />
Aoshima, M., 74, 75<br />
Arai, H., 74<br />
Arata, Y., 56<br />
Arav<strong>in</strong>d, L., 15, 16, 123<br />
Arikawa, Y., 75<br />
Aris, V., 59<br />
Arora, P., 94<br />
Arp, D.J., 16, 17<br />
Arscott, L.D., 81<br />
Artman, M., 48, 51<br />
Aslund, F., 62<br />
Asoh, S., 72<br />
Aster<strong>in</strong>ou, K., 131<br />
Atlas, R.L., 65<br />
Attarian, R., 80<br />
Av-Gay, Y., 79–81<br />
Baas, M., 13<br />
Badcock, K., 90<br />
Baden-Tillson, H., 6, 7, 10, 15, 16, 22, 34<br />
Bagwell, C., 33<br />
Bajpai, R., 91<br />
Bakalarski, C.E., 128<br />
Baker, T.A., 122, 123, 127, 128, 130<br />
Balasubramanian, R., 96<br />
Balla, G., 84, 86<br />
Balla, J., 84, 86<br />
Banaiee, N., 92<br />
Bancroft, G.J., 84<br />
Bandow, J.E., 129<br />
Banecki, B., 132<br />
Bange, F.C., 60, 84, 89
146 AUTHOR INDEX<br />
Bannant<strong>in</strong>e, J.P., 59<br />
Barbe, V., 124, 125<br />
Barletta, R.G., 78<br />
Barnes, P.J., 85, 90<br />
Barrell, B.G., 90<br />
Barry 3rd, C.E., 47–49, 52, 59, 61, 68, 75,<br />
83, 87, 90, 95, 99, 100<br />
Barry, C.E., 57<br />
Barry, W.H., 50<br />
Bartek, I.L., 75, 82, 83, 86, 87<br />
Bartossek, R., 17, 18<br />
Basaraba, R.J., 59<br />
Basham, D., 90<br />
Batto, J.-M., 124, 125<br />
Baughn, A.D., 74–77<br />
Bayer, K., 13<br />
Bayliss, C., 137<br />
Becker, P., 130<br />
Becktel, W.J., 134<br />
Beckwith, J., 62<br />
Beeson, K., 34<br />
Beja, O., 15, 16<br />
Bekierkunst, A., 48, 51<br />
Bellon, G., 59, 71, 85, 89<br />
Beltramo, C., 125, 127, 128<br />
Beman, J.M., 29, 30, 32, 33<br />
Ben-Dov, E., 33<br />
Benitez-Nelson, B.C., 6<br />
Benjam<strong>in</strong>, I.J., 50, 97<br />
Bennett, A.R., 60<br />
Bennett, G.N., 66, 68, 87, 93<br />
Bennik, M.H.J., 131, 137<br />
Benoit, S.L., 73<br />
Bensen, D.C., 15, 16<br />
Bentley, W.E., 56<br />
Berche, P., 128–131<br />
Beretti, J.L., 129, 130<br />
Berg, I.A., 19<br />
Berger, J., 59, 71, 85, 89<br />
Berks, B.C., 68, 69, 87, 89<br />
Berm<strong>in</strong>gham, E., 34<br />
Berney, M., 73, 74<br />
Bernhard, A.E., 9, 10, 12<br />
Berrios-Rivera, S.J., 66, 68, 87<br />
Berry, A., 81<br />
Berthet, F.X., 59<br />
Bertozzi, C.R., 52, 59, 68, 94, 95<br />
Berube, P.M., 10<br />
Besra, G.S., 59, 84, 90<br />
Bessieres, P., 124, 125<br />
Bewley, C.A., 79<br />
Beyer, D., 129<br />
Bhakoo, K.K., 69<br />
Billman-Jacobe, H., 80<br />
Billoud, B., 72<br />
B<strong>in</strong>trim, S.B., 4<br />
Birrer, P., 59, 71, 85, 89<br />
Bischoff, M., 130<br />
Bishai, W.R., 49, 90, 91<br />
Blackwood, K., 68<br />
Blakis, A., 30<br />
Blanchard, J.S., 81<br />
Blankenfeldt, W., 70, 72<br />
Bloch, H., 51, 54, 57, 58, 60, 99<br />
Blokker, P., 6<br />
Blomberg, A., 63<br />
Bloom, B.R., 91–93, 97<br />
Bock, E., 11<br />
Boechat, N., 60, 83<br />
Boehm, A.B., 32<br />
Boelsterli, U.A., 51<br />
Bolisetty, S., 49, 54, 61, 68, 85, 86, 89<br />
Bolla, J.M., 129, 130<br />
Bolon, D.N., 122, 123, 128<br />
Bolot<strong>in</strong>, A., 124, 125<br />
Bonch-Osmolovskaya, L., 4, 11, 23<br />
Bonec<strong>in</strong>i-Almeida Mda, G., 60, 83<br />
Bonilla-Rosso, G., 34<br />
Boon, C., 82, 83<br />
Boon, J.P., 30<br />
Boor, K.J., 124<br />
Borezee, E., 129, 130<br />
Borland, C., 85<br />
Boros, M., 49, 50, 97<br />
Bortner, C.A., 130<br />
Boschker, H.T., 6<br />
Boshoff, H.I., 48, 49, 52, 57, 59, 61, 68, 75,<br />
87, 95, 99, 100
AUTHOR INDEX 147<br />
Bossy, R., 124, 125<br />
Botste<strong>in</strong>, D., 62<br />
Bott, M., 73<br />
B€ottcher, T., 129<br />
Bottomley, P.J., 24<br />
Botzenhart, K., 59, 71, 85, 89<br />
Boucher, B.J., 97<br />
Boucher, R.C., 59, 71, 85, 89<br />
Boumann, H., 13<br />
Bourret, T.J., 68<br />
Boussau, B., 20<br />
Bowatte, S., 24, 26–28<br />
Boyle-Yarwood, S.A., 24<br />
Braff, J., 29<br />
Brahimi-Horn, M.C., 60<br />
Brandau, S., 60, 84, 89<br />
Braun, R.D., 60<br />
Bregenholt, S., 128, 129<br />
Bre<strong>in</strong>bauer, R., 70<br />
Brez<strong>in</strong>a, O., 48<br />
Brian, P., 92<br />
Bridger, S.L., 73<br />
Briley, K., 131<br />
Br<strong>in</strong>dicci, C., 85, 90<br />
Br<strong>in</strong>k, M., 29<br />
Brochier, C., 15, 16<br />
Brochier-Armanet, C., 16, 17, 20, 22, 23<br />
Brosch, R., 90<br />
Br€otz-Oesterhelt, H., 129<br />
Brovkovych, V., 60<br />
Brown, D., 90<br />
Brown, L.A., 85, 90<br />
Brown, P.O., 62<br />
Bryant, D.A., 96<br />
Bryk, R., 52, 75, 76<br />
Bryson, K., 124, 125<br />
Buchmeier, N., 80, 81<br />
Buchmeier, N.A., 80<br />
Buckel, W., 19, 94, 95<br />
Buckley, D.H., 4<br />
Buettner, G.R., 50, 78, 79<br />
Buhrke, T., 72<br />
Bukau, B., 127, 129, 132<br />
Bunch, P.K., 56<br />
Burgdorf, T., 72<br />
Burghoorn, J., 128, 130<br />
Burn, J.A., 95<br />
Burne, R.A., 128, 130, 134<br />
Burton, B.M., 122, 123, 128<br />
Burton, R.E., 122, 123, 128<br />
Buttner, M.J., 90–92, 95<br />
Bzymek, K.P., 79, 80<br />
Caceres, N.E., 78<br />
Cadena, J., 83<br />
Cadiz, V., 80<br />
Calaycay, J., 60, 83<br />
Camacho, L.R., 68, 87, 95, 99<br />
Camarasa, C., 75<br />
Cameron, K., 24, 26–28<br />
Cameron, K.C., 26<br />
Camien, M.N., 57<br />
Cammack, R., 72<br />
Campbell, J.W., 55<br />
Capozzi, V., 127<br />
Carmel-Harel, O., 62<br />
Carrillo, J., 60, 84, 86<br />
Carter, N., 127<br />
Casali, N., 84<br />
Casciotti, K.L., 29, 32<br />
Casta<strong>in</strong>g, J.P., 93<br />
Cekici, A., 59, 71, 85, 89<br />
Cellek, S., 60<br />
Cevallos, M.A., 66, 67<br />
Chaillou, S., 131<br />
Cha<strong>in</strong>, P.S.G., 16, 17<br />
Chakravarty, S., 84<br />
Chakravorty, S., 84<br />
Chamberla<strong>in</strong>, N.R., 130<br />
Chamberl<strong>in</strong>, L., 92<br />
Chan, E.D., 60<br />
Chan, J., 54, 59, 60, 68, 82, 84<br />
Chan, P.P., 16, 17<br />
Chan, W.T., 52, 54, 55<br />
Charpentier, E., 130, 132, 134<br />
Chastanet, A., 124, 125, 127–130<br />
Chater, K.F., 90–92<br />
Chatterjee, I., 130
148 AUTHOR INDEX<br />
Cheesman, M.R., 91<br />
Chen, B., 52, 54, 55, 80, 94<br />
Chen, J.Q., 33<br />
Cheng, Y., 50, 97<br />
Cherian, J., 52, 55<br />
Cheung, A.L., 129<br />
Chill<strong>in</strong>gworth, T., 90<br />
Cho, C.M., 34<br />
Choi, A.M., 60, 85<br />
Choi, M.-H., 129, 130<br />
Chora, A., 84, 86<br />
Christians, E.S., 50<br />
Chu, D., 54, 59, 68, 82<br />
Chung, K.F., 60, 83, 85<br />
Chung, S.W., 60, 85<br />
Church, M.J., 29, 30, 32<br />
Church, M.K., 60<br />
Churcher, C., 90<br />
Claiborne, A., 78<br />
Clark, D.P., 53, 56, 64, 65, 68<br />
Clausen, T., 132, 134<br />
Claverys, J.P., 127–130<br />
Clough, G.F., 60<br />
Cohen, G., 79<br />
Cohen, R.E., 123<br />
Cohn, M.L., 51<br />
Colangeli, R., 80<br />
Colbeau, A., 72<br />
Cole, J., 87<br />
Cole, S.T., 90<br />
Coleman, D.C., 3<br />
Coll<strong>in</strong>s, D.M., 91–93, 97<br />
Coll<strong>in</strong>s, M., 127<br />
Comhair, S.A., 90<br />
Connell, P., 50<br />
Connor, R., 90<br />
Conrad, R., 23, 24, 26, 28<br />
Converse, P.J., 84<br />
Cook, G.M., 73, 74<br />
Coolen, M.J., 14, 30, 32<br />
Coolen, M.J.L., 14, 15, 30<br />
Corper, H.J., 51<br />
Cosma, C.L., 47, 48<br />
Costa, K.C., 95<br />
Costello, C.M., 71, 89<br />
Coucheney, F., 125, 127<br />
Couloux, A., 124, 125<br />
Cox, A.G., 61, 86<br />
Cox, J.S., 52, 54, 59, 61, 68, 82, 85, 86,<br />
94, 95<br />
Cox, Y., 85<br />
Craig, E.A., 130<br />
Creighton, M.M., 58, 60<br />
Crick, D.C., 59<br />
Cronan Jr., J.E., 55<br />
Crossman, D.K., 49, 54, 61, 68, 85–87, 89,<br />
90, 93–99<br />
Crutz-Le Coq, A.-M., 131<br />
Cunha-Rodrigues, M., 84, 86<br />
Currenti, E., 49, 61, 76<br />
da Silva, S.M., 73<br />
Daffe, M., 93<br />
Daims, H., 11–13, 33<br />
Daldal, F., 95<br />
Damste, J.S.S., 6, 13–15, 30, 32<br />
Dang, H., 13<br />
Daniel, J., 68, 90, 94<br />
Daniel, S.L., 89<br />
Darrouzet, E., 95<br />
Dartois, V., 49, 52, 59, 61, 100, 128<br />
Darw<strong>in</strong>, K.H., 123<br />
Das, T.K., 82, 84<br />
Dasgupta, N., 82<br />
Davidge, K.S., 61, 86<br />
Davies, J., 79<br />
Davies, R., 90<br />
Davis, A.A., 4, 19<br />
Davis, B.D., 53<br />
Davis, N.K., 90<br />
Dawes, E.A., 66, 67<br />
Dawes, I.W., 62<br />
Dawes, S., 59<br />
Dawson, T.L., 50<br />
de Chastellier, C., 130<br />
de Graef, M.R., 64, 65, 68, 87<br />
De Groot, H., 89<br />
de la Torre, J., 16, 17, 29, 33<br />
de la Torre, J.R., 9–13, 33<br />
de Montellano, P.R., 83
AUTHOR INDEX 149<br />
de Nys, R., 13, 33, 34<br />
de Sieyes, N.R., 32<br />
Deb, C., 68, 90, 94<br />
DeFlaun, M., 6<br />
delCardayre, S.B., 80<br />
DeLong, E.F., 4, 10–12, 15, 16, 19, 29, 30,<br />
32, 33<br />
den Hengst, C.D., 90, 95<br />
Denizot, F., 128<br />
Deonara<strong>in</strong>, M.P., 81<br />
Deppenmeier, U., 70<br />
Dequ<strong>in</strong>, S., 75<br />
DeRiemer, K., 83<br />
Derre, I., 124, 125, 128–131<br />
Dervyn, R., 124, 125<br />
Deshane, J.S., 49, 54, 61, 68, 85, 86, 89<br />
Dev<strong>in</strong>e, K., 129, 130<br />
Devl<strong>in</strong>, K., 90<br />
Dewhirst, M.W., 60<br />
Di, H., 23, 24, 26–28<br />
Di, H.J., 26<br />
Dick, T., 49, 52, 59, 61, 76, 78, 82, 83, 86,<br />
87, 89, 100<br />
Dietrich, L.E., 70–72<br />
Dijkhuizen, L., 78<br />
Dimmeler, S., 50<br />
Dimroth, P., 73<br />
D<strong>in</strong>asquet, J., 29<br />
D<strong>in</strong>g, H., 53<br />
D<strong>in</strong>g, Z., 50, 97<br />
Doan, B., 62<br />
Dobbek, H., 76<br />
Dodsworth, J.A., 4, 95<br />
Dolganov, G.M., 49, 54, 61, 68, 82, 83, 89<br />
Donegan, N.P., 129<br />
Donohue, T.J., 4<br />
Dor<strong>in</strong>g, G., 59, 71, 85, 89<br />
Dougan, D.A., 127–129, 131, 138<br />
Dowd, C.S., 68, 87, 95, 99<br />
Drake, H.L., 89<br />
Driver, E.R., 59<br />
Drobnica, L., 48<br />
Drobnicova, I., 48<br />
Druffel, E.R., 6, 29<br />
Duan, X., 53<br />
Dubey, V.S., 68, 90, 94<br />
Dubnau, D., 128, 130, 131<br />
Dubos, R.J., 48, 60, 61, 83<br />
Duncan, K., 47<br />
Dunn, M., 66, 67<br />
Dunn, M.S., 57<br />
Dweik, R.A., 60, 86, 90<br />
Dwyer, T.J., 79<br />
Eck, J., 15<br />
Edson, N.L., 56, 57<br />
Egl<strong>in</strong>ton, T.I., 6<br />
Eguiarte, L.E., 34<br />
Ehlers, S., 60, 84, 89<br />
Ehrlich, S.D., 124, 125<br />
Ehrt, S., 49, 52, 59, 61, 68, 87, 91, 95,<br />
99, 100<br />
Eiamphungporn, W., 135<br />
Eiglmeier, K., 90<br />
Eisen, J.A., 6, 7, 10, 15, 16, 22, 34<br />
Eisen, M.B., 62<br />
Eisenberg, D., 59<br />
Eiteman, M.A., 52, 66, 68<br />
Ell<strong>in</strong>gton, M.J., 69<br />
Emmerson, P.T., 127<br />
Encarnacion, S., 66, 67<br />
Endermann, R., 129<br />
Engelmann, S., 124, 127<br />
Enger, O., 4<br />
Enomoto, K., 75<br />
Eom, C.Y., 77, 84<br />
Epiphanio, S., 84, 86<br />
Erbacher, J., 6<br />
Ernst, J.D., 92<br />
Erzurum, S.C., 86, 90<br />
Escuyer, V., 130<br />
Esser, L., 95<br />
Ett<strong>in</strong>ger-Epste<strong>in</strong>, P., 13, 33, 34<br />
Eum, S.Y., 60, 84, 86<br />
Fahey, R.C., 79–81<br />
Falcón, L.I., 34<br />
Fang, F.C., 78<br />
Farnia, P., 47<br />
Farver, C., 90
150 AUTHOR INDEX<br />
Faucet, V., 75<br />
Fauci, A.S., 47<br />
Faulkner, D.J., 79<br />
Feldman, R.A., 15, 16<br />
Feltwell, T., 90<br />
Feng, Z., 78<br />
Ferguson, S.J., 68, 69<br />
Fernandes, C.L.V., 73<br />
Ferrari, M.R., 34<br />
Ferreira, A., 84, 86<br />
Ferry, J.G., 76, 77, 87<br />
Fert, J., 124, 125, 127<br />
Fiencke, C., 11<br />
F<strong>in</strong>dlay, K.C., 90<br />
Fiocco, D., 127<br />
Firth, A., 70, 71<br />
Fitzgerald, M.X., 71, 89<br />
Fitzpatrick, A.M., 85, 90<br />
Flanagan, J.M., 123<br />
Flardh, K., 90, 92<br />
Flarsheim, C.E., 50<br />
Fleury, B., 134<br />
Florczyk, M.A., 49, 61, 76<br />
Flynn, J., 49, 52, 59, 61, 100<br />
Flynn, J.A., 84<br />
Flynn, J.M., 122, 123, 128<br />
Focks, A., 23, 25, 28<br />
Foley, J., 129<br />
Follis Jr., R.H., 61, 83, 84<br />
Fontan, P., 59<br />
Forterre, P., 20<br />
Fothergill, J.L., 70, 71<br />
Fournier, D., 93<br />
Fouts, D.E., 6, 7, 10, 15, 16, 22<br />
Fowler, A.V., 57<br />
Fox, G.E., 13<br />
Foxwell, N.A., 60<br />
Fraczkowska, K., 127, 130<br />
Francis, C.A., 29, 30, 32, 33<br />
Frazier, M., 34<br />
Fredrickson, J.K., 6<br />
Freeman, J., 34<br />
Freeman, K.H., 13, 32<br />
Frees, D., 123, 127–130, 135, 136<br />
Frehel, C., 130<br />
Freitag, T.E., 26<br />
Freundlich, J.S., 47<br />
Frey, M., 72<br />
Friedheim, E., 70<br />
Friedland, G., 47<br />
Friedman, R., 34<br />
Friedrich, B., 72<br />
Frimpong, I., 75, 83, 86, 87<br />
Fu, Z., 129<br />
Fuchs, G., 19, 74, 75<br />
Fuhrman, J.A., 4, 6, 19<br />
Fuhrmann, J., 132, 134<br />
Fujii, T., 78<br />
Fujiwara, S., 56<br />
Furst, V.W., 60<br />
Gagneux, S., 83<br />
Gahan, C.G.M., 131, 137<br />
Gaillot, O., 128, 129, 131<br />
Gallardo, V., 34<br />
Gallone, A., 127<br />
Gandhi, N.R., 47<br />
Gao, L., 33<br />
Gao, X., 50, 97<br />
Garcia, J.-L., 3<br />
Garforth, S.J., 74–77<br />
Garg, S., 91<br />
Garg, S.K., 91<br />
Garnier, T., 90<br />
Garrett, R.A., 22<br />
Garrido, E.O., 62<br />
Garrigues, C., 15<br />
Gars<strong>in</strong>, D.A., 128, 130<br />
Garton, N.J., 59, 84, 90<br />
Gas, S., 90<br />
Gasch, A.P., 62<br />
Gaston, B., 90<br />
Gatfield, J., 91<br />
Geenevasen, J.A., 13<br />
Geertman, J.M., 64<br />
Geiman, D.E., 91<br />
Geng, J., 60, 83<br />
Gengenbacher, M., 83, 89
AUTHOR INDEX 151<br />
Genghof, D.S., 79<br />
Gennaro, M.L., 59<br />
Gens<strong>in</strong>i, G., 61, 75, 77, 83<br />
Gentles, S., 90<br />
Georgopoulos, C., 132<br />
Gerbl, F.W., 33<br />
Gerth, U., 123, 124, 127–130, 132, 138<br />
Gertz, S., 124, 127<br />
Geth<strong>in</strong>g, M.J., 122<br />
Ghanavi, J., 47<br />
Ghanny, S., 59<br />
Ghyczy, M., 49, 50, 97<br />
Gibrat, J.-F., 124, 125<br />
Gicquel, B., 59<br />
Giles, G.I., 91–93<br />
Gilles-Gonzalez, M.A., 60, 83, 84<br />
Gilmour, R., 129<br />
Glockner, F., 4<br />
Gokhale, R.S., 94<br />
Golbeck, J.H., 96<br />
Gollabgir, A., 16, 17<br />
Gomez, J.E., 47, 90<br />
Gomez, L.M., 83<br />
Gonzales, J., 60, 84, 86<br />
Gonzalez, G., 60, 83, 84<br />
Gonzalez-Juarrero, M., 59<br />
Goodman, R.M., 4<br />
Goodw<strong>in</strong>, M.B., 57<br />
Gop<strong>in</strong>athan, K.P., 48<br />
Gordon, S.V., 90<br />
Gores, G.J., 50<br />
Gorovitz, B., 79, 80<br />
Gossner, A., 89<br />
Gottesman, S., 122, 123, 138<br />
G€otz, F., 89, 134<br />
Govender, T., 47<br />
Graber, J.R., 4<br />
Granath, K., 63<br />
Grandvalet, C., 125, 127, 128<br />
Grant, C.M., 62, 63<br />
Grant, R.A., 122, 123, 128<br />
Grasemann, H., 89<br />
Gray, C.T., 64, 65<br />
Green, J., 78, 96<br />
Gregoire, I.P., 84, 86<br />
Gribaldo, S., 20<br />
Grimaldi, C., 124, 125<br />
Grode, L., 59<br />
Grosset, J.H., 84<br />
Grundmeier, M., 130<br />
Guest, J.R., 53<br />
Guidry, L., 68, 87, 90–99<br />
Gustafsson, L., 63<br />
Gustafsson, L.E., 85<br />
Gutterridge, J.M.C., 53, 78, 79<br />
Guzzo, J., 125, 127, 128<br />
Gygi, S.P., 128<br />
Haapanen, J.H., 61, 75, 77, 83<br />
Hacker, J., 124, 127<br />
Hackett, M., 95<br />
Hadd, A., 15<br />
Hahn, J., 128, 130, 131<br />
Hall, S.R., 60, 85<br />
Hallam, S.J., 16, 29, 33<br />
Halliwell, B., 53, 78, 79<br />
Halpern, A.L., 6, 7, 10, 15, 16, 22, 34<br />
Hamann, C.W., 64<br />
Haml<strong>in</strong>, N., 90<br />
Hammel, J., 90<br />
Hammer, K., 129, 130, 132, 136<br />
Handelsman, J., 4, 34<br />
Hanschke, R., 123, 128, 129<br />
Hansman, R.L., 6, 29<br />
Harraghy, N., 130<br />
Harrell, M.I., 49, 54, 59, 61, 68, 82–84, 89<br />
Harris, D., 90<br />
Hartl, F.U., 122, 123, 135<br />
Hartl<strong>in</strong>g, J.A., 123<br />
Hartmann, P., 91, 96<br />
Harwood, C.R., 127<br />
Harwood, C.S., 71, 77<br />
Hashimoto, S., 78<br />
Hatzenpichler, R., 11, 12, 20, 22, 23<br />
Hayer-Hartl, M., 122, 123, 135<br />
Hayes, J.M., 6<br />
Hayes, L.G., 54, 60, 83<br />
Hazbon, M.H., 80
152 AUTHOR INDEX<br />
Hazleton, E.B., 84<br />
He, J., 23, 24, 26–28<br />
He, J.Z., 26<br />
Hecker, M., 123, 124, 127–132,<br />
134–136, 138<br />
Hedderich, R., 94, 95<br />
Hedlund, B.P., 33<br />
Heidelberg, J.F., 6, 7, 10, 15, 16, 22, 34<br />
Heidelberg, K.B., 34<br />
Heifets, L., 91<br />
Helmann, J.D., 135<br />
Hemp, J., 16, 17<br />
Henn<strong>in</strong>ger, K., 129<br />
Hentschel, U., 13, 33, 34<br />
Heo, J., 77, 84<br />
Herbaud, M.L., 128<br />
Herfort, L., 30<br />
Herman, B., 50<br />
Hernandez, M.E., 70–72<br />
Herndl, G.J., 6, 29, 30<br />
Herrmann, M., 32, 130<br />
Hersch, G.L., 122, 123, 128<br />
Hertel, C., 131<br />
Hett, E.C., 48<br />
Heuer, H., 23, 25, 28<br />
Hicks, R.E., 6<br />
Higenbottam, T., 85<br />
Hill, C., 131, 137<br />
Hill, P., 127, 130<br />
Hill, P.J., 128, 129<br />
Hill, R.T., 34<br />
H<strong>in</strong>ds, J., 47, 59, 84, 90<br />
H<strong>in</strong>ojosa, R., 83<br />
H<strong>in</strong>zen, B., 129<br />
Ho, J.L., 60, 83<br />
Hoff, D.R., 59<br />
Hoffart, L.M., 96<br />
Hoffman, J., 6, 7, 10, 15, 16, 22<br />
Hoffman, J.M., 34<br />
Hoffmann, F., 13, 33, 34<br />
Hoffner, S.E., 47<br />
Hofreiter, M., 33<br />
Hohmann, S., 63<br />
Hol, W.G., 82<br />
Holben, W., 6, 16<br />
Holgu<strong>in</strong>, F., 85, 90<br />
Holroyd, S., 90<br />
Hols, P., 127<br />
Holtappels, M., 13, 33, 34<br />
Homuth, G., 125, 132, 135<br />
Honaker, R.W., 49, 82, 83, 85, 90<br />
Hondalus, M.K., 91–93, 97<br />
Honer zu Bentrup, K., 52, 54, 55<br />
Hood, D., 137<br />
Hopmans, E.C., 13, 14, 30, 32<br />
Horn, R., 79<br />
Hornsby, T., 90<br />
Horton, E., 68<br />
Horwich, A.L., 123<br />
Howard, S.T., 59<br />
Hu, Y., 124<br />
Huang, Z., 13, 32<br />
Huang, Z.Y., 33<br />
Huet, G., 93<br />
H€ufner, E., 131<br />
H€ugler, M., 16, 17, 74, 75<br />
Humphrey, T.J., 137<br />
Hunter, G.J.E., 57<br />
Husa<strong>in</strong>, M., 68<br />
Hussey, G., 47<br />
Hutte, R., 86<br />
Hutter, B., 78<br />
Hwang, C., 62<br />
Hwang, E.H., 77, 84<br />
Ibrahim, Y.M., 129, 130<br />
Ido, Y., 50<br />
Igarashi, Y., 74<br />
Ikeda, T., 74<br />
Imlay, J.A., 53<br />
Ingalls, A.E., 6, 11–13, 29, 32, 33<br />
Ingmer, H., 123, 125, 127–131,<br />
134–136<br />
Inskeep, W.P., 33<br />
Ioannidis, I., 89<br />
Ioanoviciu, A., 83<br />
Ishii, M., 74<br />
Ishikawa, M., 72
AUTHOR INDEX 153<br />
Ito, K., 85, 90<br />
Itoh, M., 52, 75, 76<br />
Izzo, A., 82<br />
Jackson, L.S., 130<br />
Jacobs Jr., W.R., 52, 54, 55, 59, 68, 74–77,<br />
80, 82, 91–94, 97<br />
Jagels, K., 90<br />
Ja<strong>in</strong>, A., 59<br />
Ja<strong>in</strong>, M., 52, 59, 68, 94, 95<br />
Ja<strong>in</strong>, S.K., 84<br />
Jakimowicz, P., 91<br />
Jayaswal, R.K., 130<br />
Jeney, V., 84, 86<br />
Jenney Jr., F.E., 72, 73<br />
Jenn<strong>in</strong>gs, L.D., 123<br />
Jeoung, J.H., 76<br />
Jia, Z., 23, 24, 26, 28<br />
J<strong>in</strong>, W., 13<br />
Johnson, C., 80<br />
Johnson, T., 81<br />
Johnston, S.A., 59<br />
Jones, A.K., 72<br />
Jones-Carson, J., 68<br />
Jonuscheit, M., 3, 6, 7<br />
Joshi, S.A., 122, 123, 128<br />
Jovanovich, S.B., 15<br />
Juretschko, S., 4<br />
Jurgens, G., 3, 4, 6, 7<br />
Jyothisri, K., 82<br />
Kajfasz, J.K., 129, 130<br />
Kalscheuer, R., 68<br />
Kana, B.D., 59<br />
Kaneko, F., 86<br />
Kao, C.M., 62<br />
Kappler, A., 70<br />
Kapur, V., 82<br />
Karakousis, P.C., 84<br />
Karatzas, K.A.G., 131, 137<br />
Karl, D.M., 29, 30, 32, 34<br />
Karner, M.B., 29<br />
Karr, E.A., 16, 17<br />
Kass, I., 61, 75, 77, 83<br />
Kaster, A.K., 94, 95<br />
Katayama, Y., 72<br />
Katsumata, R., 78<br />
Katsura, K., 72<br />
Kaufmann, S.H., 47, 48<br />
Kaufmann, S.H.E., 59<br />
Kaupenjohann, M., 23, 25, 28<br />
Kavuru, M., 90<br />
Kawakami, R.P., 91–93, 97<br />
Keat<strong>in</strong>gs, V.M., 71, 89<br />
Kelemen, G.H., 92<br />
Keller, C., 60, 84, 89<br />
Kelley, W.L., 134<br />
Kelly, R.M., 73<br />
Kendall, S., 84<br />
Kenniston, J.A., 122, 123, 128<br />
Keough, B.P., 6<br />
Kerr, A.R., 129, 130<br />
Kesavan, A.K., 84<br />
Khan, S., 50<br />
Kharitonov, S.A., 85, 90<br />
Khodursky, A.B., 52, 66, 68<br />
Kielland-Brandt, M.C., 64<br />
Kilo, C., 50<br />
Kilstrup, M., 129, 130<br />
Kim, E., 77, 84<br />
Kim, H.J., 91<br />
Kim, J.A., 77, 84<br />
Kim, J.H., 77<br />
Kim, L., 125, 132<br />
Kim, P., 91<br />
Kim, S.W., 77, 84, 129, 130<br />
Kim, S.Y., 77, 84<br />
Kim, T.H., 91<br />
Kim, Y., 91<br />
Kim, Y.I., 127, 130<br />
Kim, Y.M., 77, 84<br />
K<strong>in</strong>g, G.M., 77, 84<br />
K<strong>in</strong>ger, A.K., 81<br />
K<strong>in</strong>kel, H., 6<br />
Kirste<strong>in</strong>, J., 128, 129, 131, 132, 138<br />
Kishan, K.V., 91<br />
Kleerebezem, M., 127<br />
Kle<strong>in</strong>, E., 60, 84, 86
154 AUTHOR INDEX<br />
Kle<strong>in</strong>, M.R., 82<br />
Kleman, G.L., 55<br />
Klenk, H.-P., 6, 7, 9, 10, 15–18, 22<br />
Kletz<strong>in</strong>, A., 6, 7, 9, 10, 15, 17, 22<br />
Klichko, V.I., 50<br />
Kl<strong>in</strong>kenberg, L.G., 84<br />
Klotz, M.G., 16, 17<br />
Knap, A.H., 6, 7, 10, 15, 16, 22<br />
Knosp, O., 67<br />
Ko, M., 80<br />
Ko, Y.F., 56<br />
Kobayashi, Y., 125<br />
Koch, C., 84<br />
Kock, H., 123, 128–130<br />
Kockelkorn, D., 19<br />
Kohno, S., 54, 59, 68, 82<br />
Kolattukudy, P.E., 68, 90, 94<br />
Koled<strong>in</strong>, T., 79, 80<br />
K€onneke, M., 9–13, 16, 17, 33<br />
Konstant<strong>in</strong>idis, K.T., 16, 29, 33<br />
Koo, H., 129, 130<br />
Koon<strong>in</strong>, E.V., 15, 16, 123<br />
Koops, H.P., 4<br />
Kosaka, K., 54<br />
Kosmiadi, G.A., 59<br />
Kotzerke, A., 23, 25, 28<br />
Kovacevic, S., 80<br />
Kowalchuk, G.A., 3, 4<br />
Kramer, N., 131<br />
Kramnik, I., 49, 54, 61, 68, 85, 86, 89<br />
Kravitz, S., 34<br />
Krebs, C., 96<br />
Krebs, W., 73<br />
Krogh, A., 90<br />
Kroll, H.-P., 129<br />
Kr€uger, E., 123, 124, 128–132, 134,<br />
136, 138<br />
Kumar, A., 49, 54, 60, 61, 68, 82–86, 89<br />
Kunst, F., 128–130<br />
Kuo, H.P., 60, 83, 85<br />
Kushmaro, A., 33<br />
Kusters, I., 123, 128–130<br />
Kuypers, M.M., 14, 30, 32<br />
Kuypers, M.M.M., 6, 13, 33, 34<br />
Kvist, T., 32<br />
Kwon, H.-Y., 129, 130<br />
Labisch<strong>in</strong>ski, H., 129<br />
LaCourse, R., 60, 83<br />
Ladel, C., 129<br />
Lai, D., 13<br />
Lalk, M., 135<br />
Lalloo, U., 47<br />
Lambert, P.H., 47<br />
Lamichhane, G., 84<br />
Lancaster Jr., J.R., 49, 60, 82–86<br />
Lancaster, J.R., 91–93<br />
Lang, D., 16, 17<br />
Lanzen, A., 17, 18<br />
Lanzen, J.L., 60<br />
Lapa e Silva, J.R., 60, 83<br />
Laskowski, D., 86<br />
Laughl<strong>in</strong>, J., 68<br />
Lavik, G., 13, 33, 34<br />
Lawton, T.J., 16, 17<br />
Leary, J.A., 52, 59, 68, 94, 95<br />
Leavell, M.D., 52, 59, 68, 94, 95<br />
Lebedeva, E., 11<br />
Lebedeva, E.V., 11, 12<br />
Lee, H.S., 91<br />
Lee, K.H., 77, 84<br />
Lee, N., 56<br />
Lee, S., 127<br />
Lee, S.M., 59, 84, 90<br />
Legan, S.K., 50<br />
Lehner, A., 132, 134<br />
Lehtovirta, L.E., 23<br />
Leichert, L.I.O., 134<br />
Leigh, J.A., 95<br />
Leija, A., 66, 67<br />
Le<strong>in</strong><strong>in</strong>ger, S., 6, 7, 9, 10, 14, 15, 22–25, 28<br />
Leistikow, R.L., 75, 82, 83, 86, 87<br />
Lemasters, J.J., 50<br />
Lemos, J.A., 129, 130<br />
Lemos, J.A.C., 128, 134<br />
Lenaerts, A.J., 59<br />
Lenz, O., 72<br />
Leone, A.M., 60, 85
AUTHOR INDEX 155<br />
Leopold, J.A., 50<br />
Levanon, S.S., 93<br />
Levchenko, I., 122, 123, 127, 128, 130<br />
Levitt, M.D., 72<br />
Levy, S., 6, 7, 10, 15, 16, 22<br />
Lew, D., 134<br />
Li, C., 50, 97<br />
Li, J., 13<br />
Li, K., 34<br />
Li, Q., 84<br />
Li, R., 82, 83<br />
Li, T., 13<br />
Li, W.J., 33<br />
Liao, R., 54, 59, 68, 82–84, 89<br />
Liao, R.P., 82<br />
Libby, S.J., 78<br />
Licht, S., 123<br />
Lie, T.J., 95<br />
Liebeke, M., 135<br />
Lieberman, R.L., 8<br />
Lienhardt, C., 47<br />
Likolammi, M., 4<br />
Lilie, H., 129<br />
L<strong>in</strong>, H., 68<br />
L<strong>in</strong>, H.C., 60, 83, 85<br />
L<strong>in</strong>, P.L., 60, 84, 86<br />
L<strong>in</strong>hares, C., 60, 83<br />
L<strong>in</strong>nane, S.J., 71, 89<br />
Liu, C.Y., 60, 83, 85<br />
Liu, L., 50, 97, 130<br />
Liu, W., 80<br />
Liu, X., 34<br />
Liu, Y., 91<br />
Liu, Z., 80<br />
Locht, C., 59<br />
Lodish, H.F., 62<br />
Lomas, M.W., 6, 7, 10, 15, 16, 22<br />
López-Garcıa, P., 15, 16<br />
Lopez-Nevot, M.A., 83<br />
Loscalzo, J., 50<br />
Losick, R., 128, 130<br />
Loss, C., 124<br />
Lothrop, W.C., 58, 60<br />
Loux, V., 124, 125<br />
Lowe, T., 16, 17<br />
Ludwig, H., 128, 131, 138<br />
Lun, D.S., 123<br />
Luzardo, Y., 130<br />
Ly, L.H., 84<br />
Lyons, R., 59<br />
Ma, K., 73<br />
Ma, Y., 50, 97<br />
Ma, Z., 47<br />
MacGregor, B.J., 6<br />
MacMick<strong>in</strong>g, J.D., 60, 83<br />
M€ader, U., 135<br />
Maglica, Z., 127<br />
Magu<strong>in</strong>, E., 124, 125<br />
Mai, D., 68, 87, 90–99<br />
Maier, R.J., 72, 73<br />
Maier, S.E., 73<br />
Malhotra, V., 82, 84<br />
Mal<strong>in</strong>ski, T., 60<br />
Mallidis, C.G., 137<br />
Malm, S., 60, 84, 89<br />
Malzan, A., 60, 84, 89<br />
Manabe, Y.C., 84<br />
Mangenot, S., 124, 125<br />
Manjunatha, U., 60, 84, 86<br />
Mann, B.E., 61, 86<br />
Mann<strong>in</strong>g, G., 16, 17<br />
Manzanillo, P., 54, 61, 82, 85, 86<br />
Manzei, S., 32<br />
Markieton, T., 131<br />
Marsh, T.L., 15<br />
Martens-Habbena, W., 10, 16, 17<br />
Marteus, H., 85<br />
Mart<strong>in</strong>, J., 83<br />
Mart<strong>in</strong>ez, A.R., 129, 130<br />
Mart<strong>in</strong>ez, G.J., 80<br />
Masjedi, M.R., 47<br />
Massana, R., 30<br />
Masuda, S., 125<br />
Masuo, S., 78<br />
Matic, I., 137<br />
Mat-Jan, F., 56<br />
Matter, E., 57
156 AUTHOR INDEX<br />
Matthews, B.W., 134<br />
Matthies, M., 23, 25, 28<br />
Maurizi, M.R., 122, 123<br />
Mavrodi, D.V., 70, 72<br />
Mayuri, Bagchi, G., 82<br />
McCallum, K., 4, 19<br />
McCollister, B.D., 68<br />
McCue, L.A., 49, 61, 76<br />
McDonough, K.A., 49, 61, 76<br />
McIlleron, H., 47<br />
McK<strong>in</strong>lay, J.B., 77<br />
McK<strong>in</strong>ney, J.D., 47, 52, 54, 55, 94<br />
McLean, J., 90<br />
McLeod, C.W., 61, 86<br />
McLoughl<strong>in</strong>, P., 71, 89<br />
McNeil, M., 94<br />
McNichol, A.P., 6<br />
Mechtler, K., 132, 134<br />
Medard, M., 123<br />
Med<strong>in</strong>a, V.G., 63<br />
Medlar, E.M., 83, 84<br />
Meena, L.S., 49<br />
Meijer, W.G., 78<br />
Mengewe<strong>in</strong>, A., 32<br />
Mentel, M., 70<br />
Merrill, M.H., 56, 57, 89<br />
Meurer, G., 6, 15, 17<br />
Meurer, G.b., 15<br />
Meyer, J., 72<br />
Meyer, K.C., 59, 71, 85, 89<br />
Meyer, M., 33<br />
Meyer, O., 77<br />
Michaelis, L., 70<br />
Michalik, S., 123, 128–130<br />
Miczak, A., 52, 54, 55<br />
Middelburg, J.J., 30<br />
Middlebrook, G., 61, 75, 77, 83<br />
Miethke, M., 128–130, 132<br />
Miller, C.C., 81<br />
Mills, G., 13<br />
Milohanic, E., 130<br />
M<strong>in</strong>, X., 50, 97<br />
M<strong>in</strong>cer, T.J., 29, 30, 32, 33<br />
M<strong>in</strong>ch, K.J., 47, 49<br />
Miranker, A.D., 123<br />
Mitchell, T.J., 129, 130<br />
Mizrahi, V., 47, 59<br />
Mockett, R.J., 50<br />
Moenne-Loccoz, P., 83<br />
Mogk, A., 125, 127, 129, 132<br />
Mohamed, N.M., 34<br />
Mohan, V.P., 54, 59, 68, 82<br />
Mohanty, D., 94<br />
Moliere, N., 128<br />
Mol<strong>in</strong>ski, T.F., 10–12, 33<br />
Moll, A., 47<br />
Mollenkopf, H., 59<br />
Monbouquette, H.G., 13<br />
Moncada, S., 60, 85<br />
Monk, C.E., 61, 86<br />
Montonen, L., 4<br />
Mora, J., 66, 67<br />
Mora, Y., 66, 67<br />
Morbidoni, H.R., 68<br />
Moreira, D., 15, 16<br />
Mori, T., 54<br />
Morowitz, H.J., 74, 75<br />
Morozk<strong>in</strong>a, E.V., 87, 89<br />
Morris, R.P., 91<br />
Morton, R.A., 75, 83, 86, 87<br />
Moser, D.P., 6<br />
Mosier, A.C., 32<br />
Mossman, M.R., 64, 65<br />
Mostertz, J., 128, 135<br />
Mota, M.M., 84, 86<br />
Motterl<strong>in</strong>i, R., 61, 86<br />
Mougous, J.D., 52, 59, 68, 94, 95<br />
Moule, S., 90<br />
Moxon, R., 137<br />
Moynihan, J.B., 71, 89<br />
Msadek, T., 124, 125, 127–131, 136<br />
Mudgett, J.S., 60, 83<br />
Muller, E.G., 62<br />
Mumford, R., 60, 83<br />
Munch, J.C., 23, 25, 28<br />
Munoz-Elias, E.J., 52, 54, 55<br />
Munster, U., 4<br />
Muratsubaki, H., 75
AUTHOR INDEX 157<br />
Murphy, L., 90<br />
Murray, A.E., 30<br />
Murray, J.F., 60<br />
Murthy, P.S., 48, 54, 58<br />
Muscariello, L., 127<br />
Muttucumaru, D.G., 47<br />
Myrold, D.D., 24<br />
Nair, S., 128–131<br />
Nakano, M.M., 128<br />
Nakano, S., 128<br />
Namsaraev, B., 11<br />
Nandi, R., 72, 73<br />
Narasimhulu, K.V., 91–93<br />
Narayanan, P.R., 84<br />
Nathan, C., 52, 60, 75, 76, 83<br />
Nathan, C.F., 60, 83<br />
Nealson, K., 6, 7, 10, 15, 16, 22, 34<br />
Nealson, K.H., 6, 16<br />
Neher, S.B., 122, 123, 128<br />
Nelson, K.E., 6, 7, 10, 15, 16, 22<br />
Nelson, W., 6, 7, 10, 15, 16, 22<br />
Neubauer, H., 89<br />
Neuwald, A.F., 123<br />
Newman, D.K., 70–72<br />
Newton, G.L., 79–81<br />
Ng, W.-L., 129<br />
Nguyen, K., 91<br />
Nguyen, L., 91, 130<br />
Nguyen, L.P., 15<br />
Nguyen, T.T.H., 135<br />
Nicholson, H., 134<br />
Nicholson, S., 60, 83<br />
Nicol, G., 17, 18, 26, 28<br />
Nicol, G.W., 6, 11, 14, 23–26<br />
Nicolas, P., 124, 125<br />
Nielsen, J., 64<br />
Niem<strong>in</strong>en, A.L., 50<br />
Nishimaki, K., 72<br />
Nissen, N., 91, 96<br />
Nissen, T.L., 64<br />
Norbeck, J., 63<br />
North, R.J., 59, 60, 83<br />
Novak, R., 130<br />
Nowag, A., 91, 96<br />
Nunn, A.J., 47<br />
O’Brien, P.J., 50<br />
O’Callaghan, M., 24, 26–28<br />
O’Connor, C.M., 71, 89<br />
O’Mullan, G.D., 30<br />
Oakes, E.C., 128<br />
Oakes, E.S.C., 122, 123, 128<br />
Oakley, B.B., 29, 32<br />
Ochsenreiter, T., 4, 6, 11, 15, 17, 23<br />
Odelberg, S.J., 50<br />
Oelgeschlager, E., 76, 77<br />
Oenema, O., 4<br />
Offre, P., 26, 28<br />
Og<strong>in</strong>o, T., 56<br />
Ogunniyi, A.D., 129, 130<br />
Ogura, M., 128<br />
Oh, J.I., 77, 84<br />
Oh, N.N., 80<br />
Ohlmeier, S., 123, 128, 129, 131<br />
Ohmori, D., 74<br />
Ohno, H., 54, 59, 68, 82<br />
Ohsawa, I., 72<br />
Ohta, S., 72<br />
Olczak, A., 72, 73<br />
Oliver, K., 90<br />
Ollivier, B., 3<br />
Olson, J., 72, 73<br />
Olson, J.W., 72, 73<br />
Oman, J., 60<br />
Onstott, T.C., 6<br />
Orosz, A., 50<br />
Orr, W.C., 50<br />
Orsi, R.H., 124<br />
Osborne, J., 90<br />
Ouverney, C.C., 6<br />
Oztas, S., 124, 125<br />
Page, W.J., 67<br />
Paget, M.S., 78, 96<br />
Paige, C., 49<br />
Palva, A., 129, 130, 132, 136<br />
Pamplona, A., 84, 86
158 AUTHOR INDEX<br />
Pan, Q., 128, 130<br />
Pancost, R.D., 6<br />
Pane-Farre, J., 124<br />
Parente, E., 131<br />
Parish, T., 47, 84<br />
Park, H., 77, 84<br />
Park, J.S., 91<br />
Park, S.-H., 129, 130<br />
Park, S.J., 77<br />
Park, S.W., 77, 84<br />
Park, T.H., 68, 87, 95, 99<br />
Parkhill, J., 90<br />
Parsons, R., 6, 7, 10, 15, 16, 22<br />
Patel, B.K.C., 3<br />
Patel, H., 62<br />
Patel, M.P., 81<br />
Patel, R.P., 49, 60, 82–86<br />
Paton, J.C., 129, 130<br />
Paulsen, H., 129<br />
Paulsen, I., 6, 7, 10, 15, 16, 22<br />
Paw<strong>in</strong>ski, R., 47<br />
Pearson, A., 6, 13, 29, 32<br />
Pelicic, V., 59<br />
Pellegr<strong>in</strong>i, E., 128–130<br />
Penaud, S., 124, 125<br />
Pereira, I.A.C., 73<br />
Perham, R.N., 81<br />
Perk<strong>in</strong>s, M.D., 47<br />
Pernthaler, A., 6<br />
Pernthaler, J., 6<br />
Perrella, M.A., 60, 85<br />
Perrone, G., 62<br />
Persson, M.G., 85<br />
Peshock, R.M., 50<br />
Peters, G., 130<br />
Petersen, A., 70<br />
Peterson, J., 6, 7, 10, 15, 16, 22<br />
Pethe, K., 68, 76, 83, 86, 87, 89, 95, 99<br />
Petzold, C.J., 52, 59, 68, 94, 95<br />
Pfannkoch, C., 6, 7, 10, 15, 16, 22<br />
Pfenn<strong>in</strong>ger-Li, X.D., 73<br />
Pickart, C.M., 123<br />
Pierre, F., 127, 128<br />
Pieters, J., 91<br />
Pillay, M., 47<br />
P<strong>in</strong>el, N., 16, 17<br />
Pitcher, A., 13, 14<br />
Platt, T., 34<br />
Plum, G., 91, 96<br />
Pommeren<strong>in</strong>g-R€oser, A., 4<br />
Poole, R.K., 61, 86<br />
Popp, B.N., 29, 30<br />
Portugal, S., 84, 86<br />
Pouyssegur, J., 60<br />
Pratt, C.W., 49, 55<br />
Preston, C., 16, 29, 30, 32, 33<br />
Preston, C.M., 10–12, 15, 16, 30, 33<br />
Price-Whelan, A., 70–72<br />
Proctor, R.A., 130, 134<br />
Pronk, J.T., 63, 64<br />
Prosser, J., 3<br />
Prosser, J.I., 14, 23–28<br />
Prudhomme, M., 127–130<br />
Purkayastha, A., 49, 61, 76<br />
Purkhold, U., 4<br />
Putnam, N., 16, 29, 33<br />
Puzewicz, J., 132<br />
Pyo, S.-N., 129, 130<br />
Qazi, S., 127, 130<br />
Qazi, S.N.A., 128, 129<br />
Qi, J., 14, 23–25<br />
Qi, R., 82, 83<br />
Qian, B., 50, 97<br />
Qoronfleh, M.W., 130<br />
Quail, M.A., 90<br />
Quaiser, A., 4, 6, 11, 15, 17, 23<br />
Quivey, R.G., 129, 130<br />
Rachman, H., 59<br />
Radax, R., 13, 33, 34<br />
Raddatz, G., 6, 15, 17<br />
Raddatz, S., 129<br />
Radha Kishan, K.V., 91<br />
Radyuk, S.N., 50<br />
Raengpradub, S., 124<br />
Raghunand, T.R., 91<br />
Ragsdale, S.W., 76
AUTHOR INDEX 159<br />
Rajakumar, K., 59, 84, 90<br />
Rajandream, M.A., 90<br />
Rajasekaran, N.S., 50<br />
Rajni, 49<br />
Ramakrishnan, L., 47, 48<br />
Ramakrishnan, T., 48, 54, 58<br />
Ramal<strong>in</strong>gam, B., 59<br />
Ramanathan, V.D., 84<br />
Rand, J.D., 63<br />
Rand, L., 76, 86, 87<br />
Randell, S., 59, 71, 85, 89<br />
Rao, S.P., 76, 83, 86, 87, 89<br />
Rapoport, G., 125, 128–131<br />
Rapp, H.T., 13, 33, 34<br />
Rasmussen, K.N., 61, 83, 84<br />
Ratjen, F., 89<br />
Ratledge, C., 47, 51, 52<br />
Rattei, T., 20, 22, 23<br />
Rawat, M., 79–81<br />
Ray, S.M., 60, 84, 86<br />
Rebr<strong>in</strong>, I., 50<br />
Redd<strong>in</strong>g, K.E., 91–93<br />
Reed, M.B., 83<br />
Reeves, R.E., 58, 60<br />
Reid, B.G., 123<br />
Reigstad, L.J., 13, 33<br />
Re<strong>in</strong>thaler, T., 6<br />
Rem<strong>in</strong>gton, K., 6, 7, 10, 15, 16, 22, 34<br />
Ren, B., 53<br />
Renfrow, M.B., 68, 87, 90, 93–99<br />
Reysenbach, A.L., 14, 32<br />
Rhee, D.-K., 129, 130<br />
Ricciardi, A., 131<br />
Rich, A.R., 61, 83, 84<br />
Richardson, A.R., 78<br />
Richardson, D.J., 68, 69, 87, 89<br />
Richardson, P.M., 16, 29, 33<br />
Richter, A., 11–13, 33<br />
Rietveld, P., 81<br />
Rieu, A., 127<br />
Rijpstra, W.I., 14, 32<br />
Riley, R.L., 61, 83<br />
Ripio, M.T., 129, 130<br />
Ritz, D., 62<br />
Rivera-Ramos, I., 129, 130<br />
Ro, Y.T., 77, 84<br />
Robert, C., 124, 125<br />
Roberts, D.M., 82<br />
Roberts, G., 47<br />
Roberts, G.P., 4<br />
Roberts, K., 33<br />
Roberts, K.J., 29, 32<br />
Robertson, G.T., 129<br />
Rob<strong>in</strong>son, N., 91, 96<br />
Robson, R., 72<br />
Rocha, E.P.C., 137<br />
Rodrigues, C.D., 84, 86<br />
Rodrıguez-Valera, F., 15, 16<br />
Rogers, J., 90<br />
Rogers, Y.H., 6, 7, 10, 15, 16, 22, 34<br />
Rohwer, F., 33<br />
Rom, W., 60, 83<br />
Romanek, C.S., 13, 32, 33<br />
Rondon, E., 59<br />
Rose, M., 129, 130<br />
Rosenzweig, A.C., 8, 16, 17<br />
Ross, C., 33<br />
Rossano, R., 131<br />
Rother, M., 76, 77<br />
Rouanet, C., 59<br />
Rouquette, C., 129, 130<br />
Rub<strong>in</strong>, B.K., 89<br />
Rub<strong>in</strong>, E.J., 47, 48<br />
Rudolph, F.B., 68<br />
Rusch, D., 6, 7, 10, 15, 16, 22<br />
Rusch, D.B., 34<br />
Russell, D.A., 87, 89<br />
Russell, D.G., 52, 54, 55<br />
Russell, G.C., 53<br />
Rustad, T.R., 47, 49, 83, 84<br />
Rutter, S., 90<br />
Ryan, G.J., 59<br />
Rybniker, J., 91, 96<br />
Ryter, S.W., 60, 85<br />
Saano, A., 4<br />
Sacchett<strong>in</strong>i, J.C., 47, 52, 54, 55, 80<br />
Sachdeva, S., 82
160 AUTHOR INDEX<br />
Sahl, H.-G., 129<br />
Sa<strong>in</strong>i, D.K., 82, 84<br />
Saito, K., 34<br />
Sambrook, J., 122<br />
San, K.Y., 66, 68, 87, 93<br />
Sanchez, A.M., 68<br />
Sandaa, R.A., 4<br />
Sangu<strong>in</strong>etti, G., 61, 86<br />
Sangurdekar, D.P., 52, 66, 68<br />
Santoro, A.E., 29, 32<br />
Santos, G.M., 6, 29<br />
Sarath, G., 78<br />
Sareen, D., 79, 80<br />
Sariyar, B., 68<br />
Sathyendranath, S., 34<br />
Sauer, R.T., 122, 123, 127,<br />
128, 130<br />
Saunders, A.M., 32<br />
Saves, I., 93<br />
Savijoki, K., 128, 129<br />
Sawers, G., 68, 69<br />
Sayavedra-Soto, L.A., 16, 17<br />
Schafer, F.Q., 78, 79<br />
Scharf, C., 124, 127, 134, 135<br />
Schauss, K., 23, 25, 28<br />
Sched<strong>in</strong>, U., 85<br />
Scheffers, W.A., 63, 64<br />
Schelle, M.W., 52, 59, 68, 94, 95<br />
Schicho, R.N., 73<br />
Schittone, S., 82<br />
Schl€appy, M.-L., 13, 33, 34<br />
Schlegel, H.G., 77<br />
Schleper, C., 3, 4, 6, 7, 9–11, 13–18, 20,<br />
22–25, 28, 29, 33, 34<br />
Schlieker, C., 127<br />
Schloter, M., 14, 23–25, 28<br />
Schlothauer, T., 129<br />
Schluger, N.W., 60<br />
Schmid, F.X., 125, 132<br />
Schmid, M.C., 4<br />
Schmid, R., 124, 127<br />
Schmidt, A., 132, 134<br />
Schmidt, T.M., 4, 6<br />
Schmitt, S., 13<br />
Schnapp<strong>in</strong>ger, D., 49, 52, 54, 59, 61, 68,<br />
82, 83, 87, 89, 91, 95, 99, 100<br />
Scholz, C., 125, 132<br />
Schoolnik, G.K., 49, 54, 59, 61, 68,<br />
81–83, 89<br />
Schouten, S., 6, 13–15, 30, 32<br />
Schramm, A., 32<br />
Schroeder, W., 129<br />
Schuchhardt, J., 59<br />
Schumann, W., 124, 125, 128, 132<br />
Schuster, S.C., 6, 7, 9, 10, 14, 15,<br />
17, 22–25<br />
Schut, G.J., 73<br />
Schwab, U., 59, 71, 85, 89, 124<br />
Schwark, L., 13, 14, 23–25, 33<br />
Schwartzberg, P., 130<br />
Scrutton, N.S., 81<br />
Sears, H.J., 68, 69<br />
Seedorf, H., 94, 95<br />
Seeger, K., 90<br />
Segal, W., 48, 51, 53, 54, 58–60, 99<br />
Seifritz, C., 89<br />
Seitz, H., 15, 16<br />
Selezi, D., 4, 11, 23<br />
Sengupta, S., 72, 73<br />
Senior, P.J., 66, 67<br />
Senner, C., 59, 84, 90<br />
Sensen, C.W., 15<br />
Seravalli, J., 76<br />
Shah, S.K., 60, 83<br />
Shah, S.R., 6, 29<br />
Shakila, H., 84<br />
Shalel-Levanon, S., 93<br />
Sharma, D., 84<br />
Sharma, S., 23, 25, 28<br />
Sheehan, H.L., 58<br />
Shen, G., 96<br />
Shen, J., 23, 24, 26–28<br />
Shen, J.P., 26<br />
Sherman, D.R., 47–49, 54, 59, 61, 68,<br />
82–84, 89<br />
Sherratt, A.L., 59, 84, 90<br />
Sherrid, A.M., 47, 49<br />
Shi, L., 59
AUTHOR INDEX 161<br />
Shiloh, M.U., 54, 61, 82, 83, 85, 86<br />
Shimizu, M., 78<br />
Sh<strong>in</strong>, M., 16, 17<br />
Shizuya, H., 15<br />
Shock, E.L., 33<br />
Siboni, N., 33<br />
Siddiqui, S.M., 122, 123, 128<br />
Sieber, S.A., 129<br />
Siegers, K., 122<br />
Sievert, S.M., 16, 17, 74, 75<br />
Silva, N.A., 129, 130<br />
Silver, R.F., 84<br />
Simon, H.M., 4<br />
S<strong>in</strong>gh, A., 68, 87, 90–99<br />
S<strong>in</strong>gh, K.K., 82<br />
S<strong>in</strong>gh, S.K., 123<br />
S<strong>in</strong>gh, V.K., 130<br />
S<strong>in</strong>ha, B., 130<br />
S<strong>in</strong>skey, A.J., 62<br />
Sirakova, T.D., 68, 90, 94<br />
Sirsi, M., 54, 58<br />
Sivan, A., 33<br />
Skelton, J., 90<br />
Small, P.M., 83<br />
Smalla, K., 23, 25, 28<br />
Smith, D.A., 84<br />
Smith, H., 34<br />
Smith, H.O., 6, 7, 10, 15, 16, 22<br />
Smith, I., 59<br />
Smith, R.J., 59, 84, 90<br />
Smittenberg, R.H., 13, 32<br />
Snoep, J.L., 64, 65, 68, 78, 87<br />
Snyder, S.A., 60<br />
Soares, M.P., 84, 86<br />
Sohal, R.S., 50<br />
Sohaskey, C.D., 59, 89<br />
Somerville, G.A., 130<br />
Song, T., 77, 84<br />
Song, Z.Q., 33<br />
Soni, V., 91<br />
Sørensen, K., 127, 130<br />
Soteropoulos, P., 59<br />
Sousa, E.H., 60, 83, 84<br />
Souza, V., 34<br />
Spang, A., 20, 22, 23<br />
Spano, G., 127<br />
Spellman, P.T., 62<br />
Spencer, J.S., 59<br />
Spencer, M.E., 53<br />
Spieck, E., 11, 12, 20, 22, 23<br />
Spiess, S., 132, 134<br />
Spiro, S., 87, 89<br />
Spouge, J.L., 123<br />
Spr<strong>in</strong>gstead, J.R., 13<br />
Squares, R., 90<br />
Squares, S., 90<br />
Sridharan, V., 94<br />
Sr<strong>in</strong>ivasan, V., 74, 75<br />
Srivastava, B.S., 59<br />
Srivastava, R., 59<br />
Srivastava, V., 59<br />
Stabler, R.A., 47<br />
Stahl, D.A., 6, 9–13, 16, 17, 20, 22, 23, 33<br />
Standfest, S., 13<br />
Stan-Lotter, H., 33<br />
Staubli, A., 51<br />
Steenkamp, D.J., 81<br />
Steffek, M., 79<br />
Steger, D., 13, 33, 34<br />
Ste<strong>in</strong>, J.L., 15, 16<br />
Ste<strong>in</strong>, L., 34<br />
Ste<strong>in</strong>buchel, A., 67, 68, 90, 94<br />
Stenzel, U., 33<br />
Stephen, J.R., 3, 4<br />
Steuber, J., 73<br />
Stevenson, T.J., 50<br />
Stewart, A., 82<br />
Stewart, C., 34<br />
Steyn, A.J., 49, 54, 60, 61, 68, 82–87,<br />
89–99<br />
Steyn, A.J.C., 49, 82, 83, 85, 90–93<br />
Stoecker, K., 11, 12<br />
Stoker, N.G., 84<br />
Stolarczyk, E., 60<br />
Storz, G., 62<br />
Stoveken, T., 67<br />
Strausberg, R.L., 34<br />
Streit, W., 20, 22, 23
162 AUTHOR INDEX<br />
Strohl, W.R., 55<br />
Strong, M., 30, 59<br />
Stuehr, D.J., 86<br />
Sturm, A.W., 47<br />
Suematsu, M., 52, 75, 76<br />
Sugahara, J., 16, 29, 33<br />
Suhail Alam, M., 91<br />
Sulston, J.E., 90<br />
Suter, E., 57<br />
Sutton, G., 34<br />
Suzuki, M.T., 15<br />
Swanson, I., 95<br />
Swanson, R.V., 15, 16<br />
Swenson, D., 52, 54, 55<br />
Switzer, R.L., 123, 128–130<br />
Ta, P., 79, 80<br />
Tabarsi, P., 47<br />
Taddei, F., 137<br />
Tahlan, K., 68, 87, 95, 99<br />
Takahashi, K., 72<br />
Takai, K., 6<br />
Takaya, N., 78<br />
Tal, Y., 34<br />
Talaat, A.M., 59<br />
Tamayo-Castillo, G., 34<br />
Tan, G., 53<br />
Tanaka, K., 84<br />
Tanaka, Y., 54<br />
Tarran, R., 59, 71, 85, 89<br />
Tascon, R.I., 129, 130<br />
Tassou, C.C., 137<br />
Tavano, C., 59<br />
Taylor, C.D., 74, 75<br />
Taylor, C.J., 87, 89<br />
Taylor, K., 60, 84, 86, 90<br />
Taylor, L.T., 15, 16, 29, 30, 32<br />
Taylor, M.W., 13, 33, 34<br />
Taylor, R.P., 50<br />
Teague, W.G., 85, 90<br />
Teal, T.K., 70, 71<br />
Teira, E., 6, 30<br />
Teixeira de Mattos, M.J., 64, 65, 68, 87<br />
Tekaia, F., 90<br />
Tepper, R., 54<br />
Tessarz, P., 127<br />
Thauer, R.K., 94, 95<br />
Thevele<strong>in</strong>, J.M., 63<br />
Thiele-Bruhn, S., 23, 25, 28<br />
Thomashow, L.S., 70, 72<br />
Thomassen, M.J., 90<br />
Thompson, E.T., 129<br />
Thompson, M.W., 123<br />
Thomson, A.J., 91<br />
Thorpe, J., 34<br />
Thunnissen, F.B., 90<br />
Tian, F., 13<br />
Tian, J., 52, 75, 76<br />
Tickoo, R., 94<br />
Tilton, R.G., 50<br />
Timmers, P., 30<br />
Tischendorf, G., 129<br />
Tischer, K., 124, 127<br />
Tischler, P., 20, 22, 23<br />
Toledo, J.C., 49, 60, 82–86<br />
Tomboulian, P., 60<br />
Tomkiewicz, R.P., 89<br />
T€ornberg, D.C., 85<br />
Torre, O., 85, 90<br />
Torsvik, V., 4<br />
Touchon, M., 137<br />
Tourna, M., 26<br />
Tran, B., 34<br />
Treangen, T.J., 137<br />
Tremblay, G.A., 84<br />
Treusch, A.H., 6, 7, 9, 10, 15, 17, 22<br />
Triccas, J.A., 59<br />
Trivedi, O.A., 94<br />
Trombley, J., 91–93<br />
Trotter, E.W., 62<br />
Tsai, F.T.F., 127<br />
Tsai, M.C., 84<br />
Tsukahara, K., 128<br />
Tuckerman, J.R., 60, 83, 84<br />
Tufariello, J.A., 84<br />
Tuomanen, E., 130<br />
Turgay, K., 128–132, 134, 138<br />
Tyagi, J.S., 81, 82, 84
AUTHOR INDEX 163<br />
Ulrich, M., 59, 71, 85, 89<br />
Ulrichs, T., 48, 59<br />
Unson, M.D., 80<br />
Upton, A.M., 52, 55, 94<br />
Urakawa, H., 10, 16, 17<br />
Urich, T., 13, 14, 23–25, 33<br />
Utaida, S., 130<br />
Utterback, T., 34<br />
Vadali, R.V., 68<br />
Valadi, A., 63<br />
Valadi, H., 63<br />
Valdramidis, V.P., 137<br />
Valente, F.M.A., 73<br />
Van Aken, H., 6<br />
Van Beusichem, M.L., 4<br />
van Bleijswijk, J., 14, 30, 32<br />
Van Damme, O., 79<br />
van de Guchte, M., 124, 125<br />
Van der L<strong>in</strong>den, E., 72<br />
van der Meer, M.T., 14, 32<br />
Van Der Merwe, M.J., 78<br />
Van Der Weijden, C.C., 78<br />
van Dijken, J.P., 63, 64<br />
van Du<strong>in</strong>, A.C., 13<br />
van Gumpel, E., 91, 96<br />
Van Keulen, G., 78<br />
van Maris, A.J.A., 63<br />
Varcamonti, M., 131<br />
Varma, K.G., 54<br />
Varmanen, P., 125, 128–132,<br />
134, 136<br />
Vats, A., 94<br />
Vaudaux, P., 134<br />
Vazquez-Boland, J.A., 129, 130<br />
Vazquez-Torres, A., 68<br />
Velayati, A.A., 47<br />
Veldhuis, M.J.W., 30<br />
Velez, L., 83<br />
Velthof, G.L., 4<br />
Vemuri, G.N., 52, 66, 68<br />
Venceslau, S.S., 73<br />
Venter, J.C., 6, 7, 10, 15, 16, 22, 34<br />
Venter, J.E., 34<br />
Veth, C., 6<br />
Via, L.E., 60, 84, 86<br />
Vignais, P.M., 72<br />
Vilchez, J.R., 83<br />
Vilcheze, C., 74–77, 80<br />
Villacorta, R., 15<br />
Villadsen, J., 64<br />
Villen, J., 128<br />
Visconti, K., 91<br />
Visconti, K.C., 49, 54, 59, 61, 68,<br />
81–83, 89<br />
Voet, D., 49, 55<br />
Voet, J.G., 49, 55<br />
Vogensen, F.K., 125, 129–132, 134–136<br />
Vogt, R.N., 81<br />
V€olker, U., 124, 130, 132, 134<br />
Voskuil, M., 54, 59, 68, 82, 89<br />
Voskuil, M.I., 49, 54, 59, 61, 68, 75,<br />
81–83, 85–87, 89, 90, 93–99<br />
Waddell, S.J., 59, 84, 90<br />
Wagner, K., 60, 75, 83, 84, 86, 87, 89<br />
Wagner, M., 4, 11–13, 20, 22, 23, 33, 34<br />
Wah, D.A., 122, 123, 128<br />
Wakeham, S.G., 14, 30, 32<br />
Waldm<strong>in</strong>ghaus, T., 128<br />
Walker, C.B., 9–13, 16, 17, 33<br />
Waltermann, M., 67<br />
Walunas, T., 124, 125<br />
Wang, C.H., 60, 83, 85<br />
Wang, H., 130<br />
Wang, J., 123<br />
Wang, T., 95, 96<br />
Wang, X., 47<br />
Ward, B.B., 30<br />
Ward, D.E., 78<br />
Ward, D.M., 14, 32<br />
Ward, S.K., 59<br />
Warner, D.F., 47<br />
Watanabe, M., 72<br />
Watanabe, Y., 16, 29, 33<br />
Waterbury, J.B., 9, 10, 12<br />
Wawrzynow, A., 132<br />
Wayne, L.G., 54, 60, 83, 89
164 AUTHOR INDEX<br />
Weber-Ban, E.U., 123, 127<br />
Wegley, L., 33<br />
Weibezahn, J., 127<br />
Weidler, G.W., 33<br />
Weidmann, S., 127<br />
Weidner, J.R., 60, 83<br />
Weigand, W.A., 56<br />
Weiss, T., 59, 71, 85, 89<br />
Weissenbach, J., 124, 125<br />
Weitzberg, E., 85<br />
Wells-Bennik, M.H.J., 137<br />
Weraarchakul, W., 130<br />
West, J.B., 61<br />
Westerhoff, H.V., 78<br />
Westermann, P., 32<br />
Whalan, S., 13, 33, 34<br />
Wheeler, P.R., 47, 51, 52<br />
White, O., 6, 7, 10, 15, 16, 22<br />
Whitehead, S., 90<br />
Whiteley, M., 70<br />
Whitman, W.B., 3<br />
Whitwell, F., 58<br />
Wickner, S., 122, 123<br />
Wiebe, W.J., 3<br />
Wiedmann, M., 124<br />
Wiegel, J., 13, 32, 33<br />
Wiegert, T., 128<br />
Wiklund, N.P., 85<br />
Wilke, B.M., 23, 25, 28<br />
Wilk<strong>in</strong>son, B.J., 130<br />
Wilk<strong>in</strong>son, R.J., 49, 52, 59, 61, 100<br />
Williams Jr., C.H., 81<br />
Williams, K.J., 68, 87, 95, 99<br />
Williamson, J.R., 50<br />
Williamson, S., 34<br />
Willms, K., 66, 67<br />
Wimpenny, J.W., 64, 65<br />
Wimpenny, J.W.T., 70, 71<br />
W<strong>in</strong>efield, C., 24, 26–28<br />
W<strong>in</strong>efield, C.S., 26<br />
W<strong>in</strong>kler, M.E., 129<br />
W<strong>in</strong>stanley, C., 70, 71<br />
Wipat, A., 127<br />
Wirsen, C.O., 74, 75<br />
Wisedchaisri, G., 82<br />
Witt, E., 123, 128, 129, 131, 138<br />
Woebken, D., 13, 33, 34<br />
Woese, C.R., 13<br />
Wolfe, A.J., 56, 64, 65<br />
Wol<strong>in</strong>, M.J., 49, 61, 76<br />
Wong, P.M., 95<br />
Wong, S.L., 125<br />
Woodruff, R.V., 127, 130<br />
Worlitzsch, D., 59, 71, 85, 89<br />
Wouters, J.A., 131, 137<br />
Wrage, N., 4<br />
Wright, D.E., 57<br />
Wu, C.W., 59<br />
Wu, D., 6, 7, 10, 15, 16, 22, 34<br />
Wu, K., 30<br />
Wu, K.-F., 130<br />
Wu, K.Y., 10–12, 15, 16, 33<br />
Wu, L., 34<br />
Wu, Y., 96<br />
Wuchter, C., 6, 14, 15, 30<br />
Xia, D., 95<br />
Xie, Q.W., 60, 83<br />
Xu, J., 84<br />
Xu, M., 23<br />
Xu, S.-X., 130<br />
Xu, W.-C., 130<br />
Xu, X., 68, 87, 95, 99<br />
Yamagata, K., 72<br />
Yamamoto, M., 74<br />
Yan, B.S., 49, 54, 61, 68, 85, 86, 89<br />
Yan, L.J., 50<br />
Yan, T., 34<br />
Yang, J., 53<br />
Yang, Y.T., 68<br />
Yankaskas, J.R., 59, 71, 85, 89<br />
Yao, Y., 50, 97<br />
Ye, Q., 33<br />
Yee, C.H., 61, 86<br />
Yi-Lian, L., 13<br />
Y<strong>in</strong>, Y.-B., 130<br />
Yooseph, S., 34
AUTHOR INDEX 165<br />
Young, D., 49, 52, 59,<br />
61, 100<br />
Young, D.B., 47<br />
Young, J.C., 122<br />
Yu, C.A., 95<br />
Yu, C.T., 60, 83, 85<br />
Yu, J.Y., 77<br />
Yu, L., 95<br />
Yuan, G., 125<br />
Yukl, E.T., 83<br />
Yun, C.S., 68, 87, 95, 99<br />
Yura, T., 124<br />
Zagorec, M., 131<br />
Zahn, R., 127<br />
Zbell, A.L., 73<br />
Zeiher, A.M., 50<br />
Zeller, K., 47<br />
Zellmeier, S., 128<br />
Zentgraf, H., 127<br />
Zervos, A., 137<br />
Zhang, C.L., 13, 32, 33<br />
Zhang, L., 23<br />
Zhang, Q., 130<br />
Zhang, X., 13, 50, 97<br />
Zhang, X.-M., 130<br />
Zhang, X.Q., 50<br />
Zhao, W.D., 33<br />
Zheng, G., 128<br />
Zheng, M., 62<br />
Zheng, R., 81<br />
Zheng, Y., 23<br />
Zhou, J., 34<br />
Zhu, G., 54, 59, 68, 82, 84<br />
Zhu, Y., 23<br />
Ziazarifi, A.H., 47<br />
Ziebandt, A.K., 124, 127<br />
Zolkiewski, M., 123<br />
Zotta, T., 131<br />
Zuber, P., 128<br />
Zuber, U., 125<br />
Z€uhlke, D., 128, 131, 132, 138<br />
Zvyagilskaya, R.A., 87, 89<br />
Zylicz, M., 132
This page <strong>in</strong>tentionally left blank
Subject Index<br />
Note: The page numbers taken from figures and tables are given <strong>in</strong> italics.<br />
A<br />
Acetyl-CoA synthase (ACS), 76<br />
Acetyl-coenzyme A (acetyl-CoA), 51<br />
N-Acetylglucosam<strong>in</strong>yl<strong>in</strong>ositol, 79, 80<br />
Aerobic ammonia oxidation, 4<br />
Aerobic archaea, 3<br />
Aerobic carboxydotrophic bacteria, 76<br />
Aerobic microorganisms, 4<br />
Allylthiourea, 11<br />
Ammonia, 3, 4<br />
competition for, 30<br />
conversion to nitrite, 33<br />
oxidation, 3, 4, 13, 17, 25, 30<br />
source of, 25<br />
Ammonia monooxygenase (AMO), 6<br />
pMMO-related prote<strong>in</strong>, 8<br />
PMO, phylogenetic relationship, 8<br />
Ammonia oxidation, 3, 4<br />
Ammonia oxidisers<br />
as dist<strong>in</strong>ct phylum with<strong>in</strong> archaea, 4,<br />
19–22<br />
phylogenetic tree, 23<br />
process<strong>in</strong>g genes, distribution,<br />
20–22<br />
uncultivated, metagenomic studies,<br />
15–16<br />
amo-related genes, 15<br />
confirm<strong>in</strong>g close relationship of, 16<br />
microheterogeneity, 16<br />
Ammonia-oxidis<strong>in</strong>g archaea (AOA), 4,<br />
6–13<br />
ammonia oxidation k<strong>in</strong>etics, 10<br />
amo/pmo genes, 8<br />
associated with mar<strong>in</strong>e <strong>in</strong>vertebrates,<br />
33–34<br />
sponge-associated<br />
communities, 34<br />
Cenarchaeum/Ax<strong>in</strong>ella<br />
association, 11<br />
contrast<strong>in</strong>g response to nitrogen<br />
deposition, 27<br />
cultivated, 12<br />
diversity, distribution and activity,<br />
22–23<br />
phylogenetic tree, 24<br />
enrichments, 11<br />
genomes and metagenomes, 15<br />
BAC-derived fosmid vectors, 15<br />
WGS approach, 15<br />
<strong>in</strong> geothermal environments, 32–33<br />
isolation, 9<br />
<strong>in</strong> mar<strong>in</strong>e environment, 28–29<br />
activity, 30–32<br />
fluxes <strong>in</strong> <strong>in</strong>organic nitrogen concentrations,<br />
31<br />
membrane lipids of, 13–15<br />
GDGT, 13, 14<br />
phospholipids, 13<br />
recovery of crenarchaeol, 13<br />
TEX 86 <strong>in</strong>dex, 14<br />
oligotrophic lifestyle, 10<br />
open read<strong>in</strong>g frames (ORFs) cod<strong>in</strong>g<br />
for, 6<br />
phylogenetic relationship, of AMO<br />
and PMO, 8<br />
predicted ORFs, on 43 kb soil fosmid<br />
54d9, 7<br />
<strong>in</strong> sediments, 32
168 SUBJECT INDEX<br />
<strong>in</strong> soil environment, 23–24<br />
activity, 25–28<br />
amoA gene abundance <strong>in</strong>, 25<br />
growth of acetylene-sensitive<br />
ammonia-oxidis<strong>in</strong>g archaea <strong>in</strong>, 28<br />
16S rRNA-based PCR surveys, 11<br />
Ammonia-oxidis<strong>in</strong>g bacteria (AOB),<br />
4, 6<br />
amoA gene abundance <strong>in</strong> soils, 25<br />
autotrophic, fixation of carbon, 19<br />
contrast<strong>in</strong>g response to nitrogen<br />
deposition, 27<br />
environmental factors, 22<br />
growth characteristics, 25<br />
growth dynamics, 26<br />
nitrification activity, 28<br />
pathways of nitrogen, oxygen and<br />
electron flow, 18<br />
ratio of AOA to AOB amoA genes,<br />
23, 25<br />
amoA genes, 9, 14, 29<br />
Anti-mycobacterial drug, 47<br />
Anti-sigma factor, SpoIIAB, 128<br />
AOA. See Ammonia-oxidis<strong>in</strong>g archaea<br />
(AOA)<br />
AOB. See Ammonia-oxidis<strong>in</strong>g<br />
bacteria (AOB)<br />
Archaea<br />
carbon metabolism, 6<br />
<strong>in</strong> moderate aerobic habitats, 4<br />
phylogenetic relationship, 5<br />
role <strong>in</strong> global cycles, 3<br />
16S rRNA gene-def<strong>in</strong>ed l<strong>in</strong>eages, 5<br />
Arthrobacter oxydans, 78<br />
Aspergillus nidulans, 78<br />
ATPase, 123, 129<br />
ATP citrate lyase, 74<br />
ATP-dependent proteases, 123<br />
ATP hydrolysis, 123<br />
ATP synthesis, 69, 89<br />
Auto-phosphorylation, 138<br />
Ax<strong>in</strong>ella mexicana, 11, 13, 16, 33<br />
Azorhizobium, 67<br />
Azotobacter beijer<strong>in</strong>ckii, 66<br />
Azotobacter v<strong>in</strong>elandii, 64, 67<br />
B<br />
BAC-derived fosmid vector, 15<br />
Bacillaceae, 127<br />
Bacillales, 124, 131<br />
Bacille Calmette-Guer<strong>in</strong> (BCG)<br />
vacc<strong>in</strong>e, 47<br />
survival, under hypoxic conditions, 61<br />
Bacillus stearothermophilus, 132<br />
Bacillus subtilis, 124, 125, 128<br />
Bacterial artificial chromosomes<br />
(BACs), 15<br />
Bacterial regulatory circuits, 138<br />
Blood–bra<strong>in</strong> barrier, 86<br />
Bone marrow-derived macrophages, 85<br />
Bradyrhizobium, 67<br />
C<br />
Calv<strong>in</strong>–Bassham–Benson cycle, 19<br />
Calv<strong>in</strong> cycle, 76, 77<br />
Candida boid<strong>in</strong>ii, 66<br />
Carbon cycle, 3<br />
Carbon dioxide (CO2), 49<br />
Carbon monoxide (CO), 49<br />
b<strong>in</strong>d<strong>in</strong>g of CO to DosS and<br />
DosT, 84<br />
Dos regulon and, 85–86<br />
as energy source, 77<br />
<strong>in</strong> Mtb persistence, 60<br />
oxidation, 77<br />
utilis<strong>in</strong>g microorganisms use enzyme<br />
CODH to, 76<br />
Carbon monoxide (CO) dehydrogenase<br />
(CODH), 76–77<br />
Carbon oxidation state (COS), 54–56<br />
Carboxydotrophic microbes, 76<br />
Cellular chaperone mach<strong>in</strong>ery, 122<br />
Cenarchaeum symbiosum, 10, 11<br />
ammonia oxidation by, 11
SUBJECT INDEX 169<br />
from concatenated dataset of<br />
ribosomal prote<strong>in</strong>s, 20<br />
fluorescent <strong>in</strong> situ hybridisation, 10<br />
genes for urease, 17<br />
genome of, 15, 16<br />
G + C content, 16<br />
pathway for carbon fixation, 19<br />
Chemolithoautotrophic ammonia<br />
oxidisers, 9<br />
Clostridium welchii, 70<br />
clpC, clpP and clpB operons, 124<br />
clpL <strong>in</strong> staphylococci, 127<br />
Clp mach<strong>in</strong>ery, 123<br />
ClpP complex, 123<br />
Clp protease, 123<br />
Clp-specific degradation, 128<br />
clpX expression, regulation of, 127<br />
13<br />
C-metabolic flux analysis, 99<br />
CoA-dependent KDH, 75<br />
Corynebacterium glutamicum, 91<br />
Crabtree effect, 65<br />
Crenarchaeol, 13, 14, 31, 32<br />
Crenarchaeota, 4, 6, 20<br />
hyperthermophilic, 13<br />
CtsR degradation<br />
McsB k<strong>in</strong>ase activity, 138<br />
role of McsB as adaptor, 138<br />
YwlE, to counteract, 138<br />
ctsR gene, 124, 125, 127, 137<br />
CtsR <strong>in</strong>activation, 124–125, 136<br />
dur<strong>in</strong>g oxidative stress, 135<br />
CtsR prote<strong>in</strong>, 134<br />
CtsR-regulated genes, 124–127<br />
cellular functions of, 127–131<br />
CtsR-regulated prote<strong>in</strong>s,<br />
distribution, 126<br />
CtsR regulon, physiological<br />
function, 127<br />
CtsR repressor, 124, 131<br />
mechanisms for <strong>in</strong>activation of, 131<br />
heat <strong>in</strong>activation of CtsR, 131–134<br />
dur<strong>in</strong>g other stress conditions,<br />
136–137<br />
dur<strong>in</strong>g oxidative stress, 134–136<br />
Cyste<strong>in</strong>e, 79<br />
Cystic fibrosis (CF), 59<br />
Cytochromes, 17<br />
D<br />
Denitrification, 4, 17, 19, 34<br />
Dihydroxyacetone phosphate<br />
(DHAP), 63<br />
Dissolved O2 tension (DOT), 65<br />
Dithiothreitol (DTT), 62<br />
DNA damage-<strong>in</strong>ducible<br />
regulator, 129<br />
Dos two-component system, 81<br />
Drosophila melanogaster, 50<br />
E<br />
Ecosystems, 3<br />
Energy-dependent prote<strong>in</strong><br />
degradation, 123<br />
Enterococcus faecalis, 78<br />
Environmental stress response<br />
(ESR), 62<br />
Escherichia coli, 15, 56, 64–66, 70, 124<br />
acetate excretion, 56<br />
fermentation, 64–66 (see also<br />
Reductive s<strong>in</strong>ks)<br />
<strong>in</strong>tracellular redox state, 65<br />
nark narU mutant, 89<br />
TCA cycle, 52<br />
Estuaries, 4<br />
Ethionamide (ETH), 80<br />
Euryarchaeota, 4, 20<br />
Extensively drug resistant<br />
(XDR), 47<br />
Extremophiles, 3<br />
F<br />
Fatty acids (FAs), 49<br />
Ferredox<strong>in</strong>s, 74<br />
Fe–S cluster prote<strong>in</strong>s, 96
170 SUBJECT INDEX<br />
Formate dehydrogenase (FDH)<br />
reaction, 64<br />
Fumarate reductase (FRD), 53, 75<br />
G<br />
Genetic mutation, 137<br />
Genomic techniques, advancement, 15<br />
Gibbs free energy, 89<br />
Glucuronic acid, 50<br />
Glucose-6-phosphate dehydrogenase<br />
(G6PD), 50<br />
Glutathione disulfide–glutathione<br />
couple (GSSG/2GSH), 78<br />
Glycerol dialkyl glycerol tetraethers<br />
(GDGTs), 13, 14<br />
Glycerol formation, 63<br />
Glycerol-phosphate dehydrogenase<br />
(GPD2) mutant, 63<br />
G3PD isoform, 63<br />
Greenhouse gas, 4<br />
Groundwater pollution, 4<br />
GSH/GSSG ratio, 50<br />
H<br />
Heat shock prote<strong>in</strong>s (Hsp), 50, 124,<br />
127, 134<br />
Hsp18, 127<br />
Hsp27, 50<br />
Hsp 100/Clp, 123, 127<br />
Helicobacter pylori, 73<br />
Helix-turn-helix (HTH), 90, 131<br />
Heme oxygenase-1 (HO-1), 60<br />
High-hydrostatic pressure (HHP)tolerant<br />
mutants, 136<br />
HrcA-dependent genes, 125<br />
HrcA repressor, 125<br />
Hsp. See Heat shock prote<strong>in</strong>s (Hsp)<br />
Hydrogenases (H2ases), 54, 72–74<br />
Hydroxylam<strong>in</strong>e oxidoreductase<br />
(HAO), 9, 17<br />
Hyperthermophilic organisms, 4<br />
Hypoxia, 50, 76, 84<br />
I<br />
Inducible nitric oxide synthase (iNOS),<br />
60, 83<br />
Isoniazid (INH), 80<br />
K<br />
a-Ketoglutarate (KG), 53<br />
a-Ketoglutarate decarboxylase<br />
(KGD), 75<br />
a-Ketoglutarate:ferredox<strong>in</strong><br />
oxidoreductase (KOR), 74<br />
Klebsiella aerogenes, 70<br />
Klebsiella pneumoniae, 73<br />
L<br />
Lactobacillaceae, 127<br />
Lactobacillales, 125, 138<br />
Lactobacillus acidophilus, 124<br />
Lactobacillus plantarum, 127<br />
Lactobacillus sakeii, 131<br />
Listeriaceae, 127<br />
Listeria monocytogenes, 124, 130, 137<br />
Lactate, 77, 78<br />
Low-molecular-weight (LMW), 79<br />
Low-molecular-weight prote<strong>in</strong> tyros<strong>in</strong>e<br />
phosphatase (LMWPTP), 132<br />
M<br />
Macrophages, 59, 60, 85, 95, 99<br />
Mar<strong>in</strong>e archaea<br />
carbon metabolism, 6<br />
complete genome sequences,<br />
predictions from, 16–19<br />
genomic fragments from, 6<br />
tractable system for study of, 11<br />
McsB adaptor, 138
SUBJECT INDEX 171<br />
McsB k<strong>in</strong>ase, 136, 138<br />
Metallosphaera sedula, 19<br />
Methanobacterium mazei Go1, 70<br />
Methanogenic archaea, 3<br />
5-N-Methyl-1-hydroxyphenaz<strong>in</strong>e, 70<br />
Methyl-malonyl CoA (MMCoA), 94<br />
Methylococcus capsulatus, 8<br />
Microarray analysis, 62, 73, 81<br />
Microautoradiography, 6<br />
Molecular chaperones dnaK and<br />
groE, 125<br />
Mono-cistronic gene, 127<br />
MSH acetyltransferase, 79<br />
MSH-dependent enzymes, 81<br />
MSH disulfide reductase, 81<br />
MSH mutants, 80, 98<br />
Msm mutant, 74, 80<br />
Mtb. See Mycobacterium tuberculosis<br />
(Mtb)<br />
Mtb dos dormancy regulon, 81<br />
biological role, and function,<br />
81–84<br />
Dos regulon and CO, 85–86<br />
and reductive stress, 86–87<br />
nitrate reductase, 87–90<br />
TAG production, 90<br />
and virulence, 84–85<br />
Mtb DwhiB3 mutant, 92, 93<br />
Mtb tgs1, expression of, 68<br />
Mtb virulence lipid, 59, 94<br />
Mtb WhiB7<br />
depict<strong>in</strong>g, sens<strong>in</strong>g and dissipat<strong>in</strong>g<br />
reductive stress to, 96<br />
as <strong>in</strong>tracellular redox sensor, 90<br />
Mtb whiB genes, 91<br />
Multidrug resistant (MDR), 47<br />
Mycobacteriophage TM4, 91<br />
Mycobacterium bovis, 76<br />
Mycobacterium smegmatis, 73<br />
Mycobacterium tuberculosis (Mtb),<br />
45, 47<br />
culture <strong>in</strong> vitro, 48<br />
Dos dormancy regulon, and role<br />
<strong>in</strong>, 48<br />
DwhiB3 deletion stra<strong>in</strong>, 92<br />
environmental factors, <strong>in</strong>fluenc<strong>in</strong>g<br />
metabolism, 52<br />
balanc<strong>in</strong>g act <strong>in</strong> vitro and <strong>in</strong> vivo,<br />
58–60<br />
carbon oxidation state (COS),<br />
54–56<br />
gaseous environment, of lung,<br />
60–61<br />
metabolites, excretion of, 56–58<br />
redox balance, 56–58<br />
TCA cycle, 52–54<br />
historical knowledge, 48, 51–52<br />
physiological characteristics, 51–61<br />
W-Beij<strong>in</strong>g l<strong>in</strong>eage, 82<br />
WhiB3 and virulence, 91–93<br />
Mycolic acid, 93<br />
Mycothiol (MSH), 79<br />
biosynthetic pathway, 80<br />
Mycothiol S-conjugate amidase<br />
(Mca), 81<br />
N<br />
NADH/NAD + ratio, 50, 66, 87<br />
NAD + /NADH-<strong>in</strong>dependent<br />
enzymes, 74<br />
NAD + /NADH ratio, 67, 71<br />
NADPH/NADP + system, 49<br />
Nitrate reductase (Nap), 69, 87–90<br />
Nitric oxide (NO), 49, 92<br />
Nitrification, 3, 4<br />
aerobic ammonia oxidation dur<strong>in</strong>g, 9<br />
Nitrifier-denitrification mechanisms, 4<br />
Nitrite, 3<br />
Nitrite-oxidis<strong>in</strong>g bacteria, 4, 9<br />
Nitrogen cycle, 3<br />
Nitrosocaldus yellowstonii, 11<br />
Nitrosopumilus maritimus, 9, 10, 17,<br />
19, 20<br />
stra<strong>in</strong> SCM1, 10
172 SUBJECT INDEX<br />
Nitrososphaera gargensis, 11<br />
belongs to l<strong>in</strong>eage of AOA dist<strong>in</strong>ct<br />
from, 11<br />
conta<strong>in</strong> crenarchaeol, 13<br />
GDGTs of, 14<br />
genomic <strong>in</strong>formation, 20<br />
Nitrosothiol reductase, 81<br />
Nitroxyl (HNO), 17<br />
NO dehydrogenase (NODH), 77<br />
O<br />
Oenococcus oeni, 125<br />
Open read<strong>in</strong>g frames (ORFs), 6<br />
b-Oxidation, 67, 76, 86, 88, 94<br />
Oxidation–reduction reactions, 97<br />
metabolic homeostasis, 45<br />
Oxidative phosphorylation, 47<br />
Oxidative stress, 45, 91, 122, 135<br />
importance, 45<br />
role, 49<br />
2-Oxoglutarate dehydrogenase complex<br />
(ODHC), 53<br />
2-Oxoglutarate:ferredox<strong>in</strong><br />
oxidoreductase (OGFO), 74–75<br />
Oxygen (O2), 49<br />
Oxygen radicals, 53<br />
P<br />
Paleothermometry, 14<br />
Paracoccus pantotrophus, 69<br />
Particulate methane monooxygenase<br />
(pMMO), 8<br />
PHB mutant, 67<br />
Phenaz<strong>in</strong>es, 69–72<br />
Poly b-hydroxybutyrate (PHB), 66, 67<br />
Polyhydroxyalkonate (PHA)<br />
biosynthesis, 66<br />
central regulator, 67<br />
support, direct evidence, 67<br />
Polyketide, 66, 68, 87, 93<br />
Polymer deposition<br />
poly-b-hydroxybutyrate (PHB),<br />
66–68<br />
polyhydroxyalkonate (PHA), 66–68<br />
triacylglycerol (TAG), 51, 59, 66–68,<br />
84, 90, 93, 94, 96<br />
Polymorphism, <strong>in</strong> iNOS, 83<br />
Proteases, 123<br />
26S proteasome system, 123<br />
Prote<strong>in</strong> degradation <strong>in</strong> eukaryotes, 123<br />
Prote<strong>in</strong> homeostasis, 122<br />
Prote<strong>in</strong> turnover, 123<br />
Proteobacteria, 4<br />
Proton motive force, 87<br />
Pseudomonas aerug<strong>in</strong>osa, 70, 71<br />
Pyocyan<strong>in</strong>, 70<br />
Pyrococcus furiosus, 73<br />
Pyruvate catabolism, 64, 65<br />
R<br />
Reactive oxygen species (ROS), 50, 72<br />
Rec-<strong>in</strong>dependent mutations, 137<br />
Redox couples, 49<br />
Redox homeostasis, 88, 98<br />
Redox reactions, 49<br />
Reductive s<strong>in</strong>ks<br />
<strong>in</strong> microbes<br />
carbon monoxide (CO)<br />
dehydrogenase (CODH), 76–77<br />
fermentation, 61–66<br />
hydrogenases, 72–74<br />
nitrate reductase, 68–69<br />
phenaz<strong>in</strong>e production, 69–72<br />
polymer deposition, 66–68<br />
reverse TCA (rTCA) cycle, 74–76<br />
<strong>in</strong> mycobacteria, 78<br />
Dos dormancy regulon, 81–90<br />
<strong>in</strong>tracellular redox environment,<br />
78–81<br />
WhiB prote<strong>in</strong>s, as <strong>in</strong>tracellular<br />
redox sensor, 90–97<br />
Reductive stress, 46, 49, 97<br />
concept of, 49–51
SUBJECT INDEX 173<br />
Regulatory AAA+ prote<strong>in</strong>, 123<br />
Regulatory prote<strong>in</strong>, 70<br />
Reverse TCA (rTCA) cycle, 74–76<br />
enzymes for, 74–75<br />
Rhizobium etli, 67<br />
Rhodococcus opacus, 68<br />
Rhodococcus ruber, 67<br />
Ribulose bisphosphate carboxylase/<br />
oxygenase (RubisCO), 19<br />
16S rRNA gene sequences, 6<br />
S<br />
Saccharomyces cerevisiae<br />
fermentation, 61–64<br />
FRDs, 75<br />
Salmonella, 55, 65, 73<br />
Sense-and-lock model, 86<br />
Shigella, 65<br />
Sigma factor, 97, 124<br />
S<strong>in</strong>gle po<strong>in</strong>t mutation, 91<br />
Sorbitol, 50<br />
ssrA-tagged prote<strong>in</strong>s, 128<br />
Staphylococcaceae, 127<br />
Staphylococcus albus, 70<br />
Staphylococcus aureus, 77, 78, 134<br />
Staphylococcus carnosus, 89<br />
Streptococcus mutans, 128<br />
Streptococcus pneumoniae, 128<br />
<strong>in</strong> mice, 129<br />
nox gene, 66<br />
Streptococcus salivarius, 125, 127<br />
Streptococcus thermophilus,<br />
125, 127<br />
Streptomyces spp., 70, 90<br />
Stress prote<strong>in</strong>, 82<br />
Succ<strong>in</strong>ate dehydrogenase (SDH), 53<br />
Succ<strong>in</strong>ic semialdehyde (SSA), 75<br />
Succ<strong>in</strong>ic semialdehyde dehydrogenase<br />
(SSADH), 75<br />
SufBCD system, 93<br />
Super-XDR (S-XDR), 47<br />
T<br />
TAG anabolism, 68<br />
TAG-produc<strong>in</strong>g bacteria, 67<br />
TAG production, 90, 93<br />
TCA cycle, 19, 33, 52–54, 67<br />
Thaumarchaeota, 20, 23, 29<br />
Thiol-oxidis<strong>in</strong>g agent diamide, 62<br />
Thioredox<strong>in</strong>, 79<br />
Transcription factors, 95, 127<br />
Transhydrogenase activity, 64<br />
a,a’-Trehalose dimycolate (TDM), 93<br />
a,a’-Trehalose monomycolate (TMM),<br />
93<br />
Tuberculosis (TB), 45<br />
<strong>in</strong>fection model, 84<br />
treatment regimes, 47<br />
Tuberculous granuloma, 48<br />
V<br />
Virulence factors, 129<br />
W<br />
Warburg manometry, 58<br />
Wastewater, treatment plants, 4<br />
Wayne model of <strong>in</strong> vitro dormancy, 54,<br />
68, 90<br />
role <strong>in</strong> Mtb survival, 83<br />
WhiB family of prote<strong>in</strong>s, 90–91<br />
and DNA b<strong>in</strong>d<strong>in</strong>g, 95–97<br />
and reductive stress, 93–95<br />
and virulence, 91–93<br />
Whole-genome shotgun (WGS), 6<br />
Wood–Ljungdahl pathway, 76<br />
X<br />
Xanthobacter flavus, 78<br />
Z<br />
Zwitterion, 70
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