POSTERS - BLAST X - University of Utah

BLAST X MEETING
CAMINO REAL SUMIYA HOTEL
CUERNAVACA, MEXICO
JANUARY 18-23, 2009
Meeting Chairperson:
Dr. David Zusman –University of California, Berkeley, CA
Meeting Vice-Chairperson:
Dr. Robert Bourret – University of North Carolina, Chapel Hill, NC
Local Arrangements Chairperson
Dr. Georges Dreyfus - Universidad Nacional Autonoma de Mexico, Mexico City
Program Committee:
Dr. John S. Parkinson (Chairperson) – University of Utah, Salt Lake City, UT
Dr. Joe Falke – University of Colorado, Boulder, CO
Robert Kadner and Robert Macnab Awards Selection Committee:
Dr. Gladys Alexandre – University of Tennessee, Knoxville, TN
Dr. Gerald Hazelbauer (Chairperson) – University of Missouri, Columbia, MO
Dr. Ruth Silversmith - University of North Carolina, Chapel Hill, NC
Dr. Alan Wolfe – Loyola University, Maywood, IL
Meeting Review Committee:
Dr. Phillip Aldridge – Newcastle University, Newcastle upon Tyne, UK
Dr. Brian Crane (Chairperson) – Cornell University, Ithaca, NY
Dr. John Kirby – University of Iowa, Iowa City, IA
Dr. Birgit Scharf – Virginia Tech University, Blacksburg, VA
Board of Directors – BLAST, Inc.:
Dr. Joe Falke – University of Colorado, Boulder, CO
Dr. Michael Manson – Texas A&M University, College Station, TX
Dr. Philip Matsumura (Chairperson) – University of Illinois at Chicago, IL
Dr. John S. Parkinson – University of Utah, Salt Lake City, UT
Administrative Assistants:
Ms. Tarra Bollinger – Molecular Biology Consortium, Chicago, IL
Ms. Peggy O’Neill – Molecular Biology Consortium, Chicago, IL
ii

BLAST X MEETING SCHEDULE
TIME EVENT LOCATION
Sunday, January 18, 2009
5:00 pm Poster room available for poster setup Orquideas Room
7:00 pm – 8:00 pm Dinner Garden
9:00 pm – 11:00 pm Welcome Reception Crisantemos Room
Monday, January 19, 2009
7:30 am – 8:30 am Breakfast Garden
8:45 am – 8:50 am Welcome -- Local Arrangements Chair (G. Dreyfus) Crisantemos Room
8:50 am – 8:55 am Welcome/ Announcements - Meeting Chair (D. Zusman) Crisantemos Room
9:00 am – 12:00 pm Meeting Session – “Two-Component Systems” Crisantemos Room
10:15 am – 10:30 am Coffee Break
12:00 pm – 1:30 pm Lunch Garden
2:00 pm – 4:00 pm Poster Session – even numbered posters Orquideas Room
6:00 pm – 7:30 pm Dinner Garden
7:30 pm – 10:00 pm Meeting Session – “Gliding Motility” Crisantemos Room
8:30 pm – 8:45 pm Coffee Break
Tuesday, January 20, 2009
7:30 am – 8:30 am Breakfast Garden
9:00 am – 12:00 pm Meeting Session – “Flagella” Crisantemos Room
10:15 am – 10:30 am Coffee Break
12:00 pm – 1:30 pm Lunch Garden
2:00 pm – 4:00 pm Poster Session – odd numbered posters Orquideas Room
6:00 pm – 7:30 pm Dinner Garden
7:30 pm – 10:00 pm Meeting Session – “Regulation” Crisantemos Room
8:30 pm – 8:45 pm Coffee Break
Wednesday, January 21, 2009
7:30 am – 8:30 am Breakfast Garden
9:00 am – 12:00 pm Meeting Session – “Chemoreceptors” Crisantemos Room
10:15 am – 10:30 am Coffee Break
12:00 pm – 1:30 pm Lunch Garden
1:30 pm Tour bus to Taxco leaves from Hotel Hotel entrance
1:45 pm Tour buses to Xochicalco leave from Hotel Hotel entrance
Thursday, January 22, 2009
7:30 am – 8:30 am Breakfast Garden
9:00 am – 12:00 pm Meeting Session – “Biofilms and Host Interactions” Crisantemos Room
10:15 am – 10:30 am Coffee Break
12:00 pm – 1:30 pm Lunch Garden
6:00 pm – 7:30 pm Dinner Garden
7:30 pm – 7:45 pm Robert Kadner and Robert Macnab Awards Presentation Crisantemos Room
7:45 pm – 10:00 pm Meeting Session – “Signaling and Behavior” Crisantemos Room
8:30 pm – 8:45 pm Coffee Break
10:00 pm – 12:00 am Reception Crisantemos Room
Friday, January 23, 2009
7:30 am – 8:30 am Breakfast Garden
Shuttle buses leave for Mexico City Airport
iii

BLAST X PROGRAM
January 19, 2009 Two-Component Systems
Monday Morning – 9:00 am – 12:00 pm Chair – Georges Dreyfus
ABSTRACT
PRESENTER TITLE
PAGE NO.
Bourret, Bob
A structural investigation of response regulator
autodephosphorylation
Intramolecular autophosphorylation of the Escherichia coli ArcB
2
Peña-Sandoval, G. sensor kinase 3
Porter, Steven
BREAK
A bifunctional kinase-phosphatase in bacterial chemotaxis
Message passing: Protein structure assembly from sequence data for
4
Szurmant, Hendrik two-component signaling proteins
Integrated control of Caulobacter cell envelope physiology by a hybrid
5
Crosson, Sean two-component/ECF sigma factor signaling network
The role of signal transduction in cell wall metabolism in Bacillus
6
Bisicchia, Paola subtilis
The WalK/WalR essential signal transduction pathway and cell wall
7
Dubrac, Sarah homeostasis in Staphylococcus aureus 8
January 19, 2009 Gliding Motility
Monday Evening – 7:30 pm – 9:30 pm Chair – Lotte Søgaard Andersen
Bulyha, Iryna
Dynamic assembly and disassembly of the Type IV molecular
machine
A “four component” signal transduction system regulates
9
Higgs, Penelope developmental progression in Myxococcus xanthus
Independence and interdependence of Dif and Frz chemosensory
10
Yang, Zhaomin
BREAK
pathways in Myxococcus xanthus chemotaxis 11
Berleman, James Predataxis behavior in Myxococcus xanthus 12
Mauriello, Emilia Dynamic localization of FrzCD in Myxococcus xanthus
Identifying novel bacterial cytoskeletal elements and cytoskeletal
13
Gitai, Zemer interactors through high-throughput imaging 14
January 20, 2009 Flagella
Tuesday Morning – 9:00 am – 12:00 pm Chair – Robert Belas
Rao, Christopher
The role of positive feedback in controlling flagella assembly
dynamics
Crystal structure of FliT, a bacterial flagellar export chaperone for the
15
Imada, Katsumi filament cap protein Hap2 (FliD)
Structural insight into active flagellar motor formation through the
16
Kojima, Seiji periplasmic region of MotB 17
Thormann, Kai
BREAK
Stator selection in Shewanella oneidensis 18
Gauthier, Mathieu Taking control of the bacterial flagellar motor
Experimental evidence for conformational spread in the bacterial
19
Branch, Richard switch complex
Do individual bacterial flagellar motors use hysteresis to maintain a
20
Reuven, Peter robust output in a noisy environment? – an experimental study 21
Tu, Yuhai Dynamics of the bacterial flagellar motor with multiple stators 22
iv

January 20, 2009 Regulation
Tuesday Evening – 7:30 pm – 9:30 pm Chair – Linda Kenney
ABSTRACT
PRESENTER TITLE
PAGE NO.
Kaserer, Alla
Partridge, John
Reimann, Sylvia
BREAK
Tran, Hoa
Martínez, Luary
Lee, Yi-Ying
Effect of osmolytes on regulating the activities of the SSK1
response regulator from Saccharomyces cerevisiae 23
Regulation of Escherichia coli motility by the nitric oxide sensitive
transcriptional repressor NsrR 24
Synthetic lethality uncovers a novel link between the MalT and
OmpR regulons 25
A chemotaxis-like signaling pathway regulates the expression of
extracellular materials in Geobacter sulfurreducens 26
The two-component regulatory system BarA/SirA is at the top of a
multi-factorial regulatory cascade controlling the expression of the
SPI-1 and SPI-2 virulence regulons in Salmonella 27
Interaction of the transcriptional regulatory complex, FlhDC, with its
target DNA 28
January 21, 2009 Chemoreceptors
Wednesday Morning – 9:00 am – 12:00 pm Chair – Sandy Parkinson
Glekas, George A novel amino acid binding structure in bacterial chemotaxis
Structure and function of the Helicobacter pylori chemoreceptor
29
Remington, James TlpB 30
Wright, Gus The TM2-HAMP connection
Structure, assembly and conformational changes in
chemoreceptors studied in intact bacterial cells using Cryo-
31
Khursigara, Cezar
BREAK
electron tomography
Discrete signal-on and -off conformations in the Aer HAMP
32
Watts, Kylie
domain
Investigating the structure of ternary complex of histidine kinase
CheA, coupling protein CheW, and chemoreceptor by pulsed
33
Bhatnagar, Jaya dipolar ESR spectroscopy 34
Erbse, Annette The chemotactic core signalling complex is ultrastable 35
Briegel, Ariane Electron cryotomography of bacterial chemotaxis arrays 36
v

January 22, 2009 Biofilms & Host Interactions
Thursday Morning – 9:00 am – 12:00 pm Chair – Alan Wolfe
ABSTRACT
PRESENTER TITLE
PAGE NO.
Belas, Robert
Two regulatory proteins control the swim-or-stick switch in
Roseobacters
Deletion analysis of RcsC reveals a novel signaling-pathway
37
Oropeza, Ricardo controlling biofilm formation in Escherichia coli 38
Vlamakis, Hera Regulation of cell fate in Bacillus subtilis biofilms
Protein misfolding done right: The biogenesis of bacterial amyloid
39
Chapman, Matt
BREAK
fibers 40
Martinez del Campo, Rhodobacter sphaeroides, a bacterium with two flagellar systems
Ana
and multiple chemotaxis gene homologs
Motility, chemotaxis and virulence of Borrelia burgdorferi, the
41
Motaleb, MD
lyme disease spirochete
Pleiotropic phenotypes of a Yersinia enterocolicia flhD mutant
42
Prüß, Birgit
include reduced lethality in a chicken embryo model
Regulation of motility by quorum sensing in Sinorhizobium meliloti
43
Gonzalez, Juan and its role in symbiosis establishment 44
January 22, 2009 Signaling & Behavior
Thursday Evening – 7:30 pm – 9:30 pm Chair – Judy Armitage
Goldberg, Shalom Engineered single- and multi-cell chemotaxis in E. coli 45
Photo-energy conversion and sensory transduction of microbial 46
Jung, Kwang-Hwan rhodopsins in photosynthetic microbes
Function of multiple chemotaxis-like pathways in mediating
changes in motility patterns and cellular morphology in
47
Bible, Amber
BREAK
Azospirillum brasilense
Probing adaptation kinetics in vivo by fluorescence resonance
48
Shimizu, Thomas energy transfer
Neumann, Silke Minor receptor signalling in E. coli 49
A systems biology approach to understanding how Bacillus
50
Chastanet, Arnaud makes up its mind
vi

POSTERS - BLAST X
Poster # Lab Presenter Title Page #
1 Phillip
Aldridge
Aldridge, Christine The characterisation of the dynamics of the
FliT:FliD:FlhD4C2 interaction and its role in
regulating flagellar assembly
52
Aldridge, Phillip Subunit feedback control of flagellar filament
assembly in Caulobacter crescentus
53
Alexandre, Gladys Function of unique domains of CheA1 from A.
Brasilense in regulating multiple cellular behaviors
54
Brown, Mostyn How does the Rhodobacter sphaeroides flagellar
motor stop – Using a clutch or a brake?
55
Delalez, Nicolas Jacques Dynamics of the flagellar motor protein FliM 56
2 Phillip
Aldridge
3 Gladys
Alexandre
4 Judith
Armitage
5 Judith
Armitage
6 Judith
Roberts, Mark A J Using control theory to elucidate connectivity in R. 57
Armitage
Sphaeroides chemotaxis
7 Howard
Berg
Yuan, Junhua Behavior of the flagellar rotary motor near zero load 58
8 David
Bolam
Diaz-Mireles, Edith Nutrient sensing by a human gut symbiont 59
9 Edmundo De la Cruz, Miguel Ángel EnvZ-OmpR and CpxA-CpxR regulate ompS1 by 60
Calva
differential promoter expression
10 Edmundo
Calva
Gallego, Ana The LeuO regulon in Salmonella 60
11 Nyles
Miller, Kelly Ann The complex hook basal body structure of the Lyme 61
Charon
disease spirochete Borrelia burgdorferi
12 Brian
Airola Airola, Michael How do PAS and HAMP domains communicate? 62
Crane
Insights from Aer2, a heme based sensor for
aerotaxis
13 Brian
Pollard, Abiola M Structure of soluble chemoreceptor suggests a 63
Crane
mechanism for propagating conformational signals
14 Georges
Castillo, David Jaime Functional analysis of a large non-conserved region 64
Dreyfus
of FlgK (HAP1) from Rhodobacter sphaeroides
15 Georges
Dreyfus
16 Thierry
Emonet
17 Joseph
Falke
18 Dimitris
Georgellis
19 Georgellis,
Dimitris
20 Juan
Gonzalez
21 Bertha
González-Pedrajo
22 Rasika
Harshey
23 Rasika
Harshey
De la Mora, Javier The flagellar muramidase from the photosynthetic
bacterium Rhodobacter sphaeroides
Alexander, Roger Modeling scaffold phosphorylation as an adaptation
mechanism in bacterial chemotaxis
Swain, Kalin Testing the Yin-Yang model of signal transduction
in a bacterial chemoreceptor cytoplasmic domain
Alvarez, Adrian Fernando Cytochrome d but not cytochrome o rescues the
toluidine blue growth sensitivity of arc mutants in E.
Coli
González, Ricardo Searching the physiological signal(s) that regulate
the activity of the sensor kinase BarA
Gurich, Nataliya The role of quorum sensing in the control of motility
and plant invasion by Sinorhizobium meliloti
García-Gómez, Elizabeth Characterization of etga, a muramidase associated
with the type III secretion system of
enteropathogenic Escherichia coli
71
Lee, Jae-Min FlhE: a periplasmic chaperone of flagellin? 72
Nieto, Vincent Michael The cyclic-di-GMP receptor protein YcgR localizes
to the flagellar basal body and changes motor bias
in Salmonella
vii
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66
67
68
69
70
73

POSTERS - BLAST X
Poster # Lab Presenter Title Page #
24 Michio
Hizukuri, Yohei Analysis of the peptidoglycan-binding domain of the 75
Homma
flagellar stator protein MotB using systematic
mutagenesis and chimeric protein in Escherichia
coli
25 Michio
Koike, Masafumi Attempt to purify the hook-basal body with C-ring 76
Homma
from the Na+-driven flagellar motor
26 Scott
Kline, Kimberly Mechanism for sortase localization and role in 77
Hultgren
efficient pilus assembly in Enterococcus faecalis
27 Akihiko
Fukuoka, Hajime Visualization of exchange of rotor component in 78
Ishijima
functioning bacterial flagellar motor
28 Akihiko
Inoue, Yuichi Torque response of the sodium-driven chimeric 79
Ishijima
flagellar motor in E.coli induced by reversible
temperature change
29 Christine
Josenhans, Christine The Helicobacter pylori flagellar anti-sigma factor 80
Josenhans
FlgM remains bacteria-associated and interacts
with FlhAc 30 Ikuro
Inaba, Takehiko The localization patterns of all histidine kinases in 81
Kawagishi
Escherichia coli cell
31 Ikuro
Nishiyama, So-ichiro Thermosensing function of Aer, a redox sensor of 82
Kawagishi
E. Coli
32 Kenji
Oosawa
Hayashi, Fumio ATPase activity of T3SS specific ATPase InvC 83
33 Kenji
Hayashi, Fumio Characterizations of the pseudorevertants from 84
Oosawa Salmonella typhimurium strain SJW1655 and
SJW1660 with the R- and the L-type straight
flagellar filaments
34 Kenji
Hayashi, Fumio Raman optical activity and vibrational circular 85
Oosawa
dichroism of flagellar filaments of Salmonella
35 Cancelled
Poster
86
36 John
Willett, Jonathan CrdC negatively regulates CheW3 and CheA3 87
Kirby
interaction during signal transduction in
Myxococcus xanthus
37 Jun
Liu, Jun Molecular architecture of intact flagellar motor 88
Liu
revealed by Cryo-Electron tomography
38 Janine
Maddock
Dobkowski, Jason Mining the E. Coli GFP fusion collection 89
39 Michael
Adase, Christopher Understanding the fundamental elements of
90
Manson
signaling in the Tar chemoreceptor
40 Michael
Manson
Crowder, Rachel Leann Linking the TM2 to HAMP—a tough nut to crack? 91
41 Michael
Seely, Andrew Electrostatic effects on signaling mutations in the C- 92
Manson
terminal region of the Escherichia coli aspartate
chemoreceptor
42 Philip
Lee, Yi-Ying Interaction of the transcriptional regulatory complex, 93
Matsumura
FlhDC, with its target DNA
43 Jonathan
McMurry, Jonathan Application of biolayer interferometry to
94
McMurry
understanding interactions among Salmonella
enterica flagellar export apparatus proteins
44 Paul
Simons, Julie A Cross-Species Comparison of Chemotactic 95
Milewski
Behavior
45 Makoto
Miyata
Nakane, Daisuke Tethered Mycoplasma 96
viii

POSTERS - BLAST X
Poster # Lab Presenter Title Page #
46 Makoto
Miyata
47 Tarek
Msadek
48 Tarek
Msadek
49 Keiichi
Namba
50 Keiichi
Namba
51 Keiichi
Namba
52 Keiichi
Namba
53 Keiichi
Namba
Nonaka, Takahiro Molecular shapes of Gli123 and Gli521 involved in
gliding motility of Mycoplasma mobile
Delauné, Aurelia The essential nature of the WalK/WalR signal
transduction pathway is linked to cell wall hydrolase
activity in Staphylococcus aureus
Falord, Mélanie The GraS/GraR two-component system and
dermaseptin resistance in Staphylococcus aureus
Che, Yong-Suk Characterization of suppressors of the MotB(D33E)
mutation, a putative proton-binding residue of the
bacterial flagellar motor
Ibuki, Tatsuya Structure of FliJ, a cytoplasmic component of the
flagellar type III; protein export apparatus of
Salmonella
Makino, Fumiaki CryoEM structure of the hook-filament junction of
Salmonella
Nakamura, Shuichi Effect of intracellular pH on the torque-speed
relationship of bacterial proton-driven flagellar
motor
Yoshimura, Shinsuke Fluorescence imaging of assembly and
disassembly of the bacterial flagellar protein export
ATPase FliL to the flagellar basal body
Ottemann, Karen M Analysis of Helicobacter pylori lacking all four
54 Karen
Ottemann chemoreceptors
55 Rebecca
Parales, Rebecca E. Chemotaxis to pyrimidines and identification of a
Parales
cytosine chemoreceptor in Pseudomonas putida
56 Chankyu
Park
57 Simon
Rainville
58 Tracy
Raivio
59 Christopher
Rao
60 Christopher
Rao
61 Kathleen
Ryan
62 Mark
Sansom
63 Florian
Schubot
64 Victoria
Shingler
65 Victor
Sourjik
66 Ann
Stock
67 Claudia
Studdert
Lee, Changhan Reactive aldehydes and motility in Escherichia coli
K12
107
Rainville, Simon Taking control of the bacterial flagellar motor 108
Malpica, Roxana Characterization of the periplasmic domain of the
sensor kinase CpxA: Role of conserved residues in
CpxA activity
Saini, Supreet Characterization of FliZ as an activator of flagellar
genes in Salmonella enterica serovar typhimurium
Wu, Kang Localization of the chemotaxis proteins in Bacillus
subtilis
Ryan, Kathleen R RcdA structure and function in regulated CtrA
proteolysis
Hall, Benjamin Modelling MCP signalling mechanisms with highthroughput
simulation of Tar TM2
Schubot, Florian D. Structural evidence suggests that antiactivator
ExsD from Pseudomonas aeruginosa is a DNA
binding protein
Herrera Seitz, María Karina A novel PAS-GGDEF-EAL protein involved in
regulation of motility in Pseudomonas putida
Sommer, Erik In vivo study of the two-component signalling
network in E. coli
Han, Hua Gene regulation by Escherichia coli response
regulator PhoB
Massazza, Diego Ariel New reporter residues of trimer formation by
Escherichia coli MCPs
ix
97
98
99
100
101
102
103
104
105
106
109
110
111
112
113
114
115
116
117
118

POSTERS - BLAST X
Poster # Lab Presenter Title Page #
68 Motohide
Kenri, Tsuyoshi Systematic detection of proteins that localize at the 119
Takahashi attachment attachment organelle regions of Mycoplasma
Mycoplasma
pneumoniae by fluorescent-protein tagging
69 Barry
Campbell, Asharie Johnson Mapping the signal transduction pathway within the 120
Taylor
PAS domain of the Aer receptor
70 Gunnar
Draheim, Roger Modulating two-component signal output with 121
von Heijne
protein-membrane interactions
71 Cancelled
Poster
122
72 Douglas
Copeland, Matthew Francis A mechanical and genetic study of Escherichia coli 123
Weibel
swarming motility
73 Douglas
Muralimohan, Abishek Probing hydrodynamic interactrions between 124
Weibel
swimming bacteria using microfulidics
74 Zhaomin
Black, Wesley P. Examination of phosphorylation in the Dif
125
Yang
chemotaxis-like system in Myxococcus xanthus
75 Igor g
(Zhulin) Jouline
Cantwell, , Brian Evolution of chemotaxis proteins p on a micro scale 126
76 Igor
(Zhulin) Jouline
Shanafield, Harold Evolution of signal transduction in a bacterial genus 127
77 David
Nan, Beiyan The chemosensory receptor FrzCD interacts with 128
Zusman
two A-motility proteins, AglZ and AgmU
78 Dimitris
Barba Ostria, Carlos Arturo Phenotypic characterization of all single mutants of 129
Georgellis
two component system proteins in Neurospora
crassa
79 Makoto
Miyata
Miyata, Makoto AFM study of Mycoplasma mobile's gliding motility 129b
x

SPEAKER ABSTRACTS
1

BLAST X Mon. Morning Session
A STRUCTURAL INVESTIGATION OF RESPONSE REGULATOR
AUTODEPHOSPHORYLATION
Yael Pazy 1 , Amy C. Wollish 1 , Stephanie A. Thomas 1 , Peter J. Miller 2 , Edward J. Collins 1,2 ,
Robert B. Bourret 1 , and Ruth E. Silversmith 1
Departments of Microbiology & Immunology 1 and Biochemistry & Biophysics 2 , University of
North Carolina, Chapel Hill, NC 27599
Assorted two-component regulatory systems are used to control a wide variety of
biological processes, which occur over a broad range of time scales. To appropriately
synchronize the adaptive responses implemented by response regulators with the
environmental stimuli detected by sensor kinases, the kinetics of biochemical signaling
reactions must be at least as fast as the biological process that they regulate. The fraction of
response regulators in the phosphorylated state is determined by the net result of
phosphorylation and dephosphorylation. Autodephosphorylation rates reported for various
wildtype response regulators span a range of at least 40,000x, consistent with timescales
ranging from about one second to one day. Furthermore, the range of rates observed suggests
that autodephosphorylation rates are an important contributor to setting the overall timescales of
two-component signaling systems.
We previously found that changing the amino acids at the variable active site positions
corresponding to residues 59 and 89 of Escherichia coli CheY could alter response regulator
autodephosphorylation rates about 100x (1). Thus, the particular amino acids found at positions
'59' and '89' in response regulators can account for two orders of magnitude in
autodephosphorylation rate, but other factors to account for an additional two to three orders of
magnitude in reaction rate must also exist and remain to be identified. To begin to characterize
the structural basis of response regulator autodephosphorylation rate, we determined highresolution
X-ray crystal structures for five CheY mutants that bear amino acid substitutions at
positions 14, 59, and 89 and consequently exhibit autodephosphorylation rates six to 40 times
slower than wildtype CheY. Each structure was determined in the presence of the phosphoryl
group analog BeF3 - and thus represents the starting point of the autodephosphorylation
reaction. Comparison of mutant and wildtype CheY structures, which are matched at all but
three residues and yet support different reaction rates, can potentially provide insight into the
mechanistic basis by which positions '59' and '89' influence autodephosphorylation rate.
Similarly, comparison of each mutant CheY structure with the structure of a wildtype response
regulator that is matched at eight (three variable and five conserved) active site residues and
yet catalyzes autodephosphorylation at rates 10-80x slower than the CheY mutants can
potentially suggest candidates for additional factors that might contribute to
autodephosphorylation rate.
Response regulator autodephosphorylation likely involves an inline attack on the
phosphoryl group by a nucleophilic water molecule. The structural comparisons outlined above
clearly suggest that autodephosphorylation rate is influenced by the extent to which amino acid
sidechains at positions '59' or '89' sterically occlude access to the phosphoryl group. Additional
factors potentially affecting autodephosphorylation rate will also be discussed.
Reference
1. Thomas, S.A., Brewster, J.A., & Bourret, R.B. (2008) Two nonconserved active site residues
affect response regulator phosphoryl group stability. Molecular Microbiology 69, 453-465.
2

BLAST X Mon. Morning Session
INTRAMOLECULAR AUTOPHOSPHORYLATION OF THE ESCHERICHIA COLI ArcB
SENSOR KINASE
Gabriela R. Peña-Sandoval, Luis A. Nuñez Oreza and Dimitris Georgellis
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México, 04510 México City, México.
The Arc two-component system is a complex signal transduction system that plays a key
role in regulating energy metabolism at the level of transcription in bacteria. This system
comprises the ArcB protein, a tripartite membrane-associated sensor kinase, and the ArcA
protein, a typical response regulator. Under anoxic growth conditions, ArcB autophosphorylates
and transphosphorylates ArcA, which in turn represses or activates the expression of its target
operons. Under aerobic conditions, the kinase activity of ArcB is silenced by the oxidation of two
cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond
formation, a reaction in which the quinones provide the source of oxidative power.
Here we present results demonstrating that the putative leucine-zipper near the second
transmembrane segment of ArcB is functional and necessary for proper ArcB signaling.
Moreover, we provide data demonstrating that in contrast to the proposed model of
intermolecular autophosphorylation, ArcB autophosphorylation is an intra-molecular reaction.
3

BLAST X Mon. Morning Session
A BIFUNCTIONAL KINASE-PHOSPHATASE IN BACTERIAL CHEMOTAXIS
Steven L. Porter, Mark A.J. Roberts, Cerys S. Manning and Judith P. Armitage 1
Oxford Centre for Integrative Systems Biology (OCISB), Department of Biochemistry, University
of Oxford, South Parks Road, Oxford OX1 3QU
Phosphorylation based signaling pathways employ dephosphorylation mechanisms for
signal termination. Histidine to aspartate phosphosignaling in the two-component system
controlling bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a
complex chemosensory pathway with multiple homologues of the Escherichia coli
chemosensory proteins, although it lacks homologues of known signal terminating CheY-P
phosphatases such as CheZ, CheC, FliY or CheX. Here we demonstrate that an unusual CheA
homologue, CheA3, is not only a phosphodonor for the principal CheY protein, CheY6, but is
also is a specific phosphatase for CheY6-P. This phosphatase activity accelerates CheY6-P
dephosphorylation to a rate that is comparable with the measured stimulus response time of ~1
s. CheA3 possesses only two of the five domains found in classical CheAs, the Hpt (P1) and
regulatory (P5) domains, which are joined by a novel 794 amino acid sequence that is required
for phosphatase activity. The P1 domain of CheA3 is phosphorylated by CheA4 and it
subsequently acts as a phosphodonor for the response regulators. A CheA3 mutant protein
deleted for the 794 amino acid region lacked phosphatase activity, retained phosphotransfer
function but did not support chemotaxis, suggesting that the phosphatase activity may be
required for chemotaxis. Using a nested deletion approach we show that a 200 amino acid
segment of CheA3 is required for phosphatase activity. The phosphatase activity of previously
identified non-hybrid histidine protein kinases depends upon the dimerization and histidine
phosphorylation (DHp) domains. CheA3, however, lacks a DHp domain, suggesting that CheA3
is a novel phosphatase.
4

BLAST X Mon. Morning Session
MESSAGE PASSING: PROTEIN STRUCTURE ASSEMBLY FROM SEQUENCE DATA FOR
TWO-COMPONENT SIGNALING PROTEINS
Hendrik Szurmant 1 , Martin Weigt 2,3 , Robert White 1,3 , Terry Hwa 3 and James A. Hoch 1
1
Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps
Research Institute, La Jolla, CA 92037
2
Institute for Scientific Interchange, I-10133 Torino, Italy
3
Center for Theoretical Biological Physics and Department of Physics, University of California at
San Diego, La Jolla, CA 92093
The crystal structure of the Bacillus subtilis response regulator Spo0F in complex with
the histidine kinase structural homologue Spo0B defined the active site of phosphotransfer and
the spatial interactions of two-component systems of microbes and plants. The limited
bioinformatic data available at the time was sufficient to deduce and understand the molecular
basis for recognition specificity between histidine kinases and response regulators (J. A. Hoch
and K. I. Varughese. 2001. J. Bacteriol. 183:4941-4949). Today, the availability of large protein
databases generated from sequences of hundreds of bacterial genomes enables more
sophisticated statistical approaches to extract interacting and specificity determining positions
between proteins from protein databases. The goal of such studies is to identify protein
interaction surfaces from sequencing data alone, without previous structural knowledge, i.e. cocrystal
data. A number of co-variance based approaches producing nearly identical results have
been applied to the highly amplified two-component systems as a means to verify mathematical
data with structural knowledge of sensor kinase/response regulator interaction; including one
presented by us at the BLAST IX in 2007 (R. A. White et al. 2007. Methods Enzymol. 422:75-
101). While producing potential specificity determining position information, these methods have
an important shortcoming in predicting spatial proximity. They cannot distinguish between
directly correlated (interacting) and indirectly correlated (non-interacting) residue positions. To
address this issue we developed a novel method that combines co-variance analysis with global
inference analysis, adopted from use in statistical physics.
When applied to a set of over 2500 representatives of the bacterial two-component
signal transduction system, the combination of covariance with global inference methods
successfully and robustly identified residue pairs that are proximal in space without resorting to
ad hoc tuning parameters, both for hetero-interactions between sensor kinase (SK) and
response regulator (RR) proteins and for homo-interactions between RR proteins. The
spectacular success of this approach illustrates the effectiveness of this combination approach
in identifying direct interaction positions based on sequence information alone. We expect this
method to be applicable for predicting interaction surfaces between proteins present in only one
copy per genome as the number of sequenced genomes continues to expand, and for
assembling multi-protein structures.
5

BLAST X Mon. Morning Session
INTEGRATED CONTROL OF CAULOBACTER CELL ENVELOPE PHYSIOLOGY BY A
HYBRID TWO-COMPONENT/ECF SIGMA FACTOR SIGNALING NETWORK
Robert Foreman, Erin Purcell, Aretha Fiebig, Dan Siegal-Gaskins & Sean Crosson
Department of Biochemistry and Molecular Biology, University of Chicago, 929 E. 57th St.,
Chicago, IL 60637
We present evidence that Caulobacter crescentus encodes a regulatory network that
integrates information about two different signals, visible light and oxidative/osmotic stress, to
regulate the cell envelope and cell adhesion. In this hybrid signaling system, light signals via
the LovK histidine kinase and oxidative/osmotic stress signals via the ECF sigma factor σ T are
integrated to regulate cell envelope physiology. Caulobacter LovK, exhibits light-controlled
autokinase activity and forms a two-component signaling system with the single-domain
receiver protein, LovR. We have shown that the LovK/LovR system can function to modulate
cell adhesion in response to blue light. LovK/LovR is a negative regulator of σ T , an envelope
stress sigma factor that is critical for cell survival under osmotic and oxidative stress. σ T , in turn,
is a positive transcriptional regulator of the lovK/lovR two-component system. This feedbackregulated
signaling network can serve as a model to probe how bacterial cells integrate and
coordinate their responses to multiple environmental queues.
6

BLAST X Mon. Morning Session
THE ROLE OF SIGNAL TRANSDUCTION IN CELL WALL METABOLISM IN BACILLUS
SUBTILIS
Paola Bisicchia, David, Noone, Efthimia Lioliou and Kevin M Devine.
Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2. Ireland.
The cell wall of Gram positive bacteria is an extracellular structure, physically removed
from the biosynthesis of the precursors used in its synthesis. Peptidoglycan and teichoic acid
precursors are synthesized within the cytoplasm and transported across the cytoplasmic
membrane where they are incorporated into the cell wall during growth and cell division. The
spatial separation of these processes implies bidirectional signaling between the cell wall and
cytoplasmic compartments, that recent work has begun to elucidate. We have shown that the
essential YycFG two-component signal transduction system of Bacillus subtilis controls cell wall
metabolism – during exponential growth, it activates expression of the YocH, YvcE and LytE
autolysins and represses expression of YoeB (IseA), an inhibitor of autolysin activity, and YjeA a
peptidoglycan deacetylase whose activity on peptidoglycan modulates its susceptibility to
autolysin digestion (Howell et al., 2003; Bisicchia et al., 2007; Salzberg and Helmann, 2007;
Yamomoto et al., 2008). Thus we propose that YycG senses some aspect(s) of the cell wall
externally, perhaps the Lipid II intermediate, and transduces this information into the cytoplasm
so that the cell wall synthetic activities in these two compartments are coordinated (Dubrac et
al., 2008). We have also demonstrated a close connection between YycFG and the PhoPR
two-component system that controls one of the phosphate limitation responses in B. subtilis
(Hulett, 2002). YycFG and PhoPR are closely related phylogenetically - hybrid YycF’-‘PhoP and
PhoP’-‘YycF response regulators are functional and there are similarities in the YycF and PhoP
DNA binding sequences. We have also shown (i) that while YycG can phosphorylate only its
cognate response regulator YycF, PhoR can phosphorylate both PhoP and YycF and (ii) that
cells depleted for YycFG cannot mount a normal PhoPR-mediated phosphate limitation
response. From these observations, we postulated that the roles of YycFG and PhoPR might
be linked during cell wall metabolism and phosphate limitation.
In this talk we will present the results of further analysis on the relationships between the
YycFG and PhoPR two-component systems and their roles in cell wall metabolism during
growth and phosphate limitation.
References:
Bisicchia et al., (2007) Molecular Microbiology 65: 180-200.
Dubrac et al., (2008) Molecular Microbiology In Press
Howell et al., (2003) Molecular Microbiology 49: 1639-1655.
Howell et al., (2006) Molecular Microbiology 59:1199-1215.
Hulett, (2002) The Pho regulon. in ‘B. subtilis and is closest relatives’ ASM Press.
Salzberg and Helmann (2007) J. Bacteriology 189: 4671-4680.
Yamamoto et al., (2008) Molecular Microbiology 70: 168-182.
7

BLAST X Mon. Morning Session
THE WalK/WalR ESSENTIAL SIGNAL TRANSDUCTION PATHWAY AND CELL WALL
HOMEOSTASIS IN STAPHYLOCOCCUS AUREUS
Sarah Dubrac 1 , Aurélia Delauné 1 , Olivier Poupel 1 , Adeline Mallet 2 , Tarek Msadek 1
Biology of Gram-positive Pathogens 1 , Department of Microbiology, Plateforme de Microscopie
Ultrastructurale 2 , Imagopole, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France
The highly conserved WalK/WalR (aka YycG/YycF) two-component system is specific to
low G+C % Gram-positive bacteria. While this system is essential for cell viability, both the
nature of its regulon and its physiological role remained mostly uncharacterized. We have
recently shown that the S. aureus WalKR system positively controls autolytic activity, in
particular that of the major autolysins, AtlA, LytM and Sle1, and identified at least ten genes
belonging to the WalKR regulon that are known or thought to be involved in S. aureus cell wall
degradation. While none of these genes appears to be essential, we have shown that their
global regulation by the WalKR system results in a drastic down regulation of cell wall dynamics,
with a complete arrest of both cell wall biosynthesis and turn over under WalKR depletion. As a
consequence of these molecular disorders TEM observations revealed that the cell wall of
WalKR depleted cells was significantly thicker and division septa were abnormally distributed.
Recent advances presented here have shown that this global regulation is directly linked to
WalKR essentiality. While the walRK genes are essential, the WalKR system is inducible since
it is assumed that the WalR response regulator is only active when phosphorylated. While the
activation signal is still unknown, several recent results suggest that it could be related to cell
wall homeostasis.
As cell wall metabolism is a major parameter influencing virulence and particularly the
innate immune response, we are now interested in characterizing the impact of WalKR on
S. aureus virulence. Beyond the regulation of genes involved in cell wall metabolism, we have
also shown that the WalKR system activates expression of at least two genes involved in
interactions with the extracellular host matrix and influences the capacity of S. aureus to adhere
to the host matrix.
References:
1. Dubrac, S., I. G. Boneca, O. Poupel, and T. Msadek. 2007. New insights into the
WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a major role in
controlling cell wall metabolism and biofilm formation in Staphylococcus aureus. J.
Bacteriol. 189:8257-69.
2. Dubrac, S., and T. Msadek. 2008. Tearing down the wall: peptidoglycan metabolism
and the WalK/WalR (YycG/YycF) essential two-component system. Adv. Exp. Med. Biol.
631:214-28.
3. Dubrac, S., P. Bisicchia, K. M. Devine and T. Msadek. 2008. A matter of life and
death: cell wall homeostasis and the WalKR (YycGF) essential signal transduction
pathway. Mol. Microbiol. 70: (in press)
8

BLAST X Mon. Evening Session
DYNAMIC ASSEMBLY AND DISASSEMBLY OF THE TYPE IV MOLECULAR MACHINE
Iryna Bulyha and Lotte Søgaard-Andersen
Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany
Myxococcus xanthus harbors two gliding motility systems, A and S. The S(ocial)-system
depends on type IV pili (T4P) and is generally active only when the cells are within contact
distance of each other. T4P undergo cycles of assembly and retraction. The force for S-motility
is generated by retraction of T4P. T4P are localized in a unipolar pattern and are present only at
the leading pole of the rod-shaped cells. M. xanthus cells periodically undergo cellular reversals
in which the old leading pole (which harbors T4P) becomes the new lagging pole (which does
not harbor T4P). These observations suggest that in parallel with a cellular reversal, the pole at
which T4P assemble switches. The molecular mechanisms regulating the T4P
extension/retraction cycle and underlying T4P pole switching remain unknown.
To investigate these mechanisms, we focused on the cellular localization of five highly
conserved T4P biogenesis proteins (PilB, PilT, PilM, PilC and PilQ), which are present in all
known T4P systems. PilB and PilT are cytoplasmic proteins and members of the secretion
ATPase superfamily of proteins; PilB is required for assembly of T4P, while PilT is necessary for
T4P retraction. PilM shows similarity to MreB/FtsA and is indispensable for T4P assembly. PilC
is an inner membrane protein and is necessary for T4P assembly. The PilQ secretin forms a
gated oligomeric channel for the pilus in the outer membrane. Using immuno-fluorescence
microscopy, and time-lapse fluorescence microscopy with functional YFP-fusions, we show that
PilQ, PilC and PilM are localized in clusters at both cell poles. These clusters have equal
intensities and they do not oscillate between the two poles during reversals. The analysis of PilB
and PilT localization revealed that both proteins are localized in polar clusters. PilB is
predominantly localized in a cluster at the leading pole and PilT is predominantly localized in a
cluster at the lagging cell pole. This localization is dynamic and the two proteins oscillate
between the poles during reversals. These observations show that T4P function depends on
two sets of proteins: one set is statically localized at both cell poles, and the other set is
dynamically localized. Based on these findings we suggest that during cellular reversals, the
T4P machinery is disassembled at the old leading pole and reassembled at the new leading
pole.
Moreover, we will present the data which suggest that the T4P assembly/retraction cycle
relies on a PilB/PilT competition-based mechanism. According to this model, PilB at the leading
cell pole stimulates T4P assembly, and the occasional accumulation of PilT at the leading cell
pole results in retraction. Therefore, the two dynamically localized ATPases determine whether
assembly or disassembly of pilus takes place.
9

BLAST X Mon. Evening Session
A "FOUR COMPONENT" SIGNAL TRANSDUCTION SYSTEM REGULATES
DEVELOPMENTAL PROGRESSION IN MYXOCOCCUS XANTHUS
Sakthimala Jagadeesan, Bongsoo Lee, and Penelope I. Higgs
Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, D35043
Marburg, Germany
Myxococcus xanthus responds to starvation by entering a multicellular developmental
program in which 10 5 cells first aggregate into mounds and then within these mounds,
differentiate into environmentally resistant spores. Under standard laboratory conditions,
formation of spores within the mounds (fruiting bodies) takes approximately 72 hours. We have
previously demonstrated that progression through the developmental program appears to be
regulated by an atypical two component signal (TCS) transduction system consisting of four
TCS homologs (RedC, RedD, RedE, and RedF). While RedC appears to be a typical
membrane bound histidine kinase, RedD consists solely of two receiver domains. RedE is a
soluble histidine kinase-like protein, and RedF is a single receiver domain response regulator.
Based on a combination of genetic and biochemical analyses, we propose a model for how
these four Red proteins function together to regulate progression through the developmental
program. Our data suggests that development is repressed when the RedC histidine kinase
phosphorylates RedF, a single domain response regulator. Developmental repression is
relieved when, in response to an unknown signal(s), RedC is instead induced to phosphorylate
the response regulator RedD. Surprisingly, the phosphoryl group is then transferred from RedD
to the histidine kinase-like protein, RedE. RedE is then likely made accessible to RedF-P,
whereupon it removes RedF’s phosphoryl group. We present the data that supports this model.
Furthermore, we will address how progression through the developmental program is modulated
by the Red system.
10

BLAST X Mon. Evening Session
INDEPENDENCE AND INTERDEPENDENCE OF Dif AND Frz CHEMOSENSORY
PATHWAYS IN MYXOCOCCUS XANTHUS CHEMOTAXIS
Qian Xu, Wesley P. Black, Christena L. Cadieux and Zhaomin Yang
Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061-0910, USA
Dif and Frz, two Myxococcus xanthus chemosensory pathways, are required in
phosphatidylethanolamine (PE) chemotaxis for excitation and adaptation, respectively. DifA and
FrzCD, the homologs of methyl-accepting chemoreceptors in the two pathways, were examined
for methylation in the context of chemotaxis and inter-pathway interactions. Evidence indicates
that DifA may not undergo methylation but signals transmitting through DifA do modulate FrzCD
methylation. Results also revealed that M. xanthus possesses Dif-dependent and Difindependent
PE sensing mechanisms. Previous studies showed that FrzCD methylation is
decreased by negative chemostimuli but increased by attractants such as PE. Results here
demonstrate that the Dif-dependent sensory mechanism suppresses the increase in FrzCD
methylation in attractant response and elevates FrzCD methylation upon negative stimulation. In
other words, FrzCD methylation is governed by opposing forces from Dif-dependent and Difindependent
sensing mechanisms. We propose that the Dif-independent but Frz-dependent PE
sensing leads to increases in FrzCD methylation and subsequent adaptation, while the Difdependent
PE signaling suppresses or diminishes the increase in FrzCD methylation to
decelerate or delay adaptation. We contend that these antagonistic interactions are crucial for
effective chemotaxis in this gliding bacterium to ensure that adaptation does not occur too
quickly relative to the slow speed of M. xanthus movement.
11

BLAST X Mon. Evening Session
PREDATAXIS BEHAVIOR IN MYXOCOCCUS XANTHUS
Jeb Berleman, Jodie Scott, Tatiana Chumley, and John R. Kirby *
University of Iowa, Department of Microbiology, Iowa City, IA 52246, USA
Spatial organization of cells is important for both multicellular development and tactic
responses to a changing environment. We find that the slow-moving, gliding bacterium,
Myxococcus xanthus, utilizes a Che-like pathway to regulate multicellular rippling during
predation of other microbial species. Tracking of GFP-labeled cells indicates directed
movement of M. xanthus cells during the formation of rippling wave structures. Quantitative
analysis of rippling indicates that ripple wavelength is adaptable and dependent on prey cell
availability. Methylation of the receptor, FrzCD, is required for this adaptation: a frzF
methyltransferase mutant is unable to construct ripples, whereas a frzG methylesterase mutant
forms numerous, tightly packed ripples. Both the frzF and frzG mutant strains are defective in
directing cell movement through prey colonies. These data indicate that the transition to an
organized multicellular state during predation in M. xanthus relies on the tactic behavior of
individual cells, mediated by a Che-like signal transduction pathway. Predataxis behavior differs
from chemotaxis behavior in that it seems to depend heavily on tactile-stimulation, as opposed
to chemical-stimulation.
12

BLAST X Mon. Evening Session
DYNAMIC LOCALIZATION OF FrzCD IN MYXOCOCCUS XANTHUS
Emilia M.F. Mauriello 1 , David P. Astling 1 , Oleksii Sliusarenko 2 and David R. Zusman 1
University of California, Department of Molecular and Cell Biology, Berkeley, CA 94720, USA 1 ;
Yale University, Department of Molecular, Cell and Developmental Biology New Haven, CT
06520, USA 2
Directional motility in the gliding bacterium Myxococcus xanthus requires controlled cell
reversals mediated by the Frz chemosensory system. FrzCD, a cytoplasmic chemoreceptor,
does not form membrane bound polar clusters typical for most bacteria, but rather cytoplasmic
clusters that are helically arranged and span the cell length. This unusual localization is
maintained in the absence of the CheA homologs FrzE or CheA4, and the CheW homologs
FrzA or FrzB. In contrast, MCPs lose their respective polar or cytoplasmic localization in
Escherichia coli and Rhodobacter spheroides strains lacking CheA and/or CheW (1, 2). The
distribution of FrzCD in living cells was found to be dynamic: FrzCD was localized in clusters
that continuously changed their size, number, and position. The number of FrzCD clusters was
correlated with cellular reversal frequency: fewer clusters were observed in hypo-reversing
mutants and additional clusters observed in hyper-reversing mutants. When moving cells made
side-to-side contacts, FrzCD clusters in adjacent cells showed transient alignments. These
events were frequently followed by one of the interacting cells reversing. These observations
suggest that FrzCD detects signals from a cell-contact sensitive signaling system and then relocalizes
as it directs reversals to distributed motility engines.
References:
1. Maddock, J.R., and Shapiro, L. (1993) Science 259: 1717-1723.
2. Wadhams, G.H., Martin, A.C., Warren, A.V., and Armitage, J.P. (2005) Mol Microbiol 58(3):
895-902.
3. Bustamante, V.H., Martínez-Flores, I., Vlamakis H.C., and Zusman, D. (2004) Mol Microbiol
58(5): 1501-1513.
13

BLAST X Mon. Evening Session
IDENTIFYING NOVEL BACTERIAL CYTOSKELETAL ELEMENTS AND CYTOSKELETAL
INTERACTORS THROUGH HIGH-THROUGHPUT IMAGING
John Werner, Michael Ingerson-Mahar, Jonathan Guberman, and Zemer Gitai
Princeton University, Department of Molecular Biology, LTL-355 Washington Rd., Princeton, NJ
08540
Bacterial cytoskeletal proteins polymerize into filamentous structures that represent key
regulators of a wide array of cellular processes, including cell shape determination, cell division,
and cell polarity. While bacterial homologs of all three major eukaryotic cytoskeletal families
have already been described, the upstream regulators of bacterial cytoskeletal assembly and
downstream effectors of cytoskeletal function remain poorly understood. In addition, recent
studies have suggested that additional filament-forming proteins remain uncharacterized.
All known cytoskeletal elements in both bacteria and eukaryotes have distinct nonuniform
subcellular distributions. Focusing on the asymmetric bacterium, Caulobacter
crescentus, we thus employed a directed high-throughput imaging approach to identify both
novel bacterial cytoskeletal proteins and proteins that act upstream or downstream of the
previously-characterized cytoskeletons. We developed a pipeline of high-throughput methods
for generating fluorescent protein fusions, expressing them in Caulobacter, imaging their
subcellular distribution at high resolution, and quantitating the resulting imaging data. By using
this pipeline to analyze over 2,800 Caulobacter proteins as both N-and C-terminal mCherry
fusions, we identified a set of ~300 localized Caulobacter proteins. This set included the three
known Caulobacter cytoskeletons (the MreB actin homolog, FtsZ tubulin homolog, and
Crescentin intermediate-filament). We also identified a novel protein that localizes to a tight line
that hugs a short region of the inner curvature of Caulobacter cells. This protein may polymerize
on its own, as it is capable of forming linear structures when expressed in heterologous systems
such as E. coli or S. pombe. Preliminary studies suggests that this previously-uncharacterized
cytoskeletal element plays a role in cell shape determination and may exhibit crosstalk with
other cytoskeletal proteins.
To identify upstream regulators of cytoskeletal assembly, we modified our pipeline to
allow us to assay the effects of overexpressing genes of interest on the localization patterns of
MreB, FtsZ, and Crescentin. A pilot screen of ~200 conserved proteins with no known function
identified four proteins that perturbed FtsZ, one that perturbed MreB, one that perturbed both
MreB and FtsZ, and one that perturbed Crescentin. The functions and mechanisms of action of
these candidate cytoskeletal regulators are currently being examined. Finally, to identify
downstream cytoskeletal effectors, we imaged our library of localized Caulobacter proteins in
the presence of the MreB-delocalizing compound, A22. We found a large number of proteins
that are either delocalized or mislocalized by A22 and are currently determining the cellular
functions of these proteins as well as the nature of their direct or indirect associations with
MreB.
14

BLAST X Tue. Morning Session
THE ROLE OF POSITIVE FEEDBACK IN CONTROLLING FLAGELLA ASSEMBLY
DYNAMICS
Supreet Saini 1 , Christy Aldridge 2 , Jonathan Brown 2 , Philip Aldridge 2 , Christopher Rao 1
1
Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana, IL 61801,
United States
2
Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place,
Newcastle upon Tyne NE2 4HH, United Kingdom
Flagellar assembly in Salmonella enterica serovar Typhimurium (S. typhimurium)
proceeds in a sequential manner, starting from the base of the flagella and concluding at the
filament tip. A key regulatory step in the assembly process is the σ 28 -FlgM checkpoint, which
prevents the activation of σ 28 -dependent Pclass3 promoters prior to completion of the hook basal
body. This regulatory checkpoint is typically assumed to involve a binary decision process:
either proceed with Pclass3 gene expression or not, depending on the state of assembly.
However, mathematical modeling suggests that this binary checkpoint may in fact result in more
subtle, rheostat-like control. σ 28 is involved in a positive feedback loop, as ιτ positively regulates
its own expression along with the expression of FliZ, an activator of Pclass2 gene expression. In
addition, the ability of σ 28 to regulate gene expression depends on the concentration of FlgM in
the cell. This suggests that the response of the σ 28 positive feedback loop is tuned by late
protein secretion. In fact, our modeling and experimental results suggests that σ 28 and FlgM are
not only involved in establishing the binary checkpoint between Pclass2 and Pclass3 gene
expression but are also involved in fine tuning the relative timing of expression. In particular, the
positive feedback loop involving σ 28 and FliZ establishes the delay between Pclass2 and Pclass3
gene expression.
In this talk, we will discuss our recent work investigating the positive feedback loops
involving σ 28 and FliZ. We have recently shown that FliZ is an FlhD4C2-dependent activator of
Pclass2 gene expression. In addition, our results indicate the FliZ speeds up the induction of Pclass2
genes in a secretion-dependent manner. With regards to σ 28 , we have found that autoregulation
controls the relative timing of gene expression. In cells lacking FlgM, both Pclass2 and Pclass3
genes are induced at the same times. Conversely, when the rate of FlgM secretion is reduced,
the delay between Pclass2 and Pclass3 gene expression is exaggerated. These results suggest that
timing is responsive to the rate of late protein secretion. Moreover, mathematical modeling
predicts that this control is due to autoregulation by σ 28 . To test this model, we have rewired the
flagellar gene circuit by replacing the native PfliA promoter with Pclass1/Pclass2/Pclass3 promoters.
Consistent with the model predictions, these promoter replacement experiments show that
autoregulation plays a key role in enforcing the timing of flagellar gene expression. Last, we also
investigated gene expression dynamics at single-cell resolution, and our preliminary results
suggest that the dynamics may exhibit bistability, consistent with control involving feedback.
Collectively, our results suggest that the regulation of flagellar gene expression is complex and
involves multiple layers of control.
15

BLAST X Tue. Morning Session
CRYSTAL STRUCTURE OF FLIT, A BACTERIAL FLAGELLAR EXPORT CHAPERONE FOR
THE FILAMENT CAP PROTREN HAP2 (FliD)
Katsumi Imada, Tohru Minamino, Miki Kinoshita and Keiichi Namba
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-
0871, Japan
The bacterial flagellum is a filamentous organelle responsible for motility. Since the
flagellum extends from the cytoplasm to the cell exterior, the external component proteins have
to be exported from the cytoplasm. The protein subunits are exported by the flagellar specific
export apparatus, which is a member of the type III secretion system. The export apparatus is
believed to be located within the C-ring of the flagellar basal body and consists of at least six
integral membrane proteins (FlhA, FlhB, FliO, FliP, FliQ, FliR) and three soluble proteins (FliH,
FliI, FliJ). In addition to these proteins, other cytoplasmic proteins (FlgN, FliA, FliS, FliT) act as
substrate-specific chaperons that facilitate the export of their substrates.
FliT is a flagellar export chaperone for FliD (HAP2), which forms a capping complex at
the distal end of the flagellar filament and promotes incorporation of flagellin subunits into the
growing filament, and prevents FliD from premature aggregation in the cytoplasm. FliT is not
only involved in protein export but also in regulation of flagellar gene expression. FliT negatively
regulates transcription of the flagellar class 2 operons by binding to FlhD4C2 complex, which is a
transcriptional activator.
We have determined a crystal structure of FliT at 3.2 Å resolution. The structure and
following genetic and biochemical studies have revealed that the C-terminal region of FliT
regulates its interactions with other flagellar proteins. We will discuss the molecular mechanisms
of protein export and gene expression based on the FliT structure.
16

BLAST X Tue. Morning Session
STRUCTURAL INSIGHT INTO ACTIVE FLAGELLAR MOTOR FORMATION THROUGH THE
PERIPLASMIC REGION OF MOTB
Seiji Kojima 1 , Mayuko Sakuma 1 , Yuki Sudo 1 , Chojiro Kojima 2 , Tohru Minamino 3 , Keiichi
Namba 3 , Michio Homma 1 and Katsumi Imada 3*
1 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan; 2 Laboratory of Biophysics, Graduate School of Biological Sciences,
Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara, 630-0192
Japan; 3 Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita,
Osaka 565-0871, Japan
Bacterial flagellar motor is a supramolecular nano-machine powered by the
transmembrane gradient of protons or sodium ions, and spins flagellar filaments to drive cell
motility. Motor torque is generated by the rotor-stator interaction that is coupled with ion flow
through the ion-channel in the stator unit, which is composed of four MotA and two MotB
subunits. About ten stators are assembled around the perimeter of the rotor and anchored to
peptidoglycan layer by the peptidoglycan-binding (PGB) domain of MotB. The rotor-stator
assembly is not a rigid complex, so each stator unit can dynamically be exchanged in the
functional motor, and is activated only when assembled around the rotor. However, the
mechanisms of the stator assembly and the activation of the proton flow remain unclear. Here,
we report the crystal structure of a C-terminal fragment of MotB (MotBC), which includes the
PGB domain and covers the whole periplasmic region essential for cell motility (PEM), at 1.75 Å
resolution. The structure, and subsequent mutational and biochemical analyses indicate that
dimer formation by the PGB domains is required for motility through the regulation of the
arrangement of the transmembrane segment. Moreover, we show that large structural changes
in the N-terminal helices should be coupled with both peptidoglycan binding and activation of
the stator. This work provides novel structural insight into the dynamic behavior of the ionchannel
complex regulated by the periplasmic domain, and the activation mechanism of the
complex coupled with completion of the assembly where it works.
17

BLAST X Tue. Morning Session
STATOR SELECTION IN SHEWANELLA ONEIDENSIS MR-1
Anja Paulick, Andrea Koerdt, Kai M. Thormann
Department of Ecophysiology, MPI für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, D-
35043 Marburg, Germany
The Gram-negative metal-ion reducing bacterium Shewanella oneidensis MR-1 is motile
by means of a single polar flagellum. We identified two potential stator systems, PomAB and
MotAB, each sufficient as a force generator to drive flagellar rotation. Physiological studies
demonstrate that PomAB is sodium-dependent while MotAB is powered by proton motive force.
Homology comparisons strongly indicate that the MotAB system has been acquired by
horizontal gene transfer, probably as a consequence of long-term adaptation from a marine to a
low-sodium freshwater environment. As in S. oneidensis MR-1, a number of bacterial species
possess more than one stator system to power a single flagellar system but it is yet unclear,
how selection of the stators is achieved.
Expression analysis at the single cell level showed that both stator systems of S.
oneidensis MR-1 are expressed simultaneously, and functional fusions of PomB and MotB to
mCherry revealed that both stator systems are present in the cell at the same time. While the
Pom system is efficiently localizing to the flagellated cell pole under all conditions, the Mot stator
is located in the cell membrane and only found at the cell pole at high abundance in media with
low sodium content. At low sodium, both stator systems are localizing to the flagellated cell pole
in the majority of the cell population, thus indicating that under such conditions a hybrid motor
may be formed. We conclude that stator selection occurs at the level of protein localization by
alterations in the localization efficiency in response to sodium levels.
In Vibrio species, two additional proteins, MotX and MotY, are involved in stator
recruitment and sodium-dependent swimming. We therefore analyzed whether S. oneidensis
MR-1 orthologs to MotX and MotY play a role in stator selection. Mutant and localization
analyses demonstrated that both proteins are required for function of the Pom as well as the
Mot stator system. As opposed to the Vibrio system, in S. oneidensis MR-1, MotX and MotY are
not required for stator recruitment and also do not play a role in stator selection in response to
sodium conditions.
18

BLAST X Tue. Morning Session
TAKING CONTROL OF THE BACTERIAL FLAGELLAR MOTOR
Mathieu Gauthier, Dany Truchon and Simon Rainville
Department of Physics, Engineering Physics and Optics and Centre d'optique photonique et
laser, Laval University, Québec, Québec, CANADA
The bacterial flagellar motor is a fairly complex machine, requiring 40-50 genes for its
expression, assembly and control. Furthermore, it is embedded in the multiple layers of the
bacterial membrane. That explains why, unlike many other molecular motors, it has not yet been
studied in vitro. As spectacular studies of linear motors (like kinesin, myosin and dynein) have
clearly demonstrated, an in vitro system provides the essential control over experimental
parameters to achieve the precise study of the motor’s physical and chemical characteristics.
Here, we report significant progress towards the development of a unique in vitro system to
study quantitatively the bacterial flagellar motor.
Our system consists of a filamentous Escherichia coli bacterium partly introduced inside
a micropipette. Femtosecond laser pulses (60 fs and ~ 15 nJ/pulse) are then tightly-focused on
the part of the bacterium that is located inside the micropipette. This vaporizes a
submicrometer-sized hole in the wall of the bacterium, thereby granting us access to the inside
of the cell and the control over the proton-motive force that powers the motor. Using a patchclamp
amplifier, we applied an external voltage between the inside and the outside of the
micropipette. If the hole in the bacterium is open, that voltage should translate into a membrane
potential powering the motors outside of the micropipette. As we varied the applied potential,
variations in the motor's rotation speed were observed. For these preliminary results, the
rotation speed was observed directly using video microscopy of fluorescently labeled filaments.
That system opens numerous possibilities to study the flagellar motor and other membrane
components.
19

BLAST X Tue. Morning Session
EXPERIMENTAL EVIDENCE FOR CONFORMATIONAL SPREAD IN THE BACTERIAL
SWITCH COMPLEX
Richard W. Branch 1 , Fan Bai 1 , Dan V. Nicolau 2 , Teuta Pilizota 1 , Bradley Steel 1 , Philip K.
Maini 2 , Richard M. Berry 1
1 Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1
3PU, UK. 2 Centre for Mathematical Biology, Mathematical Institute, University of Oxford,
St.Giles Road, Oxford OX1 3LB, UK.
The bacterial switch complex in E. coli controls the direction of rotation of the bacterial
flagellar motor between clockwise and counterclockwise modes. The complex takes the form of
a ring composed of about 110 FliN, 34 FliM and 26 FliG protein subunits. Regulation is through
binding of the signaling molecule CheY-P to FliM. FliG interfaces with the torque-generating
stator units of the motor. The precise mechanism by which the complex executes a switch is
unclear.
The complex displays the ultrasensitive nature typical of allosteric proteins, with a steep
sigmoidal relationship existing between [CheY-P] and motor rotational bias. Allosteric regulation
of proteins has classically been understood in terms of the Monod-Wyman-Changeux (MWC) or
Koshland-Nemethy-Filmer (KNF) models. However, it is unrealistic that MWC-type concerted
transitions could be responsible for quaternary conformational changes of such a large complex,
and cooperative binding studies in vitro and in vivo have precluded a KNF-type induced-fit
mechanism.
The MWC and KNF models are recognized as limiting cases of a general allosteric
scheme that has recently been described in a model of conformational spread. The model has
been shown to be capable of reproducing motor switching kinetics. A directly observable
consequence of conformational spread in the switch complex would be the variation of motor
speed associated with the conformational spread of ring subunit state. In particular, the duration
of switch events should be finite and broadly distributed due to the diffusive random walk of
conformational spread, and incomplete switches should be observed due to incomplete growth
and shrinkage of subunit state domains.
We have used high-resolution back-focal-plane interferometry of polystyrene beads
attached to truncated WT E. coli flagella to resolve intermediate states of the motor predicted by
conformational spread, and demonstrate detailed quantitative agreement between our
measurements and conformational spread simulations. Individual switch events are not
instantaneous, but follow a broad distribution of switch times with mean ~ 20 ms. The shortest
switch events are observed to last less than 1ms, while the longest require over 100ms and take
several revolutions to complete. Intervals between switches are exponentially distributed at all
values of bias. Incomplete switches reaching a range of intermediate speeds are observed. The
events are Poisson distributed in time with a bias-dependent frequency.
20

BLAST X Tue. Morning Session
DO INDIVIDUAL BACTERIAL FLAGELLAR MOTORS USE HYSTERESIS TO MAINTAIN A
ROBUST OUTPUT IN A NOISY ENVIRONMENT? – AN EXPERIMENTAL STUDY
Peter Reuven 1 , Oleg Krichevsky 2 , Michael Eisenbach 1
1 Department of Biological Chemistry, Weizmann Institute of Science, 76 100, Israel
2 Department of Physics, Ben-Gurion University, Beer Sheva, 84 105, Israel
It is known that despite imperfections of intracellular environment, flagellar motor outputs
are robust against stochastic fluctuations of CheY signal. Indirect evidence from our lab
suggests that, to maintain stability, the motor complex might damp out fluctuations in the
intracellular level of CheY by having a hysteresis feature - two different thresholds for switching.
In this case, hysteresis means that the default-state motor switches at a higher
threshold from counterclockwise to clockwise state compared to a lower threshold when
switching back. Such behavior will produce hysteretic loop in input/output characteristics of
flagellar motor.
Our aim is to pin-point the level at which the noise-filtering (via hysteresis) occurs in the
chemotactic network. We are studying flagellar rotation of single cells as a function of their
intracellular CheY-P concentration, changing the concentration up and down in order to cover
both - counterclockwise to clockwise and clockwise to counterclockwise - switching routes.
Since it is impossible to distinguish CheY from CheY-P in vivo, one has to work under
conditions that maintain CheY constantly fully phosphorylated. The challenge was how to
effectively decrease the concentration of the phosphorylated signal.
Knowing that we cannot play with the level of phosphorylation we opt to play with the
level of the CheY-P protein instead. While to increase protein concentration is a routine task, to
decrease it is less obvious. We cloned a bi-modal, inducible plasmid expressing a CheY fused
to yellow fluorescent protein (YFP) and ssrA degradation tag. YFP is used for quantifying
the signal whereas the degradation tag makes it possible to shorten the lifetime of CheY-YFP.
The core assumption of our approach is that heat-shock-induced proteases accelerate the
degradation of the ssrA-targeted CheY-YFP protein. We have verified this assumption
experimentally.
We monitor the degradation of CheY-YFP by a decrease in fluorescence intensity and
this decrease is correlated with the change of the direction of motor rotation. Input/output
characteristics of individual flagellar motors is build from these correlations.
Advancing this study will, hopefully, enable us to deeper understand how the
mechanisms of intracellular interactions affect the logic of cell's behavior.
21

BLAST X Tue. Morning Session
DYNAMICS OF THE BACTERIAL FLAGELLAR MOTOR WITH MULTIPLE STATORS
Giovanni Meacci and Yuhai Tu
IBM T. J. Watson Research Center
The bacterial flagellar motor drives the rotation of flagellar filaments and enables many
species of bacteria to swim. Torque is generated by interaction of stator units, anchored to the
peptidoglycan cell wall, with the rotor. Recent experiments [Yuan, J. & Berg, H. C. (2008) PNAS
105, 1182-1185] show that near zero load the speed of the motor is independent of the number
of stators. Here, we introduce a mathematical model of the motor dynamics that explains this
behavior based on a general assumption that the stepping rate of a stator depends on the
torque exerted by the stator on the rotor. We find that the motor dynamics can be characterized
by two time scales: the moving-time interval for the mechanical rotation of the rotor and the
waiting-time interval determined by the chemical transitions of the stators. We show that these
two time scales depend differently on the load, and that their crossover provides the
microscopic explanation for the existence of two regimes in the torque-speed curves observed
experimentally. We also analyze the speed fluctuation for a single motor using our model. We
show that the motion is smoothed by having more stator units. However, the mechanism for
such fluctuation reduction is different depending on the load. We predict that the speed
fluctuation is determined by the number of steps per revolution only at low load and is controlled
by external noise for high load. Our model can be generalized to study other molecular motor
systems with multiple power-generating units.
22

BLAST X Tue. Evening Session
EFFECT OF OSMOLYTES ON REGULATING THE ACTIVITIES OF THE SSK1 RESPONSE
REGULATOR FROM SACCHAROMYCES CEREVISIAE
Alla O. Kaserer, Paul F. Cook and Ann H. West
Department of Chemistry and Biochemistry, The University of Oklahoma, 620 Parrington Oval,
Norman, OK 73019
The multi-step His-Asp phosphorelay system in Saccharomyces cerevisiae allows cells
to adapt to osmotic, oxidative and other environmental stresses. The pathway consists of a
hybrid histidine kinase SLN1, a histidine-containing phosphotransfer (HPt) protein YPD1 and
two response regulator proteins, SSK1 and SKN7. Under non-osmotic stress conditions, the
SLN1 kinase is active and phosphoryl groups are shuttled to SSK1 via YPD1. We have
previously demonstrated that YPD1 stabilizes the phosphorylated form of SSK1. The cellular
response to hyperosmotic stress involves rapid efflux of water and change in intracellular ion
and osmolyte concentration. It is our hypothesis that these changes may affect rates of
phosphotransfer within the SLN1-YPD1-SSK1 phosphorelay system and the phosphorylated
lifetime of response regulators. Therefore, we examined the effect of different solute
concentrations on dephosphorylation of SSK1 and phosphotransfer rates within the
phosphorelay system using half-life studies and rapid quench kinetics, respectively. These
studies provide new insight and offer a better understanding of how this His-Asp multi-step
phosphorelay is environmentally regulated.
23

BLAST X Tue. Evening Session
REGULATION OF Escherichia coli MOTILITY BY THE NITRIC OXIDE SENSITIVE
TRANSCRIPTIONAL REPRESSOR NsrR
Jonathan D. Partridge and Stephen Spiro
Department of Molecular and Cell Biology, The University of Texas at Dallas, 800 W. Campbell
Road, Richardson, Texas 75080, USA
There is circumstantial evidence implicating the water-soluble free radical nitric oxide
(NO) as a regulator of motility, chemotaxis and biofilm development. Heme-containing NObinding
domains of methyl accepting chemotaxis proteins from Clostridium botulinum and
Thermoanaerobacter tengcongensis have been characterized, though the prediction that these
proteins mediate taxis towards or away from NO has not been tested. In transcriptomics
experiments, the expression of some motility genes has been observed to be perturbed by
exposure of cultures to sources of NO or nitrosative stress (imposed by S-nitrosothiols),
although both positive and negative responses have been reported, and the regulators involved
were not identified. In the non-pathogenic organism Nitrosomonas europaea, NO stimulates
biofilm formation. In Pseudomonas aeruginosa and Staphylococcus aureus, there is evidence
that NO inhibits biofilm formation, or stimulates dispersal, and NO stimulates swimming and
swarming motility in P. aeruginosa. In no case has a molecular mechanism been described
which accounts for the effects of NO on biofilm development or motility.
NO is made in bacteria either as a by-product of nitrite reduction to ammonia, or as an
intermediate of denitrification, and is made by the inducible NO synthase of host phagocytes.
Thus pathogenic bacteria can be exposed both to endogenously-generated NO, and to the NO
made by host cells. In Escerichia coli, the regulatory proteins NorR and NsrR mediate adaptive
responses to NO, by controlling the expression of genes encoding enzymes that reduce or
oxidize NO to less toxic species. The key NO detoxifying activities are the flavohemoglobin
(encoded by the hmp gene) and the flavorubredoxin (encoded by norVW), the expression of
which is regulated by NsrR and NorR, respectively. As far as is known, the norVW promoter is
the sole target for regulation by NorR, while NsrR appears to control a large regulon of genes
and operons.
The extent of the NsrR regulon of E. coli has been assessed computationally, and by a
transcriptomics analysis of a strain in which NsrR was titrated by the presence of multiple copies
of a cloned NsrR binding site. We believe that neither approach has provided a comprehensive
inventory of all of the genes regulated by NsrR. Therefore, we used chromatin
immunoprecipitation and microarray analysis (ChIP-chip) to identify NsrR binding sites in the E.
coli genome. Surprisingly, we found NsrR binding sites associated with the promoter regions of
three transcription units (mqsR-ygiT, fliAZY and fliLMNOPQR) containing genes with wellestablished
or suspected roles in motility and/or biofilm development. We have confirmed that
the fliA and fliL promoters are subject to regulation by NsrR and NO, and have identified an
NsrR binding site in the fliA promoter. We have shown that NsrR is a negative regulator of
motility in both K12 and UPEC strains of E. coli. These results provide, for the first time, a
molecular mechanism by which NO might control bacterial motility.
24

BLAST X Tue. Evening Session
SYNTHETIC LETHALITY UNCOVERS A NOVEL LINK BETWEEN THE MalT AND OmpR
REGULONS
Sylvia A. Reimann and Alan J. Wolfe
Loyola University Chicago, Maywood, Il
Synthetic lethality (SL) is a genetic term for the inviability of a double mutant combination
of two fully viable single mutants. SL is commonly interpreted as redundancy at an essential
metabolic step. However, a second, less well-known, class of SL exists: the so-called “defectdamage-repair”
(DDR) cycles that link apparently unrelated metabolic pathways (Ting et al.,
2008).
We have isolated an SL mutant of the form ompR SL(ompR), where SL(ompR) refers to
a mutation that causes death when present in a cell that lacks the two-component response
regulator OmpR. This global regulator is required for proper assembly of the cell envelope and
we reasoned that the nature of the SL(ompR) mutation would provide further insight into the role
of OmpR and its regulon.
Using a combined genetic/genomic approach, we mapped the SL(ompR) mutation to
malT, which encodes the transcription factor MalT. Because overexpression of the MalT
inhibitor MalK did not rescue growth, we proposed that the malT mutation leads to a
constitutively active form (MalT c ). To test this hypothesis, we deleted the MalT-dependent lamB,
which encodes an outer membrane porin, and found that this deletion suppressed the SL of the
ompR malT c mutant.
Since LamB and OmpR do not perform redundant functions, we propose that the
observed SL is of the DDR variety, as follows: the defect (MalT c activity) leads to damage
(constitutive expression of LamB) that is repaired by one or more members of the OmpR
regulon. To further understand the role of OmpR, we are currently seeking OmpR regulon
members that can suppress the SL of the ompR malT c mutant.
25

BLAST X Tue. Evening Session
A CHEMOTAXIS-LIKE SIGNALING PATHWAY REGULATES THE EXPRESSION OF
EXTRACELLULAR MATERIALS IN GEOBACTER SULFURREDUCENS
Hoa T. Tran 1 , Derek R. Lovley 2 , and Robert M. Weis 1
Departments of Chemistry 1 and Microbiology 2 , University of Massachusetts, Amherst, MA
01003, USA.
The chemotaxis pathway that regulates cell motility toward chemical attractants is wellstudied
in Escherichia coli. By contrast, multiple chemotaxis clusters have been found in many
other bacteria, and evidence is accumulating that these cells use chemotaxis-like pathways to
regulate diverse cellular functions. The genome of Geobacter sulfurreducens, a δ-
Proteobacterium found predominantly in the Fe(III) reducing zone of sedimentary environment
contains ~70 chemotaxis gene homologs arranged in 6 major clusters. Cluster 5 (Che5) has a
complete set of chemotaxis homologs, including the kinase cheA (1 copy), cheW (2 copies),
cheR (1), cheB (1), cheY (3) and four other non-che genes. There are 34 chemoreceptor
homologs in the genome, but none is found in the Che5 cluster. Che5-type clusters have been
identified in the genomes of several δ-Proteobacteria, yet their functions are not known. Here,
we report that G. sulfurreducens Che5 cluster regulates gene expression, and in particular the
synthesis of extracellular material that is abundant in OmcS and OmcZ, two c-type cytochromes
bound to the outer membrane. OmcS and OmcZ are essential for cell growth in insoluble
electron acceptors and for effective electricity production on electrodes. Deletion mutants of the
homologs of cheW (gsu2218, gsu2220), cheA (gsu2222) and cheR (gsu2215) increased OmcS
production and decreased OmcZ. In contrast, deletion mutants of cheB (gsu2214), one of the
three cheYs (gsu2223) and a non-che gene (gsu2216) decreased OmcS and increased OmcZ
production. The chemoreceptors that signal through these Che5 proteins are hypothesized to
belong to a single MA class. Evidence that supports this idea is based on the phenotypes of
deletion mutants in two mcp genes (gsu1704, and gsu2372), which are similar the cheA mutant.
Wherever the functional parallels can be drawn, the function of the homologues in Geobacter
Che5 signaling pathway is similar to their counterpart in the E. coli chemotaxis pathway. In
addition to changes in OmcS and OmcZ expression, the cheA, cheR and cheW (gsu2220)
mutants promote cell aggregation and overproduce non-PilA filamentous material. Moreover,
microarray data of the cheR mutant, and quantitative RT-PCR data from the other che mutants
indicate that the Che5 cluster alter the expression of ~175 genes. A substantial fraction of
these are predicted to contain export signals that will result in an to extracellular location. Taken
together, these data demonstrate that G. sulfurreducens Che5 cluster, with one class of
chemoreceptors, regulates the extracellular matrix material biosynthesis.
This research was supported by the U.S. Department of Energy Office of Science (BER)
under the Cooperative Agreement No. DE-FC02-02ER63446.
26

BLAST X Tue. Evening Session
THE TWO-COMPONENT REGULATORY SYSTEM BarA/SirA IS AT THE TOP OF A MULTI-
FACTORIAL REGULATORY CASCADE CONTROLLING THE EXPRESSION OF THE SPI-1
AND SPI-2 VIRULENCE REGULONS IN SALMONELLA
Luary C. Martínez, José L. Puente and Víctor H. Bustamante
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional
Autónoma de México. Cuernavaca, Mor., México.
Horizontal gene transfer of pathogenicity islands has been a major event in the evolution
of pathogenic bacteria. Integration of regulatory networks to control the expression of the gained
genes has represented another essential step in this process. Salmonella Pathogenicity Islands
1 and 2 (SPI-1 and SPI-2) are required at different phases during Salmonella infection in
humans and animals. A positive regulatory cascade comprising the SPI-1-encoded regulators
HilD, HilA and InvF induces expression of the SPI-1 regulon in response to conditions
resembling the intestinal environment, such as growth in Luria-Bertani (LB) rich medium. Two
global two-component regulatory systems, OmpR/EnvZ and PhoP/PhoQ, control the expression
of the SsrA/B two-component system encoded within SPI-2, which specifically induces the
expression of the SPI-2 regulon genes in response to conditions resembling the intracellular
environment, mimicked in vitro by growth at low concentrations of phosphate and magnesium.
Interestingly, we have recently shown that HilD also induces expression of the SsrA/B system,
and thus of the SPI-2 regulon, at late stationary phase in LB cultures, indicating that SPI-2
expression is also controlled by a SPI-1/SPI-2 cross-talk mechanism.
The results presented here, together with previous reports, better define the complex
and multi-factorial regulatory cascade that controls SPI-1 and SPI-2 expression through HilD.
We show that the global two-component system BarA/SirA activates the transcription of two
small RNA molecules, csrB and csrC. These molecules counteract the negative effect exerted
by the CsrA RNA binding protein on hilD mRNA stability, ensuring the synthesis of the
appropriate concentration of HilD required for the expression of HilA and SsrA/B, the central
positive regulators of the SPI-1 and SPI-2 regulons, respectively. Furthermore, we
demonstrated that growth conditions affecting SPI-1 expression in LB (e.g. low salt, acidic pH or
temperatures below 37°C), similarly repress the expression of the SPI-2 regulon. However,
while acidic pH seems to negatively regulate the regulatory cascade by affecting the activity of
the BarA sensor kinase, growth at low salt concentration or at low temperature seems to directly
repress hilD expression through an as yet unidentified mechanism.
27

BLAST X Tue. Evening Session
INTERACTION OF THE TRANSCRIPTIONAL REGULATORY COMPLEX, FlhDC, WITH ITS
TARGET DNA
Yi-Ying Lee, and Philip Matsumura
Department of Microbiology and Immunology, College of Medicine, University of Illinois at
Chicago, 835 S. Wolcott Ave., M/ C 790, Chicago, Illinois 60612-7344
The bacterial flagellum is the structure that allows bacteria to move and respond to
nutritional and chemical signals in their environment. It is a complex suborganelle and the
transcriptional regulation of the 40 plus structural genes is organized in a highly regulated
cascade. At the top of the hierarchy is the master operon which codes for FlhD and FlhC. These
two positive transcriptional regulators form a unique heteroheximeric complex which binds
upstream of the -35 region and requires sigma 70 for transcription. This complex has an
unusually large ‘footprint’ of 48 base pair and bends the DNA 110 degrees. We have proposed
that the DNA bind on the circumference of this toroid shaped FlhDC complex. Although we have
determined the sequence 3 footprints on FlhDC regulated promoters, it is not possible to
determine a consensus binding site in these 3 sequences. In this study, we have determined
which bases are important for DNA binding and activity for FlhDC regulated promoter activity.
First, we have divided the FlhDC footprint in the fliA promoter into five segments and found that
two of the segments or 40% of the footprint were not required for binding. The remaining 30
base pairs were divided into 3-5 base segments and randomly mutagenized and screened for
the ability to bind and activate the fliA promoter. Analysis of these data suggests a consensus of
12 A, 15 A, 34 T, 36 A, 37 T, 44 A, 45 T in FlhD4C2 footprint fragment were important for activity. Five of
these bases demonstrated high specificity. Finally, this consensus was tested and found to be
important in other FlhDC regulated promoter regions.
28

BLAST X Wed. Morning Session
A NOVEL AMINO ACID BINDING STRUCTURE IN BACTERIAL CHEMOTAXIS
George D. Glekas and George W. Ordal
Department of Biochemistry, University of Illinois at Urbana-Champaign
409 MSB, Urbana, IL 61801
Simple flagellated bacteria, such as Bacillus subtilis, possess the ability to sense their
environment and move to more favorable conditions, where there are, for instance, more
nutrients like amino acids, by the process of chemotaxis. The initial stage is binding of an
amino acid by receptors on the outside of the cell. This binding causes conformational changes
that affects the activity of enzymes on the inside of the cell and alters the movement of the
bacteria. The main paradigm for understanding these events is the bacterium Escherichia coli.
However, we have discovered that, in fact, the mechanism used by E. coli is not used by most
bacteria and that the mechanism used by the distantly related bacterium B. subtilis is more likely
to be the general mechanism. Unlike in E. coli, where binding of attractant causes a shift of the
receptor polypeptide that goes from the outside of the cell to the cell interior toward the cell
interior, attractant causes rotational movement of the receptors without any comparable interior
shifting
We seek to understand how this rotational movement occurs in the asparagine receptor
McpB. To do this, we have discovered that the most likely conformation of the exterior part of
the receptor is far different from that in the E. coli receptor. Molecular and homology modeling
of the McpB sensing domain has led to a structural model that reveals a dual PAS domain
structure similar to the crystal structure of the LuxQ sensor. PAS domains are known to be
conserved structures capable of binding a great many “small” molecules. Further mutagenetic
analyses of putative asparagine-binding residues have not only confirmed the validity of the
structural model, they have revealed certain residues that greatly effect chemo-attractant
binding. Using both in vivo chemotactic assays and in vitro isothermal titration calorimetry
performed on purified mutant receptor exterior regions, three residues, all in the upper PAS
domain, have been shown to lower the affinity of the McpB receptor for asparagine. Mutations
in the lower PAS domain show no such effect. Further structural and mutagenetic studies have
shown a similar dual PAS architecture in the B. subtilis proline receptor McpC, with similar
residues responsible for amino acid binding. Extensive homology modeling shows that eight of
the ten B. subtilis chemoreceptors have PAS domains in their sensing domains.
We are now in the process of modeling the consequences of binding attractant at these
residues on the expected structure of the receptor.
29

BLAST X Wed. Morning Session
STRUCTURE AND FUNCTION OF THE HELICOBACTER PYLORI CHEMORECEPTOR TlpB
John Goers, Nathan Henderson, S. James Remington, Karen Ottemann * and Karen Guillemin
Institute of Molecular Biology, University of Oregon, Eugene OR, 97403
* Environmental Toxicology, UC Santa Cruz, Santa Cruz, CA 95064
We have shown that the Helicobacter pylori chemoreceptor TlpB is required for
chemotaxis away from acid and from the quorum-sensing molecule autoinducer-2. Here we
report the determination of an atomic resolution crystal structure for the periplasmic domain of
TlpB. The structure reveals a PAS domain inserted into a pair of antiparallel helices and shows
unexpected structural homology to the LuxQ receptor from Vibrio harveyi. Furthermore, the PAS
domain tightly binds urea, suggesting that the TlpB receptor may be associated with detection of
urea. We present mutational and physical evidence for interaction of TlpB with urea and its role
in chemotaxis.
30

BLAST X Wed. Morning Session
THE TM2-HAMP CONNECTION
Gus A. Wright, Rachel L. Crowder and Michael D. Manson
Department of Biology, Texas A&M University, College Station, TX 77843
The HAMP domain—a conserved protein motif in most Histidine kinases, Adenylate
cyclases, Methyl-accepting chemotaxis proteins, and Phosphatases—typically conforms to a
helix–connector-helix domain architecture. Recently, an NMR solution structure was determined
for the Af1503 HAMP from the archaebacteria Archeoglobus fulgidis (Hulko, et al, Cell 126: 929-
940, 2006). This structure revealed that the HAMP domain is a parallel four-helix bundle with a
knob-on-knob packing. Similar structures of HAMP domains are proposed to exist in all 5
chemoreceptors of Escherichia coli, including the aspartate chemoreceptor Tar. The HAMP
domain of Tar receives information from TM2 and regulates the transmission of the signal to the
kinase signaling domain. Mutations were introduced into the TM2-HAMP connector region of
Tar to determine if manipulating the input signal into the HAMP affects the output signal to the
signaling domain. MLLT residues between R214 and P219 were deleted (-4 through -1), and
additional LLT tandem repeats were added up to 8 residues after P219 (+1 through +8).
Aspartate sensitivity, rotational bias, mean reversal frequency, and in vivo methylation were
measured for these mutants. The results suggest that adding helical turns in this region
destabilizes, or “relaxes”, the HAMP/signaling domains; while, removing helical turns from this
region stabilizes, or “tightens”, the HAMP/signaling domains. In order to determine if increasing
flexibility of the TM2/HAMP connector affects the output signal, a second set of mutants were
constructed to replace the MLLT region with four tandem glycine residues (4G). Glycine
residues were then subtracted (-4G through -1G) and added (+1G through +5G). Data collected
from this experiment suggests that increasing the flexibility of this region dampens the input
signal from TM2, in addition to destabilizing the HAMP/signaling domains.
31

BLAST X Wed. Morning Session
STRUCTURE, ASSEMBLY AND CONFORMATIONAL CHANGES IN CHEMORECEPTORS
STUDIED IN INTACT BACTERIAL CELLS USING CRYO-ELECTRON TOMOGRAPHY
Cezar M. Khursigara, Xiongwu Wu*, Peijun Zhang, Jon Lefman, Mario J. Borgnia, Yuhai Tu ‡ ,
Jacqueline Milne and Sriram Subramaniam
National Cancer Institute and *National Heart, Lung and Blood Institute, NIH, Bethesda, MD
20892.
‡ T. J. Watson Research Center, IBM, Yorktown Heights, NY 10598
Bacteria respond to changes in their chemical environment by activating an assembly of
proteins that collectively represent the bacterial chemotaxis apparatus. In Gram-negative
bacteria the core-signaling unit of the chemotaxis machinery is a ternary complex composed of
chemoreceptors, CheA and CheW that localize primarily to the poles of the cell and form
extended arrays. Using cryo-electron tomography, we describe and compare the architecture,
localization and spatial relationship between macromolecular complexes involved in chemotaxis
signaling and cellular motility in three different Gram-negative bacteria. In addition, by
combining the tomographic analysis with 3D averaging methods we demonstrate that trimeric
chemoreceptors in E. coli display two distinct conformations that differ principally in
arrangement of the HAMP domains within each trimer.
32

BLAST X Wed. Morning Session
DISCRETE SIGNAL-ON AND -OFF CONFORMATIONS IN THE AER HAMP DOMAIN
Kylie J. Watts, Mark S. Johnson and Barry L. Taylor
Dept. Microbiology and Mol. Genetics, Loma Linda University, Loma Linda, CA, USA
The PAS-FAD sensor of the aerotaxis receptor, Aer, signals through HAMP and
signaling domains that are similar to these domains in other chemoreceptors. Our previous
crosslinking studies showed that the AS-1 and AS-2 helices of the Aer-HAMP domain might
form a four-helix bundle similar to the Af1503 and Tar HAMP domains. In this study, AS-1
residues were crosslinked to AS-2′ residues in di-cys Aer mutants (using 13 proximal and 4
distal di-cys pairs). The results confirmed a parallel four-helix HAMP bundle for Aer, but one in
which AS-2 is rotated compared to the orientation of AS-2 in Af1503 or Tar.
We extended our HAMP crosslinking studies to probe for structural differences between
the signal-on (CW) and signal-off (CCW) states. In our previous crosslinking studies, we used
the oxidant copper phenanthroline, which maintains Aer in the signal-off state. In order to
generate snapshots of the signal-on state, CW lesions such as PAS-N85S were engineered into
the Aer di-cys and single-cys mutants. When the AS-1 to AS-2′ di-cys crosslinking experiments
were repeated in mutants containing N85S, several di-cys pairs showed significant increases in
dimer formation rates. These di-cys pairs were located at the distal end of the HAMP four-helix
bundle. In contrast, no significant crosslinking changes were observed at the proximal end of
the four-helix bundle. The data supports a model in which the distal ends of the HAMP helices
move closer together during signal transduction. This could be due to an inward lateral
movement of the helices, and may include some element of rotation. However, the entire Aer-
HAMP domain does not appear to rotate as has been proposed for Af1503.
In Aer, HAMP lesions that lock the receptor in the signal-on (CW) state cluster at the
distal end of a HAMP four-helix bundle, indicating a possible site for PAS-HAMP interactions
during signal transduction. We used PEG-maleimide to determine in vivo the solvent-accessible
surface of the HAMP and proximal signaling domains (residues 206-275). Solvent accessibility
was restricted for most AS-2, but not AS-1 or connector, residues in Aer. This indicates that AS-
2 residues that are exposed to solvent in Af1503 and Tar, and are predicted to be exposed in an
Aer-HAMP model, are buried in vivo in Aer. We are currently investigating whether these
residues are buried in a PAS-HAMP contact domain. We are also probing the surface of the
HAMP domain in the signal-on state (with N85S) to determine whether there are differences in
accessibility between the two signaling states.
33

BLAST X Wed. Morning Session
INVESTIGATING THE STRUCTURE OF TERNARY COMPLEX OF HISTIDINE KINASE CheA,
COUPLING PROTEIN CheW, AND CHEMORECEPTOR BY PULSED DIPOLAR ESR
SPECTROSCOPY
Jaya Bhatnagar, Peter P. Borbat, Jack H. Freed, Brian R. Crane
Cornell University, B 150 Caldwell Hall, Ithaca, NY 14853
A central question in understanding the mechanism of chemotaxis involves the nature of
interactions between histidine kinase CheA, adaptor protein CheW and receptors. Pulsed
dipolar ESR spectroscopy (PDS) has developed as a valuable technique for structural
characterization of protein complexes. PDS provides long-range distance information between
spin-labeled residues in the proteins. A set of distance measurements can be subsequently
used to model the assembly structure of the whole complex. In our previous work, we
demonstrated the success of this approach in predicting the structure of complex of CheW with
CheA. We have now applied PDS to the ternary complex formed by CheA, CheW and soluble
chemoreceptor fragments. Our results indicate changes in distance distributions from spinlabeled
sites on P4, P5 domains and CheW in the presence of unlabeled receptor. Dipolar
signals between spin-labeled receptor and CheA∆289 (domains P3, P4 and P5 together) or
CheW provide important insights about the relative position and orientation of the three
components with respect to each other. Based on this data we have developed a structural
model of the ternary complex and the conformational changes CheA undergoes upon binding to
receptor.
34

BLAST X Wed. Morning Session
THE CHEMOTACTIC CORE SIGNALLING COMPLEX IS ULTRASTABLE
Annette H. Erbse and Joseph J. Falke
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215,
Boulder, CO 80309
The chemotatic core complex, composed of the transmembrane receptor, the histidinekinase
CheA and the coupling protein CheW, is the central building block of the extensive,
highly cooperative polar signaling clusters in bacteria involved in sensing chemical gradients.
Recent studies have shown that the receptors are organized hexagonal arrays of trimers-ofdimers.
But it is still unclear how these arrays are formed and stabilized, how CheA and CheW
are incorporated into single core complexes and how the complexes are interconnected to build
the cooperative signaling network. Here we focus on the stability of the core complex. We show
that the isolated, membrane-bound core complex is stable for at least 24 hours, both when it is
assembled in vivo and in vitro. All three components are needed to achieve this ultra-stability,
which is dependent on electrostatic interactions. By contrast, the stability is independent of
ligand binding, receptor methylation or kinase activity. We propose that the assembly of the
signaling clusters is cooperative, such that interactions between CheA , CheW and the receptor
trimers-of-dimers are needed not only for receptor regulated kinase activity of individual core
complexes, but also to position neighbouring complexes during the formation of a multi-linked
network. The resulting ultra-stable network is the foundation for the ordered organization and
the hypersensitivity of the signaling patches.
35

BLAST X Wed. Morning Session
ELECTRON CRYOTOMOGRAPHY OF BACTERIAL CHEMOTAXIS ARRAYS
Ariane Briegel 1 , H. Jane Ding 1 , Zhuo Li 1 , John Werner 2 , Zemer Gitai 2 , D. Prabha Dias 1 ,
Rasmus B. Jensen 3 , Elitza Tocheva 1 and Grant Jensen 1
1 California Institute of Technology, CA
2 Princeton University, NJ
3 University of Roskilde, Denmark
Motile prokaryotes are able to sense and to respond to ambient conditions through a
process known as chemotaxis. Attractants and repellents bind to the sensing domain of methylaccepting
chemotaxis proteins (MCPs), thereby regulating the activity of the histidine kinase
CheA. Together with the linking protein CheW, CheA is located at the distal tip of the
cytoplasmic signaling domain of the MCPs. If activated, CheA phophorylates CheY (CheY-P),
which in turn controls the direction of flagellar rotation. Together with CheA and a linking protein
CheW, the MCPs form extended chemotaxis arrays at the cell poles.
Electron cryotomograhy (ECT) makes it possible to visualize chemoreceptor clusters in
prokaryotes in vivo at macromolecular resolution (4-8 nm). While high-resolution structures of
the individual chemotaxis proteins are available, their arrangement and position in the arrays
remain unclear. Understanding this "mesoscale" architecture of the clusters is critical, however,
since it is vital to the arrays' cooperative signal amplification and regulation. In order to
unambiguously identify the chemotaxis arrays inside cells, we have correlated ECT with
fluorescent light microscopy (FLM), using slightly fixed and immobilized Caulobacter crescentus
cells with a fusion of the red-fluorescent protein, mCherry, to the C-terminus of the
chemoreceptor (McpA). After plunge freezing, we imaged the same cells by ECT. In
combination with ECT of near-native wild-type and mutant cells, we used the correlated FLM
and ECT approach to identify the chemotactic array, its location and its in-vivo structure. We
demonstrate that in wild-type Caulobacter crescentus cells preserved in a near-native state, the
chemoreceptors are hexagonally packed with a lattice spacing of 12 nm, just a few tens of
nanometers away from the flagellar motor that they control. The arrays were always found on
the concave side of the cell, further demonstrating that Caulobacter cells maintain dorsal/ventral
as well as anterior/posterior asymmetry. Placing the known crystal structure of a trimer of
receptor dimers at each vertex of the lattice accounts well for the density, supporting an array
composition unlike the published models for Escherichia coli [1] or Thermotoga maritima [2].
We are now in the process of comparing the chemotaxis arrays of a wide range of bacteria to
determine the similarities and differences of these macromolecular assemblies at the
‘mesoscale’ level.
1. Shimizu T. S. et al, Nat Cell Biol 11 (2000) 792.
2. Park, S.-Y. et al, Nat Struct Mol Biol 13 (2006) 400.
36

BLAST X Thurs. Morning Session
TWO REGULATORY PROTEINS CONTROL THE SWIM-OR-STICK SWITCH IN
ROSEOBACTERS
Robert Belas
University of Maryland Biotechnology Institute, Center of Marine Biotechnology, 701 East Pratt
St., Baltimore, MD 21202
Members of the Roseobacter clade of α-Proteobacteria are among the most abundant
and ecologically relevant marine bacteria. One of the most salient features of the roseobacters
from aspects of marine ecology is their ability to enter into close physical and physiological
relationships with “red tide” phytoplankton such as dinoflagellates. For example, Silicibacter sp.
TM1040, our model roseobacter, forms a symbiosis with the dinoflagellate Pfiesteria piscicida,
such that the dinoflagellate cannot live without TM1040. Aiding TM1040 in development of the
symbiosis is a biphasic swim-or-stick lifestyle wherein a genetic regulatory circuit controls
whether the bacteria are motile and chemotactic or sessile and develop a biofilm. Bacterial
swimming and chemotaxis behavior are initial, essential steps in establishment of the symbiosis.
Once near the host surface, motility and flagellar synthesis are downregulated, while biofilm
formation and synthesis of an antibiotic are upregulated. The abilities to swim using flagella and
to form a biofilm via adhesins have been demonstrated to be important traits for both pathogenic
and symbiotic bacteria. While it is generally agreed that motility and biofilm development are
mutually exclusive, the molecular mechanisms that underlie the lifestyle switch remain virtually
unknown for most bacterial species. We have used genetic screens to search for mutants
defective in either the motile or the sessile phenotype, and have discovered many new genes
including two previously unknown and novel regulatory proteins, FlaC and FlaD that are
envisaged to act together with cyclic dimeric GMP to play important roles in the swim-or-stick
switch. FlaC is predicted to function as a response regulator protein, with homology to a protein
of Caulobacter crescentus known to be important for cell envelope function. FlaC - cells are
skewed towards the motile phase, e.g., their populations have a greater percentage of motile
cells and fewer rosettes, and have defects in antibiotic synthesis and biofilm formation. Thus,
FlaC determines whether the switch is in the swim or stick position. FlaD is predicted to be a
MarR-type DNA-binding protein. Mutations in flaD result in nonmotile cells that synthesize but
cannot rotate their flagella, i.e., they produce paralyzed flagella. We hypothesize that FlaD is
involved in the function of the flagellar motor, either by (1) acting directly to control transcription
of the class IV fliL operon or (2) acting indirectly to control transcription or activity of a protein
that acts as a ‘clutch’ to engage or disengage the flagellar motor. The implications of FlaC and
FlaD activities in the swim-or-stick strategy and their impact on the symbiosis will be discussed.
37

BLAST X Thurs. Morning Session
DELETION ANALYSIS OF RcsC REVEALS A NOVEL SIGNALING-PATHWAY
CONTROLLING BIOFILM FORMATION IN ESCHERICHIA COLI
Ricardo Oropeza, Rosalva Salgado, Ismael Hernandez-Lucas and Edmundo Calva
Departmento de Microbiologia Molecular, Instituto de Biotecnologia, Universidad Nacional
Autonoma de Mexico. Av. Universidad 2001. Col. Chamilpa. Cuernavaca, Morelos. Mexico, CP
62210
RcsC is a hybrid histidine kinase that forms part of a phosphorelay signal transduction
pathway with RcsD and RcsB. Besides the typical domains of a sensor kinase, i.e. the
periplasmic (P), linker (L), dimerization and H-containing (A), and ATP- binding (B), RcsC
possesses a receiver domain (D) at the carboxy-terminal domain.
In order to study the role played by each of the RcsC domains, four plasmids containing
several of these domains were constructed (i.e. PLAB, LAB, AB and ABD) and transformed in
Escherichia coli wild type. Different amounts of biofilm were produced, assessed by crystal
violet staining, depending on the RcsC domains expressed by the plasmid.
E. coli transformed with the plasmid expressing the ABD subdomains produced the
highest amount of biofilm, while the lowest amount of biofilm was produced under the control of
the PLAB expressing plasmid. This phenotype was observed in the same ratio when the
plasmids were transformed in a ΔrcsCDB strain.
Several mutants on genes involved in biofilm formation were transformed with this set of
plasmids. Biofilm formation was abolished in the pgaABCD and nhaR backgrounds but not in
the csrB and uvrY backgrounds. Our results suggest the existence of a signaling pathway
depending of RcsC but independent of RcsD and RcsB, activating biofilm formation by the
pgaABCD operon.
38

BLAST X Thurs. Morning Session
REGULATION OF CELL FATE IN BACILLUS SUBTILIS BIOFILMS
H. C. Vlamakis 1* , C. Aguilar 1* , R. Losick 2 , and R. Kolter 1
1 Harvard Medical School, Boston, MA, 2 Harvard University, Cambridge, MA.
*These authors contributed equally to this work.
Many microbial populations differentiate from free-living planktonic cells into surfaceassociated
multicellular communities known as biofilms. Within a biofilm, motile Bacillus subtilis
cells differentiate into non-motile chains of cells that form parallel bundles held together by an
extracellular matrix. These bundles eventually produce aerial structures that serve as
preferential sites for sporulation. By analyzing strains harboring multiple cell-type specific
promoter fusions we can visualize the spatial anatomy of at least three physiologically distinct
cell populations within mature biofilms. Motile, matrix-producing, and sporulating cells localize to
distinct regions within the biofilm and the localization and percentage of each cell type is
dynamic. Mutants unable to produce extracellular matrix form unstructured biofilms that are
deficient in sporulation. This suggests that in architecturally complex biofilms, spore formation
is coupled to the production of extracellular matrix. The coupling of matrix production and
sporulation could be explained by the phosphorylation state of the master transcriptional
regulator Spo0A. Spo0A is phosphorylated both directly and through a phosphorelay by at least
five different histidine kinase proteins. When cells have low levels of Spo0A-P, matrix genes
are expressed; however, at higher levels of Spo0A-P, sporulation commences. We have found
that a deletion of kinD, a gene encoding one of the kinases that feed into the Spo0A
phosphorelay, is sufficient to restore sporulation to matrix-deficient mutants. We hypothesize
that KinD is not acting as a kinase under these conditions, but rather functions as a
phosphatase to delay sporulation until matrix (or a matrix-encased signal) is sensed.
39

BLAST X Thurs. Morning Session
PROTEIN MISFOLDING DONE RIGHT: THE BIOGENESIS OF BACTERIAL AMYLOID
FIBERS
Xuan Wang, Neal Hammer and Matt Chapman
University of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann
Arbor, MI, 48109
Many Enterobacteriaceae spp., including E. coli, produce surface-localized amyloid
fibers called curli. Curli fibers are associated with biofilm formation, host cell adhesion and
invasion, and immune system activation. Unlike disease-associated amyloid formation, curli
biogenesis is a directed and highly regulated process. The major curli subunit protein, CsgA,
polymerizes into amyloid after interacting the CsgB nucleator protein. CsgB presents an
amyloid-like template to CsgA on the cell surface that initiates fiber formation. CsgA has five
imperfect repeating units (R1-R5) that are each predicted to form strand-loop-strand structures.
Asn and Gln residues in R1 and R5 were found to be required for efficient amyloid formation
and for interaction with the CsgB nucleator protein. Furthermore, the polymerization of CsgA
was tempered by the presence of conserved aspartic residues in R2, R3 and R4. When these
aspartic acid residues were changed to alanine (CsgA*), polymerization was significantly faster
in vitro. Even more remarkable was the observation that CsgA* assembled into an amyloid fiber
in vivo in the absence of CsgB. The ability of CsgA* to polymerize into amyloid more efficiently,
and in the absence of CsgB, was not without consequences. Cells expressing CsgA* grew more
slowly when compared to cells expressing wild type CsgA. This analysis suggests that aspartic
acid residues can potently inhibit functional amyloid formation. CsgA has apparently evolved to
efficiently assemble into an amyloid in vivo only in the presence of CsgB. This suggests an
elegant mechanism to control amyloid formation by regulating the temporal and spatial
interactions between CsgA and CsgB.
40

BLAST X Thurs. Morning Session
RHODOBACTER SPHAEROIDES, A BACTERIUM WITH TWO FLAGELLAR SYSTEMS AND
MULTIPLE CHEMOTAXIS GENE HOMOLOGS
Ana Martínez del Campo 1 , Sebastian Poggio 2 , Teresa Ballado 1 , Aurora Osorio 2 , Javier de la
Mora 1 , Laura Camarena 2 and Georges Dreyfus 1
Instituto de Fisiología Celular 1 , Instituto de Investigaciones Biomédicas 2 , Universidad Nacional
Autónoma de México, 04510 México DF, México.
Rhodobacter sphaeroides has two flagellar systems (fla1 and fla2). One of these
systems has been shown to be functional and is required for the synthesis of the wellcharacterized
single subpolar flagellum (fla1), while the other was found only after the genome
sequence of this bacterium was completed (fla2). In this work we found that the second flagellar
system of R. sphaeroides can be expressed and encodes a functional flagellum. This second
flagellar system produces polar flagella that are required for swimming. Phylogenic analysis
suggests that the flagellar system that was initially characterized, was in fact, acquired by
horizontal transfer from a γ-proteobacterium, while the second flagellar system contains the
native genes.
In addition to having two flagellar systems, this photosynthetic bacterium posses several
reiterated chemotactic genes (2 cheB, 3 cheR, 4 cheA and cheW and 6 cheY), which are
encoded in three operons (cheOp1, cheOp2 and cheOp3). In spite of this, only some of the
gene copies are required when the cell is swimming with the fla1 flagellum. The presence of a
second functional flagellum (fla2) suggests that some of these genes could be involved in its
tactic control. To test this hypothesis we proceeded to individually mutate each cheY gene. We
show evidence that CheY1, CheY2 and CheY5 control de chemotactic behavior mediated by
fla2 flagella. Additionally, we identified that open reading frame RSP6099 encodes the fla2 FliM
protein. Furthermore CheY1, CheY2 and CheY5 are located within cheOp1, which is not
essential for chemotaxis mediated by the fla1 system. This raises the question: What is the role
of cheOp1?
41

BLAST X Thurs. Morning Session
MOTILITY, CHEMOTAXIS AND VIRULENCE OF BORRELIA BURGDORFERI, THE LYME
DISEASE SPIROCHETE
M. A. Motaleb 1 , P. Stewart 2 . A. Bestor 2 , P. Rosa 2 and N. Charon 3
1 Dept of Microbiology & Immunology, East Carolina University, Greenville, NC
2 Human Bacterial Pathogenesis, NIH, RML, Hamilton, MT
3 Dept of Microbiology, Immunology & cell Biology, West Virginia University, Morgantown, WV
Borrelia burgdorferi is the causative agent of Lyme disease. It is the most prevalent
arthropod borne infection in the United States with 27,444 reported cases on 2007. The
disease is a multiple-systemic disorder with various clinical manifestations including erythema
migrans rash, arthritis, cardiac, musculoskeletal and neurological manifestations.
B. burgdorferi exists in nature in an enzootic cycle. Ixodes scapularis ticks (commonly
known as deer ticks) acquire the infection when they feed on an infected host, mainly rodents.
During subsequent tick feeding, which lasts for several days, B. burgdorferi migrate from the tick
midgut, pass through the salivary glands, and are then transmitted to the mammal through the
saliva. B. burgdorferi is highly invasive. After being deposited in the skin following a tick bite, the
spirochetes can invade many tissues including the joints, heart, and nervous system.
Motility and chemotaxis are critical for bacterial survival and adaptation in diverse
environmental conditions. In several species of bacteria, motility and chemotaxis have been
shown to be associated with the disease process. Results obtained using B. burgdorferi with
mutations in key motility and chemotaxis genes also indicate that these activities are required
for the pathogenesis of Lyme disease. These studies could lead to the development of a novel
pharmacological agent to treat/prevent Lyme disease.
42

BLAST X Thurs. Morning Session
PLEIOTROPIC PHENOTYPES OF A YERSINIA ENTEROCOLICIA FLHD MUTANT INCLUDE
REDUCED LETHALITY IN A CHICKEN EMBRYO MODEL
Birgit M. Prüß 1 , Megan K.T. Ramsett 1 , Nathan J. Carr 1 , Jyoti G. Iyer 1 , Penelope S. Gibbs 1 ,
Philip Matsumura 2 , and Shelley M. Horne 1
1
Veterinary & Microbiological Sciences Department, North Dakota State University, Fargo ND
58108
2
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago IL
60612
The goal of this study was to correlate phenotypes with gene regulation by several
flagellar regulators in Yersinia enterocolitica. FlhD/FlhC was initially described as a flagella
transcriptional activator and later recognized as a global regulator in several enteric bacteria (1,
2). In Y. enterocolitica, FlhD/FlhC positively affected the expression levels of genes of histidine
degradation and pyrimidine biosynthesis, while repressing the urease genes (1). A second
protein that is involved in the regulation of flagellar genes, the sigma factor FliA, exhibited a
negative effect upon the expression levels of seven plasmid-encoded virulence genes (3). In
addition, eight flagellar operons were regulated by FliA. Among the differences to Escherichia
coli were a 10 fold regulation of fliZ expression by FliA and a lack of FliA regulation of the flgM
operon.
Phenotypes relating to FlhD/FlhC and FliA gene regulation were investigated. These
phenotypes included growth on carbon and nitrogen sources, and virulence (4). Growth was
determined with Phenotype MicroArrays (Biolog). Compared to the wild-type strain, flhD and fliA
mutants exhibited increased growth on purines as carbon sources and decreased growth on
pyrimidines and histidine as nitrogen sources. Several dipeptides provided differential growth
conditions between the wild-type strain and both mutants. Gene regulation was determined for
the dpp (dipeptide transport) and opp (oligopeptide transport) genes and was found to correlate
with the observed phenotypes. Phenotypes relating to virulence were determined with the
chicken embryo lethality assay that was previously established and used for E. coli strains (5).
Relative to the wild-type strain, the flhD mutant caused a reduced lethality in this assay, while
the fliA mutant caused lethality similar to the wild-type. Mutants were able to colonize infected
embryo organs at levels that were comparable to the wild-type. In addition, a mutant in flhB,
encoding one component of the flagellar type III secretion system also caused a reduced
embryo lethality. Since genes of the type III secretion system are regulated by FlhD/FlhC and
not by FliA, we believe that the lethality phenotype of the flhD mutant is due to regulation of the
type III secretion genes.
1. V. Kapatral et al., Microbiol. 150, 2289 (2004).
2. B. M. Prüß et al., J. Bacteriol. 185, 534 (2003).
3. S. M. Horne, B. M. Prüß, Arch. Microbiol. 185, 115 (2006).
4. M. K. Townsend et al., BMC Microbiol. 8, 12 (2008).
5. P. S. Gibbs, J. J. Maurer, L. K. Nolan, R. E. Wooley, Avian Dis. 47, 370 (2003).
43

BLAST X Thurs. Morning Session
REGULATION OF MOTILITY BY QUORUM SENSING IN SINORHIZOBIUM MELILOTI AND
ITS ROLE IN SYMBIOSIS ESTABLISHMENT
Juan E. González*, Nataliya Gurich, Jennifer L. Morris, Konrad Mueller, and Arati V. Patankar
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas,
USA
Quorum sensing is a mechanism widely used by bacteria to coordinate their behavior in
response to a particular cell population density. Signal molecules, termed autoinducers, are
produced by bacteria, and at a high population density, accumulate in the environment. Once a
threshold level of autoinducer is reached, they bind to their cognate transcriptional regulators
and activate or repress expression of target genes, thereby preparing the bacteria for behaviors
associated with high cell density, such as interacting with eukaryotic hosts.
In Sinorhizobium meliloti, this mechanism is utilized to appropriately modulate gene
expression and permit the establishment of a nitrogen-fixing symbiosis with its host plant
Medicago sativa. S. meliloti possesses a quorum-sensing system composed of two
transcriptional regulators, SinR and ExpR, and the SinR-controlled autoinducer synthase SinI,
which is responsible for the biosynthesis of the signal molecule in the form of an N-acyl
homoserine lactone (AHL). These AHLs, in conjunction with the ExpR regulator, control a
variety of downstream genes. The concentration of AHLs varies with changes in population
density. As a result, expression of quorum-sensing-dependent genes may exhibit different
patterns during various stages of bacterial growth. Work in our laboratory has shown that the S.
meliloti ExpR/Sin quorum-sensing system regulates over 200 genes, including those involved in
exopolysaccharide synthesis, motility and chemotaxis, metal transport, and other metabolic
functions, thereby playing an important role during plant-bacteria interactions.
Inoculation of plants with a sinI-deficient strain results in a delay in invasion as well as a
significant reduction in the total number of nodules per plant when compared to the wild type,
resulting in plant development deficiencies. Concurrently, expression of most of the motility and
chemotaxis genes in the sinI mutant fail to be down-regulated by quorum sensing at high cell
population density. Microarray and real-time PCR analyses revealed that the ExpR/Sin system
adjusts the expression of the transcriptional regulators VisN/VisR and Rem, which in turn
modulate downstream motility genes in a population-density-dependent manner to decrease
motility. Recently we have shown that mutating flagellar production in a sinI mutant restores
bacterial competency for symbiosis establishment to wild type levels, suggesting that the
elimination of flagella during the invasion process is crucial. Therefore, down-regulation of
motility and chemotaxis by the ExpR/Sin quorum-sensing system plays an essential role in
successful plant invasion by S. meliloti.
44

BLAST X Thurs. Evening Session
ENGINEERED SINGLE- AND MULTI-CELL CHEMOTAXIS IN E. COLI
Shalom D. Goldberg 1 , Paige Derr 2 , William F. DeGrado 1 , and Mark Goulian 2,3
1 Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine,
Philadelphia, PA, 19104
2 Department of Physics, University of Pennsylvania, 209 South 33rd Street, Philadelphia, PA
19104
3 Department of Biology, University of Pennsylvania, 433 S University Avenue, Philadelphia, PA
19104
We have engineered the chemotaxis system of E. coli to enable responses to molecules
that are not attractants for wild-type cells. The system depends on an artificially introduced
enzymatic activity that converts the target molecule into a ligand for an E. coli chemoreceptor,
thereby allowing the cells to respond to the new attractant. Two systems, designed to respond
to asparagine and to phenylacetyl glycine respectively, showed robust chemotactic responses.
In addition, their behavior in a mixed population was suggestive of a “hitchhiker” effect in which
cells producing the ligand can induce chemotaxis of neighboring cells lacking the enzymatic
activity. This behavior was exploited to design a complex system of two strains that are mutually
interdependent for their activity, which functions as a simple microbial consortium.
45

BLAST X Thurs. Evening Session
PHOTO-ENERGY CONVERSION AND SENSORY TRANSDUCTION OF MICROBIAL
RHODOPSINS IN PHOTOSYNTHETIC MICROBES
So Young Kim, Keon Ah Lee, Ah Reum Choi, Song-I Han, and Kwang-Hwan Jung
Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang
Univeristy, Seoul 121-742, Korea (kjung@sogang.ac.kr)
Microbial rhodopsins, seven transmembrane proteins which contain all-trans/13 cis
retinal as a chromophore, have been known for three decades and extensively studies in
extreme halophiles. Photosynthetic microbes possess lots of photoactive proteins including
chlorophyll-based pigments, phytochromes, phototropin-related blue light receptors, and
cryptochromes. Surprisingly, recent genome sequencing projects discovered additional
photoactive receptors, retinal-based rhodopsins, in cyanobacterial and algal genera. Analysis of
the Anabaena and Chlamyrhodopsin revealed that they have sensory functions, which based on
our work with haloarchaeal rhodopsins, may use a variety of signaling mechanisms. Anabaena
rhodopsin is interacted with a tetramer of 14kDa soluble transducer (ASRT) and one of their
putative functions is a global regulation of phycobilin protein. The Anabaena rhodopsin shows a
visible light-absorbing pigment (540-550nm) and it has mixed photochemical reaction of all trans
and 13 cis form of retinal in ground state. Two Chlamydomonas rhodopsins are involved in
phototaxis and photophobic responses based on electrical measurements by RNAi experiment.
The rhodopsins from Gloeobacter violaceus and Acetabularia acetabulum is light-driven proton
pump coexisted with photosynthetic machinery. The genes were functionally expressed in
Escherichia coli and bound all-trans retinal to form a pigment in the presence of N- and Cterminal
MISTIC sequences. Gloeobacter and Acetabularia rhodopsin I showed a light-driven
proton pumping activity similar to proteorhodopsin.
46

BLAST X Thurs. Evening Session
FUNCTION OF MULTIPLE CHEMOTAXIS-LIKE PATHWAYS IN MEDIATING CHANGES IN
MOTILITY PATTERNS AND CELLULAR MORPHOLOGY IN AZOSPIRILLUM BRASILENSE
Amber N. Bible 1 , Zhihong Xie 1 , Matthew Russell 1 and Gladys Alexandre 1,2
1 Department of Biochemistry, Cellular and Molecular Biology and 2 Department of Microbiology,
The University of Tennessee, Knoxville, TN 37996
Molecular details on bacterial chemotaxis have been derived from studies of model
organisms such as Escherichia coli and Bacillus subtilis which genome encode for a single
chemotaxis pathway that functions to modulate changes in motility patterns. Comparative
genomics analysis indicates that the genome of many bacteria possess multiple chemotaxis-like
(Che) pathways. A. brasilense is a plant-associated bacterium that can differentiate in at least
four different cell types (swimmer, swarmer, aggregated and cyst cells). Transition from one cell
type to the other depends on the environmental (especially nutritional) conditions. One of the 4
Che-like pathways (Che1) encoded within the genome of the alphaproteobacterium A.
brasilense was recently shown to regulate changes in motility patterns, cell-to-cell aggregation
concomitant with changes in cell length (Bible et al., 2008). We will present evidence for the role
of two Che pathways and several chemoreceptors in controlling the ability of cells to modulate
multiple cellular responses, including cell length, that suggest that cross-regulation between
parallel chemotaxis pathways may function to coordinate and integrate a set of cellular
functions. Experimental evidence suggests that proteins that function in the
methylation/demethylation of chemoreceptors may have a critical role in this cross-regulation.
The implications in the lifestyle of this bacterium will also be discussed in lights of recent
experimental evidence obtained.
Bible, A. N., Stephens, B. B., Ortega, D. R., Xie, Z. and G. Alexandre (2008) Function of a
chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length
in the alphaproteobacterium Azospirillum brasilense. J Bacteriol 190: 6365-6375.
47

BLAST X Thurs. Evening Session
PROBING ADAPTATION KINETICS IN VIVO BY FLUORESCENCE RESONANCE ENERGY
TRANSFER
Thomas S. Shimizu 1 , Yuhai Tu 2 and Howard C. Berg 1
1 Department of Molecular & Cellular Biology, Harvard University, Cambridge, MA 02138.
2 T. J. Watson Research Center, IBM, Yorktown Heights, NY 10598.
Bacteria sense spatial gradients by taking time derivatives of ligand concentrations
measured during runs of a random walk 1 . The remarkable sensitivity to shallow gradients in
Escherichia coli has been explained mainly by cooperativity between receptors and
ultrasensitivity of the flagellar motor. We have revisited the experimental findings of Block,
Segall and Berg 2 , where the chemotactic response of tethered cells to time-varying stimuli were
characterized quantitatively. A simple theoretical model 3 that combines robust adaptation 4 with
an allosteric model of receptor cooperativity 5-7 can explain the general features of responses to
temporal ramps and oscillatory stimuli.
A notable feature of this model is that the steady-state amplitude of responses to
exponential ramps do not depend on the degree of receptor cooperativity (the parameter N of
an MWC-type allosteric model 5 ). The time required to reach this steady state, however, depends
inversely on N, so cooperativity speeds up computation of the derivative signal, but does not
determine its amplitude. The latter is instead determined by the adaptation kinetics, and this
relation allows us to infer quantitative characteristics of adaptation in vivo from measured rampresponse
data.
Here we present novel experiments in which the chemotactic responses of E. coli
populations during time-varying stimuli are monitored by fluorescence resonance energy
transfer 8 (FRET). This approach is far more efficient than the earlier experiments of Block et al. 2 ,
in which the chemotactic responses of individual cells were characterized through the stochastic
output of the motor. We find that the sensitivity of E. coli to gradients depends strongly on
temperature, and using our model framework, we analyze how ultrasensitvity in the adaptation
system 9 contributes to gradient sensitivity in vivo.
REFERENCES
1. Berg, H. C. & Brown, D. A. Chemotaxis in Escherichia coli analysed by three-dimensional
tracking. Nature 239, 500-4 (1972).
2. Block, S. M., Segall, J. E. & Berg, H. C. Adaptation kinetics in bacterial chemotaxis. J Bacteriol
154, 312-23 (1983).
3. Tu, Y., Shimizu, T. S. & Berg, H. C. Modeling the chemotactic response of Escherichia coli to
time-varying stimuli. Proc Natl Acad Sci U S A 105, 14855-60 (2008).
4. Barkai, N. & Leibler, S. Robustness in simple biochemical networks. Nature 387, 913-7 (1997).
5. Monod, J., Wyman, J. & Changeux, J. P. On the Nature of Allosteric Transitions: A Plausible
Model. J Mol Biol 12, 88-118 (1965).
6. Mello, B. A. & Tu, Y. An allosteric model for heterogeneous receptor complexes: Understanding
bacterial chemotaxis responses to multiple stimuli. Proc Natl Acad Sci U S A 102, 17354-9
(2005).
7. Keymer, J. E., Endres, R. G., Skoge, M., Meir, Y. & Wingreen, N. S. Chemosensing in
Escherichia coli: two regimes of two-state receptors. Proc Natl Acad Sci U S A 103, 1786-91
(2006).
8. Sourjik, V., Vaknin, A., Shimizu, T. S. & Berg, H. C. In Vivo Measurement by FRET of Pathway
Activity in Bacterial Chemotaxis. Methods Enzymol 423, 363-91 (2007).
9. Emonet, T. & Cluzel, P. Relationship between cellular response and behavioral variability in
bacterial chemotaxis. Proc Natl Acad Sci U S A 105, 3304-9 (2008).
48

BLAST X Thurs. Evening Session
MINOR RECEPTOR SIGNALLING IN E. COLI
Silke Neumann, Ned Wingreen* and Victor Sourjik
Ruprecht-Karls-Universität Heidelberg, Zentrum für molekulare Biologie Heidelberg (ZMBH), Im
Neuenheimer Feld 282, 69120 Heidelberg, Germany
* Department of Molecular Biology - Princeton University
Ligand recognition in the chemotaxis pathway of E. coli proceeds through binding of
ligands to transmembrane receptors, either directly or indirectly through periplasmic binding
proteins. E. coli has five types of receptors, with two high-abundance (or major) receptors – Tsr
for serine and Tar for aspartate and maltose – and three low-abundance (or minor) receptors –
Tap for dipeptides, Trg for ribose, galactose and glucose, and Aer for redox potential. Together
with the histidine kinase CheA, receptors form chemosensory complexes which in turn are
organized in tight clusters where receptors of different ligand specificities are intermixed. Signal
processing is thought to occur within these receptor clusters through allosteric interactions
between receptor dimers. To compare signal processing by minor and major receptors, we
systematically investigated responses mediated by Trg and Tap, and by Tar and Tsr in respect
to response sensitivity, relation between receptor occupancy and kinase inactivation, dynamic
range of the response, adaptation time to a range of stimuli, as well as integration of signals that
are sensed by different receptors using an in vivo FRET-based kinase assay. Our experimental
analysis shows that signals are amplified and integrated differently by the two receptor
populations, but in both cases signal processing can be quantitatively explained by the same
allosteric model.
49

BLAST X Thurs. Evening Session
A SYSTEMS BIOLOGY APPROACH TO UNDERSTANDING HOW BACILLUS MAKES UP ITS
MIND
Arnaud Chastanet 1 , Guocheng Yuan 2 , Thomas M. Norman 1 , Jun Liu 3 and Richard Losick 1
1 Molecular and Cellular Biology Department, Harvard University,
2 Department of Biostatistics and Computational Biology, Harvard School of Public Health,
3 Department of Statistics, Harvard University.
Understanding how cells make decisions and differentiate are key biological questions.
Mechanisms underlying such behaviors integrate multiple environmental signals in intricate
networks in order to appropriately respond to the situations. The sporulation process that takes
place in Bacillus subtilis under adverse conditions perfectly exemplifies this kind of question. For
this, numerous signals and control systems are integrated at the level of a ”decider” protein
called Spo0A, a transcriptional regulator belonging to the two-component systems family. The
decision to sporulate is taken during the first two hours after optimal sporulation conditions have
been reached. During this time, Spo0A accumulates slowly reaching a high level at hour two. It
has been previously shown that while some of its targets are activated at low concentration,
thus early on, others are switched on later, when the maximal quantity of the regulator has been
achieved. Interestingly, even in optimal conditions, only a fraction of the population will finally
decide to sporulate, a phenomenon described as bistability.
We are attempting to understand how this two-stage activation of Spo0A is achieved
through an interdisciplinary approach combining the methods of genetics and mathematics. The
time resolved picture of the regulatory process we have obtained has revealed a multiple step
process involving successive switches. First, Spo0A activity is rising during log phase, activating
some switches. Then under conditions of nutrient limitation, Spo0A is further activated to a mid
and variable extent throughout the population. During this period, “low-threshold” genes are
turned ON. In an ultimate step, a bistable switch allows Spo0A to be activated to a high level but
only in a portion of the population. These cells express high threshold genes and proceed to
sporulate.
50

POSTER ABSTRACTS
51

BLAST X ______ Poster #1
THE CHARACTERISATION OF THE DYNAMICS OF THE FliT:FliD:FlhD4C2 INTERACTION
AND ITS ROLE IN REGULATING FLAGELLAR ASSEMBLY
C. Aldridge 1,2 , K. Poonchareon 1,2 , S. Saini 3 , A. Soloyva 2 , M. Banfield 4 , T. Minamino 5,6 , C. V.
Rao 3 , and P. D. Aldridge 1,2
1: Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon
Tyne, United Kingdom, NE2 4HH. 2: Institute for Cell and Molecular Biosciences, Newcastle
University, Framlington Place, Newcastle upon Tyne, United Kingdom, NE2 4HH. 3: Department
of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana,
Illinois, United States, 61801. 4: Dept. of Biological Chemistry, John Innes Centre, Norwich,
NR4 7UH UK. 5: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka,
Suita, Osaka 565-0871, Japan. 6: Dynamic NanoMachine Project, ICORP, JST, 1-3
Yamadaoka, Suita, Osaka 565-0871, Japan.
Each bacterial cell of Salmonella enterica serovar Typhimurium produces a discrete
number of complete flagella. Flagellar gene expression in Salmonella is negatively regulated by
the Type 3 secretion chaperone FliT. FliT is known to interact with the flagellar structural subunit
FliD and the master transcriptional regulator FlhD4C2. In this regulatory circuit FliD is proposed
to act as an anti-regulator - a regulatory role similar to that observed for FlgM inhibition of s 28
activity. We were interested in determining the kinetics of the FliT:FliD:FlhD4C2 regulatory circuit
and how they influence flagellar assembly. We have shown that the FliT:FliD interaction is a 1:1
ratio while in solution FliT is a dimer. Surface plasma resonance (SPR) and analytical
ultracentrifugation (AUC) analysis of the dynamics of the FliT:FlhD4C2 interaction showed that it
was very different from FliT:FliD. A FliT:FlhD4C2 complex is only observed when excess FliT is
added to FlhD4C2. AUC analysis identified that a stable intermediate of a combination of FlhD,
FlhC and/or FliT exists. Our data suggests that FliT acts to dissociate the FlhD4C2 complex and
that this is a transient interaction allowing for the FlhD4C2 complex to reform. This suggests that
a limiting factor in the regulation of FlhD4C2 activity is the concentration of free FliT. To test this
model, we have investigated the affect of overexpressing FliT on flagellar gene expression and
basal body assembly.
52

BLAST X ______ Poster #2
SUBUNIT FEEDBACK CONTROL OF FLAGELLAR FILAMENT ASSEMBLY IN
CAULOBACTER CRESCENTUS
Phillip D. Aldridge 1,2§ Alexandra Faulds-Pain 1,2 , Christine Aldridge 1,2 , Giulia Grimaldi 1,2 ,
Christopher Birchall 1,2 , Shuichi Nakamura 3 , Tomoko Miyata 3 , Joe Gray 4 , Guanglai Li 5 , Jay
Tang 5 , Keiichi Namba 3,6 , Tohru Minamino 3,6
1: Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon
Tyne, United Kingdom, NE2 4HH 2: Institute for Cell and Molecular Biosciences, Newcastle
University, Framlington Place, Newcastle upon Tyne, United Kingdom, NE2 4HH 3: Graduate
School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871,
Japan. 4: Pinnacle, Newcastle University, Framlington Place, Newcastle upon Tyne, United
Kingdom, NE2 4HH 5: Physics Department, Brown University, 184 Hope Street, Providence, RI
02912, USA 6: Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka
565-0871, Japan.
Bacterial flagellar filaments play key roles in surface attachment and host-bacterial
interactions as well as motility. Approximately 40% of annotated flagellar systems (n = 278)
utilise multiple flagellin variants to assemble their flagellar filaments. Here we have investigated
the ability of the model flagellar system of Caulobacter crescentus to assemble its flagellar
filament from six flagellins: FljJ, FljK, FljL, FljM, FljN and FljO. A flagellin gene mutant collection
of multiple gene deletion combinations, exhibited a range of Mot phenotypes from impaired
motility (Mot -/+ ) to motile (Mot + ). Further characterisation of the mutant collection showed: 1)
there is no strict requirement for all six flagellins to assemble a filament exhibiting wild type
characteristics; 2) a correlation between slower swimming speeds and shorter filament lengths
in all ∆fljK ∆fljM Mot + mutants; and 3) the flagellins FljM – FljO are less stable than FljJ – FljL.
Our data suggests that the flagellins FljJ, FljK and FljL play both a regulatory and structural role
during filament assembly. In contrast, the flagellins FljM to FljO possess only a structural role.
We propose the model that the observed order of multiple flagellin incorporation in C.
crescentus, and plausibly other flagellar systems, is a result of the system coupling flagellin
synthesis to filament assembly.
53

BLAST X ______ Poster #3
FUNCTION OF UNIQUE DOMAINS OF CheA1 FROM A. BRASILENSE IN REGULATING
MULTIPLE CELLULAR BEHAVIORS
Amber N. Bible and Gladys Alexandre
Department of Biochemistry, Cellular, and Molecular Biology at the University of Tennessee,
Knoxville
The alpha-proteobacterium Azospirillum brasilense contains four different chemotaxis
operons. One of the 4 Che-like pathways (Che1) encoded within the genome of the
alphaproteobacterium A. brasilense was recently shown to regulate changes in motility patterns,
cell-to-cell aggregation (clumping) concomitant with changes in cell length (Bible et al., 2008).
Mutations affecting cheA1 decrease chemotaxis, cell length, but lead to an increase in
clumping relative to the wild type A. brasilense. Interestingly, CheA1 is a hybrid histidine kinase
with two P5-like domains and a REC-like receiver domain at its C-terminus. In addition, the Nterminus
of CheA1 comprises a highly conserved polytopic domain of unknown function,
suggesting that CheA1 may be a membrane-bound protein. Experimental data indicate that
CheA1 is produced as a membrane-bound protein that localizes to the cell pole, similar to other
Che1 proteins. Noticeably, the polytopic N-terminal domain of CheA1 is not required for the
localization of CheA1 neither to the cell pole, nor for changes in cell length or cell-to-cell
aggregation but it is essential for wild-type chemotaxis and aerotaxis behaviors. Data obtained
from a combination of in-frame deletions of the C-terminal domains and site-specific
mutagenesis approaches with behavioral assays suggest that the second P5-like domain of
CheA1 has a role in modulating changes in cell length. A working model for the functions of the
N- and C-terminal domains of CheA1 in modulating chemotaxis, aerotaxis, clumping and cell
length changes will be presented in lights of recent experimental data.
Bible, A. N., Stephens, B. B., Ortega, D. R., Xie, Z. and G. Alexandre (2008) Function of a
chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length
in the alphaproteobacterium Azospirillum brasilense. J Bacteriol 190: 6365-6375.
54

BLAST X ___ Poster #4
HOW DOES THE RHODOBACTER SPHAEROIDES FLAGELLAR MOTOR STOP – USING A
CLUTCH OR A BRAKE?
M. Brown, T. Pilizota, M. Leake, R. Berry, J. Armitage
University of Oxford, Oxford, UK
Unlike most species with bidirectional motors, Rhodobacter sphaeroides employs a
unidirectional stop-start flagellar motor, where stops are analogous to tumbles. By controlling
when the motor stops, cells can accumulate in areas favourable for their survival.
We asked the question, how do the CheYs stop motor rotation in R. sphaeroides; by
causing the torque-generating units to disengage from the rotor, allowing free rotational
movement, or by jamming the rotor, locking it in a particular configuration?
These hypothesises were tested by applying external force (viscous flow or optical
tweezers) to chemotactically stopped motors.
We found that the motor is stopped with a brake mechanism and that approximately 3-4
times more torque acts on the motor when stopped than when it rotates. Furthermore, by
monitoring the position of sub-micron beads attached to flagella stubs we discovered that stops
can only occur at a number of discrete angles. Analysis is underway to determine the number
of steps per revolution.
55

BLAST X ______ Poster #5
DYNAMICS OF THE FLAGELLAR MOTOR PROTEIN FliM
Nicolas J. Delalez
Microbiology Unit, Biochemistry Dept, University of Oxford, South Parks Road, Oxford OX1
3QU, UK
Escherichia coli swims by rotating 4-6 long helical filaments to propel the cell through its
environment. The flagellar motor that rotates the filament is bidirectional and composed of two
parts: the rotor and the stator, the stator being the fixed component against which the rotor
spins. The stator is composed of units of two integral membrane proteins, MotA and MotB, the
stoichiometry of each unit being (MotA4:MotB2). The rotor is composed of multiple rings,
including the C-ring which is localized at the base of the motor and is the switch complex that
gives the bidirectionality to the E. coli motor. The C-ring comprises FliG (~ 25 copies), FliM (~34
copies) and FliN (> 100 copies).
By using a TIRF microscope, and expressing GFP-MotB from the genome of E. coli in
place of the wild-type gene, it has recently been possible to monitor the protein stoichiometry,
dynamics and turnover of this stator component with single-molecule precision in functioning
bacterial flagellar motors. Repeating these experiments with different flagellar motor proteins will
greatly enhance our understanding of the mechanism of this motor.
In particular, one of the main questions for our understanding is the mechanism of the
switching process. The C-terminus of FliM, a component of the C-ring, has therefore been
tagged with Ypet and expressed from the genome. Data will be presented on the construction of
this mutant as well as data on its dynamics, turnover and stoichoimetry, and the influence of the
response regulator CheY on these features. The consequences of these results and future work
will be discussed.
56

BLAST X Poster #6
USING CONTROL THEORY TO ELUCIDATE CONNECTIVITY IN R. SPHAEROIDES
CHEMOTAXIS
Mark A. J. Roberts 1 , Elias August 2 , Judith P. Armitage 1 and Antonis Papachristodoulou 3
1
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
2
Control Group, Department of Engineering Science, Oxford University, Parks Road, Oxford,
OX1 3PJ, UK
3
Oxford Centre for Integrative Systems Biology, Department of Biochemistry, South Parks Road,
Oxford, OX1 3QU, UK
With an increasing number of sequenced bacterial genomes it becomes evident that the
chemotactic sensory mechanism of bacteria is more complex than E. coli. In this poster we
describe how ideas from engineering control theory can be used to develop a novel approach
for designing experiments in order to elucidate the biochemical network structure of signalling
pathways in general. The goal is to develop a systematic approach for finding the best
experiment that will delineate the network structure.
We then apply this method to the chemotaxis pathway of R. sphaeroides, which has
multiple homologues of the E. coli proteins. To achieve this we are constructing, in silico,
various possible models of R. sphaeroides chemotaxis that can explain experimental
observations. These models include the different possible interactions for the CheB and CheY
proteins. Applying results from optimal control theory, we determined the best input (ligand)
profile that gives an output which would allow us to discriminate best between the proposed
models, aiming to invalidate some of them. This input ligand profile is then administered to R.
sphaeroides in a flow cell and the response is measured using a tethered cell assay. We have
also developed methods to determine the best initial conditions to discriminate between the
models, based on the limitations of what can be implemented biochemically, and these were
then also tested in a tethered cell assay.
We used the experimental results from these designed tethered cell experiments to
invalidate some of the proposed network structures and hence suggest a probable network
connectivity for the multiple CheY and CheB proteins within R. sphaeroides.
This is an exciting approach to determine network structures in a fast and efficient
manner and can be applied to a wide range of signalling pathways as well as potentially
allowing chemotaxis pathways in other species using published genomes to generate the
necessary models.
57

BLAST X Poster #7
BEHAVIOR OF THE FLAGELLAR ROTARY MOTOR NEAR ZERO LOAD
Junhua Yuan and Howard Berg
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
The physiology of the flagellar rotary motor has been studied extensively in the regime of
relatively high load and low speed. Here, we describe an assay that allows systematic study of
the motor near zero load. Sixty-nanometer-diameter gold spheres were attached to hooks of
cells lacking flagellar filaments, and light scattered from a sphere was monitored at the image
plane of a microscope through a small pinhole. Resurrection experiments were carried out near
zero load. Paralyzed motors of cells carrying a motA point mutation were resurrected at 23°C by
expression of wild-type MotA, and speeds jumped from zero to a maximum value of about 300
Hz in one step. The temperature dependence of the speed near zero load also was studied and
showed a high activation enthalpy comparable to that observed previously in electrorotation
experiments. The assay has been modified so that both the speed and the direction of rotation
can be monitored near zero load. Switching properties of the flagellar motor near zero load are
under investigation.
58

BLAST X ______ Poster #8
NUTRIENT SENSING BY A HUMAN GUT SYMBIONT
Hongjun Zheng 1 , Susan Firbank 1 , Eric Martens 2 , Edith Diaz 1 , Rick Lewis 1 , Jeff Gordon 2 and
David Bolam 1
1. Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School,
Framlington Place, Newcastle upon Tyne NE2 4HH, UK
2. Center for Genome Sciences, Washington University, St Louis, USA.
The gut microbiota play an important role in human health and nutrition. Bacteroides
thetaiotaomicron (Bt) is a dominant gut symbiont whose main sources of carbon and energy are
dietary and host polysaccharides, reflected in the presence on the genome of a large number of
genes involved in the sensing, acquisition and processing of complex carbohydrates. One of the
most striking features of the Bt genome are 33 genes encoding novel hybrid two component
systems (HTCS) that contain all of the domains of a classical TCS in a single polypeptide.
Custom Genechip arrays reveal that when Bt is grown in the presence of inulin, a β-1,2 linked
fructose storage polysaccharide, a specific locus composed of eight genes predicted to be
involved in polysaccharide binding, uptake and degradation is activated. The closest regulatory
protein to this locus is an HTCS, BT1754. Genetic, biochemical and structural studies reveal
that BT1754 is the sensor that controls fructan utilisation and the identity of the signalling
molecule itself, as well as the mechanism of signal perception and insights into signal
transduction across the cytoplasmic membrane.
59

BLAST X Poster #9
THE LeuO REGULON IN SALMONELLA
Edmundo Calva, Víctor H. Bustamante, Miguel Ángel De la Cruz, Marcos Fernández-Mora,
Mario Alberto Flores-Valdez, Ana Lucía Gallego-Hernández, Carmen Guadarrama, Ismael
Hernández-Lucas, Omar Ortega, Alejandra Vázquez, and Ricardo Oropeza.
Instituto de Biotecnología UNAM, Cuernavaca, Morelos, Mexico.
LeuO is a LysR-type transcriptional regulator, which antagonizes the repressing effect by
the H-NS and StpA nucleoid proteins on the Salmonella enterica serovar Typhi ompS1
quiescent porin gene, allowing the transcriptional activation by OmpR among other regulators.
LeuO is also a positive regulator for the OmpS2 quiescent porin, for a quiescent putative
periplasmic arylsulfate sulfotransferasa (AssT), and for an open reading frame of unknown
function, STY3068. It is a negative regulator for the OmpX porin, a thiol peroxidase (Tpx), and
for STY1978. These proteins were identified by a whole-genome proteomic analysis and the
regulatory effects were confirmed on transcriptional gene reporter fusions. STY 3068 appears to
be part of an operon (5’-STY3070-STY3064-3’), as transcriptional regulation occurs upstream,
at STY 3070. The assT gene forms a LeuO-dependent operon with STY3371 and STY3372,
which are paralogues of the dsbA and dsbB genes, respectively. This operon (5’-assT-
STY3371-STY3372-3’) is repressed by OmpR.
H-NS represses the expression of ompS1, assT and STY3070 and, on the other hand,
activates the expression of tpx and STY1978. Expression of ompX was independent of H-NS.
Interestingly, ompS2 is negatively regulated by H-NS in Escherichia coli and in S. Typhimurium,
although in S. Typhi other negative regulators are involved aside from H-NS. LeuO specifically
binds to the 5´ intergenic regions of ompS1, ompS2, assT, STY3070, ompX, and tpx. LeuO did
not bind to the promoter region of STY1978, suggesting that it is regulating in an indirect
manner.
Hence, LeuO regulates a cadre of genes which appear to be mostly involved in the
response to stress and in virulence.
60

BLAST X Poster #10
EnvZ-OmpR AND CpxA-CpxR REGULATE ompS1 BY DIFFERENTIAL PROMOTER
EXPRESSION
De la Cruz, M.A, Flores-Valdez, M.A., and Calva, E.
Departamento de Microbiología Molecular, Instituto de Biotecnología UNAM Av. Universidad
2001 Col. Chamilpa C.P. 62210 Cuernavaca, Morelos México
The Salmonella enterica ompS1 gene encodes a quiescent porin that belongs to the
OmpC/OmpF family. We recently reported that LeuO, OmpR, H-NS and StpA are regulators of
ompS1 expression. We have now detailed the mechanism of OmpR regulation. In vivo,
phosphorylated OmpR (OmpR-P) showed to be determinant for the regulation of both ompS1
promoters: OmpR-P activated the P1 promoter and repressed the P2 promoter, in an EnvZdependent
manner. In vitro, OmpR-P had a seventy two-fold higher binding-affinity to the
ompS1 promoter region than OmpR, being 2 to 20-fold higher than to the major porin-encoding
ompC and ompF genes, respectively. In addition to EnvZ-OmpR, we found that CpxA-CpxR
positively regulated ompS1 expression. CpxR, together OmpR, was necessary for the activation
of the P1 promoter. CpxR also activated the P2 promoter, but only in the absence of OmpR. Our
model involves LeuO as an antagonist that relieves H-NS and StpA-mediated silencing, allowing
the binding of OmpR and CpxR. High osmolarity and acid pH, or both, stimulated ompS1
expression in an OmpR and CpxR-independent manner, yet dependent on DNA supercoiling.
61

BLAST X Poster #11
THE COMPLEX HOOK BASAL BODY STRUCTURE OF THE LYME DISEASE SPIROCHETE
BORRELIA BURGDORFERI
K. Miller 1 , R. Duda 2 , R. Hendrix 2 , M. Motaleb 1,3 , and N.W. Charon 1
1
West Virginia University Health Sciences Center, Microbiology and Immunology Department,
Box 9177, Room 2077, Morgantown, WV 26506
2
University of Pittsburgh, Department of Biological Sciences, 4249 Fifth Avenue, 357A,
Crawford Hall, Pittsburgh, PA 15260
3
East Carolina University, Department of Microbiology and Immunology, Greenville, NC 27834
FlgE is the structural protein of the flagellar hook of B. burgdorferi. Previous western blot
analysis using polyclonal FlgE antibody indicated that the majority of the hook migrated as a
high molecular weight complex in SDS-PAGE gels instead of at the position of monomeric FlgE
(46 kDa). Both the high molecular complex and a small amount of the monomer were present in
wild-type and complemented cells but absent in flgE mutants. These data suggest that FlgE
may be cross-linked into oligomers in a manner similar to that found in bacteriophage capsid
proteins and in some bacterial pili. High molecular weight FlgE complexes have been observed
in other spirochete species (Treponema phagedenis and Treponema denticola), suggesting that
this property is conserved. We are interested in determining whether or not FlgE is cross-linked,
and what amino acids are involved in the cross-linking.
The hook-basal body complexes were purified using a refined version of methods
previously reported (Sal, et al, 2008). Following purification of periplasmic flagella that contained
attached hook-basal body complexes, the filament portions were depolymerized by acidic
conditions or the use of urea. Hook-basal body complexes were then isolated by pelleting using
an ultracentrifuge recovered in a neutralizing buffer. Electron microscopy of these preparations
showed that acidic conditions resulted in more intact hook-basal body complexes than urea
treatment, although the high molecular weight forms of FlgE were found using both methods. In
order to determine results from each experiment, the experimental samples were run on 10%
SDS-PAGE gels. We are using proteolytic finger printing and peptide mapping methods to
compare the high molecular weight complexes of FlgE with the monomeric form (histidinetagged
recombinant). Hook-basal body complexes and recombinant FlgE were treated with
proteases or hydroxylamine at various temperatures (37, 45, and 65°C) and run in SDS-PAGE
gels. Western blotting was used to assess the stability of the FlgE complex as well as to
determine the size of (antigenic) peptide fragments resulting from proteolytic digestion. Digest
patterns were compared between recombinant FlgE and the isolated hook-basal body
complexes.
Refining the hook-basal body isolation procedure resulted in improved hook-basal body
preparations. The FlgE complex is stable under harsh conditions (both urea and acid treatment).
Differences and similarities in hydroxylamine digest patterns between recombinant FlgE and
purified hook-basal body complexes supports the hypothesis that FlgE is cross-linked.
62

BLAST X Poster #12
HOW DO PAS AND HAMP DOMAINS COMMUNICATE? INSIGHTS FROM Aer2, A HEME
BASED SENSOR FOR AEROTAXIS
Michael Airola (1), Joanne Widom (1), Kylie Watts (2), Brian Crane (1)
(1) Dept. of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850
(2) Dept. of Biochemistry and Microbiology, Loma Linda University, Loma Linda, CA 92350
Thousands of PAS and HAMP domains have been identified yet how these common
signal transduction domains function and communicate is still poorly understood. Structural
studies of the HAMP domain have been especially difficult due to it often being contained within
integral membrane proteins. Recently Aer2, from P. aeruginosa, has been identified as a
soluble aerotaxis receptor that binds oxygen directly through a heme moiety located in the PAS
domain. Using a combination of spectroscopic and structural techniques we sought to determine
if oxygen binding induces large-scale conformational changes. Initial results point to an
interaction between the PAS and HAMP domains of Aer2 giving insight to how these
widespread signaling modules may function.
63

BLAST X Poster #13
STRUCTURE OF SOLUBLE CHEMORECEPTOR SUGGESTS A MECHANISM FOR
PROPAGATING CONFORMATIONAL SIGNALS
Abiola Pollard and Brian Crane
Cornell University, Chemistry Research Bldg. G63, Ithaca, NY 14853
Transmembrane chemoreceptors, also known as methyl-accepting chemotaxis proteins
(MCP’s), translate extracellular signals into intracellular responses in the bacterial chemotaxis
system. MCP ligand binding domains control the activity of the CheA kinase, situated ~200 Å
away, across the cytoplasmic membrane. The 2.15 Å resolution crystal structure of a T.
maritima soluble receptor (Tm14) reveals distortions in its dimeric four-helix bundle that provide
insight into the conformational states available to MCP’s for propagating signals. A bulge in one
helix generates asymmetry between subunits that displaces the kinase-interacting tip, which
resides over 100 Å away. The maximum bundle distortion maps to the adaptational region of
transmembrane MCP’s where reversible methylation of acidic residues tunes receptor activity.
Minor alterations in coiled-coil packing geometry translates the bulge distortion to a >25 Å
movement of the tip. The Tm14 structure discloses how alterations in local helical structure,
which could be induced by changes in methylation state and/or by conformational signals from
membrane proximal regions, can reposition a remote domain that regulates the CheA kinase.
64

BLAST X Poster #14
FUNCTIONAL ANALYSIS OF A LARGE NON-CONSERVED REGION OF FlgK (HAP1) FROM
RHODOBACTER SPHAEROIDES
Castillo, D.J., Ballado, T., Camarena, L., Dreyfus, G.
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México, Apdo. Postal 70-243 Cd. Universitaria, 04510, Mexico, D.F., Mexico.
The single subpolar flagellum of Rhodobacter sphaeroides shows an enlarged hookfilamentjunction.
One of the two proteins that compose this section of the filament is HAP1Rs
(FlgKRs) it contains a central non-conserved region of 860 amino acids that makes this protein
about three times larger than its homologue in Salmonella enterica serovar Typhimurium. We
investigated the role of this central portion of the unusually large HAP1 protein of R.
sphaeroides by monitoring the effects of serial deletions in flgKRs, the gene encoding HAP1Rs,
on swimming and swarming. Two deletion mutants did not assemble functional flagella, two
were paralyzed and five exhibited reduced free-swimming speeds. Some mutants produced
unusual swarming patterns on soft agar without or with Ficoll 400. A segment of approximately
200-aa of the central region of HAP1Rs that aligns with the variable region of the flagellin
sequence from other γ- and β-proteobacteria was also found. Therefore, it is possible that the
origin of this large central domain of HAP1Rs could be associated with an event of horizontal
transfer and subsequent duplications and/or insertions.
65

BLAST X Poster #15
THE FLAGELLAR MURAMIDASE FROM THE PHOTOSYNTHETIC BACTERIUM
RHODOBACTER SPHAEROIDES
Javier de la Mora 1 , Teresa Ballado 1 , Aldo E. García-Guerrero 1 , Laura Camarena 2 and Georges
Dreyfus 1 Instituto de Fisiología Celular 1 , Instituto de Investigaciones Biomédicas 2 , Universidad
Nacional Autónoma de México, 04510, México City, México.
The biogenesis of flagellum from R. sphaeroides is a tightly regulated process and
requires the expression of more than 50 genes in a strict hierarchical manner (1). The flagellar
rod is composed by four proteins, FlgB, FlgC, FlgF and FlgG. Besides these structural
components several more proteins are required for rod assembly among these FlgJ from
Salmonella enterica, has been postulated to be a dual function protein: the N-terminal half could
function as a scaffold or cap essential for rod assembly and the C-terminal half may function as
a muramidase that degrades the peptidoglycan layer to facilitate rod penetration (2, 3).We have
previously reported that the FlgJ protein of R. sphaeroides lacks the C-terminal muramidase
domain and that mutations in this protein renders a Fla - phenotype (4).The absence of the
muramidase domain in this protein, suggests that other polypeptide must accomplish this
function. In this work we describe the characterization of an open reading frame (orf) RSP0072,
which is located within the flgG operon in R. sphaeroides. The amino acid sequence analysis of
this gene product showed the presence of a soluble lytic transglycosylase domain. The deletion
of the N-terminal region (112 aa) of the product of RSP0072 renders a non-motile phenotype as
determined by swarm assays in soft agar. Electron micrographs revealed the lack of flagellum in
the mutant cells. The purified wild-type protein showed lytic activity on extracts of M.
lysodeikticus. Interaction assays suggests that the protein encoded by RSP0072 interacts with
the flagellar rod-scaffolding protein FlgJ. We propose that the product of RSP0072 is a flagellar
muramidase that is exported to the periplasm via the Sec pathway where it interacts with FlgJ to
open a gap in the peptidoglycan layer for the subsequent penetration of the nascent flagellar
structure (5).
References:
1) Poggio S., et al., 2005, Mol. Microbiol., 58:969-83.
2) Nambu T., et al., 1999, J. Bacteriol., 181:1555-1561.
3) Hirano T., et al., 2001, J. Mol. Biol., 312:359-369.
4) Gonzalez-Pedrajo B., et al., 2002, Biochim. Biophys. Acta, 1579:55-63.
5) De la Mora J., et al., 2007, J. Bacteriol., 189:7998-04.
66

BLAST X Poster #16
MODELING SCAFFOLD PHOSPHORYLATION AS AN ADAPTATION MECHANISM IN
BACTERIAL CHEMOTAXIS
Roger Alexander, Adam Bildersee, and Thierry Emonet
Department of Molecular, Cellular, and Developmental Biology, Kline Biology Tower 1054, Yale
University, New Haven, CT 06520
Bacterial chemotaxis is one of the best-understood signaling networks in biology.
Chemotaxis in E. coli is a model system for thoroughly understanding a behavioral phenotype at
the molecular level. Chemotaxis has been studied experimentally in a variety of other species,
and modeled in B. subtilis, but no dynamical models of chemotaxis network architectures from
other species have yet been built. A key feature of the chemotaxis signal transduction network
is adaptation: after a transient response to a step change in input stimulus, its activity returns to
its pre-stimulus steady state level, allowing the system to respond to higher stimulus
concentrations without saturation. In E. coli, adaptation is mediated through two enzymes, CheR
and CheB, that methylate and demethylate the sensory receptors. In this work we consider
scaffold phosphorylation as an adaptation mechanism complementary to receptor methylation.
The receptors form a sensory array in the membrane at the cell pole; the scaffold protein
couples the receptors to the kinase CheA which they activate. In all current dynamical models of
chemotaxis, the receptors, scaffolds, and kinases are treated as a single species, a receptorkinase
complex. This is understandable, because in E. coli, the scaffold CheW is a passive
mediator of the receptor-kinase interaction. However, in other organisms, the scaffold CheV
has not only a CheW scaffold domain, but also a receiver domain that is the target of
phosphorylation by the kinase. Bacillus subtilis has both CheW and CheV scaffolds, and
phosphorylation of CheV is known to be necessary for adaptation. In B. subtilis, phospho-CheV
decouples receptor from kinase, so it actively mediates their interaction. The evolutionary
distance between B. subtilis and E. coli is wide, and there are other differences between their
chemotaxis network architectures that confound an understanding of the specific role of scaffold
phosphorylation. Therefore we choose to focus on a close relative of E. coli, Salmonella
enterica serovar typhimurium. The chemotaxis network architecture in Salmonella is almost
identical to that in E. coli, except that Salmonella has both CheW and CheV scaffolds. A mutant
Salmonella that lacks methylation-based adaptation is able to adapt partially, presumably
through its scaffold phosphorylation mechanism. We have built a dynamical model of the
chemotaxis network in E. coli that explicitly represents receptor-kinase coupling by the scaffold.
We have extended that model to include scaffold phosphorylation in Salmonella. Our model of a
Salmonella mutant that lacks methylation exhibits partial adaptation, consistent with
experimental results. This work is the first step in a long-range program to explore how
evolutionary changes in chemotaxis network architecture affects the dynamics and function of
the network. Evolution of dynamic signal transduction networks is an important emerging
research area in systems biology.
67

BLAST X Poster #17
TESTING THE YIN-YANG MODEL OF SIGNAL TRANSDUCTION IN A BACTERIAL
CHEMORECEPTOR CYTOPLASMIC DOMAIN
Ka Lin E. Swain and Joseph J. Falke
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215,
Boulder, CO, 80309
The bacterial transmembrane aspartate receptor (Tar) of E. coli and S. typhimurium
chemotaxis is a homodimer that assembles to form larger oligomers, most likely a trimer-of
dimmers. The homodimer can be divided into three modules: (i) the transmembrane signaling
module comprised of the periplasmic ligand binding domain and the transmembrane helices, (ii)
the cytoplasmic HAMP domain which serves as a signal conversion module, and (iii) the
cytoplasmic kinase control module possessing the adaptation sites and a protein interaction
region that binds the CheA kinase. The kinase control module is a 4-helix bundle essential for
transmitting the integrated signal output of the HAMP domain and the adaptation sites to the
bound histidine kinase CheA. Considerable evidence indicates that structural rearrangments of
the subunit-subunit interface within the homodimeric 4-helix bundle are important in signaling.
This study further probes the mechanism of signal transduction in the kinase control module of
the S. Typhimurium aspartate receptor. The approach mutates the conserved “knob” residues
of “sockets” that stabilize helix-helix contacts to alanines, in order to destabilize the packing of
adjacent helices in the 4- helix bundle. Knob mutations that lock the receptor in “on” and “off”
signaling states are identified by their opposite effects on CheA kinase and receptor methylation
rates. The results suggest a novel “Yin-Yang” mechanism in which the helix packing states of
the adaptation region (I) and the protein interaction region (II) have opposing effects on receptor
on-off switching: stable helix packing in region I and unstable packing in region II drive the
receptor into the kinase-activating on-state, while unstable packing in region I and stable
packing in region II favor the kinase-inactivating off-state.
68

BLAST X Poster #18
CYTOCHROME d BUT NOT CYTOCHROME o RESCUES THE TOLUIDINE BLUE GROWTH
SENSITIVITY OF arc MUTANTS IN E. COLI
Adrián F. Alvarez, Roxana Malpica, Martha Contreras, Edgardo Escamilla, Everardo Ramírez
and Dimitris Georgellis.
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México, 04510 México D.F., México
The Arc (anoxic redox control) two-component signal transduction system, consisting of
the ArcB sensor kinase and the ArcA response regulator, allows adaptive responses of
Escherichia coli to changes of O2 availability. Under anaerobic conditions, the ArcB sensor
kinase autophosphorylates and then transphosphorylates ArcA, which in this form acts as a
global transcriptional regulator that controls the expression of many operons involved in
respiratory or fermentative metabolism. Under aerobic conditions, the system is inactivated
through the inhibition of the kinase activity of ArcB by the quinone electron carriers, and the
subsequent ArcA dephosphorylation. The arcA gene was previously known as the dye gene
because null mutants were growth sensitive to the photosensitizer redox dyes toluidine blue and
methylene blue, a phenotype whose molecular basis still remains elusive. Toluidine blue is a
redox dye that, in presence of light, allows the production of reactive oxygen species (ROS). In
this study we report that the toluidine blue O effect on the arc mutants is light independent, and
only observed during aerobic growth conditions. Moreover, seventeen suppressor mutants with
restored growth were generated and analyzed. Thirteen of those possessed insertion elements
(IS) upstream the cydAB operon, rendering its expression ArcA independent. Finally, it was
found that, in contrast to cythocrome d, cythocrome o was not able to confer toluidine blue
resistance to arc mutants, thereby representing an intriguing difference between the two
terminal oxidases.
69

BLAST X Poster #19
SEARCHING THE PHYSIOLOGICAL SIGNAL(S) THAT REGULATE THE ACTIVITY OF THE
SENSOR KINASE BarA
González-Chávez, R., Rodríguez-Rangel, C., Georgellis, D.
Departamento de Genética Molecular, Instituto de Fisiología Celular, UNAM, México, D.F.
04510.Tel. 56-22-57-38. dimitris@ifc.unam.mx
The BarA/UvrY two-component system of Escherichia coli comprises the BarA protein
as the histidine sensor kinase, and UvrY as the cognate response regulator. The E. coli
BarA/UvrY two-component system (TCS) and its homologues in other gram-negative bacteria,
such as BarA/SirA of Salmonella, ExpS/ExpA of Erwinia, VarS/VarA of Vibrio, and GacS/GacA
of Pseudomonas species, have been shown to positively control expression of the noncoding
CsrB and CsrC RNAs in E. coli. These small regulatory RNAs together with the 6.8 kD CsrA
protein constitute the Csr (carbon storage regulation) system, a post-transcriptional regulatory
system that has profound effects on central carbon metabolism, motility and multicellular
behavior of E. coli.
No physiological signals able to regulate the BarA sensor kinase and thereby control the
UvrY response regulator have so far been identified for any of the orthologous TCSs. However,
in a recent study it was demonstrated that pH lower than 5.5 provides an environment that does
not allow activation of the BarA/UvrY signaling pathway, providing an important physiological
tool for further experimentation in this direction.
Here, we present experiments aiming at identifying the environmental signal(s) to which
BarA respond. Our results indicate that short fatty acids, such as formate and acetate, act as
direct signals for BarA. The implications of our findings on the overall physiology of the cell are
be discussed.
70

BLAST X Poster #20
THE ROLE OF QUORUM SENSING IN THE CONTROL OF MOTILITY AND PLANT INVASION
BY SINORHIZOBIUM MELILOTI
Nataliya Gurich* and Juan E. González
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas,
USA
Quorum sensing, a population density dependent regulation of gene expression, is used
by various bacteria to establish symbiotic or pathogenic bacterium-host associations. This
mechanism requires the production of signaling molecules called autoinducers. At high cell
population densities, autoinducer concentration in the surrounding area reaches a threshold
level, which leads to activation of specific transcriptional regulators and the control of numerous
phenotypes.
Sinorhizobium meliloti is a gram-negative soil bacterium that can form a nitrogen-fixing
symbiotic association with its host Medicago sativa. The quorum sensing system in S. meliloti is
comprised of a transcriptional regulator, SinR and an autoinducer synthase, SinI, which
specifies production of N-acyl homoserine lactone (AHL) signaling molecules. In conjunction
with AHL, an additional regulator, ExpR, controls expression of several hundred genes. Recent
microarray studies in our laboratory described the control of motility and chemotaxis in S.
meliloti by quorum sensing through the regulators VisN/VisR and Rem in a population-densitydependent
manner. The wild type strain is motile during the early log phase of growth but shuts
down flagella synthesis genes during the mid- and late log phases. In contrast, the sinI mutant
remains motile throughout all phases of growth, and at the late log phase, 35 motility and
chemotaxis genes are upregulated when compared to wild type.
This inability to repress flagella production by the sinI mutant leads to severe invasion
deficiency. Detailed analysis of microarray results suggested that the inability of the sinI mutant
to shut down flagella synthesis might be detrimental to successful plant invasion. Mutation of
flagella synthesis in the sinI strain restored invasion efficiency to wild type levels. Therefore,
control of motility and chemotaxis by the ExpR/Sin quorum sensing system plays an important
role in plant invasion and may provide a competitive edge for strains possessing it.
71

BLAST X Poster #21
CHARACTERIZATION OF EtgA, A MURAMIDASE ASSOCIATED WITH THE TYPE III
SECRETION SYSTEM OF ENTEROPATHOGENIC ESCHERICHIA COLI
Elizabeth García-Gómez, Norma Espinosa, Bertha González-Pedrajo
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México. Ap. Postal 70-243. Ciudad Universitaria, México D. F., 04510.
Tel. (5255) 56-22-59-65 egarcia@ifc.unam.mx, bpedrajo@ifc.unam.mx
Enteropathogenic E. coli (EPEC) is a human pathogen that produces severe diarrhea
and death among children from developing countries. EPEC infection is characterized by the
formation of an attaching and effacing histopathology (A/E lesion) that consists of gut epithelial
microvilli destruction, intimate adherence of the bacterium to the enterocyte and the
development of an actin-rich pedestal-like structure beneath the adherent non-invasive bacteria.
EPEC utilizes a type three secretion system (T3SS) to translocate effector proteins
directly from the bacterial cytoplasm into the host cell cytosol, subverting diverse enterocytic cell
signaling pathways and producing drastic cytoskeletal reorganization. EPEC genes required for
the assembly of the T3SS and A/E lesion development are contained on a 35.6 kb pathogenicity
island known as the locus of enterocyte effacement (LEE).
The T3SS or injectisome is a macromolecular protein complex that needs to span the
periplasmic space for its assembly; however, the peptidoglycan cell wall constitutes a barrier
that allows the free passage of only small proteins. To overcome this obstacle, lytic
transglycosylases (LTs) have been identified as specialized enzymes associated with different
transport systems. It has been proposed that during T3SS biogenesis a LT facilitates a
temporally and spatially controlled opening of the peptidoglycan layer.
In this study, we have identified and characterized a LEE open reading frame with
unknown function, rorf3 (renamed etgA), which encodes a protein with a lytic transglycosylase
domain and a signal sequence. A truncated version of EtgA lacking its signal sequence
(EtgAns) was purified by nickel chromatography as an N-terminal His-tagged recombinant
protein. Lytic activity was determined by zymograms. An invariantly conserved glutamate
residue at position 42 was replaced by alanine through site-directed mutagenesis, and its
enzymatic activity was evaluated. The effect of over-producing EtgA, EtgAns and EtgAE42A
over EPEC growth and secretion was evaluated. Furthermore, the subcellular localization of the
protein was determined. Additionally, an etgA null mutant strain was generated to evaluate the
role of EtgA in T3SS assembly.
Our results show that EtgA is a T3SS associated muralytic enzyme. The highly
conserved glutamate residue (catalytic residue) is essential for EtgA function. EPEC growth rate
was affected when EtgA was overproduced, but not with EtgAns or EtgAE42A. Our data
demonstrate that the protein is secreted by the Sec pathway and that it disrupts the
peptidoglycan layer, causing bacterial lysis. Periplasmic localization of EtgAns was determined.
Finally, we show that EtgA is essential for efficient protein secretion.
72

BLAST X Poster #22
flhE: A PERIPLASMIC CHAPERONE OF FLAGELLIN?
Jae-Min Lee* and Rasika Harshey
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Texas 78712
flhE is the last gene in the flhBAE flagellar operon whose first two members encode
components of the Type III secretion pathway in Salmonella typhimurium. The role of flhE is still
a mystery. Absence of flhE does not affect swimming motility, but reduces swarming motility. In
this study, we show that FlhE is a periplasmic protein, and have localized it within the flagellar
basal body. We have found a new ‘green’ phenotype associated with a flhE deletion mutant,
which we are using as a tool to understand flhE function. The ‘green’ phenotype refers to green
colony color when bacteria are plated on aniline blue-alizarin yellow pH indicator plates. The
green color should be indicative of a lowered pH. However, independent pH measurements
failed to confirm pH differences between wild-type and flhE strains. Curiously, mutations that
prevented assembly but not secretion of flagellar filament subunits (flgK, fliD), eliminated the
green color. Immunoblotting and immunostaining assays showed that there was more ‘free’
flagellin in flhE mutant supernatants than in wild-type bacteria isolated from swarm plates.
Based on these and other experiments, we hypothesize that FlhE is a periplasmic chaperone for
the flagellar filament subunits. We propose that improperly folded filaments interact with dyes on
the pH indicator plates giving rise to the ‘green’ phenotype, and are more easily broken when
subjected to higher viscous forces on the flagellar filament during swarming motility.
73

BLAST X Poster #23
THE Cyclic-di-GMP RECEPTOR PROTEIN YcgR LOCALIZES TO THE FLAGELLAR BASAL
BODY AND CHANGES MOTOR BIAS IN SALMONELLA
Vincent Nieto* and Rasika Harshey
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Texas 78712
Cyclic-di-GMP (cdG) is a bacterial second messenger that plays a central role in the
transition between motile and sessile states. In Salmonella enterica, absence of the cdG
phosphodiesterase YhjH is known to elevate cdG levels, inhibit motility, and promote biofilm
formation. YcgR, a cdG -binding protein, is required for this phenomenon. We show in this study
that the absence of YhjH inhibits chemotaxis, and that this inhibition is more pronounced when
YcgR is overexpressed from a plasmid. In liquid media, the bacteria were predominantly smooth
swimming. Tethering experiments showed a pronounced shift to CCW rotation with a significant
slowing of motor rotation. Inhibition of chemotaxis was therefore at the level of either production,
or activity of the chemotaxis response regulator CheY~P, which acts at the switch to change the
default CCW bias to CW. Expression of a GFP-tagged cdG-binding protein YcgR in a yhjH
mutant background, resulted in punctate fluorescent dots along the cell membrane. Presence of
the puncta was dependent on the presence of flagellar basal bodies, but not on presence of
chemotaxis signaling components, suggesting that YcgR inhibits chemotaxis by acting at the
switch. Inhibition of chemotaxis may represent a novel strategy to prepare for a sedentary
existence by disfavoring migration away from a substrate on which a biofilm is to be formed.
74

BLAST X Poster #24
ANALYSIS OF THE PEPTIDOGLYCAN-BINDING DOMAIN OF THE FLAGELLAR STATOR
PROTEIN MotB USING SYSTEMATIC MUTAGENESIS AND CHIMERIC PROTEIN IN
ESCHERICHIA COLI
Yohei Hizukuri 1 , John Frederick Morton 1 , Toshiharu Yakushi 1, 2 , Seiji Kojima 1 and Michio
Homma 1
1
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-Cho,
Chikusa-Ku, Nagoya 464-8602, Japan
2
Present address: Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi
University, Yamaguchi 753-8515, Japan
The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought
to be anchored to the peptidoglycan (PG) layer by the C-terminal conserved peptidoglycanbinding
(PGB) motif of MotB. To clarify the role of the C-terminal PGB region of MotB, we
performed systematic cysteine mutagenesis of this region in the Escherichia coli MotB.
Furthermore, we constructed three chimeric MotB proteins whose PGB regions were replaced
with corresponding regions of other PGB proteins, E. coli Pal (peptidoglycan-associated
lipoprotein), PomB (Vibrio MotB homolog) or MotY (Vibrio flagellar T-ring protein). Although
these chimeric proteins did not complement the motB mutant, we were able to isolate two
independent motile revertants from cells producing the MotB-Pal chimera. One is the F172V
mutation in the Pal region of the chimera, and this mutation did not affect the ability to bind to
PG when introduced into native Pal. The other is the P159L mutation in the MotB region, and
Pro159 mutation in native MotB has been reported to affect a spatial positioning of the
MotA/MotB stator complex relative to the motor switch complex. We speculated that the PGB
region of MotB-Pal chimera was properly aligned by the mutations and the stator and rotor could
interact properly. We tried to interpret phenotype of MotB Cys mutants by using the crystal
structure of the E. coli Pal, and found that MotB mutants that affected motility were nearly
overlapped with the predicted PG-binding residues of Pal. Our results indicate that the PGB
regions from functionally distinct proteins, MotB and Pal, works similarly to anchor the stator
complex.
75

BLAST X Poster #25
ATTEMPT TO PURIFY THE HOOK-BASAL BODY WITH C-RING FROM THE Na + -DRIVEN
FLAGELLAR MOTOR
Masafumi Koike, Seiji Kojima, Michio Homma
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan.
The flagellum is the biggest architecture in the bacterial organ. At the base of each
flagellum, there is a rotary motor embedded in the membrane whose energy source is the
electrochemical gradient of the specific ion across the membrane. E. coli and Salmonella have
the H + driven motor, and Vibrio alginolyticus has Na + driven type. The motor is composed of the
rotor and the stator, and torque is generated by the interactions between them. The hook-basal
body (HBB) is the rotor part of the motor composed of rod, LP-, MS-ring and hook. C-ring is
mounted on the cytoplasmic side of the MS-ring of HBB, and believed to be involved in torque
generation in rotor side of the motor. It is composed of three proteins; FliM, FliN, and FliG. FliG
is the most closely participated in the torque generation among them. To elucidate the
mechanism of torque generation, we undertake the isolation of C-ring from the Na + -driven polar
flagellum of V. alginolyticus to investigate rotor-stator interactions. We applied the C-ring
isolation method established for Salmonella, but that resulted in the HBB without C-ring. It
indicates that solubilization by Triton X-100 was harsh for C-ring of Vibrio. Suitable detergents
were screened and we found that CHAPS could solubilize HBB with FliG attached. Currently
immunoelectron microscopic observation is ongoing to directly detect FliG attached on the MSring.
Also, we are searching more favorable conditions to isolate remaining C-ring proteins, FliM
and FliN.
76

BLAST X Poster #26
MECHANISM FOR SORTASE LOCALIZATION AND ROLE IN EFFICIENT PILUS ASSEMBLY
IN ENTEROCOCCUS FAECALIS
Kimberly A. Kline*† Andrew L. Kau*, Swaine L. Chen*, Birgitta Henriques-Normark†, Staffan
Normark†, Michael G. Caparon* and Scott J. Hultgren*
*Department of Molecular Microbiology, Washington University School of Medicine, St. Louis,
Missouri 63110; †Department of Microbiology,Tumor and Cell Biology, Karolinska Institutet,
Solna, Sweden
Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec)
pathway for the translocation and secretion of virulence factors out of the cell. Since many of
these secreted Gram positive virulence factors are subsequently attached to the bacterial cell
surface via sortase enzymes, we sought to investigate the spatial relationship between
secretion and cell wall attachment in Enterococcus faecalis. We discovered that Sortase A
(SrtA) and Sortase C (SrtC) are co-localized with SecA at single foci in enterococcus. The SrtAprocessed
substrate aggregation substance accumulated in single foci implying a single site of
secretion for these proteins. Furthermore, we show by electron and immunofluorescent
microscopy and immunoblot analysis that in the absence of the pilus polymerizing SrtC, pilin
subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were
found to share a positively charged domain flanking a transmembrane helix. Mutation or
deletion of this domain in SrtC abolished both its retention at single foci and its function in
efficient pilus assembly. We conclude that a positively charged domain adjacent to a
transmembrane helix can act as a localization retention signal for the focal
compartmentalization of membrane proteins. Finally, we have examined the localization of the
secretion and sorting machinery and substrates throughout the bacterial cell cycle and present a
model for spatial and temporal organization of the molecular components leading to efficient
pilus biogenesis in E. faecalis.
77

BLAST X Poster #27
VISUALIZATION OF EXCHANGE OF ROTOR COMPONENT IN FUNCTIONING BACTERIAL
FLAGELLAR MOTOR
Hajime Fukuoka, Shun Terasawa, Yuichi Inoue, Akihiko Ishijima
IMRAM, Tohoku Univ., 2-1-1 Katahira, Aoba-ku, Sendai, 980-5877, Japan
Bacterial flagellum is a supramolecular complex penetrating the bacterial cell envelope,
including the cytoplasmic and the outer membranes. Bacterial flagellum consists of a basal body
(rotary motor), a helical filament (propeller), and a hook (universal joint). The flagellar motor is
driven by the electrochemical potential of H +
or Na + , and the interaction between stator and a
rotor of flagellar motor is thought to generate the motor rotation. Stator parts are exchanged in a
functioning motor and the assembly of stator to the motor requires coupling ion for the motor
rotation. In this study, we focused on protein dynamics of rotor in functioning bacterial flagellar
motors. We constructed GFP-fusions of rotor components, and investigated whether rotor
components are exchanged in a functioning motor by Fluorescent Recovery After
Photobleaching (FRAP) experiment for a single motor.
We constructed the expression systems of GFP-FliG, FliM-GFP, and GFP-FliN as GFPfusions
of rotor components. In tethered cells producing each GFP-fusions, we observed the
localization of fluorescent spot at the rotational center. Each GFP fusion was probably
incorporated into flagellar motor as a rotor component. To observe the exchange of rotor
components, we carried out FRAP experiment using evanescent illumination to the motor
located at rotational center of the tethered cell. When fluorescent spot of FliM-GFP or GFP-FliN
localized at rotational center was photobleached, the fluorescence at the rotational center
recovered with the passage of time. On the other hand, the recovery of fluorescence was not
observed in cells producing GFP-FliG. These results indicate that some rotor components, FliN
and FliM at least, assemble to the motor even after the functional motor is constructed.
Probably, in functioning motor, some components of flagellar structure are exchanged
dynamically. We would like to clarify turnover rates of rotor components until the annual
meeting.
78

BLAST X Poster #28
TORQUE RESPONSE OF THE SODIUM-DRIVEN CHIMERIC FLAGELLAR MOTOR IN
E.COLI INDUCED BY REVERSIBLE TEMPERATURE CHANGE
Yuichi Inoue (1), Kuniaki Takeda (2), Hajime Fukuoka (1), Hiroto Takahashi (1) and Akihiko
Ishijima (1).
1: IMRAM, Tohoku University; 2: Graduate School of Life Sciences, Tohoku University, Katahira
2-1-1, Aoba-ku, Sendai 980-8577, Japan
Mechanical step of motor proteins is a key to understand the molecular mechanism of
energy transduction from chemical energy into mechanical work. We have demonstrated the 26
steps in a rotation of Na + -driven chimeric flagellar motor (Sowa et al., 2005) and the torquespeed
relationship (Inoue et al., 2008) using back-focal-plane interferometry. However, present
time resolution is not enough to understand molecular mechanism of the steps of motor proteins
including not only flagellar motors but also linear motors as skeletal myosin. One possible option
would be to slow down the temperature-dependent processes in a chemo-mechanical cycle by
reducing temperature.
We report the cooling experiment with a water-cooling chip in a simple and reversible
way. A small chip of ~18mm*18mm*2mm was made with glass cover slides and plastic tubes to
lead cooling water. This chip was made direct contact on a sample chamber and placed in the
light axis of the microscope. By changing temperature of the cooling flow, sample temperature
from 23 to -8 degree Celsius was monitored using thermocouple. Temperature change could be
applied repeatedly in a time constant < 60 sec.
Simultaneous measurement of the motor speed and sample temperature showed the
speed change as reported for low temperature (Chen and Berg, 2000). With increasing
temperature with hot water, however, sudden drop of speed was measured over ~40 deg C.
When the temperature returned back to room temperature, the speed was restored mostly in
several minutes. The drop and recovery of the speed were coincided with stepwise change in
the generated torque.
These results suggest that our cooling system is useful not only for the cooling
experiment for improving time resolution but also for the heating experiment to understand heat
resistance which might be related to stator dynamics.
79

BLAST X Poster #29
THE HELICOBACTER PYLORI FLAGELLAR ANTI-SIGMA FACTOR FlgM REMAINS
BACTERIA-ASSOCIATED AND INTERACTS WITH FlhAC
Melanie Rust 1 , Sophie Borchert 1 , Eugenia Gripp 1 , Sarah Kühne 1 , Eike Niehus 1 , Sebastian
Suerbaum 1 , Kelly T. Hughes 2 , Christine Josenhans 1
1 Department for Med. Microbiology and Hospital Epidemiology, Hannover Medical School ,Carl-
Neuberg-Strasse 1, D-30625 Hannover, Germany, 2 Department of Biology, University of Utah,
USA
Helicobacter pylori persistently colonize in the human gastric mucus with the help of their
polar flagellar organelles. A particular feature of flagella in most Helicobacter species including
H. pylori is the presence of a membraneous flagellar sheath. Previously, flagellar regulators
FliA, RpoN and the functional master regulator FlhA were characterized. H. pylori also
possesses an anti-sigma factor FlgM, which has an unusually short N-terminus, yet is functional
in Salmonella. FlgM in H. pylori fulfills a similar conserved function as an antagonist and binding
partner to FliA. However, FlgM of H. pylori is unusual, since it lacks an N-terminal domain
present in other FlgM homologs, e.g. FlgM of Salmonella, whose function is intimately coupled
to its secretion through the flagellar type III secretion system.
The aim of the present study was to characterize in more detail the localization and
potential for secretion of the short H. pylori FlgM in the presence of a flagellar sheath.
Furthermore we investigated its interaction with other flagellar proteins in the basal body, which
may be required for its function in flagellar regulation. FlgM was expressed in flhA mutants and
was differentially localized in bacterial fractions of flhA mutant bacteria in comparison to wild
type bacteria. H. pylori FlgM was only released into the medium in very minor amounts in wild
type bacteria, where the bulk amount of the protein was retained in the cytoplasm, and some
FlgM was detected in the flagellar fraction. FlgM-GFP (green fluorescent protein) and FlgM-V5
translational fusions were generated and expressed in H. pylori. FlgM displayed a
predominantly polar distribution in microscopy. Evidence was gathered that it is able to interact
with the C-terminal domain of the flagellar basal body protein FlhA. We conclude that in H. pylori
and possibly in other closely related bacteria, which also possess a truncated FlgM, secretion
may not be paramount for the regulatory function of FlgM and that protein interactions at the
flagellar basal body, in particular with FlhAC, may determine the turnover and localization of
functional FlgM in H. pylori.
We gratefully acknowledge the German Research Council, grant Jo344/2-2, for financial
support.
80

BLAST X Poster #30
LOCALIZATION PATTERNS OF THE HISTIDINE KINASES IN AN ESCHERICHIA COLI CELL
Takehiko Inaba 1 , Satomi Banno 2 , Hiroyuki Sawaki 2 , Akiko Yamakawa 2 , Masayuki Yoshimoto 3
Michio Homma 3 and Ikuro Kawagishi 1,4
1: Res. Cen. Micro-Nano Tech., Hosei Univ.; 2: Natl. Inst. Infect. Dis.; 3: Div. of Biol. Science,
Nagoya Univ., 4: Dept. Frontier Biosci., Hosei Univ.
E.coli has 30 histidine kinases (HKs). In response to a specific environmental stimulus,
each HK controls the activity of the downstream response regulator (RR) by modulating its
activities of autophosphorylation on the specific His residue and transfer of the phosphoryl
group to the specific Asp residue of RR (Generally referred to as the "two-component"
regulatory system or the His-Asp phosphorelay system). The activated form of RR regulates
specific cell functions (in most cases, gene expression). Almost all HKs resemble MCPs
(methyl-accepting chemotaxis proteins) in that they have two transmembrane regions and form
homodimers. CheA and NtrB are cytosolic proteins. CheA forms a chemosensory complex with
the trasmembrane chemoreceptors (MCPs) and the adaptor CheW. This complex localizes to
the cell pole and forms a huge cluster, which plays a critical role in signal amplification.
Do the other HKs also localize to particular regions of the membrane? To answer this
question, we constructed HK-GFP fusion proteins and observed the localization via
fluorescence microscopy. As mentioned above, cytosolic CheA-GFP localized to the cell pole,
whereas the other cytosolic HK NtrB-GFP was diffused throughout the cytoplasm. Although
many of the transmembrane HK-GFPs were diffused evenly in the cytoplasmic membrane,
some HK-GFPs showed characteristic localization patterns. In particular, the GFP fusions to
the anaerobic sensors TorS and ArcB localized to the cell pole. Most of HKs that localized to
the pole are of hybrid type, i.e. they have receiver and HPt domains, which are involved in multistep
phophorelay. A series of deletion from TorS-GFP revealed that none of these domains nor
the transmitter domain was required for polar localization. Nevertheless, polar localization of
such hybrid HKs might provide a basis of signal integration and crosstalk.
81

BLAST X Poster #31
THERMOSENSING FUNCTION OF Aer, A REDOX SENSOR OF E. COLI
So-ichiro Nishiyama 1 , Shinji Ohno 2 , Noriko Ohta 3 , Akihiko Ishijima 4 1, 5
and Ikuro Kawagishi
1
Department of Frontier Bioscience, Faculty of Bioscience and Applied Chemistry, Hosei
University
2
Department of Material Chemistry, Faculty of Engineering, Hosei University
3
Division of Biological Science, Graduate School of Science, Nagoya University
4
Institute of Multidisciplinary, Research for Advanced Materials, Tohoku University
5
Department of Frontier Bioscience, Faculty of Engineering, Hosei University
Some motile bacteria can sense temperature and move to temperatures best-suited to
growth. This behavior, called thermotaxis, has been extensively studied in Escherichia coli.
Our early studies revealed that E. coli thermotaxis is mediated by chemoreceptors that also
sense amino acids or sugars: Tsr (serine), Tar (aspartate and maltose) and Trg (ribose and
galactose) function as warm sensors, producing counter-clockwise or clockwise flagellar rotation
signals upon temperature upshift and downshift, respectively. Tap (dipeptides, pyrimidines)
functions as a cold sensor, producing CW and CCW signals upon temperature increases and
decreases, respectively. Unique among these temperature sensors, Tar switches from a warm
sensor to a cold sensor after adaptation to its ligand, aspartate. Intensive studies of Tar
revealed that the receptor’s transmembrane and methylation domains play important roles in
thermotactic responses, but what part of the chemoreceptor molecule actually senses
temperature, remains unknown.
In this study, we found that the aerotaxis transducer Aer also has temperature-sensing
ability. An otherwise receptor-less strain expressing only aer showed extremely smooth
swimming and did not show any thermoresponse. However, after imposing a CW rotational
bias by adding the general repellent, glycerol (up to 10% w/v) or by co-expressing a cytoplasmic
fragment of Tar that does not mediate a thermoresponse, the cells showed CCW and CW
responses to temperature upshifts and downshifts, respectively. These results suggest that Aer
functions as a warm sensor, even though, unlike the Tar, Tap, Tsr, and Trg chemoreceptors,
Aer does not have a periplasmic ligand-binding domain. Thus, temperature sensing by E. coli
chemoreceptors may be a general attribute of their highly-conserved cytoplasmic signaling
domain (or their less conserved transmembrane domain).
82

BLAST X Poster #32
ATPase ACTIVITY OF T3SS SPECIFIC ATPase InvC
Fumio Hayashi, Eri Inobe, Kenji Oosawa
Department of Chemistry and Chemical Biology, Graduate school of Engineering, Gunma
University, 1-5-1 Tenjin, Kiryu, Gunma, 376-8515, Japan
The type III secretion systems (T3SSs) are widely used by Gram-nagative bacteria, and
there are essential systems of many bacterial pathogenic to humans, animals, and plants. The
systems are anchored to the bacterial envelope by a multi-ring, and a needle-like extracellular
structure facilitates the translocation of the virulence proteins (effectors) to a host cell from the
bacterial cytosol. An ATPase that is believed to be in close association with the basal body is
involved in the protein translocation.
The specific ATPase of T3SSs in Salmonella enterica serovar Typhimurium is InvC. We
purified InvC, determined the low kcat value of InvC-ATPase activity and the high Km value for
ATP, and found 2~3-fold stimulation of InvC-ATPase activity in the presence of phospholipid.
To examine the possibility that InvC-ATPase activity is further stimulated by the interaction with
an effector or an effecter-specific chaperon, we purified SicP (chaperon) and SptP (effector) and
measured InvC-ATPase activity in the presence of SicP or SptP. SicP- or SptP-dependent
stimulation of InvC-ATPase activity was not detected yet.
83

BLAST X Poster #33
CHARACTERIZATIONS OF THE PSEUDOREVERTANTS FROM SALMONELLA
TYPHIMURIUM STRAIN SJW1655 AND SJW1660 WITH THE R- AND THE L-TYPE
STRAIGHT FLAGELLAR FILAMENTS
Hidetoshi Tomaru, Fumio Hayashi, Eiji Furukawa, Shigeru Yamaguchi & Kenji Oosawa
Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma
University, 1-5-1 Tenjin, Kiryu, Gunma 376-8515, JAPAN
A cell of Salmonella typhimurium swims by rotating its flagellar filaments. A wild-type
cell carries a left-handed helical filament that is called a normal filament. On the other hand,
various mutants carrying different helical shapes were isolated. There are curly, coiled, semicoiled
and two kinds, L- and R-types, of straight filaments known. Transformations of filament
helical shape among normal, semi-coiled and curly were observed during tumbling of the cell.
To investigate the transformation mechanism of flagellar filaments, we isolated 95 revertants,
which recovered their swarming abilities, from Salmonella typhimurium strain SJW1655 with the
R-type straight flagellar filament. The numbers of the pseudorevertants carrying another
mutation site were 64 and the second mutation sites were 4. Similarly, we isolated 106
revertants (including 101 pseudorevertants) from SJW1660 (the L-type straight flagellar
filament). The numbers of the second mutation sites were 19. We also measured the swimming
speeds of the pseudorevertants and observed the flagellar shapes isolated from the
pseudorevertants. We will discuss, in this meeting, the differences in the distribution of the
second mutation sites, the swimming speeds, and the flagellar shapes of the pseudorevertants
from between SJW1655 and SJW1660.
84

BLAST X Poster #34
RAMAN OPTICAL ACTIVITY AND VIBRATIONAL CIRCULAR DICHROISM OF FLAGELLAR
FILAMENTS OF SALMONELLA
Tomonori Uchiyama 1 , Fumio Hayashi 1 , Masashi Sonoyama 2 , Yoshiaki Hamada 3 , Rina K.
Dukor 4 , Laurence A. Nafie 4, 5 , Kenji Oosawa 1 (kenji@nms.gunma-u.ac.jp)
1
Department of Nano-Material Systems, Gunma University, Kiryu, Japan
2
Department of Applied Physics, Nagoya University, Nagoya, Japan
3
The Open University of Japan, Chiba, Japan
4
BioTools Inc., Jupiter, FL 33458, USA
5
Department of Chemistry, Syracuse University, Syracuse, NY 13244-4100, USA
The flagellar filament of Salmonella is an assembly of a single protein, flagellin. There
are different helical and straight shape filaments from wild type and mutants. The difference
between two types of straight filaments, L- and R-type straight, is the inclination of the flagellin
monomer arrangements. The structures of the straight filaments were analyzed by X-ray
diffraction and electron microscope, while it is difficult to analyze the structure of helical flagellar
filaments by these methods, due to deficiency in the symmetric property of the molecular
assembly. On the other hand, Raman optical activity (ROA) and vibrational circular dichroism
(VCD) spectroscopies are available new techniques for studying structure and dynamics of
chiral molecules and the solution structure of biomacromolecules. In the present study, these
methods are employed for investigating the structural characteristics of bacterial flagellar
filaments.
In the ROA spectra, intensive peaks were observed only from the L-type filaments, while
no significant signals were observed from flagellin monomer and other shapes of the filaments
(R-type straight and normal helical) in our measurement conditions. The intensive signals
disappeared or were weakened when the L-type straight filaments were shortened.
In the VCD spectra, intensive peaks in the amide I region were observed from the L-type
filaments. Whereas peaks observed from the R-type filaments were different from those from Ltype
and only weak peaks were observed in the normal filaments and flagellin monomer.
From these results, it is thought that these spectra reflect the differences of structure and the
physicochemical properties of flagellin subunits in three types of filaments.
85

BLAST X Poster #35
Poster Cancelled
86

BLAST X Poster #36
CrdC NEGATIVELY REGULATES CheW3 AND CheA3 INTERACTION DURING SIGNAL
TRANSDUCTION IN MYXOCOCCUS XANTHUS
Jonathan Willett, Susanne Mueller, John Kirby
University of Iowa, Department of Microbiology, 3-403 Bowen Science Building
51 Newton Road, Iowa City, IA 52242
Previous work on the Che3 system of Myxococcus xanthus has led to the discovery that
a chemosensory signal-transduction system affects developmentally regulated gene expression.
The Che3 system contains homologs of the prototypical chemotaxis proteins such as CheA,
CheW, CheB, CheR, and MCPs but lacks a CheY homolog. The output of the system consists
of a NtrC transcriptional activator termed CrdA. There are several other unique proteins
comprising the Che3 pathway besides CrdA, with one of the more interesting being a protein
termed CrdC. CrdC is contained in the same transcriptional unit as CheW3. Preliminary data
has shown that CrdC interacts strongly CheW3 in yeast-two hybrid experiments. More
interestingly, the presence of CrdC in a yeast-three hybrid experiment has been shown to inhibit
the interaction between CheA3 and CheW3. We hypothesize that CrdC thereby presents a
unique mechanism for regulation of signal transduction through the Che3 pathway by
decoupling CheA3 and CheW3.
87

BLAST X Poster #37
MOLECULAR ARCHITECTURE OF INTACT FLAGELLAR MOTOR REVEALED BY CRYO-
ELECTRON TOMOGRAPHY
Jun Liu 1 , Tao Lin 1 , Douglas J. Botkin 1 , Erin McCrum 1 , Hanspeter Winkler 2 , Steven J. Norris 1
1
Department of Pathology and Laboratory Medicine, University of Texas Medical School at
Houston, Houston, TX 77225-0708, USA.
2
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306-4380, USA
Motility is often important for virulence of bacterial pathogens, and the flagellum is the
main organelle for motility in bacteria. Bacterial flagella are helical propellers turned by the
flagellar motor, a remarkable nano-machine embedded in the bacterial cell envelope. Powered
by the proton gradient across the cytoplasmic membrane, the motor converts electrochemical
energy into torque through an interaction between a rotating, cylindrical basal body at the end of
the flagellar filament and the stator, a surrounding protein assembly embedded in the
cytoplasmic membrane. Of the 50 genes needed to build a functional flagellum, at least 25
produce proteins essential for flagellar assembly. Although structural studies have revealed the
stunning complexity of the basal body, flagellar assembly and rotation remain poorly understood
at the molecular level, mainly because of the lack of structural information about the membranebound
stators and the torque-generating mechanism in particular. Here, we present the
structures of infectious wild-type and mutant Borrelia burgdoferi organisms and their flagella
motors in situ using high throughput Cryo-Electron Tomography (Cryo-ET). By averaging the 3-
D images of ~1280 flagellar motors, we obtained a ~3 nm resolution model of the combined
stator and rotor structure in its cellular environment. We have also been able to identify
distinctive structural changes resulting from the mutation of a flagellar gene. This is direct
mapping of a single genetic code change into the 3-D structure of a functioning molecular
machine in situ. Our results provide new insights into the motor structure and the molecular
basis for bacterial motility.
88

BLAST X Poster #38
MINING THE E. COLI GFP FUSION COLLECTION
Jason Dobkowski, Aleksandra Sikora, John Brooks, Richard Zielke and Janine Maddock
Department of MCDB, University of Michigan, 830 N. University, Ann Arbor, MI 48109
Use of green fluorescent protein (GFP) has greatly increased the ability to visualize
protein localization within bacterial cells. A library of GFP fusions, expressed from an inducible
promoter, was created with each protein from every open reading frame in the Escherichia coli
genome (Kitagawa et al, 2005). The addition of the 26.9 kDa GFP, however, can lead to
protein misfolding and aggregation and the formation of polar inclusion bodies. These polar
inclusion bodies are often mistaken for bona fide polar localization of the protein fusion. Using a
purification and screening approach, we have sorted polarly localized GFP fusions into those
that form inclusion bodies and those that are likely real polar proteins. Those that form inclusion
bodies are being used to identify interacting partners that co-aggregate with the misfolded
protein. Proteins not previously not known to be polar are being verified using
immunofluorescence microscopy and the localization of known interacting partners determined.
We are particularly interested in the spatial organization of membrane bound histidine kinases
involved in sensing environmental stress. Our current line of focus is to determine whether the
activation of the signal transduction cascade has any influence of their polar localization.
89

BLAST X Poster #39
UNDERSTANDING THE FUNDAMENTAL ELEMENTS OF SIGNALING IN THE Tar
CHEMORECEPTOR
Christopher Adase, Michael Manson
Texas A&M University, 3258 TAMU, BSBE Room 303
The Tar chemoreceptor of Escherichia coli has two different attractants: L-aspartate,
which binds directly to the receptor; and maltose, which interacts with the receptor indirectly
though maltose-binding protein (MBP). Tar also senses Ni 2+ and Co 2+ as repellents. The Tsr
chemoreceptor interacts directly with L-serine as an attractant and also senses repellents such
as indole, acetate, and benzoate in an unknown manner. Tar-Tsr and Tsr-Tar chimeras have
been created using the endogenous Nde1 site found at the end of the region encoding their
respective HAMP domains. The Tar-Tsr hybrid could sense Ni 2+ as a repellent, whereas the Tsr-
Tar hybrid could not. Thus, Ni 2+ probably interacts, directly or indirectly, with an area within the
first 256 amino acids of Tar. Using knockouts of nikA, nikB, and nikC, we tested whether
periplasmic NikA is necessary and sufficient for Ni 2+ taxis, as has been reported, and whether
Ni 2+ must be taken up into the cytoplasm. I have also created additional Tar-Tsr chimeras to
determine exactly what portion of Tar is involved in Ni 2+ sensing. Repellent taxis was assayed
using novel microfluidic techniques developed by the Jayaraman lab in the Department of
Chemical Engineering at Texas A&M.
90

BLAST X Poster #40
LINKING THE TM2 TO HAMP—A TOUGH NUT TO CRACK?
Rachel L. Crowder, Gus A. Wright, and Michael D. Manson
Department of Biology, Texas A&M University, College Station, TX 77843
The HAMP domain is a widely conserved motif found in transmembrane-signaling
proteins in prokaryotes and lower eukaryotes. It consists of a pair of amphipathic helices joined
by a flexible linker. Recently, the solution structure of the Archeoglobus fulgidis Af1503 HAMP
domain was determined using NMR (Hulko et al, Cell 126: 929-940, 2006). The domain forms a
parallel four-helix bundle that packs in a non-canonical knob-on-knob conformation. Several
models have been proposed to explain how the four helix bundle transmits the downward piston
movement of transmembrane 2 (TM2) of E. coli chemoreceptors into the signaling domain to
inhibit CheA kinase activity. The connection between TM2 and the HAMP domain is likely to be
important for transducing the input signal from TM2 to HAMP. We hypothesized that increasing
the flexibility of the connector should attenuate the output signal. To test this idea, residues
between Met-215 Thr-218 of the E. coli aspartate receptor Tar were replaced with four Gly
residues. Gly residues were then deleted (-4G through -1G) and added (+1G through +5G).
Aspartate chemotaxis, rotational bias of tethered cells, mean reversal frequencies of tethered
cells, and in vivo methylation levels were measured. These experiments suggest that increasing
flexibility between TM2 and HAMP strongly biases the receptor to the “off” (CCW-signaling)
state and decreases aspartate mediated signal transmission between TM2 and HAMP.
91

BLAST X Poster #41
ELECTROSTATIC EFFECTS ON SIGNALING MUTATIONS IN THE C-TERMINAL REGION
OF THE ESCHERICHIA COLI ASPARTATE CHEMORECEPTOR
Andrew L. Seely, Run-Zhi Lai, and Michael D. Manson, Department of Biology, Texas A&M
University, College Station, TX 77843
The Escherichia coli aspartate chemoreceptor (Tar) responds to attractant by modulating
the rotational bias of the flagellar motor. Previous studies measured the effects on Tar signaling
when positive residues in its extreme C-terminal region were either neutralized or changed to
negative residues. While this region is important as a linker to the NWETF pentapeptide where
methylation and demethylation enzymes bind, it also affects transmembrane signaling. An
R505A substitution decreased the receptor’s ability to respond to aspartate by 40%, and an
R505E charge reversal completely abolished stimulation by aspartate. It was also noted that
R505E may interact with D273 to destabilize the “on” signaling state (Lai, et al, Advanced
Publication in Biochemistry, 2008). We hypothesize that secondary mutations within the
background of the R505 mutations, and an additional R514A/E mutant, can potentially rescue or
exacerbate the ability of mutated Tar to respond to aspartate. Specific mutations will include
D273N/R, D263R, R228A/E, R505D and R509E. We will test our hypothesis in vivo through
chemotaxis swarm assays, receptor methylation assays, and tethered cell assays. Our
hypothesis will also be tested in vitro through CheA-Kinase assays.
92

BLAST X Poster #42
INTERACTION OF THE TRANSCRIPTIONAL REGULATORY COMPLEX, FlhDC, WITH ITS
TARGET DNA
Yi-Ying Lee, and Philip Matsumura
Department of Microbiology and Immunology, College of Medicine, University of Illinois at
Chicago, 835 S. Wolcott Ave., M/ C 790, Chicago, Illinois 60612-7344
The bacterial flagellum is the structure that allows bacteria to move and respond to
nutritional and chemical signals in their environment. It is a complex suborganelle and the
transcriptional regulation of the 40 plus structural genes is organized in a highly regulated
cascade. At the top of the hierarchy is the master operon which codes for FlhD and FlhC. These
two positive transcriptional regulators form a unique heteroheximeric complex which binds
upstream of the -35 region and requires sigma 70 for transcription. This complex has an
unusually large ‘footprint’ of 48 base pair and bends the DNA 110 degrees. We have proposed
that the DNA bind on the circumference of this toroid shaped FlhDC complex. Although we have
determined the sequence 3 footprints on FlhDC regulated promoters, it is not possible to
determine a consensus binding site in these 3 sequences. In this study, we have determined
which bases are important for DNA binding and activity for FlhDC regulated promoter activity.
First, we have divided the FlhDC footprint in the fliA promoter into five segments and found that
two of the segments or 40% of the footprint were not required for binding. The remaining 30
base pairs were divided into 3-5 base segments and randomly mutagenized and screened for
the ability to bind and activate the fliA promoter. Analysis of these data suggests a consensus of
12 A, 15 A, 34 T, 36 A, 37 T, 44 A, 45 T in FlhD4C2 footprint fragment were important for activity. Five of
these bases demonstrated high specificity. Finally, this consensus was tested and found to be
important in other FlhDC regulated promoter regions.
93

BLAST X Poster #43
APPLICATION OF BIOLAYER INTERFEROMETRY TO UNDERSTANDING INTERACTIONS
AMONG SALMONELLA ENTERICA FLAGELLAR EXPORT APPARATUS PROTEINS
Jonathan L. McMurry
Department of Chemistry & Biochemistry, Kennesaw State University, Kennesaw, GA
Biolayer interferometry (BLI) is an emerging optical biosensing technology for the
analysis of dynamic biomolecular interactions. Measurements are made of changes in the
interference pattern of white light reflected off of two surfaces, one of which possesses a layer
of immobilized protein (or other molecule) and the other an internal reference. Binding of
second protein to the immobilized one results in a change in distance between the two surfaces
and thus a shift in wavelength of the interference pattern. Kinetic and affinity constants can thus
be determined in real-time, without attainment of equilibrium, utilizing small, recoverable
quantities of label-free biomolecules. Using a commercial BLI biosensor, interactions among
proteins involved in flagellar export in Salmonella enterica were investigated. Among
interactions measured were those between two proteins involved in specificity switching, FliK
and FlhB. FliK binding to wild-type FlhB and two variants (N269A and P270A) surprisingly
evidenced similar kinetic profiles with submicromolar KDs resulting from fast-on and fast-off rate
constants. Other interactions between export proteins, e.g., FliH-FliI, FliH-FliJ, demonstrated
stable, low kd binding, as expected. Additional efforts include screening for interactions too
weak to allow for copurification or other traditional binding assays, attempts to demonstrate
interactions between export proteins and substrates and analysis of more-than-pairwise
interactions. Further application of BLI to the investigation of flagellar export protein dynamics
will allow for better understanding of the mechanism of export.
94

BLAST X Poster #44
A CROSS-SPECIES COMPARISION OF CHEMOTACTIC BEHAVIOR
Julie Simons and Paul Milewski
University of Wisconsin—Madison, Department of Mathematics, 480 Lincoln Dr., Madison, WI
53706
Understanding the population-level behavior of bacteria is of importance not only in
exploring how simple organisms can perform complex behavior, but also to be able to optimize
their potential for bioremediation and other uses. We are interested in modeling the
chemotactic behavior of Rhodobacter sphaeroides and the better-understood Escherichia coli
using a partial differential equation model known as the Keller-Segel model and experimental
data, with the aim of being able to make a cross-species comparison. Swarm-plate experiments
with uniform concentrations of the chemoattractant L-aspartate were performed for both bacteria
over several concentrations of aspartate. Separately, growth experiments in liquid cultures were
undertaken to quantify differences in aspartate concentration dependent growth between the
two species. From the data we are able to determine parameter values for a Keller-Segel model
and thus quantify differences between non-chemotactic diffusive behavior, growth effects, and
chemotactic behavior. This quantitative modeling work comparing population-level behavior of
these bacteria allows one to deduce metabolic function parameters in agar, which are not
possible to find experimentally and not incorporated in many previous models. We find that a
significant proportion of the E. coli wild-type population appears to be non-chemotactic whereas
the R. sphaeroides wild-type population appears to be primarily chemotactic, something not
explored in other studies. Our parameters also indicate a joint saturation of growth and
chemotaxis, which we postulate is a common evolutionary result. These findings provide a
platform from which to explore incorporating cell-level knowledge into macro-scale behavioral
models and the effects of heterogeneity of populations.
95

BLAST X Poster #45
TETHERED MYCOPLASMA
Daisuke Nakane and Makoto Miyata
Department of Biology, Graduate School of Science, Osaka City University
Mycoplasma mobile is a pathogenic flask-shaped bacterium 0.8 micron long. They bind
to solid surfaces by “legs” sticking out from the base of membrane protrusion, “neck”, and glide
by a unique mechanism. Recently, we proposed a working model, power stroke model, where
the cells are propelled by many legs repeatedly catching and releasing the solid surface, driven
by the energy of ATP hydrolysis. Here, to detect the movement of legs, we reduced the number
of working legs and amplified the leg movement.
When the cells were treated by 0.1% Tween 60, they were elongated from 0.8 micron to
2.0-4.0 micron in 15 min with the extension of cytoskeletal structures. A previous study showed
that the direct binding target of mycoplasma is a sialic acid, and the addition of free sialic acids
dissociated gliding cells from the solid surface. Here, in the presence of 0.25-1 mM of sialic
acid, the elongated cells pivoted widely, plausibly resulting from the reduction the leg number.
The pivoting ceased when strong light was applied in the presence of a fluorescence dye,
suggesting that the pivoting is caused by the gliding legs. Azide and the antibody against the
gliding machinery affected the distribution of pivoting angle differently, although both reduce the
gliding speed. On the basis of these results, the movements of legs will be discussed.
96

BLAST X Poster #46
MOLECULAR SHAPES OF Gli123 AND Gli521 INVOLVED IN GLIDING MOTILITY OF
MYCOPLASMA MOBILE
Takahiro Nonaka, Jun Adan-Kubo, Makoto Miyata
Department of Biology, Graduate School of Science, Osaka City University
3-3-138 Sugimoto Sumiyoshi-ku, Osaka-shi, JAPAN
Mycoplasma mobile has no flagella or pili, and its genome contains no genes related to
known bacterial motility. However, M. mobile binds to solid surfaces and glides smoothly and
continuously, with a unique mechanism. M. mobile cells form a membrane protrusion at the
leading pole. Three huge proteins, Gli123 (123 kDa), Gli349 (349 kDa), and Gli521 (521 kDa),
localize at neck, the base of protrusion, and form the gliding machinery. These proteins are
suggested to have the roles of scaffold for other gliding proteins, glass binding, and force
transmission, respectively. In this study, we purified Gli123 and Gli521 proteins from M. mobile
cells by biochemical procedures, rotary shadowed, and observed their molecular shapes by
transmission electron microscopy. The Gli123 molecule shaped asymmetrical oval, 31 nm long
and 16 nm wide. The Gli521 molecule consisted of three flexible arms about 130 nm long, each
of them had spherical part at the distal end. This shape is reminiscent of clathrin-triskelion
which is widely distributed in eukaryotic cells. Clathrin molecules self-assemble, control the
membrane shape and form a vesicle. The characteristic molecular shape of Gli521 may
suggest that this protein forms a two dimensional sheet like clathrin, and plays a critical role to
form the membrane protrusion in a M. mobile cell.
97

BLAST X Poster #47
THE ESSENTIAL NATURE OF THE WALK/WALR SIGNAL TRANSDUCTION PATHWAY IS
LINKED TO CELL WALL HYDROLASE ACTIVITY IN STAPHYLOCOCCUS AUREUS
Aurélia Delauné 1 , Olivier Poupel 1 , Adeline Mallet 2 , Sarah Dubrac 1 , and Tarek Msadek 1
Biology of Gram-positive Pathogens 1 , Department of Microbiology, Plateforme de Microscopie
Ultrastructurale 2 , Imagopole, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France
Staphylococcus aureus, a major Gram-positive human pathogen, is a leading cause of
both nosocomial and community infections due to its considerable capacity for adaptation. One
of the principal mechanisms involved in this process are the so-called two-component systems,
bacterial signal transduction pathways with a sensor histidine kinase that is autophosphorylated
in response to specific environmental stimuli and then transfers the phosphoryl group to its
cognate response regulator, which consequently regulates target gene expression. The WalKR
two-component system is well conserved and specific to low G+C Gram-positive bacteria,
including Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. This system
has been shown to be essential for cell viability.
We have recently demonstrated that the WalKR system positively controls autolytic
activity, and identified ten genes belonging to the WalKR regulon that appear to be involved in
S. aureus cell wall degradation. Reasoning that the essential nature of this signaling pathway
may be related to its role as a master regulatory system for cell wall metabolism, we tested
whether uncoupling autolysin gene expression from WalKR-dependent regulation could
compensate for the essential nature of the system. Several genes from the WalKR regulon were
expressed from a cadmium chloride-inducible promoter in a WalKR-independent manner.
Candidate genes were identified whose expression allowed cells to grow in the absence of
WalKR.
98

BLAST X Poster #48
THE GraS/GraR TWO-COMPONENT SYSTEM AND DERMASEPTIN RESISTANCE IN
STAPHYLOCOCCUS AUREUS
Mélanie Falord 1 , Pierre Joanne 2 , Chahrazade El Amri 2 , and Tarek Msadek 1
Biology of Gram-positive Pathogens 1 , Department of Microbiology, Institut Pasteur, 25 rue du
Dr. Roux, 75015 Paris, France
Peptidome de la peau des amphibiens 2 , FRE 2852, CNRS, Université Pierre et Marie Curie, 2
place Jussieu, 75251 Paris 75005, France
Staphylococcus aureus, a major Gram-positive human pathogen, is a leading cause of
both nosocomial and community infections due to its considerable capacity for adaptation.
S. aureus is able to resist Cationic Anti-Microbial Peptides (CAMPs) by increasing its positive
cell surface charges through D-alanylation of wall teichoic acids and lysylination of
phospholipids, leading to electrostatic repulsion of CAMPs. Synthesis of the major enzymes
involved in these mechanisms (DltA, MprF) is positively controlled by the GraS/GraR twocomponent
system. Two-component systems are bacterial signal transduction pathways with a
sensor histidine kinase that is autophosphorylated in response to specific environmental stimuli
and then transfers the phosphoryl group to its cognate response regulator which consequently
regulates target gene expression. In S. aureus, GraS is involved in CAMP sensing, promoting
bacterial resistance through GraR.
Here, we have shown that a ∆graRS mutant in S. aureus is sensitive to Colistin and
Dermaseptins. Moreover, we demonstrated that the graRS genes are part of a three-gene
operon also containing graX, a gene with unknown function but essential to the system, and
defined the operon transcriptional start site. To define the mechanism by which GraS, GraR and
GraX confer CAMP resistance to S. aureus, the three proteins were overexpressed and purified
to test their in vitro interactions. A potential GraR binding site upstream from the vraFG operon,
known to be controlled by GraS/GraR, was identified both by in silico analysis and lacZ
transcriptional fusion experiments.
99

BLAST X Poster #49
CHARACTERIZATION OF SUPPRESSORS OF THE MotB(D33E) MUTATION, A PUTATIVE
PROTON-BINDING RESIDUE OF THE BACTERIAL FLAGELLAR MOTOR
Yong-Suk Che, Shuichi Nakamura, Yusuke Morimoto, Nobunori Kami-ike, Keiichi Namba and
Tohru Minamino
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka
565-0871, Japan
MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which
conducts protons and couples proton flow to motor rotation. Asp-33 of Salmonella Typhimurium
MotB, which is a putative proton-binding site, is critical for torque generation. However, how
does the protonation of Asp could drive the conformational changes requiring for torque
generation is largely unknown.
Here, we carried out genetic and motility analysis of a slow motile motB(D33E) mutant
and its pseudorevertants. First, we confirmed that MotB(D33E) forms the complex with MotA in
the cytoplasmic membrane. Then, we isolated suppressor mutants from the motB(D33E) mutant
and identified the suppressor mutation sites. Next, we characterized the torque-speed
relationship of the flagellar motors of wild-type, motB(D33E) mutant and its suppressors by the
bead assays. As a result, we found that while the wild-type motor torque was almost constant
over a wide range of rotation rate, the MotB(D33E) mutation caused ≈40% reduction in nearstall
torque and a sharp decline in the torque-speed curve with an apparent maximal rotation
rate of ≈20 Hz. Furthermore, we also found that the second-site mutations could recover the
near-stall torque but not the sharp decline of torque-speed curve and the maximum rotation
rate. These results together suggested that MotB(D33E) mutation reduced both protonconducting
activity and torque generation step involving the stator-rotor interactions coupled
with protonation/deprotonation of Glu-33 and the second-site mutations could recover the torque
generation step but not the proton-conducting activity.
Recently, to measure the proton-conducting activity of the motB(D33E) mutant and its
pseudorevertants, we developed a novel system to monitor intracellular pH of cells
overexpressing MotA/MotB mutant proteins utilizing pH-sensitive GFP (pHluorin). Experimental
results will also be discussed.
100

BLAST X Poster #50
STRUCTURE OF FliJ, A CYTOPLASMIC COMPONENT OF THE FLAGELLAR TYPE III
PROTEIN EXPORT APPARATUS OF SALMONELLA
Tatsuya Ibuki 1, 2 , Masafumi Shimada 1, 2 , Tohru Minamino 1, 2 , Katsumi Imada 1, 2 and Keiichi
1, 2
Namba
Grad. Sch. of Frontier Biosci., Osaka Univ. 1 , Dynamic NanoMachine Project, ICORP, JST 2
The flagellum is a motile organelle composed of the basal body rings and the tubular
axial structure. The axial component proteins synthesized in the cytoplasm are transferred into
the central channel of the flagellum by the flagellar type III protein-export apparatus for selfassembly
at the growing end. The apparatus is composed of six transmembrane proteins (FlhA,
FlhB, FliO, FliP, FliQ, FliR) and three soluble components (FliH, FliI, FliJ). FliJ is an essential
component for protein export. Although FliJ is thought to be a general chaperone, its function is
still unclear. Here we report a crystal structure of FliJ. We obtained hexagonal bi-pyramid
crystals of FliJ with N-terminal extra three residues, and determined the structure at 2.1-Å
resolution using anomalous diffraction data from a mercury derivative collected at SPring-8
BL41XU.
FliJ consists of two α-helices, one is a 13-turn helix and the other a 21-turn helix, which
form a coiled-coil structure. FliJ has a remarkable structural similarity to the γ subunit of F0F1-
ATPsynthase. The other soluble components FliH and FliI are also known to have similarity to
other components of F0F1-ATPsynthase. The structure of FliI closely resembles those of the α/β
subunits, and the amino-acid sequence of FliH has two regions that are similar to those of the b
and δ subunits, respectively.
We will discuss details of the structure and possible functional mechanism of FliJ, and
similarity between the flagellar export apparatus and F0F1-ATPsynthase.
101

BLAST X Poster #51
CRYOEM STRUCTURE OF THE HOOK-FILAMENT JUNCTION OF SALMONELLA
Fumiaki Makino (1), Takayuki Kato (1), Keiichi Namba (1)
1: Grad. Sch. of Frontier Biosci., Osaka Univ.
The bacterial flagellum is a biological nanomachine for the locomotion of bacteria. The
flagellum consists of three functional parts: the basal body as a rotary motor, the filaments as a
helical propeller, and the hook as a universal joint that connects the above-mentioned two parts.
The filament is a tubular structure made of a single protein, FliC. It transforms into various
helical forms in response to mechanical perturbation by reversal of motor rotation. The hook is
also a tubular structure made of a single protein, FlgE, but is more flexible than the filament,
allowing it to transmit motor torque to the filament regardless of its orientation. The hookfilament
junction made of two proteins, FlgK and FlgL, connects these two mechanically distinct
structures. It works as a mechanical adapter, allowing the two parts to go through dynamic
conformational changes independently. To understand the adapter mechanism, we have
analyzed the structure of the hook-filament junction by electron cryomicroscopy (cryoEM). We
needed a Salmonella strain that produces short filaments for efficient data collection. We used a
strain, MMC1660, which produces short filament due to a significantly low efficiency of FliC
expression controlled by the addition of tetracycline. We established a procedure to purify the
hook-basal body with short filament, collected cryoEM images and carried out image analysis.
We will show the cryoEM structure of the hook-filament junction complex.
102

BLAST X Poster #52
EFFECT OF INTRACELLULAR pH ON THE TORQUE-SPEED RELATIONSHIP OF
BACTERIAL PROTON-DRIVEN FLAGELLAR MOTOR
Shuichi Nakamura 1,2 , Nobunori Kami-ike 2 , Jun-ichi Yokota 1,2 , Seishi Kudo 3 ,Tohru Minamino 1,2 ,
and Keiichi Namba 1,2
1 Graduate School of Frontier Bioscience, Osaka University 1-3 Yamadaoka, Suita, Osaka 565-
0871, Japan, 2 Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-
0871, Japan, 3 Faculty of Engineering, Toin University of Yokohama, 1614 Kurogane-cho, Aoba,
Yokohama 225-8502, Japan
The flagellar motor of Salmonella is a rotary nanomachine driven by proton motive force.
It has been shown that an increase in the intracellular proton concentration abolishes the motor
function, suggesting that the stator complex has intracellular proton-binding sites by which
intracellular protons kinetically interfere with the rate of proton translocation for the motor
rotation. In this study, to understand the coupling mechanism of proton flux with torque
generation, we have investigated the effect of intracellular pH on the rotation rate of the flagellar
motor. Intracellular pH was manipulated by adding potassium benzoate, which crosses the
cytoplasmic membrane in the neutral form and equilibrates the intra- and extracellular pH
without changing proton motive force. We showed that the rotation rates at low loads sharply
decreased as the external pH decreased in the presence of benzoate, suggesting that a high
intracellular proton concentration lowered the proton translocation rate. Also, computer
simulation by a simple kinetic model suggested that the decrease in intracellular pH interferes
with proton dissociation from an intracellular proton-binding site of the stator. We conclude that
the intracellular pH is a critical factor that determines the flagellar rotation rate and that proton
dissociation from the motor is a rate-limiting step in the mechanochemical reaction cycle.
103

BLAST X Poster #53
FLUORESCENCE IMAGING OF ASSEMBLY AND DISASSEMBLY OF THE BACTERIAL
FLAGELLAR PROTEIN EXPORT ATPase FliI TO THE FLAGELLAR BASAL BODY
Shinsuke Yoshimura, Tohru Minamino, Keiichi Namba
Grad. Schl. of Frontier Biosci., Osaka Univ.
For construction of the bacterial flagellum, most of the flagellar proteins are translocated
into the central channel of the growing structure by the flagellar protein export apparatus. FliI
ATPase forms a complex with its regulator FliH and facilitates the initial entry of export
substrates into the export gate made of six membrane proteins. The FliH/FliI complex also binds
to a C ring protein, FliN, through the FliH-FliN interaction for efficient export. However, it
remains unclear how these reactions proceed within the cell.
In this study, we constructed FliI-CFP and FliI-YFP fusion proteins and analyzed their
localization by fluorescence microscopy. A few bright spots within each cell suggested that
many of them are bound to the C ring, and breaching experiments showed their rapid assembly
and disassembly. We confirmed that both FliH and the C ring are required for the localization,
but faint spots observed even in the absence of the C ring suggested binding of the FliI hexamer
to the gate. FliI-YFP formed a complex with FliH∆1 missing residues 2-10 but the complex did
not show the localization. FliH∆1 interacted with neither FliN nor the gate-forming proteins.
Alanine-scanning mutagenesis of FliH revealed that only two residues, Trp-7 and Trp-10, are
responsible for these interactions. Taken all together, hydrophobic interactions of two Trp
residues of FliH with other export components seem to drive the cycling reaction of
assembly/disassembly of FliI for efficient export.
104

BLAST X Poster #54
ANALYSIS OF HELICOBACTER PYLORI LACKING ALL FOUR CHEMORECEPTORS
Karen M. Ottemann
UC Santa Cruz
Helicobacter pylori is an epsilon proteobacter that uses chemotaxis and motility to infect
human stomachs and cause ulcer disease. Based on genome sequencing and annotation, H.
pylori is predicted to have four chemoreceptors. Here we describe the construction and
characterization of mutants lacking one, two, three, or all four chemoreceptors in strain mG27,
and a comparison of chemoreceptor expression across many H. pylori strains. We find that one
chemoreceptor is sufficient to confer soft-agar migration in our standard Brucella Broth/Fetal
Bovine Serum soft agar. This finding suggests that ligands for each chemoreceptor are found in
this milieu. We also find that mutants lacking some chemoreceptor have an altered stopping
frequency, as noted by Schweinitzer et al (J Bac 2008 190:3244). Mutants lacking all four
chemoreceptors are non-chemotactic and completely smooth swimming, supporting that the
four MA-domain proteins are the only chemoreceptors in this system. Our work thus shows
experimentally that H. pylori possesses four chemoreceptors, and sets the stage for dissecting
what each chemoreceptor senses.
105

BLAST X Poster #55
CHEMOTAXIS TO PYRIMIDINES AND IDENTIFICATION OF A CYTOSINE
CHEMORECEPTOR IN PSEUDOMONAS PUTIDA
X. Liu, P.L. Wood, J.V. Parales, and R.E. Parales
Department of Microbiology, University of California, Davis, CA, 95616
Pseudomonads are motile bacteria that are widespread in nature and are known for their
catabolic versatility. These organisms have a conserved chemotaxis system that is homologous
to that present in Escherichia coli. Unlike E. coli, however, which has only one set of chemotaxis
(che) genes in a single gene cluster, Pseudomonas species have multiple che gene
homologues organized in several unlinked gene clusters. In addition, genome sequence
analyses have revealed that Pseudomonas genomes encode numerous MCP-like proteins,
suggesting that these organisms can also sense and respond to a wide range of chemicals and
environmental conditions. For example, Pseudomonas aeruginosa PAO1 has 26 MCP-like
genes and Pseudomonas putida F1 has 27. We have been studying the chemotactic responses
of P. putida strains to a variety of carbon and nitrogen sources and initiated a study in which we
have individually deleted each of the 27 MCP-like genes in P. putida F1 and tested for mutant
taxis phenotypes. We use both qualitative and quantitative chemotaxis assays to measure the
responses, including a new quantitative capillary assay carried out in 96-well plates. In this
study we demonstrated that P. putida strains F1 and PRS2000 are attracted to cytosine, but not
thymine or uracil. The chemotactic response to cytosine was constitutively expressed under all
tested growth conditions. In contrast, P. aeruginosa PAO1 was not chemotactic to any of these
pyrimidines. Chemotaxis assays with a mutant strain of P. putida F1 in which the putative
methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this
gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine.
Complementation of the F1ΔmcpC mutant with the wild-type gene restored chemotaxis to
cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to
respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.
106

BLAST X Poster #56
REACTIVE ALDEHYDES AND MOTILITY IN ESCHERICHIA COLI K12
Changhan Lee, Junghoon Lee, Jongchul Shin, Insook Kim, Kwanghee Baek, and Chankyu
Park
Department of Biological Sciences, KAIST, Daejon, Korea
Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea
The short chain carbohydrates such as glyoxal and methylglyoxal are generated in vivo
from various sugars either by an oxidative stress or by a cellular metabolism, which are believed
to be removed by the glutathione-dependent glyoxalase system. We isolated a number of E. coli
mutants conferring resistance or sensitivity to these aldehydes and found that some of them
affect motility. In the case of glyoxalse I mutant (gloA) that is deficient in the removal of
aldehyde using glutathione, significant reductions in the free-swimming as well as in swarming
behaviors were observed. When we introduced the flhDC-lacZ fusion contained in phage
Lambda into the wild-type and gloA strains, expression of LacZ was considerably reduced in the
gloA mutant compared to that of wild type, suggesting that a decrease in flagellar expression is
responsible for the impaired motility. This is further confirmed by the reduced expression of
flagellin as detected by anti-flagellin antiserum. We observed similar effects of other glyoxalrelated
genes on motility, suggesting a possibility that an intracellular level of aldehyde is
somehow associated with flagellar gene expression. Cellular redox change due to an aldehyde,
affecting motility, will be discussed.
107

BLAST X Poster #57
TAKING CONTROL OF THE BACTERIAL FLAGELLAR MOTOR
Mathieu Gauthier, Dany Truchon, Alexandre Bastien, Simon Rainville
Laval University, Physics department and COPL, Pavillon Optique Photonique, 2139, 2375, rue
de la Terrasse, Quebec, Quebec, G1V 0A6, Canada
The bacterial flagellar motor is a fairly complex machine, requiring 40-50 genes for its
expression, assembly and control. Furthermore, it is embedded in the multiple layers of the
bacterial membrane. That explains why, unlike many other molecular motors, it has not yet been
studied in vitro. As spectacular studies of linear motors (like kinesin, myosin and dynein) have
clearly demonstrated, an in vitro system provides the essential control over experimental
parameters to achieve the precise study of the motor’s physical and chemical characteristics.
Here, we report significant progress towards the development of a unique in vitro system to
study quantitatively the bacterial flagellar motor.
Our system consists of a filamentous Escherichia coli bacterium partly introduced inside
a micropipette. Femtosecond laser pulses (60 fs and ~ 15 nJ/pulse) are then tightly-focused on
the part of the bacterium that is located inside the micropipette. This vaporizes a
submicrometer-sized hole in the wall of the bacterium, thereby granting us access to the inside
of the cell and the control over the proton-motive force that powers the motor. Using a patchclamp
amplifier, we applied an external voltage between the inside and the outside of the
micropipette. If the hole in the bacterium is open, that voltage should translate into a membrane
potential powering the motors outside of the micropipette. As we varied the applied potential,
variations in the motor's rotation speed were observed. For these preliminary results, the
rotation speed was observed directly using video microscopy of fluorescently labeled filaments.
That system opens numerous possibilities to study the flagellar motor and other membrane
components.
108

BLAST X Poster #58
CHARACTERIZATION OF THE PERIPLASMIC DOMAIN OF THE SENSOR KINASE CpxA:
ROLE OF CONSERVED RESIDUES IN CpxA ACTIVITY
Malpica R and Raivio TL. Department of Biological Sciences, University of Alberta, CW405
Biological Sciences Building, Edmonton, Alberta, Canada T6G 2E9.
The Cpx two-component signal transduction system plays a major role in the
conservation of cell envelope integrity, as well in the regulation of surface structure assembly,
cellular attachment and pathogenesis in Escherichia coli. This system is comprised of the innermembrane
sensor kinase CpxA and the cytosolic response regulator CpxR. Envelope stress
caused by misfolded and mislocalized proteins activates the Cpx pathway, which also responds
to alkaline pH and overexpression of the outer membrane lipoprotein NlpE. In the presence of
these activating cues, CpxA autophosphorylates at a conserved His residue and then
transphosphorylates CpxR (CpxR-P). In turn, CpxR-P regulates the expression of several genes
such as periplasmic protein folding and degrading factors involved in envelope protein
manteinance under adverse conditions. It is known that in the absence of stress, the periplasmic
protein CpxP, whose expression is positively controlled by this pathway, inhibits the activation of
the Cpx system. The periplasmic domain of CpxA (CpxA-pd), which contains 133 residues, has
been proposed as the signal reception site and therefore as the regulatory element of the kinase
and phosphatase activities of CpxA.
To explore the role of CpxA-pd components in signal sensing and CpxA activity, we
generated mutants that carry substitution mutations in highly conserved residues of this domain.
The phenotypes of these mutants were evaluated, using alkaline pH or NlpE as inducing cues
and the activatable cpxP´-lacZ fusion as a reporter of Cpx pathway activity. Remarkably, the
substitution of Phe108, Gly130 and Pro164 by Ala lead to a constitutively active phenotype.
Also, mutants in other residues displayed a significant increase on pathway activity in the
absence of the inducing cues. Thus, we have identified relevant CpxA-pd elements for both
signalling and the overall enzymatic activity of CpxA.
109

BLAST X Poster #59
CHARACTERIZATION OF FliZ AS AN ACTIVATOR OF FLAGELLAR GENES IN
SALMONELLA ENTERICA SEROVAR TYPHIMURIUM
Supreet Saini 1 , Phillip Aldridge 2,3 , and Christopher Rao 1
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-
Champaign, Urbana, Illinois 61801, 1
Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon
Tyne NE2 4HH, United Kingdom, 2
Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place,
Newcastle upon Tyne NE2 4HH, United Kingdom 3
Flagellar assembly proceeds in a sequential manner, beginning at the base and
concluding with the filament. A critical aspect of assembly is that gene expression is coupled to
assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is
sequential, reflecting the manner in which the flagellum is made. A key mechanism for
establishing this temporal hierarchy is the σ 28 -FlgM checkpoint, which couples the expression of
late flagellar (Pclass3) genes to the completion of the hook-basal body. In this work, we
investigated the role of FliZ in coupling middle flagellar (Pclass2) gene expression to assembly
in Salmonella enterica serovar Typhimurium (Salmonella). We also demonstrate that significant
cross talk exists between different secretion systems in Salmonella with FliZ as the cross talk
element. Specifically, we show that FliZ is also an activator of Salmonella Pathogenicity Island-1
(SPI1) encoded virulence genes and the fim, lpf, std, saf, bcf, stb, stc, stf, and sth loci encoded
fimbriae in Salmonella. We demonstrate that FliZ is an FlhD4C2-dependent activator of
Pclass2/middle gene expression. Our results suggest that FliZ regulates the concentration of
FlhD4C2 posttranslationally which leads to faster induction of Pclass2/middle genes. We also
demonstrate that FliZ functions independently of the flagellum-specific sigma factor σ 28 and the
filament-cap chaperone/ FlhD4C2 inhibitor FliT. We correlate our gene expression experiments
by discussing the role of FliZ in flagellar biosynthesis during swimming and swarming motility.
FliZ was found to effect SPI1 activity levels in a HilD dependent posttranslational manner and its
effect on type I fimbriae gene expression was found to be FimZ dependent.
110

BLAST X Poster #60
LOCALIZATION OF THE CHEMOTAXIS PROTEINS IN BACILLUS SUBTILIS
Kang Wu and Christopher Rao
University of Illinois at Urbana-Champaign
The Bacillus subtilis chemotaxis pathway utilizes three proteins – CheC, CheD, and
CheV - not found in Escherichia coli. These proteins are thought to be involved in two
methylation-independent adaptation systems not found in E. coli. While the functions of these
proteins have been characterized to some degree, the details concerning their interactions
within the polar signaling complex are still unknown. To better understand these interactions, we
are investigating the factors influencing the localization of the B. subtilis chemotaxis proteins
using immunofluorescence and fluorescent protein fusions. Of significance, we have
constructed functional fluorescent protein fusions to CheC and CheD and also explored their colocalization
using two-color immunofluorescence. Using these approaches, we have quantified
the localization of these proteins in a number of different mutants. This poster will discuss some
of our preliminary results regarding the factors influencing localization of the chemotaxis
proteins in B. subtilis.
111

BLAST X Poster #61
RcdA STRUCTURE AND FUNCTION IN REGULATED CtrA PROTEOLYSIS
James A. Taylor*, Jeremy D. Wilbur † , Kathleen R. Ryan*
* Department of Plant & Microbial Biology, University of California, Berkeley, Berkeley, CA
94720-3102
†
Graduate Group in Biophysics, University of California, San Francisco, San Francisco, CA
94158
The Caulobacter crescentus master cell cycle regulator CtrA must undergo cyclic
activation and deactivation to drive orderly progression through the division cycle. CtrA is
essential for viability and directly activates or represses the transcription of ~100 genes.
However, it also blocks the initiation of DNA replication, so CtrA activity must be eliminated from
the cell before chromosome replication can occur. To this end, CtrA is rapidly degraded
specifically at the G1-S cell cycle transition by the ClpXP protease. Regulated CtrA proteolysis
in vivo requires two other factors, the single-domain response regulator CpdR and RcdA, a
conserved protein of unknown function. Although each of these proteins is cytoplasmic, they all
colocalize at one pole of the cell during CtrA degradation.
We are investigating the role of RcdA in CtrA proteolysis. RcdA was proposed to act as
an adaptor bridging CtrA and ClpXP to promote CtrA degradation. However, RcdA is not
necessary for CtrA proteolysis by ClpXP in vitro and does not change the rate of CtrA
degradation. We have therefore taken a structure-function approach to learn how RcdA
contributes to CtrA proteolysis in vivo.
Using X-ray crystallography, we have found that RcdA is a dimer, and each monomer
consists of a three-helix bundle. Peptides at the N- and C-termini and a peptide linking helices
two and three are disordered in the crystal structure. We have created point mutations and
deletions to alter conserved surface features of RcdA. We expressed these proteins in a
Caulobacter ∆rcdA mutant to identify regions of RcdA necessary for regulated CtrA degradation
and for the polar localization of CtrA and RcdA itself.
We have identified three types of rcdA mutants: 1) those that permit CtrA degradation
and protein localization, 2) those that cannot support either CtrA degradation or protein
localization, and 3), those that allow CtrA degradation without polar accumulation of either RcdA
or CtrA. Surprisingly, RcdA does not need to be stably located at the cell pole to promote CtrA
proteolysis. These results suggest that RcdA can act via transient interaction with other
degradation proteins at the pole, or that RcdA’s function can be performed anywhere in the cell.
For example, RcdA could inhibit an unknown negative regulator of CtrA proteolysis. We are
examining the rcdA mutants in further detail and are screening for additional proteins that
regulate CtrA degradation in Caulobacter.
112

BLAST X Poster #62
MODELLING MCP SIGNALLING MECHANISMS WITH HIGH-THROUGHPUT SIMULATION
OF Tar TM2
Benjamin A Hall, Mark SP Sansom
Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU
Transmembrane helices play a multiple vital roles in cell function, including intercell
signalling processes, channel gating and active transport. As such, there exists a considerable
amount of data on the biological function and structural properties of naturally occurring helices
and their mutants. Simulation studies can provide insight into the dynamics and behaviour of
biomolecular systems in a variety of environments, but however such analyses are
computationally expensive and typically difficult to automate. Coarse grain simulations are
becoming an increasingly popular tool for understanding the properties of biological systems,
overcoming canonical limits of atomistic simulations such as timescale or system size. Both
such techniques involve several manual steps, including system build, simulation set up and
analysis. Here we present Sidekick, a piece of software which automates these processes to
allow for the set up of massive numbers of coarse grain simulations on the basis of a small set
of input sequences, or a single sequence and a scanning mutation. We demonstrate the use of
this software to approach the mechanism of signalling in the MCPs, based on existing
mutational data for TM2 from different MCPs and organisms. By observing the change in
positions and orientations of the helix with different mutations, we propose that a piston model
dominates the signalling event, though there may be a lesser role for rotation of the helix.
113

BLAST X Poster #63
STRUCTURAL EVIDENCE SUGGESTS THAT ANTIACTIVATOR ExsD FROM
PSEUDOMONAS AERUGINOSA IS A DNA BINDING PROTEIN
Robert C. Bernhards, Xing Jing, Nancy J. Vogelaar, Howard Robinson#, Florian D.
Schubot*
Department of Biological Sciences, Virginia Polytechnic Institute & State University,
Washington Street, Blacksburg, VA 24060; #Biology Department, Brookhaven National
Laboratory, Upton, NY 11973-5000.
The opportunistic pathogen P.aeruginosa utilizes a type III secretion system (T3SS) to
support acute infections in predisposed individuals. In this bacterium expression of all T3SSrelated
genes is dependent on the AraC-type transcriptional activator ExsA. Prior to host
contact, the T3SS is inactive and ExsA is repressed by the antiactivator protein ExsD. The
repression, thought to occur through direct interactions between the two proteins, is relieved
upon opening of the type III secretion (T3S) channel when secretion chaperone ExsC
sequesters ExsD. We have solved the crystal structure of Δ20ExsD, a protease-resistant
fragment of ExsD that lacks only the twenty amino terminal residues of the wild type protein at
2.6 Å. Surprisingly the structure revealed similarities between ExsD and the DNA binding
domain of transcriptional repressor KorB. A model of an ExsD-DNA complex constructed on the
basis of this homology produced a realistic complex that is supported by the prevalence of
conserved residues in the putative DNA binding site and the results of differential scanning
fluorimetry studies. Our findings challenge the currently held model that ExsD solely acts
through interactions with ExsA and raise new questions with respect to the underlying
mechanism of ExsA regulation.
114

BLAST X Poster #64
A NOVEL PAS-GGDEF-EAL PROTEIN INVOLVED IN REGULATION OF MOTILITY IN
PSEUDOMONAS PUTIDA
Herrera Seitz, K 1 and Shingler, V 2
1 2
IIB, FCEyN, Univ. Nac. del Mar del Plata, Molecular Biology Department, Umeå University,
Sweden.
Chemotaxis allows motile bacteria to respond to chemical gradients to relocate
themselves near the source of nutrients. Soil Pseudomonads are known to be able to grow in a
wide variety of carbon sources, including many that are considered environmentally toxic. Unlike
previously characterized chemotactic responses in Pseudomonas strains, taxis of P. putida
CF600 and P. putida KT2440 towards methyl-phenols is dependent upon its ability to
metabolize the compound, rather than on a classical ligand-binding chemoreceptor. E. coli
metabolism-dependent taxis responses are mediated by the Aer receptor that is closely related
to chemoreceptors, but which contains a FAD-binding PAS sensory domain. P. putida
possesses three aer-like genes. During analysis of the Aer-like receptors of P. putida, Aer-1 was
found to be encoded in a dicistonic operon with PP2258, a PAS-GGDEF-EAL domain protein.
Our attention was drawn to PP2258 because a null mutant was found to exhibit a general
motility defect on solid, but not in liquid, media (Sarand et al., 2008).
GGDEF- and EAL-domains are associated with diguanylate cyclase and
phosphodiesterase activities that are involved in turnover of the near ubiquitous bacterial
second messenger c-di-GMP. The levels of c-di-GMP can modify cells behavior and motility;
therefore we reasoned that PP2258 link to motility via c-di-GMP signaling. As a first approach to
test this idea, the biochemical properties of wild type and mutant derivatives of PP2258 were
studied using over expression of the protein both in E. coli and P. putida.
When PP2258 was over expressed in E. coli or P. putida cells, c-di-GMP levels were
markedly increased compared to those of control cells, although accumulation of c-di-GMP was
much lower in E. coli than in P. putida extracts. Alanine substitutions of the GGDEF domain
associated with c-di-GMP synthesis causes a major decrease c-di-GMP accumulation, while an
alanine substitution in EAL domain associated with c-di-GMP hydrolysis led to a >7-fold
increase in accumulation. Together, these results suggest that PP2258 could be one of the rare
examples of a dual GGDEF-EAL domain protein where both domains are catalytically active.
References:
Sarand, I., Österberg, S., Holmqvist, S., Holmfeldt, P. Skärfstad, E., Parale, R. E., & Shingler, V.
(2008) Metabolism-dependent taxis towards (methyl)phenols is coupled through the most
abundant of three polar localized Aer-like proteins of Pseudomonas putida. Environ.
Microbiology. 10:1320-1334
115

BLAST X Poster #65
IN VIVO STUDY OF THE TWO-COMPONENT SIGNALLING NETWORK IN E. COLI
Erik Sommer and Victor Sourjik
Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282,
69120 Heidelberg, Germany
Contact: e.sommer@zmbh.uni-heidelberg.de
Two-component systems are the most widespread sensing systems in prokaryotes and
lower eukaryotes, with multiple members of this class being present in one organism. We are
interested in investigating the interconnection among different two-component signalling
pathways in Escherichia coli. To map interactions between the pathways in vivo and to study
relative cellular distribution of their proteins, we assay real-time dynamics of protein interactions
and their dependencies on stimulation using fluorescence imaging and fluorescence resonance
energy transfer (FRET)- and fluorescence recovery after photobleaching (FRAP)-microscopy.
Additionally, intracellular processing of sensed stimuli with regard to amplification, integration
and possible cross-talk between the systems will be investigated. Such analysis will help to
establish an integral picture of cell signalling performed by prokaryotic organisms.
116

BLAST X Poster #66
GENE REGULATION BY ESCHERICHIA COLI RESPONSE REGULATOR PhoB
Hua Han 1,2 , Timothy R. Mack 1,2 , Rong Gao 1.3 , and Ann M. Stock 1,3
1 Center for Advanced Biotechnology and Medicine, UMDNJ-Robert Wood Johnson Medical
School, Piscataway, New Jersey 08854, USA,
2 Department of Biochemistry, Graduate School of Biomedical Sciences, UMDNJ-Robert Wood
Johnson Medical School, and 3 Howard Hughes Medical Institute.
Structural analysis of the Escherichia coli response regulator transcription factor PhoB
indicates that the protein dimerizes in two different orientations, both of which are mediated by
the receiver domain. The two dimers exhibit two-fold rotational symmetry: one involves the α4β5-α5
surface and the other involves the α1/α5 surface. The α4-β5-α5 dimer is observed when
the protein is crystallized in the presence of the phosphoryl analog BeF3 - while the α1/α5 dimer
is observed in its absence. From these studies a model of the inactive and active states of
PhoB has been proposed that involves the formation of two distinct dimers. In order to gain
further insight into the roles of these dimers we have engineered a series of mutations in PhoB
intended to selectively perturb each of them. Our results indicate that perturbations to the α4β5-α5
surface disrupt phosphorylation-dependent dimerization and DNA binding as well as
PhoB mediated transcriptional activation of phoA, while perturbations to the α1/α5 surface do
not. Furthermore, experiments with a GCN4 leucine zipper/PhoB chimera protein indicate that
PhoB is activated through an intermolecular mechanism. Together these results support a
model of activation of PhoB in which phosphorylation promotes dimerization via the α4-β5-α5
face which in turn enhances DNA binding to a pair of direct-repeat half-sites – a model that we
propose to be common for most all OmpR/PhoB transcription factors. These data contrast with
a recent proposal that the α1/α5 dimer corresponds to the active form of PhoB, a conclusion
derived from structural analysis of constitutively active mutant PhoB proteins (Arribas-
Bosacoma et al., 2007 J. Mol. Biol. 366:626-641). We have also examined the kinetics of gene
expression of several PhoB-regulated genes under conditions of phosphate limitation.
117

BLAST X Poster #67
NEW REPORTER RESIDUES OF TRIMER FORMATION BY ESCHERICHIA COLI MCPs
Diego A. Massazza 1 , John S. Parkinson 2 , Claudia A. Studdert 1
1 Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Argentina
2 Biology Department, University of Utah
It is currently accepted that E. coli chemoreceptors are organized in vivo in trimer-ofdimer
arrangements. Using receptors bearing a cysteine reporter residue near the trimer axis,
we previously demonstrated efficient formation of 2- and 3-subunit crosslinking products upon
treatment of intact cells with the trifunctional maleimide reagent TMEA. In this work, we
assessed the in vivo crosslinking behavior of receptors bearing cysteine reporters at different
positions along their cytoplasmic domains.
We found that the formation of 3-subunit crosslinking products declined with increasing
distance of the reporter from the cytoplasmic tip. This result suggests that the in vivo
arrangement of the trimer of dimers resembles that observed in the crystal structure of the Tsr
cytoplasmic domain, in which the dimers contact one another at the tip and splay apart in the
regions that are farther from the tip.
We also found that stimulation with repellents immediately before the TMEA treatment
caused a slight but reproducible increase in crosslinking efficiency. This behavior is consistent
with previous observations suggesting that receptor dimers within the trimer move closer
together after repellent stimuli.
In cells co-expressing different marked receptors, we analyzed the formation of mixed
crosslinking products. When the cysteine reporters were located the same distance from the tip,
mixed crosslinking products formed with high efficiency. Mixed products did not form between
reporters at different distances from the tip. This result suggests that there are no significant
vertical displacements within the trimer of dimers, nor sufficient flexibility between the receptors
to get crosslinking between reporters at diferent levels in the dimers.
118

BLAST X Poster #68
SYSTEMATIC DETECTION OF PROTEINS THAT LOCALIZE AT THE ATTACHMENT
ORGANELLE REGIONS OF MYCOPLASMA PNEUMONIAE BY FLUORESCENT-PROTEIN
TAGGING
Tsuyoshi Kenri 1 , Atsuko Horino 1 , Mayumi Kubota 1 , Yuko Sasaki 1 , Daisuke Nakane 2 and
Makoto Miyata 2
1. Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious
Disease, 4-7-1, Gakuen, Musashimurayama, Tokyo, 208-0011, Japan 2. Graduate School of
Science, Osaka City University, Sumiyoshi-ku, Osaka, 558-8585, Japan
Mycoplasma pneumoniae is a cell wall-less bacterium with minimum range of genome
size required for self-replication. It is also known as a general cause of bronchitis and
pneumonia in humans. Pathogenicity of this bacterium is largely depends on its ability to attach
to respiratory epithelial cells (cytadherence). The M. pneumoniae cells attached to cell surface
also exhibit gliding motility. The cytadherence and gliding motility are mediated by a unique cell
terminal structure of this bacterium, the attachment organelle, which is a membrane protrusion
supported by internal cytoskeletal structures. Since the attachment organelle duplicates before
cell division, the organelle formation is thought to be coordinated with cell cycles. A number of
protein components of the attachment organelle (cytadherence-related proteins) have been
identified so far (HMW1, HMW2, HMW3, P1, P30, P65, P200, P24, P41, P90(B) and P40(C)).
However, configuration and fine structure of these proteins in the organelle are not fully
understood. In addition, the studies of Triton X-100 insoluble fraction of M. pneumoniae cells,
cross-linking analysis of P1 adhesin protein and isolation of various gliding mutants are
suggesting a possibility that there are more protein factors that associate with the attachment
organelle components. In this study, to explore whether there are more proteins that localize at
the organelle, we perform systematic fluorescent-protein tagging analysis for M. pneumoniae
ORFs. At present, we have finished the localization analysis of 620 ORFs (about 90% of total
ORFs). In this analysis, we observed that about 50 ORF products including previously known 9
cytadherence-related proteins localized at the attachment organelle region. The other ORF
products identified are 10 proteins involved in DNA, RNA and protein synthesis, 20 homologs of
variety of enzymes, 2 chaperons and 15 proteins of unknown function. Probably, some of these
proteins may be the components of the attachment organelle and have function in formation of
the organelle, cytadherence, gliding motility and coordination of cell cycle.
119

BLAST X Poster #69
MAPPING THE SIGNAL TRANSDUCTION PATHWAY WITHIN THE PAS DOMAIN OF THE
Aer RECEPTOR
Asharie J. Campbell, Kylie J. Watts, Mark S. Johnson, and Barry L. Taylor
Dept. Microbiology and Mol. Genetics, Loma Linda University, Loma Linda CA, USA
The E. coli Aer receptor senses redox changes through an FAD-binding PAS domain,
and transmits this redox status to the HAMP and signaling domains. Little is known about the
conformational changes that take place within the PAS domain. In this study, we used errorprone
PCR mutagenesis to find residues critical for PAS FAD-binding, sensing and signal
transduction. We screened 10,000 colonies for function, measured expression for 1,300 clones,
sequenced 400 mutants, and found 84 Aer aerotaxis-defective mutants that had just one amino
acid substitution. Of these, there were 72 substitutions in the PAS domain, 11 in the F1 region
and 1 in the TM region. The swimming behavior of the cells expressing these mutant proteins
included those that 1) were locked in a smooth swimming (CCW), "signal-off" state (60/84), 2)
had increased tumbling (CW) frequency (4/84) or 3) were locked in the CW "signal-on" state
(20/84). Approximately half (49/84) of the Aer mutants were functionally rescued by Tar. Mutant
proteins (11/84) that expressed at levels less than 30% of wild-type Aer showed enhanced
protein degradation rates. These replacements had altered side-chain polarity, mapped to
positions on or near loops and, with one exception, yielded a CCW (signal-off) phenotype. In
contrast, all but one of the CW (signal-on) mutants were stable, and clustered in three localized
regions: 1) in the putative FAD-binding cleft, 2) on the rear surface of the FAD-binding cleft and
3) at or near a loop in the N-terminal Cap region. When expressed at a 1:1 ratio with wild-type
Aer, three CW-locked mutants were dominant, abolishing wild-type mediated aerotaxis. Most
but not all of the FAD-binding lesions resulted in an unstable protein; those that were most
stable were located outside of the putative FAD-binding cleft.
The localization of gain-of-function (CW) lesions to three distinct clusters suggests that
conformational changes in these specific regions mimic the signal-on state of the PAS sensor.
If so, signaling within the Aer-PAS sensor would begin in the FAD-binding cleft and propagate
outward to the N-Cap loop. Previously, we found that removing part of the N-Cap mimics the
signal-on state. Thus, an attractive model is one where FAD reduction in the PAS domain
initiates a conformational change that propagates to the N-Cap loop, which, in turn, acts as a
hinge around which the N-cap moves, unmasking the signal-on state.
120

BLAST X Poster #70
MODULATING TWO-COMPONENT SIGNAL OUTPUT WITH PROTEIN-MEMBRANE
INTERACTIONS
Roger R. Draheim*, Morten H. H. Nørholm, Salomé C. Botelho, Karl Enquist, and Gunnar von
Heijne
Center for Biomembrane Research, Department of Biochemistry and Biophysics, The Arrhenius
Laboratories for Natural Sciences, Stockholm, University, 10691, Stockholm, Sweden
The most prevalent type of environmental sensor in prokaryotic organisms is the twocomponent
system (TCS). A canonical TCS consists of a membrane-spanning sensor histidine
kinase (SHK) and a cytoplasmic response regulator (RR). TCSs have been shown to regulate a
diverse array of virulence factors, therefore identifying two-component signaling pathways that
lead to enhanced pathogenicity is essential to understanding complex host-pathogen
interactions. The specific hypothesis examined is that SHK-membrane interactions can be
identified and harnessed to modulate SHK signal output in a predictable manner. If individual
SHK signal output could be directly manipulated, then two-component signaling pathways could
be rapidly unraveled in any pathogenic organism of interest.
Three discrete steps are proposed to establish a broadly-applicable methodology. The
first will identify interactions between TM2 of a well-characterized SHK (e.g., EnvZ) and the cell
membrane using a glycosylation-mapping technique. The second will identify HAMP-membrane
interactions within EnvZ using a translocon-challenge method. Protein-membrane interactions
that are identified will be confirmed using circular dichroism and fluorescent techniques. The
third will harness protein-membrane interactions identified during the first two to directly and
incrementally modulate SHK signal output. This experimentation will determine the feasibility of
coupling modulated SHK output with transcriptional profiling to rapidly unravel two-component
signaling pathways in any pathogenic organism of interest. A high-throughput approach using
fluorescent transcriptional reporter systems has been established to expedite these steps.
The long-term goal of this research is to create a method for rapidly identifying the
signaling pathways that regulate the virulence of pathogenic microorganisms. This research will
lead to a better understanding of complex host-pathogen interactions and will result in the
detection of previously unidentified therapeutic targets.
121

BLAST X Poster #71
Poster Cancelled
122

BLAST X Poster #72
A MECHANICAL AND GENETIC STUDY OF ESCHERICHIA COLI SWARMING MOTILITY
Matthew Copeland and Douglas B. Weibel
Department of Biochemistry, University of Wisconsin-Madison, WI 53706 USA
Bacterial swarming is a phenotype associated with the motility of bacteria across
surfaces in search of resources. In this abstract we describe two approaches to understand this
phenotype in Escherichia coli; a ‘mechanistic’ approach to elucidate potential flagella/flagella
interactions between adjacent swarming cells and a genetic investigation to chart the
expression of genes unique to the swarming phenotype.
In contrast to swimming motility, swarming bacteria cells migrate cooperatively across
surfaces. The role of physical interactions in the coordination of swarming motility is unknown. It
has been shown that swarming bacteria align along their long axis and move as multicellular
rafts from which the characteristic dynamic swirling patterns of swarming emerge. The
alignment of cells may facilitate or accompany the intercellular bundling of flagella between
adjacent cells and play a role in the characteristic, coordinated movement observed during
swarming motility. To test this hypothesis, we have created strains of E. coli with fluorescent
flagella and are using space- and time-resolved fluorescence resonance energy transfer (FRET)
to measure flagella/flagella interactions.
Iron starvation is known to signal swarmer cell differentiation in Vibrio parahaemolyticus
and the genes for iron uptake and metabolism have been shown to be elevated in swarming
populations of Salmonella typhimurium. We have found that iron metabolism and iron
acquisition genes are upregulated in swarming cells of E. coli versus planktonic cells. A specific
role for iron in E. coli swarming cells is unknown. Like V. parahaemolyticus, iron starvation may
be a signal for swarmer cell differentiation in E. coli or swarming development and motility may
require intracellular levels of iron in excess of those necessary for growth under vegetative
conditions. We are exploring the role of iron in E. coli swarming using a combination of chemical
and gene expression techniques and strains containing iron metabolism gene knockouts.
123

BLAST X Poster #73
PROBING HYDRODYNAMIC INTERACTRIONS BETWEEN SWIMMING BACTERIA USING
MICROFULIDICS
Abishek Muralimohan, Douglas B. Weibel
Department of Biochemistry, University of Wisconsin-Madison
433 Babcock Drive, Madison, WI 53706, U.S.A.
Populations of self propelled motile bacteria have been shown to exhibit collective
swimming behavior, leading to large scale fluid motion and fluid transport 1 . Such collective
behavior arises from hydrodynamic interactions (HI) between the individual bacterial cells. While
computational studies have been performed on HI between pairs of swimming cells 2 , there have
been no reports on experimental measurements of HI. Here, we describe a PDMS based
microfluidic device that promotes pair wise interactions between swimming bacteria. The device
generates collisions between pairs of cells and HI between them is quantified by the length
scale (persistence) over which the two cells swim together as a single unit. We have used this
device to quantify the strength of HI between pairs of filamentous E. coli hcb437 cells based on
a number of parameters including cell length, angle of initial contact, trajectory, and velocity.
Our observations indicate two subpopulations based on the persistence length of interaction:
long persisters are characterized by low angles of contact (9.0 µm); short
persisters are characterized by a wider range of contact angles (10° – 60°) and shorter cells (6
µm -9 µm). We believe that our microfluidics-based approach is well suited to the study of
bacterial interactions at a single cell level. A broader understanding of HI between motile
bacteria will shed light on other forms of collective motility, including swarming.
1. Underhill P, et. al. (2008) Phys. Rev. Lett. 100: 248101.
2. Ishikawa T, et al. (2007) Biophys. J. 93, 2217.
124

BLAST X Poster #74
EXAMINATION OF PHOSPHORYLATION IN THE Dif CHEMOTAXIS-LIKE SYSTEM IN
MYXOCOCCUS XANTHUS
Wesley P. Black and Zhaomin Yang
Department of Biological Sciences, Virginia Polytechnic and State University, Life Sciences I,
Washington Street, Blacksburg, VA 24061
Myxococcus xanthus is able to move on surfaces using social (S) gliding motility, which
is powered by the retraction of type IV pili (Tfp). Exopolysaccharides (EPS), also essential for S
motility, have been proposed to function as the anchor and trigger for Tfp retraction in M.
xanthus. EPS production in M. xanthus is regulated by the Dif chemotaxis-like signal
transduction pathway. DifA, DifC and DifE, homologous to methyl-accepting chemotaxis
proteins (MCPs), CheW and CheA respectively, are positive regulators of EPS production. DifD
and DifG, which are respective homologs of CheY and CheC, are negative regulators of EPS
production. It was demonstrated previously that DifD (CheY-like) does not function downstream
of DifE (CheA-like) in the regulation of EPS production.
The purpose of this study was to examine the phosphorylation of heterologously
expressed and purified Dif proteins in vitro. Protein phosphorylation, phosphate transfer and
dephosporylation were monitored using γ- 32 P-ATP. We demonstrate the autophosphorylation of
DifE, which is an atypical CheA-like kinase with an extra domain. Unlike observations in other
systems, the presence of both DifA (MCP-like) and DifC (CheW-like) had inhibitory rather than
stimulatory effects on DifE autophosphorylation. In addition, we show that the phosphate from
DifE-phosphate can be transferred to DifD (CheY-like). Lastly, DifG, which is similar to the CheC
phosphatase, accelerates the dephosphorylation of DifD. These results are consistent with a
model where the DifE kinase positively regulates EPS production, while DifD and DifG function
collectively to divert phosphate away from an unidentified downstream phosphorylation
substrate of DifE.
125

BLAST X Poster #75
EVOLUTION OF CHEMOTAXIS PROTEINS ON A MICRO SCALE
Brian Cantwell and Igor Zhulin
University of Tennessee, Department of Microbiology, Knoxville, TN
Oak Ridge National Laboratory, Joint Institute for Computational Sciences, Oak Ridge, TN
Computational analysis of very closely related genomes offers the advantage of more
accurate tracing of evolutionary events. The chemotaxis system of enteric bacteria Escherichia
coli and Salmonella enterica are extremely well studied, and the availability of over fifty
sequenced genomes of Enterobacteriacea offers an opportunity to determine the
microevolutionary trends in chemotaxis of enterics. Similarly, seventeen sequenced genomes
are available for the family Shewanellaceae for which Shewanella oneidensis MR-1 has been
most studied by genetic and biochemical methods. In this work we examine the evolution of
the chemotaxis systems of the Enterobacteriaceae and Shewanellaceae using computational
biology methods. Protein sequences of chemotaxis proteins and receptors were extracted from
non-redundant database by matching to domain models and organized into orthologous groups
based on reciprocal BLAST hits, genome context, and phylogenetic relationships. All
Enterobacteriacea contain a single set of chemotaxis proteins and chemoreceptors orthologous
to the E. coli Tsr, Tap, and Aer proteins. Most enteric species contain numerous additional
chemoreceptors including multiple orthologs of the E. coli Trg protein. Shewanella species have
one common set of chemotaxis proteins with some species having an additional set of
chemotaxis proteins. Two chemoreceptors are common among all seventeen Shewanella
species with an additional ten chemoreceptors found in at least fifteen of the seventeen
sequenced genomes. To examine the evolutionary trends within chemotaxis proteins, we
compared orthologous proteins by computing pairwise percentage identity and comparing
domains across species, genus, and family lines. As expected, the domains with highest
conservation include the catalytic domains of the core chemotaxis proteins as well as the
signaling subdomain of the chemoreceptors. The most divergent domains include the P2
domains of CheA, sensory domains of chemoreceptors, HAMP domains, and the methylation
subdomains of the Aer proteins.
126

BLAST X Poster #76
EVOLUTION OF SIGNAL TRANSDUCTION IN A BACTERIAL GENUS
Harold Shanafield, Luke E. Ulrich and Igor B. Zhulin
National Institute for Computational Sciences, University of Tennessee – Oak Ridge National
Laboratory
The recent completion of sequencing of multiple genomes in the Shewanella genus
provides a unique opportunity to study evolution at a much finer scale than previously possible.
Using a bi-directional best BLAST hit approach at the protein domain rather than traditional
whole-protein sequence level we analyzed the evolutionary relationships of proteins predicted to
be involved in signal transduction. Based on these relationships, we have determined a core set
of 99 proteins across the first 11 sequenced Shewanella genomes that were highly conserved in
both domain architecture and protein sequence. The core included one of the several
chemotaxis systems found in Shewanella and several two-component regulatory systems. A
large group of orthologous signal transduction proteins across multiple genomes showed some
primary sequence drift and were classified as “significant similarity”, and finally there were
several unique signal transduction proteins in each organism. We also quantified a recent
disproportionate loss of signal transduction genes in Shewanella denitrificans OS217 above and
beyond the overall reduction in that organism’s genome size, and an enrichment of signal
transduction genes in Shewanella amazonensis SB2B. Possible relationships of the observed
changes with the metabolism and environment are discussed.
127

BLAST X Poster #77
THE CHEMOSENSORY RECEPTOR FrzCD INTERACTS WITH TWO A-MOTILITY
PROTEINS, AglZ AND AgmU
Beiyan Nan and David R. Zusman
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
The Frz chemosensory system of Myxococcus xanthus controls directed motility by regulating
cellular reversals. FrzCD, the Frz system chemoreceptor, plays a central role in the Frz signal
transduction pathway. Recently, evidence was obtained that AglZ, an A-motility protein, and
FrzS, an S-motility protein, are localized in separate complexes that change their positions as
cells move forward and reverse. We were interested in studying how FrzCD might communicate
with these motility complexes. To gain information on these interactions, we have been doing
pull-down experiments using Myxococcus cell extracts and GST-tagged FrzCD as bait. The
GST-FrzCD interacting proteins were identified by mass spectrometry. Five proteins were found
to reproducibly bind to GST-FrzCD besides FrzA, FrzB and FrzE. Two of these were A-motility
associated protein, AglZ and AgmU. The interactions were confirmed using formaldehyde crosslinking,
which showed that FrzCD interacts directly with the N-terminal pseudo-receiver domain
of AglZ and two TPR clusters of AgmU. These results provide preliminary evidence for a direct
role in the control of the A-motility system by the FrzCD receptor. The roles of the other FrzCDinteracting
proteins remain to be identified.
128

BLAST X Poster #78
PHENOTYPIC CHARACTERIZATION OF ALL SINGLE MUTANTS OF TWO COMPONENT
SYSTEM PROTEINS IN NEUROSPORA CRASSA
Barba C., Chavez-Canales M., Salas G., Hernandez C., Sanchez O and Georgellis D.
Department of Molecular Genetics, Instituto de Fisiologia Celular, UNAM
Perception and response to environmental stimuli is essential for the growth and survival
of all organisms. The sensing and processing of these stimuli are carried out by molecular
circuits within the cell, which detect, amplify and integrate them into a specific response. In
prokaryotes these molecular circuits are typically organized by protein pairs, "sensory kinase"
proteins (SK) and "response regulator" proteins (RR) that belong to the large family of two
component systems (TCS). This organization implies that each SK activates its cognate RR,
and thus providing specificity to signal propagation and output. However, an interesting variation
of TCS architecture is observed in filamentous fungi where multiple SKs appear to use a single
phosphotransfer protein to relay signals to a few RRs. Therefore, the question of whether a
specific signal can generate a specific response, or whether the various signals sensed by
individual SKs result in the same response, is raised. To explore this intriguing question we
used Neurospora crassa, which has eleven SKs, one phosphotransfer protein and three RRs,
as our model. Here, by using single mutants of all SKs and RRs, we demonstrate that these
signaling cascades are involved in the regulation of various developmental processes, and in
responses to environmental conditions, such as osmotic, oxidative and fungicide stress.
129

BLAST X ____________ Poster #79
AFM STUDY OF MYCOPLASMA MOBILE’S GLIDING MOTILITY
Charles Lesoil (1) , Hiroshi Sekiguchi (1) , Takahiro Nonaka (2) , Makoto Miyata (2) , Toshiya
Osada (1) , Atsushi Ikai (3)
(1) Department of life science, Graduate school of Bioscience and Biotechnology, Tokyo
Institute of Technology.
(2) Department of Biology , Graduate school of Science, Osaka City University
(3) Innovation Research Center, Tokyo Institute of Technology
Mycoplasma mobile is a parasitic bacterium that lacks the peptidoglycan layer but still
presents recognizable flask-shape cell morphology. It glides along cell or glass surfaces at an
average speed of 2.0 to 4.5 μm/s towards the tapered end of the cell called the head, with a
unique mechanism. Recent studies have identified four proteins Gli23, Gli349 and Gli521 that
are involved in this system, and a model for gliding motility has been presented, but more
experimental data are needed to obtain the arrangement and detailed role of each protein in this
system.
In this study, we propose a novel approach to investigate the gliding motility system of
M. mobile using an AFM (Atomic Force Microscope) both as an imaging and a force
measurement device.
AFM Images of biotinylated M. mobile cells were obtained through immobilization on a
Streptavidin modified mica surface. Pictures showing the morphology of individual Gli349 and
Gli521 molecules in dried and liquid conditions were also obtained and were consistent with
previous electron micrographs of the proteins. Investigation of the interaction between Gli
molecules and Sialyllactose, the direct binding target in gliding was also conducted using AFM
tips decorated with Gli349 or Gli521 molecules and the results showed a specific interaction
between Gli349 and Sialyllactose, whereas Gli521 did not show any interaction. Nano
indentation of living cells was also performed and revealed a great variation in the local stiffness
of the cell, consistent with the available information about M. mobile’s cytoskeleton.
129b

BLAST X PARTICIPANT LIST
130

Christopher Adase
Texas A&M University
3258 TAMU
BSBE Room 303
College Station, TX 77843
Phone: (979) 845-1249
xpage@neo.tamu.edu
Michael Airola
Cornell University
G63 S.T. Olin Laboratory
Chemistry Research Building
Ithaca, NY 14853
Phone: (607) 255-4970
mva5@cornell.edu
Christine Aldridge
Newcastle University
The Medical School
Framlington Place
Newcastle upon Tyne
NE2 4HH
United Kingdom
Phone: +44 1912227704
Fax: +44 1912227424
christine.aldridge@ncl.ac.uk
Phillip Aldridge
Newcastle University
The Medical School
Framlington Place
Newcastle upon tyne
NE2 4HH
United Kingdom
Phone: +44 1912227704
Fax: +44 1912227424
p.d.aldridge@ncl.ac.uk
Roger Alexander
Yale University
Kline Biology Tower 1054
New Haven, CT 06511
Phone: (404) 217-5664
roger.alexander@yale.edu
131
Gladys Alexandre
The University of Tennessee
M407 Walters Life Sciences
1414 West Cumberland Ave.
Knoxville, TN 37996
Phone: (865) 974-0866
Fax: (865) 974-6306
galexan2@utk.edu
Adrian Alvarez
Instituto de Fisiologia Celular - UNAM
Ciudad Universitaria
México D.F. 04510
México
Phone: +525556225738
aalvarez@ifc.unam.mx
Angel Andrade
UNAM
Circuito interior Cd Universitaria
México City 05100
México
Phone: +5256225965
aandrade@live.com.mx
Judy Armitage
Oxford
South Parks Road
Oxford
OX1 3QU
United Kingdom
Phone: +44 1865 613293
armitage@bioch.ox.ac.uk
Teresa Ballado
UNAM
Instituto de Fisiologia Celular
Circuito Exterior S/N Cd. Universitaria
México City 04510
México
Phone: (5255) 56225618
Fax: (5255) 56225611
tballado@ifc.unam.mx

Rina Barak
Weizmann Institute of Science
Herzel St.
Rehovot 76100
Israel
Phone: 972-8-9342710
Fax: 972-8-9344112
rina.barak@weizmann.ac.il
Carlos Arturo Barba Ostria
Instituto de Fisiologia Celular - UNAM
Ciudad Universitaria
México D.F. 04510
México
Phone: +525556225738
cbarba@ifc.unam.mx
Robert Belas
University of Maryland
Biotechnology Institute
701 East Pratt Street
Baltimore, MD 21202
Phone: (410) 234-8876
belas@umbi.umd.edu
James Berleman
University of Iowa
51 Newton Rd.
Iowa City, IA 52242
Phone: (404) 372-4836
berleman@gmail.com
Jaya Bhatnagar
Cornell University
Chemistry Research Bldg.
Ithaca, NY 14853
Phone: (607) 255-4970
jb394@cornell.edu
132
Amber Bible
University of Tennessee, Knoxville
M407 Walters Life Sciences
1414 Cumberland Avenue
Knoxville, TN 37996
Phone: (865) 974-2364
abible@utk.edu
Paola Bisicchia
Trinity College
Lincoln Place Gate
Smurfit Institute of Genetics
Dublin 2
Ireland
Phone: 003538962447
paolabisicchia@yahoo.it
Wesley Black
Virginia Tech
Life Sciences I
Washington St.
Blacksburg, VA 24061
Phone: (540) 231-9381
weblack@vt.edu
Bob Bourret
University of North Carolina
Chapel Hill, NC 27599-7290
Phone: (919) 966-2679
Fax: (919) 962-8103
bourret@med.unc.edu
Richard Branch
University of Oxford
Clarendon Laboratory
Parks Road
Oxford
OX1 3PU
United Kingdom
Phone: +44 01865272357
r.branch1@physics.ox.ac.uk

Ariane Briegel
California Institute of Technology
1200 E. California Blvd.
Mail code 114-96
Pasadena, CA 91125
Phone: (626) 395-8848
briegel@caltech.edu
Mostyn Brown
University of Oxford
South Parks Road
Oxford
OX1 3QU
United Kingdom
Phone: +44 01865 275 298
mostyn.brown@bioch.ox.ac.uk
Iryna Bulyha
Max Planck Institute
for Terrestrial Microbiology
Karl-von-Frisch-Strasse, 8
35043 Marburg
Germany
Phone: +496421178222
Fax: +496421178209
bulyha@mpi-marburg.mpg.de
Victor Bustamante
Universidad Nacional Autonoma de México
Av. Universidad 2001 Colonia Chamilpa
Cuernavaca, Morelos 62210
México
Phone: (52) 777 329 16 27
victor@ibt.unam.mx
Edmundo Calva
Instituto de Biotecnología UNAM
Av. Universidad 2001
Cuernavaca 62210
México
Phone: +52-777-329-1645
Fax: +52-777-313-8673
ecalva@ibt.unam.mx
133
Karen Camargo
Universidad Nacional Autonoma de México
C.U. Instituto de Fisiologia Celular
México Distrito Federal 04510
México
Phone: 52 556225618
karen_100490@hotmail.com
Asharie Campbell
Loma Linda University
11021 Campus Street
Loma Linda, CA 92350
Phone: (909) 558-1000
ajohnson07b@llu.edu
Eva Campodonico
University of California, Berkeley
31 Koshland Hall
Berkeley, CA 94720
Phone: (510) 643-5457
eva.campodonico@berkeley.edu
Brian Cantwell
University of Tennessee
WLS 437
Knoxville, TN 37996-0845
Phone: (865) 974-7687
Fax: (865) 974-4007
bcantwe1@utk.edu
C. Britt Carlson
HHMI/UMDNJ
Center for Advanced Biotechnology and
Medicine
679 Hoes Lane, Room 324
Piscataway, NJ 08854
Phone: (732) 235-4206
carlson@cabm.rutgers.edu

David Castillo
Universidad Nacional Autónoma de México
IFC-UNAM México D.F., C.P. 04510
México City 04510
México
Phone: (+52) 55 562
Fax: (+52) 55 56225611
castillo@ifc.unam.mx
Matt Chapman
University of Michigan
830 North University
Ann Arbor, MI 48109
Phone: (734) 764-7592
Fax: (734) 647-0884
chapmanm@umich.edu
Arnaud Chastanet
Harvard University
16 Divinity ave
Biolabs
Cambridge, MA 02138
Phone: (617) 384-7622
Fax: (617) 496-4642
arnaud@mcb.harvard.edu
Yong-Suk Che
Osaka University
Suita1-3 Yamadaoka
Osaka 565-0871
Japan
Phone: +81-6-6879-4625
yongsuk@fbs.osaka-u.ac.jp
Matthew Copeland
University of Wisconsin-Madison
473 Biochemistry Addition
433 Babcock Drive
Madison, WI 53706
Phone: (608) 263-2636
mfcopeland@wisc.edu
134
Brian Crane
Cornell University
Baker Laboratory
Ithaca, NY 14850
Phone: (607) 254-8634
Fax: (607) 255-1248
bc69@cornell.edu
Sean Crosson
University of Chicago
929 E. 57th St.
GCIS-W138
Chicago, IL 60637
Phone: (773) 834-1926
scrosson@uchicago.edu
Rachel Crowder
Texas A&M University
3258 TAMU
College Station, TX 77843
Phone: (979) 845-1249
rcrowder@tamu.edu
Rick Dahlquist
Dept of Chemistry & Biochemistry
University of California Santa Barbara
Santa Barbara, CA 93106
Phone: (805) 893-5326
dahlquist@chem.ucsb.edu
Miguel De la Cruz
Instituto de Biotecnología UNAM
Av Universidad 2001 Col Chamilpa
Cuernavaca 62210
México
Phone: 52 777 3 29 16 27
mike@ibt.unam.mx

Javier De la Mora
Universidad Nacional Autonoma de México
C.U., Instituto de Fisiologia Celular
México, Distrito Federal 04510
México
Phone: 52 556225618
fbravo@ifc.unam.mx
Nicolas Delalez
University of Oxford
Biochemistry Dept, Microbiology Unit,
South Parks Road
Oxford
OX1 3QU
United Kingdom
Phone: +44 01865613315
nicolas.delalez@bioch.ox.ac.uk
Aurelia Delaune
Institut Pasteur
25 Rue du Dr. Roux
75015 Paris
France
Phone: 33 1 45 68 88 48
Fax: 33 1 45 68 89 38
aureliad@pasteur.fr
Kevin Devine
Trinity College Dublin
Dublin 2
Ireland
Phone: (353)-1-896-1872
Fax: (353)-1-6714968
kdevine@tcd.ie
Miguel Diaz
UNAM
Ciudad Universitaria
Apartado postal 70-243
México 04510
México
Phone: (52) 56225965
madiaz@ifc.unam.mx
135
Edith Diaz-Mireles
Newcastle University Medical School
Framlington Place
Newcastle Upon Tyne
NE2 4HH
United Kingdom
Phone: +44 0191-2228947
Fax: +44 0191-2227424
edith.diaz-mireles@ncl.ac.uk
Jason Dobkowski
University of Michigan
830 N. University
Ann Arbor, MI 48103
Phone: (734) 647-5677
jdobkow@umich.edu
Roger Draheim
Stockholm University
DBB - von Heijne Group
Svante Arrhenius vag 12, plan 4
10691 Stockholm
Sweden
Phone: +4686747656
rogerdraheim@gmail.com
Georges Dreyfus
UNAM
Instituto de Fisiologia Celular
Circuito exterior s/n Cd. Universitaria
México City 04510
México
Phone: (5255) 56225618
gdreyfus@ifc.unam.mx
Sarah Dubrac
Institut Pasteur
25 Rue du Dr. Roux
75015 Paris
France
Phone: (33) 1-45-68-88-48
Fax: (33) 1 45 68 89 38
sdubrac@pasteur.fr

Michael Eisenbach
Weizmann Institute of Science
PO Box 26
Rehovot 76100
Israel
Phone: 972-8-9343923
Fax: 972-8-9472722
m.eisenbach@weizmann.ac.il
Annette Erbse
UC Boulder
76 Chemistry
Boulder, CO 80309
Phone: (303) 492-3597
erbse@colorado.edu
Joseph Falke
University of Colorado
UCB 215
Boulder, CO 80309-0215
Phone: (303) 492-3503
falke@colorado.edu
Melanie Falord
Institut Pasteur
25 Rue du Dr. Roux
75015 Paris
France
Phone: 33 1 44 38 94 87
Fax: 33 1 45 68 89 38
mfalord@pasteur.fr
Hajime Fukuoka
Tohoku University
Sendai Katahira Aoba-Ku
980-8577
Japan
Phone: 81-22-217-5804
Fax: 81-22-217-5804
f-hajime@tagen.tohoku.ac.jp
136
Ana Gallego
Instituto de Biotecnologia, UNAM
Av Universidad 2001
Cuernavaca 62210
México
Phone: 52 777 3291627
analgh@ibt.unam.mx
Elizabeth García-Gómez
Universidad Nacional Autonoma de México,
Instituto de Fisiología Celular
Ciudad Universitaria, México D. F
Ap. Postal 70-243.
México 04510
México
Phone: (55)56225965
egarcia@ifc.unam.mx
Aldo García-Guerrero
Universidad Nacional Autónoma de México
Instituto de Fisiología Celuar
Circuito Exterior s/n Cd. Universitaria
México 04510
México
Phone: (5255) 56225618
aldog.2602@gmail.com
Mathieu Gauthier
Universite Laval
Pavillon Alexandre-Vachon
Quebec QC G1K7P4
Canada
Phone: 418-656-2131
mathieu.gauthier.5@ulaval.ca
Meztlli Gaytán
UNAM
Ap. postal 70-243
Ciudad Universitaria México D.F
México 04510
México
Phone: +52 56225965
ogaytan@ifc.unam.mx

Dimitris Georgellis
UNAM
Circuito exterior S/N
México D.F. 04510
México
Phone: +52 55 5622 5738
dimitris@ifc.unam.mx
Zemer Gitai
Princeton University
LTL-355
Washington Rd
Princeton, NJ 08540
Phone: (609) 258-9420
zgitai@princeton.edu
George Glekas
University of Illinois Urbana-Champaign
409 Medical Sciences Building
506 S. Mathews Ave.
Ubana, IL 61801
Phone: (217) 333-0268
glekas@illinois.edu
Shalom Goldberg
University of Pennsylvania School of
Medicine
422 Curie Blvd.
Philadelphia, PA 19104
Phone: (215) 898-3495
shalom@mail.med.upenn.edu
Juan Gonzalez
University of Texas at Dallas
RL11, 800 W. Campbell, Rd.
Richardson, TX 75080
Phone: (972) 883-2526
jgonzal@utdallas.edu
137
Ricardo González
UNAM
Instituto de Fisiología Celular
Ciudad Universitaria
México 04510
México
Phone: 52 5556225738
rgonzalez@ifc.unam.mx
Bertha Gonzalez-Pedrajo
National Autonomous University of México
Apartado Postal 70-243
México 04510
México
Phone: 5255 56225965
Fax: 5255 56225611
bpedrajo@ifc.unam.mx
Nataliya Gurich
University of Texas at Dallas
RL11, 800 W. Campbell, Rd.
Richardson, TX 75080
Phone: (972) 883-6291
nxg010400@utdallas.edu
Benjamin Hall
University of Oxford
South Parks Road
Oxford
OX1 3QU
United Kingdom
Phone: +441865275380
Fax: +441865275273
benjamin.hall@bioch.ox.ac.uk
Hua Han
UMDNJ/CABM
Rm 326, CABM
679 Hoes Lane
Piscataway, NJ 08854
Phone: (732) 235-4206
han@cabm.rutgers.edu

Rasika Harshey
University of Texas at Austin
1 University Station
A1000
Austin, TX 78712
Phone: (512) 471-6881
Fax: (512) 471-7088
rasika@uts.cc.utexas.edu
Caroline Harwood
University of Washington
Dept of Microbiology- Box 357242
1705 NE Pacific Street
Seattle, WA 98112
Phone: (206) 221-2848
csh5@u.washington.edu
Fumio Hayashi
Gunma University
Kiryu1-5-1 Tenjin
376-8515
Japan
Phone: +81-277-30-1663
Fax: +81-277-30-1663
hayashi@chem-bio.gunma-u.ac.jp
Gerald Hazelbauer
University of Missouri
117 Schweitzer Hall
Columbia, MO 65211
Phone: (573) 882-4845
Fax: (573) 882-5635
hazelbauerg@missouri.edu
María Herrera Seitz
Universidad Nacional de Mar del Plata
Funes 3250
Mar del Plata 7600
Argentina
Phone: 54 223 4753030
Fax: 54 223 4753150
khseitz@mdp.edu.ar
138
Penelope Higgs
Max-Planck-Institute for
Terrestrial Microbiology
Karl-von-Frisch Strasse
D35043 Marburg
Germany
Phone: +49 6421 178301
Fax: +49 6421 178309
higgs@mpi-marburg.mpg.de
Yohei Hizukuri
Nagoya University
NagoyaFuro-Cho, Chikusa-Ku
464-8602
Japan
Phone: 81-52-789-3543
Fax: 81-52-789-3001
4hiz1103@bunshi3.bio.nagoya-u.ac.jp
Shelley Horne
North Dakota State University
NDSU Vet & MIcro Sci
PO Box 6050 - Dept 7690
Fargo, ND 58108
Phone: (701) 231-6741
Fax: (701) 231-9692
sm.horne@ndsu.edu
Basarab Hosu
Harvard University
16 Divinity Avenue
Bio Labs Buliding Rom 3068
Cambridge, MA 02138
Phone: (617) 495-6127
Fax: (617) 496-1114
ghosu@mcb.harvard.edu
Colin Hughes
Cambridge University
Dept of Pathology
Cambridge
CB4 3AH
United Kingdom
Phone: 44-1223-338538
ch@mole.bio.cam.ac.uk

Tatsuya Ibuki
Osaka University
suitayamadaoka 1-3
565-0871
Japan
Phone: 81 06-6879-4625
Fax: 81 06-6879-4652
tatsuya@fbs.osaka-u.ac.jp
Katsumi Imada
Osaka University
Suita1-3 Yamadaoka
565-0871
Japan
Phone: +81-6-6879-4625
Fax: +81-6-6879-4652
kimada@fbs.osaka-u.ac.jp
Takehiko Inaba
Hosei University
Koganei Midori-cho 3-11-15
184-0003
Japan
Phone: +81-42-387-7173
takehiko.inaba.13@k.hosei.ac.jp
Yuichi Inoue
Tohoku University
Sendai Katahira 2-1-1
Aoba-ku, 980-8577
Japan
Phone: 81-22-217-5804
inoue@tagen.tohoku.ac.jp
Christine Josenhans
Medical Uiniversity Hannover
Carl-Neuberg-Strasse 1
30625 Hannover
Germany
Phone: +495115324354
Fax: +495115324354
josenhans.christine@mh-hannover.de
139
Katy Juarez
IBT-UNAM
Av. Universidad 2001, Chamilpa
Cuernavaca 62210
México
Phone: (52) 5556227240
katy@ibt.unam.mx
Jung Kwang-Hwan
Sogang University
Mapo-Gu, Shinsu-Dong 1 R1203
121-742 Seoul
Republic of Korea
Phone: 82-2-705-8795
Fax: 82-2-704-3601
kjung@sogang.ac.kr
Alla Kaserer
University of Oklahoma
620 Parrington Oval
Norman, OK 73019
Phone: (405) 325-1532
Fax: (405) 325-6111
alla@ou.edu
Linda Kenney
Univ. of Illinois-Chicago
835 S. Wolcott M/C 790
Chicago, IL 60612
Phone: (312) 413-0576
Fax: (312) 996-6415
kenneyl@uic.edu
Tsuyoshi Kenri
National Institute of Infectious Disease
Musashimurayama4-7-1
Gakuen, 208-0011
Japan
Phone: 81-42-561-0771
Fax: 81-42-565-3315
kenri@nih.go.jp

Cezar Khursigara
National Institutes of Health
50 South Drive
Bldg 50/Rm 4306
Bethesda, MD 20892
Phone: (301) 594-2236
khursigarac@mail.nih.gov
John Kirby
University of Iowa
51 Newton Road
Iowa City, IA 52242
Phone: (319) 335-7818
Fax: (319) 335-9006
john-kirby@uiowa.edu
Kimberly Kline
Washington Univ School of Medicine
Department of Molecular Microbiology
Box 8230
St. Louis, MO 63110
Phone: (314) 266-0639
k-kline@borcim.wustl.edu
Masafumi Koike
Nagoya University
Chikusa-ku
Nagoya 464-8602
Japan
Phone: 81 0527892992
Fax: 81 0527893001
4koike@bunshi4.bio.nagoya-u.ac.jp
Seiji Kojima
Nagoya University
Furo-cho 1, Chikusa-ku
Nagoya 464-8602
Japan
Phone: 81-52-789-2992
Fax: 81-52-789-3001
4seiji@bunshi4.bio.nagoya-u.ac.jp
140
Changhan Lee
KAIST
CA Daejeon 4101, BMRC, 373-1
Gusung-dong, Yusung-gu 90504
Republic of Korea
Phone: +82-42-350-2669
changhanlee@hotmail.com
Jae-Min Lee
University of Texas at Austin
1 University Station A5000
Austin, TX 78712-0162
Phone: (512) 471-6799
jaeminlee@mail.utexas.edu
Yi-Ying Lee
University of Illinoise at Chicago
835 S Wolcott (M/C 790)
Chicago, IL 60612
Phone: (312) 413-0288
yyinglee@uic.edu
Jun Liu
UT Houston Medical School
6431 Fannin, MSB 2.228
Houston, TX 77030
Phone: (713) 500-5342
jun.liu.1@uth.tmc.edu
Janine Maddock
University of Michigan
830 North University
Ann Arbor, MI 48109
Phone: (734)-936-8068
maddock@umich.edu

Fumiaki Makino
Osaka University
Suita1-3 Yamadaoka, Suita
Osaka 565-0871
Japan
Phone: 81-06-6879-4625
h1839@fbs.osaka-u.ac.jp
Roxana Malpica
University of Alberta
CW405 Biological Sciences Building
Edmonton AB T6G2E9
Canada
Phone: 780-492 4339
malpica@ualberta.ca
Mike Manson
Texas A&M University
MS 3258
BSBE 303
College Station, TX 77843
Phone: (979) 845-5158
mike@mail.bio.tamu.edu
Luary Martinez
UNAM-Instituto de Biotecnologia
Av. Universidad 2001, Colonia Chamilpa
Cuernavaca 62210
México
Phone: 52-777 329 16 27
luary@ibt.unam.mx
Ana Martinez del Campo
UNAM
Instituto de Fisiologia Celular
Circuito exterior s/n Cd. Universitaria
México City 04510
México
Phone: (5255) 56225618
acampo@ifc.unam.mx
141
Diego Massazza
Universidad Nacional de Mar del Plata
Funes 3250
Mar del Plata 7600
Argentina
Phone: 54 223 4753030
Fax: 54 223 4753150
diegomassazza@hotmail.com
Emilia Mauriello
University of California, Berkeley
31 Koshland Hall
Berkeley, CA 94720
Phone: (510) 643-5457
emilia-mauriello@berkeley.edu
Jonathan McMurry
Kennesaw State University
1000 Chastain Rd. MB #1203
Kennesaw, GA 30144
Phone: (770) 499-3238
jmcmurr1@kennesaw.edu
Paul Milewski
University of Wisconsin
480 Lincoln Dr.
Madison, WI 53706
Phone: (608) 262-3220
milewski@math.wisc.edu
Kelly Miller
West Virginia Univ., Health Sciences Center
Box 9177, Room 2077
Morgantown, WV 26506
Phone: (304) 293-5959
kmille49@mix.wvu.edu

Makoto Miyata
Osaka City University
3-3-138 Sugimoto Sumiyoshi-ku
Osaka 558-8585
Japan
Phone: +81(6)6605 3157
Fax: +81(6)6605 3158
miyata@sci.osaka-cu.ac.jp
Md Motaleb
East Carolina University
600 Moye Blvd
BT 116
Greenville, NC 27834
Phone: (252) 744-3129
Fax: (252) 744-3535
motalebm@ecu.edu
Tarek Msadek
Institut Pasteur
25 Rue du Dr. Roux
75015 Paris
France
Phone: 33 1 45 68 88 09
Fax: 33 1 45 68 89 38
tmsadek@pasteur.fr
Abishek Muralimohan
University of Wisconsin - Madison
433 Babcock Dr.
Madison, WI 53706
Phone: (608) 263-2636
muralimohan@wisc.edu
Shuichi Nakamura
Osaka Univevrsity
Suita1-3,Yamadaoka
Osaka, 565-0871
Japan
Phone: +81-6-6879-4625
naka@fbs.osaka-u.ac.jp
142
Daisuke Nakane
Osaka City University
3-3-138 Sugimoto Sumiyoshi-ku
Osaka 558-8585
Japan
Phone: +81 6 6605 3157
Fax: +81 6 6605 3158
nakane@sci.osaka-cu.ac.jp
Beiyan Nan
University of California, Berkeley
Department of Molecular and Cell Biology
Berkeley, CA 94720
Phone: (510) 643-5457
nanbeiyan@gmail.com
Silke Neumann
Ruprecht-Karls-Universität Heidelberg
Im Neuenheimer Feld 282
69120 Heidelberg
Germany
Phone: +49 6221546856
s.neumann@zmbh.uni-heidelberg.de
Vincent Nieto
University of Texas at Austin
2506 Speedway
Austin, TX 78712
Phone: (512) 471-6799
vince.nieto@gmail.com
So-ichiro Nishiyama
Hosei University
3-7-2 Kajino-cho, Koganei
Koganei 184-8584
Japan
Phone: +81-42-387-7173
Fax: +81-42-387-7002
soichiro.nishiyama.s3@k.hosei.ac.jp

Takahiro Nonaka
Osaka City University
3-3-138 Sugimoto Sumiyoshi-ku
Osaka 558-8585
Japan
Phone: +81-(0)6-6605-2575
nonaka@sci.osaka-cu.ac.jp
Luis Alberto Núñez Oreza
Instituto de Fisiologia Celular - UNAM
Ciudad Universitaria
México D.F. 04510
México
Phone: +525556225738
lanoreza@hotmail.com
Ricardo Oropeza
Instituto de Biotecnologia
Universidad Nacional Autonoma de México
Av. UNIVERSIDAD # 2001
col. Chamilpa
Cuernavaca 62210
México
Phone: 52-777-329-16-27
oropeza@ibt.unam.mx
Davi Ortega
University of Tennessee / ORNL
2856 Brock Av
Knoxville, TN 37919
Phone: (865) 384-9507
dortega@utk.edu
Karen Ottemann
UC Santa Cruz
1156 High Street
METX
Santa Cruz, CA 95064
Phone: (831) 459-3482
ottemann@ucsc.edu
143
Rebecca Parales
University of California, Davis
226 Briggs Hall
1 Shields Ave.
Davis, CA 95616
Phone: (530) 754-5233
Fax: (530) 752-9014
reparales@ucdavis.edu
Chankyu Park
KAIST
95014 CA Daejoen 4101, BMRC, KAIST
373-1, Gusung-dong, Yusung-gu
Republic of Korea
Phone: +82-42-350-2669
nasa@kaist.ac.kr
John Parkinson
University of Utah
257 South 1400 East
Salt Lake City, UT 84112-0840
Phone: (801) 581-7639
parkinson@biology.utah.edu
Jonathan Partridge
University of Texas at Dallas
800 W Campbell Road
Richardson, TX 75080
Phone: (972) 883-2519
j.partridge@gmail.com
Gabriela Peña-Sandoval
Universidad Nacional Autonoma de México
Circuito Exterior s/n
Ciudad Universitaria, Copilco
México City 04510
México
Phone: (52) 55 5622 5738
gsandova@ifc.unam.mx

Abiola Pollard
Cornell University
Chemistry Research Building
G63 S.T. Olin Laboratory
Ithaca, NY 14853
Phone: (607) 255-4970
amp65@cornell.edu
Steven Porter
University of Oxford
South Parks Road
Oxford
OX1 3QU
United Kingdom
Phone: 01865 275298
steven.porter@bioch.ox.ac.uk
Birgit Prüß
North Dakota State University
1523 Centennial Blvd.
Fargo, ND 58108
Phone: (701) 231-7848
birgit.pruess@ndsu.edu
Simon Rainville
Laval University
Pavillon d'optique photonique
2375, rue de la Terrasse
Québec QC G1V 0A6
Canada
Phone: (418) 656-2131
Fax: (418) 656-2623
rainville@phy.ulaval.ca
Everardo Ramírez
Universidad Nacional Autonoma de México
Cto. Exterior, Cd. Universitaria.
Coyoacán, D. F.
México, D. F. 04510
México
Phone: 525556225738
evergrinch@yahoo.com.mx
144
Christopher Rao
University of Illinois
211 Roger Adams Lab
Urbana, IL 61801
Phone: (217) 244-2247
chris@scs.uiuc.edu
Sylvia Reimann
Loyola University Chicago
2160 S. First Ave
Maguire Bldg 105, Rm 3822
Maywood, IL 60153
Phone: (708) 216-0845
Fax: (708) 216-9574
sreimann@lumc.edu
S. James Remington
University of Oregon
Institute of Molecular Biology
Eugene, OR 97403
Phone: (541) 346-5190
jremington@uoxray.uoregon.edu
Peter Reuven
Weizmann Institute of Science
Ullman Bldg 11-a
Rehovot 76100
Israel
Phone: 00972 8 934 2701
Fax: 00972 8 934 4112
peter.reuven@weizmann.ac.il
Mark Roberts
University of Oxford
South Parks Road
Oxford
OX1 3QU
United Kingdom
Phone: 44 1865 613367
Fax: 44 1865 613338
mark.roberts@bioch.ox.ac.uk

Claudia Rodríguez
UNAM, Instituto de Fisiología Celular
Ciudad Universitaria, Col Copilco
Distrito Federal 04510
México
Phone: 52 01 55 56 22 57 38
Fax: 52 01 55 56 22 56 11
crangel@ifc.unam.mx
Mariana Romo
Instituto de Fisiología Celular, UNAM
Circuito Externo S/N Col Copilco
México City 04510
México
Phone: 52 55 62 25 965
mromoc@ifc.unam.mx
Kathleen Ryan
UC Berkeley
Plant & Microbial Biology
251 Koshland Hall
Berkeley, CA 94720
Phone: (510) 643-9387
Fax: (510) 642-4995
kryan@nature.berkeley.edu
Supreet Saini
University of Illinois, Urbana Champaign
114 Roger Adams Lab, Box C-3
MC-712, 600 South Mathews Av
Urbana, IL 61801
Phone: (217) 244-7528
saini3@uiuc.edu
Griselda Salas
Universidad Nacional Autónoma de México
Cto. Exterior, Cd. Universitaria
Coyoacán, D. F.
México, D. F. 04510
México
Phone: 525556225738
gsalas@ifc.unam.mx
145
Oscar Sánchez
UNAM
Cto. Ext. S/N Ciudad Universitaria
México, D.F. 04510
México
Phone: 525556225738
Fax: 525556225611
oscars_69_2@hotmail.com
Birgit Scharf
Virginia Polytechnic Institute
and State University
Life Science I
Washington Street
Blacksburg, VA 24061
Phone: (617) 495-4217
Fax: (617) 496-1114
bscharf@vt.edu
Florian Schubot
Virginia Tech
Life Science I, Room 125
Washington Street
Blacksburg, VA 24061
Phone: (540) 231-2393
fschubot@vt.edu
Andrew Seely
Texas A&M University
MS 3258 TAMU
BSBE Room 303
College Station, TX 77843
Phone: (979) 845-1249
aseely@mail.bio.tamu.edu
Harold Shanafield
University of Tennessee
1414 West Cumberland
F437
Knoxville, TN 37996
Phone: (865) 974-7687
hshanafi@utk.edu

Thomas Shimizu
Harvard University
16 Divinity Ave (BL3063)
Cambridge, MA 02138
Phone: (617) 495-4217
Fax: (617) 496-1114
tshimizu@mcb.harvard.edu
Ruth Silversmith
University of North Carolina
Room 804 Mary Ellen Jones
Chapel Hill, NC 27599
Phone: (919) 966-2679
silversr@med.unc.edu
Julie Simons
University of Wisconsin
480 Lincoln Dr.
Madison, WI 53706
Phone: (608) 263-3239
simons@math.wisc.edu
Lotte Søgaard-Andersen
Max Planck Institute
for Terrestrial Microbiology
Karl-von-Frisch Str.
35043 Marburg
Germany
Phone: +49 6421 178 201
Fax: +49 6421 178 209
sogaard@mpi-marburg.mpg.de
Erik Sommer
University of Heidelberg
Im Neuenheimer Feld 282
69120 Heidelberg
Germany
Phone: +49 6221 546856
e.sommer@zmbh.uni-heidelberg.de
146
Stephen Spiro
University of Texas at Dallas
800 W Campbell Road
Richardson, TX 75080
Phone: (972) 883-6896
stephen.spiro@utdallas.edu
Diane Stassi
NIH
6701 Rockledge Dr.
Room 3202, MSC 7808
Bethesda, MD 20892
Phone: (301) 435-2514
Fax: (301) 480-0940
stassid@csr.nih.gov
Ann Stock
UMDNJ
Robert Wood Johnson Medical School
Center for Advanced Biotech. and Medicine
679 Hoes Lane
Piscataway, NJ 07976
Phone: (732) 235-4844
Fax: (732) 235-5289
stock@cabm.rutgers.edu
Claudia Studdert
Universidad Nacional de Mar del Plata
Funes 3250
Mar del Plata 7600
Argentina
Phone: 54 223 4753030
Fax: 54 223 4753150
studdert@mdp.edu.ar
Kalin Swain
University of Colorado, Boulder
Dept of Chemistry and Biochemistry
Campus Box 215
Boulder, CO 80309
Phone: (303) 492-3592
Fax: (303) 493-5894
swain@colorado.edu

Hendrik Szurmant
The Scripps Research Institute
10550 N Torrey Pines Rd
MEM-116
La Jolla, CA 92037
Phone: (858) 784-7904
Fax: (858) 784-7966
szurmant@scripps.edu
Barry Taylor
Loma Linda University
Alumni Hall
Loma Linda, CA 92350
Phone: (909) 558 4881
Fax: (909) 558 4035
bltaylor@llu.edu
Kai Thormann
Max Planck Institute
For Terrestrial Microbiology
Karl-von-Frisch-Strasse
D-35043 Marburg
Germany
Phone: +49 6421 178 302
Fax: +49 6421 178 309
thormann@mpi-marburg.mpg.de
Hoa Tran
University of Massachusetts
Morrill IV N
639 N.Plesant St.
Amherst, MA MA 01003
Phone: (413) 545-9647
htt@chem.umass.edu
Yuhai Tu
IBM Research
1101 Kichawan Rd./Rt. 134
Yorktown Heights, NY 10598
Phone: (914) 945-2762
yuhai@us.ibm.com
147
Alejandra Vazquez Ramos
Universidad Nacional Autónoma de México
Av. Universidad #2034 Col. Chamilpa
Cuernavaca 62210
México
Phone: 52 777 329 1627
Fax: 52 777 331 38673
avazquez@ibt.unam.mx
Juan-Jesus Vicente Ruiz
University of California, Berkeley
31 Koshland Hall
Berkeley, CA 94720
Phone: (510) 643-5457
juanjesus.vicente@gmail.com
Hera Vlamakis
Harvard Medical School
200 Longwood Ave.
D1-219
Boston, MA 02115
Phone: (617) 432-4359
hera_vlamakis@hms.harvard.edu
George Wadhams
Oxford University
South Parks Road
Oxford
OX1 3QU
United Kingdom
Phone: +44 01865 613329
george.wadhams@bioch.ox.ac.uk
Kylie Watts
Loma Linda University
Dept of Microbiology & Mol Genetics
AHBS 120
Loma Linda, CA 92350
Phone: (909) 558-1000
Fax: (909) 558-4035
kwatts@llu.edu

Ann West
University of Oklahoma
620 Parrington Oval
Norman, OK 73019
Phone: (405) 325-1529
Fax: (405) 325-6111
awest@ou.edu
Jonathan Willett
University of Iowa
51 Newton Road
Iowa City, IA 52242
Phone: (319) 335-7938
jonathan-willett@uiowa.edu
Alan Wolfe
Loyola University Chicago
Maguire Center
2160 South First Avenue
Maywood, IL 60153
Phone: (708) 216-5814
Fax: (708) 216-9574
awolfe@lumc.edu
Gus Wright
Texas A&M University
MS 3258 BSBE 303
College Station, TX 77843
Phone: (979) 845-1249
gwright@mail.bio.tamu.edu
Kang Wu
University of Illinois, Urbana Champaign
114 RAL Box C-3 (M/C 712)
600 S Mathews Ave,
Urbana, IL 61801
Phone: (217) 244-7528
kangwu@uiuc.edu
148
Zhaomin Yang
Virginia Tech
103 LS1 (Mailcode: 0910)
Blacksburg, VA 24061
Phone: (540) 231-1350
zmyang@vt.edu
Shinsuke Yoshimura
Osaka University
Suita 3-#601, Yamadaoka 1-chome
Osaka 565-0871
Japan
Phone: 81-06-6879-4625
Fax: 81-06-6879-4652
yoshimura@fbs.osaka-u.ac.jp
Junhua Yuan
Harvard University
16 Divinity Ave, BioLabs 3063
Cambridge, MA 02138
Phone: (617) 495-4217
jyuan@mcb.harvard.edu
David Zusman
University of California
16 Barker Hall #3204
Berkeley, CA 94720-3204
Phone: (510) 642-2293
Fax: (510) 643-6334
zusman@berkeley.edu

BLAST STAFF
Tarra Bollinger
Molecular Biology Consortium
835 S. Wolcott (M/C 790)
Chicago, IL 60612
Phone: (312) 996-1216
Fax: (312) 413-2952
tbolli1@uic.edu
149
Peggy O'Neill
Molecular Biology Consortium
835 S. Wolcott (M/C 790)
Chicago, IL 60612
Phone: (312) 996-1216
Fax: (312) 413-2952
oneill@uic.edu

INDEX
150

A
Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
Adase, Christopher, 90, 130 - Manson, Michael
Airola, Michael, 63, 130 - Crane, Brian
Aldridge, Christine, 52, 130 - Aldridge, Phillip
Aldridge, Phillip, 52, 53, 130 - Aldridge, Phillip
Alexander, Roger, 67, 130 - Emonet, Thierry
Alexandre, Gladys, 47, 54, 130 - Alexandre, Gladys
Alvarez, Adrian, 69, 130 - Georgellis, Dimitris
Andrade, Angel, 130 - González-Pedrajo, Bertha
Armitage, Judy, 4, 55, 56, 57, 130 - Armitage, Judy
B
Ballado, Teresa, 130 - Dreyfus, Georges
Barak, Rina, 132 - Eisenbach, Michael
Barba Ostria, Carlos Arturo, 129, 132 - Georgellis, Dimitris
Belas, Robert, 37, 132 - Belas, Robert
Berleman, James, 12, 132 - Kirby, John
Bhatnagar, Jaya, 34, 132 - Crane, Brian
Bible, Amber, 47, 132 - Alexandre, Gladys
Bisicchia, Paola, 7, 132 - Devine, Kevin
Black, Wesley, 125, 132 - Yang, Zhaomin
Bollinger, Tarra, 149 - Matsumura, Philip
Bourret, Bob, 2, 132 - Bourret, Bob
Branch, Richard, 20, 132 - Berry, Richard
Briegel, Ariane, 36, 133 - Jensen, Grant
Brown, Mostyn, 55, 133 - Armitage, Judith
Bulyha, Iryna, 9, 133 - Søgaard-Andersen, Lotte
Bustamante, Victor, 133 - Puente, Jose Luis
C
Calva, Edmundo, 38, 60, 61, 133 - Calva, Edmundo
Camargo, Karen, 133 - Dreyfus, Georges
Campbell, Asharie Johnson, 120, 133 - Taylor, Barry
Campodonico, Eva, 133 - Zusman, David
Cantwell, Brian, 126, 133 - Zhulin, Igor
Carlson, C. Britt, 133 - Stock, Ann
Castillo, David, 65, 134 - Dreyfus, Georges
Chapman, Matt, 40, 134 - Chapman, Matt
Chastanet, Arnaud, 50, 134 - Losick, Rich
Che, Yong-Suk, 100, 134 - Namba, Keiichi
Copeland, Matthew, 123, 134 - Weibel, Douglas
Crane, Brian, 34, 63, 64, 134 - Crane, Brian
Crosson, Sean, 6, 134 - Crosson, Sean
Crowder, Rachel, 91, 134 - Manson, Michael
D
Dahlquist, Rick, 134 - Dahlquist, Rick
151

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
De la Cruz, Miguel, 61, 134 - Calva, Edmundo
De la Mora, Javier, 66, 135 - Dreyfus, Georges
Delalez, Nicolas, 56, 135 - Armitage, Judith
Delauné, Aurelia, 98, 135 - Msadek, Tarek
Devine, Kevin, 7, 135 - Devine, Kevin
Diaz, Miguel, 135 - González-Pedrajo, Bertha
Diaz-Mireles, Edith, 59, 135 - Bolam, David
Dobkowski, Jason, 9, 135 - Maddock, Janine
Draheim, Roger, 121, 135 - von Heijne, Gunnar
Dreyfus, Georges, 41, 65, 66, 135 - Dreyfus, Georges
Dubrac, Sarah, 8, 135 - Msadek, Tarek
E
Eisenbach, Michael, 21, 136 - Eisenbach, Michael
Erbse, Annette, 35, 136 - Falke, Joseph
F
Falke, Joseph, 35, 68, 136 - Falke, Joseph
Falord, Mélanie, 99, 136 - Msadek, Tarek
Fukuoka, Hajime, 78, 136 - Ishijima, Akihiko
G
Gallego, Ana, 136 - Calva, Edmundo
García-Gómez, Elizabeth, 72, 136 - González-Pedrajo, Bertha
García-Guerrero, Aldo, 136 - Dreyfus, Georges
Gauthier, Mathieu, 19, 136 - Rainville, Simon
Gaytán, Meztlli, 136 - González-Pedrajo, Bertha
Georgellis, Dimitris, 3, 69, 70, 137 - Georgellis, Dimitris
Gitai, Zemer, 14, 137 - Gitai, Zemer
Glekas, George, 29, 137 - Ordal, George
Goldberg, Shalom, 45, 137 - DeGrado, William
Gonzalez, Juan, 44, 71, 137 - Gonzalez, Juan
González, Ricardo, 70, 137 - Georgellis, Dimitris
González-Pedrajo, Bertha, 72, 137 - González-Pedrajo, Bertha
Gurich, Nataliya, 71, 137 - Gonzalez, Juan
H
Hall, Benjamin, 113, 137 - Sansom, Mark
Han, Hua, 117, 137 - Stock, Ann
Harshey, Rasika, 73, 74, 138 - Harshey, Rasika
Harwood, Caroline, 138 - Harwood, Carrie
Hayashi, Fumio, 83, 84, 85, 138 - Oosawa, Kenji
Hazelbauer, Gerald, 138 - Hazelbauer, Gerald
Herrera Seitz, María, 115, 138 - Shingler, Victoria
Higgs, Penelope, 10, 138 - Higgs, Penelope
Hizukuri, Yohei, 75, 138 - Homma, Michio
Horne, Shelley, 138 - Prüß, Birgit
Hosu, Basarab, 138 - Berg, Howard
152

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
Hughes, Colin, 138 - Hughes, Colin
I
Ibuki, Tatsuya, 101, 139 - Namba, Keiichi
Imada, Katsumi, 16, 139 - Namba, Keiichi
Inaba, Takehiko, 81, 139 - Kawagishi, Ikuro
Inoue, Yuichi, 79, 139 - Ishijima, Akihiko
J
Josenhans, Christine, 80, 139 - Josenhans, Christine
Juarez, Katy, 139 - Juarez, Katy
Jung, Kwang-Hwan, 46, 139 - Jung, Kwang -Hwan
K
Kaserer, Alla, 23, 139 - West, Ann
Kenney, Linda, 139 - Kenney, Linda
Kenri, Tsuyoshi, 119, 139 - Takahashi, Motohide
Khursigara, Cezar, 32, 140 - Subrmaniam, Sriram
Kirby, John, 12, 86, 87, 140 - Kirby, John
Kline, Kimberly, 77, 140 - Hultgren, Scott
Koike, Masafumi, 76, 140 - Homma, Michio
Kojima, Seiji, 17, 140 - Homma, Michio
L
Lee, Changhan, 107, 140 - Park, Chankyu
Lee, Jae-Min, 73, 140 - Harshey, Rasika
Lee, Yi-Ying, 28, 93, 140 - Matsumura, Philip
Liu, Jun, 88, 140 - Liu, Jun
M
Maddock, Janine, 89, 140 - Maddock, Janine
Makino, Fumiaki, 102, 141 - Namba, Keiichi
Malpica, Roxana, 109, 141 - Raivio, Tracy
Manson, Michael, 31, 90, 91, 92, 141 - Manson, Michael
Martínez del Campo, Ana, 41, 141 - Dreyfus, Georges
Martinez, Luary, 27, 141 - Puente, Jose Luis
Massazza, Diego, 118, 141 - Studdert, Claudia
Mauriello, Emilia, 13, 141 - Zusman, David
McMurry, Jonathan, 94, 141 - McMurry, Jonathan
Milewski, Paul, 95, 141 - Milewski, Paul
Miller, Kelly, 62, 141 - Charon, Nyles
Miyata, Makoto, 96, 97, 142 - Miyata, Makoto
Motaleb, Md, 42, 142 - Motaleb, Md
Msadek, Tarek, 8, 98, 99, 142 - Msadek, Tarek
Muralimohan, Abishek, 124, 142 - Weibel, Douglas
N
Nakamura, Shuichi, 103, 142 - Namba, Keiichi
153

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
Nakane, Daisuke, 96, 142 - Miyata, Makoto
Nan, Beiyan, 128, 142 - Zusman, David
Neumann, Silke, 49, 142 - Sourjik, Victor
Nieto, Vincent, 74, 142 - Harshey, Rasika
Nishiyama, So-ichiro, 82, 142 - Kawagishi, Ikuro
Nonaka, Takahiro, 97, 143 - Miyata, Makoto
Núñez Oreza, Luis Alberto, 143 - Georgellis, Dimitri
O
O'Neill, Peggy, 149 - Matsumura, Philip
Oropeza, Ricardo, 38, 143 - Calva, Edmundo
Ortega, Davi, 143 - Zhulin, Igor
Ottemann, Karen, 105, 143 - Ottemann, Karen
P
Parales, Rebecca, 106, 143 - Parales, Rebecca
Park, Chankyu, 107, 143 - Park, Chankyu
Parkinson, John, 143 - Parkinson, John
Partridge, John, 24, 143 - Spiro, Stephen
Peña-Sandoval, Gabriela, 3, 143 - Georgellis, Dimitris
Pollard, Abiola, 64, 144 - Crane, Brian
Porter, Steven, 4, 144 - Armitage, Judith
Prüß, Birgit, 43, 144 - Prüß, Birgit
R
Rainville, Simon, 19, 108, 144 - Rainville, Simon
Ramírez, Everardo, 144 - Georgellis, Dimitris
Rao, Christopher, 15, 110, 111, 144 - Rao, Christopher
Reimann, Sylvia, 25, 144 - Wolfe, Alan
Remington, James, 30, 144 - Remington, James
Reuven, Peter, 21, 144 - Eisenbach, Michael
Roberts, Mark, 57, 144 - Armitage, Judith
Rodríguez, Claudia, 145 - Georgellis, Dimitris
Romo, Mariana, 145 - González-Pedrajo, Bertha
Ryan, Kathleen, 112, 145 - Ryan, Kathleen
S
Saini, Supreet, 110, 145 - Rao, Christopher
Salas, Griselda, 145 - Georgellis, Dimitris
Sánchez, Oscar, 145 - Georgellis, Dimitris
Scharf, Birgit, 145 - Scharf, Birgit
Schubot, Florian, 114, 145 - Schubot, Florian
Seely, Andrew, 92, 145 - Manson, Michael
Shanafield, Harold, 127, 145 - Zhulin, Igor
Shimizu, Thomas, 48, 146 - Berg, Howard
Silversmith, Ruth, 146 - Bourret, Robert
Simons, Julie, 95, 146 - Milewski, Paul
Søgaard-Andersen, Lotte, 9, 146 - Søgaard-Andersen, Lotte
154

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
Sommer, Erik, 116, 146 - Sourjik, Victor
Spiro, Stephen, 24, 146 - Spiro, Stephen
Stassi, Diane, 146 - Stassi, Diane
Stock, Ann, 117, 146 - Stock, Ann
Studdert, Claudia, 118, 146 - Studdert, Claudia
Swain, Kalin, 68, 146 - Falke, Joseph
Szurmant, Hendrik, 5, 147 - Szurmant, Hendrik
T
Taylor, Barry, 33, 120, 147 - Taylor, Barry
Thormann, Kai, 18, 147 - Thormann, Kai
Tran, Hoa, 26, 147 - Weis, Robert
Tu, Yuhai, 22, 147 - Tu, Yuhai
V
Vazquez Ramos, Alejandra, 147 - Puente, Jose Luis
Vicente Ruiz, Juan-Jesus, 147 - Zusman, David
Vlamakis, Hera, 39, 147 - Kolter, Roberto
W
Wadhams, George, 147 - Wadhams, George
Watts, Kylie, 33, 147 - Taylor, Barry
West, Ann, 23, 148 - West, Ann
Willett, Jonathan, 87, 148 - Kirby, John
Wolfe, Alan, 25, 148 - Wolfe, Alan
Wright, Gus, 31, 148 - Manson, Michael
Wu, Kang, 111, 148 - Rao, Christopher
Y
Yang, Zhaomin, 11, 125, 148 - Yang, Zhaomin
Yoshimura, Shinsuke, 104, 148 - Namba, Keiichi
Yuan, Junhua, 58, 148 - Berg, Howard
Z
Zusman, David, 13, 128, 148 - Zusman, David
155