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Japan Marine Science and Technology Center Research Activities Frontier Research System for Extremophiles Object and Outline of Research and Development Frontier Research System for Extremophiles conducts research on the organisms thriving in the deep-sea and deep-subsurface from the viewpoint of extremophiles. We contribute to the progress of science and human welfare by elucidating: ) what kinds of organisms are living in such extreme environments as the deep-sea and deep-subsurface, ) what are their distinctive features, and ) what is their usefulness in our daily life and/or industrial applications. The scientific target is to elucidate the adaptation mechanisms of extremophiles toward such extreme environments and to understand the origin of life through the discovery of the most ancient microorganisms. Another target is to develop new biotechnologies through supplying new sources of useful microorganisms. Also, The Bio- Venture Center for Extremophiles offers a forum for collaboration with researchers from industries that are interested in exploiting the biological and chemical potential of extremophiles. Isolation and cultivation of novel microorganisms from deep-sea sediments and deep-subsurface core samples are continuously carried out. In addition, multicellular organisms in the deep-sea are captured and maintained by using the DEEP AQUARIUM system. The tissue of deep-sea animals has been successfully cultured under laboratory conditions. The whole genomic sequence of thermophilic Geobacillus kaustrophilus HTA- isolated from the Mariana Trench has been mostly determined, and the comparative analysis of genomes in Bacillus species are conducted. On the other hand, piezo-phisiology is aimed to elucidate microbial life under high pressure environments e.g., the identification of genes responsible for high-pressure growth in yeast and the construction of transformants that respond to high hydrostatic pressure in bacteria are carried out. Also, research on the behavior of biomaterials and colloidal dispersions in supercritical water, one of extreme environments in deep-sea and deep-subsurface, is continuing. The second Bio-Venture forum was held. Research collaboration is successfully proceeding with companies that are interested in exploiting new biotechnologies. Research Results 1. Microbial genome analysis Aerobic endospore-forming Gram-positive Bacillus species are distributed nearly ubiquitously in nature. Recently some of these species have been reclassified as members of species belonging to new genera such as Alicyclobacillus, Amphibacillus, Brevibacillus, Geobacillus, Halobacillus, Oceanobacillus, and Salibacillus. These organisms have often been isolated from various terrestrial soils and deep-sea sediments. It is known that Bacillus-related species have a wide range of environments for growth at pH -, with temperatures around -˚C, salinity from to %- NaCl, and pressures from . MPa (atmospheric pressure) to at least MPa corresponding to the pressure at a depth of m. Thus, these extremophilic Bacillus-related species could possess adaptations to multiple extreme environments including high and low temperature, high and low pH, and high salinity. Now we are very intrigued by the questions of how these adaptive capabilities were acquired in their genome and what the original genome structure was in the ancestral progenitor Bacillus species. We initiated the microbial genome sequencing project in and have determined the complete genome sequences of Bacillus halodurans and Oceanobacillus iheyensis. An alkaliphilic Bacillus halodurans C- isolated from terrestrial soil in is the most characterized industrial strain, which produces various kinds of useful enzymes. The B.halodurans C- is the first industrial strain whose complete genome sequence was determined. Oceanobacillus iheyensis HTE isolated from the deep ocean near Nanseiisland at a depth of m is an alkaliphilic and extremely halotolerant bacterium. This strain was selected as the second bacterium for the genome 67
JAMSTEC 2002 Annual Report Frontier Research System for Extremophiles sequencing project to figure out the mechanisms for adaptation to alkaline and saline environments. In addition, we proceeded with the third whole genome sequencing project for the thermophilic Geobacillus halodurans HTA isolated from the deepest ocean at a depth of m to know the molecular diversity among extremophilic Bacillus-related species. Here, we report the determination of the whole genome sequence of thermophilic G. kaustophilus HTA, comparative genomic analysis of the two alkaliphiles, and functional analysis of the genes involved in alkaliphily. 1.1. Comparative analysis of the O. iheyensis genome. The genome consists of , Mb, encoding many proteins potentially associated with roles in regulation of intracellular osmotic pressure and pH homeostasis. The genes involved in alkaliphily were highlighted based on comparative analysis with three Bacillus species and two other Gram-positive species. The genome of O. iheyensis provides us with a unique opportunity to investigate the genes that underlie the capability to adapt to alkaline or hypersaline environment. The first issue was addressed by comparing the orthologous relationships among the proteins deduced from all CDSs identified in the five genomes of Grampositive bacteria (Fig. ). Out of , proteins identified in the O. iheyensis genome, putative proteins (.%) have no orthologous relationship to proteins encoded in the four other genomes (Fig. ). Seven hundred ninety-three proteins (.%) were orthologs identified among five Gram-positive bacterial species and (.%) were identified as common proteins only among Bacillus-related species. Two hundred forty-three putative proteins (.%) were shared only between the two alkaliphiles, O. iheyensis and B. halodurans. As shown in Fig. , the trend of orthologous relationships was almost the same in the case of a OB 838 OB/BH/BS /SA/CA 793 134 64 b (26.8%) BH/CA BH/OB 243 (6.0%) (6.6%) 267 BH/SA BH 1091 BH/BS BH/SA/CA 9 BH/OB/CA 41 BH/OB/SA 70 B. halodurans BH/BS/OB /SA/CA 793 (19.5%) BH/BS/OB/SA 373 (9.2%) BH/BS/OB/CA 283 (8.7%) (7.0%) BH/BS/OB 354 BH/OB/SA/CA 28 BH/BS/SA/CA 47 BH/BS/SA 78 BH/BS/CA 133 (22.8%) (22.7%) 66 OB/CA Ob. iheyensis OB/BH/BS/SA 73 OB/SA 373 160 OB/BS (4.6%) (10.7%) OB/BH 283 243 (7.0%) OB/BH/BS/CA 354 (8.1%) (10.1%) OB/BH/BS BS 1069 OB/SA/CA 22 OB/BS/CA 56 OB/BS/SA 87 OB/BH/CA 41 OB/BH/SA 70 OB/BH/SA/CA 28 OB/BS/SA/CA 45 BS/BH/OB /SA/CA 793 (19.3%) (26.0%) BS/BH/OB/SA B. subtilis 373 (9.1%) BS/BH/OB/CA 267 283 (3.9%) (6.9%) BS/BH BS/BH/OB 354 (6.5%) (8.6%) BS/SA/CA 34 BS/OB/CA 56 BS/BH/SA/CA 47 BS/OB/SA 87 BS/OB/SA/CA 45 BS/BH/CA 133 BS/BH/SA 78 BS/CA 143 BS/SA 105 BS/OB 160 Fig.1 Summary of orthologous relationships among all CDSs identified in the genomes of bacilli and other major Grampositive species. a, Orthologous relationships based on Ob. iheyensis genome. b, Orthologous relationships based on B. halodurans genome. c, Orthologous relationships based on B. subtilis genome. Abbreviations: OB, Oceanobacillus iheyensis; BH, Bacillus halodurans; BS, Bacillus subtilis; SA, Staphylococcus aureus; CA, Clostridium acetobutylicum. c 68
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