Genome sequence of the deep-sea ␥-proteobacterium Idiomarina loihiensis reveals amino acid fermentation as a source of carbon and energy Shaobin Hou*†, Jimmy H. Saw*, Kit Shan Lee*, Tracey A. Freitas*†‡, Claude Belisle*, Yutaka Kawarabayasi§, Stuart P. Donachie*, Alla Pikina*, Michael Y. Galperin¶, Eugene V. Koonin¶, Kira S. Makarova¶, Marina V. Omelchenko¶, Alexander Sorokin¶, Yuri I. Wolf¶, Qing X. Li储, Young Soo Keum储, Sonia Campbell储, Judith Denery储, Shin-Ichi Aizawa**, Satoshi Shibata**††, Alexander Malahoff‡‡, and Maqsudul Alam*†‡§§ *Department of Microbiology, University of Hawaii, Snyder Hall 111, 2538 The Mall, Honolulu, HI 96822; †Center for Genomics, Proteomics, and Bioinformatics Research Initiative, University of Hawaii, Keller Hall 319, Honolulu, HI 96822; ‡Maui High Performance Computing Center, 550 Lipoa Parkway, Kihei, Maui, HI 96753; §Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Higashi 1-1-1, Tsukuba-shi, Ibaraki 305-8566, Japan; ¶National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894; 储Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822; **‘‘Soft Nano-Machine’’ Project, Japan Science and Technology Corporation, 1064-18 Hirata, Takanezawa, Shioya-gun, Tochigi 329-1206, Japan; ††Department of Agriculture, Shizuoka University, Yata, Shizuoka 422-8529, Japan; and ‡‡Institute of Geological and Nuclear Sciences, 69 Gracefield Road, P.O. Box 30368, Lower Hutt, New Zealand Edited by Rita R. Colwell, University of Maryland, College Park, MD, and approved November 12, 2004 (received for review October 14, 2004)
We report the complete genome sequence of the deep-sea ␥proteobacterium, Idiomarina loihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Lo ihi submarine volcano, Hawaii. The I. loihiensis genome comprises a single chromosome of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes. A comparison of I. loihiensis to the genomes of other ␥-proteobacteria reveals abundance of amino acid transport and degradation enzymes, but a loss of sugar transport systems and certain enzymes of sugar metabolism. This finding suggests that I. loihiensis relies primarily on amino acid catabolism, rather than on sugar fermentation, for carbon and energy. Enzymes for biosynthesis of purines, pyrimidines, the majority of amino acids, and coenzymes are encoded in the genome, but biosynthetic pathways for Leu, Ile, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I. loihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. It also encodes diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery. We propose that the source of amino acids for I. loihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. loihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem. hydrothermal vent
S
teep thermal and chemical gradients at active, submarine hydrothermal vents affect the phyletic composition and metabolic activities of microbial communities at these sites (1, 2). Lo ihi is an active submarine volcano ⬇30 km south of the island of Hawaii. This youngest manifestation of the hotspot responsible for the Emperor–Hawaiian Seamount chain and Hawaiian Islands has been geophysically and geochemically characterized (1, 3). Vent waters are enriched in Fe, Li, As, Ba, Pb, Po, Mo, Sb, Te, and Tl (1). Deep-sea hydrothermal vents and their environments host diverse communities across three domains of life. Microbial communities in such habitats are adapted to high temperatures, pressure, and salinity. Complete genomes of several bacteria and archaea from submarine vents have been sequenced and offered insights into microbial evolution (4, 5). Idiomarina loihiensis sp. nov. is a ␥-proteobacterium, isolated recently from the hydrothermal vents on the Lo ‘ihi Seamount, 18036 –18041 兩 PNAS 兩 December 28, 2004 兩 vol. 101 兩 no. 52
Hawaii (6). In contrast to obligate anaerobic vent hyperthermophiles Thermotoga spp. and Pyrococcus spp. (7–9), I. loihiensis inhabits partially oxygenated cold waters at the periphery of the vent, surviving a wide range of growth temperatures (from 4°C to 46°C) and salinities (from 0.5% to 20% NaCl). We report here the complete genome sequence of I. loihiensis, deep-sea ␥proteobacterium. The 2,839,318-bp genome of I. loihiensis encodes 2,640 predicted proteins, which is larger than the typical genome size of obligate parasites of ␥-proteobacterial lineage, such as Haemophilus influenzae or Coxiella burnetii, but smaller than the genomes of facultative parasites like Escherichia coli or Vibrio cholerae or environmental organisms like Shewanella oneidensis (10). Distinguishing properties of I. loihiensis include a versatile signal transduction system, an integrated mechanism of exopolysaccharide biosynthesis, and a variety of secreted metallopeptidases. I. loihiensis appears to derive carbon and energy primarily from amino acid metabolism, rather than from sugar fermentation. Materials and Methods The I. loihiensis genome was sequenced by using both bacterial articifial chromosome (BAC) clone and whole-genome shotgun libraries (WGSL). Both ends of the BAC and WGSL clones of sizes 1, 2, and 5 kb were sequenced by Dye Terminator Cycle Sequencing using Beckman CEQ2000 sequencers (Beckman Coulter). Approximately 69,000 valid sequences were assembled into 148 contigs by using the Paracel Genome Assembler, giving ⬇10⫻ coverage of the genome. Gap-spanning forward and reverse clone pairs were used to build contig scaffolds and scaffolds were further linked to each other by using BAC end pairs. The AUTOFINISH module of CONSED (www.phrap.org) was used to automatically pick gap-closing primers between contigs for primer-walking experiments. Assembled contigs, along with the primer-walking sequences, were manually assembled by using Seqman II (DNAStar). Direct sequencing of genomic DNA was then performed to close remaining gaps (11). The entire assembly was verified by 187 long PCRs (Takara Bio, Tokyo) of 15- to 20-kb fragments with 1-kb overlapping regions. This paper was submitted directly (Track II) to the PNAS office. Abbreviation: COG, cluster of orthologous groups. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AE017340). §§To
whom correspondence should be addressed. E-mail:
[email protected].
© 2004 by The National Academy of Sciences of the USA
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Table 1. General features of the I. Ioihiensis genome Feature
I. Ioihiensis
Genome size, bp G⫹C content, % Number of predicted CDSs Average size of CDSs, bp Percentage coding, % Number of protein-coding genes tRNAs rRNA operons (16S–23S–5S) Structural RNAs Number of CDSs with known function Uncharacterized conserved proteins
2,839,318 47.04 2,711 962 92.1 2,640 56 4 3 2,230 410 (15.1%)
ORFs were identified by using GENEMARK (12), followed by BLASTX (13) searches of the remaining intergenic regions. Transfer RNAs were predicted by using TRNASCAN-SE (14). Genome annotation was performed by comparing protein sequences with those in the NCBI protein database (www.ncbi.nlm.nih.gov) and the clusters of orthologous groups (COG) database (www. ncbi.nlm.nih.gov兾COG) (15), by using BLAST and PSI-BLAST (13) with manual verification, as described (10). A maximum likelihood phylogenetic tree of ␥-Proteobacteria was constructed by using concatenated alignments of ribosomal proteins (16). Genome Organization and Analysis. The I. loihiensis genome is a single circular chromosome of 2,839,318 bp with an average G⫹C content of 47% (Table 1). The genome contains 2,640 predicted ORFs, four rRNA operons (16S-23S-5S), 56 tRNA genes, and three other RNA genes, accounting for 92.1% of the genome (Table 1 and Fig. 1; see also Fig. 6, which is published as supporting information on the PNAS web site). Phylogenetic analysis of the concatenated alignment of ribosomal proteins (Fig. 2) shows that I. loihiensis represents a distinct lineage among the ␥-Proteobacteria, which branched from the ‘‘trunk’’ of the ␥-proteobacterial tree after the Pseudomonas lineage, but before the Vibrio cluster. Pairwise genome alignments show limited conservation of gene order between Idiomarina, Vibrio, Pseudomonas, and Shewanella (Fig. 7, which is published as supporting information on the PNAS web site). Comparative genome analysis of I. loihiensis proteins showed that I. loihiensis has a typical ␥-proteobacterial proteome. Indeed, most predicted proteins in I. loihiensis have closest homologs in ␥proteobacteria (77%) or representatives of other proteobacterial subphyla (9%) (Fig. 3A). Biological roles were assigned to 1,662 (63%) ORFs; only a general functional prediction was made for 303 more (11.5%), and the rest remained functionally uncharacterized. Only 116 (4.4%) ORFs had no detectable homologs in the public protein databases. For 18 of them, homologous ORFs were found among environmental sequences from the Sargasso Sea (17), leaving 98 unique predicted proteins. Among the relatively small number of closest homologs outside the Proteobacteria, most were from cyanobacteria and Gram-positive bacteria. Most I. loihiensis proteins (83%) were assigned to COGs (15) that contained proteins from at least one other ␥-proteobacterium (Fig. 8, which is published as supporting information on the PNAS web site). Predicted I. loihiensis proteins were assigned to only 23 COGs (⬇1% of the total) that did not include representatives of other ␥-proteobacteria. Conversely, the I. loihiensis genome encodes 96% of the most conserved set of protein families (COGs) that are found in all ␥-proteobacteria. A comparison of COGs found in I. loihiensis and other free-living ␥-proteobacteria shows conservation of genes involved in translation, DNA replication and repair, and Hou et al.
Fig. 1. Circular representation of the I. loihiensis genome. From the outside inward: The first and second circles show predicted gene-coding region in plus and minus strands, respectively. By COG functional categories, red indicates translation, ribosomal structure, and biogenesis; blue indicates transcription; pink indicates DNA replication, recombination, and repair; green indicates cell division and chromosome partitioning; dark red indicates posttranslational modification; orange indicates cell envelope biogenesis, outer membrane; navy blue indicates cell motility and secretion; purple indicates inorganic ion transport and metabolism; wheat indicates signal transduction mechanisms; aquamarine indicates energy production and conversion; beige indicates carbohydrate transport and metabolism; blue-violet indicates amino acid transport and metabolism; light sky blue indicates nucleotide transport and metabolism; gold indicates coenzyme metabolism; dark khaki indicates lipid metabolism; magenta indicates secondary metabolites biosynthesis; light cyan indicates, general function prediction only; gray indicates function unknown; black indicates hypothetical. The third circle shows transposons and insertion sequence elements in red. The fourth circle shows pseudogenes in brown. The fifth and sixth circles show tRNAs and rRNAs in olive and dark green respectively.
lipid metabolism, in contrast to a substantial loss of sugar metabolism genes (Fig. 3B). Amino Acid Metabolism. The I. loihiensis genome encodes the complete set of enzymes for biosynthesis of most amino acids, except for Leu, Ile, Val, Thr, and Met (Fig. 4). The common Leu, Ile, Val pathway misses a single enzyme, the dihydroxyacid dehydratase (IlvD). The Thr and Met biosynthetic pathways miss threonine synthase (ThrC) and methionine synthase (MetE or MetH), the enzymes that catalyze the last steps in the respective pathways. These observations suggested that I. loihiensis should be auxotrophic by all or at least some of these amino acids. To test these genome annotated predictions, we measured cell growth in the absence of each suspected essential amino acid and PNAS 兩 December 28, 2004 兩 vol. 101 兩 no. 52 兩 18037
DEVELOPMENTAL BIOLOGY
Results and Discussion
Fig. 2. Phylogenetic position of I. loihiensis. Maximum likelihood phylogenetic tree of ␥-Proteobacteria constructed from concatenated alignments of ribosomal proteins. Circles indicate internal nodes with resampling of estimated log-likelihoods (RELL) bootstrap support ⬎95%. Distances are indicated in substitutions per site.
found that only Thr and Val were required (Table 2). Leucine biosynthesis might be carried out with low efficiency by enzymes of the trichloroacetic acid cycle and fatty acid biosynthesis. Leucine and isoleucine also could be produced by transamination of the corresponding oxo-acids derived from fatty acid metabolism (Fig. 4). I. loihiensis is likely to obtain Thr and Val and supplement other amino acids through hydrolysis of exogenous proteins. Indeed, the genome encodes a diverse set of predicted peptidases. These include four genes for Xaa–Pro aminopeptidases (PepQ homologs), seven genes for aminopeptidases of the S9C family, and 10 for peptidases of the M38 family (HutI homologs). Several of these proteases are not typical for ␥-proteobacteria, such as serine carboxypeptidase C (IL1750), two predicted aspartyl proteases (IL0498 and IL2346), two subtilisin-like proteases (IL0161 and IL0162), several diverged secreted peptidases of different families, namely, metallopeptidases (IL0589 and IL1025) and zinc-dependent carboxypeptidases (IL0695 and IL0199). I. loihiensis also has an extracellular exopeptidase (IL2428) similar to the recently described cyanophycinase, an enzyme that hydrolyses cyanophycin, a nonpolar reserve polymer of cyanobacteria, into aspartate-arginine dipeptides (18). The abundance of predicted metallopeptidases might be linked to the high concentations of heavy metals, including zinc, in the hydrothermal vent environment (1). I. loihiensis also encodes an extensive set of enzymes for amino acid degradation. These include the Hut system of histidine degradation (IL2450– IL2454), which is rare in ␥-proteobacteria. Sugar Metabolism. Deep-sea bacteria show limited ability to use
carbohydrates as their sole carbon and energy source (19). Indeed, I. loihiensis does not seem to have a functional PEPdependent sugar兾phosphotransferase system or any ABC-type sugar transporters, or the sugar–phosphate permease. Many enzymes of carbohydrate degradation typical of other ␥proteobacteria are also absent. Examples include such key enzymes of the pentose phosphate shunt and Entner–Doudoroff pathway as transaldolase, glucose-6-phosphate dehydrogenase, 18038 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0407638102
Fig. 3. Comparative genomic analysis of I. loihiensis proteins. (A) Phylogenetic distribution of the closest homologs of I. loihiensis proteins. The numbers indicate (in the clockwise order) best hits among ␥-proteobacteria (77%), other proteobacteria, firmicutes, cyanobacteria, other organisms, and proteins with no homologs. (B) Functional distribution of I. loihiensis proteins. The columns indicate the number of protein families (COGs) encoded in the genomes of seven free-living ␥-proteobacteria (E. coli K12, E. coli O157:H7, Salmonella typhimurium, Yersinia pestis, V. cholerae, and Pseudomonas aeruginosa), but not in I. loihiensis (Bottom); those shared by I. loihiensis and seven other ␥-proteobacteria (Middle); and those found in I. loihiensis but not in any of the others (Top). Protein functional groups represent translation (J), replication, recombination and repair (L), energy production and conversion (C), and transport and metabolism of amino acids (E), carbohydrates (G), nucleotides (F), coenzymes (H), lipids (I), and inorganic ions (P).
and 2-keto-3-deoxy-6-phosphogluconate aldolase. Still, I. loihiensis encodes the enzymes for glycolysis兾gluconeogenesis and the citric acid cycle with glyoxylate bypass (Fig. 4). Several central enzymes of sugar metabolism, including transketolase (IL2214), ribose 5-phosphate isomerase (IL2103), and ribulose5-phosphate-3-epimerase (IL2324), which are encoded by I. loihiensis, appear to be sufficient to provide intermediates for biosynthesis of nucleotides, several cofactors, and amino acids. Accordingly, I. loihiensis is capable of using glycerol and maltose, but not glucose 6-phosphate as the sole carbon source (6). Coenzymes. In oligotrophic seawater, marine bacteria are usually
self-sufficient with respect to coenzymes (20). I. loihiensis encodes all enzymes for NAD, FAD, biotin, folate, and ubiquinone biosynthesis, consistent with this organism’s ability to grow in a minimal medium without vitamins (Table 2). Biosynthesis pathways for cobalamin (B12) and molybdopterin guanine dinucleotide cofactor are missing, which correlates with the lack of B12and molybdopterin-dependent enzymes. In CoA biosynthesis, the two missing enzymes, aspartate 1-decarboxylase (PanD) and panthothenate kinase (CoaA), are the same as in the recently sequenced genome of the marine cyanobacterium Prochlorococcus marinus (20), suggesting that the two organisms might encode the same alternative enzymes. I. loihiensis also encodes a homolog of the E. coli Na⫹兾panthothenate symporter PanF (IL1528), which might be used for panthothenate uptake. Fatty Acid Metabolism. The genus Idiomarina has a unique fatty acid composition with a high percentage of iso-branched fatty acids (21). Compared to other Idiomarina spp., I. loihiensis has twice the percentage of saturated fatty acids (6). Indeed, I. loihiensis genome revealed a complete set of enzymes for fatty acid biosynthesis. However, genome sequence alone does not allow prediction of the final composition of the membrane. Therefore, we have complemented genome analysis with the characterization of the I. loihiensis membrane fatty acids. Gas chromatography/MS analysis of membrane fractions detected 11 fatty acids, dominated by 3-hydroxyoctadecanoic acid (24.7%), 3-hydroxyundecanoic acid (23.2%), 9-octadecenoic acid (19.3%), and hexadecanoic acid (11.2%) (Table 3, which is Hou et al.
DEVELOPMENTAL BIOLOGY
Fig. 4. A scheme of the principal functional systems of the I. loihiensis cell. I. loihiensis gene identifiers are shown by numbers in green. Pathways involving multiple reactions shown by double arrows. Final biosynthetic products are indicated as follows: light blue for amino acids, dark yellow for nucleotides, brown for sugars, and red for cofactors. Reactions for which no candidate enzyme was confidently identified are indicated by question marks. Missing reactions of Thr and Val biosynthesis are crossed out; the uptake of these two essential amino acids is indicated with exclamation points.
published as supporting information on the PNAS web site). These acids were identified as esters of phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC). PG and DPG were the most abundant phospholipids (45.6% and 32.2%, respectively). I. loihiensis maintains several diverged copies of genes for fatty acid synthesis. Given that the composition and modification of fatty acids plays a crucial role in maintaining cell membrane integrity in deep-sea microorganisms (22), and further analysis of these genes could help understand the regulation of membrane fluidity under constantly changing pressure and temperature. Exopolysaccharide and Biofilm. Like other deep-sea vent microor-
ganisms, I. loihiensis in vitro produces a highly viscous exopolysaccharide (19, 23). It has been suggested that vent microorganisms develop biofilms by using exopolysaccharides to ensure the formation of robust and stable communities (4, 23). Indeed, the I. loihiensis genome contains a cluster of 32 genes (IL0538– IL0569), which encode enzymes involved in the synthesis of export, modification, and polymerization of exopolysaccharides. Another gene cluster (IL0518–IL0521) encodes enzymes for sialic acid biosynthesis and sialylation of the surface polysaccharides. Sialic acid synthetase of I. loihiensis contains a C-terminal antifreeze domain, which could facilitate functioning of this enzyme at low temperatures. In addition, I. loihiensis has a Hou et al.
mannose-sensitive hemagglutinin gene cluster (IL0363–IL0379), homologous to the one that is involved in biofilm formation in V. cholerae (24). Motility, Chemotaxis, and Signal Transduction. Although biofilm
formation is essential in forming a stable population of deep-sea microorganisms, motility, and chemotaxis provide a mechanism to locate new surfaces in response to geochemical and thermal gradients (4). I. loihiensis has a single polar flagellum and encodes the full set of proteins involved in flagellar assembly and function. Most of the 45 flagella-associated genes are clustered in two large predicted operons (IL1147–IL1133 and IL1204– IL1187). Another large operon (IL1120–IL1110) combines flagellar biosynthesis ( flhAFGfliA) and chemotaxis (cheYZAB, cheW) genes. The I. loihiensis genome encodes 15 methylaccepting chemotaxis proteins (MCPs), compared to only five MCPs in E. coli. One of these MCPs (IL0176) contains a hemerythrin domain and could be involved in O2 sensing in a low O2 environment. Indeed, purified recombinant hemerythrin domain of IL0176 showed characteristic absorption spectra of oxygen binding (Fig. 9, which is published as supporting information on the PNAS web site). Hydrothermal vent systems are dynamic submarine environments, which experience constant fluctuations in temperature, pressure, and O2 availability. Therefore, in contrast to marine cyanobacteria that inhabit a stable environment and encode few PNAS 兩 December 28, 2004 兩 vol. 101 兩 no. 52 兩 18039
Table 2. Growth requirements of I. Ioihiensis strain L2-TRT Media MM* MM ⫹ Casamino acids† MM ⫹ 13 amino acids‡ MM ⫹ Asn, Cys, Gln, His, Lys, Pro, Ser MM ⫹ Arg, Met, Thr, Leu, Ile, Val MM ⫹ 12 aa ⫺ Asn MM ⫹ 12 aa ⫺ Cys MM ⫹ 12 aa ⫺ Gln MM ⫹ 12 aa ⫺ His MM ⫹ 12 aa ⫺ Lys MM ⫹ 12 aa ⫺ Pro MM ⫹ 12 aa ⫺ Ser MM ⫹ 12 aa ⫺ Arg MM ⫹ 12 aa ⫺ Met MM ⫹ 12 aa ⫺ Thr MM ⫹ 12 aa ⫺ Leu MM ⫹ 12 aa ⫺ Ile MM ⫹ 12 aa ⫺ Val
Expected from genome analysis
Experimental results
⫺ ⫹ ⫹ ⫺
⫺ ⫹ ⫹ ⫺
⫹
⫺兾⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺
*The basal minimal medium was the CHB兾E medium supplemented with SL-8 trace elements solution and 10% NaCI (6). The ⫹ sign indicates vigorous growth of I. Ioihiensis cells, ⫺ indicates no growth, and ⫺兾⫹ indicates slow growth. †Casamino acids (Difco) is a mix of 18 amino acids. ‡The 13 amino acids are listed in the two following lines.
signaling proteins (20), the vent microorganism I. loihiensis needs a versatile sensory transduction system. Indeed, it encodes 26 histidine kinases (including four hybrid kinases) and 36 response regulators. In addition, there are 34 signaling proteins containing the GGDEF domain, which have diguanylate cyclase activity and produce c-diGMP, a recently recognized secondary messenger in bacteria (25). Many of these proteins also contain the EAL domain, a likely c-diGMP phosphodiesterase (26), which is encoded in I. loihiensis in 22 copies. Several proteins containing GGDEF and EAL domains also contain periplasmic ligand-binding sensory domains, indicating their role in sensing extracellular cues. GGDEF and EAL domains are often linked to the production of extracellular polysaccharide (reviewed in ref. 26) and are likely to play a similar role in I. loihiensis. Adaptation to the Vent Environment. Survival of a bacterium in the hydrothermal vent fluids depends on effective uptake of such elements as P, N, S, and Fe and detoxification of heavy metals. Phosphate uptake appears to be mediated by a typical ABC-type Pst system (IL2106–IL2109). I. loihiensis is capable of assimilation of nitrogen from both ammonia (IL2165) and nitrate (IL0183). Although the classical ABC-type sulfate transport system is missing in I. loihiensis, it encodes three proteins that could each serve as a sulfate transporter: an MFS super family permease (IL2129), a homolog of CysZ, another putative permease (IL1703), and a homolog of the sulfur deprivation response regulator YfbS (IL0057). Iron uptake mechanisms include siderophore-mediated transport (IL0795, -1581, and -2511) and an ABC-type transport system (IL1059–IL1061). Systems for detoxification of heavy metals include enzymes for reduction and extrusion of arsenate (IL0699, IL1454, IL1465, and IL2124–IL2125), export of cadmium and other divalent cations (IL0463, IL0464, IL0632, IL0774, IL0777–IL0779, IL1222, IL1242, IL1632, IL1636, IL1640, IL1641, and IL2587), and a mercury detoxification system (IL1643–IL1646). Like other marine bacteria, I. loihiensis encodes a primary Na⫹ 18040 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0407638102
Fig. 5. A schematic diagram of lifestyle of I. loihiensis inferred from genome analysis.
pump, the Na⫹- translocating NADH兾ubiquinone oxidoreductase (IL1050–IL1045), and probably uses a sodium ion gradient as the source of energy for nutrient uptake. I. loihiensis also encodes other primary sodium pumps, namely, Na⫹-transporting oxaloacetate decarboxylase (IL1736–IL1734), ATP-dependent Na⫹ exporter NatAB (IL0274–IL0275 and IL0481–IL0482, which is unusual for ␥-proteobacteria), and an RNF system (IL1794–IL1799) (27). However, I. loihiensis membrane ATP synthetase appears to be specific for H⫹ (28), indicating that membrane energetics of I. loihiensis does not depend on the sodium ion cycle. Indeed, I. loihiensis encodes several primary H⫹ pumps, namely, succinate dehydrogenase (IL1507–IL1504), bc1 complex (IL0416–IL0418, IL0097, and IL0205), cytochrome d-type quinol oxidase (IL0041–IL0042), and cytochrome c oxidase (IL0260–IL0257). Export of Na⫹ ions at the expense of the proton gradient is performed by a variety of Na⫹兾H⫹ antiporters, including NhaC (IL0584, IL1276, IL2378), NhaD (IL0189), NhaP (IL1058 and IL1978), and the multisubunit Na⫹兾H⫹ antiporter MnhABCDEFG (IL1907–IL1912). I. loihiensis encodes a Na⫹兾proline symporter (IL0738), Na⫹兾alanine symporter (IL1268), Na ⫹ 兾glutamate symporter (IL0983 and IL1037), Na⫹兾uridine symporter, and several other predicted Na⫹-dependent permeases (IL0272, IL1178, IL1275, IL1331, IL1996, IL2299, IL2396, and IL2505). Finally, the I. loihiensis genome contains three copies of the gene encoding a Na⫹dependent multidrug efflux pump (IL0250, IL1790, and IL1842). I. loihiensis does not seem to synthesize organic compatible solutes as ectoine (ectoine synthase EctC and aminotransferase EctP are missing), trehalose (trehalose-6P synthetase OtsA and trehalose-6P phosphatase OtsB are missing, as is trehalose synthase TreS), di-inositol phosphate (myo-inositol-1-P synthase INO1 is missing), or mannosylglycerate (mannosyl-3-phosphoglycerate synthase MpgS is missing). However, in line with its proteolytic metabolic life, I. loihiensis genome encodes uptake systems for small peptides, as well as for proline, glutamate, and glycyl betaine. Examples include two paralogous PutP-like Na⫹兾 proline symporters (IL0738 and IL1528), three copies of the GltP-like glutamate transporters (IL0983, IL1037, and IL2299), and two paralogous BetT-type choline兾betaine transporters (IL1418 and IL2388). Conclusions The genome sequence of the deep-sea ␥-proteobacterium I. loihiensis suggests that I. loihiensis relies primarily on amino acid fermentation, rather than on saccharolytic pathways for carbon and energy. We propose that I. loihiensis is an opportunistic colonizer of proteinaceous particles in the deep-sea hydrothermal vent waters Hou et al.
This work was supported by the Engineering Research Center program of the National Science Foundation under Award EEC 9731725 and a University of Hawaii intramural bioinformatics grant.
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PNAS 兩 December 28, 2004 兩 vol. 101 兩 no. 52 兩 18041
DEVELOPMENTAL BIOLOGY
sequencing showed that survival and growth of I. loihiensis in the constantly changing hydrothermal vent environment requires multiple, flexible adaptation mechanisms.
(summarized in Fig. 5). Its versatile signal transduction system apparently senses dynamic changes in dissolved oxygen, temperature and pressure, and initiates synthesis of highly viscous exopolysaccharide(s) for attachment to these proteinaceous particles. Once settled, it would secrete diverse proteases to break down proteins and peptides and employ sodium-dependent transport systems to import the degradation products. Complete genome
