Nature Reviews | Microbiology

genome watch

Sink or swimLisa C. Crossman

This month’s Genome Watch discusses four unusual organisms that are found in sea and river habitats. The first two are epsi-lonproteobacteria that are associated with hydrothermal vents on the ocean floor, the third is a filamentous bacterium that is found growing in mats on the seabed and the fourth is a pathogen of salmonid fish. Comparisons of the genomes of these organisms with those of common pathogens can shed light on pathogenicity traits and define the unique features that characterize these peculiar species.

The epsilonproteobacteria group is the pre-dominant bacterial group that is found in deep-sea hydrothermal-vent environments on the seabed. Nakagawa and colleagues1 recently sequenced the complete genomes of Nitratiruptor tergarcus and Sulfuvorum litho­trophicum, both of which were isolated from an active vent at a depth of 1,000 metres in Japanese waters. Both organisms have a single, small, circular chromosome, with the 1,877,931 base pair (bp) genome of N. tergarcus being the smallest of the non-pathogenic epsilon- proteobacterial genomes to be sequenced so far and the 2,562,277 bp genome of S. litho­trophicum the largest. Considering the low G+C content of both organisms — 39.7% and 43.8%, respectively — and the remarkable environmental niche that they inhabit, it is perhaps surprising that the temperature range for growth is 10–37°C, with an optimum of 33°C for S. lithotrophicum, and 37–65°C, with an optimum of 55°C, for N. tergarcus. In keep-ing with their environmental niche, which can function either as a source or a sink for various metal ions, both species encode several heavy-metal detoxification systems and transporter proteins. S. lithotrophicum carries no genes for motility or chemotaxis, whereas N. tergarcus possesses a full complement of such genes, many of which are arranged as a single gene

cluster. The unusual G+C content of this clus-ter suggests that it was acquired by horizontal gene transfer.

S. lithotrophicum is a strict sulphur oxidizer, whereas N. tergarcus is a strict hydrogen oxi-dizer. Despite their respiratory differences, both species encode a full set of genes for carbon dioxide fixation and a complete denitrification pathway (nitrate to dinitrogen gas). Sulphur-oxidation genes are found throughout the genomes of both organisms. S. lithotrophicum encodes four alternative hydrogenases, two of the uptake type, one of the sensing type and one of the hydrogen-evolving type. N. tergarcus encodes three hydrogenases, one of the uptake type, one of the sensing type and one of the evolving type. Interestingly, Helicobacter pylori has an uptake-type hydrogenase that is essential for colonization of the host. Other pathogenic epsilonproteobacteria have a single hydrogenase, rather than an array of alternative hydrogenases such as those found in these deep-sea vent bacteria. In common with other epsilonproteobacteria, pathogenic epsilonproteobacteria lack many DNA-repair enzymes, a trait that facilitates H. pylori per-sistence during infection by enabling changes in the bacterial-cell-surface gene products that allow escape from detection by the host

immune response. In the deep-sea bacte-ria, this trait could enable these organisms to adapt to rapidly changing environmental conditions.

Remarkably, both of these proteobacte-rial genomes contain potential virulence determinants that are conserved in patho-genic epsilonproteobacteria, such as the invasion antigen CiaB, a lytic murein trans-glycosylase, the virulence factor MviN, haemolysin and an N-linked glycosylation (NLG) gene cluster. Little is known about the function of the NLG cluster, except that glycosylated proteins function in the bac-terial evasion of the host immune system in both symbiotic and pathogenic host–bacterial interactions. NLG mutants of Campylobacter spp. are less able to adhere to and invade host epithelia2. NLG might have been conserved in the genomes of these deep-branching hydrothermal-vent epsilonproteobacteria in order to maintain a symbiotic interaction with marine inver-tebrates that are present in deep-sea vent environments.

Beggiatoa spp. grow as filamentous mats in marine environments, and have not yet been isolated into pure culture3. In a unique study published in PloS Biology3, morphologically

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identical single filaments of Beggiatoa were isolated. Optical mapping of five separate fila-ments indicated a high level of diversity, so the authors opted to sequence the genomes of sin-gle filaments, rather than compile a metagen-ome of closely related species. The assumption was that a single filament represented a clonal population of cells. A single filament was ampli-fied and pyrosequenced to assemble an incom-plete sequence of high coverage depth that was arranged in contigs, the largest of which was 18.6 kilobases. A substantial amount of repeti-tive DNA was present that could not be resolved. An alternative single filament was amplified and sequenced by traditional shotgun Sanger sequencing. The Sanger method yielded a 3x coverage unfinished sequence of 1,091 contigs, with a total length of 1.3 megabases (Mb). The consensus optical map indicated the presence of a single circular chromosome of 7.4 Mb, but the large number of short overlapping contigs sug-gested that the genome could be substantially larger. The occurrence of single-copy genes was taken to indicate that a single dominant genome is present in the sequence assemblies. A compar-ative genome analysis revealed that the filaments are phylogenetically distinct, as was evident from the G+C contents of individual filaments, which differed by as much as 4%.

Beggiatoa spp. are among the largest bac-terial or archaeal cells that have been char-acterized to date, and contain a vacuole that comprises up to 90% of the cell volume. The vacuole contains nitrate (NO3

–), which might be present at concentrations of up to 500 mM. It is proposed that Beggiatoa spp. monopolize environmental nitrate by increasing the con-centration of nitrate in their vacuoles, and that this provides the organism with a competitive advantage over other denitrifiers. Beggiatoa spp. respire nitrate and sulphur, and probably release phosphates that accumulate on the sea-bed. A membrane-bound energy-conserving nitrate reductase (Nar) and a periplasmic-type nitrate reductase (Nap) are present in the Beggiatoa genome. A second nar gene was also identified that is most similar to a puta-tive nitrate reductase or nitrite oxidoreductase from Kuenenia stuttgartiensis. This is interest-ing because K. stuttgartiensis uses this enzyme for nitrite oxidation, and therefore Beggiatoa spp. might also be able to oxidize nitrite. enzymes that catalyse the final stages of deni-trification were not identified in the unfinished genome sequence.

The lithotrophic growth of Beggiatoa spp. by gaining electrons from the oxidation of hydrogen sulphide to elemental sulphur was first described in 1888. More recently, a two-step sulphur-oxidation mechanism has been described. The first step occurs in the anoxic

zone, where sulphide is oxidized to elemental sulphur using nitrate. The second step occurs in the oxic zone, where stored sulphur is oxi-dized to sulphate using oxygen. Beggiatoa spp. filaments can move between the two layers to access both zones by gliding motility. The genomes of both Beggiatoa spp. filaments con-tain proteins of the reverse dissimilatory sul-phate reductase (rDsr) pathway. However, the sulphur-oxidation pathway has not been fully elucidated in this unfinished genome sequence. By analogy with other rDsr-containing organ-isms, it is most likely that rDsrAB is responsi-ble for oxidizing stores of sulphur to sulphite. Perhaps surprisingly, genes for heterotrophic growth are present, including several enzymes of the tricarboxylic acid cycle and subunits of glycolate oxidase. Glycolate is produced by the metabolic functions of neighbouring cyanobacteria. The genome sequence indi-cates that substantial horizontal gene transfer has taken place. Coding sequences that were acquired from cyanobacteria have been iden-tified, including genes from the filamentous Nostoc spp. and Anabaena variabilis. Although many of these are conserved hypothetical genes, there are also several potential mobile elements, as well as reverse-transcriptase and element-excision-controlling-factor proteins.

Moving from the sea floor to the surface of fish, the genome of Flavobacterium psy­chrophilum, a pathogen of salmonid fish, has recently been published4. The genome is small and circular, and comprises 2,861,988 bp, with a G+C content of 32.5%. It also includes a cryp-tic plasmid. The bacterium is a psychrophile, growing in waters at temperatures of 3–15°C. Infections by F. psychrophilum cause substan-tial economic losses owing to their devastating effects on salmonid stocks, and can be spread by vertical transmission through fish eggs. The genome encodes 13 potentially secreted pro-teases that are predicted to be the main cause of host-tissue damage. A collagenase gene is unexpectedly interrupted by a mobile element insertion in the sequenced genome, and an investigation of 23 different F. psychrophilum isolates showed that those lacking collagenase activity were restricted to rainbow trout. This suggests that collagenase is not required for pathogenicity to trout. Cytolysins and haemo-lysin-like proteins are encoded in the genome sequence and are expected to be important virulence factors. Adhesion factors were also detected, and these included fibronec-tin-type domain proteins. These are prob-ably extremely important for the organism to maintain attachment to the host. No genes for type III or type Iv secretion were identi-fied, although the organism has unusual PorT and PorR proteins, which are responsible for

the transport of virulence factors in the oral pathogen Porphyromonas gingivalis5.

Adaptations to a psychrophilic lifestyle include proteins that function to increase membrane fluidity, and the production of six ATP-dependent RNA helicases that are thought to be involved in de-stabilizing the RNA secondary structures that form at low temperatures. Cold-shock proteins and chap-erones might also be involved in adaptation to these peculiar conditions. A broad range of enzymes that are capable of combating oxidative stress are present in the genome sequence. These might function in defence against the host during infection, but could also help the organism to deal with the toxic effects of the increased solubility of oxygen at low temperatures. F. psychrophilum can catabolize a range of host proteins that prob-ably serve as carbon, nitrogen and energy sources for growth. Interesting peptidases include cyanophycinase, which is used to degrade the storage compound cyanophycin, and might be deployed in times of nutrient deprivation. F. psychrophilum is a strict aerobe and carries a massive set of 24 cytochrome-oxidase genes and an extensive aerobic res-piratory chain. The genome sequence has contributed extensively to our knowledge of unusual aspects of this organism’s lifestyle and will hopefully pinpoint areas of research that will allow better control of this fish pathogen.

In conclusion, it is clear that these organ-isms represent extremes of diversity and that genomic studies will pave the way to a better understanding of their biology. Lisa C. Crossman is at the Sanger Institute, Wellcome Trust

Genome Campus, Hinxton, Cambridge CB10 1SA, UK. e-mail: microbes@sanger.ac.uk

doi:10.1038/nrmicro1777

1. Nakagawa, S. et al. Deep-sea vent epsilon-proteobacterial genomes provide insights into emergence of pathogens. Proc. Natl Acad. Sci. USA 104, 12146–12150 (2007).

2. Szymanski, C. M., Burr, D. H. & Guerry, P. Campylobacter protein glycosylation affects host cell interactions. Infect. Immun. 70, 2242–2244 (2002).

3. Mussmann, M. et al. Insights into the genome of large sulfur bacteria revealed by analysis of single filaments. PLoS Biol. 5, e230 (2007).

4. Duchaud, E. et al. Complete genome sequence of the fish pathogen Flavobacterium psychrophilum. Nature Biotechnol. 25, 763–769 (2007).

5. Sato, K. et al. Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis. J. Biol. Chem. 280, 8668–8677 (2005).

DataBaSeSEntrez Genome: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeNitratiruptor tergarcus | Sulfuvorum lithotrophicumEntrez Genome Project: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprjAnabaena variabilis | Flavobacterium psychrophilum | Helicobacter pylori | Kuenenia stuttgartiensis | Porphyromonas gingivalis

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