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Sink or swim
Lisa 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 litho­
trophicum, 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. litho­
trophicum 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
834 | November 2007 | volume 5 Nature Reviews | Microbiology
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
© 2007 Nature Publishing Group
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 epithelia 2. 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
Neil Smith
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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
nature reviews | microbiology volume 5 | November 2007 | 835
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 psy­
chrophilum, 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
© 2007 Nature Publishing Group
N e w s & a n a ly s i s
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).
DATABASES
Entrez Genome: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=genome
Nitratiruptor tergarcus | Sulfuvorum lithotrophicum
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Anabaena variabilis | Flavobacterium psychrophilum |
Helicobacter pylori | Kuenenia stuttgartiensis | Porphyromonas
gingivalis
All links are active in the online pdf