The Versatile Ε-Proteobacteria: Key Players in Sulphidic Habitats

REVIEWS

The versatile ε-proteobacteria: key players in sulphidic habitats

Barbara J. Campbell*§, Annette Summers Engel ‡§, Megan L. Porter ¶ and Ken Takai || Abstract | The ε-proteobacteria have recently been recognized as globally ubiquitous in modern marine and terrestrial ecosystems, and have had a significant role in biogeochemical and geological processes throughout Earth’s history. To place this newly expanded group, which consists mainly of uncultured representatives, in an evolutionary context, we present an overview of the taxonomic classification for the class, review ecological and metabolic data in key sulphidic habitats and consider the ecological and geological potential of the ε-proteobacteria in modern and ancient systems. These integrated perspectives provide a framework for future culture- and genomic-based studies.

Although pathogenic species such as Helicobacter Phylogenetic and ecophysiological diversity pylori have been well studied, the ε-proteobacteria, to Ideally, taxonomic classification should be performed which H. pylori is affiliated, is the most poorly charac- through a polyphasic approach using more than one terized class within the Proteobacteria1–3. In 2002, the molecular marker and phenotypic information derived International Committee on Systematics of Prokaryotes from cultured representatives5,6. However, because of Subcommittee on the taxonomy of Campylobacter and the widespread and almost exclusive use of the 16S related bacteria4 recognized the increasing number of rRNA gene for phylogenetic studies, and the dearth of unclassified and unaffiliated ε-proteobacterial 16S cultures, we compiled 1,037 16S rRNA gene sequences ribosomal RNA (rRNA) sequences deposited into (>1,200 bp) from public databases (RDPII, GenBank, the public databases and recommended that future EMBL and DDBJ) up to May 2005 and from published investigations should deal with this growing prob- reports of clones, strains or sequences described as *College of Marine Studies, lem. Despite recent culture-based investigations and ‘uncultured bacterium’ with previously determined University of Delaware, Lewes, descriptions for novel ε-proteobacterial groups, most phylogenetic affinity to the ε-proteobacteria. To con- Delaware 19958, USA. ‡Department of Geology and lineages are still without cultured representatives struct a phylogenetic foundation for more detailed Geophysics, Louisiana State or are known only from environmentally retrieved analyses, a Neighbour Joining (NJ) tree was constructed University, Baton Rouge, 16S rRNA gene sequences from PCR-based studies of in PAUP* (REF.7), calculating distances under the gen- Louisiana 70803, USA. anaerobic to microaerophilic, sulphur-rich marine and eral time-reversible model incorporating invariable ¶ Department of Biological terrestrial aquatic environments, or from symbioses sites and rate heterogeneity. The analyses revealed that Sciences, University of ε Maryland Baltimore County, with metazoans. Many of these habitats are deemed a few previously affiliated -proteobacterial 16S rRNA Baltimore, Maryland 21250, ‘extreme’ environments — from the hydrothermal flu- gene sequences were chimeric or misidentified (see USA. ids of deep-sea vents to the cold darkness of sulphidic Supplementary information S1 (table)). The four clades ||Subground Animalcule caves. that contain environmental sequences were then sub- Retrieval (SUGAR) Program, ε Extremobiosphere Research A taxonomic framework for the -proteobacteria is jected to more rigorous maximum likelihood analyses 8 Center, Japan Agency for still lacking. For lineages without cultured representa- using PHYML with the same model chosen for the NJ Marine-Earth Science & tives, this has made it difficult to fully assess the impor- analysis. To estimate nodal supports, 100 bootstrap rep- Technology, 2-15 tance of any newly discovered bacteria. In this review, licates were performed. Natsushima-cho, Yokosuka we evaluate class taxonomic structure as a frame of The ε-proteobacterial sequences currently belong to two 237-0061, Japan. ε §The authors contributed reference for placing new -proteobacterial sequences valid orders, the Nautiliales (genera Nautilia, Caminibacter 2,9–11 equally to this work. derived from 16S rRNA gene analyses in an evolutionary and Lebetimonas) and the Campylobacterales Correspondence to context. With this perspective, and to offer recommen- (families Campylobacteraceae, Helicobacteraceae and B.J.C. and A.S.E. dations for future research directions, we explore major Hydrogenimonaceae)12,13. Excluding clinical systems e-mails: [email protected] and [email protected] habitats and highlight ecophysiological diversity patterns (such as infectious associations with humans) affiliated doi:10.1038/nrmicro1414 based on phylogeny and current metabolic and genomic with the Campylobacter and Helicobacter genera, the Published online 2 May 2006 properties of cultured representatives. remaining ε-proteobacterial sequences are diagnosed

REVIEWS

Thermophile into four robust phylogenetic clusters — classified here using fumarate and can reduce nitrate to ammonia, 14 An organism that grows as the Nautiliales, Arcobacter, Sulfurospirillum and envi- with the exception of Sulfurospirillum multivorans , one optimally at high temperatures, ronmental sequence clusters — that consist of sequences feature that phylogenetically distinguishes the cultured usually above 45°C. retrieved from various marine systems (for example, sulfurospirilla is their ability to respire using alterna- heterotrophic 15 Autotroph deep-sea hydrothermal vents, vent fauna and deep-sea tive electron acceptors under conditions An organism that can use marine subsurfaces) and terrestrial systems (for example, (TABLE 1; see Supplementary information S2 (figure), carbon dioxide as the sole groundwater, caves and springs) (FIG. 1). part b). Sequences from different strains that respire source of carbon for growth. With few exceptions, ε-proteobacterial sequence using similar elements are more closely related to each affinities strongly correlate with ecotype for each of other compared with other species within the family, Heterotroph An organism that uses organic the phylogenetic clusters (denoted as coloured lines despite strains originating from different geographical compounds as nutrients to in FIG. 2 and coloured text in Supplementary informa- locations (for example, Sulfurospirillum carboxydovorans , produce energy for growth. tion S2 (figure)) and metabolic capabilities (denoted Sulfurospirillum arcachonense and Sulfurospirillum sp. as coloured symbols in Supplementary information Am-N). Chemocline S2 (figure)). Within the deeply branching group of the Although arcobacters have been implicated in A chemical gradient from high 16 to low concentrations, often Nautiliales, sequences have been retrieved exclusively human and animal enteric diseases , few studies consisting of a relatively small from hydrothermal systems, and cultured representa- have combined isolation and molecular techniques stratum where the tives of the family are thermophilic, autotrophic and can to examine their habitat range17. The type species of concentration changes rapidly reduce elemental sulphur with molecular hydrogen (see the genus Arcobacter nitrofigilis was isolated from a between the two endpoints. Supplementary information S2 (figure), part a). Even salt-marsh plant root18, but there is still significant Mesophile within the Sulfurospirillum (FIG. 1; see Supplementary diversity among the arcobacters. Similar to the sul- An organism that grows information S2 (figure), part b) and Arcobacter (FIG. 1; furospirilla, Arcobacter sequences retrieved from optimally at moderate see Supplementary information S2 (figure), part c) clus- marine and terrestrial habitats group together (FIG. 1) temperatures, ranging between ters, nearly all of the sequences are grouped based on and with ecotype (see Supplementary information S2 20°C and 45°C. environmental setting and metabolism. For instance, (figure), part c). Although the metabolic capabilities although all characterized Sulfurospirillum spp. ferment of most arcobacters have not been studied in detail, many of the cultured representatives originate from

Woli marine environments with a well defined geochemical interface between dissolved oxygen and sulphide n ampylobacter e C 17 ll concentrations . For example, ‘Candidatus Arcobacter a A rc ob sulfidicus’ was isolated from coastal marine sediments a Candidatus A. sulﬁdicus ct 19 er with an oxygen–sulphide chemocline . This bacterium

Oilﬁeld 'FWKO B' undergoes mesophilic, chemolithoautotrophic growth, and r e t c m produces filamentous sulphur with sulphide and oxygen a llu b o as the electron donor and acceptor, respectively. Based c i urospiri l lf u e S sp. Am-N on radio- and stable-isotopic experiments of carbon- H

Hydrogenimonas fixation processes, Candidatus A. sulfidicus was the first

Thioreductor ε

-proteobacterium thought to assimilate inorganic car-

Nitratiruptor

Calvin–Benson bon sources, not through the pathway, but

Caminibacter 19

by means of the reductive TCA cycle (rTCA cycle) .

Lebetimonas

Nautilia

The phylogenetic assignment of the remaining

Nautiliales sequences is problematic. Based on the bootstrap sup-

ported phylogenetic topology, there is a large group that

FIGS 1,2

is distinct from the other major clusters ( ; see

Sulfurovum

(figure), part d). This Supplementary information S2

cluster represents the largest increase in 16S rRNA gene-

sequence diversity throughout the -proteobacteria and

includes several recently described genera. Currently,

Nitratifractor

this sequence cluster has no hierarchical taxonomic clas-

sification and future taxonomic revision is required to al ent elucidate the possibility that the cluster might represent ironm Thiovulum Env Sulfuricurvum more than one hierarchical group. Thiomicrospira sp. CVO Sulfurimonas We have provisionally named this clade Thiovulgaceae Thiomicrospira denitriﬁcans fam. nov. for ease of reference throughout this review20–22. Thiovulgaceae is derived from thio meaning ‘sulphur’ ε Figure 1 | Phylogeny of 1,037 near full-length (>1,200 bp) -proteobacterial and vulgar meaning ‘of, pertaining to, common’, form- sequences collected from public databases and published research. Sequences ing Thiovulga meaning ‘pertaining to sulphur’;-aceae were aligned using Muscle v3.52 (REF. 116) followed by removal of highly divergent and ambiguous regions using Gblocks v0.91b (REF. 117). The phylogeny was reconstructed represents the ending to denote a family. The cultured using Neighbour Joining under a general time-reversible model of evolution. Major genera that belong to the family are Gram-negative taxonomic divisions, and all of the currently recognized genera, are indicated. Branches bacteria that have rod-, vibrio- or filamentous-shaped for environmental sequences are coloured to represent either marine (blue) or terrestrial non-spore-forming cells. Organisms are found in (green) habitats. mesophilic conditions, and cultured representatives are

REVIEWS

M ari ne g rou p I

Sulfurovum oup I

Terrestrial gr Nitratifractor

Lower Kane G ro u Cave group VI n d w Termite gut a t e r g r o u p I Sulfuricurvum Marine: deep-sea vents, sediments Marine: basinal sediments Lower Kane Marine: deep-sea vent G Cave group I r metazoans o u Terrestrial: water, Lower Kane n Cave group IV d contaminated water w Terrestrial: acid mine a t e drainage, lakes, springs r

Terrestrial: hydrocarbon g Lower Kane r groundwater o Cave group II/V u Terrestrial: cave microbial p

I I mats Marine: pelagic Marine: whale bone

Sulfurimonas

Thiomicrospira i

I I

Thiovulum

Figure 2 | The provisional Thiovulgaceae fam. nov. clade. This figure is expanded from FIG. 1. Branches are coloured to represent ecotype. Based on additional, more rigorous maximum likelihood analysis, terrestrial group I is placed outside of the Thiovulgaceae fam. nov. as shown in Supplementary information S2 (figure).

Chemolithoautotroph chemolithoautotrophic and can use molecular hydrogen fine-scale ecotype clade associations. Both MG I and MG An organism that obtains and/or reduced sulphur compounds as electron donors. II are composed of sequences retrieved from deep-sea energy from inorganic compounds and carbon from Members of the family have been isolated from both vents and sediments, or are associated with vent fauna;

CO2. marine and freshwater habitats. however, MG II contains a slightly broader ecotype diver- The Thiovulgaceae fam. nov. family is a member sity than MG I, as MG II contains a clade of terrestrial Calvin–Benson pathway of the order Campylobacterales and comprises the wastewater (sludge) organisms and Thiomicrospira spp. Also known as the Calvin– 23 24 25 25 Benson cycle. A series of genera Thiovulum , Nitratifractor , Sulfurovum , The newly described genera Sulfurovum and 26 27,28 29 24 biochemical, enzyme-mediated Sulfuricurvum , Thiomicrospira and Sulfurimonas , Nitratifractor are affiliated with MG I, and the sulphur- 29 23 reactions in which CO2 is which cluster into four main sequences groups, within oxidizing genera Sulfurimonas , Thiovulum and reduced and incorporated into which are two discrete ecological units — marine Thiomicrospira27,28 are affiliated with MG II. A large organic molecules. group (MG) and groundwater group (GG) (FIG. 2; see group of sequences retrieved from groundwater is Reductive TCA cycle Supplementary information S2 (figure), part d). Ecotype separated into two clusters, GG I and GG II. Whereas 26 (rTCA cycle). The TCA cycle in groups are closely related to each other (for example MG I GG I includes the genus Sulfuricurvum , Lower Kane reverse, leading to the fixation and MG II), but relatedness is not supported by bootstrap Cave groups I and IV30, and sequences isolated from of CO . Represents a putatively 2 values, which indicates that significant diversity has yet wastewater, sludge or groundwater contaminated with ancient metabolic pathway in which autotrophic carbon to be uncovered within the cluster. Unlike the phyloge- petroleum, uranium or tricholoroethene, GG II consists fixation occurs under netic and ecotype patterns within the Sulfurospirillum of sequences only from Lower Kane Cave (FIG. 2; see anaerobic conditions. and Arcobacter clusters, there are little recognizable Supplementary information S2 (figure), part d).

REVIEWS

Table 1 | Physiological characteristics of ε-proteobacteria from deep-sea hydrothermal habitats and other selected environments Isolate/phylogenetic Isolation Growth Carbon Electron Electron Sulphur/nitrate Ref. association site temperature metabolism donor acceptor reduction to: Order Nautiliales, Family Nautiliaceae

Nautilia lithotrophica Alvinella pompejana 53 ˚C Mixotroph H2, formate Sulphite, H2S10 tube, 13˚N EPR elemental sulphur

Nautilia sp. str. Am-H Alvinella pompejana 45 ˚C Mixotroph H2, formate Elemental sulphur H2S44 tube, 13˚N EPR

Caminibacter Alvinella pompejana 60 ˚C Mixotroph H2, complex Nitrate, elemental H2S/ NH3 9 hydrogeniphilus tube, 13˚N EPR organic sulphur compounds

Caminibacter Vent cap, Rainbow 55 ˚C Autotroph H2 Nitrate, oxygen H2S/ NH3 2 profundus Field, MAR (microaerobic) elemental sulphur

Caminibacter Chimney, Rainbow 55 ˚C Autotroph H2 Nitrate, elemental H2S/ NH3 46 mediatlanticus Field, MAR sulphur

Lebetimonas acidiphila In situ colonization 50 ˚C Autotroph H2 Elemental sulphur H2S11 system, TOTO, MA Order uncertain, Family Hydrogenimonaceae

Hydrogenimonas Chimney, Kairei 55 ˚C Autotroph H2 Nitrate, oxygen H2S/ NH3 13 thermophila Field, CIR (microaerobic), elemental sulphur Order uncertain, Family Nitratiruptoraceae

Nitratiruptor tergarcus Chimney, Iheya 55 ˚C Autotroph H2 Nitrate, oxygen H2S /N2 24 North Field, OT (microaerobic), elemental sulphur Order uncertain, Family Thioreductoraceae

Thioreductor Sediment, Iheya 32 ˚C Autotroph H2 Nitrate, elemental H2S/ NH3 45 micantisoli North Field, OT sulphur Order Campylobacterales, Family Campylobacteraceae

Sulfurospirillum sp. str. Alvinella 41 ˚C Heterotroph Formate, fumarate Elemental sulphur H2S44 Am-N pompejana, 13˚N EPR

– Arcobacter sp. str. Production water, 30 ˚C Autotroph H2, formate, Nitrate, oxygen H2S/NO2 28 FWKO B Coleville oil field sulphide (microaerobic), elemental sulphur Order uncertain, Family Thiovulgaceae

Sulfurovum Sediment, Iheya 30 ˚C Autotroph Elemental sulphur, Nitrate, oxygen N2 25 lithotrophicum North Field, OT thiosulphate (microaerobic)

Nitratifractor salsuginis Chimney, Iheya 37 ˚C Autotroph H2 Nitrate, oxygen N2 24 North Field, OT (microaerobic) Sulfurimonas Sediment, Hatoma 25 ˚C Autotroph Elemental sulphur, Oxygen 29 autotrophica Knoll, OT thiosulphate (microaerobic)

– Sulfuricurvum kujiense Groundwater, 25 ˚C Autotroph H2, sulphide, Nitrate, oxygen NO2 26 Japan oil storage thiosulphate, (microaerobic) cavity elemental sulphur

Thiomicrospira sp. str. Production water, 30 ˚C Mixotroph Sulphide, Oxygen N2, N2O28 CVO Coleville oil field elemental sulphur (microaerobic), nitrate, nitrite Location abbreviations: CIR, Central Indian Ridge; EPR, East Pacific Rise; MA, Mariana Volcanic Arc; MAR, Mid-Atlantic Ridge; OT, Okinawa Trough; TOTO, TOTO – caldera deep-sea hydrothermal field. Chemical abbreviations: H2S, hydrogen sulphide; NH3, ammonia; N2O, nitrous oxide; NO2 , nitric oxide.

An additional sequence cluster, representing other taxonomic diversity than previously hypothesized. terrestrial ecotypes (TG), is placed outside of the Moreover, other than these few termite-gut sequences, Thiovulgaceae fam. nov. and other families within the virtually nothing is known about the potential of order Campylobacterales (see Supplementary infor- ε-proteobacterial symbioses with terrestrial organisms. mation S2 (figure), part d). The TG I sequences from Future work in these poorly investigated or unexplored acid mine drainage, Lower Kane Cave, contaminated terrestrial systems should increase the known diversity groundwater and termite guts might represent greater of ε-proteobacterial groups.

REVIEWS

Box 1 | Integrating ecology and biogeochemistry: hydrothermal vents as a case study The ε-proteobacteria have been found at, and sometimes dominate, four main deep-sea hydrothermal vent-specific habitats: mats on the surfaces of rocks, chimneys and animal surfaces (a in the figure); discharged vent fluids and subseafloor (b); within the hydrothermal vent plume (c); and symbiotic associations with vent animals such as Alvinella pompejana, Alviniconcha aff. hessleri, and Rimicaris spp.103 (d). The metabolically versatile ε-proteobacteria are uniquely suited to thrive in deep-sea habitats and other extreme settings. These hydrothermal-vent habitats are all dynamic suboxic to anaerobic environments, and the ε-proteobacteria use many metabolic processes, including sulphur oxidation, sulphur/ – – sulphite reduction, nitrate (NO3 ) or nitric oxide (NO2 ) reduction to ammonium or nitrogen, and hydrogen and formate oxidation (see figure). In the figure, known electron donors are shown in the yellow shaded blocks and electron acceptors in the green shaded blocks. Some of the ε-proteobacteria might use complex sulphur species for sulphur oxidation or they might biotically influence the formation of iron-sulphur minerals, such as pyrite, at vents104–106. Carbon monoxide might also be used as an electron donor and metal(oid)s (iron, manganese, arsenic and selenium) might be used as electron acceptors, although these metabolisms have not been fully examined in vent habitats. These energy pathways can either be coupled with autotrophy (probably through the reductive TCA cycle), mixotrophy or heterotrophy (TABLE 1). The ε-proteobacterial groups establish themselves as the primary (and perhaps the first) colonizers in the dynamic diffuse flow vent environment because of their metabolic flexibility ( TABLE 1), specialized gene assemblages87, the possibility of 43 special modes for attachment to surfaces , rapid colonization at O2–H2S interfaces (possibly by formation of filamentous sulphur from hydrogen sulphide39) and phylotypic diversification over time as new habitats are colonized following eruptions or with titanium ring for Alvinella colonization (TRAC) deployment34,43,48,70. Ecological principles indicate that there is a tendency for the most productive species in an ecosystem to be the most dominant in a habitat, thereby pushing other species to comparatively lower densities. It is not surprising that the metabolically versatile ε-proteobacteria colonize extensive areas that are warmer (20–60oC) and have higher concentrations of sulphur species than locations where typical chemolithoautotrophic γ-proteobacteria are found.

Hydrothermal ﬂuids: Seawater: Anaerobic, >350°C Aerobic, 4°C Substrates: H , CO, CO , c Hydrothermal vent plume 2 2 0 – – Caminibacter profundus H2 S, NO3, O2 Substrates: O2, NO3 H2S, As, FeSx, Mn, 0 Se, organic acids Lebetimonas acidiphila H2 S

a Chimney structures – 0 a Vent fauna associations Caminibacter mediatlanticus H2 NO3, S – 0 Caminibacter hydrogeniphilus H, organics NO–, S0 Hydrogenimonas thermophila H2 NO3, O2, S 2 3 – Nautilia lithotrophica H, formate S0, SO2– Nitratifractor salsuginis H2 NO3, O2 2 3 – 0 Nautilia sp. Am-H H , formate S0, SO2– Nitratiruptor tergarcus H2 NO3, O2, S 2 3 Sulfurospirillium sp. Am-N Formate, S0 fumarate d Symbiotic associations No cultured representatives – –

b Hydrothermal sediments/subsurface Sea water seeping 0 2– Sulfurimonas autotrophica S, S2O3 O2 through crust and 0 2– – Sulfurovum lithotrophicum S, S2O3 NO3, O2 back to the vent – 0 system Thioreductor micantisoli H2 NO3, S Heat from magma below

In the figure, the cultured species from each hydrothermal-vent-specific habitat are shown. Coloured arrows indicate the flow of 0 2– either hot hydrothermal fluids (red) or cold sea water (blue). S , elemental sulphur; SO3 sulphite; S2O3, thiosulphate.

ε-Proteobacteria from marine systems knowledge about the microbial diversity of vent sites Hydrothermal vents and vent-associated subsurfaces. now reveals that ε-proteobacteria are probably key To interpret the possible ecological and geological sig- players in the cycling of (at least) carbon, nitrogen nificance of the uncultured ε-proteobacterial groups, and sulphur, and have important roles in symbiotic we look to the exemplary hydrothermal vent system associations with vent metazoans (BOX 2). Moreover, (BOX 1). Since the discovery of hydrothermal vents in deep-sea hydrothermal environments can be regarded 1977, the importance of microorganisms as the pre- as one of the largest reservoirs of diverse environmental vailing biological feature in these environments has ε-proteobacteria on Earth, ranging from the deeply- Mixotroph An organism that can use both been clearly established. Whereas early studies focused branching Nautiliales and Nitratiruptor groups to the heterotrophic and autotrophic on the endosymbiotic microbial assemblages of the Sulfurospirillum, Arcobacter, and the MG I and MG II metabolic processes. vent tube worm, Riftia pachyptila31, the accumulated of the Thiovulgaceae fam. nov.

REVIEWS

Although PCR biases and differences in library con- Deep-sea hydrothermal-vent habitats have also struction and screening32 might skew interpretations, most been important for obtaining pure cultures of diverse 16S rRNA gene-based studies indicate an overwhelming phylogenetic groups. The first successful isolations dominance of ε-proteobacteria in the free-living popula- were of hydrogen-oxidizing, sulphur-reducing, ther- tions in vent fluids or on, or near, the natural surfaces of mophilic chemolithoautotrophs from Alvinella pom- vent chimney structures where the most intensive hydro- pejana symbiont-associated biomass and tube samples; geochemical mixing occurs between ambient (1–4oC), these isolates belong to the Nautiliales10,44. Recently, oxygenated bottom sea water and high-temperature, several previously uncultivated, phylogenetically anoxic and sulphide-enriched vent fluid diffusing from diverse ε-proteobacterial groups were isolated from the interior portions of the chimney33–37. The full-cycle various geologically and geographically distinct deep- rRNA approach (which includes 16S rRNA gene clone sea hydrothermal fields, all with a diverse range of library construction and fluorescence in situ hybridization physiological characteristics and utilization of elec- (FISH)) has unequivocally shown that up to 90% of the tron donors (for example, hydrogen and sulphur) and microbial communities found in these hydrothermal sites acceptors (for example, sulphur and nitrogen) coupled are composed of ε-proteobacteria, predominately associ- to carbon fixation2,11,13,24,29,34,45–47 (FIG. 1; TABLE 1; see ated with the Nautilia and Sulfurimonas genera33,37. Supplementary information S2 (figure)). Other lines of evidence also point to the importance A sub-seafloor model proposed by Huber et al. of ε-proteobacteria at vents. Taylor and Wirsen38 showed indicates that ε-proteobacteria thrive in diffuse that the flocculent discharge emanating from diffuse flow areas surrounding vent chimneys and heated flow vents was similar to the filamentous sulphur pro- crustal fields and sediments, where sea water mixes duction by chemolithoautotrophic sulphur-oxidizing with hydrothermal fluids; ε-proteobacterial cells are bacteria, which were later shown to belong to the genus discharged when eruptions at mid-ocean-ridge axes Arcobacter19. Filamentous sulphur mats composed of flush fluids and resident microorganisms within the both vibrioid and filamentous sulphide-oxidizers, many seafloor crust and sediment to the seafloor surface of which were probably ε-proteobacteria, have also been and into sea water. Indeed, eruption plumes contain found on titanium devices deployed at vents39. Several diverse microbial communities, but ε-proteobacteria other research groups have retrieved ε-proteobacterial make up ~20–60% of 16S rRNA gene clone libraries 16S rRNA gene sequences from vent caps or other in situ from these plumes, with more intergroup diversity colonization devices34,40–43. For instance, ε-proteobacteria occurring from particle-attached libraries than from comprised ~81% of the total microbial community from free-living populations48. Eventually, the particles an in situ colonization device deployed into diffuse flow and microorganisms fall to the ocean floor and again vent emissions for 4 days in the Mid-Okinawa Trough34. become part of the marine sediment and subsurface In this study, water samples collected from 2 m and 10 m habitats. These hydrogeological processes have the away from the chimney structure had dramatically differ- potential to link microorganisms in all of the marine ent percentages of ε-proteobacteria, from 83% to 17.6%, habitats, and might result in a homogenized genetic respectively, indicating that the ε-proteobacteria tolerate pool of microorganisms over time. This could be one the immediate and proximal vent conditions, probably explanation why members of ε-proteobacteria from owing to the increased availability of energy sources com- the deep-sea hydrothermal vent and sediment systems pared with more distal habitats or cold sea water (BOX 1). are closely related to each other.

Box 2 | Episymbionts of Alvinella pompejana At least two endemic hydrothermal vent fauna, Alvinella pompejana (East Pacific Rise) and Rimicaris exoculata (Mid-Atlantic Ridge), contain ε-proteobacterial episymbionts107,108. A. pompejana, also known as the Pompeii worm because of its heat tolerance, builds paper-like tube colonies attached to hydrothermal-vent chimneys along the East Pacific Rise. The hydrothermal vent shrimp, R. exoculata, forms large clusters on the warmer sections of vents along the Mid-Atlantic Ridge. A. pompejana, the biology of which has been extensively reviewed109, contains two closely related ε-proteobacterial phylotypes that comprise over 65% of a 16S rRNA gene library107,110 derived from episymbiont biomass. These two groups, both within Marine Group I, are filamentous and distinctly separated horizontally on individual dorsal expansions of A. pompejana, indicating niche specialization (M. T. Cottrell and S. C. Cary, unpublished data and REF. 110). More recently, the first endosymbiotic ε-proteobacterium, represented by a single ε-proteobacterial phylotype, was discovered in a deep-sea hydrothermal-vent-endemic gastropod Alvinoconcha spp.111,112 There have been few clues as to the role of the epibionts of A. pompejana because, despite many attempts, the Phylotype ε A group of sequences that filamentous -proteobacterial symbionts have not yet been cultured. Surveys of geochemical conditions within o show some threshold of A. pompejana tubes revealed high temperatures (~20– 80 C) and anoxia, exceeding that of any known metazoan sequence similarity, usually habitat, surprisingly low or trace free hydrogen sulphide (<0.2–46.53 µM), pH values between 5.3 and 6.4, high >97%, and that also form a concentrations of potential electron acceptors (sulphate, nitrate and iron), and potentially lethal levels of heavy metals monophyletic clade. (zinc, nickel, vanadium, copper, lead, cadmium, cobalt and silver)113–115. Preliminary analysis of a metagenomic library of the A. pompejana symbionts supports the hypothesis that at least a portion of the symbiotic ε-proteobacteria detoxify Epibiont sulphide by rendering it biologically unavailable through metal-transport and sulphide-oxidation processes so that An organism that lives attached A. pompejana can thrive in this extreme microhabitat (S. C. Cary et al., unpublished data). Also, sequence analysis of a to a host organism without fosmid library revealed the potential for these symbionts to use the reductive TCA cycle, owing to the presence of a key apparent consequence (benefit gene in the pathway, aclBA (ATP citrate lyase)87. or detriment) to the host.

REVIEWS

Marine and subsurface environments. Several recent Supplementary information S2 (figure))30,68. Subsurface studies indicate that ε-proteobacteria, specifically terrestrial habitats are geographically isolated from one those affiliated with the Thiovulgaceae fam. nov., have another owing to geological structures, hydrostrati- important roles in nutrient cycling and ecosystem func- graphic connectivity and plate tectonics, and terrestrial tion at other marine interfaces and the overall marine organisms should have limited dispersal mechanisms subsurface habitat49. PCR-based studies reveal that and are not capable of atmospheric dispersal proc- ε-proteobacteria occur in high abundance at oxic– esses68,69. Therefore, high sequence similarity for the GG I anoxic interfaces, such as the chemocline-transition sequences retrieved from around the world indicates that zone where hydrogen sulphide from the sediment meets the ancestral population giving rise to the modern group oxygenated sea water. This is particularly true in both might have originated from one geographical location. the Black Sea and Cariaco Basin (southern Caribbean However, because GG II consists of only Lower-Kane- Sea), two of the largest anoxic basins on the planet50,51. Cave-derived sequences, this clade might be endemic The ε-proteobacteria are also prevalent in deep-sea sedi- only to Lower Kane Cave. More research is needed to ment push cores, the hydrocarbon-rich Guaymas Basin validate these relationships. sediment cores, methane cold seeps, gas hydrates and deltaic mud52–56. In many of these habitats, there is also Ecological significance of ε-proteobacteria a relatively high abundance of δ-proteobacteria. In one The ε-proteobacteria have significant roles in the habitats study of cold-seep sediments, δ- and ε-proteobacteria in which they thrive, as primary colonizers, primary pro- comprised >78% of the metabolically active fraction ducers or in symbiotic associations. Lopez-Garcia et al.70 (RNA), with the ε-proteobacteria dominating the suggest that, in deep-sea habitats, the ε-proteobacteria lower, sulphide-rich sediment fractions56. The strong maximize overall ecosystem function owing to their high relationship between δ- and ε-proteobacteria could be biomass and growth rates, rapid adaptations to changing due to their respective roles in the sulphur cycle (that is, geochemical conditions and metabolic versatility. These sulphate reduction for the δ-proteobacteria and sulphur factors all facilitate the colonization of new substrates oxidation for the ε-proteobacteria). and habitats39,43. For Candidatus A. sulfidicus, the formation of filamentous sulphur might also stimulate ε-Proteobacteria in terrestrial systems colonization of surfaces in marine habitats35. According to some researchers, an immense subsurface Because nearly all of the microorganisms isolated microbial biosphere might exist that is not just associated from deep-sea hydrothermal-vent or marine-sediment with marine sediments or deep-sea hydrothermal vent sys- ecotypes are chemolithoautotrophs (TABLE 1), these tems57,58. On the basis of recent PCR-based investigations colonizers also serve as one of the crucial sources for of terrestrial ecotypes, including naturally sulphur-rich organic carbon to the ecosystems (BOXES 1,2), especially environments such as oil-field brines15,27,28,59, hydrocar- at oxic–anoxic interfaces50,71,72. To assess the importance bon-contaminated groundwater15,59–61, uncontaminated of chemolithoautotrophy in marine and terrestrial set- groundwater62, sulphidic springs63–65 and limestone tings, however, in situ rates of carbon production or caves30,66–68, we are beginning to discover the importance substrate use are needed. Carbon-fixation rates estimated of ε-proteobacteria in these habitats. Although there are for Candidatus A. sulfidicus were equal to, or exceeded, terrestrial sequences belonging to the Sulfurospirillium those of known sulphur-oxidizing bacteria that use the and Arcobacter clusters, generally, most of the recently Calvin cycle19. Furthermore, based on previous work that acquired ε-proteobacterial sequences from natural, describes the organic carbon-stable-isotope compositions uncontaminated terrestrial habitats are affiliated with for some marine-vent organisms73,74 and corresponding the Thiovulgaceae fam. nov. (FIG. 2; see Supplementary carbon-isotope fractionation patterns, organic carbon is information S2 (figure)). Several chemolithoautotrophic, probably supplied from primary producers that use the nitrate-reducing, sulphur-oxidizing, microaerophilic rTCA cycle74. In Lower Kane Cave, M. L. P. estimated that ε-proteobacteria have been isolated from oil-field the rate of chemolithoautotrophic primary productivity 14 brines and oil-contaminated groundwater, including by H CO3 assimilation was 96.5 ± 6.0 mg carbon gram Arcobacter sp. strain FWKO B28, Thiomicrospira sp. dry weight per hour for the ε-proteobacterial-domi- strain CVO27,28 and Sulfuricurvum kujiense26,61 of GG I nated microbial mats24, which is comparable to rates of Push cores (FIGS 1,2; see Supplementary information S2 (figure)). So other autotrophic organisms75. Stable-carbon-isotope Soft sediment collected using a hollow plastic collection tube far, S. kujiense (isolated from hydrocarbon-contaminated analyses in Lower Kane Cave corroborate that chemolitho- 60 that is pushed into the Japanese groundwater ) is the only cultured terrestrial autotrophically produced carbon supports the otherwise sediment, after which the ends representative in the Thiovulgaceae fam. nov. nutrient-poor system30. Although the autotrophic carbon- are closed. Sulphidic caves (limestone caves with discharging fixation pathways were not evident from the study, the hydrogen-sulphide-rich groundwater) allow easy access Calvin–Benson cycle was implicated in carbon fixation. Methane cold seeps ε Areas of the deep ocean floor to the subsurface and are currently one of the best- In addition to cycling carbon, we know that -proteo- 30,63,66–68 where oil and methane gas studied natural terrestrial sites for ε-proteobacteria . bacteria metabolically convert various forms of reduced bubble up from under sea- Lower Kane Cave serves as an ideal model system for and oxidized sulphur and nitrogen compounds sediment layers at ambient understanding terrestrial ε-proteobacteria, particularly (TABLE 1, which has important bearing on the specia- temperatures, providing an energy source that can sustain the terrestrial Thiovulgaceae fam. nov., because all tion of sulphur/nitrogen within a habitat and on global deep-sea microbial of the sequences retrieved so far from Lower Kane Cave sulphur/nitrogen cycling, as well as on geochemical and communities. are affiliated with this evolutionary lineage (FIG. 2; see geological processes. Few studies have measured rates

REVIEWS

Wood–Ljungdahl pathway NTPs, Gluconeogenesis/glycolysis ) or the rTCA (or Arnon) cycle, dNTPs most closely resembles the first known autotrophic Amino pathway79–83 (FIG. 3). Until the latest studies on chemo- acids lithoautotrophic ε-proteobacteria, the rTCA cycle had PEP been described in only a few microorganisms, includ- CO ing the green sulphur bacterium Chlorobium limicola 2 Pyruvate (Chlorobiaceae), a few members of the δ-proteobacteria PS (for example Desulfobacter hydrogenophilus) and Fdox 2[H] CO2 some members of the thermophilic Aquificales and Oxaloacetate LPS 84–86 2[H] Fd archaeal Thermoproteaceae groups . Within the red ε-proteobacteria, the rTCA cycle was initially thought Malate Lipids Acetyl-CoA to be a potential CO2 fixation pathway in Candidatus A. sulfidicus19. Subsequently, two fosmids were Fumarate ATP-CL ADP + P sequenced from fosmid libraries linked to the domi- 2[H] 87 Frd nant ε-proteobacterial episymbionts of A. pompejana . ATP + Succinate Citrate CoASH Both fosmids contained the key indicator gene in the rTCA cycle, ATP citrate lyase (aclBA). Evidence for ATP, the potential presence and significance of the rTCA Isocitrate CoA Succinyl CoA cycle for autotrophic carbon fixation at deep-sea vents has accumulated from phylogenetic analysis of rTCA CO , 2[H] KGS 2-Oxoglutarate 2 genes amplified directly from hydrothermal vent CO α 2 ( -Ketoglutarate) chimney samples, from enzymatic expression analyses Fd 88–90 red of aclB , and from genetic analyses of the cultures of Fd 2[H] ox Ammonium assimilation Candidatus A. sulfidicus and the chemolithoautotrophic Nautilia sp. strain AmH19,87. Figure 3 | The reductive or reverse TCA (rTCA) cycle of carbon fixation. The two Phylogenetic evidence points to the close evolu- ferredoxin-linked (Fd) CO2-fixation reactions (green) are oxygen sensitive; therefore, this cycle is generally found in anaerobic to microaerophilic microorganisms. The net product tionary relatedness among the acl gene-encoded ATP of the cycle is one molecule of acetyl-coenzyme A (CoA) synthesized from two molecules of citrate lyases (ATP-CLs) of Persephonella marina (Aquificales), the ε-proteobacteria, and plants and CO2. Acetyl-CoA can be converted to pyruvate and phosphoenolpyruvate (PEP), which can either regenerate the intermediates of the cycle or be used for gluconeogenesis. animals87,90. The plant and bacterial ATP-CLs are Many rTCA-cycle intermediates are used in the generation of other cellular components, encoded by two subunits, aclB (the small subunit) as indicated by the green arrows. Key enzymes ATP citrate lyase (ATP-CL), pyruvate and aclA (the large subunit). Sections of the acl subu- synthase (PS, also known as pyruvate:ferredoxin oxidoreductase), ketoglutarate synthase nits have significant homologies to the large subunit (KGS, also known as 2-oxoglutarate:ferredoxin oxidoreductase) and fumarate reductase of succinyl-CoA synthetase and the small subunit of (Frd) are shown in blue ovals. ATP-CL, KGS and Frd allow the TCA cycle to operate in succinyl-CoA synthetase and citrate synthetase91. The reverse (red arrow indicates reverse direction). A shared feature of the Calvin–Benson and rTCA cycles is their bidirectionality; in the presence of small organic compounds, acl gene of C. limicola is more distantly related and 92 microorganisms can use the rTCA cycle in the forward, oxidizing direction. might be an ancestral form . The phylogenetic relationships point to the possibility of transfer of the acl gene to the eukaryotic population somewhere between of sulphur/nitrogen oxidation/reduction in cultured the Chlorobium and Aquificales split. However, there is organisms, much less in the environments in which they evidence of two types of citrate-cleaving systems within dominate. In Lower Kane Cave, an assessment of sul- the Aquificales themselves, with the citrate-cleaving phide-consumption rates in the cave revealed that, under citryl-CoA lyase/synthetase enzymes found in both microaerophilic conditions, the chemolithoautotrophic Hydrogenobacter thermophilus and Aquifex aeolicus, ε-proteobacterial Lower Kane Cave group II (GG II of the and ATP citrate lyase in P. marina87,91. Thiovulgaceae fam. nov.) consumed sulphide more rapidly than abiotic hydrogen sulphide loss mechanisms, and ε-Proteobacteria throughout Earth’s history were consequently found to be responsible for sulphu- Although more data are needed to resolve the evolution ric-acid dissolution of the cave host limestone76. These of the citrate-cleaving system in relation to the evolution results not only linked the biogeochemical carbon and of the rTCA cycle and the early evolution of life, the pres- sulphur cycles but also provided evidence for the geologi- ence and use of ATP-CL in some ε-proteobacteria clearly cal importance of the ε-proteobacteria to processes such incite some interesting questions with respect to the over- as cave development76. all evolutionary history of the ε-proteobacteria. How old is the subdivision? What role did these organisms have Wood–Ljungdahl pathway ε Also known as the acetyl- -Proteobacteria and the rTCA cycle in Early Earth habitats? Because marine deep-sea vents coenzyme A pathway. An Many of the ε-proteobacteria studied so far are chemo- are thought to be some of the most ancient colonized ancient carbon-fixation lithoautotrophs, and it is relevant to the evolutionary habitats on Earth93, it is not far-reaching to hypothesize pathway found in bacteria and history of this group that chemolithoautotrophy is that, as the ε-proteobacteria are dominant and important archaea in which CO2 is converted to acetate; the key thought to be the first type of metabolic pathway to have organisms in modern vents and similar extreme habitats, 77,78 enzyme is acetyl-coenzyme A evolved . One of two extant autotrophic pathways, this group has been significant to ecological and biogeo- synthase/CO dehydrogenase. the acetyl-coenzyme A (CoA) pathway (also called the chemical processes throughout much of Earth’s history.

REVIEWS

Aphotic Research to reconstruct the evolutionary relation- Future prospects Receiving no light or energy ships among prokaryotic genomes using comparative The ε-proteobacteria represent a unique assemblage from the sun. 16S rRNA phylogeny and protein-sequence analyses of microorganisms that, despite the attention given has resulted in mixed information regarding the origin to the pathogenic members, have had little defined of non-photosynthetic sulphur-oxidizing bacteria and taxonomic or ecological consideration. Our taxo- the ε-proteobacteria94–98. However, there is sufficient nomic considerations of the ε-proteobacteria reveal evidence to indicate that the ε-proteobacteria, along that there is a large group of environmentally relevant with the δ-proteobacteria, have closer genetic ances- ε-proteobacteria known mainly from phylogenetic try with the Chlorobiaceae and Aquificales than with studies of 16S rRNA genes, which we have provision- the other Proteobacteria94,97. Sheridan et al. estimated ally termed Thiovulgaceae fam. nov. Our phyloge- that the divergence of 16S rRNA gene sequences of netic analysis indicates that there are several clades of ε-proteobacteria dated to 1.37 billion years ago (Ga), ε-proteobacteria (for example, Thiovulgaceae fam. nov., which also corresponds to work by Brocks et al., who Thioreductor and Nitratiruptor) that will most likely detected the presence of the Chlorobiaceae, along with reveal more diversity in the future. the purple sulphur bacteria (Chromatiaceae) in rocks In many sulphidic habitats, especially at oxic– from 1.64 Ga based on unique hydrocarbon biomark- anoxic interfaces, ε-proteobacteria are not only ers (molecular fossils). However, much remains to be present in the microbial communities, but might be the explored about the evolution of the ε-proteobacteria dominant microorganisms involved in the cycling and because, unlike the Chlorobiaceae and Chromatiaceae99, recycling of carbon, nitrogen and sulphur compounds. no molecular fossils have yet been identified in the rock Quantitative measurements of ε-proteobacteria (for record for the ε-proteobacteria. example, using FISH) and biogeochemical cycling On the basis of biological and isotopic evidence98–102, (by measuring uptake or consumption rates) are few, the time period when ε-proteobacteria might have and more studies are needed to correlate the roles arisen on Early Earth, as far back as ~2 Ga, is marked by of ε-proteobacteria with their dominance in aphotic, a shift from a reducing to an oxidizing ocean and global sulphur-rich environments. Certainly, cave and ter- atmosphere owing to cyanobacterial photosynthesis98,100. restrial spring environments are more suited to these Although considerable debate surrounds the nature of types of measurements than deep-sea hydrothermal the geochemical conditions on Early Earth, and the rela- vents or the deep subsurface because these are readily tive timing of this transition is hotly deliberated101, recent accessible sites where phototrophic productivity can sulphur-isotope data from ancient mineral deposits be eliminated. indicate that free oxygen was present in the atmosphere Molecular methods, including FISH, metabolic and surface environments as early as ~2.2 Ga102, which gene presence/expression quantification and genome has implications for the evolution of oxygen-dependent sequencing, hold promise for understanding the sulphur-oxidation pathways. For present-day sulphur- biogeochemical roles of ε-proteobacteria in remote oxidizing bacteria that require aerobic or even micro- extreme environments. At present, genomes from at least aerophilic conditions (for instance, those affiliated with six environmentally relevant chemolithoautotrophic the γ-proteobacteria), the availability of free oxygen ε-proteobacteria from the Nautilia, Caminibacter, would have been crucial for growth. However, oxygen Arcobacter, Thiomicrospira, Sulfurovum and Nitratiruptor is not essential for many of the isolated sulphur-reduc- genera are being sequenced. More projects are needed, ing or sulphur-oxidizing ε-proteobacteria, especially however, to understand the metabolic flexibility of for the deeply-branching Nautiliales, which are obligate this group, and to better characterize the pathogenic anaerobes, or for other ε-proteobacteria that use vari- ε-proteobacteria. Genome projects will also further ous alternative electron acceptors (TABLE 1). Because the our understanding of ε-proteobacterial phylogenetic metabolic characteristics and ecotype preferences of diversity and ecophysiology, and will undoubtedly allow the modern ε-proteobacteria, including thermophilic for the identification of molecular markers to elucidate growth, anaerobic metabolism and autotrophy through the evolutionary history of the entire class, including the the rTCA cycle, are similar to the Chlorobiaceae and Thiovulgaceae fam. nov. Another major challenge is to Aquificales, the evolution and significance of the integrate this extensive molecular information and in situ ε-proteobacteria throughout Earth’s history are provoca- biogeochemical culture-based strategies to improve our tive avenues to pursue in future research. ability to isolate diverse metabolic groups.

1. Garrity, G. M., Bell, J. A. & Lilburn, T. In Bergey’s 3. Olsen, G. J., Woese, C. R. & Overbeek, R. The winds of how to integrate these diverse methods into a Manual of Systematic Bacteriology Vol. 2 (eds (evolutionary) change: breathing new life into unified taxonomic scheme. Brenner, D. J., Krieg, N. R., Staley, J. T. & Garrity, G. M.) microbiology. J. Bacteriol. 176, 1–6 (1994). 6. Gevers, D. et al. Re-evaluating prokaryotic 1145 Part C (Springer, New York, 2005). 4. On, S. L. W. International Committee on species. Nature Rev. Microbiol. 3, 733–739 2. Miroshnichenko, M. L. et al. Caminibacter profundus Systematics of Prokaryotes Subcommittee on (2005). sp. nov., a novel thermophile of Nautiliales ord. nov the taxonomy of Campylobacter and related 7. Swofford, D. PAUP*: Phylogenetic Analysis Using within the class ‘ε-proteobacteria’, isolated from a bacteria. Int. J. Syst. Evol. Microbiol. 54, 291–292 Parsimony (* and Other Methods) 4th edn (Sinauer deep-sea hydrothermal vent. Int. J. Syst. Evol. (2004). Microbiol. 54, 41–45 (2004). 5. Vandamme, P. et al. Polyphasic taxonomy, a consensus Associates, Sunderland, 2003). This paper erected the order Nautiliales, approach to bacterial systematics. Microbiol. Rev. 60, 8. Guindon, S., Lethiec, F., Duroux, P. & Gascuel, O. (containing the family Nautiliacea and the genera 407–438 (1996). PHYML Online — a web server for fast maximum Nautilia and Caminibacter), which is only the second An important overview of the plethora of methods likelihood-based phylogenetic inference. Nucleic Acids order to be described from the ε-proteobacteria. used to classify bacteria, including illustrations of Res. 33, W557–W559 (2005).

REVIEWS

9. Alain, K. et al. Caminibacter hydrogeniphilus gen. 26. Kodama, Y. & Watanabe, K. Sulfuricurvum kujiense 43. Alain, K. et al. Early steps in microbial colonization nov., sp. nov., a novel thermophilic, hydrogen-oxidizing gen. nov., sp. nov., a facultatively anaerobic, processes at deep-sea hydrothermal vents. Environ. bacterium isolated from an East Pacific Rise chemolithoautotrophic, sulfur-oxidizing bacterium Microbiol. 6, 227–241 (2004). hydrothermal vent. Int. J. Syst. Evol. Microbiol. 52, isolated from an underground crude oil storage cavity. 44. Campbell, B. J., Jeanthon, C., Kostka, J. E., Luther, G. W. 1317–1323 (2002). Int. J. Syst. Evol. Microbiol. 54, 2297–2300 (2004). & Cary, S. C. Growth and phylogenetic properties of 10. Miroshnichenko, M. L. et al. Nautilia lithotrophica 27. Voordouw, G. et al. Characterization of 16S rRNA novel bacteria belonging to the epsilon subdivision gen. nov., sp. nov., a thermophilic sulfur-reducing genes from oil field microbial communities indicates of the proteobacteria enriched from Alvinella ε-proteobacterium isolated from a deep-sea the presence of a variety of sulfate-reducing, pompejana and deep-sea hydrothermal vents. Appl. hydrothermal vent. Int. J. Syst. Evol. Microbiol. 52, fermentative, and sulfide-oxidizing bacteria. Appl. Environ. Microbiol. 67, 4566–4572 (2001). 1299–1304 (2002). Environ. Microbiol. 62, 1623–1629 (1996). The first study to describe characterized 11. Takai, K. et al. Lebetimonas acidiphila gen. 28. Gevertz, D., Telang, A. J., Voordouw, G. & enrichment cultures of four ε-proteobacteria from nov., sp. nov., a novel thermophilic, acidophilic, Jenneman, G. E. Isolation and characterization of A. pompejana symbionts and vent chimney hydrogen-oxidizing chemolithoautotroph within strains CVO and FWKOB, two novel nitrate-reducing, samples. the ε-proteobacteria, isolated from a deep-sea sulfide-oxidizing bacteria isolated from oil field brine. 45. Nakagawa, S., Inagaki, F., Takai, K., Horikoshi, K. hydrothermal fumarole in the Mariana Arc. Appl. Environ. Microbiol. 66, 2491–2501 (2000). & Sako, Y. Thioreductor micantisoli gen. nov., Int. J. Syst. Evol. Microbiol. 55, 183–189 This study is the first description of sp. nov., a novel mesophilic, sulfur-reducing (2005). chemoautotrophic isolates of ε-proteobacteria chemolithoautotroph within the ε-proteobacteria 12. Meinersmann, R. J., Patton, C. M., Evins, G. M., from any environment. isolated from hydrothermal sediments in the Wachsmuth, I. K. & Fields, P. I. Genetic diversity and 29. Inagaki, F., Takai, K., Hideki, K. I., Nealson, K. H. & Mid-Okinawa Trough. Int. J. Syst. Evol. Microbiol. relationships of Campylobacter species and Horikishi, K. Sulfurimonas autotrophica gen. nov., sp. 55, 599–605 (2005). subspecies. Int. J. Syst. Evol. Microbiol. 52, nov., a novel sulfur-oxidizing ε-proteobacterium 46. Voordeckers, J. W., Starovoytov, V. & Vetriani, C. 1789–1797 (2002). isolated from hydrothermal sediments in the Mid- Caminibacter mediatlanticus sp. nov., a thermophilic, 13. Takai, K., Nealson, K. H. & Horikoshi, K. Okinawa Trough. Int. J. Syst. Evol. Microbiol. 53, chemolithoautotrophic, nitrate-ammonifying Hydrogenimonas thermophila gen. nov., sp. nov., a 1801–1805 (2003). bacterium isolated from a deep-sea hydrothermal vent novel thermophilic, hydrogen-oxidizing 30. Engel, A. S., Porter, M. L., Stern, L. A., Quinlan, S. & on the Mid-Atlantic Ridge. Int. J. Syst. Evol. Microbiol. chemolithoautotroph within the ε-proteobacteria, Bennett, P. C. Bacterial diversity and ecosystem 55, 773–779 (2005). isolated from a black smoker in a Central Indian Ridge function of filamentous microbial mats from aphotic 47. Takai, K. et al. Isolation and phylogenetic diversity of ε hydrothermal field. Int. J. Syst. Evol. Microbiol. 54, (cave) sulfidic springs dominated by members of previously uncultivated -proteobacteria 25–32 (2004). chemolithoautotrophic ‘ε-proteobacteria’. FEMS in deep-sea hydrothermal fields. FEMS Microbiol. Lett. 14. Luijten, M. L. G. C. et al. Description of Microbiol. Ecol. 51, 31–53 (2004). 218, 167–174 (2003). Sulfurospirillum halorespirans sp. nov., an anaerobic, 31. Cavanaugh, C. M., Jones, M. L., Jannasch, H. W. & This study describes the isolation of metabolically ε tetrachloroethene-respiring bacterium, and transfer of Waterbury, J. B. Prokaryotic cells in the hydrothermal diverse marine -proteobacteria, predominately Dehalospirillum multivorans to the genus vent tube worm Riftia pachyptila Jones: possible from vent sites, whereby many have recently been Sulfurospirillum as Sulfurospirillum multivorans comb. chemoautotrophic symbionts. Science 213, 340–342 described as new taxa. nov. Int. J. Syst. Evol. Microbiol. 53, 787–793 (1981). 48. Huber, J. A., Butterfield, D. A. & Baross, J. A. (2003). 32. Wintzingerode, F. V., Göbel, U. B. & Stackebrandt, E. Bacterial diversity in a subseafloor habitat following a 15. Stolz, J. F. et al. Sulfurospirillum barnesii sp. nov. Determination of microbial diversity in environmental deep-sea volcanic eruption. FEMS Microbiol. Ecol. 43, and Sulfurospirillum arsenophilum sp. nov., new samples: pitfalls of PCR-based rRNA analysis. FEMS 393–409 (2003). members of the Sulfurospirillum clade of the Microbiol. Rev. 21, 213–229 (1997). 49. Kormas, K. A., Smith, D. C., Edgcomb, V. & Teske, A. ε-proteobacteria. Int. J. Syst. Bacteriol. 49, 33. Takai, K. et al. Spatial distribution of marine Molecular analysis of deep subsurface microbial communities in Nankai Trough sediments (ODP Leg 1177–1180 (1999). Crenarchaeota group I in the vicinity of deep-sea 190, Site 1176). FEMS Microbiol. Ecol. 45, 115–125 16. Snelling, W. J., Matsuda, M., Moore, J. E. & hydrothermal systems. Appl. Environ. Microbiol. 70, (2003). Dooley, J. S. G. Under the microscope: Arcobacter. 2404–2413 (2004). 50. Madrid, V. M., Taylor, G. T., Scranton, M. I. & Lett. Appl. Microbiol. 42, 7–14 (2006). 34. Nakagawa, S. et al. Distribution, phylogenetic Chistoserdov, A. Y. Phylogenetic diversity of bacterial 17. Maugeri, T. L. et al. Detection and enumeration of diversity and physiological characteristics of and archaeal communities in the anoxic zone of the Arcobacter spp. in the coastal environment of the ε-proteobacteria in a deep-sea hydrothermal field. Cariaco Basin. Appl. Environ. Microbiol. 67, Straits of Messina (Italy). New Microbiol. 28, Environ. Microbiol. 7, 1619–1632 (2005). 1663–1674 (2001). 177–182 (2005). 35. Moyer, C. L., Dobbs, F. C. & Karl, D. M. Phylogenetic 51. Vetriani, C., Tran, H. V. & Kerkhof, L. J. Fingerprinting 18. Mcclung, C. R., Patriquin, D. G. & Davis, R. E. diversity of the bacterial community from a microbial microbial assemblages from the oxic/anoxic Campylobacter nitrofigilis sp. nov., a nitrogen-fixing mat at an active, hydrothermal vent system, Loihi chemocline of the Black Sea. Appl. Environ. Microbiol. bacterium associated with roots of Spartina Seamount, Hawaii. Appl. Environ. Microbiol. 61, 69, 6481–6488 (2003). alterniflora Loisel. Int. J. Syst. Bacteriol. 33, 1555–1562 (1995). 52. Li, L., Kato, C. & Horikoshi, K. Microbial diversity in 605–612 (1983). This study is the first to focus on characterizing the sediments collected from the deepest cold-seep area, 19. Wirsen, C. O. et al. Characterization of an autotrophic genetic diversity of 16S rRNA gene sequences of a the Japan Trench. Marine Biotechnol. 1, 391–400 sulfide-oxidizing marine Arcobacter sp. that produces hydrothermal vent microbial community, finding (1999). filamentous sulfur. Appl. Environ. Microbiol. 68, that ε-proteobacteria dominate the samples. 53. Mills, H. J., Hodges, C., Wilson, K., MacDonald, I. R. & 316–325 (2002). 36. Longnecker, K. & Reysenbach, A. L. Expansion of the Sobecky, P. A. Microbial diversity in sediments The process of filamentous sulphur formation is geographic distribution of a novel lineage of associated with surface-breaching gas hydrate mounds ε attributed to the isolate Candidatus A. sulfidicus -proteobacteria to a hydrothermal vent site on the in the Gulf of Mexico. FEMS Microbiol. Ecol. 46, that was later found to use the rTCA pathway for Southern East Pacific Rise. FEMS Microbiol. Ecol. 35, 39–52 (2003). CO fixation. 287–293 (2001). 2 54. Todorov, J. R., Chistoserdov, A. Y. & Aller, J. Y. 20. De Vos, P. & Trüper, H. G. Judicial Commission 37. Nakagawa, T. et al. Geomicrobiological exploration Molecular analysis of microbial communities in of the International Committee on Systematic and characterization of a novel deep-sea hydrothermal mobile deltaic muds of Southeastern Papua New Bacteriology. IXth International (IUMS) Congress system at the TOTO caldera in the Mariana Volcanic Guinea. FEMS Microbiol. Ecol. 33, 147–155 of Bacteriology and Applied Microbiology. Minutes Arc. Environ. Microbiol. 8, 37–49 (2006). (2000). of the meetings, 14, 15 and 18 August 1999, 38. Taylor, C. D. & Wirsen, C. O. Microbiology and ecology 55. Teske, A. et al. Microbial diversity of hydrothermal Sydney, Australia. Int. J. Syst. Evol. Microbiol. 50, of filamentous sulfur formation. Science 277, sediments in the Guaymas Basin: evidence for 2239–2244 (2000). 1483–1485 (1997). anaerobic methanotrophic communities. Appl. 21. Euzeby, J. P. et al. ‘List of Changes in Taxonomic This study links the widespread occurrence of Environ. Microbiol. 68, 1994–2007 (2002). Opinion’: making use of the new lists. Int. J. Syst. Evol. filamentous sulphur formation in marine habitats to 56. Inagaki, F., Sakihama, Y., Inoue, A., Kato, C. & Microbiol. 54, 1429–1430 (2004). laboratory sulphur produced by enrichment cultures Horikoshi, K. Molecular phylogenetic analyses of 22. Euzeby, J. P. Notification of changes in taxonomic of a chemolithoautotrophic sulphur-oxidizing reverse-transcribed bacterial rRNA obtained from opinion previously published outside the IJSEM. Int. J. bacterium isolated from coastal marine seawater. deep-sea cold seep sediments. Environ. Microbiol. 4, Syst. Evol. Microbiol. 56, 11 (2006). 39. Taylor, C. D., Wirsen, C. O. & Gaill, F. Rapid microbial 277–286 (2002). 23. Wirsen, C. O. & Jannasch, H. W. Physiological and production of filamentous sulfur mats at hydrothermal 57. Whitman, W. B., Coleman, D. C. & Wiebe, W. J. morphological observations on Thiovulum sp. vents. Appl. Environ. Microbiol. 65, 2253–2255 Prokaryotes: The unseen majority. Proc. Natl Acad. J. Bacteriol. 136, 765–774 (1978). (1999). Sci. USA 95, 6578–6583 (1998). 24. Nakagawa, S., Takai, K., Inagaki, F., Horikoshi, K. & 40. Corre, E., Reysenbach, A. L. & Prieur, D. 58. Pedersen, K. Exploration of deep intraterrestrial Sako, Y. Nitratiruptor tergarcus gen. nov., sp. nov. and ε-proteobacterial diversity from a deep-sea microbial life: current perspectives. FEMS Microbiol. Nitratifractor salsuginis gen. nov., sp. nov., nitrate- hydrothermal vent on the Mid-Atlantic Ridge. FEMS Lett. 185, 9–16 (2001). reducing chemolithoautotrophs of the ε-proteobacteria Microbiol. Lett. 205, 329–335 (2001). 59. Schumacher, W., Kroneck, P. M. H. & Pfennig, N. isolated from a deep-sea hydrothermal system in the 41. Reysenbach, A. L., Longnecker, K. & Kirshtein, J. Comparative systematic study on Spirillum-5175, Mid-Okinawa Trough. Int. J. Syst. Evol. Microbiol. 55, Novel bacterial and archaeal lineages from an in situ Campylobacter and Wolinella species — description of 925–933 (2005). growth chamber deployed at a Mid-Atlantic Ridge Spirillum-5175 as Sulfurospirillum deleyianum gen. 25. Inagaki, F., Takai, K., Nealson, K. H. & Horikoshi, K. hydrothermal vent. Appl. Environ. Microbiol. 66, nov., sp. nov. Arch. Microbiol. 158, 287–293 (1992). Sulfurovum lithotrophicum gen. nov., sp. nov., a novel 3798–3806 (2000). 60. Watanabe, K., Kodama, Y., Syutsubo, K. & Harayama, S. sulfur-oxidizing chemolithoautotroph within the 42. Higashi, Y. et al. Microbial diversity in hydrothermal Molecular characterization of bacterial populations in ε-proteobacteria isolated from Okinawa Trough surface to subsurface environments of Suiyo Seamount, petroleum-contaminated groundwater discharged hydrothermal sediments. Int. J. Syst. Evol. Microbiol. Izu-Bonin Arc, using a catheter-type in situ growth from underground crude oil storage cavities. Appl. 54, 1477–1482 (2004). chamber. FEMS Microbiol. Ecol. 47, 327–336 (2004). Environ. Microbiol. 66, 4803–4809 (2000).

REVIEWS

61. Kodama, Y. & Watanabe, K. Isolation and 84. Evans, M. C. W., Buchanan, B. B. & Arnon, D. I. A new 106. Goffredi, S. K., Waren, A., Orphan, V. J., Van Dover, C. L. characterization of a sulfur-oxidizing chemolithotroph ferredoxin-dependent carbon reduction cycle in a & Vrijenhoek, R. C. Novel forms of structural growing on crude oil under anaerobic conditions. Appl. photosynthetic bacterium. Proc. Natl Acad. Sci. USA integration between microbes and a hydrothermal Environ. Microbiol. 69, 107–112 (2003). 55, 928–934 (1966). vent gastropod from the Indian Ocean. Appl. Environ.

62. Pedersen, K., Nilsson, E., Arlinger, J., Hallbeck, L. & 85. Fuchs, G., Stupperich, E. & Eden, G. Autotrophic CO2 Microbiol. 70, 3082–3090 (2004). O’Neill, A. Distribution, diversity and activity of fixation in Chlorobium limicola — evidence for the 107. Haddad, A., Camacho, F., Durand, P. & Cary, S. microorganisms in the hyper-alkaline spring waters of operation of a reductive tricarboxylic acid cycle in Phylogenetic characterization of the epibiotic bacteria Maqarin in Jordan. Extremophiles 8, 151–164 growing cells. Arch. Microbiol. 128, 64–71 associated with the hydrothermal vent polychaete (2004). (1980). Alvinella pompejana. Appl. Environ. Microbiol. 61, 63. Barton, H. A. & Luiszer, F. Microbial metabolic 86. Hugler, M., Huber, H., Stetter, K. O. & Fuchs, G. 1679–1687 (1995).

structure in a sulfidic cave hot spring: potential Autotrophic CO2 fixation pathways in archaea The first paper showing the dominance of mechanisms of biospeleogenesis. J. Cave Karst Stud. (Crenarchaeota). Arch. Microbiol. 179, 160–173 ε-proteobacterial 16S rRNA gene sequences in 67, 28–38 (2005). (2003). the episymbionts of A. pompejana. 64. Elshahed, M. S. et al. Bacterial diversity and sulfur 87. Campbell, B. J., Stein, J. L. & Cary, S. C. Evidence of 108. Polz, M. F. & Cavanaugh, C. M. Dominance of one cycling in a mesophilic sulfide-rich spring. Appl. chemolithoautotrophy in the bacterial community bacterial phylotype at a Mid-Atlantic Ridge Environ. Microbiol. 69, 5609–5621 (2003). associated with Alvinella pompejana, a hydrothermal hydrothermal vent site. Proc. Natl Acad. Sci. USA 92, 65. Rudolph, C., Moissl, C., Henneberger, R. & Huber, R. vent polychaete. Appl. Environ. Microbiol. 69, 7232–7236 (1995). Ecology and microbial structures of archaeal/bacterial 5070–5078 (2003). 109. Desbruyéres, D. et al. Biology and ecology of the strings-of-pearls communities and archaeal relatives 88. Campbell, B. J. & Cary, S. C. Abundance of reverse ‘Pompeii worm’ (Alvinella pompejana ), a normal thriving in cold sulfidic springs. FEMS Microbiol. Ecol. tricarboxylic acid cycle genes in free-living dweller of an extreme deep-sea environment: a 50, 1–11 (2004). microorganisms at deep-sea hydrothermal vents. Appl. synthesis of current knowledge and recent 66. Angert, E. R. et al. Molecular phylogenetic analysis Environ. Microbiol. 70, 6282–6289 (2004). developments. Deep Sea Res. II 45, 383–422 of a bacterial community in Sulphur River, Parker The abundance and expression of rTCA genes (1998). Cave, Kentucky. Amer. Mineral. 83, 1583–1592 in microbial communities from deep-sea 110. Cary, S. C., Cottrell, M. T., Stein, J. L., Camacho, F. & (1998). hydrothermal vents establishes the ubiquity Desbruyeres, D. Molecular identification and 67. Engel, A. S., Porter, M. L., Kinkle, B. K. & Kane, T. C. of ε-proteobacteria and chemolithoautotrophy localization of filamentous symbiotic bacteria Ecological assessment and geological significance of at vents. associated with the hydrothermal vent annelid microbial communities from Cesspool Cave, Virginia. 89. Hugler, M., Wirsen, C. O., Fuchs, G., Taylor, C. D. & Alvinella pompejana. Appl. Environ. Microbiol. 63,

Geomicrobiol. J. 18, 259–274 (2001). Sievert, S. M. Evidence for autotrophic CO2 fixation via 1124–1130 (1997). 68. Engel, A. S. et al. Filamentous ‘ε-proteobacteria’ the reductive tricarboxylic acid cycle by members of 111. Urakawa, H. et al. Hydrothermal vent gastropods from dominate microbial mats from sulfidic cave the ε subdivision of proteobacteria. J. Bacteriol. 187, the same family (Provannidae) harbour ε- and springs. Appl. Environ. Microbiol. 69, 5503–5511 3020–3027 (2005). γ-proteobacterial endosymbionts. Environ. Microbiol. (2003). 90. Takai, K. et al. Enzymatic and genetic characterization 7, 750–754 (2005). The first study to show that filamentous of carbon and energy metabolisms by deep-sea 112. Suzuki, Y. et al. Novel chemoautotrophic ε-proteobacteria are abundant and of metabolic hydrothermal chemolithoautotrophic isolates of endosymbiosis between a member of the significance in a natural, terrestrial, subterranean ε-proteobacteria. Appl. Environ. Microbiol. 71, ε-proteobacteria and the hydrothermal vent gastropod system. 7310–7320 (2005). Alviniconcha aff. hessleri (Gastropoda: Provannidae) 69. Finlay, B. J. Global dispersal of free-living microbial 91. Aoshima, M., Ishii, M. & Igarashi, Y. A novel enzyme, from the Indian Ocean. Appl. Environ. Microbiol. 71, eukaryote species. Science 296, 1061–1063 (2002). citryl-CoA lyase, catalysing the second step of the 5440–5450 (2005). 70. Lopez-Garcia, P. et al. Bacterial diversity in citrate cleavage reaction in Hydrogenobacter 113. Cary, S. C., Shank, T. & Stein, J. Worm basks in hydrothermal sediment and ε-proteobacterial thermophilus TK-6. Mol. Microbiol. 52, 763–770 extreme temperatures. Nature 391, 545 (1998). dominance in experimental microcolonizers at the (2004). 114. Di Meo-Savoie, C. A., Luther, G. W. & Cary, S. C. Mid-Atlantic Ridge. Environ. Microbiol. 5, 961–976 92. Fatland, B. L. et al. Molecular characterization of a Physicochemical characterization of the microhabitat (2003). heteromeric ATP-citrate lyase that generates cytosolic of the epibionts associated with Alvinella pompejana, 71. Casamayor, E. O., Garcia-Cantizano, J., Mas, J. & acetyl-coenzyme A in Arabidopsis. Plant Physiol. 130, a hydrothermal vent annelid. Geochim. Cosmochim. Pedros-Alio, C. Primary production in estuarine oxic/ 740–756 (2002). Acta 68, 2055–2066 (2004).

anoxic interfaces: contribution of microbial dark CO2 93. Reysenbach, A. L., Banta, A. B., Boone, D. R., Cary, S. C. 115. Luther, G. W. et al. Chemical speciation drives fixation in the Ebro River Salt Wedge Estuary. Mar. & Luther, G. W. Microbial essentials at hydrothermal hydrothermal vent ecology. Nature 410, 813–816 Ecol. Prog. Ser. 215, 49–56 (2001). vents. Nature 404, 835–835 (2000). (2001). 72. Taylor, G. T. et al. Chemoautotrophy in the redox 94. Gupta, R. S. The phylogeny of proteobacteria: 116. Edgar, R. C. MUSCLE: multiple sequence alignment transition zone of the Cariaco Basin: a significant relationships to other eubacterial phyla and with high accuracy and high throughput. Nucleic Acids midwater source of organic carbon production. Limnol. eukaryotes. FEMS Microbiol. Rev. 24, 367–402 Res. 32, 1792–1797 (2004). Oceanogr. 46, 148–163 (2001). (2000). 117. Castresana, J. Selection of conserved blocks from 73. Van Dover, C. L. & Fry, B. Stable isotopic compositions 95. Wolf, Y. I., Rogozin, I. B., Grishin, N. V., Tatusov, R. L. & multiple alignments for their use in phylogenetic of hydrothermal vent organisms. Mar. Biol. 102, Koonin, E. V. Genome trees constructed using five analysis. Mol. Biol. Evol. 17, 540–552 (2000). 257–263 (1989). different approaches suggest new major bacterial 74. Preuss, A., Schauder, R., Fuchs, G. & Stichler, W. Carbon clades. BMC Evol. Biol. 1, 8 (2001). Acknowledgements isotope fractionation by autotrophic bacteria with 3 96. Gupta, R. S. & Griffiths, E. Critical issues in bacterial B.J.C was partially supported by the National Science

different CO2 fixation pathways. Z. Naturforsch. [C] phylogeny. Theor. Popul. Biol. 61, 423–434 Foundation. A.S.E was partially supported by the College of 44, 397–402 (1989). (2002). Basic Sciences at Louisiana State University, USA. The 75. Porter, M. L. Ecosystem Energetics of Sulfidic Karst. 97. Sheridan, P. P., Freeman, K. H. & Brenchley, J. E. authors thank T.E. Hanson for his valuable input in to this Thesis, Univ. Cincinnati (1999). Estimated minimal divergence times of the major review. 76. Engel, A. S., Stern, L. A. & Bennett, P. C. Microbial bacterial and archaeal phyla. Geomicrobiol. J. 20, contributions to cave formation: new insights into 1–14 (2003). Competing interests statement sulfuric acid speleogenesis. Geology 32, 369–372 98. Canfield, D. E. & Teske, A. Late Proterozoic rise in The authors declare no competing financial interests. (2004). atmospheric oxygen concentration inferred from 77. Russell, M. J. & Hall, A. J. The emergence of life from phylogenetic and sulphur-isotope studies. Nature DATABASES iron monosulphide bubbles at a submarine 382, 127–132 (1996). The following terms in this article are linked online to: hydrothermal redox and pH front. J. Geol. Soc. London 99. Brocks, J. J. et al. Biomarker evidence for green and Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ 154, 377–402 (1997). purple sulphur bacteria in a stratified entrez/query.fcgi?db=genomeprj 78. Wachtershauser, G. The case for the chemoautotrophic Palaeoproterozoic sea. Nature 437, 866–870 Aquifex aeolicus | Chlorobium limicola | Helicobacter pylori | origin of life in an iron-sulfur world. Orig. Life Evol. (2005). Nautilia sp. strain AmH | Persephonella marina Biosph. 20, 173–176 (1990). 100. Johnson, C. M. & Beard, B. L. Geochemistry. 79. Wachtershauser, G. Evolution of the 1st metabolic Biogeochemical cycling of iron isotopes. Science 309, FURTHER INFORMATION cycles. Proc. Natl Acad. Sci. USA 87, 200–204 1025–1027 (2005). Barbara J. Campbell’s homepage: (1990). 101. Des Marais, D. J. Palaeobiology: sea change in http://www.ocean.udel.edu/cms/bcampbell This paper presents the hypothesis that a version sediments. Nature 437, 826–827 (2005). Annette Summers Engel’s laboratory: of the rTCA cycle was the first metabolic cycle to 102. Farquhar, J. & Wing, B. Multiple sulfur isotopes and http://www.geol.lsu.edu/Faculty/Engel/profile.html have evolved. the evolution of the atmosphere. Earth Planet. Sci. A metagenome of Alvinella pompejana symbiont database: 80. Smith, E. & Morowitz, H. J. Universality in Lett. 213, 1–13 (2003). http://ocean.dbi.udel.edu/index.php intermediary metabolism. Proc. Natl Acad. Sci. USA 103. Karl, D. M. The microbiology of deep-sea DDBJ: http://www.ddbj.nig.acjp 101, 13168–13173 (2004). hydrothermal vents. (CRC Press, Boca Raton, EMBL: http://www.ebi.ac.uk/embl 81. Russell, M. J. & Martin, W. The rocky roots of the 1995). ε-Proteobacteria sequence data files and alignments: acetyl-CoA pathway. Trends Biochem. Sci. 29, 104. Wirsen, C. O., Jannasch, H. W. & Molyneaux, S. J. http://geol.lsu.edu/Faculty/Engel/epsilon.htm 358–363 (2004). Chemosynthetic microbial activity at Mid-Atlantic GenBank: http://www.ncbi.nlm.nih.gov/Genbank 82. Pereto, J. G., Velasco, A. M., Becerra, A. & Lazcano, A. Ridge hydrothermal vent sites. J. Geophys. Res. 98, RDPII: http://wdcm.nig.ac.jp/RDP/html/index.html Comparative biochemistry of CO2 fixation and the 9693–9703 (1993). evolution of autotrophy. Int. Microbiol. 2, 3–10 (1999). 105. Zbinden, M., Martinez, I., Guyot, F., Cambon- SUPPLEMENTARY INFORMATION 83. Lindahl, P. A. & Chang, B. The evolution of acetyl-CoA Bonavita, M. A. & Gaill, F. Zinc-iron sulphide See online article: S1 (table) | S2 (figure) synthase. Orig. Life Evol. Biosphere 31, 403–434 mineralization in tubes of hydrothermal vent worms. Access to this links box is available online. (2001). Eur. J. Mineral. 13, 653–658 (2001).