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 458 | JUNE 2006 | VOLUME 4 www.nature.com/reviews/micro © 2006 Nature Publishing Group 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 geochemi- cal interface between dissolved oxygen and sulphide n mpylobacter e Ca 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' mesophilic chemolithoautotrophic undergoes , 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