Comparative Genomic Analysis of the Class Epsilonproteobacteria and Proposed Reclassification to Epsilonbacteraeota (phyl. nov.)

1The Epsilonproteobacteria were first described in the early 1990s as the fifth subclass of the Proteobacteria and subsequently assigned class status within this phylum. The group is widely known for its pathogenic genera Campylobacter, Helicobacter and, to a lesser extent Arcobacter. However, other members of this class are known to play ecologically important roles across a diverse range of environments in which they thrive as mesophiles or moderate thermophiles. Epsilonproteobacteria are important chemolithotrophic primary producers in deep-sea hydrothermal vent systems, where they are often the dominant bacterial lineage in vent plumes and deposits, and surrounding microbial mats. On vent chimneys, Epsilonproteobacteria can account for up to 85% of the microbial biomass. Their metabolic capacity to perform sulfur oxidation coupled to N-oxide reduction while fixing carbon via the reverse TCA cycle enables them to be early colonizers of uninhabited vent ecosystems. Non-pathogenic relatives of Campylobacter such as Sulfurospirillum and Thiovulum are often detected in sulfide-rich sediments while others show an affinity for hydrocarbon-rich environments. Host-association is also common in this class: Campylobacter, Helicobacter, and some Arcobacter species are known opportunistic pathogens of vertebrates while members of the Caminibacter, Nautilia, and Sulfurospirillum have been reported in association with deep-sea hydrothermal vent fauna. Recent metatranscriptomic data highlighted the role of Sulfurimonas-like bacteria in hydrogenase-driven sulfur oxidation and denitrification on the gills of a deep vent sea snail.

2While the Epsilonproteobacteria constitute a stable monophyletic group within the bacterial tree of life, a number of studies suggest that they do not reproducibly affiliate with other Proteobacteria, with the exception of the Desulfurellales, which are presently classified as an order of the Deltaproteobacteria (see below). Early conserved marker gene-based studies showing Epsilonproteobacteria branching immediately basal to other Proteobacteria often had inadequate outgroups and/or bootstrap values supporting this placement, while others did not resolve this association at all. More recent phylogenomic evidence based on multiple marker proteins and greater outgroup representation have largely failed to recover the Epsilonproteobacteria as reproducibly monophyletic with the rest of the Proteobacteria, further suggesting that taxonomic revision is required at the phylum level. The class Epsilonproteobacteria currently comprises two orders, Campylobacterales and Nautiliales, encompassing a number of species with ambiguous placement. Particularly problematic are the genera Nitratifractor and Nitratiruptor. The SILVA and LPSN taxonomies currently list these organisms as members of the Nautiliaceae, but this classification is not universally accepted and phylogenetic evidence suggests that they may represent novel families. In a review of the Epsilonproteobacteria, used the placeholder family Thiovulgaceae to group the Nitratifractor with Sulfurovum and Sulfurimonas, and the family Nitratiruptoraceae to describe the divergent nature of Nitratiruptor from other families. An identical taxonomy was proposed with recent phylogenomic evidence, which also revealed a stable monophyletic clade of Epsilonproteobacteria with the deltaproteobacterial genus Hippea, a member of the order Desulfurellales. Both genera belonging to this order, Desulfurella and Hippea, have low 16S rRNA gene sequence identity to other members of the Deltaproteobacteria and frequently form a clade with members of the Epsilonproteobacteria, suggesting that they should be transferred from the Deltaproteobacteria to this group.

3The widespread adoption of high-throughput sequencing technologies has resulted in the number of sequenced genomes from bacteria exceeding 70,000 in recent years1. Additionally, advances in obtaining high-quality draft genomes from metagenomic data (population genomes;; ) and single cells greatly augments genomic coverage of microbial diversity and provides the opportunity to supplant the 16S rRNA gene as the basis for microbial classification. Here, we report a phylogenomic characterization of 624 publicly available Epsilonproteobacteria and Desulfurellales isolate genomes supplemented with 33 Epsilonproteobacteria population genomes. As part of this study, we also sequenced a near-complete genome of Hydrogenimonas thermophila, and analyzed three partial genomes of single cells belonging to the genus Thioreductor. Based on our results, we propose reclassifying the Epsilonproteobacteria and Desulfurellales as a new phylum, the Epsilonbacteraeota (phyl. nov.), together with a number of subordinate changes and additions at the order and family levels.

4An ingroup comprising 619 Epsilonproteobacteria, four Hippea species and Desulfurella acetivorans were obtained from NCBI RefSeq and GenBank (Supplementary Table S1), and 33 Epsilonproteobacteria population genomes (Supplementary Table S2) were recovered from public metagenomic datasets2. The genome of H. thermophila was sequenced using the Illumina HiSeq 2500 platform (2 × 150 bp chemistry). Raw sequence data (2.4 M reads) were quality filtered using trimmomatic v0.33 in paired end mode, requiring an average quality score of Q ≥ 20 over a sliding window of four bases, and a minimum sequence length of 36 nucleotides. A draft genome was assembled using SPAdes v3.8.1 with a kmer size range of 35–75 (step size = 4) and automatic coverage cutoff. The genome was then scaffolded using FinishM v0.0.93, and scaffolds assessed for assembly errors using RefineM v0.0.134.

5Three partial Thioreductor genomes were obtained by single cell genome sequencing (Supplementary Table S2). Raw sequence data (41 M reads) were quality filtered as per H. thermophila. Quality-filtered sequences were digitally normalized using khmer v2.0 using the default two-pass approach. Normalized sequences were assembled using SPAdes, and the resulting contigs were scaffolded and refined using RefineM and FinishM as for H. thermophila. The taxonomic identity of each Thioreductor genome was confirmed by screening high-quality reads for 16S rRNA gene sequence fragments using GraftM5. Putative 16S rRNA gene fragments were aligned using the SINA web aligner and inserted into the SILVA SSU non-redundant database v123.1 using the parsimony insertion tool in ARB.

6An outgroup of 4,072 publicly available genomes representing unique species of 24 bacterial phyla were also obtained from NCBI. Completeness and contamination of all genomes was estimated using CheckM v1.0.6 with default settings.

7Ingroups for phylogenetic analyses were selected from the 653 Epsilonproteobacteria (including H. thermophila and the 33 population genomes) and five Desulfurellales genomes. The three partial Thioreductor genomes were only included in a reduced concatenated gene analysis due to their low estimated completeness (see below). To resolve the placement of the ingroup in the bacterial domain, 98 ingroup genomes representative at the species-level were selected and combined with the 4,072 outgroup genomes described above. Phylogenetic inference was performed on the 4,170 genomes using a concatenation of 120 conserved protein marker sequences. Protein sequences in each genome were identified and aligned to reference alignments using hmmer v3.1. Aligned markers were then concatenated and poorly aligned regions removed using Gblocks v0.91b.

8Maximum likelihood inference of the multiple sequence alignment was performed using the Jones-Taylor-Thornton (JTT), Whelan and Goldman (WAG), and Le and Gascuel (LG) models for amino acid evolution with gamma distributed rate heterogeneity (+Γ) implemented in FastTree v2.1.9. Neighbor joining (NJ) was performed using the Jukes-Cantor and Kimura distance corrections, and with an uncorrected distance matrix implemented in Clearcut v1.0.9. Under each model/correction, tree building was performed with all sequences included, then once with each phylum or singleton lineage removed, with the exception of Proteobacteria and ingroup genomes (a total of 186 trees). All trees were bootstrap-resampled 100 times to assess the stability of tree topologies. Robustness and reproducibility of the tree topology and association between the Epsilonproteobacteria, Desulfurellales, and Proteobacteria was assessed by manual examination of all tree topologies in ARB.

9To resolve the internal structure of Epsilonbacteraeota relationships, a slightly larger data set of 110 ingroup genomes were selected (106 Epsilonproteobacteria, four Desulfurellales) and maximum likelihood inference performed using RAxML v8.1.11. An outgroup of 10 genomes representing six bacterial phyla, including Proteobacteria and Aquificae was used to root this tree. Phylogenetic inference was performed with the WAG+Γ model and 100 bootstrap resamples. To further evaluate the robustness of Epsilonbacteraeota relationships, single gene trees were constructed using FastTree with the WAG+Γ model on the individual protein alignments and tree topologies compared to that of the concatenated alignment using the phytools and ape packages in the R software environment.

10Manual inspection of the three partial Thioreductor genomes identified 14 protein families common to all three. Phylogenetic analysis of Thioreductor was performed using the above set of 110 ingroup genomes and associated outgroup, using only these 14 protein markers. Phylogenetic inference was performed using RAxML as described above. To assess the placement of species for which genome data is not available, 16S rRNA gene analysis was performed. Epsilonbacteraeota sequences were obtained from the SILVA Living Tree Project v123. As this database does not possess a representative for the genus Thiovulum, a 16S rRNA sequence for this lineage was obtained from NCBI GenBank. Full length 16S rRNA gene sequences of Thiofractor thiocaminus, Candidatus Thioturbo danicus, Cetia pacifica, and Thioreductor species were aligned using the SINA web aligner. An outgroup comprising members of the Proteobacteria, Aquificae, and four other phyla was used to root the tree. The sequence alignment was masked using the LTP 50% SSU conservation filter prior to tree construction. Phylogenetic inference of the masked alignment was performed using RAxML with the general time reversible model with gamma distributed rate heterogeneity and 1,000 bootstrap resamples. Short sequences (<1,000 bp) belonging to Candidatus Thioturbo danicus and Thioreductor sp. Shim25-G were inserted into the resulting topology using the ARB parsimony insert tool. All tree figures were edited for publication in Inkscape v0.48.

11In order to compare our taxonomic proposals to previously proposed sequence similarity-based thresholds for taxonomic ranking, we performed 16S rRNA gene sequence and amino acid identity (AAI) comparisons between members of reclassified or newly proposed families. 16S rRNA gene sequences belonging to Epsilonproteobacteria and Desulfurellales type strains were extracted from the SILVA Living Tree Project v123 database. Sequences were aligned and hypervariable regions removed using the Lane mask. Pairwise sequence distances were calculated for 16S rRNA sequences belonging to the same family but different genera using mothur v1.39.1. AAIs of all best bi-directional diamond hits between pairwise comparisons of the 110 ingroup genomes were made using CompareM v0.0.216. AAI scores were obtained for genome pairs belonging to the same family, but different genera. Sequence similarity results for each family were visualized using R and compared to previously proposed taxonomic rank boundaries.

12Functional gene predictions for all Epsilonbacteraeota genomes were performed using Prodigal v2.6.3. Amino acid translations of predicted genes were annotated using diamond v0.8.10.72 against the Uniref 100 database (downloaded October 2015) and the accessions of target sequences mapped to their KEGG Orthology (KO) group. Annotations were transformed into an abundance matrix using a custom perl script and principal component analysis was performed using the R package vegan v2.3. Genomes were partitioned into host-associated or ‘environmental’ and indicator analysis was performed using the package indicspecies. KO groups that were significantly associated with either the host-associated or environmental lifestyle were grouped into their functional pathway, and fitted to the PCA ordination using the envfit function in vegan. Additional annotation of hydrogenase enzymes was performed using BLAST against a manually curated database. Homologous sequences were defined as greater than 30% AAI over at least 70% of the target protein length. Annotation of the reference proteins ACM93230, ACM93747, and ACM93557 of the pathway proposed to facilitate nitrite reduction to ammonium in Nautilia profundicola was performed with the same BLAST parameters as for hydrogenases.

13Phylogenetic analyses of genes involved in carbon fixation, nitrogen and sulfur cycling, and flagella structure and formation were performed using mingle v0.0.187. Protein markers for marker genes (Supplementary Table S3) were downloaded from UniProt and used for initial homolog discovery against the Genome Taxonomy Database (GTDB)8. Putative protein homologs were manually inspected for false positive matches and genes below the identity threshold or with inconsistent annotations were removed. Putative citrate lyase alpha/beta subunits sequences were also removed if a homolog of each protein in the pair was not detected in a given genome to ensure paralogs were not being directly compared. A similar strategy was applied to the Sox thiosulfate oxidation proteins (SoxA and SoxB). For each data set, protein sequences were aligned using MAFFT v7.221 using the L-INS-i algorithm. The alignment was then masked using Gblocks and phylogenetic inference performed with RAxML as described above.

14A total of 619 Epsilonproteobacteria and five Desulfurellales genomes were obtained from RefSeq version 76 and GenBank version 213 (Supplementary Table S1). Genomes were assessed for completeness and contamination by scoring the presence of conserved single-copy marker genes within each genome using CheckM. The median estimated genome completeness for this dataset is 99.4% and the minimum is 81.9%. Genomes were estimated to be less than 10% contaminated, with all but eight under 5% (Supplementary Table S1). The taxonomic annotation of the type strain Campylobacter geochelonis (GCA_900063025.1) was manually modified as the NCBI record for this genome incorrectly labels it as C. fetus. Thirty-three draft population genomes (median completeness 93.8%, contamination 1.1%) belonging to the Epsilonproteobacteria were recovered from publicly available metagenomic data sets as part of a larger study (Parks et al., submitted) and included in our analysis. In addition to the public genomes, we sequenced the type strain of H. thermophila, sole representative of the genus Hydrogenimonas and three single cells belonging to the genus Thioreductor (Supplementary Table S2). For H. thermophila, an Illumina-based assembly produced a draft genome of 96 contigs with a predicted completeness of 99.6 and 1.8% contamination. Thioreductor single cells amplifications were assembled into partial genomes with completeness estimates between 27.7 and 36.5%, and with low contamination estimates (0.3–1.2%) (Supplementary Table S2). Owing to their low completeness Thioreductor genomes were excluded from the majority of analyses, resulting in an ingroup comprising 658 quality-filtered genomes (119 complete and 539 draft) for comparative analysis. Outgroup genomes broadly representative of the bacterial domain were selected from a total of 60,258 quality controlled reference genomes available from the Genome Taxonomy Database.

15Phylogenetic affiliation(s) of the ingroup (Epsilonproteobacteria and Desulfurellales, 98 genomes) to species-level representatives of the outgroup (4,072 genomes) were assessed using two different datasets. The first dataset was a concatenation of 120 single-copy marker proteins (Parks et al., submitted) and the second was a concatenation of the 16S and 23S rRNA gene sequences. Note that the 3,144 genomes contributing to the second dataset are a subset of the first as most genome sequences derived from metagenomic data lack complete rRNA gene sequences, and is used here primarily to validate the concatenated protein tree. Based on these datasets, phylogenetic trees were inferred using Maximum Likelihood (ML) with the JTT, WAG, and LG models of amino acid substitution as well as NJ with Jukes-Cantor and Kimura distance corrections. Robustness of tree topologies was analyzed with a combination of bootstrapping and taxon resampling, implemented by removal of one phylum at a time from the outgroup dataset. The consensus of these analyses indicate that the Epsilonproteobacteria and Desulfurellales are robustly monophyletic and not reproducibly affiliated with any other phyla (Figure 1 and Table 1), which is consistent with recent reports also using concatenated protein markers. The phylum-level jackknife analysis suggests a specific association of the ingroup with the Aquificae, which is also supported by bootstrap resampling of this dataset (Figure 1). Tree topologies which suggest a common ancestry between Aquificae and Epsilonproteobacteria have been reported for several marker genes; however, this association is often not statistically robust. Phylogenomic evidence suggests that Aquificae genomes have been shaped by extensive lateral gene transfer from lineages including the Epsilonproteobacteria, a phenomenon that might have contributed to the observed association. Importantly, removal of the Aquificae in the jackknife analysis did not affect the apparent separation of the Epsilonproteobacteria from the other proteobacterial classes.

16Based on our phylogenetic analyses, we propose to reclassify the Epsilonproteobacteria and Desulfurellales as a single phylum called the Epsilonbacteraeota (phyl. nov.), consistent with a recently proposed naming convention for the phylum rank. The Epsilonproteobacteria constitute a class-level lineage within this phylum (Figure 2 and Table 2), but we propose to rename it as Campylobacteria (class. nov.) to remove any remaining association with the phylum Proteobacteria. The existing Epsilonproteobacteria orders Campylobacterales and Nautiliales are maintained within this proposed taxonomy as they represent robustly monophyletic groupings and have no direct link to the Proteobacteria in their names (Figure 2 and Supplementary Figure S1 and Table S4). Similarly, the taxon Desulfurellales is retained as the sole order within a new class, the Desulfurellia (class. nov.) (Figure 2 and Supplementary Figure S1). Family and genus level groupings are mostly consistent with existing taxonomic authorities (Table 2), and reclassified or newly proposed families fall within proposed guidelines for 16S rRNA gene similarity and AAI thresholds (Supplementary Figure S2). Proposed changes to family membership are detailed below.

17The currently defined family Campylobacteraceae, comprised of the genera Arcobacter, Campylobacter, and Sulfurospirillum, is polyphyletic in the concatenated protein tree (Figure 2), although it is monophyletic in the concatenated rRNA gene tree (Supplementary Figure S1). Inspection of individual protein tree topologies revealed that only two of the 120 markers resolve the Campylobacteraceae as monophyletic (Supplementary Figure S3). For this reason, we propose to transfer the Arcobacter and Sulfurospirillum into their own families, the Arcobacteraceae (fam. nov.) and Sulfurospirillaceae (fam. nov.), respectively. Campylobacter is a deeply divergent genus which likely requires reclassification into a number of distinct genera (indicated in Figure 2 by alphabetical suffixing). Note also that Dehalospirillum multivorans and Geospirillum barnesii have been formally transferred to the genus Sulfurospirillum, and Thiomicrospira denitrificans has been reclassified to Sulfurimonas, although these names persist in some online archives.

18The currently defined Helicobacteraceae do not form a monophyletic group to the exclusion of the Campylobacteraceae and Arcobacter; therefore, the genera Thiovulum, Sulfuricurvum, and Sulfurimonas have been removed from the Helicobacteraceae into their own family, the Thiovulaceae (fam. nov.). Monophyly of the Helicobacteraceae is also not robustly supported in the concatenated rRNA gene tree (Supplementary Figure S1). Species in the Sulfurovum and Nitratifractor genera are either currently unclassified at the family level, or classified into the families Helicobacteraceae and Nautiliaceae, respectively (Table 2). Like the Campylobacter, Helicobacter is a deeply divergent genus which likely requires reclassification into a number of distinct genera (indicated in Figure 2 by alphabetical suffixing). Sulfurovum and Nitratifractor are resolved as a robustly monophyletic group, independent of both the Helicobacteraceae and Nautiliaceae, for which we propose the name Sulfurovaceae (fam. nov., Figure 2). However, the concatenated rRNA gene tree does not support the monophyly of the Sulfurovaceae (Supplementary Figure S1). Inspection of individual protein trees reveals that the majority of markers (105/120) supports the association (Supplementary Figure S3), as well as the 16S rRNA gene by itself (Supplementary Figure S4). The revised Helicobacteraceae family comprises only species in the genera Helicobacter and Wolinella (Figure 2). Note that the genus Flexispira is not included in the Helicobacteraceae as it is a defunct basonym of Helicobacter.

19We further propose that the genus Nitratiruptor be placed into its own family Nitratiruptoraceae (fam. nov.) within the order Campylobacterales, a move that, while inconsistent with taxonomic authorities placing this genus in the Nautiliaceae (Table 2), is supported by 16S rRNA gene-based phylogenies. Analysis of the three partial single cell Thioreductor genomes confirms the 16S rRNA-based analysis that this genus is a member of the family Nautiliaceae (Supplementary Figures S4, S5). In addition to the proposed revisions in the class Campylobacteria, we propose that Hippea be transferred to its own family Hippaceae (fam. nov.) within the class Desulfurellia to reflect the depth of its relationship with the genus Desulfurella (Figure 2). A complete summary of proposed changes within the Epsilonbacteraeota are provided in Supplementary Table S3.

20There are three published genera presently classified as Epsilonproteobacteria for which genomic data are currently unavailable: Thiofractor, Candidatus Thioturbo, and Cetia. These genera are therefore provisionally placed within the Epsilonbacteraeota based on comparative analysis of 16S rRNA gene sequences alone (Supplementary Figure S4); however, their classification may require future revision as genomic information becomes available. Thiofractor and Candidatus Thioturbo appear to be members of the order Campylobacterales consistent with previous findings, however, neither are clearly resolved into the family level groupings proposed for the Epsilonbacteraeota. Thiofractor may constitute its own family and Candidatus Thioturbo may be incorporated into the Arcobacteraceae, although this latter affiliation is only based on a partial 16S rRNA sequence (Supplementary Figure S4). Cetia pacifica clusters robustly within the family Nautiliaceae, which may or may not require reclassification of the genus Caminibacter (Supplementary Figure S4; ).

21Overlaying published phenotypic information of cultured Epsilonbacteraeota on the genome-based phylogeny suggests that the ancestor of this phylum was autotrophic and thermophilic (Figure 3). Mesophily arose later in the Campylobacterales, and heterotrophic growth appears to have arisen independently in the Campylobacterales and Desulfurellia (Figure 3). To quantify the extent to which taxonomy reflects functional variation amongst Epsilonbacteraeota, we performed PERMANOVA to quantify the contribution of predicted functional profiles using KO. The largest source of variation was taxonomy (family; R = 0.68, genus; R = 0.70, p = 0.001), followed by habitat (R = 0.28, p = 0.001), indicating that while vertical inheritance is a powerful predictor of functional capacity, a large portion of variation is not captured by this process and likely reflects habitat-specific adaptation. This variation was also reflected in PCA analysis of the functional profiles, where heterotrophic Campylobacter and Helicobacter genomes were clearly separate from other Epsilonbacteraeota (Figure 4). Furthermore, there was pronounced separation within these genera by functional PCA consistent with the suggestion to classify both Campylobacter and Helicobacter into multiple genera (Figure 2). To elucidate the metabolic components driving this separation, indicator analysis was applied to the gene annotation table to highlight key features responsible for functional divergence.

22Genes involved in carbon and nitrogen fixation, assimilatory nitrate and nitrite reduction, thiosulfate oxidation and polysulfide reduction were significantly associated with the environmental ecotype indicating that carbon, nitrogen, and sulfur cycling are primary drivers of functional divergence in environmental Epsilonbacteraeota. These results were visually apparent when aligning functional genes against the concatenated protein phylogeny (Figure 3). Host-associated Epsilonbacteraeota were defined primarily by the absence of these functions, although consistent with their lifestyle the antimicrobial peptide resistance mechanism YejABEF was a key signature of host-association. The separation of Helicobacter (containing the type species, H. pylori) from Helicobacter A to D was driven by the presence of genes involved in osmoprotectant and heme transport, as well as the CAG pathogenicity island in H. pylori. Similarly, Campylobacter differentiation was in part due to the presence of additional transporters involved in lipopolysaccharide and capsular polysaccharide transport. These findings suggest that the transition toward a host-associated lifestyle in these lineages has occurred in a two-step manner. First, the loss of ‘environmental’ functions associated with the initial transition to host-association in Campylobacter and Helicobacter, followed by gene acquisition to enhance host adaptation. The distinct phylogenetic and functional differences within these genera suggest that future revision of their taxonomy may be warranted.

23Analyzing the carbon metabolism of Epsilonbacteraeota revealed a complete tricarboxylic acid (TCA) cycle in all genomes with the exception of Helicobacter (Figure 3), which functions as a directed pathway producing succinate and α-ketoglutarate in this genus. Phylogenetic analysis of the marker protein, 2-oxoglutarate oxidoreductase (KorA), support the TCA cycle being vertically inherited throughout the Epsilonbacteraeota (Supplementary Figure S6). Most genera also encode ATP citrate lyase (AclA and B, Figure 3) indicating the ability to perform autotrophic carbon fixation via reductive TCA (rTCA; ). The presence of this enzyme coincides with phenotypically established autotrophy, and genera which have lost the ability to fix carbon have adopted an exclusively heterotrophic lifestyle in carbon rich habitats (Figure 4) with the exception of a number of autotrophic Arcobacter strains for which genomes are not currently available. Phylogenetic inference of both citrate lyase subunits indicates a monophyletic history of carbon fixation in the Epsilonbacteraeota with independent losses of this capability inferred in the Campylobacter, Helicobacteraceae, and Arcobacter (Figure 3). Citrate lyase was detected in a population genome basal to the Arcobacter genus (UBA6211, Figure 3) which, combined with previous observation of citrate lyase activity and the operation of the rTCA in Arcobacter, suggests that carbon fixation was present in the common ancestor of this genus. Vertical inheritance was also observed in Epsilonbacteraeota homologs of the cytosolic [NiFe] hydrogenase group 2D, which is hypothesized to provide reducing power for the rTCA.

24No other complete carbon fixation pathways were identified in the genomes. Formate dehydrogenase is widely distributed in many autotrophs and was common in hydrothermal vent-associated lineages (Figures 2, 3), many of which are known to oxidize formate. A recent biochemical analysis of hydrothermal vents demonstrated that abiotic processes sequester available carbon dioxide as formate, and the presence of formate dehydrogenase may provide the ability to couple respiration with the creation of carbon dioxide to fuel rTCA. The Wood-Ljüngdahl (WL) pathway is more energetically favorable than the rTCA but requires strict anoxic conditions to operate, which may be incompatible with the microaerobic growth of many vent-associated Epsilonbacteraeota. Monophyletic distribution of the WL-specific marker methylene-tetrahydrofolate reductase (MetF, Figure 3 and Supplementary Figure S7) amongst the Campylobacteria suggests that this pathway was an ancestral trait which has been supplanted by the rTCA, possibly in response to the adoption of (micro)aerobic habitats. Consistent with previous analyses, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) was not detected in any genome, and the rTCA remains the only known means for carbon fixation amongst Epsilonbacteraeota. The pattern of auto- and heterotrophic lifestyles within the Epsilonbacteraeota suggests that autotrophy is an ancestral trait, punctuated by recent adaptations to heterotrophy mostly in host-associated habitats. Additionally, the Epsilonbacteraeota and Aquificae citrate lyase proteins are robustly monophyletic (Supplementary Figure S8), consistent with the inference that carbon fixation in the Epsilonbacteraeota is descended from an ancestral Aquificae phenotype.

25Anaerobic respiration using nitrate as an electron acceptor is widespread throughout the Epsilonbacteraeota, as evidenced by the presence of soluble periplasmic nitrate reductase (NapA) in most genera (Figure 3), and membrane-bound nitrate reductase (NarG) in Desulfurella. The NAP system of many Campylobacteria lack the NapC protein and where a homolog is present, it is proposed to act as the quinol-oxidizing protein component of a reversely operating hydroxylamine:quinone redox module. This pathway operates as a quinone-reducing module in ammonia-oxidizing bacteria and Thaumarchaeota, and has recently been proposed in N. profundicola. As a component of an ammonification pathway, this module produces ammonium by reduction of nitrite either directly (analogous to the NrfHA ammonification module) or via a hydroxylamine intermediate, which is thought to be transported to the cytoplasm and further reduced to ammonium by a putative hydroxylamine reductase (Har/Hcp), powered by an Fe-S ferredoxin. While hydroxylamine reduction to ammonium and nitrate-dependent expression of genes encoding the entire pathway has, so far, only been demonstrated in N. profundicola, genes for this pathway are present in Campylobacter concisus, C. curvus, and C. fetus. We further identified the complete inventory for this proposed pathway in the remaining Nautiliales genomes, as well as Campylobacter mucosalis, C. geochelonis, C. gracilis, C. hyointestinalis, and C. iguaniorum (Figure 3). Furthermore, H. thermophila and D. acetivorans lack only the predicted Fe-S ferredoxin of this pathway, but as hydroxylamine reductase is known to obtain reductant from other oxidoreductases, it is possible that these organisms are also capable of ammonification using a hydroxylamine intermediate. The NapC homolog and HaoA proteins are homologous to NrfH and NrfA, respectively and differ mainly by the infidelity of the HaoA oligomer as an ammonium-producing nitrite reductase. These homologs facilitate incomplete electron transfer and leak hydroxylamine due to looser redox coupling with the NapC homolog compared to that of the NrfA-NrfH module.

26Phylogenetic inference of the HaoA and Har/Hcp proteins suggest that this more complex nitrite ammonification pathway is monophyletic and ancestral to the Epsilonbacteraeota, despite widespread partial or complete loss of the pathway in the group, and putative lateral transfers of component genes from Epsilonbacteraeota donors to other bacteria (Supplementary Figure S9). Most Epsilonbacteraeota have multiple potential mechanisms for obtaining fixed nitrogen and the acquisition of chemoorganotrophic catabolic machinery, which provide cells with higher and more reliable reducing power, likely facilitated the functional replacement of the HaoA-type with the NrfA-type nitrite reductase. The majority of extant autotrophic Epsilonbacteraeota possess one or more ammonification pathways including dinitrogen reduction based on nitrogenase and/or a variety of pathways based on the reduction of nitrate (Figure 3). Annotation of the Thiovulum sp. ES genome did not reveal any ammonification inventory; nevertheless and consistent with its original annotation, we detected the gene encoding an ammonium transporter (AmtB) in this lineage, shared with most other Epsilonbacteraeota (Figure 3).

27Pentaheme cytochrome c nitrite reductase (NrfA) is found almost exclusively in heterotrophic Campylobacteria (Figure 3) and is known to provide protection against nitrosative stress to Campylobacter and Wolinella in their natural environments. This protein may also serve the same function in the majority of Helicobacter species, although this has not been well studied since the model species, H. pylori, lacks NrfA and has alternative mechanisms for coping with nitrosative stress. In contrast to the concatenated protein-based monophyly of the heterotrophs (Figure 3), phylogenetic inference of NrfA reveals at least two independent clades, the first comprising Sulfurospirillum, Wolinella and some Arcobacter, and the second Campylobacter and Helicobacter (Supplementary Figure S10). It is likely at least one of these clusters is the result of lateral gene transfer, which we predict to be the Campylobacter and Helicobacter clade as members of these two genera are intermingled indicating multiple lateral transfer events (Supplementary Figure S10), possibly the result of selective pressure exerted by the host immune system.

28Anaerobic respiration of Epsilonbacteraeota frequently occurs through the reduction of sulfur compounds, and is a major source of primary production in some environments. Sulfide-oxidizing sulfide:quinone oxidoreductase (SQR) catalyzes the bidirectional oxidation of hydrogen sulfide to polysulfide, and this gene is inferred to be abundant in environments where Epsilonbacteraeota (Epsilonproteobacteria) are dominant members. Consistent with this inference, we observed SQR genes in most Epsilonbacteraeota lineages (Figure 3). In contrast to the monophyletic origins of the carbon and most nitrogen cycling proteins described above, phylogenetic analysis of SQR reveals extensive polyphyly of Epsilonbacteraeota homologs, including a putative lateral transfer from a Bacteroidetes donor to the Nautilaceae ancestor (Supplementary Figure S11). An additional enzyme involved in polysulfide reduction, polysulfide reductase (PsrA) was also common in the Epsilonbacteraeota (Figure 3) and possessed polyphyletic origins (Supplementary Figure S12). These findings suggest that unlike nitrate reduction, sulfur reduction has been acquired in the Epsilonbacteraeota in response to immediate environmental needs. Polysulfide can be formed abiotically under hydrothermal vent conditions and, similar to formate, presents a resource that early colonizing Epsilonbacteraeota may exploit. It is possible that the acquisition of SQR represents a means of simultaneously detoxifying hydrogen sulfide, and utilizing it as an electron donor, as Epsilonproteobacteria have been seen to bloom in response to hydrogen sulfide releases on the African shelf.

29Oxidation of sulfur is also a well known trait of Epsilonproteobacteria, although it is only reported in a handful of genera. Sulfurimonas-like organisms have been suggested as the primary sulfur-oxidisers in deep-sea vent ecosystems and consistent with this observation, Sulfurimonas was one of the few Epsilonbacteraeota genera to possess the complete sulfur oxidation (sox) pathway (Figure 3). The absence of SoxC, and drastic reduction in electron liberation, in most genera suggests that sulfur oxidation is not the primary means of electron generation amongst Epsilonbacteraeota, with hydrogenases likely a more productive source of reducing power. The evolutionary history of sox in the Epsilonbacteraeota is convoluted. Consistent with previous analysis of sox genes, SoxA and SoxB homologs in non-Arcobacter species shared a common ancestor with Aquificae, while Arcobacter acquired a complete sox cassette through lateral gene transfer with Gammaproteobacteria (Supplementary Figure S13). However, homologs of SoxC are monophyletic in the Epsilonbacteraeota, a finding also supported by previously literature. These data suggest that after acquiring the ability to oxidize thiosulfate independently of other Epsilonbacteraeota, an ancestor of Arcobacter introduced soxCD to Sulfurimonas and the Sulfurovaceae.

30The majority of Epsilonbacteraeota possess uni- or bipolar flagella and are highly motile, with Thiovulum majus capable of achieving a speed of over 600 microns per second, or 2 m per hour. The flagella of Epsilonbacteraeota differ from that of other bacteria in several respects. For example, the C-ring of model Campylobacter jejuni and Helicobacter hepaticus differs structurally from that of other bacteria, and recent knock-out experiments identified six novel genes exclusive to flagella activity in Epsilonbacteraeota. Given the novelty of described Epsilonbacteraeota flagella, we predicted that they should be an ancestral feature of this phylum. Indeed, phylogenetic analysis of the flagellar hook protein (FlgE) and flagellin (FliC, Figure 3) indicate a monophyletic origin of this trait in the Epsilonbacteraeota (Supplementary Figure S14). Flagella are known to be absent in several Epsilonbacteraeota, including Nitratifractor salsuginis, the genus Sulfurovum, and at least two Campylobacter species, C. hominis and C. gracilis. The proposed Epsilonbacteraeota taxonomy groups the genera Nitratifractor and Sulfurovum into the novel family Sulfurovaceae, suggesting a single loss of flagella genes in the ancestor of this family. The presence of the chemotactic regulator Mcp and response regulating CheB and CheR within this family (Figure 3) provides additional evidence that the ancestor of the Sulfurovaceae possessed flagella. Campylobacter hominis and C. gracilis are sister species in the Campylobacter B clade (Figure 2) suggesting a single loss of motility occurred in their common ancestor for as yet, unclear reasons given that the rest of the genus has retained motility.

31The rise of high-throughput DNA sequencing has fundamentally changed the landscape of microbial ecology and systematics. The 16S rRNA gene has led the way in providing an evolutionary backbone for microbial taxonomy over the past 30 years, but can now be complemented by whole genome methods which use multiple genetic markers to infer evolutionary relationships. More importantly, classifications are not static. As genomically described bacterial diversity expands, historic classifications require revision as new clades are discovered and tree topologies change. The class Epsilonproteobacteria and order Desulfurellales are an excellent case in point. Comparative analysis of these groups with a broad genomic representation of the bacterial domain indicates that they constitute a monophyletic unit not specifically related to the phylum Proteobacteria. We propose the reclassification of this group as the phylum Epsilonbacteraeota, together with a small number of subordinate changes described in this manuscript. Additional changes may be required in future as, e.g., Campylobacter and Helicobacter appear to be phylogenetically and functionally diverse for genera. Metabolic reconstruction of the Epsilonbacteraeota suggests, perhaps unsurprisingly, that the ancestor to this phylum was a chemolithoautotrophic thermophile related to Aquificae, from which several heterotrophic and mesophilic lineages have evolved.

32Campylobacteria (Cam.py.lo.bac.te′ri.a. N.L. masc. n. Campylobacter type genus of the type order of the class; suff. -ia, ending to denote a class; N.L. neut. pl. n. Campylobacteria the class of the order Campylobacterales).

33Description is the same as for the order Campylobacterales. Type order: Campylobacterales, phylum: Epsilonbacteraeota phyl. nov.

34Arcobacteraceae (Ar.co.bac.ter.a.ce′ae. N.L. masc. n. Arcobacter type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Arcobacteraceae the family of the genus Arcobacter).

35Description is identical to that given by, with the acknowledgment that some species are capable of autotrophic carbon dioxide fixation via the reverse tricarboxylic acid cycle. Type genus: Arcobacter, order: Campylobacterales, class: Campylobacteria, class. nov., phylum: Epsilonbacteraeota phyl. nov.

36Description is drawn from that of, with the removal of the genera Arcobacter and Sulfurospirillum due to a lack of robust monophyly with the type genus. Type genus: Campylobacter, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov.

37The description is the same as given by for the type genus with the following changes. Currently includes only the genera Helicobacter (type genus) and Wolinella. The genera Sulfurimonas, Sulfuricurvum, Sulfurovum, and Thiovulum are removed owing to a lack of monophyly with the type genus. Type genus: Helicobacter, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov.

38Nitratiruptoraceae (Ni.tra.ti.rup.to.ra.ce′ae. N.L. masc. n. Nitratiruptor type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Nitratiruptoraceae the family of the genus Nitratiruptor).

39Described on the basis of divergent nature with all other members of Campylobacterales using both protein marker, as well as 16S and 23S rRNA gene sequence analysis. Description is the same as given by. Type genus: Nitratiruptor, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov.

40Sulfurospirillaceae (Sul.fu.ro.spir.il.la.ce′ae. N.L. neut. n. Sulfurospirillum type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Sulfurospirillaceae the family of the genus Sulfurospirillum).

41The description of the family is drawn from the description of the type genus from; cells are Gram-negative, motile, microaerobic, and mesophilic. Oxidize hydrogen or formate to reduce a range of electron acceptors. Type genus: Sulfurospirillum, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov.

42Sulfurovaceae (Sul.fur.o.va.ce′ae. N.L. neut. n. Sulfurovum type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Sulfurovaceae the family of the genus Sulfurovum).

43The family is defined based on phylogenetic analysis of protein markers (concatenated and single) and the 16S ribosomal gene sequence. Includes the genera Sulfurovum (type genus) and Nitratifractor. Cells are Gram-negative, non-motile, and mesophilic. Growth occurs in the microaerobic range. Type genus: Sulfurovum, order: Campylobacterales, class: Campylobacteria class. nov., phylum: Epsilonbacteraeota phyl. nov.

44Thiovulaceae (Thio.vu.la.ce’ae. N.L. neut. dim. n. Thiovulum type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Thiovulaceae the family of the genus Thiovulum).

45The family is defined based on phylogenetic analysis of concatenated protein marker and ribosomal gene sequences. The family currently includes the genera Thiovulum (type genus), Sulfurimonas, and Sulfuricurvum. Cells are Gram-negative, motile, microaerobic, and mesophilic. Growth occurs in the mesophilic range. Type genus: Thiovulum, order: Campylobacterales, class: Campylobacteriaclass. nov., phylum: Epsilonbacteraeota phyl. nov.

46Desulfurellia (De.sul.fu.rel′li.a. N.L. fem. n. Desulfurella type genus of the type order of the class; suff. -ia, ending to denote a class; N.L. neut. pl. n. Desulfurellia the class of the order Desulfurellales).

47Description is the same as for the order Desulfurellales. Type order: Desulfurellales, phylum: Epsilonbacteraeota phyl. nov.

48The order is as previously described but is transferred from the class Deltaproteobacteria to Desulfurellia class. nov. The order consists of the families Desulfurellaceae and Hippeaceae fam. nov. Cells are Gram-negative, thermophilic, and motile. Type family: Desulfurellaceae, class: Desulfurellia class. nov., phylum: Epsilonbacteraeota phyl. nov.

49Description is as previously, with the exception that the genus Hippea is moved to the family Hippeaceae fam. nov.. Cells are Gram-negative, obligately anaerobic, and motile. Uses short-chain fatty acids including acetate, fumarate, malate, as well as long-chain saturated fatty acids as growth substrates. Type species D. acetivorans is capable of dissimilatory sulfur reduction. Type genus: Desulfurella, order: Desulfurellales, class: Desulfurellia class. nov., phylum: Epsilonbacteraeota phyl. nov.

50Hippeaceae (Hippe.a.ce′ae. N.L. fem. n. Hippea type genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Hippeaceae the family of the genus Hippea).

51The family is defined based on phylogenetic analysis of concatenated marker proteins, and is separated from the Desulfurellaceae due to deep branching distance. Cells are Gram-negative, obligately anaerobic, thermophilic, and motile. Capable of heterotrophic growth using acetate and various other organic substrates by species. Type genus: Hippea, order: Desulfurellales, class: Desulfurellia class. nov., phylum: Epsilonbacteraeota phyl. nov.

52Epsilonbacteraeota (Ep.si.lon.bac.ter.ae.o′ta. Gr. n. epsilon, name of the fifth letter of Greek alphabet; Gr. n. baktêria, staff, cane; suff. -aeota, proposed ending to denote a phylum; N.L. neut. pl. n. Epsilonbacteraeota the phylum of bacteria sharing a common ancestry and possessing ribosomal gene sequence and protein marker similarity to those of the members of the classes Campylobacteria class. nov. and Desulfurellia class. nov)

53The phylum is described based upon concatenated protein marker and ribosomal gene sequence phylogeny. Owing to the diverse ecological range of members of this phylum there is little unifying physiology. All members are Gram-negative, and motility as well as the reduction of nitrate and polysulfide are ancestral and common traits. The phylum Epsilonbacteraeota currently comprises two classes: Campylobacteria class. nov. and Desulfurellia class. nov.; and three orders: Campylobacterales, Nautiliales, and Desulfurellales. The type class of the phylum is Campylobacteria class. nov.

54DW, DP, and PH performed the bioinformatic analysis, interpretation, and proposed taxonomic changes. KT, MK, SS, and TW sequenced additional genomes for analysis. BC, IV, JS, and MK proposed avenues for functional analysis and assisted with interpretation of data. DW and PH wrote the draft manuscript and all authors contributed to, and approved, the final manuscript.

55The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.