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. 2017 Apr 24;8:682.
doi: 10.3389/fmicb.2017.00682. eCollection 2017.

Comparative Genomic Analysis of the Class Epsilonproteobacteria and Proposed Reclassification to Epsilonbacteraeota (Phyl. Nov.)

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Free PMC article

Comparative Genomic Analysis of the Class Epsilonproteobacteria and Proposed Reclassification to Epsilonbacteraeota (Phyl. Nov.)

David W Waite et al. Front Microbiol. .
Free PMC article

Erratum in

Abstract

The Epsilonproteobacteria is the fifth validly described class of the phylum Proteobacteria, known primarily for clinical relevance and for chemolithotrophy in various terrestrial and marine environments, including deep-sea hydrothermal vents. As 16S rRNA gene repositories have expanded and protein marker analysis become more common, the phylogenetic placement of this class has become less certain. A number of recent analyses of the bacterial tree of life using both 16S rRNA and concatenated marker gene analyses have failed to recover the Epsilonproteobacteria as monophyletic with all other classes of Proteobacteria. In order to address this issue, we investigated the phylogenetic placement of this class in the bacterial domain using 16S and 23S rRNA genes, as well as 120 single-copy marker proteins. Single- and concatenated-marker trees were created using a data set of 4,170 bacterial representatives, including 98 Epsilonproteobacteria. Phylogenies were inferred under a variety of tree building methods, with sequential jackknifing of outgroup phyla to ensure robustness of phylogenetic affiliations under differing combinations of bacterial genomes. Based on the assessment of nearly 300 phylogenetic tree topologies, we conclude that the continued inclusion of Epsilonproteobacteria within the Proteobacteria is not warranted, and that this group should be reassigned to a novel phylum for which we propose the name Epsilonbacteraeota (phyl. nov.). We further recommend the reclassification of the order Desulfurellales (Deltaproteobacteria) to a novel class within this phylum and a number of subordinate changes to ensure consistency with the genome-based phylogeny. Phylogenomic analysis of 658 genomes belonging to the newly proposed Epsilonbacteraeota suggests that the ancestor of this phylum was an autotrophic, motile, thermophilic chemolithotroph that likely assimilated nitrogen from ammonium taken up from the environment or generated from environmental nitrate and nitrite by employing a variety of functional redox modules. The emergence of chemoorganoheterotrophic lifestyles in several Epsilonbacteraeota families is the result of multiple independent losses of various ancestral chemolithoautotrophic pathways. Our proposed reclassification of this group resolves an important anomaly in bacterial systematics and ensures that the taxonomy of Proteobacteria remains robust, specifically as genome-based taxonomies become more common.

Keywords: Epsilonbacteraeota; Epsilonproteobacteria; classification; evolution; genome; phylogenomics; taxonomy.

Figures

FIGURE 1
FIGURE 1
Maximum likelihood phylogenetic inference (WAG + Γ model) of Epsilonproteobacteria and Desulfurellales in the context of 4,170 bacterial genomes and based on 120 concatenated protein sequences. Robustness of the placement of the Epsilonbacteraeota was assessed using bootstrap support and per-phylum jackknifing. Symbols above each branch root (circles) represent bootstrap support of >90% (black) or ≥75% (hollow). Symbols below each root reflect jackknife reproducibility of a node, with black square representing nodes recovered with ≥90% support in ≥90% of tree topologies. Hollow squares indicate ≥75% support in ≥90% of topologies. Under every tree topology explored Epsilonproteobacteria and Desulfurellales were recovered as a strongly supported monophyletic group with no association to the remaining Proteobacteria. Key findings of tree topologies under alternate tree building models are summarized in Table 1.
FIGURE 2
FIGURE 2
Phylogenetic analysis of the Epsilonbacteraeota. Maximum likelihood tree of the Epsilonbacteraeota based on 120 concatenated protein marker sequences using RAxML. Support of internal nodes was calculated using 100 bootstrap iterations, and junctions represent nodes with 100% bootstrap support (solid), and >75% support (hollow). All taxonomic groupings are identical to those in Figure 1 (calculated with FastTree), although the branching positions of Helicobacteraceae and Sulfurovaceae differs between the two topologies. [T]: denotes type species.
FIGURE 3
FIGURE 3
Habitat and functional annotation of selected Epsilonbacteraeota pathways. Tree topology is that of Figure 2, collapsed at the genus level. Cell intensity reflects the proportion of genomes within a clade that possessed the column function. A: Trophism is inferred from genomic data. B: Although some members of Arcobacter are capable of autotrophic growth (discussed in text) all genomes in this study were obtained from heterotrophic species. Column descriptions are as follows: Carbon pathways: aconite hydratase (Acn), 2-oxoglutarate oxidoreductase alpha-, beta-, gamma-, and delta- subunits (KorABGD), succinyl-CoA synthetase alpha and beta subunits (SucCD), malate dehydrogenase (Mdh), ATP citrate lyase alpha subunit (AclA) and beta subunit (AclB), [NiFe] hydrogenase family 2D (NiFe 2D), formate dehydrogenase (Fdh), methylenetetrahydrofolate reductase (MetF) and carbon-monoxide dehydrogenase catalytic and iron sulfur subunits (CooS/CooF). Nitrogen pathways: periplasmic nitrate reductase components NapA, NapB, NapG, and NapH (NapABGH), periplasmic nitrate reductase c-type cytochrome (NapC), hydroxylamine oxidoreductase (HaoA), hydroxylamine reductase/hybrid cluster protein (Har/Hcp), predicted reductive Fe-S protein (FeS protein), ferredoxin-nitrate reductase (NarB), assimilatory nitrate reductase (catalytic subunit, NasA), ferredoxin-nitrite reductase (NirA), nitrogenase molybdenum-iron (alpha- and beta-chains) and iron protein (NifDKH), ammonium transporter (AmtB), nitrite reductase (NADH) large subunit (NirB), cytochrome c nitrite reductase (NrfA). Sulfur pathways: sulfide:quinone oxidoreductase (SQR), polysulfide reductase chain A (PsrA), thiosulfate oxidating Sox proteins (SoxXA, SoxY, SoxB, SoxCD). Motility: flagellar hook protein (FlgE), flagellin (FliC), methyl-accepting chemotaxis protein (Mcp), chemotaxis response regulator (CheB), chemotaxis methyltransferase (CheR).
FIGURE 4
FIGURE 4
Principal component analysis of Epsilonbacteraeota gene annotations. Analysis was performed using the KEGG Orthology (KO) annotation table. Circles denote host-associated individuals, and squares those from other environments. Genomes are colored by family, with exceptions of the Campylobacteraceae and Helicobacteraceae, which are separated based on suffixing described in text. Vectors represent pathway level aggregations of indicator KOs fitted to the ordination using the enfit function in vegan. Only vectors with R > 0.75 are displayed for clarity. Pathways are as follows: A, arginine biosynthesis; At, atrazine degradation; B, biotin metabolism; N, nitrogen metabolism; PTT, phenylalanine, tyrosine, and tryptophan biosynthesis; S, sulfur metabolism; Syn, synthesis and degradation of ketone bodies; T, two-component system; VLI, valine, leucine, and isoleucine degradation.

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