Phylogeny and physiology of candidate phylum 'Atribacteria' (OP9/JS1) inferred from cultivation-independent genomics

Abstract

The 'Atribacteria' is a candidate phylum in the Bacteria recently proposed to include members of the OP9 and JS1 lineages. OP9 and JS1 are globally distributed, and in some cases abundant, in anaerobic marine sediments, geothermal environments, anaerobic digesters and reactors and petroleum reservoirs. However, the monophyly of OP9 and JS1 has been questioned and their physiology and ecology remain largely enigmatic due to a lack of cultivated representatives. Here cultivation-independent genomic approaches were used to provide a first comprehensive view of the phylogeny, conserved genomic features and metabolic potential of members of this ubiquitous candidate phylum. Previously available and heretofore unpublished OP9 and JS1 single-cell genomic data sets were used as recruitment platforms for the reconstruction of atribacterial metagenome bins from a terephthalate-degrading reactor biofilm and from the monimolimnion of meromictic Sakinaw Lake. The single-cell genomes and metagenome bins together comprise six species- to genus-level groups that represent most major lineages within OP9 and JS1. Phylogenomic analyses of these combined data sets confirmed the monophyly of the 'Atribacteria' inclusive of OP9 and JS1. Additional conserved features within the 'Atribacteria' were identified, including a gene cluster encoding putative bacterial microcompartments that may be involved in aldehyde and sugar metabolism, energy conservation and carbon storage. Comparative analysis of the metabolic potential inferred from these data sets revealed that members of the 'Atribacteria' are likely to be heterotrophic anaerobes that lack respiratory capacity, with some lineages predicted to specialize in either primary fermentation of carbohydrates or secondary fermentation of organic acids, such as propionate.

Figure 1

A 16S rRNA gene phylogeny of SAG and metagenome bin data sets (underlined) within the context of cloned sequences from OP9 and JS1 and of other Bacteria. The number of SAGs used for construction of co-assemblies are indicated in parentheses. Genus, Order and Class candidate taxonomic units proposed by Yarza et al. (2014) that encompass the OP9 and JS1 data sets are indicated. Although the Sakinaw Lake JS1 metagenome bin did not contain a 16S rRNA gene, its affiliation with the Sakinaw Lake co-assembly (based on %ANI) is indicated in this figure.

Figure 2

Phylogenomic analysis of OP9 and JS1 SAGs, metagenome bin data sets and other Bacteria. Maximum likelihood phylogeny inferred with RAxML (Stamatakis, 2006) using a concatenated alignment of 31 conserved markers. The number of organisms represented by each wedge is indicated in parentheses. Etoliko Lagoon SAG 227 is not included because it did not contain any of these markers.

Figure 3

BMC gene loci in representatives of different ‘Atribacteria' lineages. Genes predicted to encode BMC shell proteins (black) and enzymes (grey) conserved in the BMC loci, as well as intervening or peripheral genes that may be involved in BMC function (white), are indicated by arrows, with corresponding RAST gene numbers below each arrow. Predicted function and COGs are indicated at the top. Homologous genes are indicated by light grey shading, and truncated contigs are indicated by vertical lines. For JS1-2, individual gene numbers and contig fragments (thin lines below gene numbers) are indicated separately in SAG 231 and the TA biofilm metagenome bin that together contain a complete set of the conserved genes in ‘Atribacteria' BMC loci on multiple, truncated contigs.

Figure 4

Predicted catabolism, BMC function and energy conservation in ‘Atribacteria' JS1-1, JS1-2 and OP9-1 lineages. (a) Catabolic degradation of propionate via the Mmc pathway in JS1-1 and JS1-2 (green arrows) and fermentation of sugars in OP9-1 (blue) converge on pyruvate, which can be further processed to acetyl-CoA, acetate and acetaldehyde (HAc) in all these lineages (grey). NADH-dependent acetyl-CoA reduction in the BMC by PduPL produces HAc, which can further serve either as an electron sink via alcohol dehydrogenase for OP9-1 (blue dotted line) or as a high energy electron source via aldehyde:Fd oxidoreductase (producing reduced Fd and acetate) for JS1-1 and JS1-2 (green dotted line). HAc and pyruvate-derived glyceraldehyde-3-phosphate (G3P) may also undergo sequential aldehyde condensation through DERA and sugar isomerase, facilitating carbon storage and later use as an electron source or sink. See text and Supplementary Table S5 for additional details. (b) Energy conservation in OP9-1 via NADH:Fd oxidoreductase (Rnf complex) and electron-confurcating hydrogenase (ECHyd). (c) Energy-conserving formate/H₂ generation in JS1-1 and JS1-2 through EBFdh, membrane-bound hydrogenase (MbhA-N), succinate dehydrogenase (Sdh) and NADH:Nqo using nicotinamide adenine dinucleotide (NAD⁺/NADH), ferredoxin (Fd), flavin (F/FH₂) and quinones (Q/QH₂) as electron carriers.