Physiological potential and evolutionary trajectories of syntrophic sulfate-reducing bacterial partners of anaerobic methanotrophic archaea

Abstract

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it.

Fig 1. Taxonomic diversity of syntropic SRB.

A concatenated gene tree of 71 ribosomal proteins from all the Desulfobacterota genomes within the GTDB database release 95 [38] was made using Anvi’o [45]. Genomes from the genus Shewanella were used as outgroup. Within this tree, the 4 most common lineages of the syntrophic partners of ANME—Seep-SRB1a, Seep-SRB1g, Seep-SRB2, and Hot-Seep1 are highlighted. While Seep-SRB1a is a genus within the order Desulfobacterales, Seep-SRB1g and Seep-SRB1c together appear to form a closely related order-level taxonomic clade within the class Desulfobacteria. Seep-SRB2 is a genus within the order Dissulfuribacterales while Hot-Seep1 is its own species within the order Desulfofervidales. The proposed type strains are identified on the tree in white with a white asterisk adjacent to the label. The list of genomes used for the generation of concatenated gene tree is listed in S1 Table and the tree is made available as a newick file in S2 Data. ANME, anaerobic methanotrophic archaea; SRB, sulfate-reducing bacteria.

Fig 2. Gene organization and distribution of the putative cluster implicated in DIET from syntrophic SRB.

(a) The syntenic blocks of genes implicated in DIET including the putative EET conduit OetABI and the operon encoding for OmcKL from HotSeep-1 (Candidatus Desulfofervidus auxilii). (b) A model of the putative EET within syntrophic SRB. ANME electrons are likely to be accepted by 1 of 3 putative nanowires formed by multiheme cytochromes homologous to OmcX, OmcS, and a cytochrome we named Apc2a. The electrons from this nanowire would then be transferred to the porin:multiheme cytochrome c conduits formed by OetABI or OmcKL and ultimately to different periplasmic cytochromes c. (c) The distribution of the putative DIET cluster in the phylum Desulfobacterota is mapped onto a whole genome phylogenetic tree of Desulfobacterota represented in Fig 1 based on the presence of OmcX, OetI, OetB, and OmcS. This cluster is not widely found except in the orders Desulfuromonadales and Geobacterales and the classes Desulfobulbia and Thermodesulfobacteria. ANME, anaerobic methanotrophic archaea; DIET, direct interspecies electron transfer; EET, extracellular electron transfer; SRB, sulfate-reducing bacteria.

Fig 3. Summary of the different electron transport chains in syntrophic SRB.

The various respiratory proteins essential for the electron transport chain within the syntrophic SRB are identified and marked within their predicted cellular compartments. Filled circles indicate their presence in each of the 4 syntrophic sulfate-reducing bacterial clades, HotSeep-1 (red), Seep-SRB2 (orange), Seep-SRB1g (blue), and Seep-SRB1a (purple). The 2 typical acceptors of electrons transferred across the inner membrane, quinols (QH₂) and DsrC, are indicated in shaded circles. These are the 2 nodes which much of the respiratory flexibility of the syntrophic SRB revolves around. SRB, sulfate-reducing bacteria.

Fig 4. cbcBA as an example of horizontal gene transfer events into Seep-SRB2.

We demonstrate one example of an important gene transfer event involving a metabolic gene. The function of cbcBA is essential for the central respiratory pathway in Seep-SRB2 and this gene was acquired by horizontal gene transfer. (A) The presence of CymA, CbcL, CbcAB, and NetBCD, commonly used electron donors to the EET conduits in Shewanella and Geobacter are mapped on to the classes Thermodesulfobacteria and Desulfobulbia. (B) CbcB protein sequences were aligned using MUSCLE [72] and then a phylogenetic tree was inferred using IQ-Tree2 [73]. The CbcB sequences from Seep-SRB2 are highlighted in orange. The phylogenetic tree is available in newick format in S2 Data. EET, extracellular electron transfer; SRB, sulfate-reducing bacteria.

Fig 5. The loss of cobalamin biosynthesis genes in the Seep-SRB1a partners of ANME-2a.

On the right, a phylogenetic tree of concatenated ribosomal proteins from all the genomes of syntrophic SRB clades—Hot-Seep1, Seep-SRB2, Seep-SRB1g, and Seep-SRB1a and related clades, Seep-SRB1c and Eth-SRB1 was made using Anvi’o [45] and made available in S2 Data. On the left, a similar concatenated protein tree (available in S2 Data) was made for ANME genomes highlighting the clades from ANME-1, ANME-2c, ANME-2b, and ANME-2a. Lines in green and light teal are used to depict the partnerships between ANME-2c and verified species of Seep-SRB1a, and ANME-2a and verified species of Seep-SRB1a, respectively. ANME-2c genomes are not separated into those belonging to partners of Seep-SRB2 and Seep-SRB1a. The presence of genes involved in cobalamin biosynthesis and nitrogen fixation are marked in light green and light blue, respectively. The proposed type strains are bolded. ANME, anaerobic methanotrophic archaea; SRB, sulfate-reducing bacteria.

Fig 6. Putative physiological factors involved in ANME/SRB aggregate formation.

Extracellular polysaccharides and protein complexes implicated in the formation of the extracellular matrix in ANME-SRB aggregates are visualized as cell-surface embedded or secreted. The capacity for biosynthesis of sulfated polysaccharides is present in 3 of the syntrophic SRB clades—Seep-SRB2, Seep-SRB1a, and Seep-SRB1g. Type VI secretion systems and eCISs are likely important for intercellular communication between ANME and SRB. ANME, anaerobic methanotrophic archaea; eCIS, extracellular contractile injection system; SRB, sulfate-reducing bacteria.

Fig 7. A summary of important gene loss and gain events in the physiological adaptation of sulfate reducing bacteria that led to a syntrophic partnership with ANME.

The presence and absence of genes involved in the electron transport chain, nutrient sharing, biofilm formation, and cellular adhesion are listed in S12 Table. We identified genes that were potentially gained, lost, or biochemically adapted using a comparative analysis of the presence a given gene in a syntrophic clade in its order-level taxonomic background. For example, if a gene is present in a syntrophic SRB clade and is present in fewer than 30% of the remaining species in a given order, this gene is considered a likely horizontally transferred gene. The likelihood of horizontal transfer is then further corroborated with a phylogenetic tree of that gene generated with close homologs from NCBI and our curated dataset. The trees are available in S1 Data. The secondary analysis of the likelihood of gene gains and losses is present in S13 Table. ANME, anaerobic methanotrophic archaea; SRB, sulfate-reducing bacteria.