Methane-Fueled Syntrophy through Extracellular Electron Transfer: Uncovering the Genomic Traits Conserved within Diverse Bacterial Partners of Anaerobic Methanotrophic Archaea

Abstract

The anaerobic oxidation of methane by anaerobic methanotrophic (ANME) archaea in syntrophic partnership with deltaproteobacterial sulfate-reducing bacteria (SRB) is the primary mechanism for methane removal in ocean sediments. The mechanism of their syntrophy has been the subject of much research as traditional intermediate compounds, such as hydrogen and formate, failed to decouple the partners. Recent findings have indicated the potential for extracellular electron transfer from ANME archaea to SRB, though it is unclear how extracellular electrons are integrated into the metabolism of the SRB partner. We used metagenomics to reconstruct eight genomes from the globally distributed SEEP-SRB1 clade of ANME partner bacteria to determine what genomic features are required for syntrophy. The SEEP-SRB1 genomes contain large multiheme cytochromes that were not found in previously described free-living SRB and also lack periplasmic hydrogenases that may prevent an independent lifestyle without an extracellular source of electrons from ANME archaea. Metaproteomics revealed the expression of these cytochromes at in situ methane seep sediments from three sites along the Pacific coast of the United States. Phylogenetic analysis showed that these cytochromes appear to have been horizontally transferred from metal-respiring members of the Deltaproteobacteria such as Geobacter and may allow these syntrophic SRB to accept extracellular electrons in place of other chemical/organic electron donors.IMPORTANCE Some archaea, known as anaerobic methanotrophs, are capable of converting methane into carbon dioxide when they are growing syntopically with sulfate-reducing bacteria. This partnership is the primary mechanism for methane removal in ocean sediments; however, there is still much to learn about how this syntrophy works. Previous studies have failed to identify the metabolic intermediate, such as hydrogen or formate, that is passed between partners. However, recent analysis of methanotrophic archaea has suggested that the syntrophy is formed through direct electron transfer. In this research, we analyzed the genomes of multiple partner bacteria and showed that they also contain the genes necessary to perform extracellular electron transfer, which are absent in related bacteria that do not form syntrophic partnerships with anaerobic methanotrophs. This genomic evidence shows a possible mechanism for direct electron transfer from methanotrophic archaea into the metabolism of the partner bacteria.

Keywords: ANME; AOM; SEEP-SRB1; anaerobic oxidation of methane; extracellular electron transfer; methane seeps; multiheme cytochrome; sulfate-reducing bacteria.

FIG 1

Phylogeny of methane seep Deltaproteobacteria and related organisms. Maximum likelihood phylogeny data were determined on the basis of an alignment of 40 universally conserved protein sequences. Internal nodes in the tree with greater than 70% or 90% bootstrap support are marked by gray or black circles, respectively. Genome bins identified in our metagenomic sequencing are highlighted in red; many of them are grouped into the Desulfobacteraceae SEEP-SRB1 or the Desulfobulbaceae SEEP-SRB4 clades. "Ca. Desulfofervidus auxilii," a previously analyzed ANME partner, is shown in bold text. Wedges represent multiple related genomes that have been collapsed for brevity. At the right, yellow squares indicate that the organism is capable of respiration using any sulfur compound; red squares indicate the ability to perform metal respiration; green circles indicate organisms that contain the same cytochrome-containing operon that is found in SEEP-SRB1. Blue, purple, and orange circles indicate that one or more of the four core genes in the cytochrome operon had been detected in the Santa Monica Mounds, Hydrate Ridge, or Eel River Basin sites.

FIG 2

Proposed model of SEEP-SRB1 metabolism in AOM consortia. All identified SRB from AOM consortia contained the canonical sulfate reduction pathway: sulfate adenylyltransferase (Sat), adenylyl-sulfate reductase (Apr), dissimilatory sulfite reductase (DsrAB), and the membrane-associated complexes Qmo and DsrMKJOP. The DsrC protein acts as a key intermediate for transferring electrons from DsrAB to other redox-active complexes, including the DsrMKJOP and Tmc membrane complexes and the soluble Flx-Hdr complex. Electrons required to reduce the Qrc or Tmc membrane complexes are proposed to be sourced from direct extracellular electron transfer from the ANME archaeon cell mediated by outer membrane c-type cytochromes. All genomes fix carbon using the Wood-Ljungdahl pathway and contain a complete tricarboxylic acid (TCA) cycle, a pentose phosphate pathway (PPP), and the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis/gluconeogenesis.

FIG 3

Representative operon structure from organisms containing large multiheme cytochromes found in SEEP-SRB1. Homologous genes are colored the same between organisms, with the exception of the cytochromes, which are colored with various intensities of red based on the number of heme binding motifs present in the gene. Genes in gray are not conserved (i.e., are unique to that genome). The NCBI locus tag identifier for the core set of four genes is shown below each operon.

FIG 4

Maximum likelihood phylogenetic trees of (A) the 16-heme cytochrome; (B) the 26-heme cytochrome; (C) the peptidyl-prolyl cis-trans-isomerase; and (D) the six-bladed beta propeller fold protein. Each tree was rooted at the midpoint branch. Internal nodes in the tree with greater than 70% or 90% bootstrap support are marked by gray or black circles, respectively. The ANME partners are labeled in red. Scale bars represent numbers of substitutions per site.