RESEARCH ARTICLE
Physiological potential and evolutionary
trajectories of syntrophic sulfate-reducing
bacterial partners of anaerobic
methanotrophic archaea
Ranjani Murali 1
ID *, Hang Yu2,3, Daan R. Speth1,4, Fabai Wu5, Kyle S. Metcalfe6,
Antoine Crémière2, Rafael Laso-Pèrez7¤, Rex R. Malmstrom8, Danielle Goudeau8,
Tanja Woyke8, Roland Hatzenpichler9, Grayson L. Chadwick2,6, Stephanie A. Connon2,
Victoria J. Orphan1,2*
a1111111111 1 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California,
a1111111111 United States of America, 2 Division of Geological and Planetary Sciences, California Institute of Technology,
a1111111111 Pasadena, California, Unites Stated of America, 3 Department of Physics and Astronomy, University of
a1111111111 Southern California, Los Angeles, California, United States of America, 4 Division of Microbial Ecology,
Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna, Austria, 5 ZJU-
a1111111111
Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, China,
6 Department of Plant and Molecular Biology, University of California, Berkeley. Berkeley, California, United
States of America, 7 Systems Biology Department, Centro Nacional de Biotecnologı́a (CNB-CSIC), Madrid,
Spain, 8 DOE Joint Genome Institute, Department of Energy, Berkeley, California, United States of America,
9 Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, United States
OPEN ACCESS of America
Citation: Murali R, Yu H, Speth DR, Wu F, Metcalfe ¤ Current address: Biogeochemistry and Microbial Ecology Department, Museo Nacional de Ciencias
KS, Crémière A, et al. (2023) Physiological Naturales (MNCN-CSIC), Madrid, Spain
potential and evolutionary trajectories of syntrophic * m.ranjani@gmail.com (RM); vorphan@caltech.edu (VJO)
sulfate-reducing bacterial partners of anaerobic
methanotrophic archaea. PLoS Biol 21(9):
e3002292. https://doi.org/10.1371/journal. Abstract
pbio.3002292
Academic Editor: Fengping Wang, School of Life Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular con-
Sciences and Biotechnology, State Key Laboratory sortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with
of Ocean Engineering, Shanghai Jiaotong
sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physi-
University, CHINA
ological basis for their adaptation and diversification is not well understood. In this work, we
Received: November 23, 2022
used comparative metagenomics and phylogenetics to investigate the metabolic adaptation
Accepted: August 8, 2023 among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and
Published: September 25, 2023 Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distin-
Peer Review History: PLOS recognizes the guish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacter-
benefits of transparency in the peer review ota. We show that the protein complexes involved in direct interspecies electron transfer
process; therefore, we enable the publication of (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic line-
all of the content of peer review and author
ages. In contrast, the proteins involved in electron transfer within the SRB inner membrane
responses alongside final, published articles. The
editorial history of this article is available here: differ between clades, indicative of convergent evolution in the adaptation to a syntrophic
https://doi.org/10.1371/journal.pbio.3002292 lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the
Copyright: This is an open access article, free of all acquisition of the DIET complexes in an ancestral clade and involve horizontal gene trans-
copyright, and may be freely reproduced, fers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also pro-
distributed, transmitted, modified, built upon, or vide evidence for unique adaptations within syntrophic SRB clades, which vary depending
otherwise used by anyone for any lawful purpose.
on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, sub-
The work is made available under the Creative
Commons CC0 public domain dedication. clades that specifically partner ANME-2a are missing the cobalamin synthesis pathway,
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 1 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
Data Availability Statement: All the metagenome suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a part-
assembled genomes (MAGs) created for this ners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features
study, along with associated metadata and
associated with DIET-based syntrophy and the adaptation of SRB towards it.
biosample data are available from the NCBI
database (BioProject: PRJNA762493).
Metagenome assemblies from sorted aggregates
that we used here are made available on the IMG
database under the following IDs – 3300036218,
3300036221, 3300036226, 3300036304,
3300036329, 3300036259. The original tree files Introduction
have been made available on the first author’s
Github page (https://github.com/ranjani-m/ Syntrophy is metabolic cooperation between microorganisms for mutual benefit. It is a com-
syntrophic-SRB) and they have also been uploaded mon adaptation in low-energy environments and enables the utilization of substrates which
as part of online supplementary data. All other neither organism could metabolize on its own [1,2]. While the driving force for different
relevant data is provided as supplementary microbial syntrophic interactions may vary, both partners benefit from sharing nutrients and
material in this manuscript.
electrons in this way, combining their resources and avoiding the need for both partners to
Funding: This research was supported by the expend energy for the synthesis of common nutrients [1,3]. Syntrophic interactions appear to
Gordon and Betty Moore Foundation Models in be specific in at least some cases, with the same organisms co-associating across different eco-
Marine Symbiosis (GBMF Grant #9324, to V.J.O),
systems and environments [4]. However, we do not yet understand the physiological basis
the U.S. Department of Energy BER(Award
Number: DE-SC0022991 to V.J.O); and a FICUS driving the specificity of interactions, often because syntrophic associations are difficult to
grant (Award doi: 10.46936/fics.proj.2017.49956/ grow in culture. Characterizing the specificity of these interactions is challenging with uncul-
60006219 to V.J.O.) through the U.S. Department tured syntrophic consortia in the environment [2]. A classic syntrophic partnership is at the
of Energy Joint Genome Institute (https://ror.org/ heart of the important biogeochemical process, sulfate-coupled anaerobic oxidation of meth-
04xm1d337), a DOE Office of Science User Facility
ane (AOM) [5]. Anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria
supported by the Office of Science of the U.S.
(SRB) coexist in multicellular consortia, with ANME performing methane oxidation coupled
Department of Energy operated under Contract No.
DE-AC02-05CH11231). Deep sea samples for this to sulfate reduction by the SRB [6–8]. Direct interspecies electron transfer (DIET) from
project were collected in part through support from ANME to SRB is predicted to be the dominant mechanism of syntrophic coupling in many
the National Science Foundation (OCE-1634002, to observed cases of sulfate-coupled AOM [9,10] though diazotrophic nitrogen is also shared
V.J.O) and the Science and Technology Center for between these partners [11–13]. There is rich ecological diversity in the observed examples of
Dark Energy Biosphere Investigations (C-DEBI,
AOM with taxonomically divergent groups of ANME coexisting with an equally diverse group
OCE-0939564, to V.J.O). R.L.-P. was funded by a
Juan de la Cierva grant (FJC2019-041362-I) from of SRB in consortia that appear morphologically different [13,14]. ANME-SRB consortia exist
the Spanish Ministerio de Ciencia e Innovación and in hydrothermal vents [15], in cold-seeps [6,7,16], in mud volcanoes [17], and a euxinic basin
by a Ramón y Cajal grant (RyC2021-031775-I) [18], and can form tight spherical aggregates [16,19], dense microbial mats [20], or loose asso-
from the Spanish Ministerio de Ciencia e ciations [19,21]. Past work has suggested that some ANME-SRB associations are more specific
Innovación (MCIN/AEI/10.13039/501100011033)
than others [13,22,23], and ecophysiological studies have demonstrated differences in genes
and the European Union («NextGenerationEU»/
expressed, even pertaining to DIET [24,25]. To investigate whether there is underlying struc-
PRTR). The funders had no role in study design,
data collection and analysis, decision to publish, or ture to this variety of ANME-SRB interactions in different ecosystems, and to infer the evolu-
preparation of the manuscript tionary trajectories that led to these extant phenomena, it is important to establish a
taxonomic, ecological, and physiological framework within which to organize our
Competing interests: The authors have declared
that no competing interests exist. observations.
Investigation of the archaeal and bacterial lineages involved in AOM identified at least 3
Abbreviations: ANI, average nucleotide identity;
divergent taxonomic groups of archaea, by analysis of 16S rRNA gene sequences and fluores-
ANME, anaerobic methanotrophic archaea; AOM,
anaerobic oxidation of methane; BIC, Bayesian cence in situ hybridization (FISH)–ANME-1 (Methanophagales), ANME-2, and ANME-3
information criterion; BONCAT, BioOrthogonal (Methanovorans) [6,17,26]. All 3 of these groups are clades within the phylum Halobacterota,
Non-Canonical Amino Acid Tagging; DIET, direct and ANME-2 is subdivided into the clades ANME-2a (Methanocomedenaceae), ANME-2b
interspecies electron transfer; EET, extracellular (Methanomarinus), ANME-2c (Methanogasteraceae), and ANME-2d (Methanoperedenaceae)
electron transfer; eCIS, extracellular contractile
[14]. In a recent paper [14], Chadwick and colleagues established a robust taxonomic frame-
injection system; FACS, fluorescence-activated cell
sorting; FBEC, flavin-based electron confurcation; work for ANME, identified key biochemical pathways in the archaea that are important for
FISH, fluorescence in situ hybridization; IMG, AOM and demonstrated that the capability for extracellular electron transfer (EET) is a signifi-
integrated microbial genomes; MAG, metagenome- cant metabolic trait that differentiates ANME from its nearest evolutionary neighbors that are
assembled genome; MDA, multiple displacement typically methanogens or alkane oxidizing microorganisms [27–29]. Within the ANME
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 2 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
amplification; MIET, mediated interspecies electron (ANME-1, ANME-2a, ANME-2b, ANME-2c, and ANME-3) that are known to partner with
transfer; PCA, principal component analysis; PGAP, SRB [6,16,19,26,30], they were also able to show that there were differences between clades
Prokaryotic Genome Annotation Pipeline; PLTS,
with respect to putative EET pathways, electron transport chains, and biosynthetic pathways
phage-like translocation system; pmf, proton
[14]. Analysis of ANME genomes and inferences from previous physiological data [24,25] also
motive force; rTCA, reductive tricarboxylic acid
cycle; SRB, sulfate-reducing bacteria. suggested differences in secretion machinery and cellular adhesion proteins in the archaeal
partner that might affect syntrophic interactions with the partner bacteria [14]. The goal of
this work is to establish a similar framework for the well-established clades of sulfate-reducing
partner bacteria, to identify important differences in biochemical pathways, and to identify
traits that might correlate with differences in ANME-SRB partnership pairing.
The key to understanding the evolutionary diversification of SRB is to understand the taxo-
nomic background of each syntrophic SRB involved in this process, since taxonomy is the
“expression of evolutionary arrangement” [31] and the phenotypic traits that differentiate the
syntrophs from their non-syntrophic evolutionary neighbors. Previous work demonstrated
that there were 4 sulfate-reducing bacterial clades within the Desulfobacterota that partner
ANME–HotSeep-1 [24,32], Seep-SRB2 [24], Seep-SRB1a [25,33], and Seep-SRB1g [13,33].
Other bacteria and archaea (seepDBB within the Desulfobulbaceae [34], alpha- and beta-pro-
teobacteria [35] and verrucomicrobia [36], Anaerolineales and Methanococcoides [37]) have
also been observed to associate with ANME. However, in this work, we investigated only these
4 clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) that are consistently and most
often found in association with ANME across different ecosystems [23]. With the use of pub-
licly available datasets and 15 metagenomes, we generated in this study from different marine
ecosystems (seeps located off the coast of Costa Rica and off the coast of S. California, as well
as hydrothermal vents in the Gulf of California), we curated a database of 46 syntrophic SRB
metagenomes with multiple representatives from each clade. With the use of the Genome Tax-
onomy Database [38], we created a taxonomic framework to reproducibly classify these syn-
trophic SRB and proposed scientific names according to the latest guidelines. Significantly,
our curated database of representative genomes and 16S rRNA sequences would allow future
studies to differentiate the known syntrophic Seep-SRB1a and Seep-SRB1g clades from the
non-syntrophic members (Seep-SRB1b, Seep-SRB1c, Seep-SRB1d, Seep-SRB1e, and Seep-
SRB1f) of the polyphyletic clade Seep-SRB1 [23,39].
To differentiate the phenotypic traits of syntrophic SRB from non-syntrophic SRB, we syn-
thesized information from prior physiological experiments [24,25] and provide a detailed bio-
chemical description of pathways that are necessary for the formation of ANME-SRB
consortia. Our analysis demonstrated that the syntrophic SRB contain all the genomic traits
consistent with their participation in DIET (including EET pathways), and with the formation
of a multispecies conductive biofilm (cellular adhesion pathways, polysaccharide biosynthesis
pathways). Comparative genome analysis between syntrophic genomes and over 550 non-syn-
trophic bacteria within the phylum Desulfobacterota, showed that these traits are rare in non-
syntrophic SRB. We also investigated the importance of partner-pairing as a meaningful eco-
logical factor that differentiates species of syntrophic SRB. We tested this by sequencing single
ANME-SRB consortia, isolated by fluorescence-activated cell sorting (FACS) [36]. We showed
that Seep-SRB1a partners of ANME-2c appear to have cobalamin biosynthesis pathways while
Seep-SRB1a partners of ANME-2a do not, indicating the latter species of Seep-SRB1a had
developed a nutritional dependence on its partner. These results indicate that there might be
characteristics that are unique to different ANME-SRB pairings and lay the groundwork for
future studies to use a species-level partnership framework to explore the co-diversification of
ANME and SRB. Our study highlights the complex evolutionary trajectory of adaptation of
these SRB to syntrophy with ANME and provides insight into the defining features of DIET-
based syntrophic interactions.
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 3 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
Results and discussion
Taxonomic diversity within syntrophic SRB partners of methanotrophic
ANME
To investigate the adaptation of SRB to a partnership with ANME, we first placed them into
their taxonomic context and assessed the phylogenetic diversity within the SRB clades (Seep-
SRB1a, Seep-SRB1g, Seep-SRB2, and HotSeep-1). For this analysis, we compiled a curated
dataset of metagenome-assembled genomes (MAGs) from these SRB clades including 34 pre-
viously published genomes [25,32,33,40–44] and 12 MAGs assembled for this study. Five of
these genomes were reconstructed from seep samples collected off the coast of California,
Costa Rica, and within the Gulf of California. We also sequenced single ANME-SRB consortia
that were sorted by FACS after they were SYBR-stained as previously described [37]. With this
technique, we could be confident of the assignment of partners that physically co-associate
within the sequenced aggregates and begin to identify partnership-specific characteristics.
From sequencing of single consortia, we obtained 2 genomes of ANME-2b associated Seep-
SRB1g, 1 genome of ANME-2a associated Seep-SRB1a, and 3 genomes of ANME-2c associated
Seep-SRB1a (Table 1). We recovered an additional 3 genomes of the nearest evolutionary
neighbors of HotSeep-1 within the order Desulfofervidales since this order of bacteria is very
poorly represented in public databases. Our dataset for comparative genomics analysis com-
prised the above mentioned 46 genomes of syntrophic SRB and over 550 other bacteria from
Desulfobacterota. Having compiled this dataset of syntrophic SRB, we also designated type
material and proposed formal names for 3 of the syntrophic SRB clades, Seep-SRB2 (Candida-
tus Desulfomithrium gen. nov.), Seep-SRB1a (Candidatus Syntrophophila gen. nov.), and
Seep-SRB1g (Candidatus Desulfomellonium gen. nov.). The genomes designated as type mate-
rial are identified in Figs 1 and S1. Further details are available in the Supporting information
as a proposal for formal nomenclature for Seep-SRB1a, Seep-SRB1g, and Seep-SRB2 (S1 Text).
Details for the phylogenetic placement of each of these clades using 16S rRNA phylogeny,
concatenated ribosomal protein phylogeny, and the Genome Taxonomy Database are pro-
vided in Materials and methods and S1–S3 Figs. HotSeep-1 is a species within the order Desul-
fofervidales, an order that is largely associated with thermophilic environments (with 1
exception, Desulfofervidales sp. DG-60 was sequenced from the White Oak Estuary [46]).
Members of HotSeep-1 are the best characterized members of this order and are known to be
syntrophic partners to thermophilic clades of methane-oxidizing ANME-1 [14,24] as well as
alkane-oxidizing archaeal relatives “Candidatus Syntrophoarchaeum butanivorans,” “Candi-
datus Syntrophoarchaeum caldarius” [27], and ethane-oxidizing “Candidatus Ethanoperedens
thermophilum” [40]. Seep-SRB2 is a genus-level clade within the order Dissulfuribacterales
[47–49] and class Dissulfuribacteria. Dissulfuribacterales include the genera Dissulfuribacter
and Dissulfurirhabdus [47–49], which are chemolithoautotrophs associated with sulfur dispro-
portionation. Seep-SRB1g is a species level clade which groups within a taxonomic order that
also includes Seep-SRB1c (Fig 1 and Table 1). This order falls within the class Desulfobacteria
along with the sister order Desulfobacterales. Like the Desulfofervidales, the order with Seep-
SRB1g is poorly characterized, yet its most well-described members are the Seep-SRB1g that
are obligate syntrophic partners of ANME, accepting electrons from the archaeal partner to
reduce sulfate [13,33]. Seep-SRB1a is a genus-level clade that along with the genus Eth-SRB1
forms a distinct family within the order Desulfobacterales (Figs 1 and S1 and S2 Table). Many
of the well-characterized members of Desulfobacterales such as Desulfococcus oleovorans,
Desulfobacter hydrogenophilus, Desulfosarcina BuS5 are known as hydrogenotrophs and
hydrocarbon degraders [50–52]. The nearest evolutionary relative of Seep-SRB1a are the Eth-
SRB1 first characterized as a syntrophic partner of ethane-degrading archaea [29]. Seep-SRB1a
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 4 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
Table 1. List of genomes from syntrophic SRB labeled by clade, generated in this study and compiled from public databases.
Assembly Organism Proposed formal name Genome size Completeness Contamination GTDB_classification Geographic location Reference
(bp)
JAJSZM000000000 Desulfofervidales_sp. 1452671 90.31 1.42 d__Bacteria;p__Desulfobacterota;c__Desulfofervidia;o__Desulfofervidales; Pescadero Basin, Gulf of This
_FWG156* f__DG-60;g__;s__ California study
JAJSZL000000000 Candidatus Desulfofervidaceae 2230686 97.97 3.46 d__Bacteria;p__Desulfobacterota;c__Desulfofervidia;o__Desulfofervidales; Pescadero Basin, Gulf of This
sp. 1* f__Desulfofervidaceae;g__;s__ California study
JAJSZS000000000 Candidatus Desulfofervidaceae 2268002 98.24 3.49 d__Bacteria; p__Desulfobacterota; c__Desulfofervidia; Hydrothermal vent Pescadero This
sp. 2* o__Desulfofervidales; f__Desulfofervidaceae; g__; s__ Basin, Gulf of California study
GCA_001577525.1 HotSeep1 2540211 96.75 4.27 d__Bacteria; p__Desulfobacterota; c__Desulfofervidia; Hydrothermal vent Guaymas [32]
o__Desulfofervidales; f__Desulfofervidaceae; g__Desulfofervidus; Basin, Gulf of California
s__Desulfofervidus auxilii
HotSeep1_draft_B50 HotSeep1 B50 2215853 95.53 9.35 d__Bacteria; p__Desulfobacterota; c__Desulfofervidia; Hydrothermal vent Pescadero This
o__Desulfofervidales; f__Desulfofervidaceae; g__Desulfofervidus; Basin, Gulf of California study
s__Desulfofervidus auxilii
CAJIMJ000000000 HotSeep1 E37 1863098 87.40 3.46 d__Bacteria; p__Desulfobacterota; c__Desulfofervidia; Guaymas Basin, Gulf of [40]
o__Desulfofervidales; f__Desulfofervidaceae; g__Desulfofervidus; California
s__Desulfofervidus auxilii
CAJIMK000000000 HotSeep1 E50 1842718 70.82 5.08 d__Bacteria; p__Desulfobacterota; c__Desulfofervidia; Guaymas Basin, Gulf of [40]
o__Desulfofervidales; f__Desulfofervidaceae; g__Desulfofervidus; California
s__Desulfofervidus auxilii
JAJSZN000000000 HotSeep1 FWG170 1777984 86.7 6.74 d__Bacteria;p__Desulfobacterota;c__Desulfofervidia;o__Desulfofervidales; Hydrothermal vent Pescadero This
f__Desulfofervidaceae;g__Desulfofervidus;s__Desulfofervidus auxilii Basin, Gulf of California study
JAAXOL000000000 Seep-SRB1a sp. 1 Syntrophophila gen. nov. 3174343 97.42 4.03 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Groundwater from Olkiluoto, [153]
(str. 013792055) sp. 1 o__Desulfobacterales; Finland
f__ETH-SRB1; g__B13-G4; s__B13-G4 sp013792055
JAJSZT000000000 Seep-SRB1a sp. 2 Syntrophophila gen. nov. 3859851 99.35 2.15 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Costa Rica Margin This
(str. CR9063A) sp. 2 o__Desulfobacterales; study
f__ETH-SRB1; g__B13-G4; s__
JAJSZU000000000 Seep-SRB1a sp. 2 Syntrophophila gen. nov. 3484925 87.56 1.94 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Costa Rica Margin This
(str. CR9063B) sp. 2 o__Desulfobacterales; study
f__ETH-SRB1; g__B13-G4; s__
JAJSZV000000000 Seep-SRB1a sp. 2 Syntrophophila gen. nov. 2980801 64.05 0.00 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Costa Rica Margin This
(str. CR9063C) sp. 2 o__Desulfobacterales; study
f__ETH-SRB1; g__B13-G4; s__
JAAXOL000000000 Seep-SRB1a sp. 3 Syntrophophila gen. nov. 3489866 98.00 1.53 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Gulf of Cadiz [154]
(str. 014237195) sp. 3 o__Desulfobacterales;
f__ETH-SRB1; g__B13-G4; s__B13-G4 sp014237365
JABZFQ000000000 Seep-SRB1a sp. 3 Syntrophophila gen. nov. 3489866 98.00 1.53 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Gulf of Cadiz [154]
(str. 014237365) sp. 3 o__Desulfobacterales;
f__ETH-SRB1; g__B13-G4; s__B13-G4 sp014237365
JAJSZO000000000 Seep-SRB1a sp. 4 Syntrophophila gen. nov. 2505125 77.03 0.65 d__Bacteria;p__Desulfobacterota;c__Desulfobacteria; Microbial mat, cold seep, Santa This
(str. FWG171) sp. 4 o__Desulfobacterales; Monica Basin, California study
f__ETH-SRB1;g__B13-G4;s__
JAJSZP000000000 Seep-SRB1a sp. 7 Syntrophophila gen. nov. 2570901 92.1 4.73 d__Bacteria;p__Desulfobacterota;c__Desulfobacteria; Microbial mat, cold seep, Santa This
(str. FWG172) sp. 7 o__Desulfobacterales; Monica Basin, California study
f__ETH-SRB1;g__B13-G4;s__
JAIORM000000000 Seep-SRB1a sp. 5 Syntrophophila gen. nov. 1938412 78.03 2.60 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Santa Monica Basin, [37]
(str. 20073_SRB) sp. 5 o__Desulfobacterales; California, USA
f__ETH-SRB1; g__B13-G4; s__
JAIORU000000000 Seep-SRB1a sp. 5 Syntrophophila gen. nov. 1678217 72.61 3.96 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Santa Monica Basin, [37]
(str. 20074_SRB) sp. 5 o__Desulfobacterales; California, USA
f__ETH-SRB1; g__B13-G4; s__
JAABVG000000000 Seep-SRB1a sp. 5 Syntrophophila gen. nov. 2766087 88.86 2.58 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Santa Monica Basin, [25]
(str. S7142MS3) sp. 5 o__Desulfobacterales; California, USA
f__ETH-SRB1; g__B13-G4; s__
JAJSZW000000000 Seep-SRB1a sp. 5 Syntrophophila gen. nov. 3861344 89.65 1.29 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Cold seep, Santa Monica Basin, This
(str. SM7059A) sp. 5 o__Desulfobacterales; California, USA study
f__ETH-SRB1; g__B13-G4; s__
QMMZ00000000 Seep-SRB1a sp. 6 Syntrophophila gen. nov. 1690800 56.35 0.97 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Hydrothermal sediment, [41]
(str. 003647525) sp. 6 o__Desulfobacterales; Guaymas Basin, Gulf of
f__ETH-SRB1; g__B13-G4; s__B13-G4 sp003647525 California
JAFCZG000000000 Seep-SRB1a sp. 8 Syntrophophila gen. nov. 5464515 52.75 9.74 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Hydrothermal sediment, [42]
(str. AB_03_Bin_172) sp. 8 o__Desulfobacterales; Guaymas Basin, Gulf of
f__ETH-SRB1; g__B13-G4; s__ California
JAFDFV000000000 Seep-SRB1a sp. 9 Syntrophophila gen. nov. 3102898 83.52 3.87 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Hydrothermal sediment, [42]
(str. Meg22_02_Bin_90) sp. 9 o__Desulfobacterales; Guaymas Basin, Gulf of
f__ETH-SRB1; g__B13-G4; s__ California
JAFDJY000000000 Seep-SRB1a sp. 9 Syntrophophila gen. nov. 2156769 62.82 5.48 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Hydrothermal sediment, [42]
(str. Meg22_24_Bin_68) sp. 9 o__Desulfobacterales; Guaymas Basin, Gulf of
f__ETH-SRB1; g__B13-G4; s__ California
JAFDKW000000000 Seep-SRB1a sp. 9 Syntrophophila gen. nov. 2114727 57.68 1.68 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; Hydrothermal sediment, [42]
(str.Meg22_46_Bin_236) sp. 9 o__Desulfobacterales; Guaymas Basin, Gulf of
f__ETH-SRB1; g__B13-G4; s__ California
JAFDAE000000000 Desulfobacterales sp. 3617733 88.46 3.59 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; o__C00003060; Hydrothermal sediment, [42]
AB_1215_Bin_34* f__C00003106; g__C00003106; s__ Guaymas Basin, Gulf of
California
MAXM00000000 Seep-SRB1g Desulfomellonium gen. 2209054 90.01 0.65 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; o__C00003060; Hydrate Ridge, Oregon, USA [33]
(str. C00003104) nov. sp. 1 f__C00003106; g__C00003106; s__C00003106 sp001751015
MANV00000000 Seep-SRB1g Desulfomellonium gen. 2149125 90.08 0.00 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; o__C00003060; Hydrate Ridge, Oregon, USA [33]
(str. C00003106) nov. sp. 1 f__C00003106; g__C00003106; s__C00003106 sp001751015
(Continued)
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 5 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
Table 1. (Continued)
Assembly Organism Proposed formal name Genome size Completeness Contamination GTDB_classification Geographic location Reference
(bp)
JAJSZX000000000 Seep-SRB1g Desulfomellonium gen. 3489866 99.17 0.65 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; o__C00003060; Cold seep, Costa Rica Margin This
(str. CR10073A) nov. sp. 1 f__C00003106; g__C00003106; s__C00003106 sp001751015 study
JAJSZY000000000 Seep-SRB1g Desulfomellonium gen. 3824408 98.71 2.15 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; o__C00003060; Cold seep, Costa Rica Margin This
(str. CR10073B) nov. sp. 1 f__C00003106; g__C00003106; s__C00003106 sp001751015 study
JAABVH000000000 Seep-SRB1g Desulfomellonium gen. 624626 36.28 0.16 d__Bacteria; p__Desulfobacterota; c__Desulfobacteria; o__C00003060; Cold seep, Santa Monica Basin, [25]
(str. S7142MS4) nov. sp. 1 f__C00003106; g__C00003106; s__C00003106 sp001751015 California, USA
PQXD00000000 Seep-SRB2 sp. 1 Desulfomithrium gen. 3571848 93.83 0.00 d__Bacteria; p__Desulfobacterota; c__Dissulfuribacteria; Hydrothermal sediment, [24]
(str. G37) nov. sp. 1 o__Dissulfuribacterales; f__UBA3076; g__UBA3076; s__UBA3076 Guaymas Basin, Gulf of
sp003194485 California
JAFDGK000000000 Seep-SRB2 sp. 1 Desulfomithrium gen. 2126229 85.79 0.6 d__Bacteria; p__Desulfobacterota; c__Dissulfuribacteria; Hydrothermal sediment, [42]
(str. Meg22_1012_Bin_255) nov. sp. 1 o__Dissulfuribacterales; f__UBA3076; g__UBA3076; s__UBA3076 Guaymas Basin, Gulf of
sp003194485 California
JAFDIH000000000 Seep-SRB2 sp. 1 Desulfomithrium gen. 2269757 91.15 0.6 d__Bacteria; p__Desulfobacterota; c__Dissulfuribacteria; Hydrothermal sediment, [42]
(str. Meg22_1214_Bin_80) nov. sp. 1 o__Dissulfuribacterales; f__UBA3076; g__UBA3076; s__UBA3076 Guaymas Basin, Gulf of
sp003194485 California
JAFDIM000000000 Seep-SRB2 sp. 1 Desulfomithrium gen. 1724888 60.53 1.98 d__Bacteria; p__Desulfobacterota; c__Dissulfuribacteria; Hydrothermal sediment, [42]
(str. Meg22_1416_Bin_176) nov. sp. 1 o__Dissulfuribacterales; f__UBA3076; g__UBA3076; s__UBA3076 Guaymas Basin, Gulf of
sp003194485 California
JAFDIU000000000 Seep-SRB2 sp. 1 Desulfomithrium gen. 1927125 76.83 2.68 d__Bacteria; p__Desulfobacterota; c__Dissulfuribacteria; Hydrothermal sediment, [42]
(str. Meg22_1618_Bin_165) nov. sp. 1 o__Dissulfuribacterales; f__UBA3076; g__UBA3076; s__UBA3076 Guaymas Basin, Gulf of
sp003194485 California
JAJSZQ000000000 Seep-SRB2 sp. 2 Desulfomithrium gen. 2393181 87.78 0.6 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Pescadero Basin, Gulf of This
(str. FWG173) nov. sp. 2 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ California study
JAJSZR000000000 Seep-SRB2 sp. 2 Desulfomithrium gen. 2622393 93.73 0.68 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Pescadero Basin, Gulf of This
(str. FWG174) nov. sp. 2 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ California study
DFBQ00000000 Seep-SRB2 sp. 3 Desulfomithrium gen. 2606474 94.03 1.98 d__Bacteria; p__Desulfobacterota; c__Dissulfuribacteria; Coal Oil Point, Santa Barbara, [44]
(str. 002367355) nov. sp. 3 o__Dissulfuribacterales; f__UBA3076; g__UBA3076; s__UBA3076 California, USA
sp002367355
PQXE00000000 Seep-SRB2 sp. 4 Desulfomithrium gen. 2549842 94.62 1.79 d__Bacteria; p__Desulfobacterota; c__Dissulfuribacteria; Marine sediment, Elba, Italy [24]
(str. E20) nov. sp. 4 o__Dissulfuribacterales; f__UBA3076; g__UBA3076; s__UBA3076
sp003194495
QNAY00000000 Seep-SRB2 sp. 5 Desulfomithrium gen. 1911994 80.95 2.13 d__Bacteria; p__Desulfobacterota; c__Dissulfuribacteria; Hydrothermal sediment, [41]
(str. 003645605) nov. sp. 5 o__Dissulfuribacterales; f__UBA3076; g__UBA3076; s__UBA3076 Guaymas Basin, Gulf of
sp003645605 California
JAFDFS000000000 Seep-SRB2 sp. 6 Desulfomithrium gen. 1600510 70.11 2.7 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Hydrothermal sediment, [42]
(str. Meg22_02_Bin_69) nov. sp. 6 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Guaymas Basin, Gulf of
California
LQBF00000000 Seep-SRB2 sp. 7 Desulfomithrium gen. 3527240 99.38 12.95 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Mahoney Lake, Canada, British [42]
(str. ML8_D) nov. sp. 7 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Columbia
JAFDGC000000000 Seep-SRB2 sp. 8 Desulfomithrium gen. 2934091 90.85 1.19 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Hydrothermal sediment, [42]
(str. Meg19_1012_Bin_147) nov. sp. 8 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Guaymas Basin, Gulf of
California
JAFDGV000000000 Seep-SRB2 sp. 8 Desulfomithrium gen. 2310472 95.49 1.19 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Hydrothermal sediment, [42]
(str.Meg22_1012_Bin_335) nov. sp. 8 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Guaymas Basin, Gulf of
California
JAFDIE000000000 Seep-SRB2 sp. 8 Desulfomithrium gen. 2351303 94.32 0.73 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Hydrothermal sediment, [42]
(str. Meg22_1214_Bin_60) nov. sp. 8 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Guaymas Basin, Gulf of
California
JAFDIO000000000 Seep-SRB2 sp. 8 (str. Desulfomithrium gen. 2557541 77.56 5.08 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Hydrothermal sediment, [42]
Meg22_1416_Bin_56) nov. sp. 8 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Guaymas Basin, Gulf of
California
JAFDIR000000000 Seep-SRB2 sp. 8 (str. Desulfomithrium gen. 2785779 95.51 3.08 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Hydrothermal sediment, [42]
Meg22_1618_Bin_149) nov. sp. 8 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Guaymas Basin, Gulf of
California
JAFDLI000000000 Seep-SRB2 sp. 8 (str. Desulfomithrium gen. 2368215 91.41 0.15 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Hydrothermal sediment, [42]
Meg22_46_Bin_87) nov. sp. 8 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Guaymas Basin, Gulf of
California
JAFDLJ000000000 Seep-SRB2 sp. 8 (str. Desulfomithrium gen. 2635042 91.64 1.92 d__Bacteria;p__Desulfobacterota;c__Dissulfuribacteria; Hydrothermal sediment, [42]
Meg22_810_Bin_10) nov. sp. 8 o__Dissulfuribacterales; f__UBA3076;g__UBA3076;s__ Guaymas Basin, Gulf of
California
*These species are closely related to the syntrophic partner but, they are not known to be partners of ANME.
ANME, anaerobic methanotrophic archaea; SRB, sulfate-reducing bacteria.
https://doi.org/10.1371/journal.pbio.3002292.t001
and Seep-SRB1g are often described as Seep-SRB1 [23,39], a historical name that refers to a
polyphyletic clade including SRB that are not partners of ANME. In order to make our analysis
more accurate, and to aid future classification of syntrophic SRB, we have been careful to dif-
ferentiate the different Seep-SRB1 clades with curated genomes and representative trees of 16S
rRNA and ribosomal proteins (Table 1 and S2 Fig). Each of the 4 syntrophic SRB clades have
evolved from taxonomically divergent ancestors with different metabolic capabilities. While
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 6 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
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.
https://doi.org/10.1371/journal.pbio.3002292.g001
the adaptation to a syntrophic partnership with ANME appears to have been convergently
evolved in these clades, their evolutionary trajectories are likely to be different.
Species diversity within each of these clades was inferred by calculating the average nucleo-
tide identity (ANI) (S1 Fig) and 16S rRNA sequence similarity (S2 Table) between different
organisms that belong to each clade, using a 95% ANI value and 98.65% similarity in 16S
rRNA as cut-offs to delineate different species. Partnership associations, as identified in previ-
ous research by our group and others, by FISH [23,24,32], magneto-FISH [53] or FACS sorting
[36], and single-aggregate sequencing [37] are depicted in Figs 1 and S1 with further details
provided in S1 Text. Briefly, HotSeep-1 has been shown to associate with ANME-1 [24] and
other archaea as described above, Seep-SRB2 associate with ANME-2c and ANME-1 [24],
SeepSRB1g appears to specifically partner ANME-2b [13] while Seep-SRB1a partners ANME-
2a and ANME-2c. All the genomes of Seep-SRB1g in our curated database belong to 1 species-
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 7 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
level clade and thus far, have been shown to partner only ANME-2b [13]. In contrast, there is
greater species diversity within the clades that are known to partner more than 1 clade of
ANME, Seep-SRB2 and Seep-SRB1a. Whether this diversification is driven by adaptation to
partnerships with multiple ANME clades remains to be seen. This pattern is also not consistent
with HotSeep-1, a species-level clade that partners multiple archaeal species. A better under-
standing of the physiological basis for syntrophic partnership formation in each of these clades
will provide a framework to understand their unique diversification trajectories.
Comparative genome analysis of syntrophic SRB
To develop insight into the adaptation of SRB to syntrophic partnerships with ANME, we used
a comparative genomics analysis approach to (1) identify the unique features of known syn-
trophic SRB partners relative to their closest non-syntrophic relatives; and (2) compare the
physiological traits that define the diversity within a given clade of syntrophic partner bacteria.
For our first objective, we placed the metabolic traits of SRB into the phylogenetic context of
the Desulfobacterota phylum, correlating the presence or absence of a physiological trait
within the context of genus, family, and order level context of each syntrophic SRB clade. As
an example, we demonstrate that the multiheme cytochrome conduit [33] implicated in DIET
between ANME and SRB is rare in non-syntrophic Desulfobacterota suggesting that this trait
is part of a required adaptation for this syntrophic relationship (Fig 2).
We also investigated the physiological differences between the species of each syntrophic
SRB clade. Two of the syntrophic SRB clades, Seep-SRB1g and HotSeep-1 have low species
diversity, while the clades Seep-SRB2 and Seep-SRB1a contain multiple species. To better
understand the genomic features underlying this diversity, we performed a comparative analy-
sis of species within the Seep-SRB1a and Seep-SRB2 to identify conserved genes across the
clade and species-specific genes. A detailed description of the analysis methods is available in
Materials and methods and Supporting information (S5 and S6 Figs and S3 and S4 Tables).
For this comparative analysis, we primarily focused on pathways that are predicted to be
important for the syntrophic interactions between ANME and SRB. In the following section,
we describe the pathways within the syntrophic SRB in greater detail and their significance for
a syntrophic lifestyle—extracellular electron transfer, inner membrane-bound electron trans-
port chain, electron bifurcation, carbon fixation, nutrient sharing, biofilm formation, cell
adhesion, and partner identification. Lastly, we explicitly compare the losses and gains of the
genes encoding for the above pathways across the syntrophic SRB and infer the evolutionary
trajectory of adaptation towards a syntrophic partnership.
1. Respiratory pathways in the 4 syntrophic SRB clades demonstrate
significant metabolic flexibility
The respiratory pathways in syntrophic SRB are defined by the necessity of ANME to transfer
the electrons derived from methane oxidation to SRB. These electrons are then transferred
across the outer membrane to periplasmic electron carriers. These periplasmic electron carri-
ers donate electrons to inner membrane complexes and ultimately, to the core sulfate reduc-
tion pathway. Some of the electrons are also used for assimilatory pathways such as carbon
fixation. Accordingly, our analysis of the respiratory pathways is split into a description of the
pathways for interspecies electron transfer, electron transfer across the inner membrane, and
carbon fixation pathways.
1.1 Multiple pathways exist for interspecies electron transfer between ANME and syn-
trophic SRB. The dominant mechanism of interspecies electron transfer between ANME
and SRB was proposed to be DIET. This hypothesis is supported by the presence of multiheme
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
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.
https://doi.org/10.1371/journal.pbio.3002292.g002
cytochromes in genomes of ANME-2a, 2b, and 2c [10], the presence of nanowire-like struc-
tures that extend between ANME-1 and its partners Hot-Seep1 [9] and Seep-SRB2 [24], and
the presence of hemes in the extracellular space between archaeal and bacterial cells in
ANME-SRB aggregates [10,24]. This hypothesis was also supported by the presence of a puta-
tive large multiheme cytochrome:porin type conduit, analogous to the conduits in Geobacter
sp. [54] and other gram-negative bacteria that have been shown to participate in EET [54], in
Seep-SRB1g [33], Seep-SRB2 [24], and Hot-Seep-1 [9]. Our analysis of a more comprehensive
dataset of syntrophic bacterial genomes confirms the presence of this porin:cytochrome c con-
duit in all the 4 syntrophic bacterial clades studied (S5 Table). Henceforth, we refer to this as
the as the (Outer-membrane bound extracellular electron transfer) or OetI-type conduit. This
conduit includes a periplasmic cytochrome c (OetA), an outer-membrane porin (OetI), and
extracellular facing cytochrome c lipoprotein (OetB) (Figs 2B and 3C). The OetI-type conduit
was first identified in G. sulfurreducens and is expressed when a Geobacter mutant of omcB is
grown on Fe(III) oxide [55]. The oetABI cassette is found in all 4 syntrophic SRB clades and
often includes 2 or 3 other putative extracellular cytochromes c, including homologs of OmcX
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 9 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
[33], OmcS (Supplementary alignment MSA1) and a 6-heme cytochrome that we termed
apc2a (S5 Table). If they are not found as part of the oet cluster, they could be found elsewhere
on the genome, possibly due to genomic rearrangement after acquisition of the cassette (S6
and S7 Tables). The omcX and omcS-like genes in the oet gene cassette are often found in an
analogous position to omcS and omcT in G. sulfurreducens (Fig 2). Based on the homology of
one of the cytochromes to OmcS, which polymerizes to form long and highly conductive fila-
ments that facilitate EET in Geobacter [56], we propose that the extracellular cytochromes c in
this gene cassette perform a similar function, forming filaments that accept electrons from
ANME. This is consistent with heme staining of the intercellular space between ANME and
SRB, and the observation of filaments that connect the partners [10,24]. This is also consistent
with the fact that different extracellular cytochromes are among the most highly expressed pro-
teins in the syntrophic SRB: ANME-1/Seep-SRB2 [24] (OmcX, OmcS-like, and Apc2a),
ANME-1/HotSeep-1 [24] (OmcX and OmcS-like), ANME-2c/Seep-SRB2 [24] (OmcX) aggre-
gates and ANME-2a/Seep-SRB1a [25] (OmcX, OmcS-like). The presence of multiple copies of
these putative filament-forming proteins in the syntrophic SRB genomes is indicative of their
importance to the physiology of syntrophic SRB. The mechanism of electron transfer from
extracellular cytochrome filaments to the interior of the cells in Geobacter is not well under-
stood. However, a porin:cytochrome c conduit is always expressed under the same conditions
as a cytochrome c containing filament in Geobacter (omcS along with extEFG or omcABC
under Fe (III) oxide reducing conditions and omcZ along with extABCD during growth on an
electrode [57]) and in ANME-SRB consortia (OmcS/OmcX with OetABI or OmcKL). These
findings suggest that each cytochrome c filament could act in concert with a porin:cytochrome
c conduit (Fig 2) to transfer electrons from the extracellular space to the periplasm.
While oetABI is conserved in all 4 syntrophic SRB clades, there are 2 other putative porin:
cytochrome c conduits in syntrophic SRB. A porin (HS1_RS02765) and extracellular cyto-
chrome c (HS1_RS02760) homologous to OmcL and OmcK from G. sulfurreducens is found
in HotSeep-1 (S6 and S7 Tables) and expressed at a 4-fold higher level than the oetABI conduit
[24]. OmcK and OmcL were also up-regulated in G. sulfurreducens when it is grown on hema-
tite and magnetite [58]. There is no gene encoding a periplasmic cytochrome c adjacent to
these genes and this is unusual for previously characterized EET conduits but, given the large
number of periplasmic cytochromes in HotSeep-1, it is conceivable that another cytochrome c
interacts with the OmcL/K homologs. This conduit is also found in Seep-SRB2 sp. 1, 2, 7, and
8 but does not appear to be expressed as highly as the OetABI in the ANME-1/Seep-SRB2 con-
sortia [24]. A different putative conduit including the porin, extracellular, and periplasmic
cytochromes c is present in the Seep-SRB1g genomes (LWX52_07950- LWX52_07960) (S6
and S7 Tables). This conduit does not have identifiable homologs in Geobacter. The presence
of multiple porin:cytochrome c conduits in the syntrophic partners suggests some flexibility in
use of electron donors, possibly from different syntrophic partners. For HotSeep-1, this obser-
vation is consistent with its ability to form partnerships with both methane and other alkane-
oxidizing archaea [28]. The role of the second conduit is less clear in Seep-SRB1g which to
date has only been shown to partner with ANME-2b [13]. Future investigation of the multiple
syntrophic SRB EET pathways and the potential respiratory flexibility it affords to their partner
archaea using transcriptomics, proteomics and possibly heterologous expression methods will
further expand our understanding of electron transfer in these diverse consortia.
While DIET is believed to be the dominant mechanism of syntrophic coupling between the
ANME and SRB partners, the potential to use diffusible intermediates such as formate and
hydrogen exists in some genomes of syntrophic SRB. Hydrogenases are present in HotSeep-1,
which can grow without ANME using hydrogen as an electron donor [32]. We also identified
periplasmic hydrogenases in Seep-SRB1a sp. 1, 5, and 8 (S7 Table) that suggest that these
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 10 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
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.
https://doi.org/10.1371/journal.pbio.3002292.g003
organisms could use hydrogen as an electron donor. However, in Seep-SRB1a, these hydroge-
nases are expressed at low levels (less than a 20th of the levels of DsrB) in the ANME-2a/Seep-
SRB1a consortia [25]. Further, previous experiments showed that the addition of hydrogen to
ANME-2/SRB consortia did not inhibit anaerobic oxidation of methane suggesting that hydro-
gen is not the predominant agent of electron transfer between ANME and SRB [35,59]. Per-
haps, hydrogenases are used by Seep-SRB1a to scavenge small amounts of hydrogen from the
environment. While membrane bound and periplasmic hydrogenases are present in non-syn-
trophic Seep-SRB1c (S7 Table), no hydrogenases are found in the syntrophic relative of Seep-
SRB1c and ANME partner, Seep-SRB1g. Similarly, periplasmic hydrogenases are present in
Dissulfuribacteriales and absent in Seep-SRB2 (one exception in 18 genomes), suggesting that
in both these partners, the loss of periplasmic hydrogenases is part of the adaptation to their
syntrophic partnership with ANME. We also identified periplasmic formate dehydrogenases
in Seep-SRB1g and Seep-SRB1a sp. 2, 3, 8, 9 (S7 Table). The periplasmic formate dehydroge-
nase from Seep-SRB1g is expressed in the environmental proteome at Santa Monica Mounds
[33], but no transcripts from the formate dehydrogenases of Seep-SRB1a were recovered in the
ANME-2a/Seep-SRB1a incubations [25]. It is possible that these syntrophic SRB scavenge for-
mate from the environment. Alternatively, a recent paper found a hybrid of electron transfer
by DIET and by diffusible intermediates (mediated interspecies electron transfer or MIET) to
be energetically favorable [60]. In this model, the bulk of electrons would still be transferred by
DIET, but up to 10% of electrons could be shared by MIET via formate [60], an intermediate
suggested in earlier studies [35,59]. This might be possible in ANME/SRB consortia with
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
HotSeep-1, some species of Seep-SRB1a and Seep-SRB1g, but not in ANME/Seep-SRB2 con-
sortia. The absence of periplasmic formate dehydrogenases and hydrogenases in Seep-SRB2 as
previously observed [24] is also true in our expanded dataset. If a diffusive intermediate should
play a role in mediating electron transfer between ANME-2c or ANME-1 and Seep-SRB2, it is
not likely to be formate or hydrogen.
1.2 Pathways for electron transfer across the inner membrane vary in different syn-
trophic SRB clades. Multiheme cytochromes c in SRB are known to mediate diverse modes
of electron transfer from different electron donors to a conserved sulfate reduction pathway
[58]. There is significant variety in the number and types of cytochromes c present in SRB
from the phylum Desulfobacterota [61] and an even greater number of large cytochromes is
present in syntrophic SRB [24,33]. To explore the potential for different routes of electron
transfer, we performed an analysis of all cytochromes c containing 4 hemes or more from the
genomes of syntrophic SRB (see Materials and methods) and identified at least 27 different
types of cytochromes c. We split these cytochromes c into those predicted to be involved in
EET, those that act as periplasmic electron carriers and those that are components of protein
complexes involved in electron transfer across the inner membrane (S6 Table). Conserved
across the syntrophic SRB partners of ANME were the cytochromes forming the core compo-
nents of the EET pathway—OetA, OetB, OmcX and OmcS-like and Apc2a extracellular cyto-
chromes, and 2 periplasmic cytochromes of the types, TpIc3 [62] and cytochrome c554 [61,63].
Beyond the conserved periplasmic cytochromes c, TpIc3 and cytochrome c554, there are also
cytochromes binding 7–8 hemes that are unique to different SRB clades (S6 Table). These
include a homolog of ExtKL [64] from G. sulfurreducens that is highly expressed in Seep-SRB2
spp. 1 and 4 during growth in a syntrophic partnership with ANME [24] and a homolog of
ExtA from G. sulfurreducens [54] protein expressed in the ANME-2a/Seep-SRB1a consortia
[25]. Previous research has suggested that the tetraheme cytochromes c are not selective as
electron carriers and play a role in transferring electrons to multiple different protein com-
plexes [65]. It is possible that these larger 7–8 heme binding cytochromes c have a more spe-
cific binding partner. Both the ExtKL and ExtA-like proteins are very similar (over 45%
sequence similarity) to their homologs in Desulfuromonadales. Since the OetI-type conduit is
also likely transferred from this order, they might act as binding partners to the OetI-type con-
duit transferring electrons to the periplasmic cytochromes c.
In SRB, the electrons from periplasmic electron donors (reduced by DIET or MIET) are
delivered through inner membrane bound complexes to quinones or directly to the heterodi-
sulfide DsrC in the cytoplasm via transmembrane electron transfer [61] (Fig 3). The electrons
from quinones or DsrC are ultimately used for the sulfate reduction pathway (including SatA,
AprAB, and DsrAB) [24,33,61]. Two conserved protein complexes are always found along
with this pathway—the Qmo complexes transfers electrons from reduced quinones to AprAB
and the DsrMKJOP complexes transfers electrons from quinones to DsrC and through DsrC
to DsrAB. Since both these complexes use electrons from reduced quinones, the source of
reduced quinones in the inner membrane is critical to different sulfate respiration pathways.
The quinol reducing complexes and complexes that reduce DsrC provide respiratory flexibility
to SRB. We also note here that the reduction of AprAB coupled to the oxidation of menaqui-
none is expected to be endergonic. There is a proposal that QmoABC might function through
flavin-based electron confurcation (FBEC), using electrons from reduced quinones and a sec-
ond electron donor such as ferredoxin to reduced AprAB [66]. Since, it is not clear what the
electron donor is likely to be, we do not explicitly consider this reaction in our analysis. A sum-
mary of all the putative complexes that are involved in the electron transport chains of the 4
syntrophic SRB is visualized in Fig 3 to detail how electron transport pathways vary among the
clades. A more detailed list of complexes present is found in S6–S8 Tables. The respiratory
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
pathways in HotSeep-1, Seep-SRB1a, and Seep-SRB1g are broadly similar in structure and are
predicted to use the Qrc complex to transfer periplasmic electrons to the quinone pool and
both DsrMKJOP and Tmc to reduce cytoplasmic DsrC. Their pathways are analogous to the
respiratory pathways in Desulfovibrio alaskensis [62,67]. Curiously, the Tmc complex in Seep-
SRB1a and Seep-SRB1g are divergent from Tmc in non-syntrophic SRB (S8 Fig) and TmcA is
absent in the operons encoding for Tmc. This absence suggests that Tmc has been adapted to
use a different electron donor than in Desulfovibrio vulgaris [67] and is consistent with the fact
that the electron donor for Seep-SRB1a and Seep-SRB1g is not hydrogen or formate but, elec-
trons from anaerobic methanotrophic archaea. It is not clear why Tmc is more divergent than
DsrMKJOP in syntrophic SRB compared to their evolutionary neighbors since they are both
likely important for DsrC reduction and are equally well expressed under methane-oxidizing
conditions [24,25]. Qrc is known to be important for energy conservation in this respiratory
pathway. Protons are translocated by Qrc from the cytoplasmic side to the periplasmic active
side. This movement of charges across the membrane leads to the generation of proton motive
force (pmf) that can be utilized by ATP synthase to generate ATP [68]. In Seep-SRB1a, Seep-
SRB1g, and HotSeep-1, Qrc likely acts with the conserved DsrMKJOP and QmoABC to gener-
ate pmf. While purified Dsr and Qmo have not yet been shown to be electrogenic, it is
expected that DsrMKJOP [69] and QmoABC [69,70] might be able to generate pmf by charge
translocation.
In Seep-SRB2, Qrc is absent and we hypothesize that CbcBA, a protein complex that
appears to be horizontally transferred from the Desulfuromonadales (Figs 3 and 4), mediates
electron transfer between periplasmic cytochromes c and quinones [71]. This is supported by
the fact that CbcBA is highly expressed during AOM between ANME-1/Seep-SRB2 and
ANME-2c/Seep-SRB2 [24]. In Geobacter sulfurreducens, which also does not have Qrc this
cytoplasmic membrane-bound oxidoreductase is expressed during growth on Fe(III) at low
potential and is important for iron reduction and growth on electrodes at redox potentials less
than −0.21 mV [71]. During AOM, the CbcBA protein in Seep-SRB2 is predicted to run in the
reverse direction, reducing quinols using electrons from DIET electrons supplied by ANME as
opposed to functioning in the metal reducing direction. While the reversibility of this complex
has not been biochemically established, the high levels of expression of this complex suggest
that this is likely functional in the electron transport chain of Seep-SRB2. It is not clear what
the likely site of energetic coupling is within the Seep-SRB2 respiratory chain. In the absence
of the Qrc complex, the most likely mechanism for energetic coupling might exist through the
action of a Q-loop mechanism [69]. In this mechanism, energy is conserved by the combined
action of 2 protein complexes that reduce and oxidize quinols, leading to the uptake and
release of protons on opposite sides of the cytoplasmic membrane. The Q-loop mechanism in
Seep-SRB2 would likely involve CbcBA and a quinol oxidizing complex such as Qmo.
In addition to the most likely pathways of electron transfer in the syntrophic SRB, as estab-
lished using transcriptomic data on ANME/SRB partnership [24,25] (Figs 2 and 3), other
inner membrane complexes exist in these genomes that may provide additional respiratory
flexibility. HotSeep-1 genomes contain a complex that involves an HdrA subunit and a protein
that also binds hemes c and contains a CCG domain similar to that found in HdrB and TmcB
predicted to interact with DsrC [70,74]. This complex a putative cytochrome c oxidoreductase
containing a CCG domain, would likely transfer electrons from cytochromes c to the DsrC
(AMM42179.1-AMM42180.1) or perhaps to a ferredoxin. The presence of HdrA might indi-
cate a role in electron bifurcation by this complex. It is highly expressed during methane oxi-
dation conditions in the ANME-1/HotSeep-1 consortia to a fifth of the level of the Tmc
complex that would play a similar role in electron transfer [24]. In some Seep-SRB1a and
Seep-SRB1g genomes, there is a homolog of Cbc6 (LWX51_14670- LWX51_14685) identified
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
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.
https://doi.org/10.1371/journal.pbio.3002292.g004
in Geobacter [75] and implicated in electron transfer from periplasmic cytochromes c to the
quinol pool. A NapC/NirT homolog [76] was conserved in Seep-SRB1g
(OEU53943.1-OEU53944.1) and some Seep-SRB2, and another conserved complex that
includes a cytochrome c and ruberythrin (AMM39991.1-AMM39993.1) is present in Seep-
SRB1g and HotSeep-1. Further research is needed to test whether there are conditions under
which these complexes are expressed. Our analysis indicates some degree of respiratory spe-
cialization in the syntrophic SRB genomes such as the loss of hydrogenases in Seep-SRB1g and
Seep-SRB2 compared to their nearest evolutionary neighbors, suggesting an adaptation
towards a partnership with ANME. However, considerable respiratory flexibility still exists
within the genomes of these syntrophic partners as is suggested by the presence of the formate
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
dehydrogenases in Seep-SRB1g and Seep-SRB1a, multiple EET conduits in HotSeep-1 and
Seep-SRB2 and multiple inner membrane complexes in Seep-SRB1a and HotSeep-1.
1.3 Cytoplasmic redox reactions, electron bifurcation, and carbon fixation pathways.
The electron transport chain outlined above would transfer electrons from periplasmic cyto-
chromes c to the cytoplasmic electron carrier DsrC or directly to the sulfate reduction pathway.
However, the electron donors for carbon or nitrogen fixation are typically NADH, NADPH,
or ferredoxin [77]. The reduction of NADH, NADPH, or ferredoxin could happen through
the transfer of electrons from DsrC [78] or through the interconversion of electrons between
cytoplasmic electron carriers through the dissipation of pmf or through the action of electron
bifurcating complexes [79]. The transfer of electrons from DsrC to these reductants likely hap-
pens through the action of protein complexes like Flx-Hdr that can oxidize 2 molecules of
NADH to reduce 1 molecule of ferredoxin and 1 molecule of DsrC. Electrons from NADH
and ferredoxin can also be exchanged with the dissipation or generation of sodium motive
force using membrane-bound Rnf and Mrp [79–81] in Seep-SRB1a, Seep-SRB1g, and Hot-
Seep1. In marine environments, the naturally occurring sodium gradient can be used to gener-
ate ferredoxin from NADH or vice versa using the Rnf complex, while the Na+/H+ antiporter,
Mrp, can transport Na+ or H+ in response to the action of Rnf [82]. The ferredoxin generated
from this process can then be used for assimilatory pathways. In Seep-SRB2, which does not
contain Rnf or Mrp (S9 Fig), the NADH needed for carbon fixation is likely obtained through
the oxidation of quinol by complex I and the dissipation of pmf [83]. In addition to Complex I
or Rnf and Mrp, there are additional cytoplasmic protein complexes that can recycle reducing
equivalents between DsrC, ferredoxin, and NADH such as the electron bifurcating Flx-Hdr
[70,78]. Several putative oxidoreductase complexes in the syntrophic SRB genomes are com-
piled in S7 Table and S10 and S11 Figs.
Syntrophic sulfate-reducing members of the Seep-SRB1a, Seep-SRB1g, and Seep-SRB2 have
been shown to fix carbon using the Wood–Ljungdahl pathway, while organisms of the clade
HotSeep-1 partnering with ANME-1 are predicted to fix carbon using the reductive tricarbox-
ylic acid cycle (rTCA) [24,33,77]. Analysis of gene synteny for a number of Seep-SRB1a, Seep-
SRB1g, and Seep-SRB2 MAGs uncovered a number of heterodisulfide (HdrA) subunits and
HdrABC adjacent to enzymes involved in the Wood–Ljungdahl pathway (S10 Fig). These sub-
units are typically implicated in flavin-based electron bifurcating reactions utilizing ferredox-
ins or heterodisulfides and NADH [79]. Specifically, Seep-SRB1g has an HdrABC adjacent to
metF that is predicted to encode for a putative metF-HdrABC, performing the reduction of
methylene tetrahydrofolate coupled to the endergonic reduction of ferredoxin to NADH, the
same reaction as the bifurcating metFV-HdrABC described below. In Seep-SRB1g, there are
also 2 copies of HdrABC next to each other whose function requires further analysis (S10 Fig).
These complexes are absent in the related group Seep-SRB1c, a lineage which has not yet been
found in physical association with ANME (S10 Fig). The presence of electron bifurcation
machinery in the carbon fixation pathways within several syntrophic SRB lineages, suggests
that they are optimized to conserve energy (S10 Fig). This is reminiscent of the MetFV-H-
drABC in the acetogen Moorella thermoacetica [79] in which the NADH-dependent methylene
tetrahydrofolate reduction within the central metabolic pathway is coupled to the endergonic
reduction of ferredoxin by NADH, allowing for the recycling of reducing equivalents. Mem-
bers of the Seep-SRB1g also have a formate dehydrogenase (fdhF2) subunit adjacent to nfnB,
the bifurcating subunit of NfnAB, which performs the NADPH-dependent reduction of ferre-
doxin (S11 Fig). This complex is predicted to function as an additional bifurcating enzyme
that would allow for the recycling of NADPH electrons. In addition, HotSeep-1, Seep-SRB2,
and Seep-SRB1g appear to have homologs of electron transfer flavoproteins, etfAB, that are
expected to be electron bifurcating. These homologs of etfAB cluster with the previously
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
identified bifurcating etfAB and possess the same sequence motif that was previously shown to
correlate with the electron bifurcating etfAB [84] (S7 Table). While the capability of electron
bifurcation by these enzyme complexes needs to be biochemically confirmed, the possibility of
a high number of bifurcating complexes, especially those connected to the carbon fixation
pathway, in the genomes of syntrophic SRB partners of ANME is compelling. It could be
argued that this is a natural adaptation to growth in very low-energy environments or to low-
energy metabolism. In fact, some of these complexes are present in non-syntrophic bacteria of
the order Desulfofervidales and genus Eth-SRB1. These adaptations could provide an addi-
tional energetic benefit for the syntrophic lifestyle, itself an adaptation to low-energy
environments.
2. Cobalamin auxotrophy and nutrient sharing in syntrophic SRB
Research on the AOM symbiosis has focused heavily on the nature of the syntrophic interme-
diates shared between ANME and SRB [9,10,12,85,86]. We currently have an incomplete
understanding of the scope of other potential metabolic interdependencies within this long-
standing symbiosis. Prior experimental research has demonstrated the potential for nitrogen
fixation and exchange in AOM consortia under certain environmental circumstances
[11,13,87,88], and in other energy limited anaerobic syntrophies between bacteria and archaea,
amino acid auxotrophies are common [89–91]. Comparative analysis of MAGs from several
lineages of ANME [14] as well as a subset of syntrophic sulfate-reducing bacterial partners [33]
lacked evidence for specific loss of pathways used in amino acid synthesis, and our expanded
analysis of SRB here is consistent with these earlier studies. Interestingly, comparative analysis
of specific pairings of ANME and their SRB partners revealed the possibility for cobalamin
dependency and exchange. Cobamides, also known as the Vitamin B12-type family of cofac-
tors, are critical for many central metabolic pathways [92]. Mechanisms for complete or partial
cobamide uptake and remodeling by microorganisms found in diverse environments are com-
mon [92]. The importance of exchange of cobamide between gut bacteria and between bacteria
and eukaryotes has been demonstrated [93,94]. In methanotrophic ANME-SRB partnerships,
ANME are dependent on cobalamin as a cofactor in their central metabolic pathway and bio-
synthetic pathways, while Seep-SRB2, Seep-SRB1a, Seep-SRB1g also have essential cobalamin-
dependent enzymes including ribonucleotide reductase, methionine synthase, and acetyl-CoA
synthase (S8 Table). This is in contrast with the HotSeep-1 clade, which appears to have fewer
cobalamin requiring enzymes and may not have an obligate dependence on vitamin B12.
However, HotSeep-1 do possess homologs of BtuBCDF and CobT/CobU, genes that are used
in cobamide salvage and remodeling [95] (S8 Table). An absence of cobalamin biosynthesis in
either ANME or these 3 clades of syntrophic SRB would thus necessarily lead to a metabolic
dependence on either the partner or external sources of cobalamin in the environment. We
observed such a predicted metabolic dependence for Seep-SRB1a within the species Seep-
SRB1a sp. 1 (n = 1 genomes), Seep-SRB1a sp. 5 (n = 4 genomes), Seep-SRB1a sp. 3 (n = 2
genomes), Seep-SRB1a sp. 7 (n = 1), and Seep-SRB1a sp. 8 (n = 1). All these genomes are miss-
ing the anaerobic corrin ring biosynthesis pathway but, some do retain genes involved in
lower ligand synthesis (BzaAB) [96] (Fig 5). Additionally, recent metatranscriptomic data
from an AOM incubation dominated by ANME-2a/Seep-SRB1a associated with Seep-SRB1a
sp. 5 (str. SM7059A) that is missing the cobalamin biosynthesis pathways confirmed active
expression of cobalamin-dependent pathways in the Seep-SRB1a including ribonucleotide
reductase and acetyl-coA synthase AcsD [25], suggesting that these syntrophs must acquire
cobalamin from their ANME partner or the environment.
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
Interestingly, the predicted cobalamin auxotrophy is not a uniform trait within the Seep
SRB1a lineage, with cobamide biosynthesis genes present in the genomes of species Seep-
SRB1a sp. 2 (n = 3), Seep-SRB1a sp. 4 (n = 1), and Seep-SRB1a sp. 9 (n = 3) (Fig 5). Of the 5
species missing cobalamin biosynthesis pathways, 2 are verified ANME-2a partners. Of the 4
species containing cobalamin biosynthesis pathways, one is a verified ANME-2c partner and
one was sequenced from a microbial mat that contains ANME-2c and ANME-2a (Fig 5).
These patterns suggest that the Seep-SRB1a partners of ANME-2a developed a nutritional aux-
otrophy that is specific to this partnership. Future experimental work will assist with testing
this predicted vitamin dependency among the ANME-2a and Seep-SRB1a and other
ANME-SRB partner pairings.
The ability to fix nitrogen is found in bacteria and archaea but is relatively rare among them
[97]. Fixed nitrogen availability can impact the productivity of a given ecosystem. Members of
the ANME-2 archaea have been demonstrated to fix nitrogen in consortia [11,12,88] and may
serve as a source of fixed nitrogen for methane-based communities in deep-sea seeps [88]. We
recently demonstrated that within the ANME-2b/Seep-SRB1g partnership, Seep-SRB1g bacte-
ria can also fix nitrogen [13]. A comparison of the nitrogen fixation ability across ANME and
SRB (Fig 5) shows that this function is present in the genome representatives of diverse ANME
and also conserved in some syntrophic bacterial partners (Seep-SRB1a and Seep-SRB1g). In
the Seep-SRB1a lineage, the nitrogenase operon is retained in both ANME-2a and ANME-2c
partners, contrasting the pattern observed with cobalamin synthesis. Interesting, the potential
to fix nitrogen occurs in species of Seep-SRB2 that come from psychrophilic deep-sea environ-
ments (Seep-SRB2 sp. 4 and Seep-SRB2 sp. 3), while earlier branching clades of Seep-SRB2
adapted to hotter environments (Seep-SRB2 sp. 1 and 2) lack nitrogenases, hinting at potential
ecophysiological adaptation to temperature (Fig 5). While the ability to fix nitrogen is retained
in several clades of syntrophic SRB, previous stable isotope labeling experiments have shown
that ANME is the dominant nitrogen fixing partner [11,13,88]. Yet, the potential to fix nitro-
gen is retained in Seep-SRB1a and Seep-SRB1g members (Fig 5), and in some cases, have been
directly linked to N2 fixation in the case of Seep SRB1g [13] or indirectly suggested from the
recovery of nifH transcripts belonging to Seep-SRB1a and Seep-SRB1g in seep sediments [12].
These observations indicate that nitrogen sharing dynamics between ANME and SRB is likely
more complicated than we have thus far observed and may correspond to differences in envi-
ronment, or perhaps to specific partnership interactions that require assessment at greater tax-
onomic resolution.
3. Pathways related to biofilm formation and intercellular communication
ANME and SRB form multicellular aggregates in which they are spatially organized in distinct
and recognizable ways [98]. ANME-2a/2b/2c and ANME-3 are known to form tight aggregates
with their bacterial partners [10,25,35,98]. Some members of ANME-1 have been observed in
tightly packed consortia with SRB [24], while others some form more loose associations
[19,26,99,100]. In these consortia, archaeal and bacterial cells are often enmeshed in an extra-
cellular polymeric substance [19,20,101]. In large carbonate associated mats of ANME-2c and
ANME-1 and SRB from the Black Sea, extractions of exopolymers consisted of 10% neutral
sugars, 27% protein, and 2.3% uronic acids [20]. This composition is consistent with the roles
played by mixed protein and extracellular polysaccharide networks shown to be important for
the formation of conductive biofilms in Geobacter sulfurreducens [102], the formation of mul-
ticellular fruiting bodies from Myxococcus xanthus [103–105], and the formation of single-spe-
cies [106] and polymicrobial biofilms [107]. Important and conserved features across these
biofilms are structural components made up of polysaccharides, cellular extensions such as
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
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.
https://doi.org/10.1371/journal.pbio.3002292.g005
type IV pili and matrix-binding proteins such as fibronectin-containing domains [108]. Func-
tional components of the biofilm matrix such as virulence factors in pathogens [109] and EET
components [102] are variable and depend on the lifestyle of the microorganism. Guided by
the molecular understanding of mechanisms and physiological adaptation to microbial growth
in biofilms, we examined the genomic evidence for similar adaptations in the syntrophic SRB
in consortia with ANME, focusing on structural and functional components of biofilms as well
as proteins implicated in partner identification (Fig 6).
3.1 Multiple polysaccharide biosynthesis pathways are found in syntrophic SRB. Our
analysis of syntrophic SRB genomes showed the presence of multiple putative polysaccharide
biosynthesis pathways in different SRB lineages including secreted extracellular polysaccharide
biosynthesis pathways and capsular polysaccharide biosynthesis pathways (S9 Table). In par-
ticular, homologs of the pel biosynthesis pathway (PelA, PelE, PelF, and PelG), first identified
in Pseudomonas aeruginosa [110,111] were present in almost all Seep-SRB1g and Seep-SRB1a
genomes (S12 and S13 Figs). These homologs are part of a conserved operon in these genomes
which includes a transmembrane protein that could perform the same function as PelD, which
along with PelE, PelF, and PelG forms the synthase component of the biosynthetic pathway
and enables transport of the polysaccharide pel across the inner membrane [111]. Metatran-
scriptomic data confirms this operon is expressed and was significantly down-regulated when
methane-oxidation by ANME-2a was decoupled from its syntrophic Seep SRB1a partner with
the addition of AQDS [25]. This biosynthesis pathway is absent in the nearest evolutionary
neighbors of Seep-SRB1a and Seep-SRB1g, Eth-SRB1 and Seep-SRB1c, respectively, suggesting
that the presence of the pel operon could serve as a better genomic marker for syntrophic
interaction with ANME-2a, ANME-2b, and ANME-2c than the presence of the oetI-type con-
duit. The pel operon was also detected in one of the Seep-SRB2 genomes but is not conserved
across this clade. In Seep-SRB2 clades, multiple capsular polysaccharide biosynthesis pathways
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
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.
https://doi.org/10.1371/journal.pbio.3002292.g006
are conserved. This includes a neuraminic acid biosynthesis pathway, a sialic acid capsular
polysaccharide widely associated with intestinal mucous glycans and used by pathogenic and
commensal bacteria to evade the host immune system [112] (S14 Fig). These differences in
polysaccharide biosynthesis pathways are likely reflected in the nature of the EPS matrix within
each ANME-SRB aggregate.
Members of the thermophilic HotSeep-1 syntrophic SRB also encode for multiple putative
polysaccharide biosynthesis pathways, including a pathway similar to the xap pathway in G.
sulfurreducens (S15 Fig). The role of polysaccharides in the formation of conductive extracellu-
lar matrices and in intercellular communication is just beginning to be understood but they
appear to be essential to its formation. For example, the mutation of the xap polysaccharide
biosynthesis pathway in G. sulfurreducens eliminated the ability of this electrogenic bacteria to
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
reduce Fe (III) in the bacterium [102] and affected the localization of key multiheme cyto-
chromes c OmcS and OmcZ and structure of the biofilm matrix [113], suggesting that the EPS
matrix contributes a structural scaffold for the localization of the multiheme cytochromes.
Similarly, the cationic polysaccharide pel in P. aeruginosa biofilms has recently been shown to
play a role in binding extracellular DNA or other anionic substrates together forming tight
electrostatic networks that provide strength to the extracellular matrix [114] and may offer a
similar role in Seep SRB1a and 1g consortia. Based on the reported chemical composition of
EPS from the Black Sea ANME-SRB biofilm [20], alongside TEM compatible staining of cyto-
chromes c in the extracellular space between ANME and SRB [9,10,24], and the genomic evi-
dence provided here of conserved polysaccharide biosynthesis pathways point to the existence
of a conductive extracellular matrix within ANME-SRB consortia that has features similar to
Geobacter biofilms [102]. While these conductive biofilms are correlated with the presence of
secreted polysaccharides, the highly conserved capsular polysaccharides common in Seep
SRB2 likely play a different role. In Myxococcus xanthus, the deletion of capsular polysaccha-
rides leads to a disruption in the formation of multicellular fruiting bodies, suggesting a possi-
ble role for capsular polysaccharides in intercellular communication [115]. This is consistent
with the universal role of O-antigen ligated lipopolysaccharides in cell recognition and the
Seep SRB2 capsular polysaccharides may serve a similar purpose in consortia with ANME,
either influencing within population interactions, or potentially mediating kin recognition.
3.2 Several putative adhesins found in syntrophic SRB are absent in free-living SRB. In
addition to polysaccharides, there are several conserved adhesion-related proteins present in
syntrophic SRB and absent in closely related SRB that are likely important for ANME-SRB bio-
film formation. These include cohesin and dockerin domain-containing proteins, similar to
those previously identified in ANME [14], immunoglobulin-like domains, cell-adhesin related
domain (CARDB) domains, bacterial S8 protease domains, PEB3 adhesin domains, cadherin,
integrin domains, and fibronectin domains (Fig 6 and S10 Table). Fibronectin domains are
found in the one of the cytochromes c, oetF that is likely part of the EET conduit. This domain
might interact with the conductive biofilm matrix itself or serve as a partnership recognition
site. PilY1 is another adhesion-related protein that appears to be important in HotSeep-1. This
is a subunit of type IV pili that is known to promote surface adhesion in Pseudomonas and
intercellular communication in multispecies Pseudomonas biofilms [116]. Our analysis of the
SRB adhesins suggests that some adhesins are conserved across a given syntrophic clade, while
others appear to be more species or partnership specific. For example, while PilY1 is conserved
across Seep-SRB2, the cohesin/dockerin complexes that are conserved in Hot-Seep1 and Seep-
SRB1g are thus far found only in Seep-SRB2 sp. 4 and 8. Analysis of gene expression data sug-
gest that in the Hot-Seep1/ANME-1 partnership, PilY1, an adhesin with an immunoglobulin-
like domain and adjacent cohesin/dockerin domains might play a role in the syntrophic life-
style [24] (S10 Table). In the ANME-2c/Seep-SRB2 partnership, PilY1, cohesin/dockerin com-
plexes and a protein with a CARDB domain are highly expressed [24] (S10 Table). Curiously,
in the Seep-SRB2 partnering with ANME-1, we could only identify 1 moderately expressed
adhesin with a fibronectin domain [24] (S10 Table). We note the presence and high levels of
expression of cohesin/dockerin domains in both ANME-2c and their verified Seep-SRB2 part-
ner [24], and the presence of fibronectin domains in both ANME-2a and their Seep-SRB1a
partner (S10 Table) suggesting that perhaps both partners within a partnership express and
secrete similar kinds of extracellular proteins. This might serve as a mechanism for partnership
sensing. While our analysis and that of earlier research into adhesins present in ANME [14]
identify a number of conserved and expressed adhesins, further work is needed to investigate
their potential role in aggregate formation.
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
3.3 Secretion systems and intercellular communication in syntrophic SRB. Extracellu-
lar contractile injection systems (eCISs) that resemble phage-like translocation systems
(PLTSs) are found in some syntrophic SRB genomes (S11 Table and S16 Fig) although they are
not as widely distributed as in ANME [14]. Typically, the eCIS bind to a target microorganism
and release effector proteins into its cytoplasm. eCIS have been shown to induce death in
worm larvae, induce maturation in marine tubeworm larvae [117] and found to mediate inter-
actions between the amoeba symbiont and its host [118]. In ANME-SRB consortia, they might
play a similar role with ANME releasing an effector protein into SRB, perhaps an effector mol-
ecule to promote the formation of a conductive biofilm or adhesins. Type VI secretion systems
(T6SS) are similar to eCIS in facilitating intercellular communication between microorgan-
isms. However, the primary distinction between them is that T6SS are membrane-bound
while eCIS appear to be secreted to the extracellular space [119,120]. Interestingly, T6SS appear
to be present in the ANME-2a partner Seep-SRB1a but absent in the ANME-2c partner Seep-
SRB1a suggesting that they might play a role in mediating partnership specificity. While secre-
tion systems are not uncommon in non-syntrophic bacteria, the high degree of their conserva-
tion in ANME and the high levels of expression of secretion systems in the ANME-2/Seep-
SRB1a [25] and ANME-2c/Seep-SRB2 [24] partnerships suggest an important role for them in
ANME-SRB syntrophy. Our analysis identified many conserved mechanisms for biofilm for-
mation and intercellular communication in SRB to complement the pathways previously iden-
tified in ANME. Significantly, several polysaccharide biosynthesis pathways and adhesins were
absent in the closest evolutionary neighbors of SRB indicating that adaptation to a syntrophic
partnership with ANME required not just metabolic specialization but adaptation to a multi-
cellular and syntrophic lifestyle.
The adaptation of syntrophic SRB to partnerships with ANME
To better understand the evolutionary adaptations acquired by syntrophic SRB to form part-
nerships with ANME, we mapped the presence and absence of the above-mentioned pathways
in central metabolism, nutrient sharing, biofilm formation, cell adhesion, and partner identifi-
cation across each of the syntrophic SRB clades and their nearest evolutionary neighbors from
the same bacterial order (S12 and S13 Tables). For example, the presence of the EET conduit
OetABI in the Seep-SRB1a clade is nearly universal but, this trait is absent in the Desulfobac-
terales order that Seep-SRB1a belongs to, suggesting strongly that this machinery was horizon-
tally acquired possibly in Seep-SRB1a or a closely related ancestor within the same family that
includes Eth-SRB1. In contrast, most genomes in the order that Seep-SRB1g belongs to contain
hydrogenases. However, hydrogenases are lacking in the syntrophic clade Seep-SRB1g imply-
ing that this trait was lost in the process of specialization to a partnership with ANME-2b. In
addition to inferring adaptation based on presence and absence, phylogenetic trees were gen-
erated for at least 1 representative gene from each identified characteristic to corroborate the
possibility of horizontal gene transfers (trees are available in S1 Data, Github (https://github.
com/ranjani-m/syntrophic-SRB)). These trees provide further insight into the adaptation of
various traits, the likely source of the genes received horizontally and in the case of Hot-Seep1
and Seep-SRB2 sp. 1 demonstrate the transfer of OetABI from one syntrophic clade to another.
With the trees, we were able to also identify those genes that were vertically acquired but
adapted for the respiratory pathways receiving DIET electrons, for example Tmc (S8 Fig). A
brief summary of the gene gains and losses is provided in Fig 7 and S13 Table. Our analysis
suggests that some traits are associated with partnerships with different ANME. The pel
operon present in Seep-SRB1g and Seep-SRB1a is more closely associated with aggregates
formed with the ANME-2a/b/c species rather than ANME-1. Similarly, the capsular
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
polysaccharide pseudaminic acid is present in those species of Seep-SRB1a that are associated
with ANME-2c but absent in those species partnering ANME-2a suggesting that this polysac-
charide might play a role in partnership identification and aggregate formation. Curiously,
many of the adhesins we identified in the syntrophic SRB genomes have few close homologs in
the NCBI NR dataset and almost no homologs in the nearest evolutionary neighbors (S13
Table), indicating that these proteins are likely highly divergent from their nearest ancestors.
This is consistent with faster adaptive rates observed in extracellular proteins [121].
With our analysis, we identified many genes and traits that are correlated with a syntrophic
partnership with ANME, but it is less easy to identify whether they are essential. The complete
conservation of the OetI-type or other EET cluster (such as OmcKL) suggests these are essen-
tial, but not sufficient, for the formation of this partnership since the multiheme cytochrome
conduits themselves are present in many organisms not forming a syntrophic partnership with
ANME. There is also a strong signature for the presence of a secreted polysaccharide pathway
such as the pel operon in Seep-SRB1a and Seep-SRB1g and a xap-like polysaccharide in Hot-
Seep1 and Seep-SRB2. With these components, a conductive biofilm matrix can be established,
but the means of partnership recognition and communication between the archaea and bacte-
ria are less clear. As suggested previously [14], the near complete conservation of the eCISs in
ANME might play a role in partnership identification. The target receptor of the eCIS is
unclear but the presence of conserved capsular polysaccharides in SRB that often are the target
of bacteriophages and pathogens is suggestive as a possible site for binding. Likewise, the high
levels of expression of cohesin and dockerin complexes by both ANME and SRB in the
ANME-2c/Seep-SRB2 partnership are indicative of a role in syntrophic partnership [24]. In
Seep-SRB1a, there are conserved fibronectin domains that likely bind the biofilm matrix and
Seep-SRB2 has a conserved cell-surface protein with a PEGA sequence motif (S12 Table).
We can infer something about the order of evolutionary adaption of syntrophic SRB from
what is essential and conserved in syntrophic SRB and what is present in their nearest evolu-
tionary ancestors. The presence of DIET complexes such as OetABI in the nearest evolutionary
neighbors of HotSeep-1 (Desulfofervidales), Seep-SRB2 (Dissulfuribacteriales), and Seep-
SRB1g (Seep-SRB1c) and the absence of adhesins (cohesins) and polysaccharide biosynthesis
(pel) in the related clades (Fig 7) suggests that the acquisition of DIET pathways in an ancestral
clade was the first and essential step towards adaptation towards a syntrophic lifestyle. Then,
the syntrophic partners likely acquired the pathways needed for aggregate formation (such as
adhesins, the pel polysaccharide biosynthesis pathway) after. Seep-SRB2 contains a respiratory
trait (CbcBA) that is absent in its nearest evolutionary neighbor (Fig 4). This indicates that
more steps were required for the adaptation of this clade to a syntrophic partnership with
ANME. The greater diversity within the clades Seep-SRB1a and Seep-SRB2 may be a result of
the larger number of partnerships with different ANME compared to a clade such as Seep-
SRB1g. However, there is insufficient evidence to rule out the possibility of promiscuous part-
nership formation with multiple ANME within each SRB species. In these cases, the observed
species diversity within Seep-SRB1a and Seep-SRB2 must be driven by other factors. Our anal-
ysis shows that the adaptation towards EET and the formation of conductive biofilms was
likely driven by a greater selection pressure than the adaptation to a specific ANME partner.
Consistent with this, the gain and loss of specific adhesin and matrix-binding proteins is more
dynamic.
Another aspect of the adaptation of syntrophic SRB is the high number of inter-clade trans-
fers. In addition to the likely transfer of OetABI between HotSeep-1 and Seep-SRB2 sp.1 (S7
Fig), we also note a high degree of similarity between the proteins of the following components
in different clades of syntrophic SRB—cohesin/dockerin modules, the OmcKL conduit, and
enzymes in the pel and xap polysaccharide biosynthesis pathways. These appear to be the result
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
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.
https://doi.org/10.1371/journal.pbio.3002292.g007
of inter-clade transfers and the high number of transfers might imply that a mechanism pro-
moting the exchange of DNA exists in this environment between ANME and SRB, either
through a viral conduit or perhaps with the eCIS carrying DNA as cargo. Further analysis is
needed to identify the number of transfer and the sources of transfers. In fact, a thorough
accounting of these horizontal gene transfers combined with molecular clock dating might
provide insight into the timeline and the relative age of the different ANME/SRB partnerships.
Our phylogenomic analysis places the verified ANME-2c partners as ancestral to the ANME-
2a partners within the Seep-SRB1a clade (Figs 1 and S1). Within the Seep-SRB2 clade, the
topology places an ANME-1 partner as basal to the remaining Seep-SRB2 and the only verified
ANME-2c partner as one of the later branching members (Figs 1 and S1). Earlier research
places ANME-1 as the deepest branching lineage of ANME [14] and this relative ancestry of
partners might suggest that Seep-SRB2 is older than Seep-SRB1a. However, it appears that
ANME-1 acquired its mcr through horizontal gene transfer [14], and we have insufficient data
to know when this occurred. Thus, we cannot know that ANME-1 was methanotrophic when
it diverged from the Methanomicrobiales. These observations suggest that we cannot constrain
the emergence of AOM solely through the relative branching patterns of the various ANME
and SRB clades. A more thorough reconstruction of the adaptive gene transfers using the
framework established for ANME and in this work for syntrophic SRB would provide insight
into the evolution of this biogeochemically important syntrophic partnership.
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
Conclusions
This comparative genomic analysis of the major ANME-partnering SRB clades provides a
valuable metabolic and evolutionary framework to understand the differences between the var-
ious syntrophic sulfate reducing partners of anaerobic methanotrophic archaea and develop
insight into their metabolic adaptation. In this work, we show that the electron transport
chains of the different syntrophic SRB partners of ANME are adapted to incorporate EET con-
duits that are needed for DIET. Groups including the Seep-SRB2 appear to have acquired cyto-
plasmic membrane complexes that can function with the EET conduits, while Seep-SRB1a
clades have adapted existing inner-membrane complexes for interaction with the EET conduit.
Electron bifurcation also appears to be common across the syntrophic lineages and is often
coupled to the cytoplasmic machinery and likely provides an advantage in low-energy envi-
ronments. We also show that the coevolution between different ANME and SRB partners may
have resulted in nutritional interdependencies, with cobalamin auxotrophy observed in at least
one of the specific syntrophic SRB subclades. Our genome-based observations provide insight
into the various adaptations that are correlated with the formation of different ANME-SRB
partnerships. These adaptive traits appear to be related with mechanisms driving other eco-
logical phenomena such as biofilm formation and non-obligate syntrophic interactions. The
identification of these traits allowed us to posit important steps in the evolutionary trajectory
of these SRB to a syntrophic lifestyle. While the full import of these observations is not yet
clear, they offer a roadmap for targeted physiological investigations and phylogenetic studies
in the future.
Materials and methods
Ethics statement
Our environmental samples constituted sediment samples collected from methane seeps and
hydrothermal vents. We followed protocol for ethical sampling and received appropriate per-
mission for collection of environmental samples when required. Sample collection and export
permits for methane seep sediment samples off the coast of Costa Rica were acquired through
the Costa Rican Ministry of the Environment and Energy (SINAC-CUSBSE-PI-R-032-2018
and Academic License SINAC-SE-064-2018). Sample collection permits for FK181031 (25/07/
2018) were granted by la Dirección General de Ordenamiento Pesquero y Acuı́cola, Comisión
Nacional de Acuacultura y Pesca (CONAPESCA: Permiso de Pesca de Fomento No. PPFE/
DGOPA-200/18) and la Dirección General de Geografı́a y Medio Ambiente, Instituto Nacio-
nal de Estadı́stica y Geografı́a (INEGI: Autorización EG0122018), with the associated Diplo-
matic Note number 18–2083 (CTC/07345/18) from la Secretarı́a de Relaciones Exteriores -
Agencia Mexicana de Cooperación Internacional para el Desarrollo/Dirección General de
Cooperación Técnica y Cientı́fica. Sample collection permit for cruise NA091 (18/04/2017)
was obtained by the Ocean Exploration Trust under permit number EG0072017.
Sampling locations and processing of samples
Push-core samples of seafloor sediment were collected from different locations on the Costa
Rica margin during the AT37-13 cruise in May 2017 (sample serial numbers: #10073, #9063),
southern Pescadero Basin [122] during the FK181031 cruise on R/V Falkor operated by the
Schmidt Ocean Institute in November 2018 (sample serial number: #PB10259, live incubation
of the top 3 cm section of push core #FK181031-S193-PC3, 123], and from Santa Monica
Basin during WF05-13 cruise in May 2013 (sample serial numbers: #7059). Sediment push-
cores retrieved from the seafloor were sectioned into 1 to 3 cm sediment horizons. At the time
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
of shipboard processing, approximately 2 mL of the sediment was sampled for DNA extraction
and FISH analysis and the rest was saved in Mylar bags under an N2 atmosphere at 4˚C for
sediment microcosm incubations. Microbial mat sample #14434 was collected from Santa
Monica Basin during the WF02-20 cruises in February 2020. Rock samples were retrieved
from South Pescadero Basin [123] during the NA091 cruise on E/V Nautilus operated by the
Ocean Exploration Trust in October to November 2017 and the FK181031 cruises (sample
serial numbers: NA091-R045, NA091-R008, and #12019, #11946, #11719, respectively) and
saved in Mylar bags under an N2 atmosphere at 4˚C.
Sediment horizons from samples 10073, 9063, and 7059 were incubated in artificial sea
water as previously described [25,36] with CH4 and 250 μM L-Homopropargylglycine (HPG)
at 4˚C. Once the presence of metabolically active ANME-SRB in these microcosms was con-
firmed by the accumulation of sulfide combined with observation of incorporation of HPG by
BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT), samples from these incuba-
tions were used for sorting of single-aggregates by FACS as described below.
#FK181031-S193-PC3 was incubated in anaerobic artificial sea water without electron donor
at 24˚C. Rock #NA091-R045 was incubated in anaerobic artificial sea water supplemented
with pyruvate at 24˚C. Rock samples from S. Pescadero Basin (#11946, #11719, and #12019)
were also incubated with artificial sea water and CH4 at 50˚C.
DNA extraction followed by metagenome sequencing for samples #11946,
#11719, #12019, #NA091-R045, #NA091-R008, #PB10259, and #14434
For incubations of carbonate samples #11946, #11719, and #12019, DNA was extracted from
approximately 500 mg of crushed rock samples using a modified version of the Zhou protocol
[124] as follows. Prior to the incubation with proteinase K, the sample was incubated with lyso-
zyme (10 mg ml-1) for 30 min at 37˚C; 10% SDS was used for incubation; after SDS incuba-
tions, the sample was extracted twice by adding 1 volume (1 mL) of phenol/chloroform/
isoamylalcohol (25:24:1) with incubation for 20 min at 65˚C followed by centrifugation; in the
final step, the DNA was eluted in 40 μl of TE 1× buffer. Approximately 250 mg of sediment
sample #PB10259 and microbial mat sample #14434 were extracted using the QIAGEN Power
Soil Kit, and 500 mg of crushed carbonate samples #NA091-R045, #NA091-R008 were also
extracted using the QIAGEN Power Soil Kit.
For samples #PB10259, #14434, NA091-R045, DNA libraries were prepared using the NEB-
Next Ultra kit and sequenced at Novogene with the instrument HiSeq4000. A library was also
prepared using the NEBNext Ultra kit for NA091-008. This sample was sequenced at Quick
Biology (Pasadena, California, United States of America) with a HiSeq2000 using a 2 × 150
protocol. DNA libraries for samples #11946, #11719, and #12019 were prepared using the Nex-
tera Flex kit and also sequenced at Novogene on the HiSeq4000. After sequencing of
NA091-R45, primers and adapters were removed from all libraries using bbduk [125] with
mink = 6 and hdist = 1 as trimming parameters and establishing a minimum quality value of
20 and a minimal length of 50 bp.
Assembly and binning of metagenomes from samples samples
#NA091-R045, #NA091-R008, #PB10259, #14434, #11946, #11719, and
#12019
Metagenomes from samples #PB10259, #14434, #11946, #11719, and #12019 were assembled
individually using SPAdes [126] v3.14.1, and each resulting assembly was binned using meta-
bat v2.15 [127]. Automatic prediction of function for genes within the various MAGs was per-
formed using prokka v.1.14.6 [128]. The reads of the DNA libraries derived from the rock
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
sample (NA091-R008) were assembled individually using SPAdes v.3.12.0. From the de novo
assemblies for NA091-R008, we performed manual binning using Anvio v.6 [45]. We assessed
the quality and taxonomy affiliation from the obtained bins using CheckM [129] and GTDB-
tk [130]. Genomes of interest affiliated to Desulfobacterota were further refined via a targeted-
reassembly pipeline. The trimmed reads for the NA091-008 assembly were mapped to the bin
of interest using bbmap [125] (minimal identity of 0.97), then the mapped reads were assem-
bled using SPAdes and finally the resulting assembly was filtered discarding contigs below
1,500 bp. This procedure was repeated for 13 to 20 cycles for each bin, until the bin quality did
not improve any further. Bin quality was assessed based on the completeness, contamination
(<5%), N50 value, and number of scaffolds of the bin using CheckM. The resulting bins were
considered as MAGs. Automatic prediction of function for genes within the various MAGs
was performed using prokka v.1.14.6 [128] and curated with the identification of Pfam [131]
and TIGRFAM [132] profiles using HMMER v.3.3 [133]; KEGG domains [134] with Kofam
[135] and of COGs and arCOGs motifs [136] using COGsoft [137].
Fluorescent-sorting of metabolically active single aggregates from samples
#10073, #9063, and #7059 followed by sequencing
Sediment-extracted consortia from samples #10073, #9063, and #7059 were analyzed. Individ-
ual ANME:SRB consortia were identified and sorted using fluorescent signal, as previously
described [36]. The SYBR-Green dye was excited using a 488-nm laser, and fluorescence was
captured with a 531-nm/30-nm filter. Gates were defined using a forward scatter (FSC) versus
531-nm emission plot, and events with a fluorescent signal brighter than >90% of aggregates
in the negative control were captured. For sample #10073, 50 consortia were sorted into 1.5
mL tubes and stored at 4˚C for sequencing. For samples #9063 and #7059, 28 and 19 consortia
were sorted, respectively.
Single consortia were lysed and DNA was amplified using multiple displacement amplifica-
tion (MDA) protocol as previously described [138]. The amplified DNA was sheared, attached
to Illumina adapters, and sequenced using the Illumina NextSeq-HO method. Only metagen-
omes from 2, 3 and 1 sorted aggregates from each of the samples# 10073, #9063, and #7059
respectively were used in this study.
Assembly and binning of single aggregate metagenomes from samples
#10073, #9063, and #7059
Metagenomes were assembled using SPAdes spades v. 3.13.0 and annotated using the inte-
grated microbial genomes (IMG) annotation pipeline. As the single aggregate metagenomes
represent extremely reduced communities, and the MDA precludes traditional contig binning
by coverage, the metagenomes were binned using a manual approach based on sequence com-
position and taxonomic assignment of the genes. Manual binning was performed using a prin-
cipal component analysis (PCA) of the tetramer frequency of the contigs, calculated using calc.
kmerfreq.pl (https://github.com/MadsAlbertsen/miscperlscripts/). Taxonomic affiliation of
the genes on the contigs was taken from the IMG annotation, and the percentage of genes on
each contig annotated as “Archaeal” or “Bacteria” was used to corroborate the clustering in the
PCA plot. Jupyter notebooks used for the binning are available at https://github.com/dspeth/
SRB_single_aggregate_bins. Automatic metabolic prediction of the MAGs was performed
using prokka v.1.14.6 [128].
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
Taxonomic classification of metagenome bins from various syntrophic
sulfate reducing bacteria
Single copy marker genes identified in the “Bacteria 71” gene set included in Anvio [120] were
extracted from each of the syntrophic SRB genomes and all genomes within the phylum Desul-
fobacterota available in release 95 of the Genome Taxonomy Database [38]. A concatenated
gene alignment was generated using MUSCLE [72] as part of the anvio script “anvi-get-
sequences-for-hmm-hits.” A phylogenetic tree was inferred using FastTree as per the Anvio-7
pipeline using the command “anvi-gen-phylogenomic-tree,” in order to provide a phyloge-
netic context for each of the 4 SRB clades. We corroborated our phylogenetic placement with
the classification provided by GTDB-tk [130]. Additionally, we assessed the extent of taxo-
nomic diversity within the 4 clades by calculating the ANI and 16S rRNA sequence similarity
between different organisms that belong to each clade using PyANI [139] in Anvio-7 [45]. A
95% ANI value of 95% [140] and 98.65% similarity in 16S rRNA [141] were used as cut-offs to
delineate different species.
Phylogenetic analysis of OetI, the outer-membrane beta barrel forming
protein in the OetI-type cluster
OetI here refers specifically to the outer-membrane beta barrel forming protein in the OetI-
type cluster implicated in DIET between ANME and SRB. All OetI sequences were identified
in the genomes of the syntrophic SRB clades by using BLASTP [142] with the query
OEU57520.1 from Seep-SRB1g sp. C00003106 and an e-value of e-30. When no OetI hits were
found in the syntrophic SRB genomes using this query, we tested for the existence of a beta
barrel within 10 genes of every multiheme cytochrome that contained more than 5 heme c
binding motifs using PRED-TMBB [143]. In this way, we identified 17 EET gene clusters in
the syntrophic SRB genomes and 5 clusters from non-syntrophic Seep-SRB1c, Desulfofervi-
dales and Dissufuribacterales. Protein sequences of OetI from each of these clusters were used
as queries to extract all the closest homologs for each of these OetI sequences from the NCBI
database. This search was performed using BLASTP with an e-value cut of 1e-5. The extracted
sequences were aligned and manually curated to eliminate sequences that were too short and
to remove nonspecific hits. A phylogenetic tree was inferred using IQ-TREE2 [73], a Dayhoff
model of substitution and 1,000 ultrafast bootstrap iterations and visualized using the iTOL
web server [144].
Phylogenetic analysis of other respiratory proteins
All sequence alignments used for analysis of respiratory proteins were made using MUSCLE
[72] and visualized using Jalview [145]. Phylogenetic trees of all proteins were inferred using
IQ-TREE2 [73] except for the following - OetB, omcX, TmcD, and TmcA. Phylogenetic trees
for OetB, omcX, TmcD, and TmcA were inferred using RAxML [146]. RAxML trees were
inferred using a Dayhoff model of rate substitution and 100 bootstraps. IQ-TREE trees were
inferred using 1,000 ultrafast bootstraps while the models were automatically selected by
IQ-TREE using the Bayesian information criterion (BIC). The models used for each specific
tree are available in S14 Table.
Sequences analysis of all cytochromes c
All cytochromes c were identified from the MAGs of syntrophic SRB by employing a word-
search method with a custom python script by querying for the commonly found “CxxCH”
motif in cytochromes c. Once these sequences were extracted, they were aligned using
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PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
MUSCLE [72]. Clusters were identified depending on the presence of well-defined regions
using visual inspection. The clusters were then tabulated in S6 Table. The cellular localization
of cytochromes c was inferred either from the cellular localization of homologous cytochromes
c from Desulfuromonadales or using Signal P-5.0 [147].
Sequences analysis of all putative adhesins
Adhesins were identified from the MAGs of syntrophic SRB by using the “all-domain” annota-
tion feature on KBase as previously described [14,148]. Once putative domains were predicted,
we extracted the coding features that corresponded to all putative adhesins based on searches
for the words “integrin,” “adhesin,” “cohesin,” “dockerin,” “fibronectin,” “PilY,” and “immu-
noglobulin in the domain descriptions.” The proteins corresponding to these results were
aligned using MUSCLE [72]. Adhesin clusters were identified depending on the presence of
well-defined regions using visual inspection and then additionally verified by use of the NCBI
Conserved Domain database [149]. The adhesins were then tabulated in S10 Table.
Identification of putative polysaccharide biosynthesis pathways
Once, the syntrophic SRB genomes were annotated using the Prokaryotic Genome Annotation
Pipeline (PGAP) [150], we identified polysaccharide biosynthesis pathways by looking for the
presence of glycosyl transferases, aminotransferases, sugar transporters, and polysaccharide
biosynthesis proteins. If gene cassette structures followed known operon structures of ABC
transporter-type, Wzx/Wzy, or synthase type pathways [151], they were retained and tabulated
in S9 Table and visualized in S12–S15 Figs.
Evolutionary analysis of important genes to identify gains, loss, and
biochemical adaptation
We tabulated the presence and absence of 33 traits that we propose are important for the for-
mation of ANME-SRB partnership in each syntrophic SRB clade and the taxonomic order
from which they originate. The presence and absence was identified using BLASTP searches
with a query sequence or HMM as listed in S13 Table. If a gene is present in over 30% of non-
syntrophic relatives in a given order, it is considered as present in this order or in a syntrophic
SRB clade, it is considered present in this taxonomic clade. If a gene is present in the order and
in the syntrophic SRB clade that belongs to this order, the gene is considered to be vertically
transferred. If a gene is present in the order but, absent in the syntrophic SRB, the gene is con-
sidered to be lost. If a gene is present in the syntrophic SRB but absent in the order it belongs
to, the gene is considered to be horizontally acquired. The last assumption is corroborated as
much as possible by gene trees deposited on Github (https://github.com/ranjani-m/
syntrophic-SRB).
Supporting information
S1 Fig. Taxonomic separation of syntrophic sulfate reducing bacteria using average nucle-
otide identity. The average nucleotide identity of genomes from each clade of the syntrophic
sulfate reducing bacteria and some related bacteria were computed using the PyANI program
available through Anvi’o [45]. The different clades of syntrophic SRB—HotSeep1, Seep-SRB2,
Seep-SRB1a, and Seep-SRB1g are colored according to the attached legend. The Seep-SRB1a
genomes in particular are differently colored depending on whether they partner ANME-2a or
ANME-2c, respectively. The geographic location from which each genome was extracted is
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 28 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
indicated on each node or clade in the tree.
(EPS)
S2 Fig. Comparison of 16S rRNA and 23S rRNA phylogeny of organisms from the phylum
Desulfobacterota. 16S rRNA and 23S rRNA sequences were extracted from all organisms
from the phylum Desulfobacterota available in GTDB release 95 [38] and from syntrophic
SRB. These sequences were aligned using MUSCLE [72] and a tree was inferred using
IQTREE2 [73]. Hot-Seep1 is placed adjacent to the order Thermodesulfbacteriales in both
these trees. The trees are available in Newick format in S2 Data.
(EPS)
S3 Fig. Phylogeny of RpoB from organisms within the phylum Desulfobacterota.
Sequences of RNA Polymerase, subunit B were extracted from all organisms from the phylum
Desulfobacterota available in GTDB release 95 [39] and from syntrophic SRB using BLASTP
[142] with an e-value cut-off of e-30 and appropriate query sequences. The sequences were
confirmed to RpoB by manual inspection of a multiple sequence alignment generated using
MUSCLE [72] and the tree was inferred using IQTREE2 [73]. In this tree, Hot-Seep1 is found
adjacent to the order Desulfovibrionales. The tree is available in Newick format in S2 Data.
(EPS)
S4 Fig. Placement of various Seep-SRB1 clades within the phylum Desulfobacterota. A
phylogenetic tree of full-length 16S sequences from various Seep-SRB1 clades including the
original 16S rRNA sequences [23] used to define the Seep-SRB1(a–f) clades.
(EPS)
S5 Fig. Pan-genome analysis of Seep-SRB1a metagenomes. Fourteen genomes from nine
Seep-SRB1a species were analyzed using the Anvi’o pan-genome analysis pipeline [45]. Five
gene cluster bins were annotated based on genes that were identified as part of the core meta-
genome, unique to Seep-SRB1a sp. 2, Seep-SRB1a sp. 3, Seep-SRB1a sp. 5, and from the Seep-
SRB1a sp. 4 and 7.
(PNG)
S6 Fig. Pan-genome analysis of Seep-SRB2 metagenomes. Nineteen genomes from eight
Seep-SRB1a species were analyzed using the Anvi’o pan-genome analysis pipeline [45]. Three
gene cluster bins were annotated based on genes that were identified as part of the core meta-
genome, present in Seep-SRB2 sp. 1 and absent in Seep-SRB2 sp. 1.
(PNG)
S7 Fig. Phylogenetic placement of the outer membrane beta barrel, OetI from the putative
DIET cluster. A multiple sequence alignment, Supplementary multiple sequence alignment
MSA2 of the OetI protein sequences extracted from the genomes of syntrophic SRB and the
NCBI database was generated using MUSCLE [72]. This alignment was used to infer a phylo-
genetic tree using IQ-Tree2 [73] and visualized on the iTOL web server [144]. (a) The phyloge-
netic placement of OetI from E20 Seep-SRB2 next to OetI from Thermodesulfobacteria and
Dissulfurirhabdus thermomarina demonstrates that it was possibly vertically acquired from a
gene transfer that was ancestral to the Seep-SRB2 and then vertically transferred. (b) The phy-
logenetic placement of Seep-SRB1a and Seep-SRB1g OetI suggests that they are related. Addi-
tionally, the placement of OetI from G37 Seep-SRB2 next to OetI from Desulfofervidales
suggests that the Seep-SRB2 partner of ANME-1 acquired its DIET cluster from HotSeep-1.
The phylogenetic tree of OetI is available in Newick format, and the presence/absence table of
OetI in Desulfobacterota is also available in S2 Data.
(EPS)
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 29 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
S8 Fig. The Tmc complex in Seep-SRB1a and Seep-SRB1g are divergent. (A) The operons
containing Tmc in Candidatus Desulfofervidus auxilii (HotSeep-1), Seep-SRB1g, and Seep-
SRB1a show that TmcA is present in the former operon while it is missing in the latter 2. (B)
The distribution of Tmc is mapped on to the phylum Desulfobacterota, showing that it is com-
mon in the order Desulfobacterales and Desulfovibrionales. (C) Phylogeny of TmcD demon-
strates that the Tmc complex in Seep-SRB1a and Seep-SRB1g cluster together and appear to be
different from the Tmc complex in other organisms from the orders Desulfobacterales and
C00003060. The phylogenetic tree of TmcD is available in Newick format, and the presence/
absence table of Tmc in Desulfobacterota is also available in S2 Data.
(EPS)
S9 Fig. Distribution of QrcABCD and RnfABCDEG in Desulfobacterota. The presence and
absence of Qrc and Rnf was demonstrated across the Desulfobacterota using BLASTP [142]
searches of different query sequences of these complexes. Both these complexes are absent
from the orders Desulfobulbales, Thermodesulfobacteriales, and Dissulfuribacteriales. The
presence/absence table of Qrc/Rnf in Desulfobacterota is also available in S2 Data.
(EPS)
S10 Fig. Gene neighborhoods of various HdrA containing complexes and carbon fixation
pathways in syntrophic SRB. HdrA homologs were identified in the 4 syntrophic SRB clades
with the following gene neighborhoods. Multiple HdrA homologs were identified adjacent to
the carbon fixation pathways in syntrophic SRB, specifically Seep-SRB1a and Seep-SRB1g.
(EPS)
S11 Fig. Operons of Flx-Hdr complexes and gene neighborhood of putative formate utiliz-
ing proteins. (a) Flx-Hdr complexes that recycle electrons between NADH, ferredoxins, and
DsrC are found in Seep-SRB1g, Seep-SRB1a, and Seep-SRB2. (b) Many putative formate utiliz-
ing proteins are found in Seep-SRB1g, Seep-SRB1a, and Seep-SRB2. In Seep-SRB1g and Seep-
SRB1a, periplasmic formate dehydrogenases (fdhAB) are found. fdhA domains as identified
here are typically found in the periplasm and have a respiratory function while fdhF2 are typi-
cally cytoplasmic.
(EPS)
S12 Fig. Putative polysaccharide biosynthesis pathways in Seep-SRB1a.
(PNG)
S13 Fig. Putative polysaccharide biosynthesis pathways in Seep-SRB1g.
(EPS)
S14 Fig. Putative Polysaccharide biosynthesis pathways in Seep-SRB2.
(PNG)
S15 Fig. Putative polysaccharide biosynthesis pathways in HotSeep-1.
(PNG)
S16 Fig. Presence of extracellular contractile injection systems (eCIS) in ANME and SRB.
The presence of eCIS conduits in ANME and SRB was identified using BLASTP [142] and
dbeCIS [119]. While the eCIS clusters are widely distributed in ANME-2a and ANME-2b, they
are only sparsely distributed in ANME-1. They are only present in the Seep-SRB1g species
found in Costa Rica and one of the Seep-SRB1a species from Santa Monica Basin. 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. Line in blue is
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 30 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
used to depict the partnership between ANME-2b and Seep-SRB1g. Underlying presence/
absence data is available in S2 Data.
(EPS)
S17 Fig. Phylogeny of afp10, the spike protein from the extracellular contractile injection
system. Afp10 is the PAAR-domain containing protein that typically interacts with the target
organism of the eCIS. Afp10 sequences were extracted from ANME and SRB using BLASTP
[142] and dbeCIS [119]. These sequences were then used as queries to repeatedly search and
identify the closest homologs from the NCBI database. A sequence alignment was then made
using MUSCLE, manually inspected and filtered, and the tree was inferred using RAxML
[146]. Seep-SRB1a sequences are related to other eCIS sequences from Desulfobacterales while
Seep-SRB2 afp10 and Seep-SRB1g afp10 sequences do not cluster with evolutionarily related
bacteria. The phylogenetic trees of afp10 are available in Newick format in S2 Data.
(EPS)
S18 Fig. Phylogeny of afp11, a base plate protein from the extracellular contractile injec-
tion system. Afp11 belongs to the baseplate of the eCIS and does not interact directly with the
target organism. Afp11 sequences were extracted from ANME and SRB using BLASTP [142]
and dbeCIS [119]. These sequences were then used as queries to repeatedly search and identify
the closest homologs from the NCBI database. A sequence alignment was then made using
MUSCLE, manually inspected and filtered, and the tree was inferred using RAxML [146].
Seep-SRB1a sequences are related to other eCIS sequences from Desulfobacterales while Seep-
SRB2 afp10 and Seep-SRB1g afp10 sequences do not cluster with evolutionarily related bacte-
ria. The phylogenetic trees of afp11 are available in Newick format in S2 Data.
(EPS)
S19 Fig. Structural model of TmcD from Seep-SRB1a. A structural model of TmcD from
Seep-SRB1a was generated using Alphafold2 [152] using the monomer option. This model
was superimposed on top of a structural model of TmcD from Olavius algarvensis available on
UniProt. The divergent sequence regions from TmcD were highlighted in red while cysteine
residues unique to Seep-SRB1a were highlighted in green. The conserved residues identified
here were observed using a multiple sequence alignment of TmcD made available in online
supplementary data.
(PNG)
S1 Table. List of genomes from the Genome Taxonomy Database [38] used for compara-
tive analysis in this study.
(XLSX)
S2 Table. 16S rRNA pairwise similarity matrix. A pairwise comparison matrix of 16S rRNA
sequence similarity between syntrophic SRB clades and their nearest evolutionary relatives was
generated using Anvi’o [45].
(XLSX)
S3 Table. Seep-SRB1a pan-genome analysis. A pan-genome analysis of Seep-SRB1a clades
was performed using Anvi’o [45] to demonstrate the conserved genes and differences within
the syntrophic SRB clade Seep-SRB1a.
(XLSX)
S4 Table. Seep-SRB2 pan-genome analysis. A pan-genome analysis of Seep-SRB1a clades was
performed using Anvi’o [45] to demonstrate the conserved genes and differences within the
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 31 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
syntrophic SRB clade Seep-SRB2.
(XLSX)
S5 Table. List of accession numbers for operons containing the OetI-type conduit in syn-
trophic SRB and their nearest evolutionary neighbors.
(XLSX)
S6 Table. List of protein accession numbers for cytochromes c identified in syntrophic
SRB and their nearest evolutionary neighbors. Cytochromes c clusters were identified by
clustering with multiple sequence alignment [72] and classified as playing roles in extracellular
electron transfer, periplasmic electron transfer, or inner membrane electron transfer.
(XLSX)
S7 Table. List of protein accession numbers for respiratory genes and nitrogen fixation in
syntrophic SRB and other organisms within Desulfobacterota.
(XLSX)
S8 Table. List of protein accession numbers for cobalamin biosynthesis pathways in syn-
trophic SRB and their nearest evolutionary neighbors.
(XLSX)
S9 Table. List of protein accession numbers for polysaccharide biosynthesis pathways in
syntrophic SRB and their nearest evolutionary neighbors.
(XLSX)
S10 Table. List of protein accession numbers for putative cellular adhesins in syntrophic
SRB and their nearest evolutionary neighbors.
(XLSX)
S11 Table. List of protein accession numbers of extracellular contractile injection systems
in syntrophic SRB and ANME.
(XLSX)
S12 Table. List of selected genes from different pathways involved in extracellular electron
transfer, respiratory pathways, and biofilm formation present and absent in syntrophic
SRB and their nearest evolutionary neighbors.
(XLSX)
S13 Table. List of genes expected to be gained by horizontal gene transfer, vertical inheri-
tance or lost, based on patterns of presence and absence in syntrophic SRB and their near-
est evolutionary neighbors. Also supported by manual inspection of trees made available
on Github (https://github.com/ranjani-m/syntrophic-SRB).
(XLSX)
S14 Table. List of protein phylogenetic models used for inference of evolutionary patterns
within proteins in this study.
(XLSX)
S1 Text. (S1_text_Proposal_for_formal_nomenclature.pdf).
(PDF)
S1 Data. Gene_trees_from_syntrophic_SRB.zip.
(ZIP)
S2 Data. Other supplementary data.zip.
(ZIP)
PLOS Biology | https://doi.org/10.1371/journal.pbio.3002292 September 25, 2023 32 / 41
PLOS BIOLOGY Adaptation of sulfate reducing bacteria to methane-fueled syntrophy
Acknowledgments
We would like to acknowledge Magdalena Mayr for her thoughtful comments on this manu-
script and Fernanda Jimenez-Otero for sharing her expertise in the field of extracellular elec-
tron transfer. We are also grateful to Alon Philosof, Aditi Narayan, Kriti Sharma, and James
Hemp for many productive discussions that assisted in the framing of this manuscript. We are
indebted to the pilots, crew, and shipboard scientists on the R/VWestern Flyer (Monterey Bay
Research Aquarium Institute), R/V Falkor (Schmidt Ocean Institute), E/V Nautilis (Ocean
Exploration Trust), and R/V Atlantis whose dedication and skills made this research possible.
Author Contributions
Conceptualization: Ranjani Murali, Hang Yu, Kyle S. Metcalfe, Grayson L. Chadwick,
Victoria J. Orphan.
Data curation: Ranjani Murali, Daan R. Speth, Fabai Wu, Rex R. Malmstrom,
Danielle Goudeau, Tanja Woyke, Roland Hatzenpichler, Stephanie A. Connon.
Formal analysis: Ranjani Murali, Daan R. Speth, Kyle S. Metcalfe, Rafael Laso-Pèrez,
Rex R. Malmstrom.
Funding acquisition: Rex R. Malmstrom, Tanja Woyke, Roland Hatzenpichler,
Victoria J. Orphan.
Investigation: Ranjani Murali, Hang Yu, Fabai Wu, Antoine Crémière, Rafael Laso-Pèrez,
Roland Hatzenpichler, Victoria J. Orphan.
Methodology: Ranjani Murali, Hang Yu, Daan R. Speth, Fabai Wu, Antoine Crémière,
Rafael Laso-Pèrez, Rex R. Malmstrom, Danielle Goudeau, Roland Hatzenpichler,
Stephanie A. Connon, Victoria J. Orphan.
Project administration: Victoria J. Orphan.
Resources: Antoine Crémière, Rex R. Malmstrom, Tanja Woyke, Victoria J. Orphan.
Software: Daan R. Speth.
Supervision: Victoria J. Orphan.
Validation: Ranjani Murali.
Visualization: Ranjani Murali, Daan R. Speth.
Writing – original draft: Ranjani Murali, Kyle S. Metcalfe, Victoria J. Orphan.
Writing – review & editing: Ranjani Murali, Hang Yu, Daan R. Speth, Fabai Wu,
Antoine Crémière, Rafael Laso-Pèrez, Rex R. Malmstrom, Tanja Woyke,
Roland Hatzenpichler, Grayson L. Chadwick, Victoria J. Orphan.
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