Physiological
adaptation of sulfate reducing bacteria in syntrophic partnership with anaerobic
1
methanotrophic archaea
2
Ranjani Murali
1
*
,
Han
g
Yu
2
,
3
, Daan
R.
Speth
4
,1
, Fabai Wu
5
,
Kyle S. Metcalfe
6
,
Antoine Crémière
2
, Rafael
3
Laso
-
Pèrez
7
,
Rex
R.
Malmstrom
8
, Danielle Goudeau
8
, Tanja Woyke
8
,
Roland Hatzenpichler
9
,
Grayson L.
4
Chadwick
2,
6
,
Victoria J. Orphan
1
,2
*
5
6
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
7
2
Division of Geological and
Planetary Sciences, California Institute of Technology, Pasadena, CA, USA
8
3
Department of Physics and Astronomy,
U
niversity of Southern California, Los Angeles, CA, USA
9
4
Max Planck Institute for Marine Microbiology, Bremen, Germany
10
5
ZJU
-
Hangzhou Global Scientific and Technological Innovation Center
,
Zhejiang, China
11
6
Department of Plant and Molecular Biology, University of California, Berkeley. Berkeley, CA, USA
12
7
National Center for Biotechnology, Madrid, Spain
13
8
DOE J
oint Genome Instit
ute, Department of Energy,
Berkeley
, CA, USA
14
9
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA
15
For correspondence, contact:
m.ranjani@gmail.com
,
vorphan@caltech.edu
16
17
18
19
20
21
22
23
24
25
26
.
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Abstract
27
Sulfate
-
coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of
28
anaerobic methanotrophic
archaea
(ANME)
in obligate syntrophic partnership with
sulfate
-
reducing
29
bacteria (SRB).
Diverse
ANME and SRB clades co
-
associate but the physiological basis for the
ir
30
adaptation and diversification is not well understood. In this work, we explore the metabolic adaptation of
31
four syntrophic SRB clades (HotSeep
-
1, Seep
-
SRB2, Seep
-
SRB1a and Seep
-
SRB1g) from a
32
phylogenomics perspective, tracing the evolution of conserved p
roteins in the syntrophic
SRB clades
, and
33
comparing the genomes of syntrophic SRB
to
their nearest evolutionary neighbors in the phylum
34
Desulfobacterota.
We note several examples of gain
,
loss
or biochemical adaptation
of proteins within
35
pathways involved
in
extracellular electron transfer
,
electron transport chain
, nutrient sharing, biofilm
36
formation and
cell adhesion
.
W
e demonstrate that
the metabolic adaptations in each of these syntrophic
37
clades
are
unique,
suggesting that they have independently evolve
d, converging to a syntrophic
38
partnership with ANME
. Within the clades we also investigated the specialization of different
syntrophic
39
SRB
species to partnerships with different ANME
clades
, using
metagenomic sequences
obtained
from
40
ANME and SRB
partners in individual consortia after fluorescent
-
sorting of cell aggregates from
41
anaerobic sediments
.
In one instance of metabolic adaptation to different partnerships, we show that
Seep
-
42
SRB1a partners of ANME
-
2c appear to lack nutritional auxotrophies,
while
the related Seep
-
SRB1a
43
partners of a different methanotrophic archaeal lineage, ANME
-
2a, are missing the cobalamin synthesis
44
pathway, suggesting that th
e
Seep
-
SRB1a
partners of ANME
-
2a
may have a
nutritional
dependence on its
45
partner.
Together, our p
aired genomic analysis of AOM consortia highlights the specific adaptation and
46
diversification of syntrophic SRB clades linked to
their
associated
ANME lineages.
47
48
Introduction
49
Syntroph
y
is
a form of metabolic cooperation between different
microorganisms that enables the
50
utilization of substrates which neither organism could metabolize on its own
[1,2]
.
Microorganisms
51
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benefit from sharing nutrients and electrons in this way, combining their resources and allowing for less
52
energy investment fo
r each partner
[1,3]
.
Syntrophic interactions appear to be specific in at least some
53
cases, with the same organisms co
-
associating across different ecosystems and environments
[4]
.
54
However, we do not yet understand the physiological basis driving the specifi
city of interactions, often
55
because syntrophic associations are difficult to grow in
the laboratory
and characterizing the specificity of
56
these interactions is challenging with uncultured syntrophic consortia in the environment
[2]
.
A classic
57
example of
a s
yntrophic relation
ship
exists between
anaerobic methanotrophic archaea (ANME)
and
58
sulfate
-
reducing bacteria
(SRB)
in
methane seeps
[5
–
7]
.
ANME oxidize methane to CO
2
anaerobically, in
59
a geologically significant process known as anaerobic oxidation of
methane(AOM)
[8]
.
AOM is a
60
thermodynamically unfavorable process that can only be completed when it is a coupled to an
61
energetically favorable
reaction such as sulfate reduction
[9]
. In multicellular consortia of ANME and
62
SRB, coupling of methane oxidation
to sulfate reduction appears to occur through direct interspecies
63
electron transfer or DIET
[10,11]
.
In addition to electron exchange, s
tudies of syntrophic ANME
-
SRB
64
consortia have
also identified
other hallmarks of syntrophy such as
diazotrophic nitrogen
exchange
[12
–
65
14]
.
The ecophysiology of ANME/SRB consortia is complex and t
here are many divergent lineages of
66
ANME and SRB that co
-
associate to form multicellular consortia.
Given the diversity of interactions in
67
ANME
-
SRB partnerships
[14,15]
, there is a pos
sibility that the basis and characteristics of some of these
68
syntrophic partnerships may differ.
Understanding
the
physiological
basis of ANME
-
SRB interactions
69
will provide insight into similar mechanisms that underpin the many syntrophic interactions in t
he
70
biosphere.
71
72
Investigation of the archaeal and bacterial lineages involved in
AOM
identified at least three divergent
73
taxonomic groups of archaea, by analysis of 16S rRNA
gene sequences
and fluorescence in situ
74
hybridization (FISH)
–
ANME
-
1, ANME
-
2 and
ANME
-
3
[5,16,17]
. All three of these groups are
clades
75
within the phylum Halobacterota. While ANME
-
1 is quite divergent
and form
a separate order called
76
Methanophagales
[18]
, all the ANME
-
2 clades
, including
ANME
-
2a (
Methanocomedenaceae
), ANME
-
2b
77
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(
Methanomarinus
), ANME
-
2c (
Methanogasteraceae
) and ANME
-
2d (
Methanoperedenaceae
) and
78
members of the ANME
-
3 (
Methanovorans
), represent family or genus level
taxa
within the order
79
Methanosarcinales
[19]
. Similar analys
e
s of ANME
-
associated SRB revealed several
clades of syntrophic
80
SRB
within the phylum Desulfobacterota
: two clades related to Desulfobacterales (Seep
-
SRB1g and
81
Seep
-
SRB1a)
[14,20
–
22]
, seepDBB within the
Desulfobulbaceae
[23]
and two more divergent groups
-
82
Seep
-
SRB2
[24]
and HotSeep
-
1
[25]
. ANME have
also been
reported
to form
spatial associations
in
83
consortia
with
additional microbial lineages including
alpha
-
and beta
-
proteobacteria
[26]
and
84
verrucomicrobia
[27]
,
Anaerolineales and Methanococcoides
[28]
.
These
partnerships and that of ANME
85
with seepDBB
,
have
however
not been physiologically well
-
characterized and we
thus
do not include
86
them in our analysis.
Our analysis of ANME
-
SRB partnerships could be used to better under the nature of
87
other
associations betw
een
ANME and non
-
SRB species
in the future
.
Previous research using FISH
88
microscopy surveys
[6,11,16,20,29,30]
magneto
-
FISH
[31,32]
,
Bioorthogonal Non
-
canonical Amino Acid
89
Tagging combined with fluorescence activated cell sorting
(BONCAT
-
FACS)
[27]
,
and network analysis
90
of statistical correlations
in 16S rRNA gene amplicon sequencing data
[14,33]
revealed the most
91
commonly observed partnerships between ANME and associated
-
SRB. Collectively, these results indicate
92
that members of the ANME
-
1 order tend
to partner with HotSeep
-
1 or Seep
-
SRB2 bacteria
[24,25,33]
,
93
ANME
-
2a with Seep
-
SRB1a
[20,22]
, ANME
-
2b with Seep
-
SRB1g
[14]
, ANME
-
2c with Seep
-
94
SRB1a
[14,20]
or Seep
-
SRB2
[24,33]
, and ANME
-
3 with Seep
-
SRB1a
[20]
and some clades of
95
Desulfobulbaceae
[29]
. These trends
suggest that some lineages of ANME and syntrophic SRB partners,
96
such as ANME
-
1, ANME
-
2c
, Seep
-
SRB2
and Seep
-
SRB1a are capable of forming partnerships with
97
multiple groups.
However, greater taxonomic resolution within currently identified clades is require
d to
98
test
whether these partnerships truly are flexible (as is often true of syntrophies based on hydrogen
99
exchange) or
if subgroups can be identified that correspond
to
specific
AOM syntrophic partnerships.
100
101
Metagenomics and comparative genomics have bee
n used to identify
metabolic
traits unique to the
102
previously identified ANME
groups
[15,19,34,35]
. Consistent with their phylogenetic distance from
103
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ANME within the Methanosarcinales, members of the ANME
-
1 differ most from the others,
as shown by
104
distinctions
in steps of the reverse methanogenesis pathway
[34]
, their
respiratory
cytochrome
s
c
[19]
and
105
in their proposed use of quinone
more often
than methanophenazine as an electron carrier
[19]
.
106
Significantly, the differences between the cytochrome
c
m
achinery of ANME
-
1 and
that of
ANME
-
107
2a/2b/2c and ANME
-
3
indicate
that the mechanism of transferring electrons out of the cell must be very
108
different
even if
it is ultimately still based on
multi
-
heme cytochromes
.
Metabolic differences within
109
ANME will directly affect the midpoint potential of the electron carrier that donates electrons to the
110
syntrophic partner, their ability to fix and share nitrogen as well as their ability to synthesize and share
111
other essential
nutrients.
As
each of the syntrophic SRB are affected by the metabolic potential of their
112
corresponding ANME partners and therefore, it is
critical
to consider the genomic traits of the SRB in the
113
context of their syntrophic partners.
114
115
Among
the syntrophi
c sulfate
-
reducing bacteria identified using 16S rRNA data and verified to be in
116
partnership with ANME by FISH, genomes exist for members of the thermally adapted HotSeep
-
1
117
clade
[24,25]
, as well as psychrophilic or mesophilic representatives of Seep
-
SRB1a
[
21,22]
, Seep
-
118
SRB1g
[14,21]
and Seep
-
SRB2
[24]
. These studies collectively confirmed that the complete pathway for
119
dissimilatory sulfate reduction was universally present in
all clades
. Additionally, a large gene cluster
120
containing multi
-
heme cytochromes (MHC
) that is hypothesized to play a role in accepting electrons from
121
ANME was detected in all genomes
[21]
. This previous work has established several defining
122
characteristics of the syntrophic partners of ANME archaea. However, a robust taxonomic framework fo
r
123
identifying syntrophic SRB and differentiating them from their non
-
syntrophic SRB relatives is lacking,
124
as is an evolutionary framework for understanding the metabolic adaptations in SRB that drove the
125
formation of a syntrophic partnership with ANME. To
bridge this knowledge gap, especially in light of
126
the recent identification of a highly specific partnership between ANME
-
2b and Seep
-
SRB1g, we
here
127
employed comparative genomics to analyze the
genomic traits
of the dominant
ANME associated SRB
128
clades
–
Seep
-
SRB1a, Seep
-
SRB1g, Seep
-
SRB2 and HotSeep
-
1. Our analysis incorporated 5
76
bacterial
129
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genomes from the phylum Desulfobacterota from the GTDB database and
46 genomes of syntrophic SRB
130
partners of ANME. This dataset include
d
15 previously unpubl
ished metagenome
-
assembled genomes
131
(MAGs) of syntrophic SRB and related clades recovered from seep sediments and mineral samples from
132
three geographically distant locations in the Pacific Ocean including seeps
located off
the coast of
Costa
133
Rica,
off the c
oast of
S. California, as well as
from hydrothermal vents
in the Gulf of California.
134
Importantly
, several of these MAGs were sourced from
fluorescent
cell sorting
and sequencing
of
single
135
AOM consortia
[27]
enabling
genomic analysis of these SRB syntrophs i
n the context of their specific
136
ANME partner. Our analysis provides a framework for using genome
-
based
phylogeny and 16S rRNA
137
similarity to identify organisms from the four syntrophic SRB clades.
In this work, we
described a
138
physiological framework
comprising
pathways that are important for the establishment of a syntrophic
139
partnership between ANME and SRB
and, w
ith our phylogenomic framework, we
identified
multiple
140
instances of metabolic adaptation
that are specific to
the syntrophic SRB
, differentiating them from their
141
nearest evolutionary neighbors. These instances
were
categorized
as likely gene gains,
losses
,
or
specific
142
cases of
biochemical adaptation to a syntrophic lifestyle.
With paired metagenomes
of archaea and
143
bacteria
from single consortia, we also demonstrate
d
that there appear to be specific instances of
144
physiological adaptation of different S
eep
-
SRB1a species to partnerships with different clades of ANME.
145
Our
study
explore
d
the
diverse
physiological strategies that underlie
the different ANME
-
SRB
146
partnerships, providing insight into the
mechanism behind the establishment of the syntrophic partnership
147
that is responsible for
AOM
.
148
149
Results and Discussion
150
Taxonomic diversity within syntrophic SRB of methanotrophic ANME
151
To invest
igate the adaptation of SRB to a partnership with ANME, we first placed them into their
152
taxonomic context and assessed the phylogenetic diversity within the SRB clades (Seep
-
SRB1a, Seep
-
153
SRB1g, Seep
-
SRB2 and HotSeep
-
1). For this analysis we compiled a curat
ed dataset of
metagenome
154
.
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assembled
genomes
(MAGs)
from these SRB clades including 34 previously published
155
genomes
[21,22,25,36
–
40]
and 12
MAGs
assembled for this study. Five of these genomes were
156
reconstructed from seep samples collected off the coast of California, Costa Rica, and within the Gulf of
157
California.
We
also sequenced single ANME
-
SRB consortia that were sorted by
FACS (Fluorescence
-
158
ac
tivated cell sorting) after
they were
SYBR
-
stained
as previously
described
[28]
. With this technique, we
159
could be confident of the assignment of partners
that
physically
co
-
associate within the sequenced
160
aggregates
and begin to identify partnership specific characteristics. From sequencing of single consortia,
161
we obtained 2 genomes of ANME
-
2b associated Seep
-
SRB1g, 1 genome of ANME
-
2a associated Seep
-
162
SRB1a and 3 genomes of ANME
-
2c associated Seep
-
SRB1a
(Table 1)
.
We
recovered an additional 3
163
genomes of the nearest evolutionary neighbors of HotSeep
-
1 within the order Desulfofervidales since this
164
order of bacteria is very poorly represented in public databases. Our
dataset for
comparative genomics
165
analysis comprised th
e above mentioned 46 genomes of syntrophic SRB and 576 other bacteria from
166
Desulfobacterota
. Having compiled this dataset
of syntrophic SRB
, we
also
designate
d
type material and
167
propose
d
formal names for three of the syntrophic SRB clades, Seep
-
SRB2
(
Candi
datus
Desulfomithrium
168
gen. nov.
)
, Seep
-
SRB1a
(
Candidatus
Syntrophophila
gen. nov.
)
, Seep
-
SRB1g
(
Candidatus
169
Desulfom
ellonium
gen. nov.
)
.
The genomes designated as type material are identified in
Figure 1
and
170
Supplementary Figure 1
. Further d
etails are available in
Supplementary information
.
171
172
Details for the phylogenetic placement of each of these clades using 16S rRNA phylogeny, concatenated
173
ribosomal protein phylogeny and the Genome Taxonomy Database are provided in
Materials and
174
Methods
and
Supplementary Information
. HotSeep
-
1 is a species within the order Desulfofervidales,
175
an order that is largely associated with thermophilic environments (with one exception
, Desulfofervidales
176
sp. DG
-
60 was sequenced
from the White Oak Estuary
[41]
)
.
Member
s of HotSeep
-
1 are
the best
177
characterized members of this order and are
known to be syntrophic partners to
thermophilic clades of
178
methane
-
oxidizing ANME
-
1
[15,24]
as well as alkane
-
oxidizing archaeal relatives
‘
Candidatus
179
Syntrophoarchaeum
butanivorans
’
,
‘
Candidatus
Syntrophoarchaeum caldarius
’
[42]
and ethane
-
oxidizing
180
.
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‘
Candidatus
Ethanoperedens thermophilum
’
[36]
.
Seep
-
SRB2 is a genus level clade within the order
181
Dissulfuribacterales
[43
–
45]
and class
Dissulfuri
bacteria. Dissulfuribacterales
include the gen
era
182
Dissulfuribacter and Dissulfurirhabdus
[44,45]
, which are chemolithoautotrophs associated with sulfur
183
disproportionation.
Seep
-
SRB1g is a species level clade which groups within a
taxonomic
order that also
184
includes Seep
-
SRB1c
(
Figure 1,
Table 1
).
This order falls within the class Desulfobacteria along with the
185
sister order Desulfobacterales.
Like the Desulfofervidales, th
e
order
with Seep
-
SRB1g
is poorly
186
characterized,
yet its most well
-
described members
are the Seep
-
SRB1g that
are obliga
te syntrophic
187
partners of ANME, accepting electrons from the archaeal partner to reduce sulfate
[14,21]
.
Seep
-
SRB1a is
188
a genus level clade that along with the genus Eth
-
SRB1 forms a distinct family
within the order
189
Desulfobacterales
(
Figure 1
,
Supplementary
Figure 1
,
Supplementary Table 2
).
Many of the well
-
190
characterized members of Desulfobacterales such as
Desulfococcus oleovorans
,
Desulfobacter
191
hydrogenophilus
,
Desulfosarcina
BuS5 are known as
hydrogenotrophs and
hydrocarbon
degraders
[46
–
192
48]
. The nearest evolutionary relative of Seep
-
SRB1a are the Eth
-
SRB1 first characterized as a syntrophic
193
partner
of
ethane
-
degrading archaea
[49]
. Each of the four syntrophic SRB clades have evolved from
194
taxonomically divergent ancestors with different metabo
lic capabilities
.
While the adaptation to a
195
syntrophic partnership with ANME appears to have been convergently evolved in these clades, their
196
evolutionary trajectories are likely to be different.
197
198
Species diversity within each of these clades was inferred
by calculating the average nucleotide identity
199
(ANI) (
Supplementary
Figure
1
) and 16S rRNA sequence similarity (
Supplementary Table 2
) between
200
different organisms that belong to each clade, using a 95 % ANI value and 98.65 % similarity in 16S
201
rRNA as cut
-
offs to delineate different specie
s. Partnership associations, as identified in previous research
202
by our group and others
,
by FISH
[20,24,25]
, magneto
-
FISH
[32]
or FACS sorting
[27]
and single
-
203
aggregate sequencing
[28]
are depicted in
Figure 1
and
Supplementary Figure 1
with further details
204
provided in
Supplementary Information
. All the genomes of Seep
-
SRB1g in our curated database
205
belong to one species
-
level clade and thus far, have been shown to partner only ANME
-
2b
[14]
.
In
206
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contrast, there is grea
ter species diversity within the clades that are known to partner more than one clade
207
of ANME, Seep
-
SRB2 and Seep
-
SRB1a. Whether this diversification is driven by adaptation to
208
partnerships with multiple ANME clades remains to be seen. This pattern is also
not consistent with
209
HotSeep
-
1, a species level clade that partners multiple archaeal species. A better understanding of the
210
physiological basis for syntrophic partnership formation in each of these clades will provide a framework
211
to understand their uniqu
e diversification trajectories.
212
213
Comparative genome analysis of syntrophic
SRB
214
To
develop
insight into the adaptation of
SRB
to syntrophic partnerships with ANME, we use
d
a
215
comparative genomics analysis
approach
to 1) identify
the unique
features of known syntrophic SRB
216
partners relative to their closest non
-
syntrophic relatives and 2)
compare the physiological traits that
217
define the diversity within a given taxonomic clade of syntrophic partner bacteria.
For our first objective,
218
we placed
the metabolic traits of SRB into the
phylogenetic
context of the
Desulfobacterota
phylum,
219
correlating the presence or absence of a physiological trait
within the context of
genus, family and
order
220
level context of each syntrophic SRB
clade
. As an example,
we demonstrate that the multi
-
heme
221
cytochrome conduit
[21]
implicated in
DIET
between ANME and SRB is rare in non
-
syntrophic
222
Desulfobacterota suggesting that this trait is part of a required adaptation for this syntrophic relationship
223
(
Figure 2
)
.
224
225
We also
investigated the physiological differences between the species of each syntrophic SRB clade.
226
Two
of the syntrophic SRB clades, Seep
-
SRB1g and HotSeep
-
1
have low
diver
sity
,
with
representatives
227
from different seep and vent ecosystems
each
belong
ing
to
a
single
species
-
level clade
. The clades Seep
-
228
SRB2 and Seep
-
SRB1a
in contrast,
contain multiple species. To
better understand
the
genomic features
229
underlying
this diversity, we perf
ormed a
comparative analysis of species within the Seep
-
SRB1a and
230
Seep
-
SRB2 to identify conserved genes across the clade and species
-
specific genes.
A detailed
231
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description of the analysis methods is available in
Materials and Methods
and
Supplementary
232
Info
rmation
(
Supplementary Figure
s
5, 6, Supplementary Tables 3, 4
)
. For this comparative analysis,
233
we primarily focused on pathways that are predicted to be important for the syntrophic interactions
234
between ANME and SRB. In the following section, we describe
the pathways within the syntrophic SRB
235
in greater detail and their significance for a syntrophic lifestyle
–
extracellular electron transfer,
236
membrane
-
bound electron transport chain, electron bifurcation, carbon fixation, nutrient sharing, biofilm
237
formatio
n, cell adhesion and partner identification.
Lastly
, we explicitly compare the losses and gains of
238
the genes encoding for the above pathways across the syntrophic SRB and infer the evolutionary
239
trajectory of adaptation towards a syntrophic partnership.
240
241
Re
spiratory
pathways in the four syntrophic
SRB
clades
demonstrate significant metabolic
242
flexibility
243
The respiratory pathways in syntrophic SRB are defined by the necessity
of ANME to transfer the
244
electrons derived from methane oxidation
to SRB.
These electrons are then transferred across the outer
245
membrane to periplasmic electron carriers. These periplasmic electron carriers donate electrons to inner
246
membrane complexes and ultimately, to the core sulfate reduction pathway. Some of the electrons a
re
247
also used for assimilatory pathways such as carbon fixation. Accordingly, our analysis of the respiratory
248
pathways is split into a description of the pathways for interspecies electron transfer, electron transfer
249
across the inner membrane, and carbon fi
xation pathways. In several instances we also note the potential
250
for multiple complexes having redundant functionality which may afford respiratory flexibility within
251
these pathways and emphasize the steps or reactions at which energy conservation likely o
ccurs.
252
a.
Multiple p
athways for interspecies electron transfer between ANME and SRB
253
The dominant mechanism of interspecies electron transfer between ANME and SRB
was proposed to be
254
direct interspecies electron transfer (DIET)
.
This
hypothesis
is
supported by the presence of multi
-
heme
255
cytochromes in
genomes of
ANME
-
2a, 2b and 2c
[11]
, the presence of nano
-
wire like structures that
256
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extend between ANME
-
1 and its partners Hot
-
Seep1
[10]
and Seep
-
SRB2
[24]
, and
the presence of hemes
257
in the extracellular
space between archaeal and bacterial cells in ANME
-
SRB aggregates
[11,24]
. This
258
hypothesis was also supported by the presence of a putative large multi
-
heme cytochrome:porin type
259
conduit, analogous to the conduits in
Geobacter sp.
[50]
and other gram
-
negati
ve bacteria
that
have been
260
shown
to participate in extracellular electron transfer (EET)
[50]
, in Seep
-
SRB1g
[21]
, Seep
-
SRB2
[24]
and
261
Hot
-
Seep
-
1
[10]
. Our analysis of a more comprehensive dataset of syntrophic bacterial genomes confirms
262
the presence of this po
rin:cytochrome
c
conduit in all the four syntrophic bacterial clades
studied
263
(
Supplementary Table 5
). Henceforth, we refer to this as the as the (
O
uter
-
membrane bound
264
e
xtracellular electron
t
ransfer) or Oet
-
type
conduit (OetI
was verified to
be capable of EET
, data not
265
shown)
.
This conduit includes
a
periplasmic cytochrome
c
(OetA)
,
an outer
-
membrane porin (OetI), and
266
extracellular facing
cytochrome
c
lipoprotein (OetB) (
Figure
2
b
, Figure 3
). The Oet
I
-
type conduit
was
267
first
identified in
G. sulfurreducens
and is expressed when a Geobacter mutant of omcB is grown on
268
Fe(III) oxide
[51]
.
The
o
et
A
B
I cassette is found in all
four syntrophic SRB clades
, and often
includes two
269
or three other putative extracell
ular cytochromes
c
, including
homolog
s
of OmcX
[21]
,
OmcS
270
(Supplementary alignment
MSA1
)
and
a 6
-
heme cytochrome that we
termed
apc2a
(
Supplementary
271
Table
5
).
If they are not found as part of the oet cluster, they
could
be found elsewhere on the genome
,
272
possibly due to genomic rearrangement after acquisition of the
cassette
(
Supplementary Table
s
6
, 7
).
273
The omcX and omcS
-
like genes in the oet gene cassette are
often
found in an analogous position to omcS
274
and omcT in G.
sulfurreducens
(
Figure
2
)
. Based on the homology of one of the cytochromes to OmcS,
275
which polymerizes to form long and highly conductive filaments that facilitate extracellular electron
276
transfer in
Geo
bacter
[52]
, we propose that the extracellular cytochromes
c
in this gene cassette perform a
277
similar function, forming filaments that accept electrons from ANME. This is consistent with
heme
278
staining
of the
intercellular space between ANME and SRB, and
the
observation
of
filaments that connect
279
the partners
[11,24]
. This is also consistent with the fact that different extracellular cytochromes are
280
amongst the most highly
-
expressed proteins in
the syntrophic SRB
: ANME
-
1/Seep
-
SRB2
[24]
(OmcX,
281
OmcS
-
like and apc2a), ANME
-
1/HotSeep
-
1
[24]
(OmcX
and OmcS
-
like
), ANME
-
2c/Seep
-
SRB2
[24]
282
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(OmcX) aggregates and ANME
-
2a/Seep
-
SRB1a
[22]
(OmcX, OmcS
-
like).
The presence of m
ultiple
283
copies of these putative filament forming proteins in the syntrophic SRB genomes is indicative of their
284
importance to the
physiology of
syntrophic SRB.
The m
echanism of electron transfer from extracellular
285
cytochrome
filaments to the interior of the cells in
Geobacter
is not well understood. However, a
286
porin:cytochrome
c
conduit is always expressed under the same conditions as a cytochrome
c
containing
287
filamen
t
in
Geobacter
(omcS along
with
extEFG or omcABC
under Fe (III) oxide reducing conditions
288
and omcZ along with extABCD during growth on an electrode
[53]
)
and in ANME
-
SRB consortia
289
(OmcS/OmcX with OetABI or OmcKL).
These findings
suggest that
each
cytochrome
c
filament
could
290
act
in concert with a porin:cytochrome
c
conduit
(
Figure 2
)
to transfer electrons from the extracellular
291
space to the periplasm.
292
293
While oetABI
is conserved in all four syntrophic SRB clades, there are two other putative
294
porin:cytochrome
c
conduits in syntrophic SRB. A porin (HS1_RS02765) and extracellular cytochrome c
295
(HS1_RS02760) homologous to OmcL and OmcK from
G
.
sulfurreducens
is found in
HotSeep
-
1
296
(
Supplementary Table 6, 7
)
and expressed at a
four
-
fold higher
level than the oetABI conduit
[24]
.
297
OmcK and OmcL were also upregulated in
G
.
sulfurreducens
when it is grown on hematite and
298
magnetite
[54]
. There is no
gene encoding a
periplasmic cytochrome
c
adjacent to these genes and this is
299
unusual for previously characterized
EET
conduits but, given the large number of periplasmic
300
cytochromes in HotSeep
-
1, it is conceivable that another cytochrome
c
interacts with the OmcL/K
301
homol
ogs
. This conduit is also found in Seep
-
SRB2 sp. 1, 2, 7 and 8 but does not appear to be expressed
302
as highly
as
the OetABI
in the ANME
-
1/Seep
-
SRB2 consortia
[24]
. A different putative conduit including
303
the porin, extracellular and periplasmic cytochromes
c
is present in the Seep
-
SRB1g genomes
304
(LWX52_07950
-
LWX52_07960)
(
Supplementary Table 6, 7
)
. This conduit does not have identifiable
305
homologs in
Geobacter.
The pr
esence of multiple porin:cytochrome
c
conduits in the syntrophic partners
306
suggests some flexibility in use of electron donors, possibly from different syntrophic partners. For
307
HotSeep
-
1, this observation is consistent with its ability to form partnerships
with both methane and other
308
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alkane
-
oxidizing archaea
[55]
. The role of the second conduit is less clear in Seep
-
SRB1g which to date
309
has only been shown to partner with ANME
-
2b, and currently lacks representation in enrichment
310
cultures
[14]
.
Future investigat
ion of the multiple syntrophic SRB extracellular electron transfer pathways
311
and the potential respiratory flexibility it affords to their partner archaea using transcriptomics,
312
proteomics and possibly heterologous expression methods will further expand our
understanding of
313
electron transfer in these diverse consortia.
314
315
While direct interspecies electron transfer is believed to be the dominant mechanism of syntrophic
316
coupling between the ANME and SRB partners,
the potential to use diffusible intermediates su
ch as
317
formate and hydrogen exists in some genomes of syntrophic SRB.
Hydrogenases are present in
HotSeep
-
318
1
, which
can grow without ANME using hydrogen as an electron
donor
[25]
.
We
also
identified
319
periplasmic
hydrogenases
in
Seep
-
SRB1a sp. 1, 5 and 8 (
Supplementary Table
s 7
)
which
suggest that
320
these organisms could use hydrogen as an electron donor
.
However, in
Seep
-
SRB1a
these hydrogenases
321
are
expressed at low levels (less than a twentieth of the levels of DsrB) in the ANME
-
2a/Seep
-
SRB1a
322
consortia
[22]
.
Further, previous experiments showed that the addition of hydrogen to ANME
-
2/SRB
323
consortia did not inhibit anaerobic oxidation of methane suggesting
that
hydrogen
is not the predominant
324
agent of electron transfer between ANME a
nd SRB
[26,56]
.
Perhaps,
hydrogenases
are used by Seep
-
325
SRB1a to scavenge small amounts of hydrogen from the environment.
While
membrane bound
and
326
periplasmic
hydrogenase
s
are
present in
non
-
syntrophic
Seep
-
SRB1c (
Supplementary Table 7
)
,
no
327
hydrogenases are
found
in the syntrophic relative
of Seep
-
SRB1c
and ANME partner
,
Seep
-
SRB1g.
328
Similarly periplasmic hydrogenases are present in Dissulfuribacteriales and absent in Seep
-
SRB2 (one
329
exception in 18 genomes), suggesting that in both these pa
rtners, the loss of periplasmic hydrogenases is
330
part of the adaptation to their syntrophic partnership with ANME.
We also identified
periplasmic formate
331
dehydrogenases in Seep
-
SRB1g and Seep
-
SRB1a sp. 2, 3, 8, 9 (
Supplementary Table
7
). The
332
periplasmic for
mate dehydrogenase from Seep
-
SRB1g
is
expressed in the environmental proteome at
333
Santa Monica Mounds
[21]
,
but no transcripts from the formate dehydrogenases of Seep
-
SRB1a were
334
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recovered in the ANME
-
2a/Seep
-
SRB1a incubations
[22]
.
It is possible that these s
yntrophic SRB
335
scavenge formate from the environment. Alternatively,
a recent paper
found
a hybrid of electron transfer
336
by DIET and
by
diffusible intermediates (mediated interspecies electron transfer or MIET) to be
337
energetically favorable
[57]
. In
this
model, the bulk of electrons would still be transferred by DIET, but up
338
to 10 % of electrons could be shared by MIET
via
formate
[57]
, an intermediate suggested in earlier
339
studies
[26,56]
.
This might be possible in
ANME/SRB consortia with HotSeep
-
1, some sp
ecies of Seep
-
340
SRB1a and Seep
-
SRB1g
,
but not in consortia with Seep
-
SRB2
.
The absence of periplasmic formate
341
dehydrogenases and hydrogenases in Seep
-
SRB2 as previously observed
[24]
is also true in our expanded
342
dataset. If a diffusive
intermediate
should pla
y a role in mediating electron transfer between ANME
-
2c or
343
ANME
-
1 and Seep
-
SRB2, it is not likely to be formate or hydrogen.
344
b.
Different pathways for electron transfer across the inner membrane in syntrophic
345
SRB
346
Multiheme cytochromes
c
in
SRB
are known
to mediate diverse modes of electron transfer from different
347
electron donors to a conserved sulfate reduction pathway
[58]
. There is significant
variety
in the number
348
and types
of cytochromes
c
present in sulfate reducing bacteria from the phylum
Desulfobacterota
[58]
349
and an even greater number of large cytochromes is present in syntrophic SRB
[21,24]
. To explore the
350
potential for different routes of electron transfer, we performed an analysis of all cytochromes
c
351
containing four hemes or more from t
he
genomes of
syntrophic SRB
(
see
Materials and Methods)
and
352
identified at least 27 different types of cytochromes
c
.
We
split
these
cytochromes
c
into those
predicted
353
to be
involved in extracellular electron transfer, those that act as periplasmic electron carriers and those
354
that are
components of protein complexes
involved in electron transfer across the inner membrane
355
(
Supplementary Table
6
, Supplementary Information
). Cons
erved across the syntrophic SRB
partners
356
of ANME
were the cytochromes forming the core components of the EET pathway
–
OetA, OetB, OmcX
357
and OmcS
-
like and Apc2a extracellular cytochromes, and two periplasmic cytochromes of the types,
358
TpI
c
3
[59]
and cytochrome
c
554
[58,60]
. Beyond the conserved periplasmic cytochromes
c
,
TpI
c
3
and
359
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cytochrome
c
554
,
there are
also
cytochromes binding 7
-
8 hemes that are unique to different
SRB
clades
360
(
Supplementary Table
6
).
These include a
homolog of ExtKL
[61]
from
G. sulfurreducens
that
is highly
361
expressed in Seep
-
SRB2 sp
p
.
1 and
4 during growth in a syntrophic partnership with ANME
[24]
,
and a
362
homolog of ExtA from
G. sulfurreducens
[50]
protein expressed in the ANME
-
2a/Seep
-
SRB1a
363
consortia
[22]
. Previous research
has suggested that the tetraheme cytochromes
c
are not selective as
364
electron carriers and play a role in transferring electrons to multiple different protein complexes
[62]
. It is
365
possible that these larger 7
-
8 heme binding cytochromes
c
have a more specif
ic binding partne
r.
Both the
366
ExtKL and ExtA
-
like proteins are very similar (over 45% sequence similarity) to their homologs in
367
Desulfuromonadales
. S
ince the OetI
-
type conduit is also likely transferred from this order, they might act
368
as binding
partners
.
369
370
In
SRB
, the electrons from periplasmic electron donors (reduced by DIET or MIET) are delivered through
371
inner membrane bound complexes to quinones or directly to
the heterodisulfide
DsrC in the cytoplasm via
372
transmembrane electron transfer
[58]
(
Figure 3
).
The electrons from quinones or DsrC are ultimately used
373
for
the
sulfate reduction pathway
(including SatA, AprAB and DsrAB)
[58,21,24]
. Two conserved protein
374
complexes are always found along with this pathway
–
the Qmo complexes transfers electro
ns from
375
reduced quinones to AprAB and the DsrMKJOP complexes transfers electrons from quinones to DsrC
376
and through DsrC to DsrAB
. Since both
these complexes use electrons from reduced quinones, the source
377
of reduced quinones in the inner membrane
is critic
al to different sulfate respiration pathways. Th
e
quinol
378
reducing
complex
es
and complexes that reduce DsrC provide respiratory flexibility to sulfate reducing
379
bacteria.
We also note here that the reduction of AprAB
coupled to the oxidation of menaquinone is
380
expected to be endergonic
.
T
here is a proposal that QmoABC might function through flavin
-
based
381
electron confurcation (FBEC),
using electrons from reduced quinones and a
second electron donor such
382
as ferredoxin
to
reduced AprAB
[63]
,
Since, it is not clear what the electron donor is likely to be, we do
383
not explicitly consider this reaction in our analysis.
A summary of
all
the putative complexes
that are
384
involved in the electron transport chains of the four syntroph
ic SRB is visualized in
Figure
3
to detail
385
.
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