of 102
1
Title:
Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating
1
methanotrophic and methanogenic archaea
2
Short Title: Evolution of anaerobic methanotrophic archaea
3
4
Grayson L
Chadwick
a
,&
*
#
,
Connor T
Skennerton
a
*
,
Rafael Laso
-
Pérez
b
,
c
, Andy O
Leu
d
, Daan R
5
Speth
a
, Hang
Yu
a
, Connor Morgan
-
Lang
e
, Roland
Hatzenpichler
a
,$
, Danielle
Goudeau
f
, Rex
6
Malmstrom
f
,
William J
Brazelton
g
,
Tanja
Woyke
f
, Steven
J
Hallam
e
,
h
,
i
,
j
, Gene W
Tyson
d
, Gunter
7
Wegener
b
,
c
, Antje
Boetius
b
,
c
,
k
, Victoria J
Orphan
a
,#
8
9
a
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA,
10
USA 91125
11
b
Max
-
Planck Institute for Marine Microbiology, 28359 Bremen, Germany
12
c
MARUM, Center for Marine Environmental Science, 28359
and Department of Geosciences,
13
University of Bremen, Bremen, Germany
14
d
Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, University
15
of Queensland, Brisbane, Queensland, Australia
16
e
Graduate Program in Bioinformatics, University of British Columbia, Genome Sciences Centre,
17
100
-
570 West 7th Avenu
e, Vancouver, British Columbia V5Z 4S6, Canada
18
f
US Department of Energy Joint Genome Institute,
Berkeley
, CA, USA
94720
19
g
School of Biological Sciences, University of Utah, Salt Lake City, Utah, USA
20
h
Department of Microbiology & Immunology, University of Br
itish Columbia, 2552
-
2350 Health
21
Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada
22
i
Genome Science and Technology Program, University of British Columbia, 2329 West Mall,
23
Vancouver, BC V6T 1Z4, Canada
24
j
ECOSCOPE Training Program, University of Brit
ish Columbia, Vancouver, British Columbia,
25
Canada V6T 1Z3
26
k
Alfred Wegener Institute
, Helmholtz Center
for Polar and Marine Research
,
Bremerhaven
27
28
Running Head:
29
30
#Address correspondence to Grayson L Chadwick,
ch
adwick@berkeley.edu
;
Victoria J Orphan
,
31
vorpha
n@
gps
.c
a
l
t
e
c
h.e
du
32
* T
he
s
e
a
ut
hors
c
ont
ri
but
e
d e
qua
l
l
y t
o t
hi
s
w
ork
33
$
Curre
nt
a
ffi
l
i
a
t
i
on:
D
e
pa
rt
m
e
nt
of
Che
m
i
s
t
ry
a
nd
Bi
oc
he
m
i
s
t
ry,
T
he
rm
a
l
Bi
ol
ogy
Ins
t
i
t
ut
e
,
a
nd
34
Ce
nt
e
r for Bi
ofi
l
m
E
ngi
ne
e
ri
ng, M
ont
a
na
S
t
a
t
e
U
ni
ve
rs
i
t
y, Boz
e
m
a
n, M
T
, U
S
A
59717
35
&
Curre
nt
a
ffi
l
i
a
t
i
on:
D
e
pa
rt
m
e
nt
of M
ol
e
c
ul
a
r a
nd Ce
l
l
Bi
ol
ogy, U
ni
ve
rs
i
t
y of Ca
l
i
forni
a
36
Be
rke
l
e
y, Be
rke
l
e
y, CA
, U
S
A
94720
-
3220
37
38
39
.
CC-BY-ND 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 26, 2021.
;
https://doi.org/10.1101/2021.09.25.461819
doi:
bioRxiv preprint
2
Abstract
40
T
he
a
na
e
robi
c
oxi
da
t
i
on
of
m
e
t
ha
ne
c
oupl
e
d
t
o
s
ul
fa
t
e
re
duc
t
i
on
i
s
a
m
i
c
robi
a
l
l
y
m
e
di
a
t
e
d
proc
e
s
s
41
re
qui
ri
ng
a
s
ynt
rophi
c
pa
rt
ne
rs
hi
p
be
t
w
e
e
n
a
na
e
robi
c
m
e
t
ha
not
rophi
c
(A
N
M
E
)
a
rc
ha
e
a
a
nd
s
ul
fa
t
e
42
re
duc
i
ng
ba
c
t
e
ri
a
(S
RB).
Ba
s
e
d
on
ge
nom
e
t
a
xonom
y,
A
N
M
E
l
i
ne
a
ge
s
a
re
pol
yphyl
e
t
i
c
w
i
t
hi
n
43
t
he
phyl
um
H
al
obac
t
e
r
ot
a
,
none
of
w
hi
c
h
ha
ve
be
e
n
i
s
ol
a
t
e
d
i
n
pure
c
ul
t
ure
.
H
e
re
w
e
re
c
ons
t
ruc
t
44
28
A
N
M
E
ge
nom
e
s
from
e
nvi
ronm
e
nt
a
l
m
e
t
a
ge
nom
e
s
a
nd
fl
ow
s
ort
e
d
s
ynt
rophi
c
c
ons
ort
i
a
.
45
T
oge
t
he
r
w
i
t
h
a
re
a
na
l
ys
i
s
of
pre
vi
ous
l
y
publ
i
s
he
d
da
t
a
s
e
t
s
,
t
he
s
e
ge
nom
e
s
e
na
bl
e
a
c
om
pa
ra
t
i
ve
46
a
na
l
ys
i
s
of
a
l
l
m
a
ri
ne
A
N
M
E
c
l
a
de
s
.
W
e
re
vi
e
w
t
he
ge
nom
i
c
fe
a
t
ure
s
w
hi
c
h
s
e
pa
ra
t
e
A
N
M
E
from
47
t
he
i
r
m
e
t
ha
noge
ni
c
re
l
a
t
i
ve
s
a
nd
i
de
nt
i
fy
w
ha
t
di
ffe
re
nt
i
a
t
e
s
A
N
M
E
c
l
a
de
s
.
L
a
rge
m
ul
t
i
he
m
e
48
c
yt
oc
hrom
e
s
a
nd
bi
oe
ne
rge
t
i
c
c
om
pl
e
xe
s
pre
di
c
t
e
d
t
o
be
i
nvol
ve
d
i
n
nove
l
e
l
e
c
t
ron
bi
furc
a
t
i
on
49
re
a
c
t
i
ons
a
re
w
e
l
l
-
di
s
t
r
i
but
e
d
a
nd
c
ons
e
rve
d
i
n
t
he
A
N
M
E
a
rc
ha
e
a
,
w
hi
l
e
s
i
gni
fi
c
a
nt
va
ri
a
t
i
ons
i
n
50
t
he
a
na
bol
i
c
C1
pa
t
hw
a
ys
e
xi
s
t
s
be
t
w
e
e
n
c
l
a
de
s
.
O
ur
a
na
l
ys
i
s
ra
i
s
e
s
t
he
pos
s
i
bi
l
i
t
y
t
ha
t
51
m
e
t
hyl
ot
rophi
c
m
e
t
ha
noge
ne
s
i
s
m
a
y ha
ve
e
vol
ve
d from
a
m
e
t
ha
not
rophi
c
a
nc
e
s
t
or.
52
53
.
CC-BY-ND 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 26, 2021.
;
https://doi.org/10.1101/2021.09.25.461819
doi:
bioRxiv preprint
3
Table of Contents
54
Abstract
................................
................................
................................
................................
..
2
55
Introduction
................................
................................
................................
...........................
4
56
Results
................................
................................
................................
................................
...
5
57
Genome
-
resolved diversity of the methanotrophic archaea
................................
.....................
5
58
AN
ME energy metabolism
................................
................................
................................
......
8
59
Energy metabolism phase 1: The conserved C1 machinery of the methanogenesis pathway in
60
ANME archaea
................................
................................
................................
...............................
9
61
Function of the methanogenesis pathway in ANME archaea
................................
................................
........
9
62
Differing roles for MetF in ANME archaea
................................
................................
................................
..
14
63
Energy metabolis
m phase 2: Cytoplasmic electron carrier oxidation and energy conservation
...
16
64
F
420
H
2
oxidation
................................
................................
................................
................................
...............
17
65
Ferredoxin oxidation
................................
................................
................................
................................
.......
18
66
CoM
-
SH/CoB
-
SH oxidation
................................
................................
................................
............................
19
67
Novel gene clusters encoding elect
ron bifurcation/confurcation complexes
................................
...............
20
68
Energy metabolism phase 3: Genomic evidence for mechanisms of syntrophic electron transfer
24
69
Hydrogen transfer
................................
................................
................................
................................
...........
24
70
Formate transfer
................................
................................
................................
................................
..............
25
71
Other soluble electron carriers
................................
................................
................................
.......................
26
72
Direct interspecies electr
on transfer
................................
................................
................................
..............
26
73
Potential methanophenazine:cytochrome
c
oxidoreductase complexes
................................
......................
28
74
Multiheme cytochrome
c
protein abundance and expression
................................
................................
......
29
75
S
-
layer conduits
................................
................................
................................
................................
...............
31
76
Duplication of Cytochrome
c
maturation m
achinery
................................
................................
...................
33
77
Anabolic pathways
................................
................................
................................
................
35
78
Anabolic C1 metabolism
................................
................................
................................
................................
.
36
79
Apparent amino acid prototrophy
................................
................................
................................
.................
39
80
Incomplete partial TCA cycles for 2
-
oxoglutarate synthesis
................................
................................
.......
40
81
Additional ANME genomic features of interest
................................
................................
.....
41
82
Nitrogenase in ANME
................................
................................
................................
................................
.....
41
83
ANME
-
1 genomes harbor many FrhB/FdhB/FpoF paralogs
................................
................................
......
43
84
Extensive Dockerin/Cohesin domain
-
containing proteins
................................
................................
...........
45
85
Phag
e
-
like protein translocation structures
................................
................................
................................
..
46
86
Discussion
................................
................................
................................
.............................
47
87
The evolution and conserved metabolic features of marine ANME archaea
................................
.............
47
88
The “Methanoalium” group of ANME
-
1 and the potential for methanogenesis in ANME
......................
49
89
Anabolic independence of the ANME archaea from their syntrophic partner
................................
..........
50
90
Biogeochemical and microbiological c
onsideration of ANME carbon signatures
................................
.....
50
91
MetF, F
420
-
dependent NADP reductase and electron bifurcation complexes
................................
.............
52
92
An energetic argument for both chemical diffusion and direct electron transfer in ANME
-
SRB
93
syntrophy
................................
................................
................................
................................
..........................
53
94
Conclusion
................................
................................
................................
................................
........................
55
95
Materials and Methods
................................
................................
................................
..........
55
96
.
CC-BY-ND 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted September 26, 2021.
;
https://doi.org/10.1101/2021.09.25.461819
doi:
bioRxiv preprint
4
Acknowledgments
................................
................................
................................
.................
62
97
References
................................
................................
................................
............................
62
98
Supporting Information
................................
................................
................................
........
84
99
100
I
ntroduction
101
A
na
e
robi
c
oxi
da
t
i
on
of
m
e
t
ha
ne
(A
O
M
)
c
oupl
e
d
t
o
s
ul
fa
t
e
re
duc
t
i
on
i
s
a
ke
y
m
i
c
ro
bi
ol
ogi
c
a
l
102
proc
e
s
s
i
n
oc
e
a
n
s
e
di
m
e
nt
s
t
ha
t
c
ont
rol
s
t
he
a
m
ount
of
m
e
t
ha
ne
re
l
e
a
s
e
d
i
nt
o
ove
rl
yi
ng
w
a
t
e
rs
a
nd
103
t
he
a
t
m
os
phe
re
.
H
ow
e
ve
r,
de
s
pi
t
e
t
he
gl
oba
l
re
l
e
va
nc
e
a
nd
di
s
t
ri
but
i
on
of
t
hi
s
proc
e
s
s
,
t
he
re
a
re
104
c
urre
nt
l
y
no
s
t
ra
i
n
i
s
ol
a
t
e
s
t
ha
t
w
i
l
l
c
a
rry
out
AOM
w
i
t
h
s
ul
fa
t
e
.
Th
us
,
our
unde
rs
t
a
ndi
ng
of
t
he
105
phys
i
ol
ogi
c
a
l
a
nd
bi
oc
he
m
i
c
a
l
ba
s
i
s
for
A
O
M
ha
s
a
dva
nc
e
d
m
uc
h
m
ore
s
l
ow
l
y
t
ha
n
i
t
ha
s
for
106
m
a
ny
ot
he
r
m
i
c
robi
a
l
ly
-
m
e
di
a
t
e
d
bi
oge
oc
he
m
i
c
a
l
proc
e
s
s
e
s
.
T
w
e
nt
y
ye
a
rs
a
go,
s
t
rong
e
vi
de
nc
e
107
e
m
e
rge
d
t
ha
t
a
rc
ha
e
a
m
a
y
be
i
nvol
ve
d
i
n
A
O
M
ba
s
e
d
on
s
t
a
bl
e
i
s
ot
ope
m
e
a
s
ure
m
e
nt
s
of
a
rc
ha
e
a
l
108
l
i
pi
ds
a
nd
s
m
a
l
l
s
ubuni
t
ri
bos
om
a
l
RN
A
(
S
S
U
or
16S
rRN
A
)
ge
ne
c
l
one
l
i
bra
ri
e
s
from
m
a
ri
ne
109
m
e
t
ha
ne
s
e
e
ps
(1)
.
S
hort
l
y
t
he
re
a
ft
e
r
fl
uore
s
c
e
nc
e
i
n
s
i
t
u
hybri
di
z
a
t
i
on
from
m
e
t
ha
ne
s
e
e
p
110
e
nvi
ronm
e
nt
s
provi
de
d
m
i
c
ros
c
opi
c
e
vi
de
nc
e
for
t
he
e
xi
s
t
e
nc
e
of
a
pre
va
l
e
nt
i
nt
e
rdom
a
i
n
111
c
ons
ort
i
a
c
ons
i
s
t
i
ng
of
a
n
a
rc
ha
e
on
re
l
a
t
e
d
t
o
know
n
m
e
t
ha
noge
ns
a
nd
a
ba
c
t
e
ri
um
re
l
a
t
e
d
t
o
112
s
ul
fa
t
e
re
duc
i
ng
ba
c
t
e
ri
a
(2,
3)
.
Th
e
s
e
di
s
c
ove
ri
e
s
l
e
d
t
o
t
he
c
urre
nt
pa
ra
di
gm
t
ha
t
s
ul
fa
t
e
-
113
de
pe
nde
nt
AOM
is
c
a
rri
e
d
out
by
a
na
e
robi
c
m
e
t
ha
not
rophi
c
(A
N
M
E
)
a
rc
ha
e
a
i
n
a
s
ynt
rophi
c
114
pa
rt
ne
rs
hi
p w
i
t
h s
ul
fa
t
e
re
duc
i
ng ba
c
t
e
ri
a
(S
RB).
115
116
S
ubs
e
que
nt
w
ork
ha
s
e
xpa
nde
d
our
unde
rs
t
a
ndi
ng
di
ve
rs
i
t
y
a
nd
a
c
t
i
vi
t
i
e
s
of
t
he
ANME
a
rc
ha
e
a
117
a
nd
l
e
a
d
t
o
va
ri
ous
hypot
he
s
e
s
pe
rt
a
i
ni
ng
t
o
t
he
bi
oc
he
m
i
c
a
l
m
e
c
ha
ni
s
m
s
unde
rl
yi
ng
t
he
118
s
ynt
rophi
c
i
nt
e
ra
c
t
i
ons
be
t
w
e
e
n
A
N
M
E
a
nd
S
RB.
D
i
ve
rs
i
t
y
s
urve
ys
ha
ve
s
ugge
s
t
e
d
t
ha
t
A
N
M
E
119
a
re
pol
yphyl
e
t
i
c
w
i
t
h
t
hre
e
di
s
t
i
nc
t
c
l
a
de
s
(A
N
M
E
-
1,
2,
a
nd
3)
w
i
t
hi
n
t
he
H
al
obac
t
e
r
ot
a
.
120
Inve
s
t
i
ga
t
i
ons
of
16S
rRN
A
ge
ne
phyl
oge
ni
e
s
s
upport
A
N
M
E
-
1
a
s
a
fa
m
i
l
y
-
l
e
ve
l
c
l
a
de
,
w
hi
l
e
121
ANME
-
2
i
s
c
om
pri
s
e
d
of
t
w
o
di
s
t
i
nc
t
fa
m
i
l
i
e
s
w
i
t
hi
n
t
he
Me
t
hanos
ar
c
i
nal
e
s
,
a
nd
m
e
m
be
rs
of
122
ANME
-
3
a
re
a
nove
l
ge
nus
c
l
os
e
l
y
re
l
a
t
e
d
t
o
Me
t
hanoc
oc
c
oi
de
s
w
i
t
hi
n
t
he
fa
m
i
l
y
123
Me
t
hanos
ar
c
i
nac
e
ae
(4,
5)
.
Ini
t
i
a
l
‘om
i
c
a
na
l
ys
i
s
of
fos
m
i
d
l
i
bra
ri
e
s
from
A
N
M
E
orga
ni
s
m
s
124
su
pport
ed
re
ve
rs
e
m
e
t
ha
noge
ne
s
i
s
a
s
t
he
bi
oc
he
m
i
c
a
l
m
ode
l
for
t
he
m
e
t
ha
ne
oxi
da
t
i
on
pa
t
hw
a
y
125
i
n
AOM
(6
8)
.
S
ubs
e
que
nt
a
na
l
ys
i
s
of
m
ore
c
om
pl
e
t
e
A
N
M
E
ge
nom
e
s
from
e
nri
c
hm
e
nt
c
ul
t
ure
s
126
(9
11)
,
or
m
e
t
a
ge
nom
e
a
s
s
e
m
bl
e
d
ge
nom
e
s
(M
A
G
s
)
from
A
O
M
ha
bi
t
a
t
s
ha
ve
a
dde
d
t
o
our
127
unde
rs
t
a
ndi
ng
of
s
om
e
of
t
he
m
a
j
or
groups
of
A
N
M
E
,
a
nd
furt
he
r
re
fi
ne
d
t
he
re
ve
rs
e
128
m
e
t
ha
noge
ne
s
i
s
hypot
he
s
i
s
(12, 13)
.
129
130
T
he
l
i
m
i
t
e
d
num
be
r
of
A
N
M
E
ge
nom
e
s
c
urre
nt
l
y
a
va
i
l
a
bl
e
re
l
a
t
i
ve
t
o
t
he
i
r
16S
rRN
A
ge
ne
131
di
ve
rs
i
t
y
l
e
a
ds
t
o
que
s
t
i
ons
a
bout
w
he
t
he
r
t
he
obs
e
rva
t
i
ons
m
a
de
i
n
pre
vi
ous
s
t
udi
e
s
re
pre
s
e
nt
132
c
ons
e
rve
d
fe
a
t
ure
s
of
t
he
A
N
M
E
a
rc
ha
e
a
,
or
a
re
s
ke
w
e
d
by
t
he
re
l
a
t
i
ve
l
y
s
m
a
l
l
s
a
m
pl
e
s
i
z
e
an
d
133
t
he
i
nc
om
pl
e
t
e
or
bi
a
s
e
d
na
t
ure
of
m
e
t
a
ge
nom
i
c
bi
nni
ng
m
e
t
hods
.
In
orde
r
t
o
de
ve
l
op
a
be
t
t
e
r
134
m
ode
l
for
t
he
e
vol
ut
i
on
a
nd
m
e
t
a
bol
i
c
c
a
pa
bi
l
i
t
i
e
s
of
t
he
A
N
M
E
a
rc
ha
e
a
we
pe
rform
e
d
a
l
a
rge
135
c
om
pa
ra
t
i
ve
a
na
l
ys
i
s
of
t
he
m
os
t
c
om
pl
e
t
e
s
e
t
of
A
N
M
E
ge
nom
e
s
t
o
da
t
e
,
e
nc
om
pa
s
s
i
ng
39
136
re
c
ons
t
ruc
t
e
d
MAGs
,
bi
nne
d
fos
m
i
d
l
i
bra
ri
e
s
,
a
m
pl
i
fi
e
d
s
i
ngl
e
a
ggre
ga
t
e
ge
nom
e
s
(A
S
A
G
s
)
,
a
nd
137
c
om
bi
ne
-
a
s
s
e
m
bl
e
d
s
i
ngl
e
a
m
pl
i
fi
e
d
ge
nom
e
s
(Co
-
S
A
G
s
),
m
ore
t
ha
n
doubl
i
ng
t
he
pre
vi
ous
l
y
138
a
va
i
l
a
bl
e
ge
n
om
i
c
i
nform
a
t
i
on
.
T
hi
s
a
na
l
ys
i
s
i
nc
l
ude
s
re
pre
s
e
nt
a
t
i
ve
s
from
all
re
c
ogni
z
e
d
m
a
ri
ne
139
A
N
M
E
groups
i
nc
l
udi
ng
ANME
-
1,
2a
,
2b,
2c
,
a
nd
3.
U
s
i
ng
t
hi
s
e
xpa
nde
d
da
t
a
s
e
t
w
e
c
ons
t
ruc
t
a
140
.
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