Hindawi Publishing Corporation Archaea Volume 2017, Article ID 1654237, 22 pages http://dx.doi.org/10.1155/2017/1654237
Review Article Reverse Methanogenesis and Respiration in Methanotrophic Archaea Peer H. A. Timmers,1,2,3 Cornelia U. Welte,3,4 Jasper J. Koehorst,5 Caroline M. Plugge,1,2 Mike S. M. Jetten,3,4,6 and Alfons J. M. Stams1,3,7 1
Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, Netherlands Wetsus, European Centre of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911 MA Leeuwarden, Netherlands 3 Soehngen Institute of Anaerobic Microbiology, Heyendaalseweg 135, 6525 AJ Nijmegen, Netherlands 4 Department of Microbiology, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, Netherlands 5 Laboratory of Systems and Synthetic Biology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, Netherlands 6 TU Delft Biotechnology, Julianalaan 67, 2628 BC Delft, Netherlands 7 Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 2
Correspondence should be addressed to Peer H. A. Timmers; [email protected] Received 3 August 2016; Revised 11 October 2016; Accepted 31 October 2016; Published 5 January 2017 Academic Editor: Michael W. Friedrich Copyright © 2017 Peer H. A. Timmers et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., “trace methane oxidation”). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.
1. Introduction 1.1. Anaerobic Methane-Oxidizing Archaea (ANME). Anaerobic methane-oxidizing archaea (ANME) perform anaerobic oxidation of methane (AOM) via reversal of the methanogenic pathway. ANME were first discovered in marine sediments where AOM was coupled to sulfate reduction (SR) (Table 1, reaction (1)). Here, ANME formed metabolically interdependent consortia with sulfate-reducing bacteria (SRB) that belong to the Deltaproteobacteria [1–3]. Three distinct methanotrophic groups were identified: ANME-1 (subclusters a and b), ANME-2 (subclusters a, b, and c),
and ANME-3. The ANME-1 cluster is related to Methanomicrobiales and Methanosarcinales but forms a separate cluster [2], ANME-2 are related to cultivated members of the Methanosarcinales [4], and ANME-3 are more related to Methanococcoides spp. [5] (Figure 1). The ANME clades are not monophyletic with each other and the phylogenetic distance between the subgroups is large, with 16S rRNA gene sequence similarity of only 75–92% [6]. Subclusters ANME2a and ANME-2b form a coherent clade that is distinguished from ANME-2c [7] and are therefore often grouped together as ANME-2a/b (Figure 1). The wide phylogenetic distribution is reflected in the ecological niche adaptation of the different
2
Archaea Methanomicrobiales
ANME-1 Archaeoglobales
Methanosaeta
Methanococcales
10%
Methanolobus Methanohalophilus
AAA GoM-Arc I ANME-2d ANME-2a/b ANME-2c
Methanococcoides ANME-3
Methanosarcina
Figure 1: Phylogenetic tree of full length archaeal 16S rRNA sequences showing all methanotrophic clades so far described (grey) and other archaeal clades used in our domain based (meta)genome comparison (black). The tree was constructed with the ARB software package (version arb-6.0.1.rev12565) [49] using 2800 sequences from the SILVA SSURef NR 99 database (release 119.1) [50]. Trees were calculated by maximum likelihood analysis (RAxML, PHYML) and the ARB neighbor-joining method with terminal filtering and the Jukes-Cantor correction. Resulting trees were compared manually and a consensus tree was constructed. Sulfolobales as outgroup was removed after tree calculations. The scale bar represents the percentage of changes per nucleotide position. Table 1: Gibbs free energy changes under standard conditions (Δ𝐺0 ) for anaerobic methane oxidation coupled to different electron acceptors (possibly) performed by ANME. Reaction (1) CH4 + SO4 2− → HCO3 − + HS− + H2 O (2) CH4 + 4 NO3 − → HCO3 − + 4 NO2 − + H2 O + H+ (3) CH4 + 8 Fe(OH)3 + 16 H+ → CO2 + 8 Fe2+ + 22 H2 O (4) CH4 + 4 MnO2 + 8 H+ → CO2 + 4 Mn2+ + 6 H2 O (5) CH4 + 4/3 Cr2 O7 2− + 32/3 H+ → 8/3 Cr3+ + CO2 + 22/3 H2 O
ANME clades. ANME clades involved in sulfate-dependent AOM (S-AOM) co-occur in many different marine environments, except for ANME-3 that was mainly found in mud volcanoes and in some seep sediments [6, 8, 9]. In marine sediments, a distinct zonation occurs where ANME2a/b dominate upper layers and ANME-2c and/or ANME1 abundance increases in deeper zones, indicating ecological niche separation [10–15]. ANME also form a versatile partnership with non-SRB such as beta-proteobacteria [16] and Verrucomicrobia [17]. ANME, and especially ANME-1, have also been observed without a (closely associated) bacterial partner [5, 12, 13, 18–22]. It was therefore suggested that ANME could perform AOM alone, using electron acceptors such as metal oxides, or perform other processes such as methanogenesis [23, 24]. Indications exist that AOM can
Gibbs free energy (Δ𝐺0 , kJ mol−1 ) −16.3 −517.2 −571.2 −763.2 −841.4
be coupled to the reduction of different metal (oxides) (Table 1, reactions (3)–(5)), but limited experimental evidence exists to date that ANME are responsible for this reaction (discussed in Section 3.3). Besides marine environments, ANME involved in S-AOM can be found in terrestrial [25, 26] and freshwater ecosystems [27]. A member of a fourth subcluster, “Candidatus (Ca.) Methanoperedens nitroreducens,” was recently discovered to perform nitrate-dependent AOM (N-AOM) [28] (Table 1, reaction (2)). This cluster was named “ANME-2d” [29] but later renamed to “GOM Arc I” [30] and “AOM-associated archaea (AAA)” [6]. Phylogenetic analysis shows that the ANME-2d cluster is monophyletic with “Ca. M. nitroreducens” and other AAA/GoM Arc I sequences, but distinct from other ANME-2 subclusters (Figure 1). ANME-2d were
Archaea initially enriched in bioreactors inoculated with freshwater samples [28, 31–33]. As ANME-2d archaea were only recently recognized, their environmental preferences remain insufficiently studied. So far they have been found in freshwater canals [31], soils and rice paddy fields [34–36], lakes and rivers [35], and wastewater treatment plants [33]. In situ AOM activity of ANME-2d was determined recently for the first time [36]. More ANME phylotypes in different environments and possibly new archaeal clades involved in AOM may yet have to be discovered. For example, methyl coenzyme M reductase A genes (mcrA) from Bathyarchaeota (formerly known as Miscellaneous Crenarchaeota Group) and from the new archaeal phylum Verstraetearchaeota were recently found, indicating their involvement in methane metabolism [37, 38]. This review focusses on archaea performing AOM through the reversal of the methanogenesis pathway. We describe the reversibility of the central methanogenic pathway, including the key enzyme in methanogenesis and anaerobic methanotrophy (i.e., methyl coenzyme M reductase, Mcr). The possibility of methanogens to perform methane oxidation and of ANME to perform methanogenesis is also addressed. Lastly, the physiological adaptations of ANME to perform respiration using different electron acceptors during AOM are discussed. 1.2. ANME versus Methanogens: Domain Based (Meta)Genome Comparison. In order to find additional differences between archaeal methanotrophs and related methanogens that could validate and complement findings in the literature, we performed domain based (meta)genome comparison between selected metagenomes of ANME and genomes of methanogens, as done previously for bacterial genomes [39]. For archaeal methanotrophs, we used the metagenomes of ANME-1 [40, 41], ANME-2a [42], and ANME-2d [28, 43]. For methanogens, we used genomes of closely and distantly related species able to perform acetoclastic methanogenesis (A: Methanosaeta concilii GP6), methylotrophic methanogenesis (M-1: Methanococcoides burtonii DSM6242, M-2: Methanolobus tindarius DSM2278, and M3: Methanohalophilus mahii DSM5219), hydrogenotrophic methanogenesis (H-1: Methanospirillum hungatei JF-1, H-2: Methanobacterium formicicum DSM3637, H-3: Methanococcus maripaludis C5, and H-4: Methanoregula formicica SMSP), and both acetoclastic and methylotrophic methanogenesis (AM: Methanosarcina acetivorans C2A). The genome of a sulfate-reducing archaeon that contained most enzymes for methanogenesis except for Mcr (S: Archaeoglobus fulgidus DSM 4304) was also included in the comparison. For each dataset the protein domains were obtained through InterProScan 5.17-56.0 using the TIGRFAM, ProDom, SMART, PROSITE, PfamA, PRINTS, SUPERFAMILY, COILS, and Gene3D domain databases. Results of the analysis are given in Table 2 and Table S1 of the Supplementary Material available online at https://doi.org/10.1155/2017/1654237. Since the ANME-1 metagenome assembled by Stokke et al. 2012 [40] contained many bacterial genes, we did not refer to this data for the domain based (meta)genome comparison but only used the ANME-1 metagenome described by Meyerdierks
3 et al., 2010 [41]. We included both ANME-1 metagenomes to analyze the organization of genes for the formaldehydeactivating enzyme (Table S2) and the iron-only hydrogenase (Table S3).
2. Reversal of the Methanogenesis Pathway 2.1. The Central Methanogenesis Pathway. ANME are described to perform “reverse methanogenesis” [44] which implies the complete reversal of methanogenesis from H2 and CO2 , that is, hydrogenotrophic methanogenesis (for kinetic and thermodynamic considerations, the reader is referred to [45]). During “forward” hydrogenotrophic methanogenesis, CO2 is reduced to CH4 with reducing equivalents derived from H2 (Figure 2). During methylotrophic methanogenesis, this pathway is already partly reversed. Methylotrophic methanogens utilize one-carbon compounds such as methylamines, methanol or methylated sulfur compounds (methanethiol, dimethyl sulfide) that are activated to methyl coenzyme M. About 75% of the methyl coenzyme M (CH3 -CoM) is reduced to produce CH4 and about 25% of CH3 -CoM is oxidized to CO2 using the methanogenesis pathway in reverse during methylotrophic growth. The oxidative part provides reducing equivalents that are needed for the generation of the proton motive force in the methanogenic respiratory chain and the reduction of CH3 CoM by methyl coenzyme M reductase (Mcr) [46] (Figure 3). In all methanogens, the Mcr reaction operates in the forward reaction and yields methane and the heterodisulfide of coenzyme B and coenzyme M (CoB-S-S-CoM): CH3 -CoM + CoB-SH → CH4 + CoB-S-S-CoM; Δ𝐺0 = −30 kJ mol−1 [52]
(1)
The heterodisulfide is a central intermediate and acts as terminal electron acceptor in all methanogens. In hydrogenotrophic methanogens without cytochromes, it is the electron acceptor of the cytoplasmic electron-bifurcating CoB-S-S-CoM reductase (HdrABC) and F420 -nonreducing hydrogenase (MvhADG) complex [47, 48] that is needed to provide reduced ferredoxin for the first step in methanogenesis; the reduction of CO2 to a formyl group. Within the methanogens with cytochromes, only a few members of the genus Methanosarcina are able to grow on H2 /CO2 . They use a ferredoxin-dependent hydrogenase (Ech) for ferredoxin reduction and an additional membrane bound methanophenazine-dependent hydrogenase (Vho) for H2 oxidation coupled to the reduction of the heterodisulfide by the membrane bound CoB-S-S-CoM reductase (HdrDE). F420 cycling can be accomplished using the F420 -dependent hydrogenase (Frh) and F420 H2 : phenazine oxidoreductase (Fpo) complex [48] (Figure 2). For methanogens it is of crucial importance that Mcr operates in the forward reaction to yield methane and the heterodisulfide. If all reactions of the methanogenic pathway are reversed such as during AOM, the pathway requires the input of energy and produces electron donors (Figures 4–6). Therefore, during AOM, an external electron acceptor is needed which makes the
Central pathway Mcr, alpha subunit, N-terminal Mcr, alpha subunit, N-terminal subdomain 1 Mcr, alpha subunit, N-terminal subdomain 2 Mcr, alpha subunit, C-terminal Mcr, alpha/beta subunit, C-terminal Mcr, alpha/beta subunit, domain 2, C-terminal Mcr, beta subunit Mcr, beta subunit, C-terminal Mcr, beta subunit, N-terminal Mcr, gamma subunit Mcr, protein C Mcr, protein C-like Mcr, protein D Mcr, ferredoxin-like fold 5,10methylenetetrahydromethanopterin reductase Acetoclastic methanogenesis CO dehydrogenase/acetyl-CoA synthase complex alpha subunit CO dehydrogenase/acetyl-CoA synthase complex beta subunit CO dehydrogenase/acetyl-CoA synthase delta subunit CO dehydrogenase/acetyl-CoA synthase delta subunit, TIM barrel CO dehydrogenase b subunit/acetyl-CoA synthase epsilon subunit 4 2 2 3 6 9 1 3 4 14 2 5 0 12 0
0 13 5 11 3
IPR015811
IPR015823
IPR009047 IPR008924
IPR022681
IPR003179 IPR022679 IPR022680 IPR003178 IPR007687 IPR026327 IPR003901 IPR009024
IPR019946
IPR004460
IPR004461
IPR004486
IPR016041
IPR003704
ANME-1 S
IPR003183
InterPro ID
2
2
1
4
1
0
2 2 2 8 1 2 0 6
6
2 4
2
3
2
ANME-1 M
2
5
1
2
1
2
1 1 1 4 1 2 3 3
3
1 2
2
1
1
ANME-2a
2
3
1
2
1
1
1 1 1 4 1 2 6 3
3
1 2
2
1
1
ANME-2d H
2
2
1
2
1
1
1 1 1 4 1 2 6 3
3
1 2
2
1
1
ANME-2d A
4
4
2
4
3
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
AM
2
2
1
4
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
A
2
2
1
2
1
1
1 1 1 4 1 1 3 3
3
1 2
2
1
1
M-1
2
3
1
2
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
M-2
2
3
1
2
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
M-3
2
3
1
2
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
H-1
2
2
1
2
1
1
2 2 2 8 1 2 9 6
6
2 4
4
2
2
H-2
2
2
1
2
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
H-3
2
5
1
4
1
1
2 2 2 8 1 1 6 6
6
2 4
4
2
2
H-4
4
2
1
2
2
1
0 0 0 0 0 0 0 0
0
0 0
0
0
0
S
Table 2: Domain based (meta)genome comparison of selected metagenomes of methanotrophs and selected genomes of other archaea. Domain abundance in every (meta)genome is indicated by numbers. S-AOM performing ANME include ANME-1-s [40], ANME-1-m [41], and ANME-2a [42]. N-AOM performing ANME include ANME-2d-h [28] and ANME-2da [43]. The acetoclastic (A) and methylotrophic (M) methanogens include Methanosarcina acetivorans C2A (AM), Methanosaeta concilii GP6 (A), Methanococcoides burtonii DSM6242 (M-1), Methanolobus tindarius DSM2278 (M-2), and Methanohalophilus mahii DSM5219 (M-3). Hydrogenotrophic methanogens (H) include Methanospirillum hungatei JF-1 (H-1), Methanobacterium formicicum DSM3637 (H-2), Methanococcus maripaludis C5 (H-3), and Methanoregula formicica SMSP (H-4). The sulfate-reducing archaeon (S) is Archaeoglobus fulgidus DSM 4304.
4 Archaea
Methylotrophic methanogenesis Methyltransferase MtaA/CmuA Methanol-cobalamin methyltransferase, B subunit Monomethylamine methyltransferase MtmB Trimethylamine methyltransferase Dimethylamine methyltransferase MtbB Trimethylamine methyltransferase, Methanosarcina Methylamine methyltransferase corrinoid protein reductive activase C-type cytochromes Di-haem cytochrome, transmembrane, nitrate reduction Doubled CXXCH motif Cytochrome c-like domain Class III cytochrome C (tetraheme cytochrome) Tetraheme cytochrome domain, flavocytochrome c3 (Shewanella) Octaheme c-type cytochrome Methanogenesis multiheme c-type cytochrome Multiheme cytochrome S-layer domains S-layer family duplication domain Sarcinarray family protein S-layer homology domain Cell export and cell adhesion HYR domain CARDB domain Collagen-binding surface protein Cna-like, B-type domain von Willebrand factor, type A VWA N-terminal Adhesion lipoprotein Adhesin B 13 0 1 1 64 4 55 1 8 0
IPR006457 IPR026476 IPR001119
IPR003410 IPR011635
IPR008454
IPR002035 IPR013608 IPR006128 IPR006129
4
IPR012286
78
2
IPR020942
IPR011031
33 2
IPR010177 IPR009056
1
2
IPR016174
0
0
IPR026339
IPR027594
0
IPR012740
IPR024673
0
IPR012653
28 2 4 3
1
5 14
0 0 0
15
0
0
0
0
0 0
0
0
0
0
0
0
3 11
IPR008031
IPR010426
0
0
IPR021079
0
ANME-1 M
0
ANME-1 S
IPR006360
InterPro ID
17 0 4 4
2
2 15
29 6 2
52
1
2
4
0
3 6
4
0
0
0
0
0
0
0
ANME-2a
32 0 5 5
1
0 6
16 0 0
80
0
0
5
3
2 0
3
0
0
0
0
0
0
0
ANME-2d H
Table 2: Continued.
14 0 0 0
8
0 6
26 4 0
73
0
0
4
3
3 17
7
0
0
0
0
0
0
0
ANME-2d A
37 0 0 0
0
0 30
34 1 0
3
1
0
2
0
0 15
4
4
2
9
4
12
3
7
AM
23 0 5 3
6
0 4
19 0 0
0
0
0
0
0
0 0
0
0
0
0
0
0
0
0
A
17 0 3 0
0
0 1
16 0 0
6
1
0
0
0
0 4
0
2
1
6
5
12
2
4
M-1
42 0 0 0
2
0 4
44 2 0
8
1
2
1
0
0 8
4
2
1
6
2
21
2
5
M-2
9 0 4 4
2
0 0
17 1 0
14
1
2
0
0
0 6
0
2
1
6
2
15
2
6
M-3
28 0 4 0
0
0 9
0 0 0
0
0
0
1
0
0 0
1
0
0
0
0
0
0
0
H-1
8 0 5 3
0
0 0
0 0 0
0
0
0
0
0
0 0
1
0
0
0
0
0
0
0
H-2
0 0 0 0
0
0 0
0 0 0
0
0
0
0
0
0 0
0
0
0
0
0
0
0
1
H-3
27 0 0 0
0
0 3
0 0 0
1
0
0
0
0
0 0
0
0
0
0
0
0
0
0
H-4
7 0 0 0
0
0 8
0 0 0
7
0
1
2
0
0 4
1
0
0
0
0
0
0
0
S
Archaea 5
InterPro ID Invasin/intimin cell-adhesion IPR008964 fragments Putative cell wall binding repeat 2 IPR007253 Archaeosortase A IPR014522 Archaeosortase B IPR026430 Archaeosortase C IPR022504 Exosortase/archaeosortase domain IPR026392 Exosortase, EpsH IPR013426 Exosortase EpsH-related IPR019127 Archaeosortase family protein ArtE IPR026485 Cell wall hydrolase/autolysin, IPR002508 catalytic PEF-CTERM protein sorting IPR017474 domain PGF-pre-PGF domain IPR026453 PGF-CTERM archaeal IPR026371 protein-sorting signal LPXTG cell wall anchor domain IPR019931 VPXXXP-CTERM protein sorting IPR026428 domain Cellulosome-related/Dockerin Cellulosome anchoring protein, IPR002102 cohesin domain Dockerin domain IPR016134 Dockerin type I repeat IPR002105 Carbohydrate-binding domains Carbohydrate-binding domain IPR008965 Carbohydrate-binding-like fold IPR013784 Carbohydrate-binding, CenC-like IPR003305 Carbohydrate-binding/sugar IPR006633 hydrolysis domain Carbohydrate-binding domain, IPR010502 family 9 Bacteroidetes-associated carbohydrate-binding often IPR024361 N-terminal Carbohydrate binding module, IPR031768 xylan-binding domain Galactose-binding domain-like IPR008979
ANME-1 M 0 2 0 0 0 0 0 2 0 2 0 1 4 0 0
77 112 0 42 5 0 22 0 0 0 1
ANME-1 S 17 2 2 0 0 5 1 4 0 2 0 1 9 3 0
80 149 1 50 14 1 27 1 0 0 12
17
0
1
0
5
39 3 7
44 5
52
0
1
6
18
3
1
0 1 0 1 2 0 2 0
3
ANME-2a
6
0
0
0
2
2 10 0
4 0
4
0
0
2
9
4
0
0 2 0 2 5 1 3 0
2
ANME-2d H
Table 2: Continued.
39
1
0
2
2
6 19 17
6 0
12
0
0
1
8
1
0
0 2 0 1 3 0 2 0
6
ANME-2d A
4
0
0
0
27
2 0 0
5 0
2
0
0
2
24
0
0
0 1 0 0 1 0 1 0
7
AM
4
0
0
0
3
0 0 0
17 0
0
0
1
0
0
0
0
0 1 0 0 1 0 1 0
0
A
2
0
0
0
7
3 0 0
2 0
4
0
1
2
7
10
0
0 1 0 1 2 0 2 0
2
M-1
1
0
0
0
8
13 0 0
4 0
15
0
0
1
21
19
0
0 1 0 1 2 0 1 0
7
M-2
3
0
0
0
8
2 1 0
0 0
3
5
0
2
11
0
0
0 1 1 0 2 0 1 0
0
M-3
0
0
0
0
0
3 3 0
9 0
1
0
0
1
0
0
0
0 1 0 0 1 0 1 0
1
H-1
0
0
0
0
5
0 0 0
0 0
1
0
0
0
0
0
0
0 0 0 0 0 0 0 0
2
H-2
0
0
0
0
2
1 0 0
0 0
0
0
0
0
0
0
0
0 0 0 0 1 0 1 1
0
H-3
0
0
0
0
1
0 6 0
0 0
0
0
0
1
1
0
1
0 2 0 0 2 0 2 0
2
H-4
0
0
0
0
8
2 1 0
2 1
4
0
0
3
1
0
0
0 1 0 0 1 0 1 0
2
S
6 Archaea
Menaquinones 3-Demethylubiquinone-9 3-methyltransferase Succinate dehydrogenase/fumarate reductase, flavoprotein subunit UbiE/COQ5 methyltransferase UbiE/COQ5 methyltransferase, conserved site NrfD family Futalosine hydrolase Cyclic dehypoxanthine futalosine synthase Aminodeoxyfutalosine synthase Menaquinone synthesis (chorismate dehydratase & naphthoate synthase) FO synthase, subunit 2 Phenazines Phenazine biosynthesis PhzF protein 0 0 3 3 3 0 1 0 8 3 1
IPR014006
IPR004033
IPR023576
IPR005614 IPR019963
IPR022431
IPR022432
IPR003773
IPR020050
IPR003719
ANME-1 S
IPR028973
InterPro ID
1
3
6
0
1
0 0
0
0
0
0
ANME-1 M
4
1
0
0
0
0 0
0
0
1
0
0
3
2
1
1
4 1
2
2
2
3
ANME-2d H
Table 2: Continued. ANME-2a
0
3
2
1
1
3 1
2
2
1
3
ANME-2d A
2
2
0
0
0
0 0
0
1
0
0
AM
0
1
0
0
0
0 0
0
0
0
0
A
0
2
0
0
0
0 0
0
1
0
0
M-1
0
1
0
0
0
0 0
1
0
0
0
M-2
0
2
0
0
0
0 0
0
0
1
0
M-3
0
2
0
0
0
0 0
0
0
0
0
H-1
0
2
0
0
0
0 0
0
0
0
1
H-2
0
2
0
0
0
0 0
0
0
0
0
H-3
2
2
0
0
0
0 0
0
0
0
0
H-4
0
3
2
0
1
3 0
0
0
1
0
S
Archaea 7
8
Archaea
CH4
2H+
CoB-S-S-CoM HdrDE
Mcr
CoB-SH
MPhH2 H2
CoM-SH CH3 -CoM
MPh Vho
Mtr
2Na+
CH3 -H4 MPT F420 H2
Mer
F420
H2
Frh
CH2 =H4 MPT F420 H2 Mtd
F420
H2
CH≡H4 MPT H2 O
Mch
CHO-H4 MPT Ftr
H2 H+
Ech
CHO-MFR Fdred Fdox
Fmd CO2
Figure 2: Hydrogenotrophic methanogenesis in cytochrome containing Methanosarcina barkeri. Black lines represent presence of conversions. See Table 3 for nomenclature.
reaction thermodynamically favourable (Table 1). The reverse reaction of Mcr is therefore an essential step in AOM and is discussed in Section 2.2. The respiratory chain that is needed for using different terminal electron acceptors will be discussed in Section 3. Evidence that ANME use the reverse methanogenesis pathway during AOM is derived from metagenomic and metatranscriptomic analyses. This showed that all genes for the (reverse) methanogenic pathway were present and expressed in ANME-2a [42] (Figure 4) and ANME-2d [28] (Figure 5). ANME-1 were consistently lacking the gene encoding N5 ,N10 -methylene- tetrahydromethanopterin (H4 MPT) reductase (Mer), which is an enzyme needed to oxidize methyl-H4 MPT during methane oxidation [40, 41, 44, 51] (Figure 6, Table 2). Possible explanations could be that (1) the mer gene is present but not yet detected, (2) ANME1 used a bypass of this step, and (3) Mer was replaced by a structural analogue. The first possibility is highly unlikely. Although no closed genome of ANME-1 is publicly available to date, all ANME-1 metagenomes consistently only lack Mer and no other methanogenic genes. The second possibility was proposed previously where ANME-1 uses a bypass of
Mer via the formation of methanol or methylamine [41], as was detected in deletion mutants of Methanosarcina [52, 53]. Here, CH3 -CoM was presumably converted to methanol by a methyltransferase and subsequently to formaldehyde by a methanol dehydrogenase (Mdh). Then, formaldehyde would be converted to N5 ,N10 methylene-H4 MPT using a fusion protein of the formaldehyde-activating enzyme (Fae) and a hexulose-6-phosphate synthase (Hps) [53] (Figure 6). Both Fae and Hps were found in the ANME-1 metagenome [41] and metaproteome [40, 51]. However, no genes coding for enzymes involved in methanol metabolism were detected in these ANME-1 datasets [40, 41, 51] (Table 2), indicating that this alternative pathway probably does not occur. The presence of the Fae/Hps fusion protein in ANME-1 during AOM could also be explained by its involvement in ribose phosphate synthesis and not in AOM [54]. Indeed, the Fae gene domains of ANME-1 were located in between ribulosephosphate binding barrel and ribosomal protein S5 domains (Table S2). The third possibility of a structural analogue is most likely since an analogue of N5 ,N10 -methylene tetrahydrofolate (H4 HF) reductase (MetF) was expressed by ANME1 during AOM which could replace Mer [40] (Figure 6).
Archaea
9 Table 3: Nomenclature.
Central methanogenic pathway Fmd Ftr Mch Mtd Mer Mtr Mcr Mdh Fae/Hps MetF Electron transport Mvh Vho Fpo Fqo Hdr Frh Ech MePh/MePhH2 MQ/MQH2 Cyt b Cyt c MHC Rieske Orf7 Nar Nap Nrf
Formylmethanofuran (CHO-MFR) dehydrogenase Formylmethanofuran-tetrahydromethanopterin (H4 MPT) formyltransferase N5 ,N10 -methenyl-H4 MPT cyclohydrolase F420 H2 -dependent methylene -H4 MPT dehydrogenase N5 ,N10 -methylene-H4 MPT reductase 5 N -methyl-H4 MPT:coenzyme M (CoM) methyltransferase Methyl coenzyme M (CH3 -CoM) reductase Methanol dehydrogenase Fusion protein of formaldehyde activating enzyme/ hexulose-6-phosphate synthase N5 ,N10 -methylene tetrahydrofolate (H4 HF) reductase analogue F420 -nonreducing hydrogenase Methanophenazine-dependent hydrogenase F420 H2 :phenazine oxidoreductase F420 H2 :quinone oxidoreductase Coenzyme B-coenzyme M heterodisulfide (CoB-S-S-CoM) reductase F420 -dependent hydrogenase Ferredoxin-dependent hydrogenase Methanophenazine Menaquinone Cytochrome b Cytochrome c Multiheme c-type cytochrome Rieske cytochrome b complex Pseudoperiplasmic b-type cytochrome Nitrate reductase Periplasmic nitrate reductase Nitrite reductase
2.2. Methyl Coenzyme M Reductase (Mcr). The enzymatic reaction of a purified Mcr from ANME has not been measured to date. The key question is whether a methanogenic Mcr can explain the observed in situ AOM rates or if the Mcr of ANME is structurally altered. There are three main factors to be considered: the kinetic limitations as defined by enzyme properties (i.e., half-maximal activity at a specific 𝐾𝑀 value), the thermodynamic constraints of the enzymatic reaction, and the maximal or ambient rate of the enzymatic reaction. For the Mcr from Methanothermobacter marburgensis, kinetic parameters have been determined to illustrate the reversibility of reaction (1). In the methanogenic reaction, the purified Mcr isoform I [55] catalyzes the production of methane with a 𝑉max of 30 U mg−1 and a 𝐾𝑀 of 5 mM for CH3 -CoM. The same (methanogenic) enzyme was able to oxidize methane to CH3 -CoM with a rate of 0.0114 U mg−1 and a 𝐾𝑀 for methane of ∼10 mM [56]. To answer if the observed AOM rates are in accordance with the measured methane oxidation rate for the purified Mcr enzyme from M. marburgensis, the Mcr activity during AOM is needed. Estimates for AOM rates in terms of activity (per cell dry mass) range between
Review Article Reverse Methanogenesis and Respiration in Methanotrophic Archaea Peer H. A. Timmers,1,2,3 Cornelia U. Welte,3,4 Jasper J. Koehorst,5 Caroline M. Plugge,1,2 Mike S. M. Jetten,3,4,6 and Alfons J. M. Stams1,3,7 1
Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, Netherlands Wetsus, European Centre of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911 MA Leeuwarden, Netherlands 3 Soehngen Institute of Anaerobic Microbiology, Heyendaalseweg 135, 6525 AJ Nijmegen, Netherlands 4 Department of Microbiology, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, Netherlands 5 Laboratory of Systems and Synthetic Biology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, Netherlands 6 TU Delft Biotechnology, Julianalaan 67, 2628 BC Delft, Netherlands 7 Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 2
Correspondence should be addressed to Peer H. A. Timmers; [email protected] Received 3 August 2016; Revised 11 October 2016; Accepted 31 October 2016; Published 5 January 2017 Academic Editor: Michael W. Friedrich Copyright © 2017 Peer H. A. Timmers et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., “trace methane oxidation”). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.
1. Introduction 1.1. Anaerobic Methane-Oxidizing Archaea (ANME). Anaerobic methane-oxidizing archaea (ANME) perform anaerobic oxidation of methane (AOM) via reversal of the methanogenic pathway. ANME were first discovered in marine sediments where AOM was coupled to sulfate reduction (SR) (Table 1, reaction (1)). Here, ANME formed metabolically interdependent consortia with sulfate-reducing bacteria (SRB) that belong to the Deltaproteobacteria [1–3]. Three distinct methanotrophic groups were identified: ANME-1 (subclusters a and b), ANME-2 (subclusters a, b, and c),
and ANME-3. The ANME-1 cluster is related to Methanomicrobiales and Methanosarcinales but forms a separate cluster [2], ANME-2 are related to cultivated members of the Methanosarcinales [4], and ANME-3 are more related to Methanococcoides spp. [5] (Figure 1). The ANME clades are not monophyletic with each other and the phylogenetic distance between the subgroups is large, with 16S rRNA gene sequence similarity of only 75–92% [6]. Subclusters ANME2a and ANME-2b form a coherent clade that is distinguished from ANME-2c [7] and are therefore often grouped together as ANME-2a/b (Figure 1). The wide phylogenetic distribution is reflected in the ecological niche adaptation of the different
2
Archaea Methanomicrobiales
ANME-1 Archaeoglobales
Methanosaeta
Methanococcales
10%
Methanolobus Methanohalophilus
AAA GoM-Arc I ANME-2d ANME-2a/b ANME-2c
Methanococcoides ANME-3
Methanosarcina
Figure 1: Phylogenetic tree of full length archaeal 16S rRNA sequences showing all methanotrophic clades so far described (grey) and other archaeal clades used in our domain based (meta)genome comparison (black). The tree was constructed with the ARB software package (version arb-6.0.1.rev12565) [49] using 2800 sequences from the SILVA SSURef NR 99 database (release 119.1) [50]. Trees were calculated by maximum likelihood analysis (RAxML, PHYML) and the ARB neighbor-joining method with terminal filtering and the Jukes-Cantor correction. Resulting trees were compared manually and a consensus tree was constructed. Sulfolobales as outgroup was removed after tree calculations. The scale bar represents the percentage of changes per nucleotide position. Table 1: Gibbs free energy changes under standard conditions (Δ𝐺0 ) for anaerobic methane oxidation coupled to different electron acceptors (possibly) performed by ANME. Reaction (1) CH4 + SO4 2− → HCO3 − + HS− + H2 O (2) CH4 + 4 NO3 − → HCO3 − + 4 NO2 − + H2 O + H+ (3) CH4 + 8 Fe(OH)3 + 16 H+ → CO2 + 8 Fe2+ + 22 H2 O (4) CH4 + 4 MnO2 + 8 H+ → CO2 + 4 Mn2+ + 6 H2 O (5) CH4 + 4/3 Cr2 O7 2− + 32/3 H+ → 8/3 Cr3+ + CO2 + 22/3 H2 O
ANME clades. ANME clades involved in sulfate-dependent AOM (S-AOM) co-occur in many different marine environments, except for ANME-3 that was mainly found in mud volcanoes and in some seep sediments [6, 8, 9]. In marine sediments, a distinct zonation occurs where ANME2a/b dominate upper layers and ANME-2c and/or ANME1 abundance increases in deeper zones, indicating ecological niche separation [10–15]. ANME also form a versatile partnership with non-SRB such as beta-proteobacteria [16] and Verrucomicrobia [17]. ANME, and especially ANME-1, have also been observed without a (closely associated) bacterial partner [5, 12, 13, 18–22]. It was therefore suggested that ANME could perform AOM alone, using electron acceptors such as metal oxides, or perform other processes such as methanogenesis [23, 24]. Indications exist that AOM can
Gibbs free energy (Δ𝐺0 , kJ mol−1 ) −16.3 −517.2 −571.2 −763.2 −841.4
be coupled to the reduction of different metal (oxides) (Table 1, reactions (3)–(5)), but limited experimental evidence exists to date that ANME are responsible for this reaction (discussed in Section 3.3). Besides marine environments, ANME involved in S-AOM can be found in terrestrial [25, 26] and freshwater ecosystems [27]. A member of a fourth subcluster, “Candidatus (Ca.) Methanoperedens nitroreducens,” was recently discovered to perform nitrate-dependent AOM (N-AOM) [28] (Table 1, reaction (2)). This cluster was named “ANME-2d” [29] but later renamed to “GOM Arc I” [30] and “AOM-associated archaea (AAA)” [6]. Phylogenetic analysis shows that the ANME-2d cluster is monophyletic with “Ca. M. nitroreducens” and other AAA/GoM Arc I sequences, but distinct from other ANME-2 subclusters (Figure 1). ANME-2d were
Archaea initially enriched in bioreactors inoculated with freshwater samples [28, 31–33]. As ANME-2d archaea were only recently recognized, their environmental preferences remain insufficiently studied. So far they have been found in freshwater canals [31], soils and rice paddy fields [34–36], lakes and rivers [35], and wastewater treatment plants [33]. In situ AOM activity of ANME-2d was determined recently for the first time [36]. More ANME phylotypes in different environments and possibly new archaeal clades involved in AOM may yet have to be discovered. For example, methyl coenzyme M reductase A genes (mcrA) from Bathyarchaeota (formerly known as Miscellaneous Crenarchaeota Group) and from the new archaeal phylum Verstraetearchaeota were recently found, indicating their involvement in methane metabolism [37, 38]. This review focusses on archaea performing AOM through the reversal of the methanogenesis pathway. We describe the reversibility of the central methanogenic pathway, including the key enzyme in methanogenesis and anaerobic methanotrophy (i.e., methyl coenzyme M reductase, Mcr). The possibility of methanogens to perform methane oxidation and of ANME to perform methanogenesis is also addressed. Lastly, the physiological adaptations of ANME to perform respiration using different electron acceptors during AOM are discussed. 1.2. ANME versus Methanogens: Domain Based (Meta)Genome Comparison. In order to find additional differences between archaeal methanotrophs and related methanogens that could validate and complement findings in the literature, we performed domain based (meta)genome comparison between selected metagenomes of ANME and genomes of methanogens, as done previously for bacterial genomes [39]. For archaeal methanotrophs, we used the metagenomes of ANME-1 [40, 41], ANME-2a [42], and ANME-2d [28, 43]. For methanogens, we used genomes of closely and distantly related species able to perform acetoclastic methanogenesis (A: Methanosaeta concilii GP6), methylotrophic methanogenesis (M-1: Methanococcoides burtonii DSM6242, M-2: Methanolobus tindarius DSM2278, and M3: Methanohalophilus mahii DSM5219), hydrogenotrophic methanogenesis (H-1: Methanospirillum hungatei JF-1, H-2: Methanobacterium formicicum DSM3637, H-3: Methanococcus maripaludis C5, and H-4: Methanoregula formicica SMSP), and both acetoclastic and methylotrophic methanogenesis (AM: Methanosarcina acetivorans C2A). The genome of a sulfate-reducing archaeon that contained most enzymes for methanogenesis except for Mcr (S: Archaeoglobus fulgidus DSM 4304) was also included in the comparison. For each dataset the protein domains were obtained through InterProScan 5.17-56.0 using the TIGRFAM, ProDom, SMART, PROSITE, PfamA, PRINTS, SUPERFAMILY, COILS, and Gene3D domain databases. Results of the analysis are given in Table 2 and Table S1 of the Supplementary Material available online at https://doi.org/10.1155/2017/1654237. Since the ANME-1 metagenome assembled by Stokke et al. 2012 [40] contained many bacterial genes, we did not refer to this data for the domain based (meta)genome comparison but only used the ANME-1 metagenome described by Meyerdierks
3 et al., 2010 [41]. We included both ANME-1 metagenomes to analyze the organization of genes for the formaldehydeactivating enzyme (Table S2) and the iron-only hydrogenase (Table S3).
2. Reversal of the Methanogenesis Pathway 2.1. The Central Methanogenesis Pathway. ANME are described to perform “reverse methanogenesis” [44] which implies the complete reversal of methanogenesis from H2 and CO2 , that is, hydrogenotrophic methanogenesis (for kinetic and thermodynamic considerations, the reader is referred to [45]). During “forward” hydrogenotrophic methanogenesis, CO2 is reduced to CH4 with reducing equivalents derived from H2 (Figure 2). During methylotrophic methanogenesis, this pathway is already partly reversed. Methylotrophic methanogens utilize one-carbon compounds such as methylamines, methanol or methylated sulfur compounds (methanethiol, dimethyl sulfide) that are activated to methyl coenzyme M. About 75% of the methyl coenzyme M (CH3 -CoM) is reduced to produce CH4 and about 25% of CH3 -CoM is oxidized to CO2 using the methanogenesis pathway in reverse during methylotrophic growth. The oxidative part provides reducing equivalents that are needed for the generation of the proton motive force in the methanogenic respiratory chain and the reduction of CH3 CoM by methyl coenzyme M reductase (Mcr) [46] (Figure 3). In all methanogens, the Mcr reaction operates in the forward reaction and yields methane and the heterodisulfide of coenzyme B and coenzyme M (CoB-S-S-CoM): CH3 -CoM + CoB-SH → CH4 + CoB-S-S-CoM; Δ𝐺0 = −30 kJ mol−1 [52]
(1)
The heterodisulfide is a central intermediate and acts as terminal electron acceptor in all methanogens. In hydrogenotrophic methanogens without cytochromes, it is the electron acceptor of the cytoplasmic electron-bifurcating CoB-S-S-CoM reductase (HdrABC) and F420 -nonreducing hydrogenase (MvhADG) complex [47, 48] that is needed to provide reduced ferredoxin for the first step in methanogenesis; the reduction of CO2 to a formyl group. Within the methanogens with cytochromes, only a few members of the genus Methanosarcina are able to grow on H2 /CO2 . They use a ferredoxin-dependent hydrogenase (Ech) for ferredoxin reduction and an additional membrane bound methanophenazine-dependent hydrogenase (Vho) for H2 oxidation coupled to the reduction of the heterodisulfide by the membrane bound CoB-S-S-CoM reductase (HdrDE). F420 cycling can be accomplished using the F420 -dependent hydrogenase (Frh) and F420 H2 : phenazine oxidoreductase (Fpo) complex [48] (Figure 2). For methanogens it is of crucial importance that Mcr operates in the forward reaction to yield methane and the heterodisulfide. If all reactions of the methanogenic pathway are reversed such as during AOM, the pathway requires the input of energy and produces electron donors (Figures 4–6). Therefore, during AOM, an external electron acceptor is needed which makes the
Central pathway Mcr, alpha subunit, N-terminal Mcr, alpha subunit, N-terminal subdomain 1 Mcr, alpha subunit, N-terminal subdomain 2 Mcr, alpha subunit, C-terminal Mcr, alpha/beta subunit, C-terminal Mcr, alpha/beta subunit, domain 2, C-terminal Mcr, beta subunit Mcr, beta subunit, C-terminal Mcr, beta subunit, N-terminal Mcr, gamma subunit Mcr, protein C Mcr, protein C-like Mcr, protein D Mcr, ferredoxin-like fold 5,10methylenetetrahydromethanopterin reductase Acetoclastic methanogenesis CO dehydrogenase/acetyl-CoA synthase complex alpha subunit CO dehydrogenase/acetyl-CoA synthase complex beta subunit CO dehydrogenase/acetyl-CoA synthase delta subunit CO dehydrogenase/acetyl-CoA synthase delta subunit, TIM barrel CO dehydrogenase b subunit/acetyl-CoA synthase epsilon subunit 4 2 2 3 6 9 1 3 4 14 2 5 0 12 0
0 13 5 11 3
IPR015811
IPR015823
IPR009047 IPR008924
IPR022681
IPR003179 IPR022679 IPR022680 IPR003178 IPR007687 IPR026327 IPR003901 IPR009024
IPR019946
IPR004460
IPR004461
IPR004486
IPR016041
IPR003704
ANME-1 S
IPR003183
InterPro ID
2
2
1
4
1
0
2 2 2 8 1 2 0 6
6
2 4
2
3
2
ANME-1 M
2
5
1
2
1
2
1 1 1 4 1 2 3 3
3
1 2
2
1
1
ANME-2a
2
3
1
2
1
1
1 1 1 4 1 2 6 3
3
1 2
2
1
1
ANME-2d H
2
2
1
2
1
1
1 1 1 4 1 2 6 3
3
1 2
2
1
1
ANME-2d A
4
4
2
4
3
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
AM
2
2
1
4
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
A
2
2
1
2
1
1
1 1 1 4 1 1 3 3
3
1 2
2
1
1
M-1
2
3
1
2
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
M-2
2
3
1
2
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
M-3
2
3
1
2
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
H-1
2
2
1
2
1
1
2 2 2 8 1 2 9 6
6
2 4
4
2
2
H-2
2
2
1
2
1
1
1 1 1 4 1 2 3 3
3
1 2
2
1
1
H-3
2
5
1
4
1
1
2 2 2 8 1 1 6 6
6
2 4
4
2
2
H-4
4
2
1
2
2
1
0 0 0 0 0 0 0 0
0
0 0
0
0
0
S
Table 2: Domain based (meta)genome comparison of selected metagenomes of methanotrophs and selected genomes of other archaea. Domain abundance in every (meta)genome is indicated by numbers. S-AOM performing ANME include ANME-1-s [40], ANME-1-m [41], and ANME-2a [42]. N-AOM performing ANME include ANME-2d-h [28] and ANME-2da [43]. The acetoclastic (A) and methylotrophic (M) methanogens include Methanosarcina acetivorans C2A (AM), Methanosaeta concilii GP6 (A), Methanococcoides burtonii DSM6242 (M-1), Methanolobus tindarius DSM2278 (M-2), and Methanohalophilus mahii DSM5219 (M-3). Hydrogenotrophic methanogens (H) include Methanospirillum hungatei JF-1 (H-1), Methanobacterium formicicum DSM3637 (H-2), Methanococcus maripaludis C5 (H-3), and Methanoregula formicica SMSP (H-4). The sulfate-reducing archaeon (S) is Archaeoglobus fulgidus DSM 4304.
4 Archaea
Methylotrophic methanogenesis Methyltransferase MtaA/CmuA Methanol-cobalamin methyltransferase, B subunit Monomethylamine methyltransferase MtmB Trimethylamine methyltransferase Dimethylamine methyltransferase MtbB Trimethylamine methyltransferase, Methanosarcina Methylamine methyltransferase corrinoid protein reductive activase C-type cytochromes Di-haem cytochrome, transmembrane, nitrate reduction Doubled CXXCH motif Cytochrome c-like domain Class III cytochrome C (tetraheme cytochrome) Tetraheme cytochrome domain, flavocytochrome c3 (Shewanella) Octaheme c-type cytochrome Methanogenesis multiheme c-type cytochrome Multiheme cytochrome S-layer domains S-layer family duplication domain Sarcinarray family protein S-layer homology domain Cell export and cell adhesion HYR domain CARDB domain Collagen-binding surface protein Cna-like, B-type domain von Willebrand factor, type A VWA N-terminal Adhesion lipoprotein Adhesin B 13 0 1 1 64 4 55 1 8 0
IPR006457 IPR026476 IPR001119
IPR003410 IPR011635
IPR008454
IPR002035 IPR013608 IPR006128 IPR006129
4
IPR012286
78
2
IPR020942
IPR011031
33 2
IPR010177 IPR009056
1
2
IPR016174
0
0
IPR026339
IPR027594
0
IPR012740
IPR024673
0
IPR012653
28 2 4 3
1
5 14
0 0 0
15
0
0
0
0
0 0
0
0
0
0
0
0
3 11
IPR008031
IPR010426
0
0
IPR021079
0
ANME-1 M
0
ANME-1 S
IPR006360
InterPro ID
17 0 4 4
2
2 15
29 6 2
52
1
2
4
0
3 6
4
0
0
0
0
0
0
0
ANME-2a
32 0 5 5
1
0 6
16 0 0
80
0
0
5
3
2 0
3
0
0
0
0
0
0
0
ANME-2d H
Table 2: Continued.
14 0 0 0
8
0 6
26 4 0
73
0
0
4
3
3 17
7
0
0
0
0
0
0
0
ANME-2d A
37 0 0 0
0
0 30
34 1 0
3
1
0
2
0
0 15
4
4
2
9
4
12
3
7
AM
23 0 5 3
6
0 4
19 0 0
0
0
0
0
0
0 0
0
0
0
0
0
0
0
0
A
17 0 3 0
0
0 1
16 0 0
6
1
0
0
0
0 4
0
2
1
6
5
12
2
4
M-1
42 0 0 0
2
0 4
44 2 0
8
1
2
1
0
0 8
4
2
1
6
2
21
2
5
M-2
9 0 4 4
2
0 0
17 1 0
14
1
2
0
0
0 6
0
2
1
6
2
15
2
6
M-3
28 0 4 0
0
0 9
0 0 0
0
0
0
1
0
0 0
1
0
0
0
0
0
0
0
H-1
8 0 5 3
0
0 0
0 0 0
0
0
0
0
0
0 0
1
0
0
0
0
0
0
0
H-2
0 0 0 0
0
0 0
0 0 0
0
0
0
0
0
0 0
0
0
0
0
0
0
0
1
H-3
27 0 0 0
0
0 3
0 0 0
1
0
0
0
0
0 0
0
0
0
0
0
0
0
0
H-4
7 0 0 0
0
0 8
0 0 0
7
0
1
2
0
0 4
1
0
0
0
0
0
0
0
S
Archaea 5
InterPro ID Invasin/intimin cell-adhesion IPR008964 fragments Putative cell wall binding repeat 2 IPR007253 Archaeosortase A IPR014522 Archaeosortase B IPR026430 Archaeosortase C IPR022504 Exosortase/archaeosortase domain IPR026392 Exosortase, EpsH IPR013426 Exosortase EpsH-related IPR019127 Archaeosortase family protein ArtE IPR026485 Cell wall hydrolase/autolysin, IPR002508 catalytic PEF-CTERM protein sorting IPR017474 domain PGF-pre-PGF domain IPR026453 PGF-CTERM archaeal IPR026371 protein-sorting signal LPXTG cell wall anchor domain IPR019931 VPXXXP-CTERM protein sorting IPR026428 domain Cellulosome-related/Dockerin Cellulosome anchoring protein, IPR002102 cohesin domain Dockerin domain IPR016134 Dockerin type I repeat IPR002105 Carbohydrate-binding domains Carbohydrate-binding domain IPR008965 Carbohydrate-binding-like fold IPR013784 Carbohydrate-binding, CenC-like IPR003305 Carbohydrate-binding/sugar IPR006633 hydrolysis domain Carbohydrate-binding domain, IPR010502 family 9 Bacteroidetes-associated carbohydrate-binding often IPR024361 N-terminal Carbohydrate binding module, IPR031768 xylan-binding domain Galactose-binding domain-like IPR008979
ANME-1 M 0 2 0 0 0 0 0 2 0 2 0 1 4 0 0
77 112 0 42 5 0 22 0 0 0 1
ANME-1 S 17 2 2 0 0 5 1 4 0 2 0 1 9 3 0
80 149 1 50 14 1 27 1 0 0 12
17
0
1
0
5
39 3 7
44 5
52
0
1
6
18
3
1
0 1 0 1 2 0 2 0
3
ANME-2a
6
0
0
0
2
2 10 0
4 0
4
0
0
2
9
4
0
0 2 0 2 5 1 3 0
2
ANME-2d H
Table 2: Continued.
39
1
0
2
2
6 19 17
6 0
12
0
0
1
8
1
0
0 2 0 1 3 0 2 0
6
ANME-2d A
4
0
0
0
27
2 0 0
5 0
2
0
0
2
24
0
0
0 1 0 0 1 0 1 0
7
AM
4
0
0
0
3
0 0 0
17 0
0
0
1
0
0
0
0
0 1 0 0 1 0 1 0
0
A
2
0
0
0
7
3 0 0
2 0
4
0
1
2
7
10
0
0 1 0 1 2 0 2 0
2
M-1
1
0
0
0
8
13 0 0
4 0
15
0
0
1
21
19
0
0 1 0 1 2 0 1 0
7
M-2
3
0
0
0
8
2 1 0
0 0
3
5
0
2
11
0
0
0 1 1 0 2 0 1 0
0
M-3
0
0
0
0
0
3 3 0
9 0
1
0
0
1
0
0
0
0 1 0 0 1 0 1 0
1
H-1
0
0
0
0
5
0 0 0
0 0
1
0
0
0
0
0
0
0 0 0 0 0 0 0 0
2
H-2
0
0
0
0
2
1 0 0
0 0
0
0
0
0
0
0
0
0 0 0 0 1 0 1 1
0
H-3
0
0
0
0
1
0 6 0
0 0
0
0
0
1
1
0
1
0 2 0 0 2 0 2 0
2
H-4
0
0
0
0
8
2 1 0
2 1
4
0
0
3
1
0
0
0 1 0 0 1 0 1 0
2
S
6 Archaea
Menaquinones 3-Demethylubiquinone-9 3-methyltransferase Succinate dehydrogenase/fumarate reductase, flavoprotein subunit UbiE/COQ5 methyltransferase UbiE/COQ5 methyltransferase, conserved site NrfD family Futalosine hydrolase Cyclic dehypoxanthine futalosine synthase Aminodeoxyfutalosine synthase Menaquinone synthesis (chorismate dehydratase & naphthoate synthase) FO synthase, subunit 2 Phenazines Phenazine biosynthesis PhzF protein 0 0 3 3 3 0 1 0 8 3 1
IPR014006
IPR004033
IPR023576
IPR005614 IPR019963
IPR022431
IPR022432
IPR003773
IPR020050
IPR003719
ANME-1 S
IPR028973
InterPro ID
1
3
6
0
1
0 0
0
0
0
0
ANME-1 M
4
1
0
0
0
0 0
0
0
1
0
0
3
2
1
1
4 1
2
2
2
3
ANME-2d H
Table 2: Continued. ANME-2a
0
3
2
1
1
3 1
2
2
1
3
ANME-2d A
2
2
0
0
0
0 0
0
1
0
0
AM
0
1
0
0
0
0 0
0
0
0
0
A
0
2
0
0
0
0 0
0
1
0
0
M-1
0
1
0
0
0
0 0
1
0
0
0
M-2
0
2
0
0
0
0 0
0
0
1
0
M-3
0
2
0
0
0
0 0
0
0
0
0
H-1
0
2
0
0
0
0 0
0
0
0
1
H-2
0
2
0
0
0
0 0
0
0
0
0
H-3
2
2
0
0
0
0 0
0
0
0
0
H-4
0
3
2
0
1
3 0
0
0
1
0
S
Archaea 7
8
Archaea
CH4
2H+
CoB-S-S-CoM HdrDE
Mcr
CoB-SH
MPhH2 H2
CoM-SH CH3 -CoM
MPh Vho
Mtr
2Na+
CH3 -H4 MPT F420 H2
Mer
F420
H2
Frh
CH2 =H4 MPT F420 H2 Mtd
F420
H2
CH≡H4 MPT H2 O
Mch
CHO-H4 MPT Ftr
H2 H+
Ech
CHO-MFR Fdred Fdox
Fmd CO2
Figure 2: Hydrogenotrophic methanogenesis in cytochrome containing Methanosarcina barkeri. Black lines represent presence of conversions. See Table 3 for nomenclature.
reaction thermodynamically favourable (Table 1). The reverse reaction of Mcr is therefore an essential step in AOM and is discussed in Section 2.2. The respiratory chain that is needed for using different terminal electron acceptors will be discussed in Section 3. Evidence that ANME use the reverse methanogenesis pathway during AOM is derived from metagenomic and metatranscriptomic analyses. This showed that all genes for the (reverse) methanogenic pathway were present and expressed in ANME-2a [42] (Figure 4) and ANME-2d [28] (Figure 5). ANME-1 were consistently lacking the gene encoding N5 ,N10 -methylene- tetrahydromethanopterin (H4 MPT) reductase (Mer), which is an enzyme needed to oxidize methyl-H4 MPT during methane oxidation [40, 41, 44, 51] (Figure 6, Table 2). Possible explanations could be that (1) the mer gene is present but not yet detected, (2) ANME1 used a bypass of this step, and (3) Mer was replaced by a structural analogue. The first possibility is highly unlikely. Although no closed genome of ANME-1 is publicly available to date, all ANME-1 metagenomes consistently only lack Mer and no other methanogenic genes. The second possibility was proposed previously where ANME-1 uses a bypass of
Mer via the formation of methanol or methylamine [41], as was detected in deletion mutants of Methanosarcina [52, 53]. Here, CH3 -CoM was presumably converted to methanol by a methyltransferase and subsequently to formaldehyde by a methanol dehydrogenase (Mdh). Then, formaldehyde would be converted to N5 ,N10 methylene-H4 MPT using a fusion protein of the formaldehyde-activating enzyme (Fae) and a hexulose-6-phosphate synthase (Hps) [53] (Figure 6). Both Fae and Hps were found in the ANME-1 metagenome [41] and metaproteome [40, 51]. However, no genes coding for enzymes involved in methanol metabolism were detected in these ANME-1 datasets [40, 41, 51] (Table 2), indicating that this alternative pathway probably does not occur. The presence of the Fae/Hps fusion protein in ANME-1 during AOM could also be explained by its involvement in ribose phosphate synthesis and not in AOM [54]. Indeed, the Fae gene domains of ANME-1 were located in between ribulosephosphate binding barrel and ribosomal protein S5 domains (Table S2). The third possibility of a structural analogue is most likely since an analogue of N5 ,N10 -methylene tetrahydrofolate (H4 HF) reductase (MetF) was expressed by ANME1 during AOM which could replace Mer [40] (Figure 6).
Archaea
9 Table 3: Nomenclature.
Central methanogenic pathway Fmd Ftr Mch Mtd Mer Mtr Mcr Mdh Fae/Hps MetF Electron transport Mvh Vho Fpo Fqo Hdr Frh Ech MePh/MePhH2 MQ/MQH2 Cyt b Cyt c MHC Rieske Orf7 Nar Nap Nrf
Formylmethanofuran (CHO-MFR) dehydrogenase Formylmethanofuran-tetrahydromethanopterin (H4 MPT) formyltransferase N5 ,N10 -methenyl-H4 MPT cyclohydrolase F420 H2 -dependent methylene -H4 MPT dehydrogenase N5 ,N10 -methylene-H4 MPT reductase 5 N -methyl-H4 MPT:coenzyme M (CoM) methyltransferase Methyl coenzyme M (CH3 -CoM) reductase Methanol dehydrogenase Fusion protein of formaldehyde activating enzyme/ hexulose-6-phosphate synthase N5 ,N10 -methylene tetrahydrofolate (H4 HF) reductase analogue F420 -nonreducing hydrogenase Methanophenazine-dependent hydrogenase F420 H2 :phenazine oxidoreductase F420 H2 :quinone oxidoreductase Coenzyme B-coenzyme M heterodisulfide (CoB-S-S-CoM) reductase F420 -dependent hydrogenase Ferredoxin-dependent hydrogenase Methanophenazine Menaquinone Cytochrome b Cytochrome c Multiheme c-type cytochrome Rieske cytochrome b complex Pseudoperiplasmic b-type cytochrome Nitrate reductase Periplasmic nitrate reductase Nitrite reductase
2.2. Methyl Coenzyme M Reductase (Mcr). The enzymatic reaction of a purified Mcr from ANME has not been measured to date. The key question is whether a methanogenic Mcr can explain the observed in situ AOM rates or if the Mcr of ANME is structurally altered. There are three main factors to be considered: the kinetic limitations as defined by enzyme properties (i.e., half-maximal activity at a specific 𝐾𝑀 value), the thermodynamic constraints of the enzymatic reaction, and the maximal or ambient rate of the enzymatic reaction. For the Mcr from Methanothermobacter marburgensis, kinetic parameters have been determined to illustrate the reversibility of reaction (1). In the methanogenic reaction, the purified Mcr isoform I [55] catalyzes the production of methane with a 𝑉max of 30 U mg−1 and a 𝐾𝑀 of 5 mM for CH3 -CoM. The same (methanogenic) enzyme was able to oxidize methane to CH3 -CoM with a rate of 0.0114 U mg−1 and a 𝐾𝑀 for methane of ∼10 mM [56]. To answer if the observed AOM rates are in accordance with the measured methane oxidation rate for the purified Mcr enzyme from M. marburgensis, the Mcr activity during AOM is needed. Estimates for AOM rates in terms of activity (per cell dry mass) range between
