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Methanosaeta, the Forgotten Methanogen?

Article  in  Trends in Microbiology · May 2007

DOI: 10.1016/j.tim.2007.02.002 · Source: PubMed

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150 Update TRENDS in Microbiology Vol.15 No.4

Genome Analysis

Methanosaeta, the forgotten methanogen?

Kerry S. Smith and Cheryl Ingram-Smith
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634-0318, USA

Although the aceticlastic methanoarchaea Methanosar-
cina and Methanosaeta employ different enzymes to
catalyze the first step of aceticlastic methanogenesis,
it has long been assumed that the remainder of the
py
Methanosarcina and Methanosaeta employ different
enzymes to catalyze the first step of aceticlastic metha-
nogenesis but the remainder of the pathway was assumed
to be the same in both genera. Although analysis of the

pathway was the same. Analysis of the recently com-
pleted genome sequence of Methanosaeta thermophila
confirms that the majority of core steps of the pathway
are similar in both genera, but striking differences have
been discovered in electron transfer and energy conser-
vation. In addition, the presence of genes encoding
enzymes for the CO2 reduction pathway in the Msa.
co
recently completed genome sequence of Methanosaeta
thermophila confirms that the majority of core steps of
the pathway are similar in both genera, important differ-
ences have been discovered in electron transfer and
energy conservation.
Unexpectedly, the genome sequence suggests that the
first step of methanogenesis in Msa. thermophila might be

thermophila genome suggests the possibility that
Methanosaeta might be more metabolically diverse than
previously thought. Thus, genome analysis of Msa. ther-
mophila presents new research avenues for this forgot-
on enzymes for the CO2 reduction pathway in the Msa. ther-
ten methanogen and reminds us of the questions that
still remain unanswered about aceticlastic methanogen-
esis in both Methanosaeta and Methanosarcina.
rs
al
highly regulated at both the transcriptional and post-
translational levels, enabling adaptation to environmental
changes. In addition, the presence of genes encoding
mophila genome suggests the possibility that Methano-
saeta could be more metabolically diverse than previously
thought. The genome sequence of Methanosaeta concilii
has been completed (University of Washington Genome

Center) and is currently being annotated for deposit into

Methanosaeta might be the principal methane GenBank (K.S. Smith, C. Ingram-Smith and R.D. Barber,
producer on earth personal communication). Except as noted, the genome
pe

The role of acetate as a key intermediate in the anaerobic sequence of Msa. concilii confirms the findings for Msa.
food chain is underscored by estimates that as much as thermophila discussed here.
two-thirds of biologically produced methane released to the
atmosphere each year is derived from the methyl group of
Amplification of ACS genes in Methanosaeta
acetate [1]. Surprisingly, only two genera of methanoarch-
The first step of aceticlastic methanogenesis is activation of
aea are known to use acetate as a substrate for methano-
acetate to acetyl-CoA (Figure 1). This reaction is catalyzed
genesis. Unlike Methanosarcina (abbreviated in species
by AMP-forming acetyl-CoA synthetase (ACS) or the com-
names as Msr), which prefers methylated compounds such
r's

bined actions of acetate kinase (AK) and phosphotransace-

as methanol and methylamines to acetate, Methanosaeta
tylase (PTA). Expression studies in Methanosarcina
(abbreviated in species names as Msa) is a specialist that
thermophila [7] and gene knockouts in Msr. acetivorans
uses only acetate. A minimum threshold concentration of
[8] provide evidence for use of the AK-PTA pathway in
1 mM acetate is required for Methanosarcina growth,
o

Methanosarcina, and a gene encoding ACS is not present

whereas that for Methanosaeta is in the 5–20 mM range
in the completed Methanosarcina genome sequences. How-
[2]. Methanosaeta are widely distributed in nature and,
ever, ACS has been purified from both Methanothrix soehn-
th

owing to their high affinity for acetate, prevail over Metha-

genii and Msa. thermophila CALS-1 (formerly Methanothrix
nosarcina in the low acetate environments of rice paddies
sp. strain CALS-1) and characterized [9,10]. (Note: M. soehn-
[3] and anaerobic waste digesters [4], both major sources of
genii strain Opfikon was probably impure, and hence was
biogenic methane. Thus, Methanosaeta is probably the
Au

not renamed as Methanosaeta.) AK and PTA activities were

predominant methane producer on earth.
not detected in M. soehngenii [10] and genes encoding AK
In recent years, the field of aceticlastic methanogenesis
and PTA are absent from the Msa. thermophila genome.
has focused primarily on Methanosarcina, which has a
Surprisingly, four acs genes were identified in Msa.
much faster growth rate and higher yield than Methano-
thermophila, three of which are tandemly positioned
saeta. The completion of the Methanosarcina acetivorans
(Figure 2). Msa. concilii has five putative acs genes, of
and Methanosarcina mazei genome sequences [5,6] and
which four are tandemly positioned (Figure 2). ACS phy-
development of several genetic tools have fueled this
logeny and gene organization indicate that the closest
preference for Methanosarcina as the model for aceticlas-
homologs between the two species generally align in the
tic methanogenesis. It has long been understood that
same position within the tandem repeat (Figure 2).
Corresponding author: Smith, K.S. (kssmith@clemson.edu). The first gene of the Msa. concilii acs tandem corresponds
Available online 21 February 2007. to the previously identified gene in M. soehngenii [11].
www.sciencedirect.com
 Update TRENDS in Microbiology Vol.15 No.4 151

py
co
al
on
rs

Figure 1. The proposed pathways for aceticlastic methanogenesis in Methanosarcina mazei and Methanosaeta thermophila. Enzymes common to both species are shown
in blue. The enzymes for activation of acetate to acetyl-CoA differ between Methanosarcina and Methanosaeta (purple). (a) Methanosarcina mazei. In the case of
Methanosarcina, the proposed pathway differs between Msr. mazei and Methanosarcina acetivorans in the electron transport pathway (red). Msr. mazei uses ECH
pe

hydrogenase to reduce H+ to H2 whereas Msr. acetivorans uses RNF (see main text). Note that MP does not span the entire membrane but is a small electron carrier present
within it. (b) Methanosaeta thermophila. The proposed pathway is common to both Msa. thermophila and Methanosaeta concilii, with the exception of the level of
amplification of ACS (see main text). The putative acetate transporter ADY2 is shown in green. Abbreviations: ACS, acetyl-CoA synthetase; AK, acetate kinase; CA, carbonic
anhydrase; CH3-THSPt, methyltetrahydrosarcinapterin; CODH, carbon monoxide dehydrogenase/acetyl-CoA decarbonylase complex; FD, ferredoxin; HDR, heterodisulfide
(CoM-S-S-CoB) reductase; MCR, methyl-CoM methylreductase; MP, methanophenazine; MTR, methyltetrahydrosarcinapterin:CoM methyltransferase; PPase, inorganic
pyrophosphatase; PTA, phosphotransacetylase. GI numbers for Msa. thermophila genes: ACS: 88950500, 88950501, 88950502, 88951644; PPase: 88951143; CODH,
88951192, 88951193, 88951196, 88951197, 88951198; CA, 88952123; MTR, 88951610, 88951611, 88951612, 88951613, 88951614, 88951615, 88951616, 88951617; MCR,
88952087, 88952088, 88952090; HDR, 88951091, 88951092; Ady2, 88951170.
r's

The characterized ACS1 from Methanothermobacter
thermautotrophicus (MT-ACS1) has a strong preference
for acetate, and propionate is the only other acyl subst-
rate that can be used. Trp416 of MT-ACS1 has been shown
o
differs from both MT-ACS1 and AF-ACS2 at one or more of
the other three positions, suggesting that acs gene ampli-
fication might have resulted in functional divergence of
this enzyme. Thus, differential acs expression could pro-

to be a key determinant of substrate specificity and range vide Methanosaeta with the ability to adapt to changes in
[12] and is invariant among ACSs with the exception of its environment. Alternatively, equifunctionalization [14]
MSC-ACS4 and MST-ACS4, which both have Phe at the through acs gene amplification could elevate activated
th

corresponding position. Three highly conserved residues – acetate levels in the cell; this is considered less likely
Ile312, Thr313 and Val388 of MT-ACS1 – have been shown because these enzymes have diverged and the Msa. ther-
to influence acyl substrate selection and affinity [12] and mophila ACSs share only 64–80% similarity at the amino
Au

are conserved among all nine Methanosaeta ACSs. This
suggests that the Methanosaeta ACSs have a preference
for acetate.
Archaeoglobus fulgidus ACS2 (AF-ACS2) has all four of
these conserved residues yet has been shown to use buty-
rate, valerate and isobutyrate in addition to acetate and
propionate [13]. Sequence alignment and comparison of
models of AF-ACS2 and MT-ACS1 suggested five residues
that differ between the two enzymes that could have a role
in the expanded substrate range of AF-ACS2 [13]. The
Methanosaeta ACSs are identical to MT-ACS1 at two of
these positions. However, each of the Methanosaeta ACSs
www.sciencedirect.com
acid level.
The presence of multiple ACSs in Methanosaeta raises
the question of whether one or more are regulated by post-
translational modification. The activity of bacterial and
eukaryotic ACSs has been shown to be regulated by acety-
lation [15–17]. In Salmonella enterica, acetylation of the
completely conserved Lys609 of ACS by the Pat acetyl-
transferase inactivates the enzyme [18] and deacetylation
by the sirtuin Sir-2 ortholog CobB reactivates it [19]. A
BLAST search of the Msa. thermophila genome revealed
the presence of three open reading frames (ORFs) with
>32% identity to the C-terminal GNAT (GCN5-related
152 Update TRENDS in Microbiology Vol.15 No.4

py
co
al
Figure 2. ACS phylogeny and gene organization in Methanosaeta. (a) Methanosaeta thermophila (MST) and Methanosaeta concilii (MSC) ACS sequences were aligned with
ClustalX [37] and phylogenetic analysis was performed with MEGA [38] using the neighbor-joining algorithm with a gamma distance estimation (g = 2). The gamma
distance corrects for the inequality of the substitution rates among sites. The phylogenetic tree was constructed based on pairwise distance estimates of the expected
on
number of amino acid replacements per site (0.05 in the scale bar). One thousand bootstrap replicates were performed and values >80% are shown. (b) Organization of acs
genes in Msa. concilii (top) and Msa. thermophila (bottom). Closest homologs between the two species are indicated by identical colors.

N-acetyltransferase) functional domain of the S. enterica (MCR) [21]. The two electrons required for this reduction are
rs

Pat. One homolog contains only the GNAT domain
whereas the other two have an additional N-terminal
acetyl-CoA hydrolase/acetate-CoA transferase domain. A
homolog of the CobB deacetylase was not identified.
pe
derived from the sulfur atoms of CH3-S-CoM and HSCoB,
yielding the heterodisulfide CoM-S-S-CoB [21]. Heterodi-
sulfide reductase (HDR) reduces this mixed disulfide to the
sulfhydryl forms of each cofactor using electrons derived

The Bacillus subtilis AcuA and AcuC, unrelated to the S.
enterica Pat and CobB, have been shown to acetylate and
deacetylate ACS, respectively [20]. Although an AcuA
homolog was not identified in Msa. thermophila, four
putative deacetylases with >35% identity to the B. subtilis
AcuC have been identified. Thus, Methanosaeta might be
using parts of two different acetylation/deacetylation sys-
r's
from the oxidation of the carbonyl group to CO2 [21]. Genes
encoding the enzymes from CODH through MCR are pre-
sent in Msa. thermophila (Figure 1). A gene encoding a Cam
homolog has also been identified; however, the predicted
enzyme lacks an external loop containing Glu84, a key
proton-shuttle residue in the proton transfer step of the
Methanosarcina g class carbonic anhydrase [23]. Although it

tems. The presence of four ACSs in addition to multiple
putative acetylases and deacetylases in Msa. thermophila
suggests that acetate activation and its regulation are
much more complex than expected.
o
would be expected that Methanosaeta has a cofactor similar
to H4SPT, such a cofactor has not yet been isolated.
Electron transfer and energy conservation differ in the

two genera
Core steps of the pathway are similar in Methanosaeta Energy for the activation of acetate by ACS in Methanosaeta
and Methanosarcina is provided by hydrolysis of ATP to AMP and PPi. Adenylate
th

A key step of aceticlastic methanogenesis (Figure 1) is kinase uses an additional ATP in the conversion of AMP to
cleavage of the carbon–carbon and carbon–sulfur bonds of ADP [10]. Thus, Methanosaeta must gain at least enough
acetyl-CoA by the five-subunit carbon monoxide dehydro- energy from the conversion of acetyl-CoA to methane and
Au

genase/acetyl-CoA decarbonylase complex (CODH/ACDS)
[21]. The methyl group is transferred to the cofactor tetra-
hydrosarcinapterin (H4SPT) and the carbonyl group is oxi-
dized to CO2, which diffuses out of the cell and is converted to
bicarbonate by Cam, a g class carbonic anhydrase prefer-
entially expressed during growth on acetate [22].
Methyltetrahydrosarcinapterin:CoM methyltransferase
(MTR), an integral membrane protein complex, transfers
the methyl group from H4SPT to HSCoM. Reductive
demethylation of CH3-S-CoM, the final step of methane
production common to methanogenic pathways for all sub-
strates, is catalyzed by the methyl-CoM methylreductase
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CO2 to synthesize more than the two ATP expended. How
this is accomplished has been a key question for many years
and might be partially answered by the Msa. thermophila
genome sequence.
Transfer of electrons from the CODH/ACDS complex to
HDR in Methanosarcina has been under intense study in
recent years. A 2-[4Fe-4S] ferredoxin accepts electrons
generated from the oxidation of the carbonyl group to
CO2 by the CODH/ACDS complex (Figure 1). Biochemical
experiments in Msr. mazei suggest that the electrons
passed to ferredoxin are used to reduce H+ to H2 through
ECH hydrogenase [24]. The H2 generated is proposed to
 Update TRENDS in Microbiology
diffuse out of the cell where it is recaptured by the
F420-non-reducing hydrogenase VHO [24]. The electrons
then flow to HDR through methanophenazine, a redox-
active 2-hydroxyphenazine derivative isolated from cyto-
plasmic membranes of Msr. mazei [25].
In Msr. mazei, the electrochemical gradient generated
by the membrane-bound ECH is used for ATP synthesis
during growth on acetate. In Msr. acetivorans, ECH
is absent and is replaced by the RNF complex, which
has been hypothesized to transfer electrons directly to
HDR through methanophenazine without H2 production
and generate an electrochemical gradient that drives ATP
synthesis [26]. An ORF with identity to the Msr. mazei
ferredoxin has been found in the Msa. thermophila genome
but genes encoding ECH and RNF are absent (Figure 1). So
how does Methanosaeta generate energy during growth on
acetate? Genomic analysis suggests that F420-reducing
hydrogenase is not only the most likely but also possibly
the only candidate enzyme for creating an electrochemical
gradient to drive ATP synthesis. The presence of a proton-
translocating ATP synthetase supports the hypothesis that
F420-reducing hydrogenase is involved in generating the
electrochemical gradient.
Hydrolysis of PPi by inorganic pyrophosphatase (PPase)
drives the ACS reaction forward (Figure 1). The PPase
isolated from M. soehngenii was soluble but up to 5% was
membrane associated [27], raising the possibility that PPi
hydrolysis could be coupled to proton translocation. A gene
rs
encoding a PPase has been identified in Msa. thermophila.
However, the PPase encoded by this ORF belongs to the
type II soluble PPases, which argues against coupling of
PPi hydrolysis and proton translocation.
pe
Transport of acetate by aceticlastic methanoarchaea
Analysis of the Msa. thermophila genome has raised new
questions about the aceticlastic pathway. One key question
is what enables Methanosaeta to outcompete Methanosar-
cina in low acetate environments? The ACS pathway of
Methanosaeta has much higher affinity for acetate than
r's
the AK-PTA pathway employed by Methanosarcina, but
is this explanation sufficient? The discovery of a putative
acetate transporter in methanoarchaea could provide an
additional answer.
o
Acetate in the undissociated form is believed to diffuse
freely across the cytoplasmic membrane of bacteria [28].
In the yeast Saccharomyces cerevisiae, an acetate trans-
th
porter Ady2 was recently characterized and disruption
of the gene abolished active transport of acetate [29].
Inspection of the Msa. thermophila genome uncovered
Au
the presence of an ORF with identity to Ady2. BLAST
searches revealed Ady2 homologs in all methanoarchaea
that are capable of using acetate as a carbon and/or energy
source. In the genomes of the archaea Thermoplasma
volcanium, Thermoplasma acidophilum, Sulfolobus solfa-
taricus and the methanoarchaeon M. thermautotrophicus,
an Ady2 homolog is found adjacent to acs, providing
circumstantial evidence for Ady2 as an acetate transpor-
ter in archaea.
Both Msr. acetivorans and Msr. mazei have three ORFs
with identity to the deduced Ady2 sequence; however,
these were annotated as transcription factors despite
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Vol.15 No.4 153
the presence of multiple predicted transmembrane
domains. Expression of one of the Msr. mazei Ady2 ORFs
was ten-fold greater in cells grown in acetate than in
cells grown in methanol [30]. It is possible that although
genomics indicates the presence of an acetate transporter
in both Methanosaeta and Methanosarcina, differences
in the affinities of these transporters might provide
the key to why Methanosaeta dominates in low acetate
environments.
py
Methanosaeta might be more metabolically diverse
than previously thought
Genes encoding the enzymes required for reduction of CO2
co
to methane have been identified in both the Msa. thermo-
phila and the Msr. acetivorans genomes. So why can’t
either species obtain energy for growth from reduction of
CO2 to methane? The answer might lie in the absence of
ECH. A knockout of ech in Msr. barkeri prevents growth on
CO2, presumably because H2 cannot be used as an electron
donor [31]. RNF in Msr. acetivorans and F420-reducing
hydrogenase in Methanosaeta do not produce H2 and
al
cannot substitute for ECH in this function.
Msr. acetivorans is capable of growth on CO and
produces methane, acetate and formate as products [8].
on
It has been postulated that CO is oxidized to CO2, then
reduced to methane through the CO2 reduction pathway
[32]. Recent proteomic analysis confirms that enzymes
used for CO2 reduction are preferentially expressed during
growth on CO [33]. In addition, AK and PTA, used to
produce acetate from acetyl-CoA and to generate ATP
by substrate level phosphorylation, are also expressed
during growth on CO [33] and are required for this mode
of growth [8]. Whether Methanosaeta can grow on CO to
produce methane, acetate and formate has not been
reported, but this remains a distinct possibility because
genes encoding the enzymes thought to be necessary for
growth are present in Msa. thermophila. Because AK and
PTA are absent, ACS must function in the direction of
breakdown of acetyl-CoA to ATP plus acetate. This would
require the PPase and adenylate kinase activities to be
absent.
Several methanoarchaea have been shown to perform
trace methane oxidation under diverse growth conditions
[34]. ‘Reverse methanogenesis’ by operation of the pathway
for CO2 reduction to methane in the reverse direction has
also been postulated in the anaerobic methane-oxidizing
archaea [35]. Two groups of putative anaerobic methane-
oxidizing archaea (ANME-1 and ANME-2) that are phylo-
genetically very closely related to Methanosaeta have
been isolated [36]. The close phylogenetic relationship
with these anaerobic-methane oxidizers and the presence
of the genes for the CO2 reduction pathway raises the
possibility that Methanosaeta could have the capacity for
oxidation of methane, although the conditions under which
this would occur are not known.
Concluding remarks
Aceticlastic methanogenesis has been widely studied in
Methanosarcina and, to a lesser extent, in Methanosaeta.
Although the key enzymes of this pathway have been
known for some time, a full understanding of methanogen-
154 Update TRENDS in Microbiology Vol.15 No.4
Box 1. Key unanswered questions about aceticlastic
methanogenesis and Methanosaeta
 Why are some steps of methanogenesis and electron transport
conserved in the two genera of aceticlastic methanoarchaea but
others are not?
 Is there another genus of aceticlastic methanoarchaea that has yet
to be isolated?
 Is acetate transported into Methanosarcina and Methanosaeta?
 How is energy conserved in Methanosaeta?
 Are Methanosaeta more metabolically diverse that previously
thought?
esis is still lacking. Completion of genome sequences of
species from both genera has greatly increased our knowl-
edge of their physiology and metabolism, and has enabled
us to identify similarities and differences between these
ecologically important methanoarchaea. Such comparisons
also remind us of questions that still remain unanswered
(Box 1).
Finally, although difficulties with growth and yield
have discouraged studies in Methanosaeta, the completed
genome sequences of two species of Methanosaeta now
enable more directed studies to answer pertinent questions
regarding aceticlastic methanogenesis in both genera.
Additionally, these genome sequences open up exciting
new avenues of research and permit proteomic and micro-
array approaches to studying the metabolic capabilities
rs
of this forgotten methanogen.
Acknowledgements
This work was supported by NSF Award # 0333210 to K.S.S and by Clemson
pe
University. The genome of Methanosaeta thermophila was sequenced at the
Joint Genome Institute of the U.S. Department of Energy (DOE-JGI) as
part of the Microbial Genome Program. This sequence is available in
GenBank through the National Center for Biotechnology Information
(NCBI) or the DOE-JGI website (www.jgi.doe.gov). The genome sequence of
Methanosaeta concilii is complete and currently undergoing annotation and
will be publicly released in GenBank.
r's
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doi:10.1016/j.tim.2007.02.002

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Elsevier celebrates two anniversaries with
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a gift to university libraries in the developing world

In 1580, the Elzevir family began their printing and bookselling business in the Netherlands, publishing
works by scholars such as John Locke, Galileo Galilei and Hugo Grotius. On 4 March 1880, Jacobus
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George Robbers founded the modern Elsevier company intending, just like the original Elzevir family, to
reproduce fine editions of literary classics for the edification of others who shared his passion, other
‘Elzevirians’. Robbers co-opted the Elzevir family printer’s mark, stamping the new Elsevier products
with a classic symbol of the symbiotic relationship between publisher and scholar. Elsevier has since
become a leader in the dissemination of scientific, technical and medical (STM) information, building a
reputation for excellence in publishing, new product innovation and commitment to its STM
communities.
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In celebration of the House of Elzevir’s 425th anniversary and the 125th anniversary of the modern
Elsevier company, Elsevier donated books to ten university libraries in the developing world. Entitled
‘A Book in Your Name’, each of the 6700 Elsevier employees worldwide was invited to select one of
the chosen libraries to receive a book donated by Elsevier. The core gift collection contains the
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company’s most important and widely used STM publications, including Gray’s Anatomy, Dorland’s
Illustrated Medical Dictionary, Essential Medical Physiology, Cecil Essentials of Medicine, Mosby’s
th

Medical, Nursing and Allied Health Dictionary, The Vaccine Book, Fundamentals of Neuroscience, and
Myles Textbook for Midwives.

The ten beneficiary libraries are located in Africa, South America and Asia. They include the Library of
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the Sciences of the University of Sierra Leone; the library of the Muhimbili University College of Health
Sciences of the University of Dar es Salaam, Tanzania; the library of the College of Medicine of the
University of Malawi; and the University of Zambia; Universite du Mali; Universidade Eduardo
Mondlane, Mozambique; Makerere University, Uganda; Universidad San Francisco de Quito, Ecuador;
Universidad Francisco Marroquin, Guatemala; and the National Centre for Scientific and Technological
Information (NACESTI), Vietnam.

Through ‘A Book in Your Name’, these libraries received books with a total retail value of
approximately one million US dollars.

For more information, visit www.elsevier.com

www.sciencedirect.com

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