methane is a powerful greenhouse gas , and its atmospheric concentration has been steadily increasing over the past 300 years .
there are two major ways in which methane is removed from the environment : aerobic oxidation by a specialized group of bacteria and anaerobic oxidation by a specialized group of archaea .
the former is important for keeping methane concentrations balanced in freshwater sediments and soils , whereas the latter is the major process in anoxic marine environments .
the biochemistry of aerobic methane oxidation is relatively well understood , following intensive research efforts with a number of model organisms , but the biochemistry of anaerobic methane oxidation is not yet fundamentally understood and no anaerobic methane - oxidizer has been isolated in pure culture so far .
three recent studies using global approaches [ 1 - 3 ] have shed new light on both aerobic and anaerobic systems . here , we first review background information on the two metabolic systems involving methane and then discuss the insights revealed through the three recent studies [ 1 - 3 ] , as well as a fourth   that is useful for interpreting the new results on anaerobic methane oxidation .
type i methanotrophs are -proteobacteria that have stacked membranes harboring methane monooxygenase ( pmmo ) , the enzyme for primary methane oxidation , and that use the ribulose monophosphate ( rump ) cycle , which converts formaldehyde into multicarbon compounds , for building cell biomass .
type ii methanotrophs belong to the -proteobacteria , have rings of pmmo - harboring membranes at the periphery of the cells , and use the serine cycle , an alternative pathway for converting formaldehyde into biomass ; these bacteria also often contain a soluble ( s ) mmo in addition to pmmo .
the third type , type x methanotrophs , belong to the genus methylococcus ( -proteobacteria ) and combine features characteristic of the other two types : they have stacked membranes and the rump cycle , but they also have elements of the serine cycle and smmo .
the type x methanotroph methylococcus capsulatus has been a favorite model for research because of its robust growth on methane and its relative ease of use as a genetic system [ 6 - 9 ] .
two almost identical gene clusters have been identified encoding the subunits of pmmo , which are expressed simultaneously and are functionally redundant , and another gene cluster encodes the subunits of smmo .
copper has been shown to play an essential role in expression of the pmmo operons , whereas the smmo operon appears to be expressed only in low - copper conditions .
the catalytic mechanisms for both pmmo and smmo   are understood on a sophisticated level , but until recently no whole - genome sequence has been available for m. capsulatus or for any other methanotroph .
two recent studies   have used a whole - genome - shotgun sequencing approach to complement the mounting dataset on the biochemistry and regulation of aerobic methane oxidation .
in contrast , understanding of the process of anaerobic methane oxidation is in its infancy .
microbes involved in this process have been identified recently as archaea related to methanosarcinales that fall phylogenetically into two distinct groups , anme - i and anme - ii ; these are normally found in association with sulfate - reducing bacteria .
there is no clear concept of how methane oxidation is linked to sulfate reduction ; figure 1 shows a possible model .
this co - metabolism has to be viewed in the light of the thermodynamic constraints , however ; the free energy ( g ) for anaerobic methane oxidation in situ is estimated at -20 to -40 kj / mol ) , the lowest value described that enables microbial growth .
there is agreement on the hypothesis that reverse methanogenesis plays a key role in the methane oxidation process : most enzymes of methanogenesis are easily reversible , and part of the methanogenesis pathway operates in reverse for energy generation in methanosarcina species growing on such substrates as methanol or methylamine .
but the last step of methanogenesis and presumably the first in anaerobic methane oxidation ( step 1 in figure 1 ) , catalyzed by methyl - coenzyme m reductase ( mcr ) , presents a mechanistic challenge given the fact that methane is chemically unreactive .
nevertheless , data have been obtained showing that methanotrophic archaea have homologs of the genes for all three subunits of mcr , suggesting that mcr or a similar enzyme may indeed be responsible for anaerobic methane oxidation .
two recent studies   describe efforts to establish the roles of mcr homologs and of other genes potentially involved in reverse methanogenesis by directly assessing environmental dna and protein pools .
in a paper recently published in plos biology , ward et al .   describe the complete genomic sequence of methylococcus capsulatus ( bath ) .
they annotate the genome in terms of the specific adaptations this organism has evolved in order to succeed at a lifestyle solely dependent on utilization of methane .
the genome of m. capsulatus ( 3.3 megabases , mb ) is much smaller than the genome of a model facultative methylotroph , methylobacterium extorquens am1 ( 7 mb ) , a bacterium with a much more versatile lifestyle , but is comparable in size to the genome of another obligate methylotroph , methylobacillus flagellatus ( 2.9 mb ) , suggesting that the degree of specialization in methylotrophy may correlate with genome size .
the tricarboxylic acid ( tca ) cycle is the pathway that converts acetyl - coa to co2 and is the major source of reducing equivalents during growth on multicarbon compounds ; the long - held hypothesis that m. capsulatus lacks a complete tca cycle   has not been proven true by genome sequencing , as putative genes for all the enzymes of the cycle were identified in the recent study .
in addition , the organism seems to encode an array of enzymes that could metabolize sugars , so the inability of m. capsulatus to grow on sugars remains enigmatic .
analysis of the genes encoding enzymes involved in the metabolism of single - carbon compounds in m. capsulatus ( figure 2 ) has been greatly simplified by the addition of data available from pre - genomic analyses [ 7 - 9,21 ] and from the initial analysis of the genome of m. extorquens . as expected ,
all the genes encoding enzymes of the rump pathway have been identified . in accordance with previous observations ,
most of the genes for the serine cycle were also found , as were the genes for the calvin - benson - bassham ( cbb ) cycle , the pathway that reduces co2 and converts it into biomass ( figure 2f ) .
the potential to operate all three known pathways for the assimilation of single - carbon compounds that are found in various methylotrophs makes this organism unique , but further analysis involving knockout mutations is needed to understand the functions of each of the three pathways .
the first glimpses into the expression patterns of pathways enabling methanotrophy are coming from a proteomic analysis of m. capsulatus by a group that has independently sequenced the m. capsulatus genome to 8x coverage . in this work ,
quantitative proteomic analysis was performed in order to compare the response of m. capsulatus to low - copper and high - copper conditions .
identified a total of 682 differentially expressed proteins using a cleavable isotope - coded affinity tag ( cicat ) technique .
the authors   demonstrated that , as expected , pmmo is overexpressed in conditions of high copper whereas smmo is expressed at low copper levels .
equally interesting data from this work concern the expression of proteins other than mmos , indicating that , indeed , all three assimilatory pathways are simultaneously expressed .
the oxidative pathway linked to tetrahydromethanopterin ( h4mpt ) is one of the pathways by which formaldehyde can be oxidized to co2 ( figure 2b ) ; all the enzymes in this pathway were identified , pointing to the importance of this pathway , as suggested previously by enzyme - activity measurements .
peptides for the oxidative branch of the rump cycle were also identified , suggesting that it is operational in m. capsulatus ( figure 2a ) .
some of the major serine - cycle enzymes were found to be overexpressed under high - copper conditions .
it is unlikely , however , that their expression would be directly regulated by copper ; it is more likely that they are responding to the higher flux of formaldehyde that occurs during growth under high - copper conditions .
it is important to note that the serine cycle can not operate as a major assimilatory pathway in m. capsulatus unless the two - carbon compound glyoxylate that is depleted during the cycle can be regenerated , but no genes have been identified in the genome that potentially encode either of the enzyme systems that can convert acetyl - coa into glyoxylate : the isocitrate lyase and the glyoxylate - regeneration cycle .
given these considerations , what might the function of the serine cycle ( and the interconnected tca cycle ) be in m. capsulatus ?
we suggest that a possible role for this pathway could be to handle the extra flux of formaldehyde that the organism may encounter under certain growth conditions ( figure 2c ) .
the excess of formate generated in the h4mpt - linked pathway ( figure 2b ) could also be redirected into the serine cycle after reduction to methylene - tetrahydrofolate ( methylene - h4f ; figure 2d ) .
acetyl - coa and other intermediates generated in this way could serve as building blocks for cell biomass .
the role of the cbb cycle in m. capsulatus ( figure 2f ) is not clear at present . given that the fixation of co2 is a far less efficient mechanism of carbon sequestration than the rump or serine cycles , a significant amount of carbon shunted through the cbb cycle would be predicted to decrease growth yield .
it is possible , however , that it serves to reduce the local concentration of co2 and/or to generate intermediates for biomass production . once again
to provide support for the hypothesis that reverse methanogenesis is important in anaerobic methanotrophy , a consortium of researchers focused on identifying the enzyme potentially involved in the initial step of anaerobic methane oxidation ; this enzyme is hypothesized to be similar to the bacterial mcr ( figure 1 , step 1 ) .
a microbial mat in the black sea largely consisting of anme-1-type archaea was chosen as a source of this hypothetical enzyme .
, a conspicuous protein consisting of three subunits similar to the  ,  , and  subunits of mcr is abundantly present in this microbial mat ( 7% of the total extracted protein ) , suggesting that it has an important role in anaerobic methane oxidation .
the protein contains a variant of f430 , a cofactor used by the classical mcr , but the two cofactors differ in molecular weight as determined by mass spectrometry .
the genes encoding this protein were sequenced as a part of an insert detected in an environmental dna library .
alignment of amino - acid sequences translated from these genes with the respective sequences of methanogen mcr subunits showed that residues involved in active - site formation in the  and  subunits were conserved , but one of the important residues in the active site of the  subunit was substituted .
it is interesting to speculate that this modification of the active site and the use of a modified f430 cofactor could provide a mechanism for the biochemical activation of methane and could make the first step of reverse methanogenesis thermodynamically and kinetically possible .
in a recent paper published in science , hallam et al .   describe a large environmental sequencing effort which aimed to provide further evidence for the hypothesis of reverse methanogenesis .
the group   isolated dna from a 52o - meter - deep sediment of eel river basin in california , known for a high abundance of anme-1 and anme - ii archaea , and used it for both whole - genome shotgun analysis and fosmid ' walking ' ( fosmids are large - insert plasmids ) .
a total of 111.3 mb of non - redundant sequence was generated by shotgun sequencing and another 4.6 mb more were generated by fosmid - end sequencing .
fosmids containing either 16s rrna genes belonging to anme - i or anme - ii archaea or homologs of the mcra gene were analyzed in detail , producing an additional 7.4 mb of sequence .
the main conclusion from this work   is that anme archaea contain most of the genes involved in methanogenesis , with one exception : mer , the gene encoding methylene - h4mpt reductase ( step 3 of reverse methanogenesis ; see figure 1 ) .
on the basis of the apparent lack of mer , the authors propose a model in which parts of the methanogenesis pathway function in two opposite directions : a novel mcr - like enzyme oxidizes methane to methyl - com ( step 1 ) , and methyl - h4mpt : com methyl - transferase catalyzes a reverse reaction to produce methyl - h4mpt ( step 2 ) , while the rest of the enzymes reduce co2 to methylene - h4mpt ( steps 4 to 7 in reverse ) ; that is , contrary to previous models , methane is not oxidized to co2 by anme archaea .
firstly , the model aggravates the thermodynamic constraints mentioned earlier , given that reduction of co2 to formyl - methanofuran ( step 7 ) is an energy - consuming reaction ( g = + 16 kj / mol ) .
secondly , the fate of the methylene - h4mpt produced in steps 4 to 7 is proposed to involve either the assimilatory serine cycle or formaldehyde oxidation , but the high energy cost of such schemes would suggest they could operate only as minor pathways , not as major assimilatory or detoxification pathways .
thirdly , there is no discussion by hallam et al .   of how net co2 would be produced from methane .
are an attempt to explain how methane metabolism might function in the absence of mer , they highlight the many aspects of this metabolic mode that are still unknown .
two different explanations might be that either mer has simply not been detected because of incomplete sequence data , or that the function of mer is fulfilled by a novel enzyme ( a non - homologous substitution ) , possibly involving a cofactor different from f420 , so the reverse - methanogenesis pathway might in fact be complete ( as in figure 1 ) .
an example of such a non - homologous substitution is seen in methylotrophic bacteria , in which a version of the ' reverse methanogenesis ' pathway has been found to operate where an nad(p)-linked methylene - h4mpt dehydrogenase acts in place of unrelated f420-linked or h2-forming enzymes . in conclusion ,
recent studies involving both organismal and environmental genomics shed new light on the biochemical details of the two processes important for methane balance on earth - aerobic and anaerobic methane oxidation - and suggest that these processes have more in common than just the substrate , methane , and the final oxidation product , co2 .
both processes involve common cofactors , such as h4mpt , common single - carbon intermediates bound to h4mpt , and common or similar enzymes for core reactions . although some enzymes involved in reactions that shift single - carbon compounds between different levels of oxidation are evolutionarily related in both processes , the primary methane oxidation enzymes , mmo and the newly identified mcr homolog , must have evolved independently and are fundamentally different .
a proposed pathway for anaerobic oxidation of methane involving the homolog of methyl - com reductase and a novel methylene - tetrahydromethanopterin ( h4mpt ) reductase ( mer ) , and its connection with the sulfate reduction pathway .
solid arrows represent enzymes predicted from the sequences found by hallam et al . ; the dotted arrow represents the one enzyme that was not predicted , methylene h4mpt - reductase ( mer ) .
enzymes performing steps 1 - 7 : 1 , methyl - com reductase - like protein ( mcr ) ; 2 , methyl - h4mpt : coenzyme m ( com ) methyl - transferase ( mtr ) ; 3 , methylene - h4mpt reductase ( mer ) ; 4 , f420-dependent methylene - h4mpt dehydrogenase ( mtd ) ; 5 , methenyl - h4mpt cyclohydrolase ( mch ) ; 6 , formyl - mfr : h4mpt formyltransferase ( ftr ) ; 7 , formyl - mfr dehydrogenase ( fmd ) .
( b ) reverse methanogenesis is thought to be connected to sulfate reduction through an unknown intermediate ( x ) ; erepresents an electron .
hallam et al .   suggest that steps 1 and 2 in ( a ) function in the down direction and methyl - h4mpt is used for biomass generation ( c ) , while steps 4 to 7 function in the up direction and the methylene - h4mpt produced is either converted to biomass through the serine cycle or is oxidized to co2 .
pathways in the aerobic methanotrophic bacterium methylococcus capsulatus involved in the metabolism of single - carbon compounds , as determined by genome sequencing and proteome analysis .
formaldehyde produced from methane can be metabolized in the following alternative ways : ( a ) through the ribulose monophosphate ( rump ) cycle , which can either generate biomass ( via the assimilatory ( a ) rump cycle ) or co2 ( via the dissimilatory ( d ) rump cycle ) ; ( b ) by conversion to formate via intermediates containing tetrahydromethanopterin ( h4mpt ) ; ( c ) via methylene - tetrahydrofolate ( methylene - h4f ) to the serine cycle and from there into biomass . under certain conditions
, there can be an excess of formaldehyde and formate ; the former can be used up through pathway ( c ) and the latter by reduction to methylene - h4f ( d ) and thus directed into the serine cycle .
co2 produced in any of these reactions can be converted to biomass by either ( e ) the serine cycle or ( f ) the calvin - benson - bassham ( cbb ) cycle .
 recent sequencing of the genome and proteomic analysis of a model aerobic methanotrophic bacterium , methylococcus capsulatus ( bath ) has revealed a highly versatile metabolic potential . in parallel , 
 environmental genomics has provided glimpses into anaerobic methane oxidation by certain archaea , further supporting the hypothesis of reverse methanogenesis . 
None
Aerobic and anaerobic methanotrophs
Genomic insights into the aerobic methanotrophy of 
Proteomics of 
A novel MCR-like enzyme and anaerobic methane oxidation
The environmental genomics of reverse methanogenesis
Acknowledgements
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