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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

A mutation in the mch gene, encoding the enzyme 5,10-methenyl tetrahydromethanopterin (H4MPT) cyclohydrolase, was constructed in vitro and recombined onto the chromosome of the methanogenic archaeon Methanosarcina barkeri. The resulting mutant does not grow in media using H2/CO2, methanol, or acetate as carbon and energy sources, but does grow in media with methanol/H2/CO2, demonstrating its ability to utilize H2 as a source of electrons for reduction of methyl groups. Cell suspension experiments showed that methanogenesis from methanol or from H2/CO2 is blocked in the mutant, explaining the lack of growth on these substrates. The corresponding mutation in Methanosarcina acetivorans C2A, which cannot grow on H2/CO2, could not be made in wild-type strains, but could be made in strains carrying a second copy of mch, suggesting that M. acetivorans is incapable of methyl group reduction using H2. M. acetivorans mch mutants could also be constructed in strains carrying the M. barkeri ech hydrogenase operon, suggesting that the block in the methyl reduction pathway is at the level of H2 oxidation. Interestingly, the ech-dependent methyl reduction pathway of M. acetivorans involves an electron transport chain distinct from that used by M. barkeri, because M. barkeri ech mutants remain capable of H2-dependent methyl reduction.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

The biologically mediated formation of methane (methanogenesis) is the terminal step in the carbon cycle in many anaerobic environments and is exclusively mediated by members of the domain Archaea. These methanogenic archaea are found in extremely diverse anaerobic environments and catalyse methane production via four distinct, but overlapping, pathways (for recent reviews, see Thauer, 1998; Deppenmeier et al., 1999).

Most methanogens isolated to date possess the hydrogenotrophic pathway, in which CO2 is sequentially reduced via coenzyme-bound intermediates to methane using H2 as the electron donor (Fig. 1A). In Methanosarcina species, electrons from H2 are acquired via three types of hydrogenases. The Ech hydrogenase uses H2 to reduce ferredoxin, which then is used to reduce CO2 to the formyl level (Meuer et al., 1999; Stojanowic and Hedderich, 2004). The F420-dependent hydrogenases (Frh and Fre) reduce F420, a hydride carrier analogous to NAD (Jacobson et al., 1982). Reduced F420 then provides the electrons needed for reduction of methenyl and methylene groups to methylene and methyl groups respectively. The third hydrogenase, the F420-non-reducing hydrogenase, also known as the Vho hydrogenase, reduces methanophenazine, a membrane-bound electron carrier analogous to quinone (Brodersen et al., 1999). These electrons pass through an electron transport chain, ultimately resulting in the reduction of the methyl group to methane. A second pathway, aceticlastic methanogenesis, proceeds first by the activation of acetate to acetyl-CoA (Fig. 1B). Subsequently, the carbonyl group of acetyl-CoA is oxidized to CO2, generating the electrons needed for reduction of the methyl group to methane (Ferry, 1992). In the third pathway, the methylotrophic pathway, methyl groups from C-1 (one-carbon) compounds, such as methanol or methylamine, are funnelled into the methanogenic pathway by transfer to coenzyme M (CoM) (Fig. 1C; Keltjens and Vogels, 1993). Methyl-CoM is then disproportionated in a 3:1 ratio: one mole of methyl-CoM is oxidized to CO2 to provide the electrons needed to reduce three moles of methyl-CoM to methane. In the methylotrophic pathway, methyl-CoM oxidation is believed to require the same enzymes used for CO2 reduction, but running in the opposite direction. However, this has never been demonstrated in vivo. Finally, the methyl reduction pathway is a hybrid between the hydrogenotrophic and the methylotrophic pathways (Fig. 1D). In this fourth pathway, methyl groups derived from C-1 compounds are completely reduced to methane using H2 as the electron donor. This was first discovered as a distinct pathway in Methanosphaera stadtmaniae, which cannot disproportionate methanol or reduce CO2 to methane using H2, but which can grow on a combination of methanol and H2 (Miller and Wolin, 1985).

image

Figure 1. Four overlapping methanogenic pathways found in M. barkeri. All four pathways share a common step in the reduction of methyl-CoM to methane; however, they differ in the pathway used to form methyl-CoM, and in the source of the electrons used for its reduction to methane. Many methanogens reduce CO2 to methane using electrons derived from the oxidation of H2 (hydrogenotrophic pathway, A, shown in red). Alternatively, acetate can be split into a methyl group and an enzyme-bound carbonyl moiety. The latter is oxidized to CO2 to provide the electrons required for reduction of the methyl group to methane (aceticlastic pathway, B shown in blue). C-1 compounds such as methanol or methyl-amines can also be disproportionated to CO2 and methane. In this pathway one molecule of the C-1 compound is oxidized to provide electrons for reduction of three additional molecules to methane (methylotrophic pathway, C, shown in green). Finally, C-1 compounds can be reduced using electrons derived from hydrogen oxidation (methyl reduction pathway, D, shown in orange). The step catalysed by the Mch protein is indicated: note that this enzyme is predicted to be required for both the hydrogenotrophic and methylotrophic pathways. Abbreviations: Ech, ferredoxin-dependent hydrogenase; Frh, F420-dependent hydrogenase; Vho, methanophenazine-dependent hydrogenase; Fpo, F420 dehydrogenase; CHO-MF, formyl-methanofuran; CHO-H4MPT, formyl-tetrahydromethanopterin; CH≡H4MPT, methenyl-tetrahydromethanopterin; CH2=H4MPT, methylene-tetrahydromethanopterin; CH3-H4MPT, methyl-tetrahydromethanopterin; CH3-CoM, methyl-coenzyme M; CoM, coenzyme M; CoB, coenzyme B; CoM-CoB, mixed disulphide of CoM and CoB; Mph/MphH2, oxidized and reduced methanophenazine; F420/F420H2, oxidized and reduced Factor 420; Fd(ox)/Fd(red), oxidized and reduced ferredoxin; Ac, acetate; Ac-Pi, acetyl-phosphate; Ac-CoA, acetyl-coenzyme A.

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Methanosarcina species are the most metabolically diverse of the methanogens, with some species being able to utilize each of the pathways described above. Because methanogenesis is the only energy-conserving mechanism known to be used by methanogens, the capacity to utilize alternative routes for methanogenesis makes Methanosarcina species particularly attractive for genetic analysis. Accordingly, Methanosarcina mutants blocked in one pathway remain viable because of the existence of the other pathways (Zhang et al., 2000; Meuer et al., 2002). In contrast, most other methanogens utilize only a single pathway and, thus, are inviable when their single pathway is blocked (Pfeiffer et al., 1998).

Among the more versatile of Methanosarcina species is Methanosarcina barkeri, which is able to utilize methylated compounds, acetate and H2/CO2 for growth. Physiological studies have demonstrated that M. barkeri can reduce methanol to methane using H2 in stoichiometric amounts, suggesting that it can also grow via the methyl reduction pathway (Blaut and Gottschalk, 1984; Müller et al., 1986). Methanosarcina acetivorans, on the other hand, is incapable of growth on H2/CO2, but can grow on methylated compounds or acetate. Despite its inability to grow on H2/CO2, analysis of the M. acetivorans genome revealed the presence of three putative hydrogenase operons (a putative F420-reducing Frh hydrogenase and two putative F420-non-reducing hydrogenases of the Vho type) and a putative hydrogenase maturation operon, although no ech-type hydrogenase operon could be identified in the genome. Therefore, although this hypothesis has never been tested experimentally, it was argued that M. acetivorans might still be capable of utilizing the methyl reduction pathway (Galagan et al., 2002).

To examine this possibility that M. acetivorans can grow by methyl reduction, and to gain further insight into the in vivo roles of known methanogenesis genes, we constructed and characterized M. barkeri and M. acetivorans mutants lacking the mch gene, which encodes methylene-tetrahydromethanopterin (H4MPT) cyclohydrolase. This enzyme catalyses the reversible hydrolysis of 5,10-methylene-H4MPT to 5-formyl-H4MPT and, as described above, is believed to be required for both the hydrogenotrophic and methylotrophic pathways. However, Mch should not be required for the methyl reduction pathway (see Fig. 1). Thus, the ability to mutate the mch gene could serve to indicate the presence of the methyl reduction pathway because methyl reduction should be required for growth on H2/CO2/methanol when the hydrogenotrophic and methylotrophic pathways are blocked. Additionally, heterologous expression of the ech hydrogenase operon was used to gain insight into why M. acetivorans is unable to utilize H2, the most common reductant used by methanogens for growth.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Deletion of mch in M. barkeri abolishes 5,10-methenyl-H4MPT cyclohydrolase activity

A mutation with an insertion of a puromycin-resistance gene cassette (pac) into the mch gene was constructed in vitro and recombined onto the chromosome of the M. barkeri as described. The mutant strain, M. barkeri mch1, was verified by DNA hybridization (data not shown) and tested for Mch activity by following hydrolysis of 5,10-methenyl-H4MPT to 5-formyl-H4MPT. Whereas wild-type extracts contained significant Mch activity (0.370 µmol min−1 mg−1), only background levels could be detected in M. barkeri mch1 extracts (0.006 µmol min−1 mg−1). Thus, Mch is solely responsible for this reaction in M. barkeri, a conclusion fully consistent with the growth phenotype described in the next section.

M. barkeri mch1::pac is capable of growth by only methyl reduction

The M. barkeri mch1 mutant is severely limited with respect to its ability to utilize alternate growth substrates (Table 1). Although the mutant grew on methanol/H2/CO2, the conditions under which the mutant was isolated, it did not grow, even after prolonged incubation (up to 6 months), in media with methanol, acetate or H2/CO2. Thus, as predicted, mch is required for both the hydrogenotrophic and methylotrophic pathways, but not for the H2-dependent methyl reduction pathway. The inability to grow on acetate was somewhat surprising, but may be consistent with a defect in biosynthetic reactions (see Discussion).

Table 1.  Growth and methane production from M. barkeri strainsa.
 SubstrateGeneration time (h)CH4 produced (µmol)b
  • a

    . Growth and methane production in resting cell suspensions were determined as described in Experimental procedures. The average and standard deviation of three trials are shown. NG, no growth.

  • b

    . Cells were harvested from cultures grown to mid-log phase in HS medium with methanol plus H2/CO2. Substrate amounts added were: methanol (500 µmol), acetate (500 µmol), H2/CO2 (2678 µmol H2, 670 µmol CO2). Values were adjusted for methane production from no substrate control, which ranged from 0.3 µmol to 0.8 µmol.

M. barkeri Methanol 7.2 ± 0.4359.9 ± 14.7
H2/CO2 8.6 ± 0.9303.6 ± 42.1
Methanol/H2/CO2 5.2 ± 0.2730.0 ± 86.0
Acetate37.9 ± 6.1  1.8 ± 0.5
M. barkeri mch1::pac MethanolNG  2.0 ± 1.2
H2/CO2NG  1.1 ± 0.3
Methanol/H2/CO2 7.1 ± 0.6476.8 ± 18.7
AcetateNG  0.5 ± 0.1

To test whether the observed growth phenotypes of M. barkeri mch1 were due to a defect in methanogenesis, we measured the conversion of various growth substrates to methane in resting suspensions of methanol/H2/CO2-grown cells (Table 1). M. barkeri converted 72% of methanol to methane, in accordance with the expected 3:1 ratio. M. barkeri mch1, on the other hand, did not produce methane from either methanol or H2/CO2, suggesting that the lack of growth on these substrates is due to a block in methanogenesis. When mutant cells were incubated with methanol/H2/CO2 nearly 100% of the methanol was reduced to methane, conclusively demonstrating that M. barkeri mch1 is capable of methanogenesis via the methyl reduction pathway. Only background levels of methane were produced from acetate; however, this is consistent with previous results (Meuer et al., 2002) and the observation that the enzymes needed for aceticlastic methanogenesis are highly regulated in response to growth on acetate (Singh-Wissmann and Ferry, 1995). Because the mutant is unable to grow on acetate, it was not possible to perform the appropriate cell-suspension experiments needed to address the role of mch in aceticlastic methanogenesis.

The mch gene is essential in M. acetivorans

Although M. acetivorans is incapable of growth on H2/CO2, it has been proposed that M. acetivorans may be able to utilize H2 for reduction of methylated substrates (Galagan et al., 2002). To test this hypothesis, we attempted to disrupt the mch gene in M. acetivorans by transformation with a linearized plasmid containing an mch::pac construct similar to that used for construction of the M. barkeri mch mutant. Multiple attempts were made to isolate puromycin-resistant mutants on media with methanol/H2/CO2 as growth substrates (permissive conditions for the M. barkeri mch1 mutant); however, no mch mutants were obtained. Similar attempts in media supplemented with pyruvate, to overcome potential biosynthetic blocks, also failed, although a similar supplementation regime successfully overcame the biosynthetic block in M. barkeri ech mutants (Meuer et al., 2002).

As an alternative strategy, a markerless exchange method using an hpt counter-selection (Pritchett et al., 2004) was also used to attempt construction of an M. acetivorans mch mutant (Fig. 2). For this approach, a partial diploid strain was constructed that carries both the wild-type mch gene and the Δmch allele. Subsequently, recombinants that had resolved the merodiploid state were selected. If both alleles were equally fit under the growth conditions used, then the recombinants should have been comprised of equal numbers of mutant and wild-type strains. However, if there was a strong selection against the mutant allele (such as for an essential gene), then mutants would not be obtained among the recombinants. We examined 25 recombinants obtained on methanol/H2/CO2 medium and an additional 120 recombinants obtained on the same medium supplemented with acetate, pyruvate, casamino acids and yeast extract. All 145 recombinants possessed the wild-type mch allele. Therefore, it is very likely that mch is essential in M. acetivorans under the growth conditions that we examined.

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Figure 2. A selection/counter-selection strategy suggests that mch is essential in M. acetivorans. (A) pAMG5 was integrated into the region upstream of mch in the M. acetivorans chromosome via a single recombination event, which was selected by puromycin resistance. The resulting mch merodiploid (B) can then be resolved (C), either regenerating the wild-type locus or leaving a deletion of mch. Resolved recombinants are selected on the basis of 8-ADP resistance, which occurs by loss of the hpt gene encoded by pAMG5. Without selective pressure against the mutant, this resolution should result in 50% wild type, 50%Δmch; however, in this experiment 100% of 8-ADPR recombinants were wild type.

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To demonstrate that these results were not dependent on our methods, we constructed a strain carrying the mch allele from M. barkeri inserted into the hpt locus of M. acetivorans. The native mch could subsequently be deleted in this strain with no apparent effect on growth (Table 2). The ability to delete mch only in the presence of a complementing copy further supports the idea that mch is essential for M. acetivorans. Moreover, given the results obtained with the M. barkeri mch mutant, these data strongly suggest that M. acetivorans is incapable of growth via the H2-dependent methyl reduction pathway.

Table 2.  Growth of M. acetivorans strains containing complementing copy of mcha.
GenotypeGeneration time (h)
MethanolMethanol/H2/CO2Acetate
  • a

    . Growth rates were determined as described.

Δhpt::mch + (barkeri)7.2 ± 0.47.6 ± 0.433.7 ± 4.8
Δhpt::mch + (barkeri), Δmch7.1 ± 0.27.4 ± 0.333.8 ± 6.4

Heterologous expression of ech hydrogenase operon in M. acetivorans allows growth via methyl reduction pathway, but not via the hydrogenotrophic pathway

Hydrogenase activity should be required for growth via both the methyl reduction pathway and hydrogenotrophic pathways. Although one F420- and two methanophenazine-dependent hydrogenase operons are apparently encoded in the M. acetivorans genome (Galagan et al., 2002), this organism is incapable of growth on H2/CO2. Moreover, previous reports showed little hydrogenase activity in extracts of acetate-grown M. acetivorans cells (Nelson and Ferry, 1984). However, to our knowledge, hydrogenase activity has never been measured in extracts of cells grown in the presence of H2.

Because hydrogenases are often induced only in the presence of their substrate (Schwartz et al., 1998), we attempted to detect hydrogenase activity in extracts of cells grown on methanol/H2/CO2. Although we could readily detect hydrogenase activity in M. barkeri extracts (11.22 ± 1.67 µmol min−1 mg−1), none was detected in extracts of M. acetivorans (<0.02 µmol min−1 mg−1). Thus, the hydrogenase operons of M. acetivorans are not functionally expressed under these growth conditions. This result suggests that the inability of M. acetivorans to grow via the methyl reduction pathway is due to a lack of hydrogenase activity.

Although the M. acetivorans genome encodes three putative hydrogenases, it does not encode an Ech hydrogenase homologue. To examine whether the absence of Ech hydrogenase was responsible for the growth defects of M. acetivorans, we constructed a transgenic strain with the echABCDEF operon from M. barkeri inserted into the M. acetivorans chromosome at the ssuBCA locus. (This locus is useful as a permissive site for integration of foreign DNA because recombinant strains with insertions into this operon can be selected on the basis of their resistance to the inhibitor 2-bromoethane sulphonic acid (BES) (Zhang et al., 2000). In contrast to the parental strain, the ech+ recombinant strain, hereafter designated M. acetivorans ech+, exhibited substantial hydrogenase activity (2.25 ± 0.42 µmol min−1 mg−1). While this level of hydrogenase activity is only c. 20% of that observed in M. barkeri, it is important to note that the viologen dye-based assay used here does not discriminate between the three classes of hydrogenase. If one assumes that Ech activity comprises c. one-fourth of the total M. barkeri hydrogenase activity, then the level of Ech activity in M. acetivorans ech+ is comparable to that in M. barkeri.

Despite the presence of a functionally expressed ferredoxin-dependent hydrogenase, M. acetivorans ech+ was not able to grow on H2/CO2 (Table 3). Therefore, the inability of wild-type M. acetivorans to grow on this substrate cannot be solely attributable to the lack of Ech. M. acetivorans ech+ grew at a rate comparable to the parent strain on methanol or methanol/H2/CO2 and somewhat slower on acetate. Interestingly, the growth yield of M. acetivorans ech+ increased by c. 20% when growing on methanol/H2/CO2 as compared to growth on methanol alone and increased 20% as compared to the wild-type on methanol/H2/CO2. Possible explanations for this increased growth yield are discussed below.

Table 3.  Growth and methane production of M. acetivorans strains encoding the M. barkeri ech hydrogenase operon.
Relevant genotypeaSubstrateGeneration time (h)bMaximum growth (OD600)bMethane production (µmol)d
  • a

    . All strains are derivatives of M. acetivorans WWM19, which carries an insertion of pWM357 into the hpt locus. This mutation does not effect growth or methane production (data not shown). The echABCDEF genes were derived from M. barkeri Fusaro.

  • b

    . Growth rate was measured by following the optical density of the cultures. The maximum optical density is reported. NG, no growth; N/A, not applicable; ND, not determined.

  • c

    . Maximum OD600 was not determined under these conditions because a white precipitate formed in the tube upon reaching an OD600c. 0.4. This precipitation was presumably caused by an increase in pH due to consumption of acetic acid.

  • d

    . Methane production was measured in suspensions of methanol/H2/CO2-grown cells as described. The substrate amounts added were: methanol (500 µmol), acetate (500 µmol), H2/CO2 (2678 µmol H2, 670 µmol CO2). Values were adjusted for methane production from no substrate control, which ranged from 0.3 µmol to 0.8 µmol, and represent the average of at least three trails.

Wild typeMethanol 7.1 ± 0.60.74 ± 0.02356.2 ± 16.5
H2/CO2NGN/A  1.2 ± 0.6
Methanol/H2/CO2 7.5 ± 0.50.76 ± 0.02363.8 ± 17.0
Acetate30.1 ± 3.0NDc  0.6 ± 0.1
Δssu::echABCDEF+Methanol 7.0 ± 0.30.75 ± 0.02359.6 ± 23.6
H2/CO2NGN/A 22.8 ± 7.5
Methanol/H2/CO2 7.2 ± 0.10.93 ± 0.02458.9 ± 17.0
Acetate42.0 ± 3.9NDc  0.8 ± 0.1
Δssu::echABCDEF+mch::pacMethanolNGN/A  1.8 ± 0.7
H2/CO2NDND  0.7 ± 0.1
Methanol/H2/CO2 7.1 ± 0.60.85 ± 0.03407.9 ± 15.8
AcetateNGN/A<0.1

Methane production by resting suspensions of methanol/H2/CO2-grown cells was measured to determine the effect of ech on methanogenesis in M. acetivorans (Table 3). Methylotrophic methanogenesis in M. acetivorans ech+ did not differ from the wild type: 73% of the methanol was converted to methane (as expected for a 3:1 disproportionation). With methanol/H2/CO2, however, the wild type converted only 73% of the methanol to methane, whereas M. acetivorans ech+ reduced over 90% of the methanol to methane. This excess methyl group reduction was presumably driven by electrons derived from H2 rather than those derived from oxidation of methyl-CoM, which demands the 3:1 disproportionation ratio. Thus, the methyl reduction pathway is functional in M. acetivorans ech+, whereas the wild type can only use the methylotrophic pathway.

Introduction of ech into M. acetivorans allows for deletion of mch

Genetic studies provided further evidence for the presence of a functional methyl reduction pathway in M. acetivorans ech+. In contrast to the wild type, mch could be readily disrupted in M. acetivorans ech+ by pac insertion, demonstrating that mch is dispensable in M. acetivorans ech+. The phenotype of the M. acetivorans ech+mch::pac strain is similar to that of the M. barkeri mch1::pac mutant described above: it is unable to grow on either methanol or acetate, but can grow on the combination of methanol/H2/CO2 (Table 3). Methane production by resting cells was also similar to the M. barkeri mch1::pac mutant. In M. acetivorans ech+Δmch resting cells methanogenesis was blocked from methanol and from H2/CO2; however, methane production was readily observed from methanol/H2/CO2. These data strongly support the idea that mch is essential in M. acetivorans because the organism lacks a functional H2-dependent methyl reduction pathway.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

The data reported here show that mutational analysis of the mch gene can be a powerful tool to address questions concerning H2 metabolism in Methanosarcina species. Accordingly, our data show that Mch is required for methanogenesis by the methylotrophic and hydrogenotrophic pathways, but dispensable for the H2-dependent methyl reduction pathway, as evidenced by the ability of M. barkeri Δmch to grow on methanol/H2/CO2, but not on methanol or H2/CO2. Consistent with this finding, mch is essential for growth of M. acetivorans, which lacks hydrogenase activity and therefore cannot utilize the methyl reduction or CO2 reduction pathways for methanogenesis. Hydrogenase activity could, however, be provided to M. acetivorans by heterologous expression of the ech genes from M. barkeri, thus allowing utilization of H2 for methyl reduction, as demonstrated by the change in stoichiometry of methanogenesis in resting cell suspensions. Further confirmation that expression of a functional hydrogenase led to acquisition of methyl reduction capability was provided by subsequent deletion of the no longer essential mch gene in the M. acetivorans ech+ strain.

It is interesting to note that acquisition of a functional methyl reduction pathway in M. acetivorans ech+ led to increased growth yields on media with methanol/H2/CO2 relative to media with methanol alone, or relative to strains without ech in the same methanol/H2/CO2 medium. The simplest explanation for this observation is that the strains were experiencing a higher effective substrate concentration during growth via the methyl reduction pathway. Thus, during growth via the methylotrophic pathway, 25% of the methyl groups are oxidized to provide the reducing equivalents needed to reduce the remainder to methane. However, during H2-dependent methyl reduction 100% of the methyl groups are available for reduction to methane. Therefore, even though the methanol concentration is the same in both media, the effective number of methyl groups available for reduction to methane is 25% higher when growth occurs via methyl reduction. It is probably no coincidence that the increase in growth yield is c. 25%. Interestingly, this implies that the most, if not all, of the energy conservation that occurs during methanogenesis from methanol occurs during the methyl reduction steps, which are presumably identical in the methylotrophic and methyl reduction pathways.

The ferredoxin-dependent Ech hydrogenase, which is absent in M. acetivorans, was previously shown to be required for the reduction of CO2 to formyl-methanofuran in M. barkeri (Meuer et al., 2002). However, introduction of a functional Ech hydrogenase to M. acetivorans was insufficient to allow growth on H2/CO2. It is likely that this growth defect is due to the lack of functional Frh- and Vho-type hydrogenases in this organism (despite the presence of genes which appear to encode these enzymes). Based on the known enzymology of the hydrogenotrophic pathway, both reduced F420 and reduced methanophenazine are probably required for CO2 reduction (see Fig. 1). Thus, both F420- (Frh) and methanophenazine-dependent hydrogenases (Vho) should be required when H2 is the sole source of reducing equivalents. Despite the growth defect on H2/CO2, small amounts of methane were produced by resting cells M. acetivorans ech+. This suggests that there is a biochemical connection between the reduced ferredoxin pool generated by Ech and the pools of reduced F420 and methanophenazine needed to reduce CO2 to methane. It is evident, however, that this connection is insufficient to support growth.

While M. acetivorans ech+ uses the Ech hydrogenase for the methyl reduction pathway, Ech is clearly not required for the methyl reduction pathway in M. barkeri. Thus, when the ech operon is deleted in M. barkeri, methanogenesis from methanol/H2 is unaffected (Meuer et al., 2002). A variety of experimental evidence (reviewed in Deppenmeier, 2004) suggests that the methanophenazine-reducing Vho hydrogenase is used for methyl reduction in M. barkeri. In this organism, an energy-conserving electron transport chain delivers reducing equivalents from H2 through methanophenazine to provide the reduced coenzyme B (CoB-SH) used in the terminal step of methanogenesis (Fig. 3A). In the absence of a functional Vho hydrogenase, M. acetivorans ech+ must have an alternate mechanism to generate reduced CoB-SH. Therefore, given that Ech produces reduced ferredoxin from H2 oxidation, there must be an alternative electron transport chain from ferredoxin to the heterodisulphide CoB-S-S-CoM in M. acetivorans (Fig. 3B). Moreover, this alternative electron transport chain must involve energy-conserving steps, because it replaces an energy-conserving electron transport chain that is thought to be the sole energy conservation mechanism in the H2-dependent methyl reduction pathway (Deppenmeier, 2004). Additional evidence for differences between the electron transport chains of the two Methanosarcina species was recently provided based on the role of Ech in aceticlastic methanogenesis in M. barkeri (Meuer et al., 2002). Analysis of M. barkeri ech mutants, which failed to grow on acetate, led to the suggestion that H2 is an obligate intermediate in aceticlastic methanogenesis. In this scheme, reduced ferredoxin produced by oxidation of the carbonyl group of acetate is oxidized by Ech to produce H2, which is subsequently channelled into the electron transport chain via the methanophenazine-reducing Vho hydrogenase (Fig. 1B). However, this electron transport scheme cannot apply to M. acetivorans, which grows well on acetate, but which lacks both Ech and any measurable hydrogenase activity. Because reduced ferredoxin is almost certainly produced by acetate oxidation in M. acetivorans, there must be an electron transport chain that links reduced ferredoxin to CoB, which is needed in the final step of methanogenesis. The nature of this electron transport chain remains to be elucidated, but may be related to the ability, noted above, of M. acetivorans ech+ to perform limited H2-dependent CO2 reduction.

image

Figure 3. Model for alternative electron transport chain in M. acetivorans resulting in methyl reduction. In both M. barkeri(A) and M. acetivorans ech+(B) electrons from H2 must be used to regenerate CoB-SH and CoM-SH from the CoM-S-S-CoB heterodisulphide that is formed in the final step of the methanogenic pathway. Abbreviations: CH3-CoM, methyl-coenzyme M; CM, cytoplasmic membrane; CoB-SH, reduced coenzyme B; CoM-SH, reduced coenzyme M; CoM-S-S-CoB, heterodisulphide of coenzyme M and coenzyme B; Ech, Ech hydrogenase; Fd, reduced ferredoxin; Hdr, heterodisulphide reductase; Mph, oxidized methanophenazine; MphH2, reduced methanophenazine; Vho, viologen-dependent hydrogenase; +, outside of membrane; −, cytoplasmic side of membrane.

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Also worthy of note is the failure of mch mutants to grow on acetate. Although not predictable from examination of the aceticlastic pathway alone, this result is fully consistent with published findings and is not particularly surprising. Aceticlastic methanogenesis, as shown in Fig. 1B, is a balanced redox reaction with one carbonyl moiety being oxidized for every methyl group reduced. Nevertheless, it has been reported that during growth on acetate a significant fraction of acetate methyl groups is oxidized to CO2 (Krzycki et al., 1982). It was proposed that this oxidation proceeds via a reversal of the CO2 reduction pathway in order to generate the reducing equivalents needed for anabolic reactions during growth (Jablonski et al., 1990). Deletion of mch would block this oxidative pathway and thus impair growth on acetate. Unfortunately, we were unable to provide experimental support for this hypothesis. Attempts to supplement this supposed biosynthetic block by providing yeast extract, casamino acids and methanogen cell extract failed (data not shown), despite the observation that similar biosynthetic blocks in the ech mutant of M. barkeri could be overcome by such supplementation (Meuer et al., 2002). This failure may be due to an inability of the organism to take up the required intermediate. With the idea that H2 might be able to provide the required reducing equivalents, we also attempted, again unsuccessfully, to grow the mutant in media with both H2 and acetate (data not shown). This result is consistent with in vitro data showing that the presence of H2 inhibits the oxidative pathway (Meuer et al., 2002). We were unable to grow the cells under the conditions needed to induce the enzymes of the aceticlastic pathway, thus we could not accurately assess the ability of resting cells to produce methane from acetate.

Finally, it is tempting to speculate that the absence of H2 metabolism in M. acetivorans is an adaptation to growth on acetate in a marine environment. The involvement of H2 as an obligate intermediate in aceticlastic methanogenesis presents a significant problem. Because H2 is a freely diffusible gas that is membrane-permeable, it is likely that a significant fraction of this intermediate would be lost to the producing organism. Thus, the processes involving H2 as an obligate intermediate might be fairly inefficient. However, M. barkeri strains typically grow in large multicellular aggregates. Thus, any H2 lost to one cell would likely be taken up by neighbouring cells (which would be genetic siblings), thus preventing loss of H2 to the aggregate as a whole. M. acetivorans, on the other hand, isolated from a marine environment, grows as disaggregated single cells (Sowers et al., 1984) and would likely be subject to diffusive H2 losses. Selection for alternate, non-H2-dependent, electron transport chains would prevent such losses and increase the fitness of organism during growth on acetate. The price for this increase in fitness would be the inability to grow on H2/CO2. However, the loss of hydrogenotrophic methanogenesis is not likely to be of consequence in marine environments where sulphate reducers are expected to out-compete methanogens for external H2 (Zinder, 1993).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

Methanosarcina strains and plasmids

Methanosarcina strains used in this study were constructed by standard methods (Metcalf et al., 1997; Boccazzi et al., 2000; Zhang et al., 2001; Pritchett et al., 2004) and are listed in Table 4. Details of the strain constructions are provided in the supplementary online material (Table S1). All Methanosarcina strains were grown as single cells (Sowers et al., 1993) at 37°C under strictly anaerobic conditions in HS media (Metcalf et al., 1996). Growth substrates provided were methanol (125 mM) or acetate (120 mM or 40 mM in combination with methanol) under either a N2/CO2 (80/20%) or H2/CO2 (80/20%) headspace as indicated. All cultures were grown with gentle agitation to enhance transfer of gaseous substrates to the aqueous phase. Cultures were supplemented as indicated with yeast extract (0.1%), casamino acids (0.1%), pyruvate (50 mM), and Methanosarcina extract (0.05%) prepared as described (Pritchett et al., 2004). Growth on agar-solidified media was as described (Sowers et al., 1993) with the modifications from Boccazzi et al. (2000). Puromycin was added to a final concentration of 2 µg ml−1 to select for the presence of pac (Possot et al., 1988). 2-bromoethane sulphonic acid (BES) was added to a final concentration of 1 mM to select against the presence of the ssuBCA operon. 8-aza-2,6-diaminopurine (8ADP) was added to a final concentration of 20 µg ml−1 to select against the presence of hpt (Pritchett et al., 2004). All additions were from sterile, anaerobic stock solutions. For growth rate determinations a c. 3% inoculum of a mid-log phase (OD600c. 0.5) culture was transferred to fresh media and incubated at 37°C. Growth was quantified by measuring the optical density at 600 nm (OD600); generation times were calculated during exponential growth phase.

Table 4.  Methanosarcina strains and plasmids.
Methanosarcina strainsGenotypeSource
M. barkeri (DSM 804)Wild typeLaboratory stocks
M. barkeri WWM68 mch1::pac This study
M. acetivorans (DSM 2834)Wild typeLaboratory stocks
M. acetivorans WWM1Δhpt(Pritchett et al., 2004)
M. acetivorans WWM19Δhpt::pWM357This study
M. acetivorans WWM65Δhpt::mch (barkeri)This study
M. acetivorans WWM64Δssu::echABCDEF (barkeri)This study
M. acetivorans WWM67Δhpt:: mch (barkeri), Δmch::pacThis study
M. acetivorans WWM66Δssu::echABCDEF (barkeri), mch::pacThis study

Plasmids used in the study, and their construction, are described in the supplementary online materials (Table S2). Standard techniques were used throughout for isolation and manipulation of plasmid DNA in E. coli (Ausebel et al., 1992). DH5α/λpir (Miller and Mekalanos, 1988) was used as a host strain for plasmids containing the oriR6K origin of replication, DH5α and DH10B (Invitrogen, Carlsbad, CA, USA) were the host strains used for other plasmids. The inferred plasmid sequences are available upon request.

Methane production by cell suspensions

Cells from mid-log phase (OD600c. 0.5) HS-methanol/H2/CO2-grown cells were collected by centrifugation, washed once in plain HS medium (without any substrate) and resuspended at a concentration of 109 cells ml−1 in HS medium containing 25 µg ml−1 sparsomycin (Sigma, St. Louis, MO) to inhibit protein synthesis. Cells were kept on ice and supplemented with substrate as indicated. The 25 ml headspace was exchanged to 200 kPa of overpressure either N2/CO2 or H2/CO2 (80:20, v/v). Cell suspensions were then incubated at 37°C on a Roller Drum (New Brunswick Scientific, Edison, NJ) until CH4 production ceased. This generally took c. twice as long for M. acetivorans ech+Δmch on methanol/H2/CO2 as for the other strains. CH4 was measured by gas chromatography in a Hewlett Packard gas chromatograph (5890 Series II) equipped with a flame ionization detector. Methane production is reported as the amount produced corrected for the amount produced without substrate.

Enzymatic activities

All steps were carried out under strictly anaerobic conditions in an anaerobic chamber (Coy Laboratories, Ann Arbor, MI). H4MPT was purified from M. thermoautotrophicum as described (Escalante-Semerena et al., 1984). 5,10-methenyl-H4MPT was synthesized enzymatically as described (Donnelly et al., 1985). Cells from a late logarithmic growth phase culture were subjected to osmotic lysis by resuspension in lysis buffer (100 mM Tris-HCl pH 8, 1 mM PMSF, and 5 µg ml−1 DNaseI), and cleared by centrifugation at 9000 g for 10 min. The resulting supernatant was removed and assayed for 5,10-methenyl-H4MPT cyclohydrolase activity under anaerobic conditions as described (DiMarco et al., 1986), with the following modifications. Each cuvette was filled with 100 mM Tris-HCl pH 8 to a final reaction volume of 0.7 ml and heated to 37°C. Cell extract was added, and the reaction was initiated by the addition of 5,10-methenyl-H4MPT to a concentration of 30 µM. UV-Vis absorbance spectra were recorded in 2 s intervals with a Hewelett Packard 8453 diode array spectrophotometer. Enzyme activity was calculated by following loss of 5,10-methenyl-H4MPT at 340 nm (ɛ = 21.6 mM−1 cm−1).

For hydrogenase activity measurements, strains were grown to mid-log phase as above. Cell extracts were prepared by osmotic lysis in reaction buffer (see below) + DNaseI, followed by clearing of the lysate by centrifugation at 14 000 g for 5 min. Hydrogenase activity was determined in sealed 1.4 ml anaerobic cuvettes filled with 0.7 ml of reaction buffer containing 50 mM 3-(N-morpholino) propane sulphonic acid (MOPS) pH 7.0 and 2 mM dithiothreitol (DTT), and were purged with either 100% N2 (negative control) or 100% H2. Cell extract was added, followed by addition of benzyl viologen (BV) to 2 mM final concentration. Hydrogenase activity was followed by BV reduction at 576 nm (ɛ = 8.65 mM−1 cm−1) using a Hewelett Packard 8453 Spectrophotometer. BV reduction was measured in the absence of H2 to correct for non-specific activity. Protein concentration was determined by the Bradford method (Bradford, 1976) using bovine serum albumin as the standard.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

We would like to thank P. Welander for construction of pPW1 and pPW2 and A. Eliot and M. Rother for critical review of the manuscript. This work was supported by grants from the Department of Energy (DE-FG02-02ER15296) and the National Science Foundation (MCB 0212466) to W.W.M. Further support for A.G. was provided by NIH Cell and Molecular Biology Training Grant ♯ PHS2T32GMO7283.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary material
  9. References
  10. Supporting Information

The following supplementary material is available for this article: Tables S1-S2. Tables S1-S2.

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