Journal of Bacteriology, December 2003, p . 7160-7168, Vol . 185, No . 24

In the facultative methylotroph Methylobacterium extorquens AM1, primary oxidation of C₁ substrates such as methanol or methylamine occurs in the periplasm through the action of methanol dehydrogenase (1) and methylamine dehydrogenase (8) (Fig. 1) . Formaldehyde that enters the cytoplasm condenses with one of two pterin cofactors, tetrahydrofolate (H₄F) or tetrahydromethanopterin (H₄MPT), to form the respective methylene derivatives . The reaction of formaldehyde with H₄F seems to occur spontaneously (14), and no enzyme has been found thus far that is capable of accelerating this reaction (33). Methylene-H₄F can either be assimilated through the serine cycle or may be oxidized to methenyl-H₄F, formyl-H₄F, and ultimately formate (reviewed in reference 17) . Formate can then be oxidized to CO₂ through the action of formate dehydrogenases (16; L . Chistoserdova and M . E . Lidstrom, unpublished data) .

 
Alternatively, formaldehyde can be oxidized through a similar C₁ transfer pathway that is linked to the folate analog H₄MPT. H₄MPT-dependent C₁ transfers were thought to be unique to methanogenic and sulfate-reducing archaea until their unexpected discovery in M . extorquens AM1 (6) . Subsequently, this pathway has been found in most gram-negative methylotrophs with a few exceptions (32) . In methylotrophic bacteria the initial step in this pathway is the reaction of formaldehyde with H₄MPT to form methylene-H₄MPT (Fig. 1) . While this reaction can occur spontaneously, as is apparently the case for H₄F, a specific formaldehyde-activating enzyme (Fae) has been shown to catalyze this condensation, and this enzyme is required for methylotrophic growth (33) . The resulting methylene-H₄MPT is oxidized to methenyl-H₄MPT through the action of one of two methylene-H₄MPT dehydrogenases, MtdA and MtdB . MtdA, which also catalyzes the oxidation of methylene-H₄F, is strictly NADP dependent (31), whereas MtdB can use either NAD⁺ or NADP⁺ but is specific for methylene-H₄MPT (10). Methenyl-H₄MPT is converted to formyl-H₄MPT by methenyl-H₄MPT cyclohydrolase (Mch) (24) . Finally, formate is released from formyl-H₄MPT through the action of the formyltransferase/hydrolase complex (Fhc) (22, 23) . Formate can then be oxidized to CO₂ by formate dehydrogenase, as for the H₄F-linked pathway . Fhc contains formyltransferase activity that is active with methanofuran purified from the methanogen Methanothermobacter marburgensis (23) . The identity and function of the methanofuran analog present in M . extorquens AM1 has not yet been determined, however, and so this pathway simply will be referred to here as the H₄MPT pathway .

A number of mutants defective for known or suspected H₄MPT pathway functions have been generated and, based on their growth phenotype, a function in energy generation during methylotrophic growth has been proposed . Null mutants lacking mtdB (10), fae (33), and dmrA (which encodes a putative dihydromethanopterin reductase [20]) have been reported to be both incapable of growth on methanol and inhibited by either methanol or formaldehyde during growth on succinate . This methanol-sensitive mutant phenotype is thus far unique to the mutants of M . extorquens AM1 defective for the H₄MPT pathway and has been proposed to be due to an inability to detoxify the formaldehyde produced from methanol . Double mutants lacking both MDH activity and MtdB activity were no longer sensitive to methanol (10), lending further support to the concept that methanol sensitivity is a proxy for formaldehyde detoxification deficiency . A null mutant lacking Orf4, a homolog of the first enzyme in the H₄MPT biosynthesis pathway, ß-ribofuranosylaminobenzene 5'-phosphate (ß-RFAP) synthase (28), has also been generated and was incapable of growth on methanol (6) . However, efforts to obtain null mutants in other genes, such as mtdA (7), mch, and fhcBADC (6), have not been successful . Mutants resulting from an incomplete allelic exchange event that separated a wild-type copy of the gene from its native promoter by the integrated vector were obtained for these genes and, where examined, this led to reduced enzymatic activity (6, 7) . In all cases, this class of mutants exhibited defective growth on methanol, indicating a specific role in methylotrophy in addition to apparent essentiality. MtdA activity is likely required to produce formyl-H₄F for biosynthetic needs (7) . It has not been clear, however, why the other genes for which null mutants could not be obtained and have a known or predicted role in the H₄MPT pathway for formaldehyde oxidation would be required for growth on a multicarbon substrate such as succinate . Therefore, the role of the H₄MPT pathway has been uncertain, and in this study we have carried out experiments to define that role .

Here we present a comparative physiological analysis of H₄MPT pathway mutants, analyzing the methanol-sensitive phenotype in more detail and demonstrating that it is due to formaldehyde production from methanol . In addition, through the complementation of H₄MPT pathway mutants with an alternative formaldehyde oxidation system and by demonstrating that the H₄MPT pathway is in fact not essential, we have defined the role of the H₄MPT pathway as the primary formaldehyde oxidation and detoxification route in M. extorquens AM1 .

Generation of mutant strains. M. extorquens AM1 deletion mutants lacking mxaF, orf4, mtdB, mch, or the fhcBADC cluster were generated using the allelic exchange vector pCM184 (18) . Approximately 0.5-kb regions upstream and downstream of these genes or gene clusters were amplified by PCR and cloned into pCR2.1 (Invitrogen) as follows. Cloning of the mxaF upstream and downstream flanks resulted in pCM191 and pCM192, the orf4 flanks resulted in pCM250 and pCM251, the mtdB flanks resulted in pCM255 and pCM256, the mch flanks resulted in pCM260 and pCM261, and the flank downstream of fhcC resulted in pCM264 . The construct to generate mxaF::kan mutants was generated by introducing the 0.5-kb ApaI-SacI fragment from pCM192 between the corresponding sites of pCM184 to produce pCM193 and, subsequently, the 0.6-kb EcoRV-Asp718I fragment from pCM191 was ligated between the PvuII and Asp718I sites of pCM193 to produce pCM194 . The construct to generate orf4::kan mutants was generated by introducing the 0.5-kb EcoRI-Asp718I fragment from pCM250 into the same sites of pCM184 to produce pCM252 and, subsequently, the 0.7-kb ApaI-SacI fragment from pCM251 was ligated into the same sites of pCM252 to produce pCM253 . The construct to generate mtdB::kan mutants was generated by introducing the 0.6-kb SacII-SacI fragment from pCM256 into the same sites of pCM184 to produce pCM257 and, subsequently, the 0.5-kb AatII-Asp718I fragment from pCM255 was ligated into the same sites of pCM257 to produce pCM258 . The construct to generate mch::kan mutants was generated by introducing the 0.6-kb AatII-Asp718I fragment from pCM260 into the same sites of pCM184 to produce pCM262 and, subsequently, the 0.7-kb ApaI-SacI fragment from pCM261 was ligated into the same sites of pCM262 to produce pCM263. Finally, the construct to generate fhcBADC::kan mutants was generated by introducing the 0.5-kb EcoRI-NcoI fragment from pCM250 into the same sites of pCM184 to produce pCM265 and, subsequently, the 0.5-kb SacII-AgeI fragment from pCM264 was ligated into the same sites of pCM265 to produce pCM266 .

Mutant strains of M . extorquens AM1 were generated by introducing the appropriate donor constructs by conjugation from E . coli S17-1 (29) as previously described (4) . Unmarked deletion strains were generated using the cre-expressing plasmid pCM157 as described elsewhere (18), allowing the generation of double mutant strains . All mutants were confirmed by diagnostic PCR analysis . All strains and plasmids utilized in this study are described in Table 1 .

 
Phenotypic analyses of mutant strains. In order to compare the growth of wild-type M . extorquens AM1 with that of mutants in liquid medium, cultures were grown to mid-exponential phase, centrifuged, and then resuspended into fresh medium containing the carbon source described . To test for sensitivity to methanol, methanol was added to one set of succinate flasks to the reported final concentration after 2 h . Mutant phenotypes were also assessed on solid medium by comparing the relative rate of colony formation. Sensitivity to methanol or formaldehyde was assayed using succinate medium to which methanol or formaldehyde was added immediately before pouring plates at the following tested concentrations: 125, 10, and 1 mM and 100, 10, 1, and 0.1 µM for methanol; 1, 0.5, 0.1, 0.05, 0.01, and 0.005 mM for formaldehyde . Because an undetermined fraction of the methanol will volatilize, the reported MIC for methanol is a maximum value . All phenotypic analyses were performed at least twice .

Generation of a plasmid overexpressing mtdA. The coding region of mtdA was amplified by PCR and cloned into pCR2.1 (Invitrogen) to produce pCM254 . The 1.0-kb HindIII-XbaI fragment of pCM254 was cloned between the same sites of the expression plasmid pCM80 (19) to generate pCM259. Plasmids were introduced into appropriate strains using the helper strain pRK2073 (9) .

Construct for the heterologous expression of the GSH-dependent formaldehyde oxidation pathway from Paracoccus denitrificans. The two primary genes comprising the glutathione (GSH)-dependent formaldehyde oxidation pathway of P. denitrificans, flhA (26) and fghA (11), were amplified by PCR using pWRxox451 (25) as a template and cloned into pCR2.1 (Invitrogen) to produce pCM102 and pCM103 . The 1.0-kb EcoRI fragment of pCM103 was introduced into the EcoRI site of pCM80 to generate pCM104, into which the 1.4-kb XbaI fragment from pCM102 was inserted into the corresponding site to produce pCM106 .

Enzyme assays. The activities of MtdA (31), FlhA (26), and FghA (11) were assayed in two to three replicates as described using cell extracts prepared using a French press from cell material harvested from exponential-phase cultures . Variation in enzyme activities between cultures was less than 20% . Total protein concentration in the extracts was assayed spectrophotometrically (13, 34) using a Beckmann DU 640B spectrophotometer .

All four mutants in the H₄MPT pathway employed in this study grew with wild-type characteristics in liquid medium containing succinate, indicating that the respective functions are not required for general heterotrophic growth (Fig. 2A), but they were unable to grow in medium containing methanol (Fig. 2D) . Analogous results were observed for growth on solid medium . Additionally, the H₄MPT pathway mutant strains grew like wild type on formate, indicating that they are not required for the metabolism of the more-oxidized C₁ compound formate . In order to compare the inhibitory effect of methanol on the growth of mutants defective for the H₄MPT pathway on succinate, methanol was added to a set of succinate flasks after 2 h to either a 1 or 125 mM final concentration (Fig . 2B and C) . Under these conditions, the mtdB mutant CM258K.1 grew like wild type . However, the three other mutants were inhibited at both methanol concentrations . Addition of methanol at 1 mM caused a more severe inhibition of the dmrA and orf4 mutant strains relative to the fae mutant strain, whereas 125 mM methanol caused cessation of growth in all three strains .

 
The MICs of methanol or formaldehyde during growth on succinate plates were also examined for the four H₄MPT pathway mutants . On solid medium a distinct inhibitory effect of methanol was observed for the mtdB mutant CM258K.1, with an MIC of 10 mM and an MIC of formaldehyde of 0.5 mM . In comparison, the wild type had an MIC of formaldehyde of 1 mM and was not inhibited by 125 mM methanol . The other mutants were observed to be significantly more sensitive, with MICs for methanol and formaldehyde, respectively, of 10 and 100 µM for the fae mutant CM198K.1 and 1 and 10 µM for the dmrA and orf4 mutants CM212K.1 and CM253K.1 .

Overexpression of mtdA provides partial complementation of the mtdB mutant phenotype. We hypothesized that the relatively moderate sensitivity of the mtdB mutant strain CM258K.1 to methanol may be due to the presence of another enzyme, MtdA, whose substrate specificity overlaps with that of MtdB. Even though the presence of MtdA is insufficient for wild-type resistance to methanol, it may contribute to the removal of formaldehyde by converting methylene-H₄MPT to methenyl-H₄MPT . To test this hypothesis, the region encoding mtdA was cloned and introduced into the expression vector pCM80 (19) to allow for overexpressed levels of MtdA . The plasmid containing mtdA expressed from the strong promoter P_mxaF resulted in an over-sevenfold increase in MtdA activity from 270 to 1,970 mU during growth on methanol . Neither CM258K.1 bearing the empty vector pCM80 nor pCM259 was capable of growth on methanol plates . However, the MIC for methanol in the presence of succinate was 125 mM for CM258K.1 containing pCM259, compared to 10 mM for CM258K.1 with pCM80. Therefore, a substantial increase in MtdA activity provides partial complementation of the mtdB mutant phenotype .

Methanol sensitivity of H₄MPT pathway mutants requires formaldehyde production. Previous work (10) on mtdB mutants indicated that the sensitivity to methanol could be alleviated if a mtdB::kan mutant was generated in a strain that contained a mutation in the gene (mxaF) encoding the large subunit of MDH (21) . This demonstrated that the sensitivity of this strain required the production of formaldehyde and was not simply a consequence of methanol itself . We extended this analysis to characterize the other three mutants with greater sensitivity to methanol . A series of double mutants were constructed in the mxaF strain CM194.1 . The resulting strains were resistant to the addition of 125 mM methanol to succinate cultures, with only the mxaF dmrA::kan and mxaF orf4::kan mutants showing even a slight growth inhibition (Fig. 3) . Similar results were obtained using solid media . The mxaF mtdB::kan strain CM194-258K.1 was not inhibited by 125 mM methanol, compared to an MIC of 10 mM for the mtdB::kan strain CM258K.1 . The mxaF fae::kan strain CM194-198K.1 had an MIC of methanol of 10 mM, compared to 10 µM for the fae::kan strain CM198K.1 . Finally, the mxaF dmrA::kan strain CM194-212K.1 and the mxaF orf4::kan strain CM194-253K.1 exhibited an MIC for methanol of 100 µM, compared to 1 µM for the corresponding MDH⁺ strains. This residual sensitivity to methanol suggests either a low-level alternate methanol oxidation activity or a direct effect of methanol at higher levels . However, these data suggest that the extreme sensitivity to methanol observed in all tested H₄MPT pathway mutants is not due to methanol itself but, rather, requires the production of formaldehyde .

 
Methanol sensitivity of H₄MPT pathway mutants is alleviated and growth on C₁ compounds is achieved by expressing a heterologous GSH-dependent formaldehyde oxidation pathway. The data presented thus far suggest that the methanol-sensitive phenotype observed for H₄MPT pathway mutants is due to an inability to detoxify the formaldehyde produced from methanol, either directly or as one of its derivatives. As a final test of this hypothesis, a heterologous formaldehyde oxidation system was cloned and expressed in H₄MPT pathway mutants . The two primary genes of the glutathione (GSH)-dependent formaldehyde oxidation pathway of P . denitrificans, flhA (encodes GSH- and NAD-dependent formaldehyde dehydrogenase [26]) and fghA (encodes S-formyl-GSH hydrolase [11]) were cloned by PCR amplification and introduced together into the expression vector pCM80 (19) to generate the plasmid pCM106 . Introduction of pCM106 resulted in activities of 2,500 and 2,300 mU for FlhA and FghA, respectively, whereas these activities were undetectable in wild-type M. extorquens AM1 carrying pCM80 without an insert . Mutants defective for mtdB, fae, dmrA, and orf4 bearing pCM106 were insensitive to 125 mM methanol present in succinate plates . Additionally, the presence of pCM106 allowed the mtdB mutant to grow like wild type in the presence of 1 mM formaldehyde and raised the MIC of formaldehyde to 0.5 mM for fae, dmrA, and orf4 mutant strains . The protective effect of expressing the GSH-dependent formaldehyde oxidation pathway was also tested in liquid medium for two representative mutants that lacked either fae or dmrA . Addition of 125 mM methanol to succinate growth medium did not inhibit growth in strains bearing pCM106 (Fig . 4A and B) . Finally, beyond alleviating methanol sensitivity, the expression of the GSH pathway in the H₄MPT pathway mutants allowed growth in methanol liquid medium (Fig. 4C) and on methanol plates, albeit the complementation of the dmrA and orf4 mutants was less robust than for the other two mutants. The ability of the heterologous GSH-dependent formaldehyde oxidation system to alleviate the methanol sensitivity of H₄MPT pathway mutants provides strong evidence that the cause of the methanol-sensitive phenotype is the inability to detoxify intracellular formaldehyde produced from methanol .

 
Null mutants lacking mch or fhcBADC can only be obtained in an H₄MPT biosynthesis-negative background. One important question regarding the role of the H₄MPT pathway in M . extorquens AM1 is why it has not been possible to obtain null mutations in genes that encode the two enzymes catalyzing the final reactions of the pathway, Mch and Fhc (6) . A few scenarios may be suggested to explain this phenomenon: (i) a C₁-H₄MPT intermediate is required for growth on multicarbon compounds; (ii) these mutants are even more sensitive to methanol, such that ambient concentrations are lethal; or (iii) accumulation of an intermediate of the H₄MPT pathway causes a toxic effect or a regulatory problem . To test these hypotheses, we attempted constructing deletion versions of these mutations in various backgrounds . No mutants were obtained in the wild type using the deletion constructs for Mch (mch::kan) or FhcBADC (fhcBADC::kan), in agreement with our laboratory's previous results for insertion mutants (6). Likewise, no deletion mutants were obtained in the backgrounds lacking MDH (mxaF strain CM194.1), Fae (fae strain CM198.1), or MtdB (mtdB strain CM258.1) . Both deletions were readily generated, however, in the orf4 strain CM253.1 . The orf4 mch::kan strain CM253-263K.1 and orf4 fhcBADC::kan strain CM253-266K.1 grew normally on succinate or formate but exhibited defective growth on methanol or methylamine, as had been observed for the orf4 mutant CM253K.1 . Furthermore, the MICs of methanol and formaldehyde during growth on succinate were the same as those observed for CM253K.1 . These data indicate that none of the H₄MPT pathway enzymes are essential for metabolism of multicarbon compounds . The inability to generate mch or fhcBADC mutants in a wild-type background is discussed below .

Mutants defective for fae exhibit an intermediate level of sensitivity to methanol or formaldehyde . Fae catalyzes the condensation of formaldehyde with H₄MPT, but this reaction also proceeds nonenzymatically at a lower rate (33) . The fact that the fae mutant has a less severe phenotype than the H₄MPT biosynthesis mutants is consistent with the nonenzymatic condensation of formaldehyde with H₄MPT occurring at sufficient levels to allow a low level of formaldehyde oxidation through this pathway in the absence of Fae activity, but not enough to handle the full formaldehyde flux of methylotrophic growth .

Mutants lacking MtdB activity have the least severe phenotype of the H₄MPT pathway mutants investigated in this work . Two methylene-H₄MPT dehydrogenases are present in M . extorquens AM1, MtdA (NADP dependent, but also utilizes methylene-H₄F) and MtdB (H₄MPT specific, but utilizes either NAD⁺ or NADP⁺). The sensitivity of mtdB mutants to methanol or formaldehyde and the inability to grow on methanol indicated that this enzyme plays a critical role in formaldehyde oxidation (10) . The relatively moderate sensitivity of the mtdB mutant compared to that of either the fae or H₄MPT biosynthesis mutants indicates that, despite being insufficient for growth on C₁ compounds or complete resistance to methanol or formaldehyde, MtdA activity can support a moderate formaldehyde flux in the absence of MtdB . To further address this question, mtdA was cloned and overexpressed to levels 7.4-fold higher than in the wild type . This level of MtdA activity was insufficient to allow growth on methanol; however, it largely alleviated the sensitivity to methanol . These data suggest that despite the normal high level of MtdA activity in the wild type, the enzyme level is limiting in the absence of MtdB activity . It has been suggested that the requirement for MtdA to use NADP⁺, rather than NAD⁺, in methylene-H₄MPT reduction limits its in vivo activity (31) .

The mutant phenotypes discussed above are correlated with the magnitude of the decreased formaldehyde flux through the H₄MPT pathway . For the mutants with the greatest defect, the H₄MPT biosynthesis mutants (dmrA and orf4), the impact is remarkable considering that the MIC drops at least 5 orders of magnitude compared to the wild type . Our demonstration that this phenotype can be at least partially compensated with an alternate NAD- and GSH-linked formaldehyde oxidation system demonstrates that this H₄MPT pathway not only serves as the main energy-generating pathway during methylotrophic growth, it also must be the major formaldehyde detoxification pathway . It is notable that an analogous methanol-sensitive phenotype has been observed for P. denitrificans mutants lacking flhA (26) or fghA (11), which demonstrates the widespread importance for methylotrophic bacteria to maintain the capacity for formaldehyde detoxification . The growth inhibition observed for M . extorquens AM1 H₄MPT pathway mutants may be due directly to formaldehyde accumulation. Alternatively, growth inhibition may be caused by a reactive conjugate of formaldehyde with another compound, analogous to what has been described previously for GSH-dependent oxidation of dichloromethane (15), or perhaps even a regulatory circuit poised to sense an imbalance of formaldehyde production and utilization .

Our results suggest that in these mutants, formaldehyde may accumulate in the cytoplasm . The relative resistance of the wild type to formaldehyde added to the medium, as well as the ability of a cytoplasmic formaldehyde oxidation system to alleviate the phenotype, support the idea of cytoplasmic formaldehyde rather than periplasmic formaldehyde being responsible for toxicity. However, it is not possible at this time to measure cytoplasmic formaldehyde distinct from periplasmic formaldehyde . In addition, proteins and nucleic acids inside the cell will serve as a large sink for formaldehyde, and it is likely that formaldehyde will damage the cell substantially before it accumulates internally .

Our demonstration that it is possible to obtain null mutants in the H₄MPT pathway shows that this pathway is not required for growth on multicarbon compounds . We suggest that the explanation for the inability to completely block the H₄MPT-dependent formaldehyde oxidation pathway in wild-type cells is due to the accumulation of a C₁-H₄MPT intermediate(s), since the identical mutations are tolerated in the absence of H₄MPT biosynthesis . This scenario implies that accumulation of a C₁-H₄MPT intermediate(s) is either toxic and/or interferes with normal regulatory circuits . Further work will be required to test this hypothesis and distinguish between these possibilities .

The work presented here demonstrates that M. extorquens AM1 relies on the H₄MPT pathway to oxidize formaldehyde both during growth on C₁ substrates and to detoxify formaldehyde during growth on multicarbon compounds. Remarkably, the heterologous GSH-dependent pathway from P. denitrificans is able to largely replace this function . This result indicates that these pathways comprise analogous metabolic modules (5, 12) . Although they use entirely different enzymes and cofactors, they can fulfill the same cellular function, namely, the NAD(P)-dependent oxidation of formaldehyde to formate .

This work was supported by a grant from the National Institutes of Health (GM 36296) .

Present address: 2215 Biomedical Physical Sciences, Michigan State University, East Lansing, MI 48824-4320 .

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