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Stéphane Vuilleumier - One of the best experts on this subject based on the ideXlab platform.
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chlorine isotope fractionation of the major Chloromethane degradation processes in the environment
Environmental Science & Technology, 2020Co-Authors: Frank Keppler, Thierry Nadalig, Markus Greule, Jaime D Barnes, Axel Horst, Enno Bahlmann, Jing Luo, Christoph S Hartmann, Stéphane VuilleumierAbstract:Chloromethane (CH3Cl) is an important source of chlorine in the stratosphere, but detailed knowledge of the magnitude of its sources and sinks is missing. Here, we measured the stable chlorine isot...
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Methylotrophs and Methylotroph Populations for Chloromethane Degradation.
Current issues in molecular biology, 2019Co-Authors: Frana Oise Bringel, Stéphane Vuilleumier, Frank Keppler, Ludovic Besaury, Pierre Amato, Eileen Kröber, Steffen Kolb, Thierry NadaligAbstract:Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of Chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of Chloromethane by methylotrophic communities in terrestrial environments is a major underestimated Chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using Chloromethane as the sole carbon source. In addition to anaerobes that degrade Chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use Chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'Chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of Chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with Chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of Chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated Chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of Chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for Chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial Chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global Chloromethane cycle.
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Transfer of a Catabolic Pathway for Chloromethane in Methylobacterium Strains Highlights Different Limitations for Growth with Chloromethane or with DiChloromethane
Frontiers in microbiology, 2016Co-Authors: Joshua K. Michener, Stéphane Vuilleumier, Françoise Bringel, Christopher J. MarxAbstract:Chloromethane is an ozone-depleting gas, produced predominantly from natural sources, that provides an important carbon source for microbes capable of consuming it. Chloromethane catabolism has been difficult to study owing to the challenging genetics of its native microbial hosts. Since the pathways for Chloromethane catabolism show evidence of horizontal gene transfer, we reproduced this transfer process in the laboratory to generate new Chloromethane-catabolizing strains in tractable hosts. We demonstrate that six putative accessory genes improve Chloromethane catabolism, though heterologous expression of only one of the six is strictly necessary for growth on Chloromethane. In contrast to growth of Methylobacterium strains with the closely-related compound diChloromethane, we find that chloride export does not limit growth on Chloromethane and, in general, that the ability of a strain to grow on diChloromethane is uncorrelated with its ability to grow on Chloromethane. This heterologous expression system allows us to investigate the components required for effective Chloromethane catabolism and the factors that limit effective catabolism after horizontal transfer.
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Hydrogen and carbon isotope fractionation during degradation of Chloromethane by methylotrophic bacteria.
MicrobiologyOpen, 2013Co-Authors: Thierry Nadalig, Stéphane Vuilleumier, Markus Greule, Françoise Bringel, Frank KepplerAbstract:Chloromethane (CH3Cl) is a widely studied volatile halocarbon involved in the destruction of ozone in the stratosphere. Nevertheless, its global budget still remains debated. Stable isotope analysis is a powerful tool to constrain fluxes of Chloromethane between various environmental compartments which involve a multiplicity of sources and sinks, and both biotic and abiotic processes. In this study, we measured hydrogen and carbon isotope fractionation of the remaining untransformed Chloromethane following its degradation by methylotrophic bacterial strains Methylobacterium extorquens CM4 and Hyphomicrobium sp. MC1, which belong to different genera but both use the cmu pathway, the only pathway for bacterial degradation of Chloromethane characterized so far. Hydrogen isotope fractionation for degradation of Chloromethane was determined for the first time, and yielded enrichment factors (e) of −29‰ and −27‰ for strains CM4 and MC1, respectively. In agreement with previous studies, enrichment in 13C of untransformed CH3Cl was also observed, and similar isotope enrichment factors (e) of −41‰ and −38‰ were obtained for degradation of Chloromethane by strains CM4 and MC1, respectively. These combined hydrogen and carbon isotopic data for bacterial degradation of Chloromethane will contribute to refine models of the global atmospheric budget of Chloromethane.
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Hydrogen and carbon isotope fractionation during degradation of Chloromethane by methylotrophic bacteria.
MicrobiologyOpen, 2013Co-Authors: Thierry Nadalig, Stéphane Vuilleumier, Markus Greule, Françoise Bringel, Frank KepplerAbstract:Chloromethane (CH3 Cl) is a widely studied volatile halocarbon involved in the destruction of ozone in the stratosphere. Nevertheless, its global budget still remains debated. Stable isotope analysis is a powerful tool to constrain fluxes of Chloromethane between various environmental compartments which involve a multiplicity of sources and sinks, and both biotic and abiotic processes. In this study, we measured hydrogen and carbon isotope fractionation of the remaining untransformed Chloromethane following its degradation by methylotrophic bacterial strains Methylobacterium extorquens CM4 and Hyphomicrobium sp. MC1, which belong to different genera but both use the cmu pathway, the only pathway for bacterial degradation of Chloromethane characterized so far. Hydrogen isotope fractionation for degradation of Chloromethane was determined for the first time, and yielded enrichment factors (ε) of -29‰ and -27‰ for strains CM4 and MC1, respectively. In agreement with previous studies, enrichment in (13) C of untransformed CH3 Cl was also observed, and similar isotope enrichment factors (ε) of -41‰ and -38‰ were obtained for degradation of Chloromethane by strains CM4 and MC1, respectively. These combined hydrogen and carbon isotopic data for bacterial degradation of Chloromethane will contribute to refine models of the global atmospheric budget of Chloromethane.
Thomas Leisinger - One of the best experts on this subject based on the ideXlab platform.
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Chloromethane-Induced Genes Define a Third C1 Utilization Pathway in Methylobacterium chloromethanicum CM4
Journal of bacteriology, 2002Co-Authors: Alex Studer, Craig Mcanulla, Thomas Leisinger, Rainer Büchele, Stéphane VuilleumierAbstract:Methylobacterium chloromethanicum CM4 is an aerobic α-proteobacterium capable of growth with Chloromethane as the sole carbon and energy source. Two proteins, CmuA and CmuB, were previously purified and shown to catalyze the dehalogenation of Chloromethane and the vitamin B12-mediated transfer of the methyl group of Chloromethane to tetrahydrofolate. Three genes located near cmuA and cmuB, designated metF, folD and purU and encoding homologs of methylene tetrahydrofolate (methylene-H4folate) reductase, methylene-H4folate dehydrogenase-methenyl-H4folate cyclohydrolase and formyl-H4folate hydrolase, respectively, suggested the existence of a Chloromethane-specific oxidation pathway from methyl-tetrahydrofolate to formate in strain CM4. Hybridization and PCR analysis indicated that these genes were absent in Methylobacterium extorquens AM1, which is unable to grow with Chloromethane. Studies with transcriptional xylE fusions demonstrated the Chloromethane-dependent expression of these genes. Transcriptional start sites were mapped by primer extension and allowed to define three transcriptional units, each likely comprising several genes, that were specifically expressed during growth of strain CM4 with Chloromethane. The DNA sequences of the deduced promoters display a high degree of sequence conservation but differ from the Methylobacterium promoters described thus far. As shown previously for purU, inactivation of the metF gene resulted in a CM4 mutant unable to grow with Chloromethane. Methylene-H4folate reductase activity was detected in a cell extract of strain CM4 only in the presence of Chloromethane but not in the metF mutant. Taken together, these data provide evidence that M. chloromethanicum CM4 requires a specific set of tetrahydrofolate-dependent enzymes for growth with Chloromethane.
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Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4.
European journal of biochemistry, 2001Co-Authors: Alex Studer, Stéphane Vuilleumier, Erhard Stupperich, Thomas LeisingerAbstract:The cmuA and cmuB genes are required for growth of Methylobacterium chloromethanicum strain CM4 with Chloromethane as the sole carbon source. While CmuB was previously shown to possess methylcobalamin:tetrahydrofolate methyltransferase activity, sequence analysis indicated that CmuA represented a novel and so far unique two-domain methyltransferase/corrinoid-binding protein involved in methyl transfer from Chloromethane to a corrin moiety. CmuA was purified from wild-type M. chloromethanicum strain CM4 and characterized as a monomeric, cobalt-containing and zinc-containing enzyme of molecular mass 67 kDa with a bound vitamin B12 cofactor. In combination, CmuA and CmuB proteins catalyze the in vitro transfer of the methyl group of Chloromethane to tetrahydrofolate, thus affording a direct link between Chloromethane dehalogenation and core C1 metabolism of Methylobacterium. Chloromethane dehalogenase activity in vitro is limited by CmuB, as formation of methyltetrahydrofolate from Chloromethane displays apparent Michaelis-Menten kinetics with respect to methylated CmuA, with an apparent Km of 0.27 microM and a Vmax of 0.45 U x mg(-1). This contrasts with sequence-related systems for methyl transfer from methanogens, which involve methyltransferase and corrinoid protein components in well-defined stoichiometric ratios.
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Chloromethane Utilization Gene Cluster from Hyphomicrobium chloromethanicum Strain CM2T and Development of Functional Gene Probes To Detect Halomethane-Degrading Bacteria
Applied and Environmental Microbiology, 2001Co-Authors: Craig Mcanulla, Alex Studer, Stéphane Vuilleumier, Thomas Leisinger, Ian R Mcdonald, Claire A Woodall, J. Colin MurrellAbstract:Chloromethane (CH3Cl) is a volatile organic compound with an average concentration in the atmosphere of 540 ppt (vol/vol) (22). Chloromethane is of environmental concern because it may be responsible for about 13% of the destruction of the stratospheric ozone layer (3). The primary sources of Chloromethane are thought to be biological and nonbiological processes that occur in nature. The major sources of Chloromethane to date include oceans, biomass burning, wood-rotting fungi, and salt marshes (20, 36). The main sink for Chloromethane is thought to be the reaction with tropospheric and stratospheric hydroxyl radicals. Soils have also been shown to be a potentially significant sink for Chloromethane (23). Chloromethane can be cometabolized by bacteria, both by oxidation (35, 40) and by hydrolysis (21). In addition, several methylotrophic bacteria which are able to use Chloromethane as a growth substrate have been characterized. These include the strictly anaerobic homoacetogenic bacterium Acetobacterium dehalogenans (31) and several aerobic methylotrophs of the genera Hyphomicrobium and Methylobacterium (6). Anoxic dehalogenation of Chloromethane by A. dehalogenans has been shown to be catalyzed by enzymes that transfer the methyl group of Chloromethane by means of a corrinoid protein to tetrahydrofolate to yield chloride and methyl tetrahydrofolate, an intermediate in the acetyl coenzyme A pathway (48). Doronina et al. (6) initially isolated eight strains from industrially contaminated Russian soils; however, 16S rRNA sequencing showed that only two distinct strains had been isolated, and these strains were recently designated Hyphomicrobium chloromethanicum CM2T and Methylobacterium chloromethanicum CM4T (29). Physiological and genetic studies exploring the mechanism of Chloromethane metabolism in M. chloromethanicum CM4T recently suggested a pathway for Chloromethane utilization (45, 46). It was shown that two polypeptides (67 and 35 kDa) were induced during growth on Chloromethane (46). Growth yields and oxygen electrode stoichiometries suggested that Chloromethane was completely oxidized to CO2 and that a total of six electrons were produced by this oxidation. Chloromethane-grown cells were also capable of dehalogenating bromomethane and iodomethane but not diChloromethane and higher chloroalkanes, such as chloroethane. This suggested that the enzyme(s) responsible for Chloromethane degradation was specific for monohalomethanes. No growth was observed with bromomethane as the sole carbon and energy source, presumably due to the greater toxicity of this compound. Transposon mutagenesis was used to create Methylobacterium mutants unable to grow on Chloromethane. Genes containing the transposon insertion were then cloned and sequenced, and the resulting information was used to develop biochemical assays. Based on the results, a pathway for Chloromethane degradation was suggested (Fig. (Fig.1),1), which represents a novel catabolic pathway for aerobic methylotrophs (45). FIG. 1 Proposed pathway for Chloromethane metabolism in M. chloromethanicum CM4T. The pathway was modified from that of Vannelli et al. (45). CmuA, methyltransferase I; CmuB, methyltransferase II; MetF, putative 5,10-methylene-tetrahydrofolate reductase; FolD, ... The first step of this pathway involves CmuA, a 67-kDa polypeptide which has a methyltransferase domain and a corrinoid-binding domain. The methyltransferase domain transfers the methyl group of Chloromethane to the Co atom of the enzyme-bound corrinoid group (methyltransferase I activity). A second polypeptide, CmuB, then transfers the methyl group to tetrahydrofolate, forming methyl tetrahydrofolate (methyltransferase II activity). This folate-linked methyl group is then progressively oxidized to formate and then to CO2 to provide reducing equivalents. Carbon assimilation presumably occurs at the level of methylene tetrahydrofolate, which can feed directly into the serine cycle. This pathway was postulated on the basis of physiological, genetic, and biochemical evidence. Four genes, cmuA, cmuB, cmuC, and purU, were shown to be essential for growth on Chloromethane but not on other C1 substrates. Two other facultative methylotrophs capable of growth on both Chloromethane and bromomethane as sole carbon and energy sources have been isolated. Strain IMB-1 was isolated from soil which had been fumigated with bromomethane (4, 32), while strain CC495 was isolated from topsoil in a pristine woodland site (5). These two strains were shown by 16S rRNA sequence analysis to be closely related to each other and to the genus Aminobacter (5, 18). Growth of IMB-1 on bromomethane was shown to be inducible (39). Growth was observed with bromomethane, Chloromethane, and iodomethane as sole carbon and energy sources. Cells grown on bromomethane were capable of oxidizing Chloromethane and vice versa, suggesting that a single inducible enzyme system was responsible for the oxidation of monohalomethanes. The physiology and biochemistry of Chloromethane degradation by CC495 were investigated by Coulter et al. (5). Growth on Chloromethane was inducible, and two polypeptides with apparent molecular masses of 67 and 29 kDa were expressed. The 67-kDa polypeptide was purified and identified as a halomethane: bisulfide/halide ion methyltransferase. This enzyme is a corrinoid protein, and its reported N-terminal sequence showed 81% identity to the N-terminal sequence of CmuA from M. chloromethanicum CM4T. A fifth isolate, strain MB2, isolated from a marine environment, was capable of growth on bromomethane as a sole carbon and energy source (14) but has not been characterized further. Here we report on the sequence and an analysis of the cmu gene cluster from H. chloromethanicum CM2T. Biochemical evidence for degradation of Chloromethane by CM2T is also presented. Development of specific PCR primers for detection of cmuA genes in isolates and enrichments is also described.
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Properties of the methylcobalamin:H4folate methyltransferase involved in Chloromethane utilization by Methylobacterium sp. strain CM4
European journal of biochemistry, 1999Co-Authors: Alex Studer, Stéphane Vuilleumier, Thomas LeisingerAbstract:Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with Chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of Chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of Chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA96, 4615–4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol·min−1·(mg protein)−1. The apparent kinetic constants for H4folate were: Km = 240 µM, Vmax = 28.5 nmol·min−1·(mg protein)−1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mm, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored Chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.
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A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with Chloromethane
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Todd Vannelli, Alex Studer, Stéphane Vuilleumier, Michael Messmer, Thomas LeisingerAbstract:Methylobacterium sp. strain CM4, an aerobic methylotrophic α-proteobacterium, is able to grow with Chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with Chloromethane, were previously obtained by miniTn5 mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of Chloromethane to formate. The methyl group of Chloromethane is first transferred by the protein CmuA (cmu: Chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from Chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium. Remarkable features of this pathway for the catabolism of Chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from Chloromethane into central metabolism.
Alex Studer - One of the best experts on this subject based on the ideXlab platform.
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Chloromethane-Induced Genes Define a Third C1 Utilization Pathway in Methylobacterium chloromethanicum CM4
Journal of bacteriology, 2002Co-Authors: Alex Studer, Craig Mcanulla, Thomas Leisinger, Rainer Büchele, Stéphane VuilleumierAbstract:Methylobacterium chloromethanicum CM4 is an aerobic α-proteobacterium capable of growth with Chloromethane as the sole carbon and energy source. Two proteins, CmuA and CmuB, were previously purified and shown to catalyze the dehalogenation of Chloromethane and the vitamin B12-mediated transfer of the methyl group of Chloromethane to tetrahydrofolate. Three genes located near cmuA and cmuB, designated metF, folD and purU and encoding homologs of methylene tetrahydrofolate (methylene-H4folate) reductase, methylene-H4folate dehydrogenase-methenyl-H4folate cyclohydrolase and formyl-H4folate hydrolase, respectively, suggested the existence of a Chloromethane-specific oxidation pathway from methyl-tetrahydrofolate to formate in strain CM4. Hybridization and PCR analysis indicated that these genes were absent in Methylobacterium extorquens AM1, which is unable to grow with Chloromethane. Studies with transcriptional xylE fusions demonstrated the Chloromethane-dependent expression of these genes. Transcriptional start sites were mapped by primer extension and allowed to define three transcriptional units, each likely comprising several genes, that were specifically expressed during growth of strain CM4 with Chloromethane. The DNA sequences of the deduced promoters display a high degree of sequence conservation but differ from the Methylobacterium promoters described thus far. As shown previously for purU, inactivation of the metF gene resulted in a CM4 mutant unable to grow with Chloromethane. Methylene-H4folate reductase activity was detected in a cell extract of strain CM4 only in the presence of Chloromethane but not in the metF mutant. Taken together, these data provide evidence that M. chloromethanicum CM4 requires a specific set of tetrahydrofolate-dependent enzymes for growth with Chloromethane.
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Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4.
European journal of biochemistry, 2001Co-Authors: Alex Studer, Stéphane Vuilleumier, Erhard Stupperich, Thomas LeisingerAbstract:The cmuA and cmuB genes are required for growth of Methylobacterium chloromethanicum strain CM4 with Chloromethane as the sole carbon source. While CmuB was previously shown to possess methylcobalamin:tetrahydrofolate methyltransferase activity, sequence analysis indicated that CmuA represented a novel and so far unique two-domain methyltransferase/corrinoid-binding protein involved in methyl transfer from Chloromethane to a corrin moiety. CmuA was purified from wild-type M. chloromethanicum strain CM4 and characterized as a monomeric, cobalt-containing and zinc-containing enzyme of molecular mass 67 kDa with a bound vitamin B12 cofactor. In combination, CmuA and CmuB proteins catalyze the in vitro transfer of the methyl group of Chloromethane to tetrahydrofolate, thus affording a direct link between Chloromethane dehalogenation and core C1 metabolism of Methylobacterium. Chloromethane dehalogenase activity in vitro is limited by CmuB, as formation of methyltetrahydrofolate from Chloromethane displays apparent Michaelis-Menten kinetics with respect to methylated CmuA, with an apparent Km of 0.27 microM and a Vmax of 0.45 U x mg(-1). This contrasts with sequence-related systems for methyl transfer from methanogens, which involve methyltransferase and corrinoid protein components in well-defined stoichiometric ratios.
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Chloromethane Utilization Gene Cluster from Hyphomicrobium chloromethanicum Strain CM2T and Development of Functional Gene Probes To Detect Halomethane-Degrading Bacteria
Applied and Environmental Microbiology, 2001Co-Authors: Craig Mcanulla, Alex Studer, Stéphane Vuilleumier, Thomas Leisinger, Ian R Mcdonald, Claire A Woodall, J. Colin MurrellAbstract:Chloromethane (CH3Cl) is a volatile organic compound with an average concentration in the atmosphere of 540 ppt (vol/vol) (22). Chloromethane is of environmental concern because it may be responsible for about 13% of the destruction of the stratospheric ozone layer (3). The primary sources of Chloromethane are thought to be biological and nonbiological processes that occur in nature. The major sources of Chloromethane to date include oceans, biomass burning, wood-rotting fungi, and salt marshes (20, 36). The main sink for Chloromethane is thought to be the reaction with tropospheric and stratospheric hydroxyl radicals. Soils have also been shown to be a potentially significant sink for Chloromethane (23). Chloromethane can be cometabolized by bacteria, both by oxidation (35, 40) and by hydrolysis (21). In addition, several methylotrophic bacteria which are able to use Chloromethane as a growth substrate have been characterized. These include the strictly anaerobic homoacetogenic bacterium Acetobacterium dehalogenans (31) and several aerobic methylotrophs of the genera Hyphomicrobium and Methylobacterium (6). Anoxic dehalogenation of Chloromethane by A. dehalogenans has been shown to be catalyzed by enzymes that transfer the methyl group of Chloromethane by means of a corrinoid protein to tetrahydrofolate to yield chloride and methyl tetrahydrofolate, an intermediate in the acetyl coenzyme A pathway (48). Doronina et al. (6) initially isolated eight strains from industrially contaminated Russian soils; however, 16S rRNA sequencing showed that only two distinct strains had been isolated, and these strains were recently designated Hyphomicrobium chloromethanicum CM2T and Methylobacterium chloromethanicum CM4T (29). Physiological and genetic studies exploring the mechanism of Chloromethane metabolism in M. chloromethanicum CM4T recently suggested a pathway for Chloromethane utilization (45, 46). It was shown that two polypeptides (67 and 35 kDa) were induced during growth on Chloromethane (46). Growth yields and oxygen electrode stoichiometries suggested that Chloromethane was completely oxidized to CO2 and that a total of six electrons were produced by this oxidation. Chloromethane-grown cells were also capable of dehalogenating bromomethane and iodomethane but not diChloromethane and higher chloroalkanes, such as chloroethane. This suggested that the enzyme(s) responsible for Chloromethane degradation was specific for monohalomethanes. No growth was observed with bromomethane as the sole carbon and energy source, presumably due to the greater toxicity of this compound. Transposon mutagenesis was used to create Methylobacterium mutants unable to grow on Chloromethane. Genes containing the transposon insertion were then cloned and sequenced, and the resulting information was used to develop biochemical assays. Based on the results, a pathway for Chloromethane degradation was suggested (Fig. (Fig.1),1), which represents a novel catabolic pathway for aerobic methylotrophs (45). FIG. 1 Proposed pathway for Chloromethane metabolism in M. chloromethanicum CM4T. The pathway was modified from that of Vannelli et al. (45). CmuA, methyltransferase I; CmuB, methyltransferase II; MetF, putative 5,10-methylene-tetrahydrofolate reductase; FolD, ... The first step of this pathway involves CmuA, a 67-kDa polypeptide which has a methyltransferase domain and a corrinoid-binding domain. The methyltransferase domain transfers the methyl group of Chloromethane to the Co atom of the enzyme-bound corrinoid group (methyltransferase I activity). A second polypeptide, CmuB, then transfers the methyl group to tetrahydrofolate, forming methyl tetrahydrofolate (methyltransferase II activity). This folate-linked methyl group is then progressively oxidized to formate and then to CO2 to provide reducing equivalents. Carbon assimilation presumably occurs at the level of methylene tetrahydrofolate, which can feed directly into the serine cycle. This pathway was postulated on the basis of physiological, genetic, and biochemical evidence. Four genes, cmuA, cmuB, cmuC, and purU, were shown to be essential for growth on Chloromethane but not on other C1 substrates. Two other facultative methylotrophs capable of growth on both Chloromethane and bromomethane as sole carbon and energy sources have been isolated. Strain IMB-1 was isolated from soil which had been fumigated with bromomethane (4, 32), while strain CC495 was isolated from topsoil in a pristine woodland site (5). These two strains were shown by 16S rRNA sequence analysis to be closely related to each other and to the genus Aminobacter (5, 18). Growth of IMB-1 on bromomethane was shown to be inducible (39). Growth was observed with bromomethane, Chloromethane, and iodomethane as sole carbon and energy sources. Cells grown on bromomethane were capable of oxidizing Chloromethane and vice versa, suggesting that a single inducible enzyme system was responsible for the oxidation of monohalomethanes. The physiology and biochemistry of Chloromethane degradation by CC495 were investigated by Coulter et al. (5). Growth on Chloromethane was inducible, and two polypeptides with apparent molecular masses of 67 and 29 kDa were expressed. The 67-kDa polypeptide was purified and identified as a halomethane: bisulfide/halide ion methyltransferase. This enzyme is a corrinoid protein, and its reported N-terminal sequence showed 81% identity to the N-terminal sequence of CmuA from M. chloromethanicum CM4T. A fifth isolate, strain MB2, isolated from a marine environment, was capable of growth on bromomethane as a sole carbon and energy source (14) but has not been characterized further. Here we report on the sequence and an analysis of the cmu gene cluster from H. chloromethanicum CM2T. Biochemical evidence for degradation of Chloromethane by CM2T is also presented. Development of specific PCR primers for detection of cmuA genes in isolates and enrichments is also described.
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Properties of the methylcobalamin:H4folate methyltransferase involved in Chloromethane utilization by Methylobacterium sp. strain CM4
European journal of biochemistry, 1999Co-Authors: Alex Studer, Stéphane Vuilleumier, Thomas LeisingerAbstract:Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with Chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of Chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of Chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA96, 4615–4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol·min−1·(mg protein)−1. The apparent kinetic constants for H4folate were: Km = 240 µM, Vmax = 28.5 nmol·min−1·(mg protein)−1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mm, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored Chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.
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A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with Chloromethane
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Todd Vannelli, Alex Studer, Stéphane Vuilleumier, Michael Messmer, Thomas LeisingerAbstract:Methylobacterium sp. strain CM4, an aerobic methylotrophic α-proteobacterium, is able to grow with Chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with Chloromethane, were previously obtained by miniTn5 mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of Chloromethane to formate. The methyl group of Chloromethane is first transferred by the protein CmuA (cmu: Chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from Chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium. Remarkable features of this pathway for the catabolism of Chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from Chloromethane into central metabolism.
Thierry Nadalig - One of the best experts on this subject based on the ideXlab platform.
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chlorine isotope fractionation of the major Chloromethane degradation processes in the environment
Environmental Science & Technology, 2020Co-Authors: Frank Keppler, Thierry Nadalig, Markus Greule, Jaime D Barnes, Axel Horst, Enno Bahlmann, Jing Luo, Christoph S Hartmann, Stéphane VuilleumierAbstract:Chloromethane (CH3Cl) is an important source of chlorine in the stratosphere, but detailed knowledge of the magnitude of its sources and sinks is missing. Here, we measured the stable chlorine isot...
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Methylotrophs and Methylotroph Populations for Chloromethane Degradation.
Current issues in molecular biology, 2019Co-Authors: Frana Oise Bringel, Stéphane Vuilleumier, Frank Keppler, Ludovic Besaury, Pierre Amato, Eileen Kröber, Steffen Kolb, Thierry NadaligAbstract:Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of Chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of Chloromethane by methylotrophic communities in terrestrial environments is a major underestimated Chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using Chloromethane as the sole carbon source. In addition to anaerobes that degrade Chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use Chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'Chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of Chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with Chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of Chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated Chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of Chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for Chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial Chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global Chloromethane cycle.
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probing the diversity of Chloromethane degrading bacteria by comparative genomics and isotopic fractionation
Frontiers in Microbiology, 2014Co-Authors: Thierry Nadalig, Frana Oise Bringel, Markus Greule, Frank Keppler, Sta Phane VuilleumierAbstract:Chloromethane (CH3Cl) is produced on earth by a variety of abiotic and biological processes. It is the most important halogenated trace gas in the atmosphere, where it contributes to ozone destruction. Current estimates of the global CH3Cl budget are uncertain and suggest that microorganisms might play a more important role in degrading atmospheric CH3Cl than previously thought. Its degradation by bacteria has been demonstrated in marine, terrestrial and phyllospheric environments. Improving our knowledge of these degradation processes and its magnitude is thus highly relevant for a better understanding of the global budget of CH3Cl. The cmu pathway, for Chloromethane utilisation, is the only microbial pathway for CH3Cl degradation elucidated so far, and was characterised in detail in aerobic methylotrophic Alphaproteobacteria. Here, we reveal the potential of using a two-pronged approach involving a combination of comparative genomics and isotopic fractionation during CH3Cl degradation to newly address the question of the diversity of Chloromethane-degrading bacteria in the environment. Analysis of available bacterial genome sequences reveals that several bacteria not yet known to degrade CH3Cl contain part or all of the complement of cmu genes required for CH3Cl degradation. These organisms, unlike bacteria shown to grow with CH3Cl using the cmu pathway, are obligate anaerobes. On the other hand, analysis of the complete genome of the Chloromethane-degrading bacterium Leisingera methylohalidivorans showed that this bacterium does not contain cmu genes. Isotope fractionation experiments with L. methylohalidivorans suggest that the unknown pathway used by this bacterium for growth with CH3Cl can be differentiated from the cmu pathway. This result opens the prospect that contributions from bacteria with the cmu and Leisingera-type pathways to the atmospheric CH3Cl budget may be teased apart in the future.
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Probing the diversity of Chloromethane-degrading bacteria by comparative genomics and isotopic fractionation
Frontiers in microbiology, 2014Co-Authors: Thierry Nadalig, Frana Oise Bringel, Markus Greule, Frank Keppler, Sta Phane VuilleumierAbstract:Chloromethane (CH3Cl) is produced on earth by a variety of abiotic and biological processes. It is the most important halogenated trace gas in the atmosphere, where it contributes to ozone destruction. Current estimates of the global CH3Cl budget are uncertain and suggest that microorganisms might play a more important role in degrading atmospheric CH3Cl than previously thought. Its degradation by bacteria has been demonstrated in marine, terrestrial, and phyllospheric environments. Improving our knowledge of these degradation processes and their magnitude is thus highly relevant for a better understanding of the global budget of CH3Cl. The cmu pathway, for Chloromethane utilisation, is the only microbial pathway for CH3Cl degradation elucidated so far, and was characterized in detail in aerobic methylotrophic Alphaproteobacteria. Here, we reveal the potential of using a two-pronged approach involving a combination of comparative genomics and isotopic fractionation during CH3Cl degradation to newly address the question of the diversity of Chloromethane-degrading bacteria in the environment. Analysis of available bacterial genome sequences reveals that several bacteria not yet known to degrade CH3Cl contain part or all of the complement of cmu genes required for CH3Cl degradation. These organisms, unlike bacteria shown to grow with CH3Cl using the cmu pathway, are obligate anaerobes. On the other hand, analysis of the complete genome of the Chloromethane-degrading bacterium Leisingera methylohalidivorans MB2 showed that this bacterium does not contain cmu genes. Isotope fractionation experiments with L. methylohalidivorans MB2 suggest that the unknown pathway used by this bacterium for growth with CH3Cl can be differentiated from the cmu pathway. This result opens the prospect that contributions from bacteria with the cmu and Leisingera-type pathways to the atmospheric CH3Cl budget may be teased apart in the future.
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Fluorescence-based bacterial bioreporter for specific detection of methyl halide emissions in the environment.
Applied and Environmental Microbiology, 2013Co-Authors: Muhammad Farhan Ul Haque, Frana Oise Bringel, Thierry Nadalig, Hubert Schaller, Sta Phane VuilleumierAbstract:Methyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, Chloromethane (CH3Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include Chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics of Methylobacterium extorquens CM4, an aerobic alphaproteobacterium which utilizes Chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the Chloromethane dehalogenase gene cmuA fused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced into M. extorquens CM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model Chloromethane-producing plant Arabidopsis thaliana in particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions.
Sören Svensson - One of the best experts on this subject based on the ideXlab platform.
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Vibrational structure of the Chloromethane series, CH4−nCln, studied by core photoelectron spectroscopy and ab initio calculations
The Journal of Chemical Physics, 1999Co-Authors: S. Sundin, Leif J. Sæthre, Stacey L. Sorensen, A. Ausmees, Sören SvenssonAbstract:Vibrationally resolved C1s photoelectron spectra of the Chloromethane series, CH4-nCln, are reported. The spectra are compared with results from ab initio calculations at the MP2 level using the Z+ ...
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vibrational structure of the Chloromethane series ch4 ncln studied by core photoelectron spectroscopy and ab initio calculations
Journal of Chemical Physics, 1999Co-Authors: S. Sundin, Leif J. Sæthre, Stacey L. Sorensen, A. Ausmees, Sören SvenssonAbstract:Vibrationally resolved C1s photoelectron spectra of the Chloromethane series, CH4-nCln, are reported. The spectra are compared with results from ab initio calculations at the MP2 level using the Z+ ...