Abstract
Chloromethane (CH3Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization.
Highlights
Chloromethane (CH3Cl) is a volatile organic compound emitted by oceans, plants, wood-rotting fungi and biomass burning, estimated to account for 17% of chlorine-catalyzed ozone degradation in the stratosphere [1]
We found that growth with chloromethane elicits a specific adaptive response in M. extorquens CM4
The repertoire of known cmu genes and genes conserved in chloromethane-degrading strains includes genes essential for dehalogenation of chloromethane, genes essential for growth with chloromethane as the sole carbon and energy source, and genes found in the vicinity of genes cmuA, cmuB and cmuC in methylotrophic chloromethane-degrading strains [2]
Summary
Chloromethane (CH3Cl) is a volatile organic compound emitted by oceans, plants, wood-rotting fungi and biomass burning, estimated to account for 17% of chlorine-catalyzed ozone degradation in the stratosphere [1]. The only known microbial aerobic utilization pathway for chloromethane is tetrahydrofolate (H4F)-dependent [6]. This pathway was identified in the alpha-Proteobacterium M. extorquens CM4 using minitransposon random mutagenesis [7] and its chloromethane dehalogenase activity characterized in detail [8,9]. The first step of the cmu (chloromethane utilization) pathway is catalyzed by the two-domain methyltransferase/corrinoid-binding CmuA protein that transfers the methyl group from chloromethane to a corrinoid cofactor [9,10]. The methylcobalamin:H4F methyltransferase CmuB enzyme subsequently catalyzes the transfer of the methyl group from the corrinoid cofactor to H4F [8].
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