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Bacterial adaptation to growth with toxic halogenated chemicals was explored in the context of methylotrophic metabolism of Methylobacterium extorquens, by comparing strains CM4 and DM4, which show robust growth with chloromethane and dichloromethane, respectively. Dehalogenation of chlorinated methanes initiates growth-supporting degradation, with intracellular release of protons and chloride ions in both cases. The core, variable and strain-specific genomes of strains CM4 and DM4 were defined by comparison with genomes of non-dechlorinating strains. In terms of gene content, adaptation toward dehalogenation appears limited, strains CM4 and DM4 sharing between 75 and 85% of their genome with other strains of M. extorquens. Transcript abundance in cultures of strain CM4 grown with chloromethane and of strain DM4 grown with dichloromethane was compared to growth with methanol as a reference C1 growth substrate. Previously identified strain-specific dehalogenase-encoding genes were the most transcribed with chlorinated methanes, alongside other genes encoded by genomic islands (GEIs) and plasmids involved in growth with chlorinated compounds as carbon and energy source. None of the 163 genes shared by strains CM4 and DM4 but not by other strains of M. extorquens showed higher transcript abundance in cells grown with chlorinated methanes. Among the several thousand genes of the M. extorquens core genome, 12 genes were only differentially abundant in either strain CM4 or strain DM4. Of these, 2 genes of known function were detected, for the membrane-bound proton translocating pyrophosphatase HppA and the housekeeping molecular chaperone protein DegP. This indicates that the adaptive response common to chloromethane and dichloromethane is limited at the transcriptional level, and involves aspects of the general stress response as well as of a dehalogenation-specific response to intracellular hydrochloric acid production. Core genes only differentially abundant in either strain CM4 or strain DM4 total 13 and 58 CDS, respectively. Taken together, the obtained results suggest different transcriptional responses of chloromethane- and dichloromethane-degrading M. extorquens strains to dehalogenative metabolism, and substrate- and pathway-specific modes of growth optimization with chlorinated methanes.

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.