Functional genomics of dichloromethane utilization in Methylobacterium extorquens DM4

Abstract

Dichloromethane (CH(2)Cl(2) , DCM) is a chlorinated solvent mainly produced by industry, and a common pollutant. Some aerobic methylotrophic bacteria are able to grow with this chlorinated methane as their sole carbon and energy source, using a DCM dehalogenase/glutathione S-transferase encoded by dcmA to transform DCM into two molecules of HCl and one molecule of formaldehyde, a toxic intermediate of methylotrophic metabolism. In Methylobacterium extorquens DM4 of known genome sequence, dcmA lies on a 126 kb dcm genomic island not found so far in other DCM-dechlorinating strains. An experimental search for the molecular determinants involved in specific cellular responses of strain DM4 growing with DCM was performed. Random mutagenesis with a minitransposon containing a promoterless reporter gfp gene yielded 25 dcm mutants with a specific DCM-associated phenotype. Differential proteomic analysis of cultures grown with DCM and with methanol defined 38 differentially abundant proteins. The 5.5 kb dcm islet directly involved in DCM dehalogenation is the only one of seven gene clusters specific to the DCM response to be localized within the dcm genomic island. The DCM response was shown to involve mainly the core genome of Methylobacterium extorquens, providing new insights on DCM-dependent adjustments of C1 metabolism and gene regulation, and suggesting a specific stress response of Methylobacterium during growth with DCM. Fatty acid, hopanoid and peptidoglycan metabolisms were affected, hinting at the membrane-active effects of DCM due to its solvent properties. A chloride-induced efflux transporter termed CliABC was also newly identified. Thus, DCM dechlorination driven by the dcm islet elicits a complex adaptive response encoded by the core genome common to dechlorinating as well as non-dechlorinating Methylobacterium strains.

Genes of the N -Methylglutamate Pathway Are Essential for Growth of Methylobacterium extorquens DM4 with Monomethylamine

“…Mutants with an MMA-dependent phenotype were then further assayed for growth and induction of GFP fluorescence in M3 medium containing MMA (20 and 80 mM), dichloromethane (1.4 mM), methanol or formate (20 mM), pyruvate or glycerol (7.5 mM), acetate or betaine (10 mM), succinate (5 mM), and combinations thereof. Growth and fluorescence were tested after 7 days of incubation at 30°C of 5-l drops of serial dilutions of bacterial cultures by a two-step PCR method combining semidegenerate primers together with minitransposon-specific primers as previously described (10). The sequences obtained were compared with the DM4 genome sequence by using BLAST (11) to determine the minitransposon insertion site.…”

“…Insert sequences were verified to be of the wild type. Plasmids were transferred into Methylobacterium by conjugation as previously described (10).…”

Phylogeny Poorly Predicts the Utility of a Challenging Horizontally Transferred Gene in Methylobacterium Strains

“…1). First, the strain must accommodate the protons and chloride produced intracellularly as a by-product of DCM dehalogenation (22)(23)(24). Additionally, the S-chloromethylglutathione intermediate formed during the dehalogenation reaction is highly reactive and mutagenic (25)(26)(27).…”

Complete Genome Sequences of Six Strains of the Genus Methylobacterium

“…Five of the strains possess conserved clusters of genes associated with photosynthesis, including genes encoding the light-harvesting complex and the reaction center, and genes involved in biosynthesis of bacteriochlorophyll and carotenoids. Further analyses of these six genomes will include comparisons to the two Methylobacterium genomes already reported (18), i.e., M. extorquens AM1, a major model strain in studies of methylotrophy (2) and genome evolution (5), and the dichloromethanedegrading strain M. extorquens DM4 (14). This will define both core-and strain-specific features of Methylobacterium strains and provide new insights into the metabolic flexibility of these facultative methylotrophs and into the modes of bacterial adaptation to specific ecological niches.…”