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Dichloromethane (CH2Cl2, 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/glutathioneS-transferase encoded bydcmAto transform DCM into two molecules of HCl and one molecule of formaldehyde, a toxic intermediate of methylotrophic metabolism. InMethylobacterium extorquensDM4 of known genome sequence,dcmAlies on a 126 kbdcmgenomic 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 reportergfpgene yielded 25dcmmutants 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 kbdcmislet directly involved in DCM dehalogenation is the only one of seven gene clusters specific to the DCM response to be localized within thedcmgenomic island. The DCM response was shown to involve mainly the core genome ofMethylobacterium extorquens, providing new insights on DCM-dependent adjustments of C1 metabolism and gene regulation, and suggesting a specific stress response ofMethylobacteriumduring 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 thedcmislet elicits a complex adaptive response encoded by the core genome common to dechlorinating as well as non-dechlorinatingMethylobacteriumstrains.