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.

bMonomethylamine (MMA, CH 3 NH 2 ) can be used as a carbon and nitrogen source by many methylotrophic bacteria. Methylobacterium extorquens DM4 lacks the MMA dehydrogenase encoded by mau genes, which in M. extorquens AM1 is essential for growth on MMA. Identification and characterization of minitransposon mutants with an MMA-dependent phenotype showed that strain DM4 grows with MMA as the sole source of carbon, energy, and nitrogen by the N-methylglutamate (NMG) pathway. Independent mutations were found in a chromosomal region containing the genes gmaS, mgsABC, and mgdABCD for the three enzymes of the pathway, ␥-glutamylmethylamide (GMA) synthetase, NMG synthase, and NMG dehydrogenase, respectively. Reverse transcription-PCR confirmed the operonic structure of the two divergent gene clusters mgsABC-gmaS and mgdABCD and their induction during growth with MMA. The genes mgdABCD and mgsABC were found to be essential for utilization of MMA as a carbon and nitrogen source. The gene gmaS was essential for MMA utilization as a carbon source, but residual growth of mutant DM4gmaS growing with succinate and MMA as a nitrogen source was observed. Plasmid copies of gmaS and the gmaS homolog METDI4690, which encodes a protein 39% identical to GMA synthetase, fully restored the ability of mutants DM4gmaS and DM4gmaS⌬metdi4690 to use MMA as a carbon and nitrogen source. Similarly, chemically synthesized GMA, the product of GMA synthetase, could be used as a nitrogen source for growth in the wild-type strain, as well as in DM4gmaS and DM4gmaS⌬metdi4690 mutants. The NADH:ubiquinone oxidoreductase respiratory complex component NuoG was also found to be essential for growth with MMA as a carbon source. M onomethylamine (MMA; methylamine) is a nitrogen-containing C 1 compound released by natural sources such as the breakdown of proteins and amine osmolytes, as well as by humanmade nitrogen-containing pesticides, pharmaceuticals, and herbicides (1). MMA is ubiquitous in the environment and can serve as the sole source of carbon and energy for methylotrophic bacteria, which grow on compounds with no C-C bonds (2) but also as a nitrogen source for a large variety of bacteria (3). MMA utilization by Gram-negative bacteria occurs either by oxidation of MMA into formaldehyde by MMA dehydrogenase (MADH) encoded by mau genes (4) found only in methylotrophic bacteria so far (5) or by the N-methylglutamate (NMG) pathway, the genes for which were first identified in the betaproteobacterium Methyloversatilis universalis FAM5 (6). This metabolism effects the condensation of MMA with glutamate to NMG, with ␥-glutamylmethylamide (GMA) a possible intermediate (Fig. 1). The genes gmaS, mgsABC, and mgdABCD encode GMA synthetase (GMAS), NMG synthase, and NMG dehydrogenase, respectively (6). Of these, only gmaS was found not to be required for MMA metabolism in M. universalis FAM5 (6). In contrast, gmaS was also required for MMA utilization in the facultative methane utilizer Methylocella silvestris BL2 (1). In addition, the NMG pathway was recently ...