Dichloromethane (DCM) is efficiently utilized as a carbon and energy source by aerobic, Gram-negative, facultative methylotrophic bacteria. It also serves as a sole carbon and energy source for a nitrate-respiring Hyphomicrobium sp. and for a strictly anaerobic co-culture of a DCM-fermenting bacterium and an acetogen. The first step of DCM utilization by methylotrophs is catalyzed by DCM dehalogenase which, in a glutathione-dependent substitution reaction, forms inorganic chloride and S-chloromethyl glutathione. This unstable intermediate decomposes to glutathione, inorganic chloride and formaldehyde, a central metabolite of methylotrophic growth. Genetic studies on DCM utilization are beginning to shed some light on questions pertaining to the evolution of DCM dehalogenases and on the regulation of DCM dehalogenase expression. DCM dehalogenase belongs to the glutathione S-transferase supergene family. Analysis of the amino acid sequences of two bacterial DCM dehalogenases reveals 56% identity, and comparison of these sequences to those of glutathione S-transferases indicates a closer relationship to class Theta eukaryotic glutathione S-transferases than to a number of bacterial glutathione S-transferases whose sequences have recently become available. dcmA, the structural gene of the highly substrate-inducible DCM dehalogenase, is carried in most DCM utilizing methylotrophs on large plasmids. In Methylobacterium sp. DM4 its expression is governed by dcmR, a regulatory gene located upstream of dcmA, dcmR encodes a trans-acting factor which negatively controls DCM dehalogenase formation at the transcriptional level. Our working model thus assumes that the dcmR product is a repressor which, in the absence of DCM, binds to the promoter region of dcmA and thereby inhibits initiation of transcription.

Dichloromethane (DCM) dehalogenases enable facultative methylotrophic bacteria t o utilize DCM as sole carbon and energy source. DCM-degrading aerobic methylotrophic bacteria expressing a type A DCM dehalogenase were previously shown to share a Conserved 4.2 kb BamHl DNA fragment containing the dehalogenase structural gene, dcmA, and dcmR, the gene encoding a putative regulatory protein. Sequence analysis of a 10 kb DNA fragment including this region led to the identification of three types of insertion sequences identified as 157354, IS7355 and 157357, and also two ORFs, orf353and orf792, of unknown function. Two identical copies of element 157354 flank the conserved 4.2 kb fragment as a direct repeat. The occurrence of these newly identified 15 elements was shown to be limited to DCM-utilizing methylotrophs containing a type A DCM dehalogenase. The organization of the corresponding dcm regions in 12 DCM-utilizing strains was examined by hybridization analysis using IS-specif ic probes. Six different groups could be defined on the basis of the occurrence, position and copy number of IS sequences. All groups shared a conserved 5.6 kb core region with dcmA, dcmR, orf353 and orf192 as well as 157357. One group of strains including Pseudomonss sp. DM1 contained two copies of this conserved core region. The high degree of sequence conservation observed within the genomic region responsible for DCM utilization and the occurrence of clusters of insertion sequences in the vicinity of the dcm genes suggest that a transposon is involved in the horizontal transfer of the DCM-utilization character among methylotrophic bacteria.

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