Identification of dcmR, the regulatory gene governing expression of dichloromethane dehalogenase in Methylobacterium sp. strain DM4

Abstract

The genes for dichloromethane utilization by Methylobacterium sp. strain DM4 are encoded on a 2.8-kb sequenced DNA fragment, the dcm region. This fragment contains dcmA, the structural gene of dichloromethane dehalogenase and, upstream of dcmA, a 1.5-kb region responsible for inducibility of dichloromethane dehalogenase by dichloromethane. A fragment of the dcm region covering dcmA and 230 bp of its upstream region was integrated into the chromosome of a Methylobacterium sp. strain DM4 mutant deleted for the dcm region. This yielded a strain expressin dichloromethane dehalogenase constitutively at the induced level. Plasmids carrying various segments of the 1.5-kb regulatory region were tested for their ability to restore regulation. The data obtained led to the identification of dcmR, the structural gene of a putative dcm-specific repressor. Transcription of dcmR was divergent from dcmA. dcmR encoded a 30-kDa protein with a helix-turn-helix motif near the amino terminus. The transcription start sites of dcmA and dcmR were identified by nuclease S1 mapping. The promoter regions of these genes contained nearly identical 12-bp sequences covering positions -14 to -25 relative to the mRNA start sites. Experiments with dcmR'-'lacZ fusions demonstrated that dcmR expression was markedly autoregulated at the level of transcription and less so at the protein level. These findings are compatible with both dcmA and dcmR expression being negatively controlled at the transcriptional level by the DcmR protein.