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. (20). In all of the aerobic methylotrophs tested, the catalytic activity of DCM dehalogenase is low and the enzyme is 50-to 80-fold induced by DCM (18,26). The genes responsible for DCM utilization have been examined in Methylobacterium sp. strain DM4, a pinkpigmented facultative methylotrophic bacterium. They are encoded on a 2.8-kb BamHI-PstI DNA fragment located on the chromosome or on an undetected megaplasmid of this organism (19), and three cryptic plasmids carried by strain DM4 are unrelated to DCM metabolism (10). A 0.9-kb segment of the cloned and sequenced 2.8-kb BamHI-PstI fragment encodes dcmA, the structural gene of DCM dehalogenase. dcmA has been shown to belong to the glutathione S-transferase supergene family. DCM dehalogenase expression is subject to negative control at the transcriptional level, and our preliminary observations suggested that a regulatory protein responsible for this control is also encoded on the sequenced fragment, in the upstream region of dcmA (19).In this communication, we report the functional analysis of the 1.5-kb region upstream of dcmA. We present evidence that this region contains dcmR, the structural gene of a trans-active protein controlling dcmA expression as well as its own synthesis.

Dichloromethane metabolism and C1 utilization genes in Methylobacterium strains The GenBank accession numbers for the sequences determined in this work are AJ421476 and AJ421477.

“…Plasmid pME8220 was constructed by cloning the 1n5 kb HindIII-PstI fragment containing dcmA without its negative transcriptional regulator dcmR (La Roche & Leisinger, 1991), from plasmid pME1540 (Schmid-Appert, 1996), into HindIII\PstI-digested and dephosphorylated broad-hostrange cloning vector pCM62 (Marx & Lidstrom, 2001). Plasmid pME8221 was obtained by cloning the 2 kb BamHI fragment containing dcmA and its negative transcriptional regulator dcmR from plasmid pME1518 (La Roche & Leisinger, 1990) into BamHI digested and dephosphorylated pCM62.…”

“…Plasmid pME8221 (Fig. 2c) allows expression of the structural gene dcmA under the control of its upstream negative transcriptional regulator dcmR (La Roche & Leisinger, 1991), while plasmid pME8220, containing dcmA with a truncated upstream DNA region lacking the dcmR regulator gene, leads to constitutive expression of DCM dehalogenase (Fig. 2b).…”

Evolution of Enzymes for the Metabolism of New Chemical Inputs into the Environment

“…First, dcmA is seen to be associated with a dcmR regulatory gene, which tightly controls expression of DcmA under conditions of dichloromethane exposure (25). Second, methylotrophs containing dcmA may have other adaptations that facilitate growth on dichloromethane (26,27).…”

Analysis of a Rhizobium leguminosarum gene encoding a protein homologous to glutathione S-transferases

“…It has been shown that the transcription of the dcmA gene (whose product has similarity to GST) was stimulated by growing the cells in the presence of the substrate dichloromethane (LaRoche & Leisinger, 1991). This regulation depended upon the presence of the dcmR gene that is transcribed divergently from the 'structural' gene dcmA.…”

“…GSTs (Hofer et al, 1994;Masai et a/., 1991Masai et a/., , 1993LaRoche & Leisinger, 1991). However, the ability to condense GSH to substrates such as CDNB appears to be quite widespread in prokaryotes (Zablotowicz et al, 1995) and there have been reports of GSH-binding proteins from other bacteria .…”