Plasmid Analysis and Cloning of the Dichloromethane-utilization Genes of Methylobacterium sp. DM4

Abstract

The dichloromethane (DCM)-utilizing facultative methylotroph Methylobacterium sp. DM4 was shown to contain three plasmids with approximate sizes of 120 kb, 40 kb and 8 kb. Curing experiments suggested that the DCM-utilization character was correlated with the possession of an intact 120 kb plasmid. The DCM-utilization genes were cloned on the broad-host-range vector pVK100. Plasmid pME1510, a recombinant plasmid carrying a 21 kb Hind111 fragment complemented DCM-utilization-negative derivatives of Methylobacterium sp. DM4 and conferred the DCM-utilization-positive phenotype to a number of Gram-negative methylotrophic bacteria. In Southern hybridization experiments with pME1510 as a probe, chromosomal DNA from Methylobacterium sp. DM4 gave definite signals while purified plasmid DNA did not. Plasmid pME15lO did not hybridize with total DNA from a cured DCMnon-utilizing derivative of Methylobacterium sp. DM4. It is concluded that the DCM-utilization genes are located on the chromosome or on a megaplasmid. Curing procedures thus led to the formation of a chromosomal or megaplasmid deletion larger than 21 kb and covering the DCMutilization genes or to the loss of an undetected megaplasmid.

Reclassification of Methylobacterium chloromethanicum and Methylobacterium dichloromethanicum as later subjective synonyms of Methylobacterium extorquens and of Methylobacterium lusitanum as a later subjective synonym of Methylobacterium rhodesianum

“…In the previous reports (Doronina et al, 2000;McDonald et al, 2001) (Galli and Leisinger, 1988). At the same time, it suggests that dichloromethane utilization could be lost if the growth condition of M. dichloromethanicum had been changed and the plasmid was lost from the cell.…”

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

“…Enzyme activity was determined at 30°C by following the rate of formaldehyde production in a colorimetric test as described previously (19,20 regulated DCM dehalogenase synthesis in the DCM-nonutilizing strain DM4-2cr (19). In this strain, a DNA fragment larger than 21 kb and spanning the dcm region is deleted (10). Introduction of plasmids with deletions in the dcmA upstream region into strain DM4-2cr led to transconjugants with constitutive expression of DCM dehalogenase.…”

“…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.…”

Interactive Analytics for Complex Cognitive Activities on Information from Annotations of Prokaryotic Genomes

“…A finding from the box plot visual representation (Figure 3) is that methanol-oxidizing Methylobacterium nodulans ORS 2060, the legume (Crotalaria) root-nodule-forming and nitrogen-fixing bacteria [28], has at least 7 sequenced plasmids [29]. The possession of an intact 120kb megaplasmid correlated with ability of Methylobacterium extorquens DM4 to utilize dichloromethane as sole source of carbon and energy [30]. Comparative analysis of the genes in the plasmids of Methylobacterium species could improve understanding of methylotrophy and nitrogen-fixation.…”