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

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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 kg plasmid. The DCM-utilization genes were cloned on the broad-host-range vector pVK100. Plasmid pME1510, a recombinant plasmid carrying a 21 kb HindIII 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 did not. Plasmid pME1510 did not hybridize with total DNA from a cured DCM-non-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 DCM-utilization genes or to the loss of an undetected megaplasmid.