Transformation of Mycobacterium smegmatis with E. coli plasmids

One limiting factor in the studies of tuberculosis and leprosy is the lack of a versatile system for genetic analysis and manipulation of mycobacteria. One strategy used in constructing a plasmid vector for transforming Mycobacterium smegmatis was to insert fragments of a mycobacterial plasmid into an Escherichia coli plasmid. We found that the parental E. coli plasmid is capable of self-replication in M. smegmatis yielding chloramphenicol-resistant colonies. Plasmids from different passages of one M. smegmatis transformant were recovered and characterised by restriction digest analysis. The plasmid from the earlier passage was found to be indistinguishable from the original plasmid by restriction analysis. Plasmids from later preparations, however, were found to have undergone modifications in the M. smegmatis host resulting in an apparent increase in transformation efficiency for M. smegmatis. These plasmids can be used as a shuttle vector for the genetic manipulation of mycobacterial species.