A stable Escherichia coli-Mycobacterium smegmatis plasmid shuttle vector containing the mycobacteriophage D29 origin

Abstract A plasmid shuttle vector for Escherichia coli and mycobacteria was constructed from an E. coli plasmid containing the ColE1 origin, a 2.6-kb Pst I fragment from bacteriophage D29 that grows in numerous mycobacterial species, and the kanamycin resistance gene either of Tn 903 or of Tn 5 . The resultant plasmid is 7.63 kb and can be introduced via transformation into Mycobacterium smegmatis with high efficiency. In M. smegmatis the plasmid is stable and apparently present in multiple copies. Bioluminescence ( luxA and luxB of Vibrio harveyi and fischeri ) has been expressed in M. smegmatis from the aminoglycoside transferase promoter of Tn 5 . The D29 fragment should carry an origin of replication and some associated genes that act on it since various mutations destroy the ability of this fragment to replicate in M. smegmatis . The fragment was localized on the D29 genome map.