MYCOBACTERIOPHAGE D29 CONTAINS AN INTEGRATION SYSTEM SIMILAR TO THAT OF THE TEMPERATE MYCOBACTERIOPHAGE L5

A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmi d that transforms Mycobacterium smegmatis, was sequenced. The determin ed sequence was 2592 nucleotides long and had a mean G+C content of 63 .7 mol%, similar to that of mycobacterial DNA. Four ORFs were identifi ed: one with strong homology to dCMP deaminase genes; one homologous t o mycobacteriophage L5 gene 36, whose function is unknown; one encodin g a possible excisase; and one encoding an integrase. The intergenic r egion between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same att P core sequence as mycobacteriophage L5. Transformation experiments us ing subclones of pRM64 indicated that the integrase gene and all the i ntergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to t ransform but recombinants were highly unstable. Southern analysis of t otal DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bact erial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at differe nt sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally held view of D29 as a virulent phage must be reviewed.