TARGETS FOR PSAM2 INTEGRASE-MEDIATED SITE-SPECIFIC INTEGRATION IN THEMYCOBACTERIUM-SMEGMATIS CHROMOSOME

An improved integrative cassette from plasmid pSAM2 has been construct ed containing plasmid int and attP genes but excluding the xis gene, w hich should result in increased stability by suppression of the excisi on reaction. This cassette was included in both suicide and thermosens itive plasmids and used for integration in Mycobacterium smegmatis. Su icide plasmids containing this cassette integrated at a single site (a ttB1) in the M. smegmatis chromosome. The sequence of the attB1 site h as been determined and was identified as a putative tRNA(Pro) gene. Th ermosensitive plasmids containing the cassette integrated both at the same attB1 site and at other different sites, often giving rise to sim ultaneous integration at two sites. A second integration site (attB2) has been sequenced, which was located in the region encoding 16S rRNA of one of the two rrn operons of M. smegmatis.