Analysis of the internal replication region of a mycobacterial linear plasmid

Linear plasmids have previously been identified by the authors in mycobacteria, the telomeres of which have terminal inverted repeats and covalently attached proteins. In this study, the replication of these unusual molecules was investigated by studying a 25 kb linear plasmid from the slow-growing species Mycobacterium celatum called pCLP. An internal region of pCLP responsible for replication in Mycobacterium smegmatis was identified. The nucleotide sequence of the minimum replication region of pCLP, which was 2.8 kb long, contained a putative replication gene, rep, and a putative origin of replication consisting of an 18 bp direct repeat and an AT-rich region. A short section of the pCLP replication region was also found to have sequence identity with the replication regions of mycobacterial circular plasmids, suggesting that these linear and circular plasmids are related. It was found that pCLP replicated in Mycobacterium bovis BCG and was compatible in M. smegmatis with pAL5000- and pJAZ38-derived plasmids from Mycobacterium fortuitum, which belong to two different compatibility groups. Thus, this new Escherichia coli-mycobacteria shuttle vector may be used in both slow- and fast-growing mycobacteria and in co-transformation experiments with other mycobacterial vectors.