P. Stolt et Ng. Stoker, FUNCTIONAL DEFINITION OF REGIONS NECESSARY FOR REPLICATION AND INCOMPATIBILITY IN THE MYCOBACTERIUM-FORTUITUM PLASMID PAL5000, Microbiology, 142, 1996, pp. 2795-2802
Different parts of the Mycobacterium fortuitum plasmid pAL5000 necessa
ry for plasmid replication and incompatibility were defined and studie
d. Two ORFs, named repA and repB, were defined which are necessary for
replication. A pAL5000 derivative deleted in these genes can be made
to replicate by providing the gene products in trans. A 435 bp fragmen
t was defined which was necessary in cis for replication and which had
an influence on copy number. This region (inc), which contains severa
l repeated motifs, was also able to confer a degree of incompatibility
when cloned into an otherwise unrelated mycobacterial replicon. pAL50
00-derived plasmids carrying two copies of the inc region had a lower
copy number and were less stable than the wild-type. These effects wer
e only observed when the two regions were in the same orientation. Pla
smids carrying only the inc region and no other parts of pAL5000 could
be made to replicate if repA and repB were supplied in trans from ano
ther plasmid. Based on these findings, systems for selectively curing
cells of one plasmid of a pair were designed and shown to be functiona
l in Mycobacterium smegmatis. These have potential as a simple deliver
y system for achieving transposon mutagenesis or gene replacement in m
ycobacteria.