Different parts of the plasmid pAL5000 necessary for plasmid replication and incompatibility were defined and studied. Two ORFs, named and were defined which are necessary for replication. A pAL5000 derivative deleted in these genes can be made to replicate by providing the gene products A 435 bp fragment was defined which was necessary for replication and which had an influence on copy number. This region (), which contains several repeated motifs, was also able to confer a degree of incompatibility when cloned into an otherwise unrelated mycobacterial replicon. pAL5000-derived plasmids carrying two copies of the region had a lower copy number and were less stable than the wild-type. These effects were only observed when the two regions were in the same orientation. Plasmids carrying only the region and no other parts of pAL5000 could be made to replicate if and were supplied from another plasmid. Based on these findings, systems for selectively curing cells of one plasmid of a pair were designed and shown to be functional in These have potential as a simple delivery system for achieving transposon mutagenesis or gene replacement in mycobacteria.


Article metrics loading...

Loading full text...

Full text loading...


Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error