Plasmid vectors for gram-positive bacteria switching from high to low copy number.

ABSTRACT

A set of vectors for Gram-positive bacteria was constructed with a new feature which enables the switching down of their copy number per cell. These vectors carry the replication region of pAM beta 1, containing a gene essential for replication, repE, and its regulator, copF. The latter gene was inactivated by inserting a linker into its unique KpnI site. Since copF downregulates the expression of repE, its inactivation leads to an increase in the plasmid copy number per cell. The original low copy state can be restored by removal of the linker via KpnI cleavage and ligation. The new replicon was used to build (i) vectors for studying gene regulation by transcriptional or translational fusion with the bacterial luciferase gene, (ii) vectors for gene expression, and (iii) cassettes of the replicon with different multiple cloning sites, which would facilitate construction of vectors for novel purposes.