Vectors for Lactobacilli and other Gram-positive bacteria based on the minimal replicon of pRV500 from Lactobacillus sakei.

The low-copy-number plasmid pRV500, belonging to the pUCL287 group of theta-type plasmids, was previously isolated from Lactobacillus sakei and characterized. We show here that the replicon of this plasmid enables replication also in Enterococcus faecalis and Bacillus subtilis but not in Lactococcus lactis. A 1.25 kb region encompassing the iterons and the repA gene was sufficient for replication, copy-number control and relative stable maintenance in L. sakei. Functional implications of host or plasmid-borne factors in the maintenance of pUCL287-type plasmids are discussed. The minimal replicon from pRV500 was fused to pBluescript for constructing the shuttle E. coli/lactobacilli cloning vector pRV610. pRV610 enables the white/blue lacZ alpha-complementation in E. coli. The cassettes for selection (erythromycin resistance) and replication (iterons and repA gene) are each bordered by unique restriction sites for easy replacement if needed. Derivatives in which chloramphenicol or tetracycline resistance replaced erythromycin resistance were constructed. In order to allow inducible gene expression, a copper-inducible promoter was placed on the pRV613 derivative. Expression of the downstream reporter gene lacZ was shown to be induced by 30 microM CuSO(4).