HIGH-COPY-NUMBER AND LOW-COPY-NUMBER LACTOCOCCUS SHUTTLE CLONING VECTORS WITH FEATURES FOR CLONE SCREENING

High- and low-copy-number shuttle cloning vectors were constructed by incorporating the Escherichia coli P15A plasmid origin of replication into the pAM beta 1-derived vectors, pIL252 and pIL253. The resulting vectors were structurally stable in Lactococcus, which is a common feature of theta-replicating plasmids, and also displayed good structural stability in E. coli, possibly due to lack of a resolvase-encoding gene. All the vectors expressed erythromycin resistance (Er-R) in both; brain heart infusion medium allowed clear selection of Er-R in E. coli. Some of the vectors provided insertional inactivation of a cat (pTRKH1; pTRKL1) or tet (pTRKH1; pTRKH3; pTRKH5) gene to facilitate screening for clones. Multiple cloning sites in a lacZ gene, which expresses beta-galactosidase in lacZ alpha-complementing E. coli strains, were included in some vectors (pTRKH2/H5 and pTRKL2) to enable blue/white screening of clones on XGal plates. The 'H' and 'L' prefixes signify if the vector exists at high (H) or low (L) copy number in Lactococcus. Successful introduction of these vectors into Lactococcus, Enterococcus, Streptococcus and Lactobacillus highlights their utility for expanding the possibilities for genetic manipulation of these industrially significant bacteria.