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 fea ture of theta-replicating plasmids, and also displayed good structural stability in E. coli, possibly due to lack of a resolvase-encoding ge ne. 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 (pTRK H1; pTRKL1) or tet (pTRKH1; pTRKH3; pTRKH5) gene to facilitate screeni ng for clones. Multiple cloning sites in a lacZ gene, which expresses beta-galactosidase in lacZ alpha-complementing E. coli strains, were i ncluded in some vectors (pTRKH2/H5 and pTRKL2) to enable blue/white sc reening 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. S uccessful introduction of these vectors into Lactococcus, Enterococcus , Streptococcus and Lactobacillus highlights their utility for expandi ng the possibilities for genetic manipulation of these industrially si gnificant bacteria.