Cloning vectors for lactococci based on a plasmid encoding resistance to cadmium

An 8.8-kb plasmid (pND302) was identified in Lactococcus lactis spp lactis M71 which encodes cadmium resistance (Cd-R). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, Cd-R should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L. lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (Nis(R)) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing Nis(R) and Cd-R, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10(3)/mu g DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci.