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1 Department of Food Science and Nutrition, University of Minnesota, St. Paul 55108
An origin of replication and a nisin resistance determinant on a 7.6-kb EcoRI fragment, capable of existing as an independent replicon when circularized, was used to construct a vector for cloning homologous DNA in Lactococcus lactis ssp. lactis. A medium, designated M17-GTN, was developed to identify L. lactis ssp. lactis cells containing pFM011 from a mixed population of nisin-sensitive resistant transformants produced by electroporation or by protoplast transformation. To demonstrate the usefulness of nisin resistance as a selectable marker, a 6.3-kb EcoRI fragment encoding reduced bacteriophage sensitivity was cloned into the EcoRI site of pFM011. Separately, the erythromycin resistance marker from pGB301 was cloned into the HindIII site of pFM011. Both plasmids were introduced into L. lactis. ssp. lactis. The origin of replication of pFM011 was localized to a 2.2-kb HindIII-HaeIII fragment. Seven additional unique restriction sites added by inserting a multiple cloning site. Results indicated that pFM011 and its recombinant derivatives were relatively stable in the L. lactis ssp. lactis background. As pFM011 was derived entirely from lactococcal DNA, this study demonstrates its use as a food-grade cloning vector for lactococci.
Key Words: cloning • vector • nisin • lactococci
Submitted on September 15, 1990
Accepted on December 3, 1990
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