Improvement of plasmid encoded lactococcal bacteriophage resistance by mutator strain Epicurian coli XL1-Red

A random mutation strategy using mutator strain, Epicurian coli XL1-Red, wa s applied to a plasmid, pND018, constructed by inserting a Lactococcus laci s bacteriophage resistance gene (abil) into a L. lactis/E. coli shuttle vec tor (pDL278), to introduce random mutations throughout the plasmid. Followi ng transformation of the mutated plasmid library to a plasmid free and phag e sensitive strain of L. lactis (LM0230), mutated plasmids were screened by cross-streaking and efficiency of plaquing (EOP) assays. Two strains with enhanced resistance were obtained, as well as several phage sensitive strai ns. Repeated transformation of the mutated plasmids to LM0230 confirmed tha t the observed phenotypes were caused by mutations located on the plasmids. The EOP values and plaque morphology of two enhanced phage resistance muta nts were characterized at 30 degrees C and 37 degrees C. These results indi cate that this simple procedure can be applied to generate modified plasmid s with improved phage resistance, which may be of commercial value.