ISOLATION OF LACTOCOCCUS-LACTIS NONSENSE SUPPRESSORS AND CONSTRUCTIONOF A FOOD-GRADE CLONING VECTOR

Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two s uppressor strains and the DNA sequence determined, One suppressor is a n ochre suppressor with an altered tRNA(gin) and the other an amber su ppressor with an altered tRNA(ser). The nonsense suppressors allowed i solation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food-grade cloning vector based totally on DNA from Lactococcos and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following electroporation of a sup pressible purine auxotroph, can be done on purine-free medium. The pep N gene from L. lactis Wg2 was subcloned resulting in a food-grade plas mid giving a four- to fivefold increase in lysine aminopeptidase activ ity.