PHENOTYPIC CONSEQUENCES OF ALTERING THE COPY NUMBER OF ABIA, A GENE RESPONSIBLE FOR ABORTING BACTERIOPHAGE INFECTIONS IN LACTOCOCCUS-LACTIS

The abiA gene (formerly hsp) encodes an abortive phage infection mecha nism which inhibits phage DNA replication. To analyze the effects of v arying the abiA gene dosage on bacteriophage resistance in Lactococcus lactis, various genetic constructions were made. An IS946-based integ ration vector, pTRK75, was used to integrate a single copy of abiA int o the chromosomes of two lactococcal strains, MG1363 and NCK203. In bo th strains, a single copy of abiA did not confer any significant phage resistance on the host except for one of the MG1363 integrants, NCK62 5, which exhibited a slightly higher level of resistance to phages sk1 and p2. Hybridization of the total cellular RNA from NCK625 to an abi A-specific probe indicated that the integration took place downstream of a promoter causing stronger expression of abiA in this integrant. T hree abiA-containing plasmids of various copy numbers were introduced into both strains, and the recombinants were evaluated for resistance to phages c2, p2, sk1, and phi31. Plasmid pTRK18 has a copy number of approximately six (cn = 6) and caused a decreased plaque size for all phages evaluated. Integration of pTRK75 into a native plasmid of NCK20 3 generated pTRK362 (cn = 13), which caused a reduced efficiency of pl aquing (EOP = 10(-2) and reduced plaque size. A high-copy-number abiA plasmid (pTRK363), based on the pAMbeta1 origin of replication, was al so constructed (cn = 100). Plasmid pTRK363 caused a significant reduct ion in EOP (10(-4) to 10(-8)) and plaque size for all phages tested, a lthough in some cases, this plasmid caused the evolution of AbiA-resis tant phage derivatives. Altering the gene dosage or expression level o f abiA significantly affects the phage resistance levels.