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Agar surface conjugal matings were used to introduce heat-sensitive phage resistance (Hsp+ ) determinants carried on the conjugal plasmid pTR2030 intoStreptococcus cremoris KH, HP, 924, and TDM1. Lactose-fermenting (Lac+ ) transconjugants were selected from matings of Lac− variants ofS. cremoris KH, HP, 924, and TDM1 withStreptococcus lactis ME2 or a high-frequency donor,S. lactis T-EK1 (pTR1040, Lac+ ; pTR2030, Hsp+ ). For all of theS. cremoris strains examined, select Lac+ transconjugants were completely resistant to plaquing by their homologous lytic phages. In all cases the plaquing efficiencies were less than 10−9 . Acquisition of a 30-megadalton plasmid (pTR2030) in theS. cremoris phage-resistant transconjugants was demonstrated by direct plasmid analysis, by hybridization with32 P-labeled probes, or by conjugal transfer of pTR2030 out of the phage-resistant transconjugants into a plasmid-cured recipient,S. lactis LM2302. Acid production, coagulation ability, and proteolytic activity of phage-resistant transconjugants in milk were comparable to those of their phage-sensitive parents. Further,S. cremoris phage-resistant transconjugants were not attacked by phage in starter culture activity tests, which included a 40°C incubation period. The results demonstrated that phage resistance determinants on pTR2030 could be conjugally transferred to a variety ofS. cremoris strains and confer resistance to phage under conditions encountered during cheese manufacture. Phage-resistant transconjugants ofS. cremoris M43 and HP were also constructed without the use of antiblotic markers to select conjugal recipients from mating mixtures.

Conjugal Transfer of Bacteriophage Resistance Determinants on pTR2030 into Streptococcus cremoris Strains