Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Abstract

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC4 strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac+), lactococcin-producing (Lap+), lactococcin-resistant (Lapr), sucrosefermenting (Suc+) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Eryr) at the frequencies of 104 transformants/μg of DNA. PC4 protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac−Lap−Eryr, Lac+Suc+Lap+LaprEryr, and Lac−Suc+Lap+ LaprLapr, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc+Lap+Lapr and Lac+ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC4 transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.