Transductional evidence for plasmid linkage of lactose metabolism in Streptococcus lactis C2

Abstract

A lactose negative (Lac-), proteinase negative (Prt-) mutant, designated C145, was isolated S. lactis C2 after treatment with nitrosoguanidine and ultraviolet irradiation. The mutant appeared to be cured of the prophage(s) present in S. lactis C2 based on non inducibility by ultraviolet irradiation or mitomycin C. When cleared lysate material from C145 was subjected to cesium chloride ethidium bromide (EB) density gradient centrifugation, no plasmid peak was observed, suggesting that C145 was cured of plasmid deoxyribonucleic acid (DNA). A histogram showing distribution of contour lengths of circular molecules of DNA from C145, however, revealed the presence of a greatly diminished number of DNA molecules as compared with the parent culture and indicated the absence of the 30 x106 plasmid. Cesium chloride ethidium bromide gradient profiles from Lac+Prt- and Lac+prt+ transductants of C145 revealed no plasmid peak, but electron microscopy of the fractions normally possessing the satellite band of DNA showed the presence of a new plasmid species having a molecular weight from 20 x 106 to 22 x 106. This plasmid was lost when the transductants became Lac-. Examination of a plasmid histogram from a spontaneous Lac-Prt- mutant of S. lactis C2 resembled that of C145 with the absence of the 30 x106 plasmid and the presence of the 22 x 106 plasmid in Lac+Prt+ transductants. The results suggest that lactose metabolism is mediated through the 30 x 106 plasmid in S lactis C2 and that the transducing bacteriophage, which is too small to accommodate the entire plasmid, is transferring about two thirds of the original plasmid through a process termed transductional shortening.