Proteinase PI and lactococcin A genes are located on the largest plasmid inLactococcus lactissubsp.lactisbv. diacetylactis S50

Abstract

Lactococcus lactis subsp. lactis bv. diacetylactis S50 produces a lactococcin A-like bacteriocin named bacteriocin S50, and cell envelope-associated PI-type proteinase activity. This strain harbours 3 small size plasmids: pS6 (6.3 kb), pS7a (7.31 kb), and pS7b (7.27 kb). Plasmid curing using a combination of novobiocin treatment (10 µg·mL–1) and sublethal temperature (40 °C) resulted in a very low yield (0.17%) of Prt–, Bac–, Bacsderivatives, which retained all 3 small size resident plasmids. Pulsed-field gel electrophoresis of DNA isolated from the strain S50 and cured derivatives in combination with restriction enzyme analysis and DNA–DNA hybridization revealed that S50 contains 2 additional large plasmids: pS140 (140 kb) and pS80 (80 kb). Conjugation experiments using strain S50 as a donor and various lactococcal recipients resulted in Prt+, Bac+, Bacrtransconjugants. Analysis of these transconjugants strongly indicated that plasmid pS140 harbours the prt and bac genes encoding proteinase and bacteriocin production, and immunity to bacteriocin, since each Prt+, Bac+, Bacrtranconjugant contained pS140. Accordingly, none of the Prt–, Bac–, Bacstransconjugants contained this plasmid. pS140 was a self-transmissible conjugative plasmid regardless of the host lactococcal recipient used in the test. Frequency of conjugation of plasmid pS140 did not depend on either the donor or recipient strain.Key words: Lactococcus, plasmids, conjugation, bacteriocin, proteinase.