Characterization of Restriction-Modification Plasmids from Lactococcus lactis ssp. cremoris and Their EffectsWhen Combined with pTR2030

Abstract

Three different restriction-modification plasmids (pTRK12, pTRK30, pTRK317) were isolated from an industrial starter strain, Lactococcus lactis ssp. cremoris TDM1. A lactose-fermenting transconjugant, Lactococcus lactis ssp. lactis NCK40, was isolated from matings between L. lactis ssp. cremoris TDM1 and a plasmid-free recipient. The NCK40 transconjugant contained a 100-kb self-transmissible plasmid (pTRK11) encoding for restriction-modification and a 13.5-kb plasmid (pTRK10) encoding proteolytic activity. Following isolation of lactosenegative derivatives from NCK40, a 30.5-kb plasmid, pTRK12, was identified that encoded proteolytic and restriction-modification of the identical specificity as pTRK11. Restriction analyses and hybridization experiments indicated that pTRK12 contained sequences from pTRK11 and all of pTRK10. Cotransformation of tatal plasmid DNA from L. lactis ssp. cremoris TDM1 with vector pVS2 identified two other restriction-modification plasmids, pTRK30 (28.0kb) and pTRK317 (15.5kb). Efficiencies of plaquing for phage c2 on restriction-modification transconjugants and transformants was 10–2 to 10–4. The specificity of restriction-modification activities conferred by each of the three plasmids was different. When the abortive infection plasmid pTR2030 was combined with pTRK30, both phage inhibition phenotypes were expressed. However, when pTR2030 was combined with pTRK12, the abortive infection phenotype was not fully expressed. Significant cell death occurred when abortive infection cells containing only pTR2030 were infected with phage. Combining the restriction-modification system of p-0 with pTR2030 significantly improved cell survival following phage infection. Operation of restriction-modification systems in conjunction with the abortive defense mechanism maximized cell survival. The data suggest that cell death is minimized when the lytic cycle is halted by restriction before abortive infection responses induce phage abortion and kill the cell.