Evidence for a Plasmid-Linked Restriction-Modification System in Lactobacillus helveticus

The presence of a restriction-modification (R/M) system against two bacteriophages, 328-B1 and hv, was demonstrated in three Lactobacillus helveticus strains, CNRZ 1094, CNRZ 1095, and CNRZ 1096. In addition, the burst size of phage 328-B1 in the three restrictive strains CNRZ 1094, CNRZ 1095, and CNRZ 1096 was reduced with respect to the values obtained in its propagating strain, CNRZ 328. Heating at 60°C did not inactivate the R/M system. Nonrestrictive variants from CNRZ 1094 were easily obtained under several culture conditions, but treatment with novobiocin at 42°C followed by storage at −20°C resulted in drastic elimination of the R⁺/M⁺ phenotype from all clones tested. Electrophoretic analysis of CNRZ 1094 nonrestrictive variants revealed the concomitant loss of a 34-kb plasmid. Four EcoRI fragments from the 34-kb plasmid were cloned in the Escherichia coli vector pACYC184. The use of one or several of these fragments as probes confirmed the plasmidic location of the genes responsible for the R/M system. These probes also showed the presence of R/M plasmids in the two other restrictive strains, CNRZ 1095 and CNRZ 1096. Lactose-fermenting ability and/or proteolytic capacity was not linked to the 34-kb plasmid.