Genetic evidence for plasmid-encoded lactococcin production inLactococcus lactis subsp.lactis 484

Abstract

Lactococcus lactis subsp.lactis 484 produced a proteinaceous antibacterial substance designated as ‘lactococcin’ capable of inhibiting members of theLactococcus group,Bacillus cereus, Staphylococcus aureus, andSalmonella typhi. Growth of this culture in the presence of 2–30 μg/ml of ethidium bromide or acriflavin or novobiocin, and at elevated temperature (39° and 41°C), could not produce any lactococcin-negative (Lap⁻) variants. However, protoplast-induced curing with lysozyme was successful in developing Lap⁻ derivatives. Two types of cured derivatives, namely Lac⁻ Lap⁺ and Lac⁻ Lap⁻, were obtained. Lap⁻ variants were also lacking sucrose-fermenting ability (Suc⁺) and lactococcin resistance (Lap^r). The lactose-negative (Lac⁻) variants and Lap⁺ were clearly lacking the largest (65 Md) plasmid. However, Lap⁻ Suc⁻ Lap^s variants lost a 2 Md plasmid.L. lactis subsp.lactis 484 transferred lactose-fermenting ability as well as Lap⁺ Suc⁺ Lap^r phenotypes simultaneously toL. lactis subsp.lactis LM 2306 and LM 0230 by surface mating at a frequency of 10⁻⁴ and 10⁻¹ per donor respectively. However, cured Lac⁻ Lap⁻ transconjugants could not transfer Lac⁺ Lap⁺ Suc⁺ Lap^r phenotypes to any of these recipient strains. Our results indicate that Lac⁺ and Lap⁺ Suc⁺ Lap^r phenotypes are associated with 65 Md and 2 Md plasmids respectively. Conjugal transfer of 2 Md plasmid is possible only in the presence of a conjugative 65 Md plasmid.