A Strategy for Rotation of Different Bacteriophage Defenses in a Lactococcal Single-Strain Starter Culture System

Abstract

A new strategy for starter culture rotations was developed for a series of phage-resistant clones genetically derived from a single strain of Lactococcus lactis subsp. lactis. Phage-resistant derivatives carrying different defense systems were constructed via conjugation with various plasmids encoding abortive infection (Abi/Hsp) and/or restriction and modification (R/M) systems of different specificity. The plasmids included pTR2030 (Hsp + R + /M + ), pTN20 (Abi + R + /M + ), pTRK11 (R + /M + ), and pTRK68 (R + /M + ). Selected phage-resistant transconjugants or transformants were evaluated in different rotation sequences through cycles of the Heap-Lawrence starter culture activity test in milk contaminated with phage and whey from the previous cycle. When used in consecutive sequence, derivative strains carrying the R/M systems encoded by pTN20, pTRK11, and pTRK68 retarded phage development when the initial levels of phage contamination were below 10 2 PFU/ml but not when levels were increased to 10 3 PFU/ml. Use of a derivative bearing pTR2030 (Hsp + R + /M + ) at the beginning of the rotation prevented phage development, even when the initial levels of phage contamination were high (10 6 PFU/ml). Alternating the type and specificity of R/M and Abi defenses through the rotation prevented phage proliferation and in some cases eliminated contaminating phages. A model rotation sequence for the phage defense rotation strategy was developed and performed successfully over nine cycles of the Heap-Lawrence starter culture activity test in the presence of high-titer commercial phage composites. This phage defense rotation strategy is designed to protect a highly specialized Lactococcus strain from phage attack during continuous and extended use in the dairy industry.