Cloning of genomic DNA of Lactococcus lactis that restores phage sensitivity to an unusual bacteriophage sk1-resistant mutant

An unusual, spontaneous, phage ski-resistant mutant (RMSK1/1) of lactococcu s lactis C2 apparently blocks phage DNA entry into the host. Although no vi sible plaques formed on RMSK1/1, this host propagated phage at a reduced ef ficiency, This was evident from center-of-infection experiments, which show ed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sen sitive parental strain, C2, Moreover, viable cell counts 0 and 4 h after in fection were not significantly different from those of an uninfected cultur e. Further characterization showed that phage adsorption was normal, but bu rst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages, Ph age sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterizat ion of the DNA that restored phage sensitivity revealed an open reading fra me with similarity to sequences encoding lysozymes (beta -1,4-N-acetylmuram idase) and lysins from various bacteria, a fungus, and phages of Lactobacil lus and Streptococcus and also revealed DNA homologous to noncoding sequenc es of temperate phage of L, lactis, DNA similar to a region of phage ski, a gene with similarity to tRNA genes, a prophage attachment site, and open r eading frames with similarities to san and to sequences encoding phosphopro tein phosphatases and protein kinases, Mutational analyses of the cloned DN A showed that the region of homology with lactococcal temperate phage was r esponsible for restoring the phage-sensitive phenotype, The region of homol ogy with DNA of lactococcal temperate phage was similar to DNA from a previ ously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal tem perate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strai n, The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with component s of the resistance mechanism.