Improvement and optimization of two engineered phage resistance mechanismsin Lactococcus lactis

Homologous replication module genes were identified for four P335 type phag es, DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes,designated rep(2009) and orf17. On e of these genes, rep(2009), codes for a putative replisome organizer prote in and contains an assumed origin of phage DNA replication (ori(2009)), whi ch was identical for all four phages. DNA fragments representing the ori(20 09) sequence confer a phage-encoded resistance (Per) phenotype on lactococc al hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori(2009) sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmi ds targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep(2009) and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strateg ies for inhibiting bacteriophage proliferation, and they also identify a no vel gene, orf17, which appears to be important for phage DNA replication. F urthermore, these results indicate that although the actual mechanisms of D NA replication are very similar, if not identical, for all four phages, exp ression of the replication genes is significantly different in each ease.