Effect of increasing the copy number of bacteriophage origins of replication, in trans, on incoming-phage proliferation

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in trans in Lactococcus lactis NCK203, Per50, the cloned origin of replication from phage φ50, interferes with φ50 replication. The per50 fragment was found to afford negligible protection to NCK203 against φ50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected φ50 infection, reducing the efficiency of plaquing (EOP) to 2.5 x 10^-4 and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage φ31 and demonstrated to function as an origin of replication by enabling replication of per31- containing plasmids, in NCK203, on φ31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of φ31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 x 10^-7 but the plaques were large and contained Per31-resistant phages. Characterization of these 'new' phages revealed at least two different types that were similar to φ31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.