A novel antivirulence element in the temperate bacteriophage HK022

Lysogens of the temperate lambdoid phage HK022 are immune to superinfection by HK022. Superinfection immunity is conferred in part by the action of the HK022 CI repressor at the O.R operators. In this work, we have identified an additional regulatory element involved in immunity. This site, termed OFR (operator far right), is located just downstream of the cro gene, more than 250 nucleotides distant from OR. The behavior of phage containing a mutation in OFR suggests that the wild-type site functions as an antivirulence element. HK022 OFR- mutants were able to form turbid plaques indistinguishable from those of the wild type. However, they gave rise to virulent derivatives at a far higher frequency than the wild type (approximately 10(-5) for OFR- versus about 10(-9) for the wild type). This frequency was so high that cultures of HK022 OFR- lysogens were rapidly overgrown by virulent derivatives. Whereas virulent mutants arising from a wild-type OFR+ background contained mutations in both OR1 and OR2, virulent derivatives of the OFR- mutant phage contained a single mutation in either OR1 or OR2. We conclude that the wild-type OFR site functions to prevent single mutations in OR from conferring virulence. The mechanism by which OFR acts is not yet clear. Both CI and Cro bound to OFR and repressed a very weak rightward promoter (PFR). It is unlikely that repression of PFR by CI or Cro binding to OFR can account in full for the antivirulence phenotype conferred by this element, since PFR is such a weak promoter. Other models for the possible action of OFR are discussed.