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 O-FR (operator far right), is located just downstream of the cro gene, more than 250 nucleotides distant from O-R. The behavior of phage containing a mutation in O-FR suggests that the wild-type site functions as an antivirulence element. HK022 O-FR(-) 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 (approximate to 10(-5) for O-FR(-) versus about 10(-9) for the wild type). This frequency was so high that cultures of HK022 O-FR(-) lysogens were rapidly overgrown by virulent derivatives. Whereas virulent mutants arising from a wild-type O-FR(+) background contained mutations in both O(R)1 and O(R)2, virulent derivatives of the O-FR(-) mutant phage contained a single mutation in either O(R)1 or O(R)2. We conclude that the wild-type O-FR site functions to prevent single mutations in O-R from conferring virulence. The mechanism by which O-FR acts is not yet clear. Both CI and Cro bound to O-FR and repressed a very weak rightward promoter (P-FR). It is unlikely that repression of P-FR by CI or Cro binding to O-FR can account in full for the antivirulence phenotype conferred by this element, since P-FR is such a weak promoter. Other models for the possible action of O-FR are discussed.