The growth of bacteriophage HK75 is inhibited by specific mutations in the zinc binding domain of the host RNA polymerase beta prime subunit. It shares this rare property with bacteriophage HK022 and other phages that use RNA mediated antitermination to promote early gene expression. Recent genomic analysis of HK75 and HK022 has confirmed the relatedness of these two phages and place HK75 in the lambdoid family of bacteriophages. Lambdoid phages are temperate and can adopt a lytic or lysogenic lifestyle upon infection of a suitable host. However, HK75 only forms clear plaques and thus appears to be defective in its ability to form lysogens. Based on published analyses of other lambdoid phages, a clear plaque phenotype is commonly due to a mutation in one of 5 phage genes: cI, cII, cIII, int, xis or the phage repressor DNA binding sites. To determine which mutation is responsible for the clear plaque phenotype of HK75, we cloned the cI and cIII genes and assayed their activities. The HK75 cI gene clone prevented super-infection by HK75. This result demonstrated repressor functionality and thus the clear plaque phenotype cannot be due to a mutation in the HK75 cI gene. Several amino acid differences were noted between the HK022 and HK75 CIII proteins. To determine if the clear plaque phenotype was due to mutations in the HK75 cIII gene, we cloned it into an expression vector. Only under conditions of cIII gene overexpression were lysogens of HK75 recovered. The phage CIII protein normally protects CII from proteolysis. Stabilization of CII by mutations in specific host proteases has been shown to suppress a clear plaque phenotype caused by mutations in the cIII gene. When HK75 was plated on a protease deficient strain of E. coli, turbid plaques were formed and lysogens were recovered. These results support the idea that the clear plaque phenotype of HK75 is due to a defect in the expression of the phage cIII gene.

Lambdoid phages suppress transcription termination to fully express their genes. Antitermination of early gene expression in most lambdoid phages is mediated by an interaction between the N protein and a number of host-encoded factors. Bacteriophage HK022 does not rely on a protein for antitermination. To promote full expression of early phage genes, the transcripts of the HK022 put sites interact directly with RNA polymerase to convert it to a termination resistant form. Bacteriophage HK639 also uses RNA-mediated antitermination. However, it only possesses a single put-like element in its left operon. Because most lambdoid phages, including HK022, have antiterminator elements in each of their early operons, the presence of a single antitermination site in HK639 was unexpected. We have shown that host genes involved in promoting protein-mediated antitermination are not required for HK639 growth. We have also shown that expression of the left operon is essential for lytic growth. Replacement of the left operon promoter, PL, and the putL antitermination sequence prevented HK639 phage release. A similar construct that only replaced putL also prevented phage release. These results suggest that antitermination is required for HK639 excision and/or lytic growth. To distinguish between a defect in phage excision versus a defect in lytic growth, the mutations were crossed onto lytically growing phage. Recombinant phages could not be recovered which suggests a defect in lytic growth is preventing phage release. Additional replacements of left operon sequences suggest that antitermination is not the only requirement for lytic growth. A 2,161bp deletion (HK639 genome coordinates 30,888-33,048) and a 1,736bp deletion (HK639 genome coordinates 29,152- 30,887) downstream of the HK639 putL site also prevented phage release, whereas a 1,746bp deletion (HK639 genome coordinates 29,151-27,406) did not. These results suggest that the deleted HK639 left operon sequences are required for lytic growth. BLAST analysis did not provide insight into the function of the deleted genes. Although the function of many of the HK639 left operon genes is unknown, their importance in phage growth can now be verified by complementation analysis. Our results suggest that HK639 may use a novel mechanism to control the expression of its early genes.