Analysis of the Clear Plaque Phenotype of the Bacteriophage HK75

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: <i>cI, cII, cIII, int, xis</i> or the phage repressor DNA binding sites. To determine which mutation is responsible for the clear plaque phenotype of HK75, we cloned the <i>cI</i> and <i>cIII</i> genes and assayed their activities. The HK75 <i>cI</i> 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 <i>cI</i> 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 <i>cIII</i> gene, we cloned it into an expression vector. Only under conditions of <i>cIII</i> 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 <i>cIII</i> gene. When HK75 was plated on a protease deficient strain of <i>E. coli</i>, 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 <i>cIII</i> gene.

Name thesis:degree_name

Master of Biology

Discipline thesis:degree_discipline

Department of Biology

Year

2010

Author dc:creator

• Kunapuli, Phani Chandrika

Contributors dc:contributor

• Dr. Rodney King (Director), Dr. Claire Rinehart, Dr. Sigrid Jacobshagen

• lambdoid phages
• bacteriophages
• molecular genetics
• Bacteriology
• Pathogenic Microbiology

Repository record dc:identifier

https://digitalcommons.wku.edu/theses/219

OAI identifier oai:identifier

oai:digitalcommons.wku.edu:theses-1220