Investigating the Role of CP80 and CP84 Genes in Phage 186

Abstract

Bacteriophage, one of the most widespread viruses, infect bacteria cells for reproduction. There are range of different phage strains, such as 186, lambda (λ) and P2. Current research focuses on identifying genomic function of bacteriophages, especially phage 186, with the aim to identify proteins with novel functions, such as developing new antimicrobials or molecular biology tools. This project aims to investigate the role of two uncharacterised genes, CP80 and CP84, from bacteriophage 186. Initial studies indicated that CP80 showed homology to the gene regulator DksA, a transcription factor that requires cofactors, such as ppGpp and RNA polymerase, to control expression of a range of genes. CP84 showed homology to PAPS reductase, an enzyme important for catalysing the transfer of electrons for sulphur reduction during cysteine biosynthesis. In order to confirm the activities of these genes, the open reading frames of CP80 and CP84 were cloned into pBAD30 and pBADHis/A, respectively, and used to study the function and phenotype of the CP80 and CP84 proteins. Coomassie-stained SDS- PAGE was unable to confirm successful expression of CP80, with expression of CP84 confirmed by Western immunoblot. The mutant E. coli DnaK∆ strain showed growth with filamentation. Moreover, a previous study shows that E. coli with with deleted PAPS reductase (CysH) does not grown in M9 + 0.2% glucose media. In this study, CP80 was co- expressed in the DnaK∆ E. coli strain; however, no suppression of filamentation was observed. Moreover, CP84 was co-expressed in E. coli ∆cysH, but this did not restore growth. Further work is required to confirm the functions of these proteins and how they contribute to the life cycle of bacteriophage 186.