Identification and Preliminary Characterization of a Novel Single-Stranded DNA Binding Protein of Staphylococcus aureus Phage Phi11 Expressed in Escherichia coli.

Bacteriophage Phi11 harbors a gene, gp13, encoding the putative SSB protein (GenBank accession no. NC_004615.1). SSB proteins bind to and protect the single-stranded DNA molecules from nuclease digestion and are essential for the growth and metabolic activities of the organisms encoding them. In this investigation, we have carried out the cloning, recombinant expression, and purification of rGp13 for the first time in Escherichia coli. EMSA data indicated that the purified recombinant Gp13 protein was capable of binding to single-stranded DNA. The protein exhibited maximum binding activity at 32 °C. Furthermore, our bioinformatic analysis has revealed that Gp13 consists of an OB-fold, a characteristic of SSB proteins. However, the arrangement of the OB-fold is unique, being located in the C-terminal domain of Gp13. Despite the importance of SSB proteins in various metabolic processes as well as in various types of PCR, there are no reports on the purification and characterization of SSB proteins from staphylococcal bacteriophages. We expect that the purification and characterization of recombinant Gp13 will help us gain a better insight into its biological activity and make it available in large quantities for molecular biology work.

Studies on the gene regulation involved in the lytic-lysogenic switch in Staphylococcus aureus temperate bacteriophage Phi11.

Antirepressor proteins of bacteriophages are chiefly involved in interfering with the function of the repressor protein and forcing the bacteriophage to adopt the lytic cycle. The genome of Staphylococcus aureus phage, Phi11 has already been sequenced; from the genome sequence, we amplified gp07 gene and analysed its involvement in the developmental pathway of Phi11. Our results indicate that Gp07 functions as a novel antirepressor and regulates the developmental pathway of Phi11 by enhancing the binding of the Cro repressor protein to its cognate operator. We also report our finding that the CI repressor protein of Phi11 binds to the putative operator of Gp07 and regulates its expression. We further report that S.aureus transcriptional repressor LexA and coprotease RecA play a crucial role in the lytic-lysogenic switching in Phi11. We also identified that the N-terminal domain (Bro-N) of Gp07 is actually responsible for enhancing the binding of Cro repressor to its cognate operator. Our results suggest that Phi11 prophage induction is different from other bacteriophages. This study furnishes a first-hand report regarding the regulation involved in the developmental pathway of Phi11.