Purification and characterization of repressor of temperate S-aureus phage phi 11(M-T-2007)

Abstract

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage 11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-Cl) by affinity chromatography. A similar to 19 kDa protein copurified with intact His-Cl (similar to 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At similar to 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi 11 cl-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O-L and O-R, respectively. Equilibrium binding studies indicate that His-Cl binds to OR with a little more strongly than OL and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O-L and O-R harbor a nearly identical inverted repeat and studies show that 11 repressor binds to each repeat efficiently. Additional analyses indicate that 11 repressor, like, repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi 11 CI even nearly resembles to that of lambda phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi 11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.