title: Purification and characterization of repressor of temperate S. aureus phage phi11.
abstract: To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-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-CI binds to O(R) with a little more strongly than O(L) 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 phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 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 phi11 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.
authors.affiliation: Department of Biochemistry, Bose Institute, P1/12-CIT Scheme VII M, Calcutta 700 054, India.
authors.forename: Malabika
authors.initials: M
authors.lastname: Das
authors.forename: Tridib
authors.initials: T
authors.lastname: Ganguly
authors.forename: Partho
authors.initials: P
authors.lastname: Chattoraj
authors.forename: Palas Kumar
authors.initials: PK
authors.lastname: Chanda
authors.forename: Amitava
authors.initials: A
authors.lastname: Bandhu
authors.forename: Chia Yen
authors.initials: CY
authors.lastname: Lee
authors.forename: Subrata
authors.initials: S
authors.lastname: Sau
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000235:genetics; D010802:Phylogeny; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D012097:Repressor Proteins; D014764:Viral Proteins