Purification and characterization of repressor of temperate S. aureus phage phi11.
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.
Das
Malabika
M
Department of Biochemistry, Bose Institute, P1/12-CIT Scheme VII M, Calcutta 700 054, India.
Ganguly
Tridib
T
Chattoraj
Partho
P
Chanda
Palas Kumar
PK
Bandhu
Amitava
A
Lee
Chia Yen
CY
Sau
Subrata
S
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*
D012097:Repressor Proteins; D014764:Viral Proteins