In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS.
Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length SaeR could be restored by sensor kinase SaeS-induced phosphorylation. Phosphorylated SaeR is more resistant to digestion by trypsin, suggesting conformational changes. DNase I footprinting assays revealed that the SaeR protection region in the P1 promoter contains a direct repeat sequence (GTTAAN(6)GTTAA [where N is any nucleotide]). This sequence is critical to the binding of phosphorylated SaeR. Mutational changes in the repeat sequence greatly reduced both the in vitro binding of SaeR and the in vivo function of the P1 promoter. From these results, we concluded that SaeR recognizes the direct repeat sequence as a binding site and that binding requires phosphorylation by SaeS.
Sun
Fei
F
Department of Chemistry, University of Chicago, Chicago, Illinois, USA.
Li
Chunling
C
Jeong
Dowon
D
Sohn
Changmo
C
He
Chuan
C
Bae
Taeok
T
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I; D024202:Electrophoretic Mobility Shift Assay; D008954:Models, Biological; D008957:Models, Genetic; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011494:Protein Kinases / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
D001426:Bacterial Proteins; C433805:SaeR protein, Staphylococcus aureus; D014157:Transcription Factors; D011494:Protein Kinases; C527351:SaeS protein, Staphylococcus aureus; D003850:Deoxyribonuclease I