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. 2010 Apr;192(8):2111-27.
doi: 10.1128/JB.01524-09. Epub 2010 Feb 19.

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

Fei Sun  1 , Chunling Li, Dowon Jeong, Changmo Sohn, Chuan He, Taeok Bae
Affiliations

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

Fei Sun et al. J Bacteriol. 2010 Apr.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
The sae locus of S. aureus. Alternative names of the P1 and P3 promoters are in parentheses. The saeQ ORF contains a smaller ORF (shown in gray). The transcript species are indicated under the ORF map along with their sizes. This map is adapted from a report by Geiger et al. (17).
FIG. 2.
FIG. 2.
DNA binding activities of unphosphorylated SaeR (A), the C-terminal effector domain of SaeR (B), and phosphorylated SaeR (C) to the P1 promoter region. In a typical assay, 2 ng of a γ-32P-end-labeled P1 promoter fragment was incubated with the indicated concentration of protein in the presence of 3 μg/ml salmon sperm DNA at room temperature for 15 min. Free DNA is indicated by a white arrowhead, and bound DNA is indicated by a black arrowhead. SaeR, unphosphorylated SaeR; SaeRC, the C-terminal effector domain of SaeR; P-SaeR, phosphorylated SaeR; + cold DNA, addition of a 10-fold excess of unlabeled DNA.
FIG. 3.
FIG. 3.
SaeR phosphorylation by the cytoplasmic domain of SaeS and strain Newman cell lysates. (A) SaeSC (2 μM) or cell lysates from strains Newman (NM), ΦΝΞ-9725 (NMsaeS-), and USA300-0114 (USA) were mixed with SaeR (10 μM) in the presence of [γ-32P]ATP. All of the reactions were performed for 10 min at room temperature. (B) SaeSC (3 μM) was phosphorylated with [γ-32P]ATP, and then SaeR (9 μM) was added. P-SaeSC, phosphorylated SaeSC; P-SaeR, phosphorylated SaeR. The three additional protein bands phosphorylated in the strain Newman cell lysates are indicated by white arrowheads.
FIG. 4.
FIG. 4.
Limited trypsin digestion analysis of SaeR and P-SaeR. (A) SaeR (50 μM) was phosphorylated by SaeSC (1 μM) in the presence of 1 mM ATP. An equal amount of SaeSC (1 μM) was added to unphosphorylated SaeR (50 μM) but without ATP. The proteins were mixed with 0.2 μg/μl trypsin and incubated at 37°C. Aliquots of 10 μl were removed from the reaction mixtures at different time intervals and quenched by the addition of 10 μl 2× SDS loading buffer, followed by heating at 90°C for 5 min. Samples were analyzed by 14% SDS-PAGE and Coomassie straining. Fr, a 10-kDa SaeR fragment resistant to trypsin digestion. (B) The staining results were quantified by Quantity One (Bio-Rad). The error bars represent the standard deviations calculated from two independent experiments.
FIG. 5.
FIG. 5.
Identification of SaeR binding sequences. (A) DNase I footprinting analysis of the P1 promoter with SaeRC and P-SaeR. Sequencing of the DNA probe was carried out by the Maxam-Gilbert method. The nucleotide positions are indicated to the left of the footprinting image. The regions protected by SaeRC are in bold brackets, and the regions protected by P-SaeR are in plain brackets. Values represent distances from the transcription start site, which was set to +1. (B) P1 promoter sequence with a summary of the DNase I footprinting assay results. The −10 and −35 promoter regions are indicated by solid lines above the sequence. SaeRC-protected regions are in solid boxes, and the P-SaeR-protected regions are in dotted-line boxes. The direct repeat sequences are in boldface. The transcription start site is indicated by a right-angled arrow; and the corresponding nucleotide is in boldface italics.
FIG. 6.
FIG. 6.
DNA binding of P-SaeR to various staphylococcal promoters. (A) Schematic map of the hla promoter. The perfect repeat sequence is represented by a black box, and the imperfect repeat sequence is represented by a gray box. The sequences of the repeats are also shown above the map, where N represents any nucleotide. Values represent distances from the ATG start codon, which is shown to the right. The transcription start site (G) is shown by a right-angled arrow. The two DNA probes used for EMSA are shown as solid lines with the names under them. (B) DNA binding of P-SaeR to two different regions of the hla promoter. The concentration of P-SaeR is indicated at the top. Unbound free DNA probe is indicated by a white arrowhead. I and II represent the hla promoter regions shown in panel A. (C) The direct repeat sequences in the promoters of emp, map/eap, vwb, and arlR. The transcription sites are indicated by a boldfaced letter and a right-angled arrow. The −10 and −35 regions of the promoters are indicated by solid boxes. The direct repeat sequences are in boldface and shaded gray. (D) DNA binding of P-SaeR to the emp, map/eap, vwb, and arlR promoters. Protein concentrations are indicated at the top. For clarity, only unbound free DNA is indicated.
FIG. 7.
FIG. 7.
Effects of P1 promoter mutations on binding to P-SaeR and promoter function. (A) Summary of the mutations in the P1 promoter. Each mutation is indicated by an arrow with the name under the sequence. The −10 and −35 regions are in a solid box. The direct repeat sequence is in boldface and shaded gray. +1, transcription start site; Δ, a deletion mutation. (B) Binding of the mutant promoters to P-SaeR. Increasing amounts of P-SaeR (0, 1, 2, 4, and 8 μM) were used for the binding assay. The mutated nucleotides of the direct repeat and mutated promoter regions are shown at the top. Unbound free DNA probe is indicated by a white arrowhead. Δ, a deletion mutation. (C) Activities of the mutated P1 promoters represented by LacZ expression. The values presented are relative LacZ expression, where the LacZ expression from the wild-type promoter was set to 100%. The lacZ assays were repeated two or three times with similar results. Error bars represent standard deviations. WT, wild type.
FIG. 8.
FIG. 8.
Model of P1 promoter activation by the SaeRS system. RNAP, RNA polymerase.
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