Molecular interactions between two global regulators, sar and agr, in Staphylococcus aureus

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Virulence determinants in Staphylococcus aureus are regulated by loci like agr and sar.
• The SarA protein is known to be essential for optimal transcription of RNAII and RNAIII within the agr locus.

Purpose of the Study:

• To elucidate the specific molecular interaction between the SarA protein and the agr regulatory locus.
• To identify the DNA binding site and essential domains of SarA for agr promoter activity.

Main Methods:

• Overexpression and purification of SarA as a glutathione S-transferase (GST) fusion protein.
• Gel shift assays and supershift assays using purified SarA and monoclonal antibodies.
• DNase I footprinting to map the SarA binding site on the agr promoter.
• Serial in-frame deletion analysis of the sarA gene and Northern blot analysis.

Main Results:

• Purified SarA specifically binds to the P2, P3, and combined P2-P3 promoter fragments of the agr locus.
• SarA was confirmed to be part of the DNA-protein complex via supershift assays.
• DNase I footprinting identified a 29-bp binding site on the agr promoter between the P2 and P3 regions.
• Residues 16-124 of SarA, excluding the N-terminal 15 residues, were found to be essential for DNA binding and transcriptional activation.

Conclusions:

• SarA is a DNA-binding protein that specifically targets the interpromoter region of the agr P2 and P3 promoters.
• This specific binding by SarA is critical for the activation of RNAII and RNAIII transcription, thereby controlling virulence in Staphylococcus aureus.