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Related Experiment Videos

Characterization of Staphylococcus aureus SarA binding sites.

Kristen M Sterba1, Samuel G Mackintosh, Jon S Blevins

  • 1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.

Journal of Bacteriology
|July 18, 2003
PubMed
Summary

Researchers identified the specific DNA sequence recognized by the staphylococcal accessory regulator (SarA) protein. This finding clarifies how SarA controls gene expression in Staphylococcus aureus, advancing our understanding of bacterial virulence.

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • The staphylococcal accessory regulator (SarA) protein influences over 100 genes in Staphylococcus aureus.
  • The precise mechanism of SarA's gene regulation, specifically its DNA binding site characteristics, remains largely undefined.
  • Understanding SarA binding is crucial for elucidating Staphylococcus aureus pathogenesis.

Purpose of the Study:

  • To definitively identify the DNA binding site characteristics of the SarA protein.
  • To investigate the direct interaction between SarA and cis-regulatory elements of its target genes.
  • To establish a consensus binding motif for SarA.

Main Methods:

  • Electrophoretic mobility shift assays (EMSAs) were employed to study SarA-DNA interactions.

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  • Systematic evolution of ligands by exponential enrichment (SELEX) was performed using purified recombinant SarA.
  • DNA fragments selected via SELEX were sequenced, cloned, and analyzed by EMSA.

    • Main Results:

    • Multiple high-affinity SarA binding sites were detected upstream of the cna gene.
    • SELEX identified AT-rich DNA fragments that bind SarA with high affinity.
    • A 7-bp consensus binding motif (ATTTTAT) was identified in 46 of 56 sequenced clones and found upstream of several key S. aureus genes.

    Conclusions:

    • The study defines a 7-bp consensus DNA motif (ATTTTAT) as the high-affinity binding site for SarA.
    • This identified motif is conserved upstream of numerous SarA-regulated genes, including cna, spa, fnbA, sspA, agr, and hla.
    • The findings provide a molecular basis for SarA-mediated gene regulation in Staphylococcus aureus.