Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2003
Publication Date: 8/10/2003
Citation: Sterba, K.M., Mackintosh, S.G., Blevins, J.S., Hurlburt, B.K., Smeltzer, M.S. 2003. Characterization of staphylococcus aureus sara binding sites. Journal of Bacteriology. 185(15):4410-4417. Interpretive Summary: The SarA protein of Staphylococcus aureus binds DNA and regulates the expression of a large number of virulence factors. There is disagreement in the literature as to the definition of the binding site requirements for SarA which could be important in the development of DNA-based anti-staphylococcal therapies. In this work we have used the SELEX (systematic evolution of ligands by exponential enrichment) strategy to DNA molecules that are bound by SarA with high affinity. These DNAs, as well as DNA fragments containing putative regulatory sequences from genes controlled by SarA were tested for binding affinity. As a result of this work we propose different requirements for defining a SarA binding site.
Technical Abstract: The staphylococcal accessory regulator (sarA) locus encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSA) were used to identify SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using SELEX (systematic evolution of ligands by exponential enrichment) and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA-binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes including cna, fnbA and sspA. The composition of SarA-selected DNAs was AT-rich, which is consistent with the nucleotide composition of the S. aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. Using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla and cna. With the exception of cna, which has not been previously examined, this motif was within the putative SarA binding site previously associated with each gene.