Characterization of Staphylococcus aureus SarA binding sites

The staphylococcal accessory regulator locus (sarA) 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 (EMSAs) were used to identify a 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 a SELEX (systematic evolution of ligands by exponential enrichment) procedure 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 Staphylococcus 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. By 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 7-bp motif was within the putative SarA binding site previously associated with each gene.     

Characterization of the SarA virulence gene regulator of Staphylococcus aureus

Staphylococcus aureus is a potent human pathogen that expresses a large number of virulence factors in a temporally regulated fashion. Two pleiotropically acting regulatory loci were identified in previous mutational studies. The agr locus comprises two operons that express a quorum-sensing system from the P2 promoter and a regulatory RNA molecule from the P3 promoter. The sar locus encodes a DNA-binding protein that activates the expression of both agr operons. We have cloned the sarA gene, expressed SarA in Escherichia coli and purified the recombinant protein to apparent homogeneity. The purified protein was found to be dimeric in the presence and absence of DNA and to consist mostly of alpha-helices. DNase I footprinting of SarA on the putative regulatory region cis to the agr promoters revealed three high-affinity binding sites composed of two half-sites each. Quantitative electrophoretic mobility shift assays (EMSAs) were used to derive equilibrium binding constants (KD) for the interaction of SarA with these binding sites. An unusual ladder banding pattern was observed in EMSA with a large DNA fragment including all three binding sites. Our data indicate that SarA regulation of the agr operons involves binding to multiple half-sites and may involve other sites located downstream of the promoters.     

Staphylococcus aureus accessory regulators: expression within biofilms and effect on adhesion.

Many of the genes encoding the virulence factors for Staphylococcus aureus are controlled by the accessory gene regulator (agr) and staphylococcal accessory regulator (sar). This regulation may be affected by the environment in which the organisms are grown. In the majority of ecosystems, bacteria grow attached to surfaces and form biofilms. We used S. aureus strains containing mutations inactivating agr and sar to determine whether the presence of these genes influences the attachment of the bacterium to a surface. We also used strains harbouring reporter constructs of the agr and sar operons to determine their expression in biofilms. The attachment study results showed that the sarA mutant strain adhered better to glass than did the agrA mutant or the wild type. There was an increased adherence to fibronectin-coated glass for all three strains compared to glass. Thus, these adhesion studies demonstrate that agr and sar have pleiotrophic effects on the surface expression of molecules responsible for binding to different substrata. In the biofilms higher numbers of bacteria and the greatest expression were observed at the base, but there were no observable differences between the reporter constructs. Expression of the agr and sar reporter fusions was significantly higher in the deepest layers of the biofilms where the greatest numbers of bacteria were also observed, perhaps as one might expect for genes that are regulated in a cell density dependent fashion.     

Cloning and sequencing of sarA of Staphylococcus aureus, a gene required for the expression of agr.

To evaluate the effect of a sar mutation on the agr locus, Northern (RNA) blotting was performed to determine the levels of RNAIII, the agr regulatory molecule, in two isogenic pairs of Staphylococcus aureus strains. Our results demonstrated that RNAIII was either significantly diminished or absent in both sar mutants compared with the parents. The RNAIII level was partially restored in sar mutants complemented with an intact sar gene (designated sarA). Additionally, we were able to complement selected sar phenotypes with a plasmid carrying RNAIII (pRN6735). These studies suggest that the sarA gene is necessary for the optimal expression of agr. The sarA gene of strain RN450 was subsequently cloned and sequenced. Sequence analysis revealed an open reading frame of 372 bp with a predicted molecular size of 14,718 Da and a deduced pI of 8.52. The deduced protein sequence has a predominance of charged residues (33%) and shares sequence similarity with the virF gene of Shigella flexneri.     

Evolutionary genetics of the accessory gene regulator (agr) locus in Staphylococcus aureus

The accessory gene regulator (agr) locus influences the expression of many virulence genes in the human pathogen Staphylococcus aureus. Four allelic groups of agr, which generally inhibit the regulatory activity of each other, have been identified within the species. Interference in virulence gene expression caused by different agr groups has been suggested to be a mechanism for isolating bacterial populations and a fundamental basis for subdividing the species. To test the hypothesis that the species is phylogenetically structured according to agr groups, we mapped agr groups onto a clone phylogeny inferred from partial sequences of 14 genes from 27 genetically diverse strains. Shimodaira-Hasegawa and parametric bootstrap tests rejected the hypotheses that the species is subdivided into three or five monophyletic agr groups but failed to reject the hypothesis that the species is subdivided into two groups that each consist of multiple clonal complexes and multiple agr groups. Additional evidence for agr recombination is found from clustered polymorphisms in complete agr sequences. However, agr recombination has not occurred frequently or randomly through time, because the topology and branch lengths of the clone phylogeny are reflected within each agr group. To account for these observations, we propose a new evolutionary model that involves a genetically polymorphic ancestral population of S. aureus that horizontally transferred agr groups between two subspecies groups near the time that these subspecies groups diverged.