Characterizing the Effect of theStaphylococcus aureusVirulence Factor Regulator, SarA, on Log-Phase mRNA Half-Lives

Abstract

Bacterial pathogens regulate virulence factor expression at both the level of transcription initiation and mRNA processing/turnover. Within Staphylococcus aureus, virulence factor transcript synthesis is regulated by a number of two-component regulatory systems, the DNA binding protein SarA, and the SarA family of homologues. However, little is known about the factors that modulate mRNA stability or influence transcript degradation within the organism. As our entree to characterizing these processes, S. aureus GeneChips were used to simultaneously determine the mRNA half-lives of all transcripts produced during log-phase growth. It was found that the majority of log-phase transcripts (90%) have a short half-life (<5 min), whereas others are more stable, suggesting that cis-and/or trans-acting factors influence S. aureus mRNA stability. In support of this, it was found that two virulence factor transcripts, cna and spa, were stabilized in a sarA-dependent manner. These results were validated by complementation and real-time PCR and suggest that SarA may regulate target gene expression in a previously unrecognized manner by posttranscriptionally modulating mRNA turnover. Additionally, it was found that S. aureus produces a set of stable RNA molecules with no predicted open reading frame. Based on the importance of the S. aureus agr RNA molecule, RNAIII, and small stable RNA molecules within other pathogens, it is possible that these RNA molecules influence biological processes within the organism.

Characterization of the Staphylococcus aureus Heat Shock, Cold Shock, Stringent, and SOS Responses and Their Effects on Log-Phase mRNA Turnover

“…1) indicate that log-phase transcripts are degraded rapidly within mock-treated cells; 89.7% of all transcripts had half-lives of Յ5 min, 206 transcripts (9.2%) had intermediate half-lives (Ͼ5 min but Յ30 min), and 25 (1.1%) RNA species were stable (half-lives of Ͼ30 min). These results are in agreement with previous studies using custom-made S. aureus GeneChips (Saur2a), which found that the half-lives of 89.6% of all UAMS-1 log-phase transcripts were Ͻ5 min, those of 9.5% of transcripts were intermediate, and those of 0.7% of transcripts were stable (Ͼ60 min) (46). Induction of the SOS response did not appreciably affect global RNA turnover properties, whereas induction of the heat shock, cold shock, and stringent responses appeared to dramatically stabilize RNA species (Fig.…”

“…To do so, either log-phase UAMS-1 cells were mock treated or the corresponding stress response was induced (conditions described above). Rifampin was then added to inhibit de novo transcript synthesis, as previously described (46). Aliquots were removed at 0, 2.5, 5.0, 15, and 30 min post-transcriptional arrest, and cell viability and rifampin resistance were measured (see Materials and Methods).…”

“…Aliquots were removed at 0, 2.5, 5.0, 15, and 30 min post-transcriptional arrest, and cell viability and rifampin resistance were measured (see Materials and Methods). Total bacterial RNA was isolated, and the mRNA half-lives of transcripts produced in mock-treated, cold-shocked, heat-shocked, stringent response-induced, and SOS response-induced cells were determined using Affymetrix S. aureus GeneChips as previously described (46,49).…”

“…We have previously shown that logphase UAMS-1 cells produce a set of SSR (half-lives of Ͼ60 min) molecules that are not expected to code for protein products (46). Given the importance of the S. aureus agr-encoded RNAIII molecule and small noncoding RNAs within other pathogens, it is likely that many of these molecules play an important role(s) in S. aureus biological processes.…”

The SarA protein family of Staphylococcus aureus

“…DNA binding and profiling studies suggest that the SarA protein may regulate target genes by directly binding to target gene promoters or indirectly via downstream effects on regulons (e.g. binding to the agr promoter) or by stabilizing mRNA during log phase (Cheung et al, 2004;Roberts et al, 2006).…”