The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up‐regulated by agr whereas surface proteins are down‐regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr‐null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.

The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable, but can nevertheless be controlled and must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of thirty scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, May 16-18, 2011. From these discussions emerged a priority list of steps that need to be taken to resolve this global crisis.

The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and agr, sarA, and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up-or downregulated in an agrand/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by agr and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organism's pathogenesis also appeared to be regulated by each effector, suggesting that products of both the agr and the sarA locus are more-global transcription regulators than previously realized.Staphylococcus aureus is a major cause of human disease. The organism causes a variety of clinical manifestations, ranging from localized skin infections to severe sepsis, and is a leading cause of hospital-acquired infection (3). Despite advances in antibacterial chemotherapy, S. aureus strains have demonstrated resistance to all currently available antibiotics. Due in part to the immense clinical importance of this organism, an enormous amount of effort has been directed toward identifying the genes and regulatory mechanisms associated with S. aureus pathogenesis. Collectively, this work has demonstrated that the organism's pathogenesis can be attributed to its capacity to produce a variety of virulence factors (29).The identification of virulence factors and the regulatory networks that influence their expression has been facilitated by the observation(s) that many, if not most, virulence genes are expressed in laboratory cultures. While there is currently a substantial list of staphylococcal virulence factors, it is likely that this list is incomplete and is skewed by the limitations of the experiments used to identify them. Virulence factors that have already been identified generally include (i) bacterial surface proteins that are involved in processes such as adhesion and evasion of the host immune response and (ii) secreted exoproteins that degrade host tissue(s) and inactivate host defensive mechanisms (29).The genes encoding most virulence factors belong to an extensive regulon that is coordinately regulated in response to a variety of intra-and extracellular signals (1,5,21). Octapeptide signaling m...

We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71: [4206][4207][4208][4209][4210][4211] 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential-and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.

Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by B activity. While B was found to positively control 198 genes by a factor of >2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of B . Gene products that were found to be influenced by B are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by B were preceded by a nucleotide sequence that resembled the B consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by B activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the B of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.Transcription of DNA into RNA is catalyzed by RNA polymerase. In bacteria, one RNA polymerase generates nearly all cellular RNAs, including ribosomal, transfer, and mRNA. This enzyme consists of six subunits, ␣ 2 ␤␤Ј, with ␣ 2 ␤␤Ј forming the catalytically competent RNA polymerase core enzyme (E). The core is capable of elongation and termination of transcription, but it is unable to initiate transcription at specific promoter sequences. The subunit, which when bound to E forms the holoenzyme (E-), directs the multisubunit complex to specific promoter elements and allows efficient initiation of transcription (reviewed in references 5 and 6). Therefore, factors provide an elegant mechanism in eubacteria to allow simultaneous transcription of a variety of genetically unlinked genes, provided all of these genes share the same promoter specificities.In addition to the housekeeping sigma subunit, 70 or A , most bacteria produce one or more additional subunits, termed alternative factors, which direct the respective Ecomplex to distinct classes of promoters that contain alternative factor-specific sequences. Alternative factors have been shown in various bacteria to be of importance for survival under extreme conditions (7,14,23,31,38,44,49,60,68,73,78,79,80) and to influence virulence and pathogenicity (8,13,32,35,37,42,51,57,61,71,75,78,81).At least six alternative factors are produced by the enteric bacterium Escherichia coli (reviewed in reference 6). Genomic sequence analysis suggests that many alternative factors also exist in a number of other pathogenic species such as Treponema palladium (4 alternative f...