More than 200 direct CodY target genes in Staphylococcus aureus were identified by genome-wide analysis of in vitro DNA binding. This analysis, which was confirmed for some genes by DNase I footprinting assays, revealed that CodY is a direct regulator of numerous transcription units associated with amino acid biosynthesis, transport of macromolecules, and virulence. The virulence genes regulated by CodY fell into three groups. One group was dependent on the Agr system for its expression; these genes were indirectly regulated by CodY through its repression of the agr locus. A second group was regulated directly by CodY. The third group, which includes genes for alpha-toxin and capsule synthesis, was regulated by CodY in two ways, i.e., by direct repression and by repression of the agr locus. Since S. aureus CodY was activated in vitro by the branched chain amino acids and GTP, CodY appears to link changes in intracellular metabolite pools with the induction of numerous adaptive responses, including virulence.Bacterial survival depends upon the ability to sense and respond to environmental stresses, such as changes in temperature, pH, osmolarity, cell population density, and nutrient availability. Staphylococcus aureus has a well-characterized ability to survive when faced with suboptimal conditions, highlighted by the ability of S. aureus to persist in mammalian hosts both as a commensal and as a pathogen. Many regulators of S. aureus virulence gene expression have been characterized (6). With the exception of the stress-dependent activation of B and the link between CcpA (catabolite control protein A) and select virulence factor expression (46), however, the specific mechanisms of virulence regulation in response to changes in nutrient availability are largely unknown. The best-characterized regulator of S. aureus virulence in response to environmental changes is the Agr (accessory gene regulator) system. This system, encoded at the agr locus, includes a quorumsensing mechanism that activates a two-component system that controls synthesis of a regulatory RNA, RNAIII (for a review, see reference 33).CodY, a highly conserved regulatory protein of stationaryphase adaptation in low-GϩC Gram-positive bacteria, is emerging as a regulator of virulence in S. aureus (28,38,47) as well as in other Gram-positive pathogens (4,13,19,20,(28)(29)(30). First discovered in two nonpathogenic species, Bacillus subtilis and Lactococcus lactis, CodY senses nutrient availability by direct interaction with metabolite effectors. CodY homologs define a unique, winged helix-turn-helix-containing family of transcription factors. For B. subtilis CodY, as well as for CodY proteins from Clostridium difficile, Listeria monocytogenes, and Bacillus cereus, the effectors are GTP and the branched chain amino acids (BCAAs; isoleucine, leucine, and valine) (4,13,20,31,39,43). GTP and the BCAAs increase synergistically the affinity of CodY for its DNA target sites (17, 50). CodY proteins from L. lactis and Streptococcus pneumoniae, however, respond ...

CodY is a global regulatory protein that was first discovered in Bacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G؉C gram-positive bacteria, including Staphylococcus aureus. The introduction of a codY-null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (hla), the accessory gene regulator (agr) (RNAII and RNAIII/hld), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus.

MgrA has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance. To comprehensively identify the target genes regulated by mgrA, we employed a microarray method to analyze the transcription profiles of S. aureus Newman, its isogeneic mgrA mutant, and an MgrA-overproducing derivative. We compared genes that were differentially expressed at exponential or early stationary growth phases. Our results showed that MgrA affected an impressive number of genes, 175 of which were positively regulated and 180 of which were negatively regulated in an mgrA-specific manner. The target genes included all functional categories. The microarray results were validated by real-time reverse transcription-PCR quantitation of a set of selected genes from different functional categories. Our data also indicate that mgrA regulates virulence factors in a fashion analogous to that of the accessory gene regulatory locus (agr). Accordingly, exoproteins are upregulated and surface proteins are downregulated by the regulator, suggesting that mgrA may function in concert with agr. The fact that a large number of genes are regulated by mgrA implies that MgrA is a major global regulator in S. aureus.Staphylococcus aureus can cause a diverse range of diseases, from superficial skin infections to serious infections such as osteomyelitis, septic arthritis, pneumonia, infected implant failure, and toxic shock syndrome (23). This bacterium is the prominent cause of nosocomial infections. A large number of virulence factors including secreted proteins, cell wall-tethered proteins, and cytoplasmic and integrated membrane proteins are believed to be involved in the infection processes. These virulence factors are coordinately regulated by a network of regulatory genes, which can be grouped into two major classes, two-component systems (TCSs) and small transcription regulators. The sensors of the 16 TCSs found in the S. aureus genome are likely to be responsible for sensing various environmental cues and transmitting the information to the responders, which either alone or in conjunction with other small transcription regulators regulate "downstream" genes (1,7,31).Among the TCSs, the accessory gene regulator (agr) system is the best-characterized system. agr is a quorum-sensing system that is activated by responding to the accumulation of an autoinducing peptide during cell growth (reviewed in reference 31). The autoinducing peptide is processed from AgrD by AgrB (40), which is also believed to export the peptide. The signal is then sensed by AgrC and transmitted to responder AgrA, which binds to the agr promoters (18). Activation results in the upregulation of the agrDCBA operon and RNAIII, a small RNA effector molecule of the agr system. RNAIII activates the production of many exoproteins and represses several cell surface proteins at the transcriptional level. Interestingly, RNAIII has also been shown to regulate alpha-toxin and protein A production by an antisense mechanism at the translational leve...

The virulence determinants of Staphylococcus aureus are coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, named mgr, was linked by transposon Tn917 and mapped by three-factorial transduction crosses. The region containing the mgr locus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene, mgrA. Interestingly, mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream of mgrA that resulted in a four-to eightfold increase in mgrA transcription in strain CYL5614. Thus, these results indicate that mgrA is an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the mgr locus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helixturn-helix DNA-binding motif, with the Escherichia coli MarR family of transcriptional regulators. Northern slot blot analyses suggested that mgr affected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.Staphylococcus aureus is an important human pathogen responsible for a wide range of diseases. The pathogenicity of the organism is largely determined by its ability to coordinately produce a plethora of extracellular toxins, enzymes, and surface antigens under various environmental conditions. Recently, several regulatory loci which globally affect the expression of many of these virulence genes have been identified. Among these global regulators, agr and sarA have been studied most extensively.agr is a complex quorum-sensing regulatory system consisting of two divergent transcriptional units, P2 and P3. The P2 operon contains four genes, agrBDCA, of which the agrBD genes are involved in the production and export of an autoinducing peptide. As the cell density increases to a certain level, the accumulated peptide activates, through the two-component system encoded by agrCA, both the P2 and P3 promoters. The P3 operon encodes an RNA effector, RNAIII, which then regulates the target genes (reviewed in reference 21). RNAIII has been shown to control target gene expression largely at the transcriptional level; however, the mechanism is unknown. At the translational level, RNAIII has been shown to regulate alpha-toxin by an antisense mechanism (21).The sarA locus consists of three overlapping transcripts initiating from three different promoters but terminating at a common 3Ј end. All three transcripts contain the major open reading frame (ORF), sarA, within the overlapping region. The SarA protein has been shown to be required ...

Both Staphylococcus aureus and S. epidermidis are capable of forming biofilm on biomaterials. We used Tn917 mutagenesis to identify a gene, rbf, affecting biofilm formation in S. aureus NCTC8325-4. Sequencing revealed that Rbf contained a consensus region signature of the AraC/XylS family of regulators, suggesting that Rbf is a transcriptional regulator. Insertional duplication inactivation of the rbf gene confirmed that the gene was involved in biofilm formation on polystyrene and glass. Phenotypic analysis of the wild type and the mutant suggested that the rbf gene mediates the biofilm formation of S. aureus at the multicellular aggregation stage rather than at initial attachment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the mutation resulted in the loss of an ϳ190-kDa protein. Biofilm production by the mutant could be restored by complementation with a 2.5-kb DNA fragment containing the rbf gene. The rbf-specific mutation affected the induction of biofilm formation by glucose and a high concentration of NaCl but not by ethanol. The mutation did not affect the transcription of the ica genes previously shown to be required for biofilm formation. Taken together, our results suggest that the rbf gene is involved in the regulation of the multicellular aggregation step of S. aureus biofilm formation in response to glucose and salt and that this regulation may be mediated through the 190-kDa protein.