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.

Acyl-homoserine lactone-mediated quorum sensing (QS) regulates diverse activities in many species of Proteobacteria. QS-controlled genes commonly code for production of secreted or excreted public goods. The acyl-homoserine lactones are synthesized by members of the LuxI signal synthase family and are detected by cognate members of the LuxR family of transcriptional regulators. QS affords a means of population density-dependent gene regulation. Control of public goods via QS provides a fitness benefit. Another potential role for QS is to anticipate overcrowding. As population density increases and stationary phase approaches, QS might induce functions important for existence in stationary phase. Here we provide evidence that in three related species of the genus Burkholderia QS allows individuals to anticipate and survive stationary-phase stress. Survival requires QS-dependent activation of cellular enzymes required for production of excreted oxalate, which serves to counteract ammonia-mediated alkaline toxicity during stationary phase. Our findings provide an example of QS serving as a means to anticipate stationary phase or life at the carrying capacity of a population by activating the expression of cytoplasmic enzymes, altering cellular metabolism, and producing a shared resource or public good, oxalate.Burkholderia carrying capacity | glumae | pseudomallei | thailandensis | cell death A cyl-homoserine lactone (AHL)-mediated quorum sensing (QS) regulates diverse activities, including bioluminescence, biofilm formation, motility, and virulence factor formation, in many Proteobacteria (1-3). AHLs are synthesized most typically by members of the LuxI family signal synthases and detected by members of the LuxR family of transcriptional regulators (1-3). A large body of work has characterized the molecular mechanisms of bacterial QS; demonstrating the population-wide benefits that drive QS-mediated cooperative behavior has proven difficult, however. Cooperative activities benefit individuals within a group (4, 5).QS-controlled genes commonly code for the production of extracellular public goods that can be shared by all members of the group regardless of which members produce them. These extracellular products are often important for nutrient acquisition, interspecies competition, or virulence (6-8). In Pseudomonas aeruginosa, QS control of secreted proteases provides fitness benefits, because the proteases are produced only when they can be used efficiently (9). Other potential roles of QS in bacteria have been proposed, including the hypothesis that QS enables bacteria to anticipate population carrying capacity in a given environment. Anticipation of stationary phase might allow individuals to modify their physiology in preparation for survival at population carrying capacity.Here we address the question of whether QS is involved in anticipation of stationary-phase stress in three closely related bacteria: the rice pathogen Burkholderia glumae, the opportunistic human pathogen Burkholderia pseudomallei, and the...

c Burkholderia thailandensis contains three acyl-homoserine lactone quorum sensing circuits and has two additional LuxR homologs. To identify B. thailandensis quorum sensing-controlled genes, we carried out transcriptome sequencing (RNA-seq) analyses of quorum sensing mutants and their parent. The analyses were grounded in the fact that we identified genes coding for factors shown previously to be regulated by quorum sensing among a larger set of quorum-controlled genes. We also found that genes coding for contact-dependent inhibition were induced by quorum sensing and confirmed that specific quorum sensing mutants had a contact-dependent inhibition defect. Additional quorum-controlled genes included those for the production of numerous secondary metabolites, an uncharacterized exopolysaccharide, and a predicted chitin-binding protein. This study provides insights into the roles of the three quorum sensing circuits in the saprophytic lifestyle of B. thailandensis, and it provides a foundation on which to build an understanding of the roles of quorum sensing in the biology of B. thailandensis and the closely related pathogenic Burkholderia pseudomallei and Burkholderia mallei.

Burkholderia thailandensis uses acyl-homoserine lactone-mediated quorum sensing systems to regulate hundreds of genes. Here we show that cell-cell contact-dependent type VI secretion (T6S) toxin-immunity systems are among those activated by quorum sensing in B. thailandensis. We also demonstrate that T6S is required to constrain proliferation of quorum sensing mutants in colony cocultures of a BtaR1 quorum-sensing signal receptor mutant and its parent. However, the BtaR1 mutant is not constrained by and outcompetes its parent in broth coculture, presumably because no cell contact occurs and there is a metabolic cost associated with quorum sensing gene activation. The increased fitness of the wild type over the BtaR1 mutant during agar surface growth is dependent on an intact T6SS-1 apparatus. Thus, quorum sensing activates B. thailandensis T6SS-1 growth inhibition and this control serves to police and constrain quorum-sensing mutants. This work defines a novel role for T6SSs in intraspecies mutant control.DOI: http://dx.doi.org/10.7554/eLife.14712.001