Many bacteria use a cell-cell communication system called quorum sensing to coordinate population density-dependent changes in behavior. Quorum sensing involves production of and response to diffusible or secreted signals, which can vary substantially across different types of bacteria. In many species, quorum sensing modulates virulence functions and is important for pathogenesis. Over the past half-century, there has been a significant accumulation of knowledge of the molecular mechanisms, signal structures, gene regulons, and behavioral responses associated with quorum-sensing systems in diverse bacteria. More recent studies have focused on understanding quorum sensing in the context of bacterial sociality. Studies of the role of quorum sensing in cooperative and competitive microbial interactions have revealed how quorum sensing coordinates interactions both within a species and between species. Such studies of quorum sensing as a social behavior have relied on the development of “synthetic ecological” models that use nonclonal bacterial populations. In this review, we discuss some of these models and recent advances in understanding how microbes might interact with one another using quorum sensing. The knowledge gained from these lines of investigation has the potential to guide studies of microbial sociality in natural settings and the design of new medicines and therapies to treat bacterial infections.

Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Δeep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.

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...

The genome of Burkholderia thailandensis codes for several LuxR-LuxI quorum-sensing systems. We used B. thailandensis quorum-sensing deletion mutants and recombinant Escherichia coli to determine the nature of the signals produced by one of the systems, BtaR2-BtaI2, and to show that this system controls genes required for the synthesis of an antibiotic. BtaI2 is an acyl-homoserine lactone (acyl-HSL) synthase that produces two hydroxylated acyl-HSLs, N-3-hydroxy-decanoyl-HSL (3OHC 10 -HSL) and N-3-hydroxyoctanoyl-HSL (3OHC 8 -HSL). The btaI2 gene is positively regulated by BtaR2 in response to either 3OHC 10 -HSL or 3OHC 8 -HSL. The btaR2-btaI2 genes are located within clusters of genes with annotations that suggest they are involved in the synthesis of polyketide or peptide antibiotics. Stationary-phase cultures of wild-type B. thailandensis, but not a btaR2 mutant or a strain deficient in acyl-HSL synthesis, produced an antibiotic effective against gram-positive bacteria. Two of the putative antibiotic synthesis gene clusters require BtaR2 and either 3OHC 10 -HSL or 3OHC 8 -HSL for activation. This represents another example where antibiotic synthesis is controlled by quorum sensing, and it has implications for the evolutionary divergence of B. thailandensis and its close relatives Burkholderia pseudomallei and Burkholderia mallei. Burkholderia thailandensis, Burkholderia pseudomallei, andBurkholderia mallei are closely related betaproteobacteria. These three species show extensive genome sequence identity (37, 72). B. mallei is an obligate mammalian pathogen that infects solipeds (horses, mules, and donkeys) and can also cause human infections (66, 69). B. pseudomallei is an emerging human pathogen found in soil and stagnant water, and it infects rice farmers in Southeast Asia and people of Northern Australia (8,70). Once thought to be a nonpathogenic saprophyte endemic to the water and soil environments of central and northeastern Thailand (4), B. thailandensis has recently been found in the central United States and Australia (25,34). Sufficiently high doses of B. thailandensis can cause mammalian infections (4,11,56).We have been interested in acyl-homoserine lactone (acyl-HSL) quorum sensing among the B. mallei-B. pseudomallei-B. thailandensis group because quorum sensing has been implicated in the virulence of B. mallei and B. pseudomallei and because each member of this group has multiple quorumsensing systems (60-62). Quorum sensing involves production of and response to a self-produced extracellular signal that allows individuals to monitor the group population density (3,21,67). Proteobacteria often use acyl-HSL signals for quorum sensing. The signals are produced by members of the LuxI family of acyl-HSL synthases. The proteins that respond to acyl-HSLs are members of the LuxR family of transcription factors (22). Dozens of species possess acyl-HSL quorum-sensing systems. The genes coding for LuxR and LuxI homologs are often adjacent on a chromosome and are considered cognate pairs. The nature of the...

Acyl-homoserine lactone (acyl-HSL) quorum-sensing signaling is common to many Proteobacteria. AcylHSLs are synthesized by the LuxI family of synthases, and the signal response is mediated by members of the LuxR family of transcriptional regulators. Burkholderia thailandensis is a member of a closely related cluster of three species, including the animal pathogens Burkholderia mallei and Burkholderia pseudomallei. Members of this group have similar luxI and luxR homologs, and these genes contribute to B. pseudomallei and B. mallei virulence. B. thailandensis possesses three pairs of luxI-luxR homologs. One of these pairs, BtaI2-BtaR2, has been shown to produce and respond to 3OHC 10 -HSL and to control the synthesis of an antibiotic. By using a markerless-exhange method, we constructed an assortment of B. thailandensis quorum-sensing mutants, and we used these mutants to show that BtaI1 is responsible for C 8 -HSL production and BtaI3 is responsible for 3OHC 8 -HSL production. We also show that a strain incapable of acyl-HSL production is capable of growth on the same assortment of carbon and nitrogen sources as the wild type. Furthermore, this mutant shows no loss of virulence compared to the wild type in mice. However, the wild type self-aggregates in minimal medium, whereas the quorum-sensing mutant does not. The wild-type aggregation phenotype is recovered by addition of the BtaI1-R1 HSL signal C 8 -HSL. We propose that the key function of the BtaR1-BtaI1 quorum-sensing system is to cause cells to gather into aggregates once a sufficient population has been established.