Eunhye Goo

Bacterial quorum sensing, cooperativity, and anticipation of stationary-phase stress

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.

Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase

Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In Gram-negative bacteria, QS is often mediated by N-acyl-l-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the Gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-l-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5′-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-l-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

Proteomic analysis of quorum sensing-dependent proteins in Burkholderia glumae.

Burkholderia glumae, the causal agent of bacterial rice grain rot, utilizes quorum sensing (QS) systems that rely on N-octanoyl homoserine lactone (synthesized by TofI) and its cognate receptor TofR to activate toxoflavin biosynthesis genes and an IclR-type transcriptional regulator gene, qsmR. Since QS is essential for B. glumae pathogenicity, we analyzed the QS-dependent proteome by 2-dimensional gel electrophoresis. A total of 79 proteins, including previously known QS-dependent proteins, were differentially expressed between the wild-type BGR1 and the tofI mutant BGS2 strains. Among this set, 59 proteins were found in the extracellular fraction, and 20 were cytoplasmic. Thirty-four proteins, including lipase and proteases, were secreted through the type II secretion system (T2SS). Real-time RT-PCR analysis showed that the corresponding genes of the 49 extracellular and 13 intracellular proteins are regulated by QS at the transcriptional level. The T2SS, encoded by 12 general secretion pathway (gsp) genes with 3 independent transcriptional units, was controlled by QS. beta-Glucuronidase activity analysis of gsp::Tn3-gusA gene fusions and electrophoretic mobility shift assays revealed that the expression of gsp genes is directly regulated by QsmR. T2SS-defective mutants exhibited reduced virulence, indicating that the T2SS-dependent extracellular proteins play important roles in B. glumae virulence.

Control of bacterial metabolism by quorum sensing

Bacterial quorum sensing (QS)-dependent gene expression is a dynamic response to cell density. Bacteria produce costly public goods for the benefit of the population as a whole. As an example, QS rewires cellular metabolism to produce oxalate (a public good) to enable survival during the stationary phase in Burkholderia glumae, Burkholderia thailandensis, and Burkholderia pseudomallei. Recent reports showed that QS serves as a metabolic brake to maintain homeostatic primary metabolism in B. glumae and readjusts the central metabolism of Pseudomonas aeruginosa. In this review, we emphasize the dynamics and complexity of the control of gene expression by QS and discuss the metabolic costs and possible metabolic options to sustain cooperativity. We then focus on how QS influences bacterial central metabolism.

Complete Genome Sequence of Burkholderia gladioli BSR3

We report the complete genome sequence of Burkholderia gladioli BSR3, isolated from a diseased rice sheath in South Korea.

The Quorum Sensing-Dependent Gene katG of Burkholderia glumae Is Important for Protection from Visible Light

Quorum sensing (QS) plays important roles in the pathogenicity of Burkholderia glumae, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in B. glumae. The QS-defective mutant of B. glumae exhibited less catalase activity than wild-type B. glumae. A beta-glucuronidase assay of a katG::Tn3-gusA78 reporter fusion protein revealed that katG expression is under the control of QS. Furthermore, katG expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates katG expression. The katG mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 micromol(-1) m(-2), the survival rate of the katG mutant was 10(5)-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the katG mutant.

Bacterial quorum sensing and metabolic slowing in a cooperative population

Significance Quorum sensing (QS) is a coordinated gene-regulation system that controls bacterial social behaviors, such as virulence, motility, biofilm formation, and toxin production, in response to cell density. Acyl-homoserine lactone-mediated QS coordinates cooperativity between individual cells of many Proteobacteria species. QS may also control nutrient acquisition and help maintain the homeostatic primary metabolism of individuals in a cooperative population. Here, we show that QS restricts glucose uptake and slows primary metabolism of individual cells in crowded conditions. QS-deficient cells experienced serious physiological challenges under similar conditions, indicating that QS functions as a means to ensure efficient energy and resource utilization of individuals in crowded environments. Acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) controls the production of numerous intra- and extracellular products across many species of Proteobacteria. Although these cooperative activities are often costly at an individual level, they provide significant benefits to the group. Other potential roles for QS include the restriction of nutrient acquisition and maintenance of metabolic homeostasis of individual cells in a crowded but cooperative population. Under crowded conditions, QS may function to modulate and coordinate nutrient utilization and the homeostatic primary metabolism of individual cells. Here, we show that QS down-regulates glucose uptake, substrate level and oxidative phosphorylation, and de novo nucleotide biosynthesis via the activity of the QS-dependent transcriptional regulator QsmR (quorum sensing master regulator R) in the rice pathogen Burkholderia glumae. Systematic analysis of glucose uptake and core primary metabolite levels showed that QS deficiency perturbed nutrient acquisition, and energy and nucleotide metabolism, of individuals within the group. The QS mutants grew more rapidly than the wild type at the early exponential stage and outcompeted wild-type cells in coculture. Metabolic slowing of individuals in a QS-dependent manner indicates that QS acts as a metabolic brake on individuals when cells begin to mass, implying a mechanism by which AHL-mediated QS might have evolved to ensure homeostasis of the primary metabolism of individuals under crowded conditions.

Regulation of Universal Stress Protein Genes by Quorum Sensing and RpoS in Burkholderia glumae

Burkholderia glumae possesses a quorum-sensing (QS) system mediated by N-octanoyl-homoserine lactone (C(8)-HSL) and its cognate receptor TofR. TofR/C(8)-HSL regulates the expression of a transcriptional regulator, qsmR. We identified one of the universal stress proteins (Usps), Usp2, from a genome-wide analysis of QS-dependent proteomes of B. glumae. In the whole genome of B. glumae BGR1, 11 usp genes (usp1 to usp11) were identified. Among the stress conditions tested, usp1 and usp2 mutants died 1 h after heat shock stress, whereas the other usp mutants and the wild-type strain survived for more than 3 h at 45°C. The expressions of all usp genes were positively regulated by QS, directly by QsmR. In addition, the expressions of usp1 and usp2 were dependent on RpoS in the stationary phase, as confirmed by the direct binding of RpoS-RNA holoenzyme to the promoter regions of the usp1 and usp2 genes. The expression of usp1 was upregulated upon a temperature shift from 37°C to either 28°C or 45°C, whereas the expression of usp2 was independent of temperature stress. This indicates that the regulation of usp1 and usp2 expression is different from what is known about Escherichia coli. Compared to the diverse roles of Usps in E. coli, Usps in B. glumae are dedicated to heat shock stress.

Quorum Sensing Controls Flagellar Morphogenesis in Burkholderia glumae

Burkholderia glumae is a motile plant pathogenic bacterium that has multiple polar flagella and one LuxR/LuxI-type quorum sensing (QS) system, TofR/TofI. A QS-dependent transcriptional regulator, QsmR, activates flagellar master regulator flhDC genes. FlhDC subsequently activates flagellar gene expression in B. glumae at 37°C. Here, we confirm that the interplay between QS and temperature is critical for normal polar flagellar morphogenesis in B. glumae. In the wild-type bacterium, flagellar gene expression and flagellar number were greater at 28°C compared to 37°C. The QS-dependent flhC gene was significantly expressed at 28°C in two QS-defective (tofI::Ω and qsmR::Ω) mutants. Thus, flagella were present in both tofI::Ω and qsmR::Ω mutants at 28°C, but were absent at 37°C. Most tofI::Ω and qsmR::Ω mutant cells possessed polar or nonpolar flagella at 28°C. Nonpolarly flagellated cells processing flagella around cell surface of both tofI::Ω and qsmR::Ω mutants exhibited tumbling and spinning movements. The flhF gene encoding GTPase involved in regulating the correct placement of flagella in other bacteria was expressed in QS mutants in a FlhDC-dependent manner at 28°C. However, FlhF was mislocalized in QS mutants, and was associated with nonpolar flagellar formation in QS mutants at 28°C. These results indicate that QS-independent expression of flagellar genes at 28°C allows flagellar biogenesis, but is not sufficient for normal polar flagellar morphogenesis in B. glumae. Our findings demonstrate that QS functions together with temperature to control flagellar morphogenesis in B. glumae.

A novel light-dependent selection marker system in plants.

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.