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Bonnie L. Bassler - One of the best experts on this subject based on the ideXlab platform.
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the vibrio harveyi master quorum sensing regulator luxr a tetr type protein is both an activator and a repressor dna recognition and binding specificity at target promoters
Molecular Microbiology, 2008Co-Authors: Audra J Pompeani, Bonnie L. Bassler, Joseph J Irgon, Michael F Berger, Martha L Bulyk, Ned S WingreenAbstract:Quorum sensing is the process of cell-to-cell communication by which bacteria communicate via secreted signal molecules called autoinducers. As cell population density increases, the accumulation of autoinducers leads to co-ordinated changes in gene expression across the bacterial community. The marine bacterium, Vibrio harveyi, uses three autoinducers to achieve intra-species, intra-genera and inter-species cell–cell communication. The detection of these autoinducers ultimately leads to the production of LuxR, the quorum-sensing master regulator that controls expression of the genes in the quorum-sensing regulon. LuxR is a member of the TetR protein superfamily; however, unlike other TetR repressors that typically repress their own gene expression and that of an adjacent operon, LuxR is capable of activating and repressing a large number of genes. Here, we used protein binding microarrays and a two-layered bioinformatics approach to show that LuxR binds a 21 bp consensus operator with dyad symmetry. In vitro and in vivo analyses of two promoters directly regulated by LuxR allowed us to identify those bases that are critical for LuxR binding. Together, the in silico and biochemical results enabled us to scan the genome and identify novel targets of LuxR in V. harveyi and thus expand the understanding of the quorum-sensing regulon.
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the luxs dependent autoinducer ai 2 controls the expression of an abc transporter that functions in ai 2 uptake in salmonella typhimurium
Molecular Microbiology, 2008Co-Authors: Michiko E. Taga, Julie Lee Semmelhack, Bonnie L. BasslerAbstract:Summary In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers. A novel autoinducer called AI-2, originally discovered in the quorum-sensing bacterium Vibrio harveyi ,i s made by many species of Gram-negative and Grampositive bacteria. In every case, production of AI-2 is dependent on the LuxS autoinducer synthase. The genes regulated by AI-2 in most of these luxS-containing species of bacteria are not known. Here, we describe the identification and characterization of AI2-regulated genes in Salmonella typhimurium. We find that LuxS and AI-2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)-type transporter. We have named this operon the lsr (luxS regulated) operon. The Lsr transporter has homology to the ribose transporter of Escherichia coli and S. typhimurium .A gene encoding a DNA-binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI-2 detection to operon expression. This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI-2. Mutations in the lsr operon render S. typhimurium unable to eliminate AI-2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI-2 into the cells. It is intriguing that an operon regulated by AI-2 encodes functions resembling the ribose transporter, given recent findings that AI-2 is derived from the ribosyl moiety of S-ribosylhomocysteine.
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quorum sensing controls biofilm formation in vibrio cholerae through modulation of cyclic di gmp levels and repression of vpst
Journal of Bacteriology, 2008Co-Authors: Christopher M Waters, Joshua D. Rabinowitz, Wenyun Lu, Bonnie L. BasslerAbstract:Two chemical signaling systems, quorum sensing (QS) and 3,5-cyclic diguanylic acid (c-di-GMP), reciprocally control biofilm formation in Vibrio cholerae. QS is the process by which bacteria communicate via the secretion and detection of autoinducers, and in V. cholerae, QS represses biofilm formation. c-di-GMP is an intracellular second messenger that contains information regarding local environmental conditions, and in V. cholerae, c-di-GMP activates biofilm formation. Here we show that HapR, a major regulator of QS, represses biofilm formation in V. cholerae through two distinct mechanisms. HapR controls the transcription of 14 genes encoding a group of proteins that synthesize and degrade c-di-GMP. The net effect of this transcriptional program is a reduction in cellular c-di-GMP levels at high cell density and, consequently, a decrease in biofilm formation. Increasing the c-di-GMP concentration at high cell density to the level present in the low-celldensity QS state restores biofilm formation, showing that c-di-GMP is epistatic to QS in the control of biofilm formation in V. cholerae. In addition, HapR binds to and directly represses the expression of the biofilm transcriptional activator, vpsT. Together, our results suggest that V. cholerae integrates information about the vicinal bacterial community contained in extracellular QS autoinducers with the intracellular environmental information encoded in c-di-GMP to control biofilm formation. Vibrio cholerae, the causative agent of the diarrheal disease cholera, exists primarily in marine environments, and infection of humans usually occurs through ingestion of contaminated water (45). After traversing the stomach, V. cholerae expresses an array of virulence factors that enable colonization of the host intestinal epithelium. The major colonization factor, toxin coregulated pilus, promotes adherence to the intestinal lining, and the subsequent secretion of cholera toxin leads to severe diarrhea and release of the bacterium into the environment (14). Critical to its infection cycle is V. cholerae’s ability to alternate between expression of virulence traits essential for survival inside the host and expression of traits such as biofilm formation that are necessary for survival in its ex vivo marine environment (13, 22, 23, 51, 59). Two chemical signaling systems, quorum sensing (QS) via autoinducer (AI) molecules (see below) and 3,5-cyclic diguanylic acid (c-di-GMP) signaling, control the transition between these two lifestyles. QS is a cell-cell communication process involving the production, secretion, and detection of chemical signal molecules known as AIs that allows bacteria to synchronize the behavior of the population (54). V. cholerae produces two AIs and responds to them by using parallel phosphorelay signaling systems (36). The two AI molecules are termed CAI-1, (S)-3hydroxytridecan-4-one (19), and AI-2, (2S,4S)-2-methyl2,3,3,4-tetrahydroxytetrahydrofuran borate (5, 36). In the lowcell density state (i.e., when AI levels are low), the AI receptors function as kinases, and funnel phosphate to the response regulator, LuxO (Fig. 1). LuxOP activates the expression of
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The Quorum-Sensing Molecule Autoinducer 2 Regulates Motility and Flagellar Morphogenesis in Helicobacter pylori
Journal of Bacteriology, 2007Co-Authors: Bethany A. Rader, Bonnie L. Bassler, Shawn R Campagna, Martin F. Semmelhack, Karen GuilleminAbstract:The genome of the gastric pathogen Helicobacter pylori contains a homologue of the gene luxS, which has been shown to be responsible for production of the quorum-sensing signal autoinducer 2 (AI-2). We report here that deletion of the luxS gene in strain G27 resulted in decreased motility on soft agar plates, a defect that was complemented by a wild-type copy of the luxS gene and by the addition of cell-free supernatant containing AI-2. The flagella of the luxS mutant appeared normal; however, in genetic backgrounds lacking any of three flagellar regulators—the two-component sensor kinase flgS, the sigma factor σ28 (also called fliA), and the anti-sigma factor flgM—loss of luxS altered flagellar morphology. In all cases, the double mutant phenotypes were restored to the luxS+ phenotype by the addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Furthermore, in all mutant backgrounds loss of luxS caused a decrease in transcript levels of the flagellar regulator flhA. Addition of DPD to luxS cells induced flhA transcription in a dose-dependent manner. Deletion of flhA in a wild-type or luxS mutant background resulted in identical loss of motility, flagella, and flagellar gene expression. These data demonstrate that AI-2 functions as a secreted signaling molecule upstream of FlhA and plays a critical role in global regulation of flagellar gene transcription in H. pylori.
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Phosphorylation and processing of the quorum-sensing molecule Autoinducer-2 in enteric bacteria.
ACS Chemical Biology, 2007Co-Authors: Karina B. Xavier, Stephen T Miller, Wenyun Lu, Joshua D. Rabinowitz, István Pelczer, Martin F. Semmelhack, Bonnie L. BasslerAbstract:Quorum sensing is a process of chemical communication that bacteria use to assess cell population density and synchronize behavior on a community-wide scale. Communication is mediated by signal molecules called autoinducers. The LuxS autoinducer synthase produces 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor to a set of interconverting molecules that are generically called Autoinducer-2 (AI-2). In enteric bacteria, AI-2 production induces the assembly of a transport apparatus (called the LuxS regulated (Lsr) transporter) that internalizes endogenously produced AI-2 as well as AI-2 produced by other bacterial species. AI-2 internalization is proposed to be a mechanism enteric bacteria employ to interfere with the signaling capabilities of neighboring species of bacteria. We have previously shown that Salmonella enterica serovar Typhimurium binds a specific cyclic derivative of DPD. Here we show that following internalization, the kinase LsrK phosphorylates carbon-5 of the open form of DPD. Phosphoryl...
Karina B. Xavier - One of the best experts on this subject based on the ideXlab platform.
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Methods for analysis of bacterial Autoinducer-2 production.
Current protocols in microbiology, 2020Co-Authors: Michiko E. Taga, Karina B. XavierAbstract:Quorum sensing is a cell-cell signaling process that many bacteria use to regulate gene expression as a function of the density of the population. This phenomenon involves the production, release, and response to small chemical molecules termed autoinducers. Most autoinducers are species-specific; however, one autoinducer called Autoinducer-2 (AI-2) is produced and detected by many species of bacteria and thus can foster inter-species communication. This unit describes two assays to detect and quantify AI-2 from biological samples. The first uses a bacterial reporter strain, which produces bioluminescence in response to AI-2. The second is an in vitro assay based on a modified version of an AI-2 receptor fused to a cyan fluorescent protein and a yellow fluorescent protein. Binding of AI-2 to this fusion protein induces a dose-dependent decrease in fluorescence resonance energy transfer (FRET), enabling quantification of the AI-2 concentration in the samples.
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Synthesis of d-desthiobiotin-AI-2 as a novel chemical probe for Autoinducer-2 quorum sensing receptors
Bioorganic Chemistry, 2019Co-Authors: Vanessa Miranda, Karina B. Xavier, Inês M. Torcato, M. Rita VenturaAbstract:Abstract In processes regulated by quorum sensing (QS) bacteria respond to the concentration of autoinducers in the environment to engage in group behaviours. Autoinducer-2 (AI-2) is unique as it can foster interspecies communication. Currently, two AI-2 receptors are known, LuxP and LsrB, but bacteria lacking these receptors can also respond to AI-2. In this work, we present an efficient and reproducible synthesis of a novel chemical probe, d -desthiobiotin-AI-2. This probe binds both LuxP and LsrB receptors from different species of bacteria. Thus, this probe is able to bind receptors that recognise the two known biologically active forms of AI-2, presenting the plasticity essential for the identification of novel unknown AI-2 receptors. Moreover, a protocol to pull down receptors bound to d -desthiobiotin-AI-2 with anti-biotin antibodies has also been established. Altogether, this work highlights the potential of conjugating chemical signals to biotinylated derivatives to identify and tag signal receptors involved in quorum sensing or other chemical signalling processes.
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Synthesis and biological activity of a potent optically pure Autoinducer-2 quorum sensing agonist
Bioorganic Chemistry, 2018Co-Authors: Osvaldo S. Ascenso, Karina B. Xavier, Inês M. Torcato, M. Rita Ventura, Ana Sofia Miguel, João C. Marques, Christopher D. MaycockAbstract:Abstract Quorum sensing (QS) regulates population-dependent bacterial behaviours, such as toxin production, biofilm formation and virulence. Autoinducer-2 (AI-2) is to date the only signalling molecule known to foster inter-species bacterial communication across distantly related bacterial species. In this work, the synthesis of pure enantiomers of C4-propoxy-HPD and C4-ethoxy-HPD, known AI-2 analogues, has been developed. The optimised synthesis is efficient, reproducible and short. The (4S) enantiomer of C4-propoxy-HPD was the most active compound being approximately twice as efficient as (4S)-DPD and ten-times more potent than the (4R) enantiomer. Additionally, the specificity of this analogue to bacteria with LuxP receptors makes it a good candidate for clinical applications, because it is not susceptible to scavenging by LsrB-containing bacteria that degrade the natural AI-2. All in all, this study provides a new brief and effective synthesis of isomerically pure analogues for QS modulation that include the most active AI-2 agonist described so far.
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Current Protocols in Microbiology - Methods for analysis of bacterial Autoinducer-2 production.
Current protocols in microbiology, 2011Co-Authors: Michiko E. Taga, Karina B. XavierAbstract:Quorum sensing is a cell-cell signaling process that many bacteria use to regulate gene expression as a function of the density of the population. This phenomenon involves the production, release, and response to small chemical molecules termed autoinducers. Most autoinducers are species-specific; however, one autoinducer called Autoinducer-2 (AI-2) is produced and detected by many species of bacteria and thus can foster inter-species communication. This unit describes two assays to detect and quantify AI-2 from biological samples. The first uses a bacterial reporter strain, which produces bioluminescence in response to AI-2. The second is an in vitro assay based on a modified version of an AI-2 receptor fused to a cyan fluorescent protein and a yellow fluorescent protein. Binding of AI-2 to this fusion protein induces a dose-dependent decrease in fluorescence resonance energy transfer (FRET), enabling quantification of the AI-2 concentration in the samples. Curr. Protoc. Microbiol. 23:1C.1.1-1C.1.15. © 2011 by John Wiley & Sons, Inc. Keywords: AI-2; Autoinducer-2; luminescent proteins luminescent measurements; Vibrio harveyi; bacterial LuxS protein; 4,5-dihydroxy-2,3-pentanedione; quorum sensing; BB170; FRET; CFP-LuxP-YFP
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Identification of functional LsrB-like Autoinducer-2 receptors.
Journal of Bacteriology, 2009Co-Authors: C Pereira, Stephen T Miller, Anna K. De Regt, Patrícia H. Brito, Karina B. XavierAbstract:Although a variety of bacterial species have been reported to use the interspecies communication signal Autoinducer-2 (AI-2) to regulate multiple behaviors, the molecular mechanisms of AI-2 recognition and signal transduction remain poorly understood. To date, two types of AI-2 receptors have been identified: LuxP, present in Vibrio spp., and LsrB, first identified in Salmonella enterica serovar Typhimurium. In S. Typhimurium, LsrB is the ligand binding protein of a transport system that enables the internalization of AI-2. Here, using both sequence analysis and structure prediction, we establish a set of criteria for identifying functional AI-2 receptors. We test our predictions experimentally, assaying key species for their abilities to import AI-2 in vivo, and test their LsrB orthologs for AI-2 binding in vitro. Using these experimental approaches, we were able to identify AI-2 receptors in organisms belonging to phylogenetically distinct families such as the Enterobacteriaceae, Rhizobiaceae, and Bacillaceae. Phylogenetic analysis of LsrB orthologs indicates that this pattern could result from one single origin of the functional LsrB gene in a gammaproteobacterium, suggesting possible posterior independent events of lateral gene transfer to the Alphaproteobacteria and Firmicutes. Finally, we used mutagenesis to show that two AI-2-interacting residues are essential for the AI-2 binding ability. These two residues are conserved in the binding sites of all the functional AI-2 binding proteins but not in the non-AI-2-binding orthologs. Together, these results strongly support our ability to identify functional LsrB-type AI-2 receptors, an important step in investigations of this interspecies signal.
Donald R Demuth - One of the best experts on this subject based on the ideXlab platform.
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Autoinducer-2 and QseC Control Biofilm Formation and In Vivo Virulence of Aggregatibacter actinomycetemcomitans
Infection and Immunity, 2010Co-Authors: Elizabeth A. Novak, Hanjuan Shao, Carlo Amorin Daep, Donald R DemuthAbstract:Biofilm formation by the periodontal pathogen Aggregatibacter actinomycetemcomitans is dependent upon Autoinducer-2 (AI-2)-mediated quorum sensing. However, the components that link the detection of the AI-2 signal to downstream gene expression have not been determined. One potential regulator is the QseBC two-component system, which is part of the AI-2-dependent response pathway that controls biofilm formation in Escherichia coli. Here we show that the expression of QseBC in A. actinomycetemcomitans is induced by AI-2 and that induction requires the AI-2 receptors, LsrB and/or RbsB. Additionally, inactivation of qseC resulted in reduced biofilm growth. Since the ability to grow in biofilms is essential for A. actinomycetemcomitans virulence, strains that were deficient in QseC or the AI-2 receptors were examined in an in vivo mouse model of periodontitis. The ΔqseC mutant induced significantly less alveolar bone resorption than the wild-type strain (P < 0.02). Bone loss in animals infected with the ΔqseC strain was similar to that in sham-infected animals. The ΔlsrB, ΔrbsB, and ΔlsrB ΔrbsB strains also induced significantly less alveolar bone resorption than the wild type (P < 0.03, P < 0.02, and P < 0.01, respectively). However, bone loss induced by a ΔluxS strain was indistinguishable from that induced by the wild type, suggesting that AI-2 produced by indigenous microflora in the murine oral cavity may complement the ΔluxS mutation. Together, these results suggest that the QseBC two-component system is part of the AI-2 regulon and may link the detection of AI-2 to the regulation of downstream cellular processes that are involved in biofilm formation and virulence of A. actinomycetemcomitans.
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autoinducer 2 is required for biofilm growth of aggregatibacter actinobacillus actinomycetemcomitans
Infection and Immunity, 2007Co-Authors: Hanjuan Shao, Richard J Lamont, Donald R DemuthAbstract:Autoinducer 2 (AI-2) is required for the growth of Aggregatibacter (Actinobacillus) actinomycetemcomitans in culture under conditions of iron limitation. However, in vivo this organism thrives in a complex multispecies biofilm that forms in the human oral cavity. In this report, we show that adherent growth of A. actinomycetemcomitans on a saliva-coated surface, but not planktonic growth under iron-replete conditions, is defective in a LuxS-deficient background. Biofilm growth of the luxS mutant exhibited lower total biomass and lower biofilm depth than those for the wild-type strain. Normal biofilm growth of the luxS mutant was restored genetically by introduction of a functional copy of luxS and biochemically by addition of partially purified AI-2. Furthermore, introduction of S-adenosylhomocysteine hydrolase, which restores the metabolism of S-adenosylmethionine in the absence of LuxS, into A. actinomycetemcomitans did not complement the luxS mutation unless AI-2 was added in trans. This suggests that AI-2 itself is required for biofilm growth by A. actinomycetemcomitans. A biofilm growth deficiency similar to that of the LuxS-deficient strain was also observed when a gene encoding the AI-2-interacting protein RbsB or LsrB was inactivated. Biofilm formation by A. actinomycetemcomitans was virtually eliminated upon inactivation of both rbsB and lsrB. In addition, biofilm growth by wild-type A. actinomycetemcomitans was reduced in the presence of ribose, which competes with AI-2 for binding to RbsB. These results suggest that RbsB and LsrB function as AI-2 receptors in A. actinomycetemcomitans and that the development of A. actinomycetemcomitans biofilms requires AI-2.
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differential interaction of aggregatibacter actinobacillus actinomycetemcomitans lsrb and rbsb proteins with autoinducer 2
Journal of Bacteriology, 2007Co-Authors: Hanjuan Shao, Richard J Lamont, Deanna James, Donald R DemuthAbstract:Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A. actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.
Frederick M Hughson - One of the best experts on this subject based on the ideXlab platform.
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Regulation of LuxPQ Receptor Activity by the Quorum-Sensing Signal Autoinducer-2
Molecular Cell, 2005Co-Authors: Matthew B. Neiditch, Bonnie L. Bassler, Stephen T Miller, Michael J. Federle, Frederick M HughsonAbstract:Summary The extracellular signaling molecule Autoinducer-2 (AI-2) mediates quorum-sensing communication in diverse bacterial species. In marine vibrios, binding of AI-2 to the periplasmic receptor LuxP modulates the activity of the inner membrane sensor kinase LuxQ, transducing the AI-2 information into the cytoplasm. Here, we show that Vibrio harveyi LuxP associates with LuxQ in both the presence and absence of AI-2. The 1.9 A X-ray crystal structure of apoLuxP, complexed with the periplasmic domain of LuxQ, reveals that the latter contains two tandem Per/ARNT/Simple-minded (PAS) folds. Thus, although many prokaryotic PAS folds themselves bind ligands, the LuxQ periplasmic PAS folds instead bind LuxP, monitoring its AI-2 occupancy. Mutations that disrupt the apoLuxP:LuxQ interface sensitize V. harveyi to AI-2, implying that AI-2 binding causes the replacement of one set of LuxP:LuxQ contacts with another. These conformational changes switch LuxQ between two opposing enzymatic activities, each of which conveys information to the cytoplasm about the cell density of the surrounding environment.
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salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum sensing signal ai 2
Molecular Cell, 2004Co-Authors: Stephen T Miller, Karina B. Xavier, Bonnie L. Bassler, Michiko E. Taga, Shawn R Campagna, M F Semmelhack, Frederick M HughsonAbstract:Abstract Bacterial populations use cell-cell communication to coordinate community-wide regulation of processes such as biofilm formation, virulence, and bioluminescence. This phenomenon, termed quorum sensing, is mediated by small molecule signals known as autoinducers. While most autoinducers are species specific, Autoinducer-2 (AI-2), first identified in the marine bacterium Vibrio harveyi , is produced and detected by many Gram-negative and Gram-positive bacteria. The crystal structure of the V. harveyi AI-2 signaling molecule bound to its receptor protein revealed an unusual furanosyl borate diester. Here, we present the crystal structure of a second AI-2 signal binding protein, LsrB from Salmonella typhimurium . We find that LsrB binds a chemically distinct form of the AI-2 signal, (2 R ,4 S )-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran ( R -THMF), that lacks boron. Our results demonstrate that two different species of bacteria recognize two different forms of the autoinducer signal, both derived from 4,5-dihydroxy-2,3-pentanedione (DPD), and reveal new sophistication in the chemical lexicon used by bacteria in interspecies signaling.
Michiko E. Taga - One of the best experts on this subject based on the ideXlab platform.
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Methods for analysis of bacterial Autoinducer-2 production.
Current protocols in microbiology, 2020Co-Authors: Michiko E. Taga, Karina B. XavierAbstract:Quorum sensing is a cell-cell signaling process that many bacteria use to regulate gene expression as a function of the density of the population. This phenomenon involves the production, release, and response to small chemical molecules termed autoinducers. Most autoinducers are species-specific; however, one autoinducer called Autoinducer-2 (AI-2) is produced and detected by many species of bacteria and thus can foster inter-species communication. This unit describes two assays to detect and quantify AI-2 from biological samples. The first uses a bacterial reporter strain, which produces bioluminescence in response to AI-2. The second is an in vitro assay based on a modified version of an AI-2 receptor fused to a cyan fluorescent protein and a yellow fluorescent protein. Binding of AI-2 to this fusion protein induces a dose-dependent decrease in fluorescence resonance energy transfer (FRET), enabling quantification of the AI-2 concentration in the samples.
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Methods for analysis of bacterial Autoinducer-2 production.
Current protocols in microbiology, 2020Co-Authors: Michiko E. TagaAbstract:The quorum-sensing signal molecule Autoinducer-2 (AI-2) is produced by over 50 diverse bacterial species and controls many different processes, including antibiotic production, biofilm formation, and virulence. AI-2 production often varies according to growth phase, media conditions, and the presence of specific factors. This unit describes a biological assay for AI-2 activity produced by a bacterial strain of interest. The assay employs an AI-2 reporter strain, Vibrio harveyi BB170, which produces light in response to AI-2. In the first stage of the assay, culture fluids of the bacterial strain of interest are collected over a time course of growth and filtered to remove cells. In the next stage, these culture fluids are mixed with BB170, and the light produced in response to AI-2 in the culture fluids is measured using a luminometer. BB170 is exquisitely sensitive to AI-2, and therefore, even low amounts of AI-2 can be detected using this bioassay.
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Current Protocols in Microbiology - Methods for analysis of bacterial Autoinducer-2 production.
Current protocols in microbiology, 2011Co-Authors: Michiko E. Taga, Karina B. XavierAbstract:Quorum sensing is a cell-cell signaling process that many bacteria use to regulate gene expression as a function of the density of the population. This phenomenon involves the production, release, and response to small chemical molecules termed autoinducers. Most autoinducers are species-specific; however, one autoinducer called Autoinducer-2 (AI-2) is produced and detected by many species of bacteria and thus can foster inter-species communication. This unit describes two assays to detect and quantify AI-2 from biological samples. The first uses a bacterial reporter strain, which produces bioluminescence in response to AI-2. The second is an in vitro assay based on a modified version of an AI-2 receptor fused to a cyan fluorescent protein and a yellow fluorescent protein. Binding of AI-2 to this fusion protein induces a dose-dependent decrease in fluorescence resonance energy transfer (FRET), enabling quantification of the AI-2 concentration in the samples. Curr. Protoc. Microbiol. 23:1C.1.1-1C.1.15. © 2011 by John Wiley & Sons, Inc. Keywords: AI-2; Autoinducer-2; luminescent proteins luminescent measurements; Vibrio harveyi; bacterial LuxS protein; 4,5-dihydroxy-2,3-pentanedione; quorum sensing; BB170; FRET; CFP-LuxP-YFP
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the luxs dependent autoinducer ai 2 controls the expression of an abc transporter that functions in ai 2 uptake in salmonella typhimurium
Molecular Microbiology, 2008Co-Authors: Michiko E. Taga, Julie Lee Semmelhack, Bonnie L. BasslerAbstract:Summary In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers. A novel autoinducer called AI-2, originally discovered in the quorum-sensing bacterium Vibrio harveyi ,i s made by many species of Gram-negative and Grampositive bacteria. In every case, production of AI-2 is dependent on the LuxS autoinducer synthase. The genes regulated by AI-2 in most of these luxS-containing species of bacteria are not known. Here, we describe the identification and characterization of AI2-regulated genes in Salmonella typhimurium. We find that LuxS and AI-2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)-type transporter. We have named this operon the lsr (luxS regulated) operon. The Lsr transporter has homology to the ribose transporter of Escherichia coli and S. typhimurium .A gene encoding a DNA-binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI-2 detection to operon expression. This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI-2. Mutations in the lsr operon render S. typhimurium unable to eliminate AI-2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI-2 into the cells. It is intriguing that an operon regulated by AI-2 encodes functions resembling the ribose transporter, given recent findings that AI-2 is derived from the ribosyl moiety of S-ribosylhomocysteine.
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salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum sensing signal ai 2
Molecular Cell, 2004Co-Authors: Stephen T Miller, Karina B. Xavier, Bonnie L. Bassler, Michiko E. Taga, Shawn R Campagna, M F Semmelhack, Frederick M HughsonAbstract:Abstract Bacterial populations use cell-cell communication to coordinate community-wide regulation of processes such as biofilm formation, virulence, and bioluminescence. This phenomenon, termed quorum sensing, is mediated by small molecule signals known as autoinducers. While most autoinducers are species specific, Autoinducer-2 (AI-2), first identified in the marine bacterium Vibrio harveyi , is produced and detected by many Gram-negative and Gram-positive bacteria. The crystal structure of the V. harveyi AI-2 signaling molecule bound to its receptor protein revealed an unusual furanosyl borate diester. Here, we present the crystal structure of a second AI-2 signal binding protein, LsrB from Salmonella typhimurium . We find that LsrB binds a chemically distinct form of the AI-2 signal, (2 R ,4 S )-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran ( R -THMF), that lacks boron. Our results demonstrate that two different species of bacteria recognize two different forms of the autoinducer signal, both derived from 4,5-dihydroxy-2,3-pentanedione (DPD), and reveal new sophistication in the chemical lexicon used by bacteria in interspecies signaling.
