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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 cholerae Quorum-Sensing Protein VqmA Integrates Cell Density, Environmental, and Host-Derived Cues into the Control of Virulence
'American Society for Microbiology', 2020Co-Authors: Ameya A. Mashruwala, Bonnie L BasslerAbstract:Quorum sensing (QS) is a process of chemical communication that bacteria use to orchestrate collective behaviors. QS communication relies on chemical signal molecules called Autoinducers. QS regulates virulence in Vibrio cholerae, the causative agent of the disease cholera. Transit into the human small intestine, the site of cholera infection, exposes V. cholerae to the host environment. In this study, we show that the combination of two stimuli encountered in the small intestine, the absence of oxygen and the presence of host-produced bile salts, impinge on V. cholerae QS function and, in turn, pathogenicity. We suggest that possessing a QS system that is responsive to multiple environmental, host, and cell density cues enables V. cholerae to fine-tune its virulence capacity in the human intestine.Quorum sensing is a chemical communication process in which bacteria use the production, release, and detection of signal molecules called Autoinducers to orchestrate collective behaviors. The human pathogen Vibrio cholerae requires quorum sensing to infect the small intestine. There, V. cholerae encounters the absence of oxygen and the presence of bile salts. We show that these two stimuli differentially affect quorum-sensing function and, in turn, V. cholerae pathogenicity. First, during anaerobic growth, V. cholerae does not produce the CAI-1 Autoinducer, while it continues to produce the DPO Autoinducer, suggesting that CAI-1 may encode information specific to the aerobic lifestyle of V. cholerae. Second, the quorum-sensing receptor-transcription factor called VqmA, which detects the DPO Autoinducer, also detects the lack of oxygen and the presence of bile salts. Detection occurs via oxygen-, bile salt-, and redox-responsive disulfide bonds that alter VqmA DNA binding activity. We propose that VqmA serves as an information processing hub that integrates quorum-sensing information, redox status, the presence or absence of oxygen, and host cues. In response to the information acquired through this mechanism, V. cholerae appropriately modulates its virulence output
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mechanism underlying Autoinducer recognition in the vibrio cholerae dpo vqma quorum sensing pathway
bioRxiv, 2019Co-Authors: Xiuliang Huang, Jon E. Paczkowski, Brad R. Henke, Justin E Silpe, Jian Ping Cong, Olivia P Duddy, Bonnie L BasslerAbstract:Quorum sensing is a bacterial communication process whereby bacteria produce, release and detect the accumulation of extracellular signaling molecules called Autoinducers to coordinate collective behaviors. In Vibrio cholerae, the quorum-sensing Autoinducer, DPO (3,5-dimethyl-pyrazin-2-ol), binds the receptor-transcription factor, VqmA. In response, the DPO-VqmA complex activates transcription of the vqmR gene encoding the VqmR small RNA. VqmR represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA has DPO-dependent and DPO-independent activity. We solved the DPO-VqmA crystal structure and compared it to existing structures to understand the conformational changes the protein undergoes upon DNA binding. Analysis of DPO analogs reveals that a hydroxyl or carbonyl group at the 29 position is critical for binding. The proposed DPO precursor, a linear molecule, Ala-AA (N-alanyl-aminoacetone), also binds and activates VqmA. DPO and Ala-AA occupy the same binding site as judged by site-directed mutagenesis and competitive ligand binding analyses.
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an Autoinducer analogue reveals an alternative mode of ligand binding for the lasr quorum sensing receptor
ACS Chemical Biology, 2019Co-Authors: Jon E. Paczkowski, Bonnie L Bassler, Frederick M. Hughson, Chari D Smith, Amelia R. Mccready, Brad R. Henke, Jian Ping Cong, Philip D JeffreyAbstract:Bacteria use a cell–cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called Autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone Autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical Autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of...
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An Autoinducer Analogue Reveals an Alternative Mode of Ligand Binding for the LasR Quorum-Sensing Receptor
2019Co-Authors: Jon E. Paczkowski, Frederick M. Hughson, Chari D Smith, Amelia R. Mccready, Brad R. Henke, Jian Ping Cong, Philip D Jeffrey, Bonnie L BasslerAbstract:Bacteria use a cell–cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called Autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone Autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical Autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors
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Structural determinants driving homoserine lactone ligand selection in thePseudomonas aeruginosaLasR quorum-sensing receptor.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Amelia R. Mccready, Jon E. Paczkowski, Brad R. Henke, Bonnie L BasslerAbstract:Quorum sensing is a cell–cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by signal molecules called Autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use the Pseudomonas aeruginosa LasR quorum-sensing receptor to explore signal discrimination. The cognate Autoinducer, 3OC12 homoserine lactone (3OC12HSL), is a more potent activator of LasR than other homoserine lactones. However, other homoserine lactones can elicit LasR-dependent quorum-sensing responses, showing that LasR displays ligand promiscuity. We identify mutants that alter which homoserine lactones LasR detects. Substitution at residue S129 decreases the LasR response to 3OC12HSL, while enhancing discrimination against noncognate Autoinducers. Conversely, the LasR L130F mutation increases the potency of 3OC12HSL and other homoserine lactones. We solve crystal structures of LasR ligand-binding domains complexed with noncognate Autoinducers. Comparison with existing structures reveals that ligand selectivity/sensitivity is mediated by a flexible loop near the ligand-binding site. We show that LasR variants with modified ligand preferences exhibit altered quorum-sensing responses to Autoinducers in vivo. We suggest that possessing some ligand promiscuity endows LasR with the ability to optimally regulate quorum-sensing traits.
William E Bentley - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of the lsrr proteins complexed with phospho ai 2 and two signal interrupting analogues reveal distinct mechanisms for ligand recognition
Journal of the American Chemical Society, 2013Co-Authors: Alexander Grishaev, Min Guo, Jacqueline A I Smith, Herman O Sintim, Eunhee Kim, Haekap Cheong, William E Bentley, Kyoungseok RyuAbstract:Quorum sensing (QS) is a cell-to-cell communication system responsible for a variety of bacterial phenotypes including virulence and biofilm formation. QS is mediated by small molecules, Autoinducers (AIs), including AI-2 that is secreted by both Gram-positive and -negative microbes. LsrR is a key transcriptional regulator that governs the varied downstream processes by perceiving AI-2 signal, but its activation via Autoinducer-binding remains poorly understood. Here, we provide detailed regulatory mechanism of LsrR from the crystal structures in complexes with the native signal (phospho-AI-2, D5P) and two quorum quenching antagonists (ribose-5-phosphate, R5P; phospho-isobutyl-AI-2, D8P). Interestingly, the bound D5P and D8P molecules are not the diketone forms but rather hydrated, and the hydrated moiety forms important H-bonds with the carboxylate of D243. The D5P-binding flipped out F124 of the binding pocket, and resulted in the disruption of the dimeric interface-1 by unfolding the α7 segment. Howeve...
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autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of e coli
Metabolic Engineering, 2010Co-Authors: Chenyu Tsao, William E Bentley, Sara Hooshangi, James J ValdesAbstract:Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (Autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the Autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and beta-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions.
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autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of e coli
Metabolic Engineering, 2010Co-Authors: Chenyu Tsao, William E Bentley, Sara Hooshangi, James J ValdesAbstract:Abstract Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or “program” entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (Autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the Autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and β-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions.
Frederick M. Hughson - One of the best experts on this subject based on the ideXlab platform.
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an Autoinducer analogue reveals an alternative mode of ligand binding for the lasr quorum sensing receptor
ACS Chemical Biology, 2019Co-Authors: Jon E. Paczkowski, Bonnie L Bassler, Frederick M. Hughson, Chari D Smith, Amelia R. Mccready, Brad R. Henke, Jian Ping Cong, Philip D JeffreyAbstract:Bacteria use a cell–cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called Autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone Autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical Autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of...
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An Autoinducer Analogue Reveals an Alternative Mode of Ligand Binding for the LasR Quorum-Sensing Receptor
2019Co-Authors: Jon E. Paczkowski, Frederick M. Hughson, Chari D Smith, Amelia R. Mccready, Brad R. Henke, Jian Ping Cong, Philip D Jeffrey, Bonnie L BasslerAbstract:Bacteria use a cell–cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called Autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone Autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical Autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors
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The Vibrio cholerae quorum-sensing Autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA
Nature Chemical Biology, 2009Co-Authors: Robert C Kelly, Wenyun Lu, Martin F. Semmelhack, Frederick M. Hughson, Joshua D Rabinowitz, Douglas A Higgins, Philip D Jeffrey, Megan E Bolitho, Wai-leung Ng, Bonnie L BasslerAbstract:Vibrio cholerae , the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called Autoinducers. The strongest Autoinducer, called CAI-1 (for cholera Autoinducer-1), was previously identified as ( S )-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as ( S )-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate–dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release ≥100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anticholera treatment.
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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, Michiko E. Taga, Bonnie L Bassler, Martin F. Semmelhack, Shawn R Campagna, 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.
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structural identification of a bacterial quorum sensing signal containing boron
Nature, 2002Co-Authors: Xin Chen, Bonnie L Bassler, Istvan Pelczer, Stephan Schauder, Noelle Potier, Alain Van Dorsselaer, Frederick M. HughsonAbstract:Cell–cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules called Autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression. Community cooperation probably enhances the effectiveness of processes such as bioluminescence, virulence factor expression, antibiotic production and biofilm development1,2,3,4. Unlike other Autoinducers, which are specific to a particular species of bacteria, a recently discovered Autoinducer (AI-2)5 is produced by a large number of bacterial species. AI-2 has been proposed to serve as a ‘universal’ signal for inter-species communication1,2,6,7. The chemical identity of AI-2 has, however, proved elusive. Here we present the crystal structure of an AI-2 sensor protein, LuxP, in a complex with Autoinducer. The bound ligand is a furanosyl borate diester that bears no resemblance to previously characterized Autoinducers. Our findings suggest that addition of naturally occurring borate to an AI-2 precursor generates active AI-2. Furthermore, they indicate a potential biological role for boron, an element required by a number of organisms but for unknown reasons.
Karina B. Xavier - One of the best experts on this subject based on the ideXlab platform.
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unit 1c 1 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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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.
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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, Wenyun Lu, Martin F. Semmelhack, Istvan Pelczer, Joshua D Rabinowitz, Stephen T. Miller, 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...
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interference with ai 2 mediated bacterial cell cell communication
Nature, 2005Co-Authors: Karina B. Xavier, Bonnie L BasslerAbstract:In a process called quorum sensing, bacteria communicate with each other using chemical signal molecules. This allows bacterial populations to synchronize their behaviour, and to act in some respects as multicellular organisms. One chemical communication molecule, called Autoinducer-2 (AI-2), is a universal molecule that bacteria use to communicate between species. Some species of bacteria are now shown to interfere with AI-2-directed communication. This may give them an advantage in mixed-species communities, in the human gut microflora for instance. Bacteria communicate by means of chemical signal molecules called Autoinducers. This process, called quorum sensing, allows bacteria to count the members in the community and to alter gene expression synchronously across the population. Quorum-sensing-controlled processes are often crucial for successful bacterial–host relationships—both symbiotic and pathogenic. Most quorum-sensing Autoinducers promote intraspecies communication, but one Autoinducer, called AI-2, is produced and detected by a wide variety of bacteria and is proposed to allow interspecies communication1,2. Here we show that some species of bacteria can manipulate AI-2 signalling and interfere with other species' ability to assess and respond correctly to changes in cell population density. AI-2 signalling, and the interference with it, could have important ramifications for eukaryotes in the maintenance of normal microflora and in protection from pathogenic bacteria.
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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, Michiko E. Taga, Bonnie L Bassler, Martin F. Semmelhack, Shawn R Campagna, 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.
E P Greenberg - One of the best experts on this subject based on the ideXlab platform.
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quorum sensing in vibrio fischeri evidence that s adenosylmethionine is the amino acid substrate for Autoinducer synthesis
Journal of Bacteriology, 1996Co-Authors: Brian L. Hanzelka, E P GreenbergAbstract:Synthesis of the Autoinducer signal involved in the cell density-dependent activation of Vibrio fischeri luminescence is directed by luxI. The Autoinducer is N-(3-oxohexanoyl)homoserine lactone, and little is known about its synthesis. We have measured Autoinducer synthesis by amino acid auxotrophs of Escherichia coli that contained luxI on a high-copy-number plasmid. Experiments with cell suspensions starved for methionine or homoserine show that either methionine or S-adenosylmethionine but not homoserine or homoserine lactone is required for Autoinducer synthesis. The S-adenosylmethionine synthesis inhibitor cycloleucine blocks methionine-dependent Autoinducer synthesis. Thus, it appears that S-adenosylmethionine rather than methionine is the molecule required for Autoinducer synthesis. The amount of 15N-labeled methionine incorporated into the Autoinducer by growing cultures of a homoserine and a methionine auxotroph was measured by mass spectrometry. The labeling studies show that even in the presence of homoserine, almost all of the Autoinducer produced contains the 15N label from methionine. Thus, it appears that S-adenosylmethionine serves as the amino acid substrate in the luxI-dependent synthesis of the V. fischeri Autoinducer.
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quorum sensing in vibrio fischeri probing Autoinducer luxr interactions with Autoinducer analogs
Journal of Bacteriology, 1996Co-Authors: Amy L Schaefer, Brian L. Hanzelka, A Eberhard, E P GreenbergAbstract:The Vibrio fischeri luminescence genes are activated by the transcription factor LuxR in combination with a diffusible signal compound, N-(3-oxohexanoyl) homoserine lactone, termed the Autoinducer. We have synthesized a set of Autoinducer analogs. Many analogs with alterations in the acyl side chain showed evidence of binding to LuxR. Some appeared to bind with an affinity similar to that of the Autoinducer, but none showed a higher affinity, and many did not bind as tightly as the Autoinducer. For the most part, compounds with substitutions in the homoserine lactone ring did not show evidence of binding to LuxR. The exceptions were compounds with a homocysteine thiolactone ring in place of the homoserine lactone ring. Many but not all of the analogs showing evidence of LuxR binding had some ability to activate the luminescence genes. None were as active as the Autoinducer. While most showed little ability to induce luminescence, a few analogs with rather conservative substitutions had appreciable activity. Under the conditions we employed, some of the analogs showing little or no ability to induce luminescence were inhibitors of the Autoinducer.
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cell to cell signaling in the symbiotic nitrogen fixing bacterium rhizobium leguminosarum autoinduction of a stationary phase and rhizosphere expressed genes
Journal of Bacteriology, 1996Co-Authors: K M Gray, James P Pearson, J A Downie, B E A Boboye, E P GreenbergAbstract:The Sym plasmid pRL1JI encodes functions for the formation of nitrogen-fixing pea root nodules by Rhizobium leguminosarum. Some of the nodulation genes are involved in recognition of chemical signals produced by the plant root, and others are required for production of chemical signals recognized by the plant. pRL1JI also contains a regulatory gene, rhiR, that is homologous to luxR, the transcriptional activator of luminescence genes in Vibrio fischeri. LuxR requires a signal compound, an Autoinducer, for its activity. We have identified an R. leguminosarum Autoinducer that, together with RhiR, is required to activate both the rhizosphere-expressed rhiABC operon and a growth-inhibiting function encoded by pRL1JI. This intercellular signal is an N-acylated homoserine lactone structurally related to the V. fischeri and other Autoinducers. These findings indicate a new level of intercellular communication in root nodule formation.
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evidence that the n terminal region of the vibrio fischeri luxr protein constitutes an Autoinducer binding domain
Journal of Bacteriology, 1995Co-Authors: Brian L. Hanzelka, E P GreenbergAbstract:The Vibrio fischeri luminescence genes are regulated by the LuxR protein and an N-acyl homoserine lactone compound termed the Autoinducer. The C-terminal one-third of LuxR contains a domain that can interact with the transcription complex and activate the luminescence genes. On the basis of limited evidence it has been suggested that the N-terminal two-thirds of LuxR constitutes a domain that serves to bind the Autoinducer. We show that tritium-labeled Autoinducer binds to Escherichia coli cells in which LuxR is overexpressed. We also show that tritium-labeled Autoinducer binds to E. coli in which truncated LuxR proteins missing portions of the C-terminal domain are expressed but does not bind to E. coli cells in which truncated LuxR proteins missing portions of the N-terminal region are expressed. Our results provide evidence that the Autoinducer binds to LuxR and that in E. coli the N-terminal two-thirds of LuxR can fold into a polypeptide capable of binding the Autoinducer in the absence of the C-terminal domain.
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interchangeability and specificity of components from the quorum sensing regulatory systems of vibrio fischeri and pseudomonas aeruginosa
Journal of Bacteriology, 1994Co-Authors: K M Gray, Barbara H Iglewski, Luciano Passador, E P GreenbergAbstract:Autoinduction is a conserved mechanism of cell density-dependent gene regulation that occurs in a variety of gram-negative bacteria. Autoinducible luminescence in Vibrio fischeri requires a transcriptional activator, LuxR, while a LuxR homolog, LasR, activates elastase expression in Pseudomonas aeruginosa. Both LuxR and LasR require specific signal molecules, called Autoinducers, for activity. We show here the activation in Escherichia coli of the V. fischeri luminescence (lux) operon by LasR and of the P. aeruginosa elastase gene (lasB) by LuxR when each is in the presence of its cognate Autoinducer. Neither LuxR nor LasR showed appreciable activity with the heterologous V. fischeri or P. aeruginosa Autoinducer. This supports the view that there is a direct interaction of each transcriptional activator with its proper Autoinducer and suggests that there are conserved, autoinduction-related elements within the promoter regions of these genes.
