Academic literature on the topic 'Vibrio. luxR'
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Journal articles on the topic "Vibrio. luxR"
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Bazhenov, Sergey, Olga Melkina, Vadim Fomin, Ekaterina Scheglova, Pavel Krasnik, Svetlana Khrulnova, Gennadii Zavilgelsky, and Ilya Manukhov. "LitR directly upregulates autoinducer synthesis and luminescence in Aliivibrio logei." PeerJ 9 (September 21, 2021): e12030. http://dx.doi.org/10.7717/peerj.12030.
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LitR is a master-regulator of transcription in the ainS/R and luxS/PQ quorum sensing (QS) systems of bacteria from Vibrio and Aliivibrio genera. Here, we for the first time directly investigated the influence of LitR on gene expression in the luxI/R QS system of psychrophilic bacteria Aliivibrio logei. Investigated promoters were fused with Photorhabdus luminescens luxCDABE reporter genes cassette in a heterological system of Escherichia coli cells, litR A. logei was introduced into the cells under control of Plac promoter. LitR has been shown to upregulate genes of autoinducer synthase (luxI), luciferase and reductase (luxCDABE), and this effect doesn’t depend on presence of luxR gene. To a much lesser degree, LitR induces luxR1, but not the luxR2 — the main luxI/R regulator. Enhanced litR expression leads to an increase in a LuxI-autoinducer synthesis and a subsequent LuxR-mediated activation of the luxI/R QS system. Effect of LitR on luxI transcription depends on lux-box sequence in luxI promoter even in absence of luxR (lux-box is binding site of LuxR). The last finding indicates a direct interaction of LitR with the promoter in the lux-box region. Investigation of the effect of LitR A. logei on luxI/R QS systems of mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio salmonicida showed direct luxR-independent upregulation of luxI and luxCDABE genes. To a lesser degree, it induces luxR A. fischeri and luxR1 A. salmonicida. Therefore, we assume that the main role of LitR in cross-interaction of these three QS systems is stimulating the expression of luxI.
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Antunes, Luis Caetano M., Rosana B. R. Ferreira, C. Phoebe Lostroh, and E. Peter Greenberg. "A Mutational Analysis Defines Vibrio fischeri LuxR Binding Sites." Journal of Bacteriology 190, no. 13 (December 14, 2007): 4392–97. http://dx.doi.org/10.1128/jb.01443-07.
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ABSTRACT Vibrio fischeri quorum sensing involves the LuxI and LuxR proteins. The LuxI protein generates the quorum-sensing signal N-3-oxohexanoyl-l-homoserine lactone (3OC6-HSL), and LuxR is a signal-responsive transcriptional regulator which activates the luminescence (lux) genes and 17 other V. fischeri genes. For activation of the lux genes, LuxR binds to a 20-base-pair inverted repeat, the lux box, which is centered 42.5 base pairs upstream of the transcriptional start of the lux operon. Similar lux box-like elements have been identified in only a few of the LuxR-activated V. fischeri promoters. To better understand the DNA sequence elements required for LuxR binding and to identify binding sites in LuxR-regulated promoters other than the lux operon promoter, we have systematically mutagenized the lux box and evaluated the activity of many mutants. By doing so, we have identified nucleotides that are critical for promoter activity. Interestingly, certain lux box mutations allow a 3OC6-HSL-independent LuxR activation of the lux operon promoter. We have used the results of the mutational analysis to create a consensus lux box, and we have used this consensus sequence to identify LuxR binding sites in 3OC6-HSL-activated genes for which lux boxes could not be identified previously.
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Urbanowski, M. L., C. P. Lostroh, and E. P. Greenberg. "Reversible Acyl-Homoserine Lactone Binding to Purified Vibrio fischeri LuxR Protein." Journal of Bacteriology 186, no. 3 (February 1, 2004): 631–37. http://dx.doi.org/10.1128/jb.186.3.631-637.2004.
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ABSTRACT The Vibrio fischeri LuxR protein is the founding member of a family of acyl-homoserine lactone-responsive quorum-sensing transcription factors. Previous genetic evidence indicates that in the presence of its quorum-sensing signal, N-(3-oxohexanoyl) homoserine lactone (3OC6-HSL), LuxR binds to lux box DNA within the promoter region of the luxI gene and activates transcription of the luxICDABEG luminescence operon. We have purified LuxR from recombinant Escherichia coli. Purified LuxR binds specifically and with high affinity to DNA containing a lux box. This binding requires addition of 3OC6-HSL to the assay reactions, presumably forming a LuxR-3OC6-HSL complex. When bound to the lux box at the luxI promoter in vitro, LuxR-3OC6-HSL enables E. coli RNA polymerase to initiate transcription from the luxI promoter. Unlike the well-characterized LuxR homolog TraR in complex with its signal (3-oxo-octanoyl-HSL), the LuxR-30C6-HSL complex can be reversibly inactivated by dilution, suggesting that 3OC6-HSL in the complex is not tightly bound and is in equilibrium with the bulk solvent. Thus, although LuxR and TraR both bind 3-oxoacyl-HSLs, the binding is qualitatively different. The differences have implications for the ways in which these proteins respond to decreases in signal concentrations or rapid drops in population density.
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Egland, Kristi A., and E. P. Greenberg. "Conversion of the Vibrio fischeriTranscriptional Activator, LuxR, to a Repressor." Journal of Bacteriology 182, no. 3 (February 1, 2000): 805–11. http://dx.doi.org/10.1128/jb.182.3.805-811.2000.
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ABSTRACT The Vibrio fischeri luminescence (lux) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the lux box at a position centered 42.5 bases upstream of the transcriptional start of the lux operon. LuxR has proven difficult to study in vitro. A truncated form of LuxR has been purified, and together with ς70 RNA polymerase it can activate transcription of the lux operon. Both the truncated LuxR and RNA polymerase are required for binding tolux regulatory DNA in vitro. We have constructed an artificial lacZ promoter with the lux box positioned between and partially overlapping the consensus −35 and −10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant Escherichia coli. Furthermore, multiplelux boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind tolux boxes independently of RNA polymerase binding to the promoter region. A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a nativelux operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in E. coli. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the lux promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to lux box-containing DNA.
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Antunes, Luis Caetano M., Amy L. Schaefer, Rosana B. R. Ferreira, Nan Qin, Ann M. Stevens, Edward G. Ruby, and E. Peter Greenberg. "Transcriptome Analysis of the Vibrio fischeri LuxR-LuxI Regulon." Journal of Bacteriology 189, no. 22 (September 7, 2007): 8387–91. http://dx.doi.org/10.1128/jb.00736-07.
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ABSTRACT The Vibrio fischeri quorum-sensing signal N-3-oxohexanoyl-l-homoserine lactone (3OC6-HSL) activates expression of the seven-gene luminescence operon. We used microarrays to unveil 18 additional 3OC6-HSL-controlled genes, 3 of which had been identified by other means previously. We show most of these genes are regulated by the 3OC6-HSL-responsive transcriptional regulator LuxR directly. This demonstrates that V. fischeri quorum sensing regulates a substantial number of genes other than those involved in light production.
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Zhang, Jun, Bing Liu, Dan Gu, Yuan Hao, Mo Chen, Yue Ma, Xiaohui Zhou, David Reverter, Yuanxing Zhang, and Qiyao Wang. "Binding site profiles and N-terminal minor groove interactions of the master quorum-sensing regulator LuxR enable flexible control of gene activation and repression." Nucleic Acids Research 49, no. 6 (March 8, 2021): 3274–93. http://dx.doi.org/10.1093/nar/gkab150.
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Abstract LuxR is a TetR family master quorum sensing (QS) regulator activating or repressing expression of hundreds of genes that control collective behaviors in Vibrios with underlying mechanism unknown. To illuminate how this regulator controls expression of various target genes, we applied ChIP-seq and DNase I-seq technologies. Vibrio alginolyticus LuxR controls expression of ∼280 genes that contain either symmetric palindrome (repDNA) or asymmetric (actDNA) binding motifs with different binding profiles. The median number of LuxR binding sites for activated genes are nearly double for that of repressed genes. Crystal structures of LuxR in complex with the respective repDNA and actDNA motifs revealed a new mode of LuxR DNA binding that involves contacts of its N-terminal extension to the minor groove. The N-terminal contacts mediated by Arginine-9 and Arginine-11 differ when LuxR binds to repDNA vs actDNA, leading to higher binding affinity at repressed targets. Moreover, modification of LuxR binding sites, binding profiles, and N-terminal extension have important consequences on QS-regulated phenotypes. These results facilitate fundamental understanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in Vibrio QS, which may facilitate to design QS inhibiting chemicals that interfere with LuxR regulation to effectively control pathogens.
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Callahan, Sean M., and Paul V. Dunlap. "LuxR- and Acyl-Homoserine-Lactone-Controlled Non-luxGenes Define a Quorum-Sensing Regulon in Vibrio fischeri." Journal of Bacteriology 182, no. 10 (May 15, 2000): 2811–22. http://dx.doi.org/10.1128/jb.182.10.2811-2822.2000.
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ABSTRACT The luminescence (lux) operon (luxICDABEG) of the symbiotic bacterium Vibrio fischeri is regulated by the transcriptional activator LuxR and two acyl-homoserine lactone (acyl-HSL) autoinducers (the luxI-dependent 3-oxo-hexanoyl-HSL [3-oxo-C6-HSL] and the ainS-dependent octanoyl-HSL [C8-HSL]) in a population density-responsive manner called quorum sensing. To identify quorum-sensing-regulated (QSR) proteins different from those encoded by lux genes, we examined the protein patterns of V. fischeri quorum-sensing mutants defective in luxI, ainS, andluxR by two-dimensional polyacrylamide gel electrophoresis. Five non-Lux QSR proteins, QsrP, RibB, AcfA, QsrV, and QSR 7, were identified; their production occurred preferentially at high population density, required both LuxR and 3-oxo-C6-HSL, and was inhibited by C8-HSL at low population density. The genes encoding two of the QSR proteins were characterized: qsrP directs cells to synthesize an apparently novel periplasmic protein, andribB is a homolog of the Escherichia coli gene for 3,4-dihydroxy-2-butanone 4-phosphate synthase, a key enzyme for riboflavin synthesis. The qsrP and ribBpromoter regions each contained a sequence similar to thelux operon lux box, a 20-bp region of dyad symmetry necessary for LuxR/3-oxo-C6-HSL-dependent activation oflux operon transcription. V. fischeri qsrP andribB mutants exhibited no distinct phenotype in culture. However, a qsrP mutant, in competition with its parent strain, was less successful in colonizing Euprymna scolopes, the symbiotic host of V. fischeri. The newly identified QSR genes, together with the lux operon, define a LuxR/acyl-HSL-responsive quorum-sensing regulon in V. fischeri.
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Qin, Nan, Sean M. Callahan, Paul V. Dunlap, and Ann M. Stevens. "Analysis of LuxR Regulon Gene Expression during Quorum Sensing in Vibrio fischeri." Journal of Bacteriology 189, no. 11 (March 30, 2007): 4127–34. http://dx.doi.org/10.1128/jb.01779-06.
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ABSTRACT The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-l-homoserine lactone (3-oxo-C6-HSL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and 3-oxo-C6-HSL or instead was due to indirect control via an unidentified regulatory element, promoters of interest were cloned into a lacZ reporter and tested for their LuxR and 3-oxo-C6-HSL dependence in recombinant Escherichia coli. The promoters for qsrP, acfA, and ribB were found to be directly activated via LuxR-3-oxo-C6-HSL. The sites of transcription initiation were established via primer extension analysis. Based on this information and the position of the lux box-binding site near position −40, all three promoters appear to have a class II-type promoter structure. In order to more fully characterize the LuxR regulon in V. fischeri MJ-100, real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. Taken together, the results demonstrate that regulation of the production of QsrP, RibB, and AcfA is controlled directly by LuxR at the level of transcription, thereby establishing that there is a LuxR regulon in V. fischeri MJ-100 whose genes are coordinately expressed during mid-exponential growth.
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McDougald, Diane, Scott A. Rice, and Staffan Kjelleberg. "SmcR-Dependent Regulation of Adaptive Phenotypes inVibrio vulnificus." Journal of Bacteriology 183, no. 2 (January 15, 2001): 758–62. http://dx.doi.org/10.1128/jb.183.2.758-762.2001.
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ABSTRACT Vibrio vulnificus contains homologues of the V. harveyi luxR and luxS genes. A null mutation insmcR (luxR) resulted in a defect in starvation survival, inhibition of starvation-induced maintenance of culturability that occurs when V. vulnificusis starved prior to low-temperature incubation, and increased expression of stationary-phase phenotypes.
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Miyamoto, Carol M., Paul V. Dunlap, Edward G. Ruby, and Edward A. Meighen. "LuxO controls luxR expression in Vibrio harveyi: evidence for a common regulatory mechanism in Vibrio." Molecular Microbiology 48, no. 2 (April 4, 2003): 537–48. http://dx.doi.org/10.1046/j.1365-2958.2003.03453.x.
Full textDissertations / Theses on the topic "Vibrio. luxR"
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Faini, Marie Annette. "Transcriptional Control during Quorum Sensing by LuxR and LuxR Homologues." Thesis, Virginia Tech, 2003. http://hdl.handle.net/10919/31994.
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Quorum sensing is a mechanism used by many proteobacteria to regulate expression of target genes in a population-dependent manner. The quorum sensing system of Vibrio fischeri activates the luminescence (lux) operon when the autoinducer signaling molecule (N-3-oxohexanoyl homoserine lactone) is recognized and bound by the activator protein LuxR. LuxR subsequently binds to the lux box centered at à 42.5 bp upstream of the transcription initiation site and activates transcription from the lux operon promoter, resulting in the emission of light at high cell densities. LuxR consists of 250 amino acids arranged into an N-terminal (regulatory) domain and a C-terminal (activation) domain, and is thought to function as an ambidextrous activator capable of making multiple contacts with the alpha and sigma subunits of RNA polymerase (RNAP). Published work describing the results of alanine scanning mutagenesis performed on the C-terminal domain of LuxR (residues 190-250) has identified residues (K198, W201 and I206) that appear to play a role in positive control of transcription initiation. Additional mutagenesis of residues 180-189 has been undertaken via a three-primer or four-primer PCR-based method in this study. Variants of LuxR were screened for their ability to activate luciferase production and to repress transcription from an artificial promoter, and production of full-length LuxR was measured, in an attempt to identify additional positive control variants. No additional positive control variants were found in this study. Work has also been undertaken to identify intergenic suppressors between positive control variants of LuxR and the RNAP alpha subunit, RpoA. Starting with a recombinant Escherichia coli strain encoding the lux operon and LuxR variant I206E, a random chemical mutagenesis was performed on a vector encoding RpoA. Following transformation of the mutated plasmids encoding RpoA, high throughput luminescence assays were used to identify isolates with phenotypes brighter than the control. Isolation of an intergenic suppressor will confirm the existence of protein-protein interactions between LuxR and RpoA within the transcription initiation complex. The ability of other LuxR family members to establish productive protein-protein interactions with RNAP necessary for transcription initiation was also examined. LuxR homologues EsaR of Pantoea stewarti ssp. stewartii, a repressor of known function, and ExpR of Erwinia carotovora subsp. carotovora were also analyzed for their ability to activate the lux operon, as well as to repress transcription from an artificial promoter containing the lux box.Master of Science
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Qin, Nan. "Analysis of the Regulons Controlled by Transcriptional Regulators LuxR and LitR in Vibrio fischeri." Diss., Virginia Tech, 2008. http://hdl.handle.net/10919/28433.
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Quorum sensing is a bacterial signaling system that controls gene expression in a population density-dependent manner. In Gram-negative proteobacteria, the cell density control of luminescence was first observed in the symbiotic marine bacterium Vibrio fischeri and this system is one of the best studied quorum sensing systems. Two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). A lacZ reporter was used to show that the promoters for qsrP, acfA, and ribB were directly activated via LuxR-3-oxo-C6-HSL in recombinant Escherichia coli. The sites of transcription initiation were established via primer extension analysis. Based on the position of the lux box-binding site near position â 40, all three promoters appear to have a class II-type promoter structure. Real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation.
In order to fully characterize the LuxR regulon in V. fischeri ES114, microarray analysis was performed in the Greenberg lab (University of Washington) and 18 LuxR-3-oxo-C6-HSL regulated promoters were found including 2 genes (qsrP and acfA) identified previously in MJ-100 in addition to the well-studied lux operon. In collaboration with them, full-length purified LuxR protein was used to show direct interaction between the LuxR protein and 7 genes/operons newly identified out of 13 genes/operons examined. The binding affinity between LuxR proteins and those genes was also measured.
Based on the sequence of the lux boxes of the known genes regulated by LuxR and LitR, a position specific weight matrix (PSWM) was created and used to search through the intergenic regions of the V. fischeri ES114 genome. Some potential LuxR and LitR-regulated genes with high score were tested experimently to confirm direct activation. For the LuxR regulon, these possible LuxR-regulated promoters were cloned into a lacZ reporter and tested for their LuxR dependence. Beyond the genes found in microarray, the promoter of the intergenic region VFA0658-0659 was found to be activated by LuxR and 3-oxo-C6-HSL. For the LitR regulon, two LitR-regulated genes found in the microarray were also identified using PSWM and confirmed by real-time PCR to be dependent on LitR for expression. EMSA experiments showed that LitR can specifically bind to the litR boxes of LitR-regulated genes, litR and VF0170 which confirmed that the regulation is direct.Ph. D.
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Callahan, Sean M. (Sean Michael) 1966. "The quorum-sensing regulation of Vibrio fischeri : novel components of the autoinduce/LuxR regulatory circuit." Thesis, Massachusetts Institute of Technology, 1999. http://hdl.handle.net/1721.1/85290.
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Williams, Joshua W. "Multi-tiered Regulation of luxR Provides Precise Timing and Maintenance of the Quorum Sensing Response of Vibrio fischeri." Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/38580.
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The quorum-sensing response of Vibrio fischeri involves a complex network of genes (encoding regulatory proteins as well as sRNAs), that govern host-association and production of bioluminescence. A key regulator of this system is LuxR, which is the transcriptional activator of the lux operon as well as several other genes in. LuxR also autoregulates its own transcription, which we have shown causes bistability and hysteresis in the quorum-sensing response. This behavior allows the system to maintain a stable and robust response in the face of environmental fluctuation or decreases in external autoinducer concentration caused by other sources. There are many factors that are known to regulate luxR expression, including the ArcA redox-responsive regulator, the cAMP-CRP secondary metabolism regulator, and components of the quorum-sensing pathway like LitR. Because of this, LuxR levels are critical in both the timing of quorum-sensing induction, as well as the maintenance of the response over time. This makes it a potential target for multiple levels of regulation in response to factors such as environmental and metabolic conditions, as well as other components of the quorum-sensing network.
Another important global regulatory protein in V. fischeri (and most other species of Gram-negative proteobacteria) is the post-transcriptional regulator CsrA. CsrA controls processes involved in carbon storage and utilization, as well as the transition from exponential to stationary phase growth. We have demonstrated that CsrA is regulated by two sRNAs (CsrB1 and CsrB2) in V. fischeri. Because CsrA regulates changes in cell behavior and is an important metabolic regulator, there is a good possibility that it has some interactions with the quorum-sensing regulon, whose endproduct, bioluminescence, creates a large metabolic demand from the cell.
In an effort to determine at which point in the quorum-sensing regulatory network CsrA regulation is important, epistasis experiments were designed using factorial design, which is a subset of statistical analysis of variance (ANOVA). This method was used to generate a high degree of confidence in the data, so that even minor interactions in the regulatory networks could be established. By altering the levels of CsrA expression in various mutant strains of V. fischeri, we have demonstrated that CsrA acts by an unknown mechanism to increase the transcription of luxR when the quorum-sensing regulator LitR is absent. Our results also demonstrated that CsrA mediates this effect through repression of ArcA activity, which is known to act directly on the luxR and luxI intergenic region as a repressor. This indicates that CsrA may bypass the upstream parts of the quorum-sensing regulatory cascade that lead to litR activation, so that LitR and LuxR may be regulated differently in response to certain conditions.
This work has shown that the interactions between global regulons can coordinately control the amount of quorum-sensing induction by affecting the level of LuxR in the cell. The balance of these regulatory networks allows the cell to tightly regulate the quorum-sensing response. Thus, LuxR serves as a critical regulatory hub in the cell, at which multiple signals can be integrated in order to generate the appropriate cellular response.Ph. D.
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Chatterjee, Jaidip. "The luminescence induction point of Vibrio harveyi is an integration of multiple regulatory controls : LuxR, MetR, and CRP." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36889.
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Luminescence is a secondary metabolic process that can be detected and assayed in a non-disruptive, in vivo, and real-time manner. As such the Vibrio harveyi luminescence (lux) system constitutes a model system that can be used to delineate the relatively complex transcriptional control mechanisms that are inherent to secondary metabolic processes. Molecular insights into the control of luminescence induction in V. harveyi is limited to the two-component quorum-sensing system and LuxR. While the quorum-sensing system is thought to facilitate luminescence induction through a mechanism of derepression, LuxR has been shown to be an essential activator of the process by virtue of the absence of luminescence in a luxR null mutant. Additionally, it has been suggested that LuxR activates its own expression. Data presented here demonstrates that LuxR is involved in autorepression. Moreover, it is argued that LuxR may not be a typical activator in that it may mediate activation of lux gene expression through a mechanism of repression. An implication of this proposal is that a second unidentified activator is required for luminescence induction to occur. The search of candidates to fill this proposed activation role focused on two highly conserved and extensively studied activators, MetR and CRP. Both are involved in nutrient dependent pathways as MetR is an activator of methionine biosynthesis genes and CRP is implicated in the regulation of metabolic pathways in response to the presence of preferred carbohydrates. In the specific case of the V. harveyi lux system, MetR is shown to act as a repressor, while CRP is shown to be a critical activator of luminescence induction. The evolutionary and ecological implications of these findings are discussed.
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Trott, Amy Elizabeth. "Amino Acid Residues in LuxR Critical for its Mechanism of Transcriptional Activation during Quorum Sensing." Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/34070.
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Vibrio fischeri, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in V. fischeri, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or lux operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the lux operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An in vivo luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the lux operon in recombinant Escherichia coli. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the lux box) or to establish associations with RNA polymerase (RNAP) at the lux operon promoter necessary for transcriptional initiation. A third in vivo assay was used to test the ability of the altered forms of LuxR to bind to the lux box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the lux box in the DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the lux box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the lux box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the lux operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the lux operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.Master of Science
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McDougald, S. Diane School of Microbiology & Immunology UNSW. "Regulation of starvation and nonculturability in the marine pathogen, Vibrio vulnificus." Awarded by:University of New South Wales. School of Microbiology and Immunology, 2000. http://handle.unsw.edu.au/1959.4/19118.
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Vibrio vulnificus is a model environmental organism exhibiting a classical starvation response during nutrient limitation as well as a non-culturable state when exposed to low temperatures. In addition to these classic global responses, this organism is an opportunistic pathogen that exhibits numerous virulence factors. This organism was chosen as the model organism for the identification of regulators of the viable but nonculturable response (VBNC) and the starvation-induced maintenance of culturability (SIMC) that occurs when cells are starved prior to low temperature incubation. In order to accomplish this, three indirect approaches were used; proteomics, investigation of intercellular signalling pathways and genetic analysis of regulators involved in these responses. Two-dimensional gel electrophoresis was used to identify proteins expressed under conditions that induced SIMC. It was determined that carbon and long-term phosphorus starvation were important in the SIMC response. V. vulnificus was shown to possess genes, luxS and smcR, that are homologues of genes involved in signalling system system 2 in Vibrio harveyi. Signal molecules were produced upon starvation and the entry to stationary phase in V. vulnificus. Furthermore, a null mutation in smcR, a transcriptional regulator was shown to have pleiotropic effects in V. vulnificus, including up-regulation of numerous virulence factors and a defect in starvation survival and development of the SIMC response. We propose that V. vulnificus possesses a signalling system analogous to that of system 2 in V. harveyi, and that this system is involved in the regulation of stationary phase and starvation adaptation in this organism.
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Johnson, Deborah Cumaraswamy. "Role of region 4 of the sigma 70 subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing." Thesis, Virginia Tech, 2002. http://hdl.handle.net/10919/31680.
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The mechanism of gene regulation used by Gram-negative bacteria during quorum sensing is well understood in the bioluminescent marine bacterium Vibrio fischeri. The cell-density dependent activation of the luminescence (lux) genes of V. fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl) homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. LuxR, a 250 amino acid polypeptide, binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site. During transcriptional activation of the lux operon, LuxR is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli sigma 70 subunit of RNAP in LuxR-dependent transcriptional activation of the luxI promoter has been investigated. Rich in basic amino acids, this conserved portion of sigma 70 is likely to be surface-exposed and available to interact with transcription factors bound near the -35 element. The effect of 16 single and 2 triple alanine substitution variants of sigma 70 between amino acid residues 590 and 613, was determined in vivo by measuring the rate of transcription from a luxI-lacZ translational fusion via b-galactosidase assays in recombinant E. coli. In vitro work was performed with LuxRDN, the autoinducer-independent C-terminal domain (amino acids 157 to 250) of LuxR because purified, full length LuxR is unavailable. Single-round transcription assays were performed in the presence of LuxRDN and 19 variant RNAPs, one of which contained a C-terminally truncated sigma 70 subunit devoid of region 4. Results indicate that region 4 is essential for LuxRDN-dependent luxI transcription with two specific amino acid residues, E591 and K597, having negative effects on the rate of LuxRDN-dependent luxI transcription in vivo and in vitro. None of the residues tested were identified as having any effect on LuxR-dependent luxI transcription in vivo. These findings suggest that region 4.2 is most likely to be in close proximity to LuxR when bound to the luxI promoter. However, unlike the situation found for other ambidextrous activators, no single residue within region 4.2 of sigma 70 may be critical by itself for LuxR-dependent during transcriptional activation.Master of Science
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Finney, Angela H. "Role of the C-terminal domain of the a subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing." Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/36285.
Full textAbstract:
Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, Vibrio fischeri. In V. fischeri, the luminescence or lux genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the lux operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the aCTD of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effect of seventy alanine substitution variants of the a subunit was determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by in vitro assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxRDN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRDN, and fourteen residues in the aCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxRDN-dependent activation in vivo and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type aCTD is capable of interacting with the lux DNA fragment tested, all five of the variant forms of the aCTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that aCTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the lux operon during quorum sensing.Master of Science
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Odenbach, Tina. "Charakterisierung der Hybridsensorkinase LuxN und des Antwortregulators LuxO des Quorum sensing-Systems in Vibrio harveyi." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-100461.
Full textBooks on the topic "Vibrio. luxR"
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Callahan, Sean M. The quorum-sensing regulon of Vibrio fischeri: Novel components of the autoinducer/LuxR regulatory circuit. Cambridge, Mass: Massachusetts Institute of Technology, 1999.
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Callahan, Sean M. The quorum-sensing regulon of Vibrio fischeri: Novel components of the autoinducer/LuxR regulatory circuit. Cambridge, Mass: Massachusetts Institute of Technology, 1999.
Find full textBook chapters on the topic "Vibrio. luxR"
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Nyholm, Spencer V. "Fiat Lux: The Squid–Vibrio Association as a Model for Understanding Host–Microbe Associations." In Advances in Environmental Microbiology, 295–315. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-28068-4_11.
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Visick, Karen L., and Edward G. Ruby. "Temporal Control of Lux Gene Expression in the Symbiosis between Vibrio Fischeri and Its Squid Host." In New Developments in Marine Biotechnology, 277–79. Boston, MA: Springer US, 1998. http://dx.doi.org/10.1007/978-1-4757-5983-9_59.
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Newman, Jane D., and Julia C. van Kessel. "Purification of the Vibrio Quorum-Sensing Transcription Factors LuxR, HapR, and SmcR." In Methods in Molecular Biology. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/7651_2020_306.
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Thomas, Michael D., and Anita Van Tilburg. "Overexpression of foreign proteins using the Vibrio fischeri lux control system." In Bioluminescence and Chemiluminescence Part C, 315–29. Elsevier, 2000. http://dx.doi.org/10.1016/s0076-6879(00)05497-5.
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Wakabayashi, Kenichi, and Masayuki Yamamura. "The Enterococcus faecalis Information Gate." In Cellular Computing. Oxford University Press, 2004. http://dx.doi.org/10.1093/oso/9780195155396.003.0011.
Full textAbstract:
Information exchange between cellular compartments allows us to engineer systems based around cooperative principles. In this chapter we consider a unique bacterial communication system, the conjugative plasmid transfer of Enterococcus faecalis. Using these bacteria, we describe how to engineer a logically controlled information gate and build a logical inverter based upon it. Cellular computing is an alternative computing paradigm based on living cells. Microscale organisms, especially bacteria, are well suited for computing for several reasons. A small culture provides an almost limitless supply of bacterial “hardware.” Bacteria can be stored and easily modified by gene recombination. In addition, and important for our purposes, bacteria can produce various signal molecules that are useful for computation. DNA-binding proteins recognize specific regulatory regions of DNA, bind them, and regulate their genetic expression. These proteins are available for use as computing signals inside the cell. Weiss et al. have shown, for example, how to construct logic circuits based on gene expression regulated by DNA-binding proteins. Some signal molecules are associated with intercellular communications between individuals. Intercellular communication is one of the fundamental characteristics of multicellular organisms, but it is also found in single-celled microorganisms, including bacteria. Communication mediated by homoserine lactones can widely be seen in various Gram-negative bacteria. The mechanism of this behavior was well characterized in Vibrio fischeri, due to their bioluminescent activity mediated by homoserine lactones. It has been shown that bacterial information transfer can be engineered as an extension of Escherichia coli into which the lux genes of Vibrio fischeri are transformed. The communication abilities of bacteria therefore allow us to build microbial information processors for cellular computing. Communication mechanisms in Gram-positive bacteria are not yet well understood. One of the exceptions to this is the conjugative plasmid transfer system in Enterococcus faecalis. E. faecalis conjugate in response to a pheromone is released by other cells. Pheromones are seven- or eight-residue amino peptides produced in E. faecalis. In the case of cPD1, the pheromone is produced by truncation of a 22-residue precursor that is the signal peptide of a lipoprotein.
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Devine, Jerry H., and Gerald S. Shadel. "Assay of autoinducer activity with luminescent Escherichia coli sensor strains harboring a modified Vibrio fischeri lux regulon." In Bioluminescence and Chemiluminescence Part C, 279–87. Elsevier, 2000. http://dx.doi.org/10.1016/s0076-6879(00)05494-x.
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Weiss, Ron, and Thomas F. ,Jr ,. Knight. "Cellular Computation and Communication Using Engineered Genetic Regulatory Networks." In Cellular Computing. Oxford University Press, 2004. http://dx.doi.org/10.1093/oso/9780195155396.003.0012.
Full textAbstract:
In this chapter we demonstrate the feasibility of digital computation in cells by building several operational in vivo digital logic circuits, each composed of three gates that have been optimized by genetic process engineering. We have built and characterized an initial cellular gate library with biochemical gates that implement the NOT, IMPLIES, andANDlogic functions in E. coli cells. The logic gates perform computation using DNA-binding proteins, small molecules that interact with these proteins, and segments of DNA that regulate the expression of the proteins. We also demonstrate engineered intercellular communications with programmed enzymatic activity and chemical diffusions to carry messages, using DNA from the Vibrio fischeri lux operon. The programmed communications is essential for obtaining coordinated behavior from cell aggregates. This chapter is structured as follows: the first section describes experimental measurements of the device physics of in vivo logic gates, as well as genetic process engineering to modify gates until they have the desired behavior. The second section presents experimental results of programmed intercellular communications, including time–response measurements and sensitivity to variations in message concentrations. Potentially the most important element of biocircuit design is matching gate characteristics. Experimental results in this section demonstrate that circuits with mismatched gates are likely to malfunction. In generating biology’s complex genetic regulatory networks, natural forces of selection have resulted in finely tuned interconnections between the different regulatory components. Nature has optimized and matched the kinetic characteristics of these elements so that they cooperatively achieve the desired regulatory behavior. In building de novo biocircuits, we frequently combine regulatory elements that do not interact in their wild-type settings. Therefore, naive coupling of these elements will likely produce systems that do not have the desired behavior. In genetic process engineering, the biocircuit designer first determines the behavioral characteristics of the regulatory components and then modifies the elements until the desired behavior is attained. Below, we show experimental results of using this process to convert a nonfunctional circuit with mismatched gates into a circuit that achieves the correct response.