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Expt 1: The contribution of vjbR and N-dodecanoyl homoserine lactone on gene regulation in Brucella melitensis

ABSTRACT: Quorum sensing (QS) is a communication system in bacteria that regulates gene expression in response to a small diffusible signal indicative of the bacterial population present. QS has been shown to regulate virulence genes, biofilm formation, cell division and secretion systems in a diverse set of bacteria. Brucella melitensis produces a QS signaling molecule, N-dodecanoyl homoserine lactone (C12-HSL), that interacts with a LuxR transcriptional regulator, VjbR. Deletion of vjbR has been found to highly attenuate intracellular survival of B. melitensis. The importance of QS in the regulation of genes necessary for the establishment and maintenance of B. melitensis infection has already been described for the virB and fla operons, however, a complete description of the vjbR regulon has not been described. Genome mining revealed seven luxR-like genes in B. melitensis, only one of which, vjbR, was confirmed to be attenuated for intracellular survival using macrophage assays. Using custom B. melitensis microarrays, we compared transcripts from wild type and ∆vjbR strains in the presence and absence of exogenous C12-HSL. Comparison of the transcriptomes obtained from culture-grown bacteria revealed bi-phasic gene regulation with expression peaks during exponential and early stationary growth phase; characterized by the regulation of 226 and 246 genes (respectively) by VjbR and 349 and 146 (respectively) altered by the addition of C12-HSL. Comparison of the VjbR and C12-HSL regulated genes provided confirmation that expression of 134 genes are regulated by both conditions with all but 3 genes inversely regulated. Overall, these results indicate that VjbR is an activator of gene expression at the exponential growth phase and exerts an equal effect on gene expression at the stationary growth phase; while C12-HSL has a repressive effect on gene expression at both growth stages examined and acts as an antagonist to VjbR activity. Direct transcript analysis comparing ∆vjbR with and without the addition of C12-HSL revealed that the AHL signal is able to direct gene expression in the absence of VjbR and exclusively exerted a positive influence on the expression of 56 genes, including a 93-fold increase in the expression of BabR, a second LuxR homologue. Genes regulated by these QS components included adhesins, proteases, antibiotic and toxin resistance genes, stress survival aids, including DNA repair genes and protein chaperones, transporters and porins, cellular membrane biogenesis genes, amino acid metabolism and transport, transcriptional regulators, and energy production genes. From these data, we conclude that QS is a global regulator of gene expression, and present potential virulence genes that may provide insight into the bacteria’s ability to establish and maintain the replicative vacuole (BCV) within the host cell. Keywords: Microarray comparison of a genetic deletion mutant and the addition of an effector molecule.

ORGANISM(S): Brucella melitensis bv. 1 str. 16M Brucella melitensis

PROVIDER: GSE13527 | GEO | 2010/04/23

SECONDARY ACCESSION(S): PRJNA113989

REPOSITORIES: GEO

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Project description:Quorum sensing (QS) is a communication system in bacteria that regulates gene expression in response to a small diffusible signal indicative of the bacterial population present. QS has been shown to regulate virulence genes, biofilm formation, cell division and secretion systems in a diverse set of bacteria. Brucella melitensis produces a QS signaling molecule, N-dodecanoyl homoserine lactone (C12-HSL), that interacts with a LuxR transcriptional regulator, VjbR. Deletion of vjbR has been found to highly attenuate intracellular survival of B. melitensis. The importance of QS in the regulation of genes necessary for the establishment and maintenance of B. melitensis infection has already been described for the virB and fla operons, however, a complete description of the vjbR regulon has not been described. Genome mining revealed seven luxR-like genes in B. melitensis, only one of which, vjbR, was confirmed to be attenuated for intracellular survival using macrophage assays. Using custom B. melitensis microarrays, we compared transcripts from wild type and ∆vjbR strains in the presence and absence of exogenous C12-HSL. Comparison of the transcriptomes obtained from culture-grown bacteria revealed bi-phasic gene regulation with expression peaks during exponential and early stationary growth phase; characterized by the regulation of 226 and 246 genes (respectively) by VjbR and 349 and 146 (respectively) altered by the addition of C12-HSL. Comparison of the VjbR and C12-HSL regulated genes provided confirmation that expression of 134 genes are regulated by both conditions with all but 3 genes inversely regulated. Overall, these results indicate that VjbR is an activator of gene expression at the exponential growth phase and exerts an equal effect on gene expression at the stationary growth phase; while C12-HSL has a repressive effect on gene expression at both growth stages examined and acts as an antagonist to VjbR activity. Direct transcript analysis comparing ∆vjbR with and without the addition of C12-HSL revealed that the AHL signal is able to direct gene expression in the absence of VjbR and exclusively exerted a positive influence on the expression of 56 genes, including a 93-fold increase in the expression of BabR, a second LuxR homologue. Genes regulated by these QS components included adhesins, proteases, antibiotic and toxin resistance genes, stress survival aids, including DNA repair genes and protein chaperones, transporters and porins, cellular membrane biogenesis genes, amino acid metabolism and transport, transcriptional regulators, and energy production genes. From these data, we conclude that QS is a global regulator of gene expression, and present potential virulence genes that may provide insight into the bacteria’s ability to establish and maintain the replicative vacuole (BCV) within the host cell. Keywords: Microarray comparison of a genetic deletion mutant and the addition of an effector molecule. Samples consist of RNA isolated from Brucella melitensis grown to logarithmic or stationary phase. RNA was extracted from wild type with and without exogenously added C12-HSL, and a ∆vjbR mutant. There are three biological replicates of each sample. Every Brucella melitensis open reading frame was printed in quadruplicate on each microarray. Each replicate was normalized against labeled Brucella melitensis 16M genomic DNA.

Project description:Quorum sensing (QS) is a communication system in bacteria that regulates gene expression in response to a small diffusible signal indicative of the bacterial population present. QS has been shown to regulate virulence genes, biofilm formation, cell division and secretion systems in a diverse set of bacteria. Brucella melitensis produces a QS signaling molecule, N-dodecanoyl homoserine lactone (C12-HSL), that interacts with a LuxR transcriptional regulator, VjbR. Deletion of vjbR has been found to highly attenuate intracellular survival of B. melitensis. The importance of QS in the regulation of genes necessary for the establishment and maintenance of B. melitensis infection has already been described for the virB and fla operons, however, a complete description of the vjbR regulon has not been described. Genome mining revealed seven luxR-like genes in B. melitensis, only one of which, vjbR, was confirmed to be attenuated for intracellular survival using macrophage assays. Using custom B. melitensis microarrays, we compared transcripts from wild type and ∆vjbR strains in the presence and absence of exogenous C12-HSL. Comparison of the transcriptomes obtained from culture grown bacteria revealed bi-phasic gene regulation with expression peaks during exponential and early stationary growth phase; characterized by the regulation of 226 and 246 genes (respectively) by VjbR and 349 and 146 (respectively) altered by the addition of C12-HSL. Comparison of the VjbR and C12-HSL regulated genes provided confirmation that expression of 134 genes are regulated by both conditions with all but 3 genes inversely regulated. Overall, these results indicate that VjbR is an activator of gene expression at the exponential growth phase and exerts an equal effect on gene expression at the stationary growth phase; while C12-HSL has a repressive effect on gene expression at both growth stages examined and acts as an antagonist to VjbR activity. Direct transcript analysis comparing ∆vjbR with and without the addition of C12-HSL revealed that the AHL signal is able to direct gene expression in the absence of VjbR and exclusively exerted a positive influence on the expression of 56 genes, including a 93-fold increase in the expression of BabR, a second LuxR homologue. Genes regulated by these QS components included adhesins, proteases, antibiotic and toxin resistance genes, stress survival aids, including DNA repair genes and protein chaperones, transporters and porins, cellular membrane biogenesis genes, amino acid metabolism and transport, transcriptional regulators, and energy production genes. From these data, we conclude that QS is a global regulator of gene expression, and present potential virulence genes that may provide insight into the bacteria’s ability to establish and maintain the replicative vacuole (BCV) within the host cell. Keywords: Microarray comparison of a genetic deletion mutant and the addition of an effector molecule. Samples consist of RNA isolated from Brucella melitensis grown to logarithmic or stationary phase. RNA was extracted from a ∆vjbR mutant with and without exogenously added C12-HSL. There are three biological replicates of each sample. Every Brucella melitensis open reading frame was printed in quadruplicate on each microarray. Each replicate was normalized against labeled Brucella melitensis 16M genomic DNA.

Project description:Quorum sensing (QS) is a communication system in bacteria that regulates gene expression in response to a small diffusible signal indicative of the bacterial population present. QS has been shown to regulate virulence genes, biofilm formation, cell division and secretion systems in a diverse set of bacteria. Brucella melitensis produces a QS signaling molecule, N-dodecanoyl homoserine lactone (C12-HSL), that interacts with a LuxR transcriptional regulator, VjbR. Deletion of vjbR has been found to highly attenuate intracellular survival of B. melitensis. The importance of QS in the regulation of genes necessary for the establishment and maintenance of B. melitensis infection has already been described for the virB and fla operons, however, a complete description of the vjbR regulon has not been described. Genome mining revealed seven luxR-like genes in B. melitensis, only one of which, vjbR, was confirmed to be attenuated for intracellular survival using macrophage assays. Using custom B. melitensis microarrays, we compared transcripts from wild type and ∆vjbR strains in the presence and absence of exogenous C12-HSL. Comparison of the transcriptomes obtained from culture grown bacteria revealed bi-phasic gene regulation with expression peaks during exponential and early stationary growth phase; characterized by the regulation of 226 and 246 genes (respectively) by VjbR and 349 and 146 (respectively) altered by the addition of C12-HSL. Comparison of the VjbR and C12-HSL regulated genes provided confirmation that expression of 134 genes are regulated by both conditions with all but 3 genes inversely regulated. Overall, these results indicate that VjbR is an activator of gene expression at the exponential growth phase and exerts an equal effect on gene expression at the stationary growth phase; while C12-HSL has a repressive effect on gene expression at both growth stages examined and acts as an antagonist to VjbR activity. Direct transcript analysis comparing ∆vjbR with and without the addition of C12-HSL revealed that the AHL signal is able to direct gene expression in the absence of VjbR and exclusively exerted a positive influence on the expression of 56 genes, including a 93-fold increase in the expression of BabR, a second LuxR homologue. Genes regulated by these QS components included adhesins, proteases, antibiotic and toxin resistance genes, stress survival aids, including DNA repair genes and protein chaperones, transporters and porins, cellular membrane biogenesis genes, amino acid metabolism and transport, transcriptional regulators, and energy production genes. From these data, we conclude that QS is a global regulator of gene expression, and present potential virulence genes that may provide insight into the bacteria’s ability to establish and maintain the replicative vacuole (BCV) within the host cell. Keywords: Microarray comparison of a genetic deletion mutant and the addition of an effector molecule.

Project description:Many pathogenic bacteria use a regulatory process termed Quorum Sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection and response to these molecules depend on transcriptional regulators belonging to the LuxR family. Such a system have been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacteria responsible for brucellosis, a word-wide zoonosis remaining a serious public health concern in endemic countries. Two LuxR-type regulators, VjbR and BabR, have been identified in the genome of this pathogen. The vjbR mutant is highly attenuated in all tested models suggesting a crucial role of QS in the virulence of Brucella. This attenuation is at least due to the involvement of VjbR in the activation of the virB operon coding for a type four secretion system essential for Brucella to reach its intracellular replication compartment. At present, no function has been attributed to BabR. To assess the role of both Brucella QS-regulators, we performed in tandem comparative transcriptomic and proteomic analyses of vjbR and babR mutants. These experiments revealed that 10% of Brucella genome is regulated through those regulators, revealing that QS is a global regulatory system in this intracellular pathogen. The overlapping between BabR and VjbR targets suggest an unexpected cross-talk between these two regulators. Moreover, our results demonstrate that VjbR and BabR regulate many gene and/or proteins involved in stress response, metabolism and virulence. These targets are potentially involved in the adaptation of Brucella to the oxidative, pH and nutritional stresses encountered within the host. These findings highlight the involvement of QS in the virulence of Brucella and led us to suggest that this regulatory system could be implied in the spatial and sequential adaptation of Brucella to the host environment. Keywords: Quorum Sensing, Comparative gene expression, Brucella melitensis

Project description:Quorum-sensing (QS) is a cell-cell communication system that controls gene expression in many bacterial species, mediated by diffusible signal molecules. While the intracellular regulatory mechanisms of QS are often well-understood, the functional roles of QS remain controversial. In particular, the use of multiple signals by many bacterial species poses a serious challenge to current functional theories. Here we address this challenge by showing that bacteria can use multiple QS signals to infer both their social (density) and physical (mass-transfer) environment. Analytical and evolutionary simulation models show that the detection of and response to complex social/physical contrasts requires multiple signals with distinct half-lives and combinatorial (non-additive) responses to signal concentrations. We test these predictions using the opportunistic pathogen Pseudomonas aeruginosa, and demonstrate significant differences in signal decay between its two primary signal molecules as well as diverse combinatorial responses to dual signal inputs. QS is associated with the control of secreted factors, and we show that secretome genes are preferentially controlled by synergistic M-bM-^@M-^XAND-gateM-bM-^@M-^Y responses to multiple signal inputs, ensuring the effective expression of secreted factors in high density and low mass-transfer environments. Our results support a novel functional hypothesis for the use of multiple signals and, more generally, show that bacteria are capable of combinatorial communication. The two primary signal molecules of P. aeruginosa are the homoserine lactones N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butyryl-homoserine lactone (C4-HSL). Effects of the different signal molecules was assessed using a double QS synthase mutant of Pseudomonas aeruginosa PAO1 lasI/rhlI grown at 37M-BM-0C in 25 ml LB broth and 250 ml flasks with shaking at 200 r.p.m. in four treatments, each with a replicate: (a) no addition; (b) 3-oxo- C12-HSL; (c) C4-HSL; and (d) both 3-oxo-C12-HSL and C4-HSL.

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Expt 1: The contribution of vjbR and N-dodecanoyl homoserine lactone on gene regulation in Brucella melitensis

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