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: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.

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Quorum Sensing regulation in Brucella melitensis 16M

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