Project description:Many Gram-negative bacteria employ cell-to-cell communication mediated by N-acyl homoserine lactones (quorum sensing) to control expression of a wide range of genes including, but not limited to, genes encoding virulence factors. Outside the laboratory, the bacteria live in complex communities where signals may be perceived across species. We here present a newly found natural quorum sensing inhibitor, produced by the pseudomonads Pseudomonas sp. B13 and Pseudomonas reinekei MT1 as a blind end in the biodegradation of organochloride xenobiotics, which inhibits quorum sensing in P. aeruginosa in naturally occurring concentrations. This catabolite, 4-methylenebut-2-en-4-olide, also known as protoanemonin, has been reported to possess antibacterial properties, but seems to have dual functions. Using transcriptomics and proteomics, we found that protoanemonin significantly reduced expression of genes and secretion of proteins known to be under control of quorum sensing in P. aeruginosa. Moreover, we found activation of genes and gene products involved in iron starvation response. It is thus likely that inhibition of quorum sensing, as the production of antibiotics, is a phenomenon found in complex bacterial communities.
Project description:The gene PA2384 in Pseudomonas aeruginosa PAO1, annotated originally as unknown function, has been previously shown to be dramatically responsive to iron limitation. In the present study, PA2384 is shown by bioinformatics analysis to have a weak similarity to the N-termini DNA binding domain of fur, a well-known ferric uptake regulator. To experimentally investigate the function of PA2384 P. aeruginosa PAO1 recombinant (pUCP20::PA2384) overexpressing PA2384 and PA2384 knock-out mutant PAO1-23844 are constructed and studied. Physiological characterization in a carefully controlled cultivation system shows that the knockout strain needed a longer lag phase to grow and exhibited a significantly reduced production speed of siderophore (pyoverdine and pyochelin) in the stationary phase. Genome-scale transcriptional profiles at different growth stages are compared between the wild type and ∆PA2384 mutant grown under iron-limiting conditions. The expression of more than 350 genes is affected. Among them, seventy-one genes involved in iron uptake are significantly induced by PA2384. 102 quorum sensing (QS) dependent genes exhibited differential transcription. They include genes related to the biosynthesis of some important virulence factors such as pyocyanin, rhamnolipids and hydrogen cyanide. Transcription of genes responsible for the synthesis of Pseudomonas Quinolone Signal (PQS) is greatly advanced by the knockout of PA2384. We postulate that PA2384 affects QS via PQS. Furthermore, the knockout of PA2384 also results in altered expression of genes involved in electron transfer, central metabolism, phosphorus starvation and translation. It implies that PA2384 may affect more basic physiology than only respond to iron uptake and is a versatile global regulator in P. aeruginosa under iron starvation. Keywords: iron starvation, quorum sensing, time course
Project description:Many Gram-negative bacteria employ cell-to-cell communication mediated by N-acyl homoserine lactones (quorum sensing) to control expression of a wide range of genes including, but not limited to, genes encoding virulence factors. Outside the laboratory, the bacteria live in complex communities where signals may be perceived across species. We here present a newly found natural quorum sensing inhibitor, produced by the pseudomonads Pseudomonas sp. B13 and Pseudomonas reinekei MT1 as a blind end in the biodegradation of organochloride xenobiotics, which inhibits quorum sensing in P.M-bM-^@M-^Caeruginosa in naturally occurring concentrations. This catabolite, 4-methylenebut-2-en-4-olide, also known as protoanemonin, has been reported to possess antibacterial properties, but seems to have dual functions. Using transcriptomics and proteomics, we found that protoanemonin significantly reduced expression of genes and secretion of proteins known to be under control of quorum sensing in P.M-bM-^@M-^Caeruginosa. Moreover, we found activation of genes and gene products involved in iron starvation response. It is thus likely that inhibition of quorum sensing, as the production of antibiotics, is a phenomenon found in complex bacterial communities. Strain P. aeruginosa MPAO1 was cultivated under defined ABT medium. The orginal culture was divided in the early exponential phase into two parts. One was treated with 125M-NM-<M Protoanemonin and the other served as a control. The experiment was performed in duplicates.
Project description:The protein Aldehyde responsive quorum-sensing Inhibitor (ARQL) was exposed to increasing levels of glyoxal, to identify the glycation modifications.
Project description:Comparison of 3 yeast strains grown under nitrogen starvation, nitrogen rich conditions, with and without quorum sensing molecule 2-phenylethanol
Project description:Transcriptional profiling of DSF regulon under iron starvation in Xanthomonas oryzae pv. oryzicola (Xoc; BXOR1) using wild type, rpfF mutant and rpfF mutant with complementing plasmid pSC9. Cell-cell signalling mediated by quorum sensing molecule known as Diffusible signalling factor (DSF) is required for the virulence of Xanthomonas group of plant pathogens. The transcriptional profiling in this study is to elucidate the role of DSF in iron acquisition under the iron limitting environment which would lead to successful colonization and pathogenesis inside host.
Project description:To better understand the role of QscR in P. aeruginosa gene regulation and to better understand the relationship between QscR, LasR and RhlR control of gene expression we used transcription profiling to identify a QscR-dependent regulon. Our analysis revealed that QscR activates some genes and represses others. Some of the repressed genes are not regulated by the LasR-I or RhlR-I systems while others are. The LasI-generated 3-oxododecanoyl-homoserine lactone serves as a signal molecule for QscR. Thus QscR appears to be an integral component of the P. aeruginosa quorum sensing circuitry. QscR uses the LasI-generated acyl-homoserine lactone signal and controls a specific regulon that overlaps with the already overlapping LasR and RhlR-dependent regulons. Keywords: Quorum sensing regulon, Direct activation
Project description:Transcriptional profiling of DSF regulon under iron starvation in Xanthomonas oryzae pv. oryzicola (Xoc; BXOR1) using wild type, rpfF mutant and rpfF mutant with complementing plasmid pSC9. Cell-cell signalling mediated by quorum sensing molecule known as Diffusible signalling factor (DSF) is required for the virulence of Xanthomonas group of plant pathogens. The transcriptional profiling in this study is to elucidate the role of DSF in iron acquisition under the iron limitting environment which would lead to successful colonization and pathogenesis inside host. Agilent one-color experiment, Organism: Xanthamonus oryzicola, (AMADID-041087) Genotypic Technology Pvt. Ltd. designed Custom Xanthamonus oryzicola 8x15k Gene expresssion Array, Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:The gene PA2384 in Pseudomonas aeruginosa PAO1, annotated originally as unknown function, has been previously shown to be dramatically responsive to iron limitation. In the present study, PA2384 is shown by bioinformatics analysis to have a weak similarity to the N-termini DNA binding domain of fur, a well-known ferric uptake regulator. To experimentally investigate the function of PA2384 P. aeruginosa PAO1 recombinant (pUCP20::PA2384) overexpressing PA2384 and PA2384 knock-out mutant PAO1-23844 are constructed and studied. Physiological characterization in a carefully controlled cultivation system shows that the knockout strain needed a longer lag phase to grow and exhibited a significantly reduced production speed of siderophore (pyoverdine and pyochelin) in the stationary phase. Genome-scale transcriptional profiles at different growth stages are compared between the wild type and âPA2384 mutant grown under iron-limiting conditions. The expression of more than 350 genes is affected. Among them, seventy-one genes involved in iron uptake are significantly induced by PA2384. 102 quorum sensing (QS) dependent genes exhibited differential transcription. They include genes related to the biosynthesis of some important virulence factors such as pyocyanin, rhamnolipids and hydrogen cyanide. Transcription of genes responsible for the synthesis of Pseudomonas Quinolone Signal (PQS) is greatly advanced by the knockout of PA2384. We postulate that PA2384 affects QS via PQS. Furthermore, the knockout of PA2384 also results in altered expression of genes involved in electron transfer, central metabolism, phosphorus starvation and translation. It implies that PA2384 may affect more basic physiology than only respond to iron uptake and is a versatile global regulator in P. aeruginosa under iron starvation. Experiment Overall Design: mutant and wilde type biological replicates were analyzed at 3 timepoints
Project description:Vibrio campbellii BB120 (ATCC BAA-1116, previously designated as Vibrio harveyi) is a fundamental model strain for studying population density-based cell-to-cell communication, known as quorum sensing, among gram-negative bacteria. In V. campbellii BB120, sensing of autoinducers at high cell densities activates the expression of the master transcriptional regulator, LuxR, which controls the expression of genes involved in group behaviors. Unlike BB120, the Vibrio campbellii environmental isolate DS40M4 was recently shown to be capable of natural transformation, a process by which bacteria take up exogenous DNA and incorporate it into their genome via homologous recombination. Here, we compare other phenotypes between DS40M4 and BB120. We find that DS40M4 has a faster growth rate and stronger type VI secretion-mediated cell killing, whereas BB120 forms more robust biofilms and is bioluminescent. We exploited the power of natural transformation to rapidly generate >30 mutant strains to explore the function of DS40M4-encoded homologs of the BB120 quorum-sensing system. Our results show that DS40M4 has a similar quorum-sensing circuit to BB120 but with three distinct differences: 1) DS40M4 lacks the canonical HAI-1 autoinducer LuxM synthase but has an active LuxN receptor, 2) the quorum regulatory small RNAs (Qrrs) are not solely regulated by autoinducer signaling through the response regulator LuxO, and 3) the DS40M4 LuxR regulon is <100 genes, which is relatively small compared to the >400 genes regulated in BB120. This work illustrates that DS40M4 is a tractable and relevant model strain for studying quorum-sensing phenotypes in Vibrio campbellii.