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.
Project description:Vibrio campbellii BAA-1116 was used as a Harveyi clade model organism to determine the impact of indole signaling on virulence. Gene expression analysis of V. campbellii grown in LB35 broth with or without 100 μM indole revealed that indole decreased: (1) V. campbellii virulence in shrimp and prawn challenge assays, (2) exopolysaccharide production, and (3) swimming motility. The results also indicated that indole inhibits quorum sensing-regulated bioluminescence and blocks the three-channel quorum sensing system by interfering with quorum sensing signal transduction.
Project description:Acyl-homoserine lactone (acyl-HSL) quorum sensing was first discovered in Vibrio fischeri where it serves as a key control element of the seven-gene luminescence (lux) operon. Since this initial discovery, other bacteria have been shown to control hundreds of genes by acyl-HSL quorum sensing. Until recently, it has been difficult to examine the global nature of quorum sensing in V. fischeri. However, the complete genome sequence of V. fischeri is now available and this has enabled us to use transcriptomics to identify quorum-sensing regulated genes and to study the quorum-controlled regulon of this bacterium. In this study, we used DNA microarray technology to identify over two-dozen V. fischeri genes regulated by the quorum sensing signal N-3-oxohexanoyl-L-homoserine lactone (3OC6-HSL). Keywords: Comparison of transcriptome profiles
Project description:Production of pyomelanin in the Vibrio campbellii hmgA mutant results in the repression of quorum sensing, bioluminescence and virulence
Project description:The selective advantage of bioluminescence in bacterial cells that do not form symbiotic relationships with aquatic animals is still not known. Some evidence suggests that bioluminescence plays a role in DNA repair by a photoreactivation process (Czyz 2000) and that non-bioluminescent strains are less virulent than their bioluminescent isogenic counterparts (Ruwandeepika 2010). All hypotheses to date suggest bioluminescence associated or mediated changes in gene expression, yet the evidence for this does not exist. In this study, we generated an in-frame luxAB deletion mutant (the two contiguous genes that encode for bacterial luciferase) and compared its mid-log phase gene expression profile with that of the wild type spontaneous streptomycin resistant (STR) V. campbellii BAA-1116 parental strain from which it was derived. Both mid-log phase transcriptomes were elucidated using custom designed whole genome microarrays (520694F, Affymetrix) to determine the effect luciferase has on V. campbellii gene expression. The virulence phenotypes of both strains were also subsequently tested in Artemia franciscana challenge experiments.
Project description:The selective advantage of bioluminescence in bacterial cells that do not form symbiotic relationships with aquatic animals is still not known. Some evidence suggests that bioluminescence plays a role in DNA repair by a photoreactivation process (Czyz 2000) and that non-bioluminescent strains are less virulent than their bioluminescent isogenic counterparts (Ruwandeepika 2010). All hypotheses to date suggest bioluminescence associated or mediated changes in gene expression, yet the evidence for this does not exist. In this study, we generated an in-frame luxAB deletion mutant (the two contiguous genes that encode for bacterial luciferase) and compared its mid-log phase gene expression profile with that of the wild type spontaneous streptomycin resistant (STR) V. campbellii BAA-1116 parental strain from which it was derived. Both mid-log phase transcriptomes were elucidated using custom designed whole genome microarrays (520694F, Affymetrix) to determine the effect luciferase has on V. campbellii gene expression. The virulence phenotypes of both strains were also subsequently tested in Artemia franciscana challenge experiments. Three biological replicates of the wild type (STR) and luxAB deletion mutant of V. campbellii BAA-1116 were grown to mid-log phase (15 h, 200 rpm, 30M-BM-0C, in 25 mL autoinducer bioassay medium) and total RNA was extracted from 1.0E+9 cells. Messenger RNA was isolated from the total RNA extracts treated with DNase, labeled with biotin, fragmented and hybridized to V. campbellii BAA-1116 whole genome microarrays (520694F, Affymetrix).
Project description:Vibrio campbellii BAA-1116 was used as model organism from the Harveyi clade to understand how melanization affected cellular phenotype, metabolism and virulence. An in-frame deletion of the homogentisate-1,2-dioxygenase (hmgA) gene resulted in the overproduction of a pigment in cell culture supernatants and cellular membranes that was identified as pyomelanin. Unlike previous demonstrations in Vibrio cholerae, Burkholderia cepacia and Pseudomonas aeruginosa, the pigmented V. campbellii mutant did not show increased UV or hydrogen peroxide resistance and was found to be ~2.7 times less virulent then the wild type strain in Penaeus monodon shrimp virulence assays. Microarray-based transcriptomic analyses revealed that the deletion of the hmgA gene and subsequent pyomelanin production negatively effected the expression of 129 genes involved in protein translation, cell division, membrane transport, electron transfer and amino acid utilization. The response was mediated in part by an impairment of the quorum sensing regulon as transcripts of the quorum sensing high cell density master regulator LuxR and other operonic members of this regulon were significantly repressed in the hmgA mutant. Taken together, the results suggest that the pyomelanization of V. campbellii sufficiently impairs the metabolic activities of this organism and renders it less fit and virulent then its isogenic wild type strain.