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: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 is a gram-negative bacterial pathogen that is both free-living and a pathogen of marine organisms and exhibits swimming motility via a single, polar flagellum. Swimming motility is a critical virulence factor in V. campbellii pathogenesis, and disruption of the flagellar motor significantly decreases host mortality. However, while V. campbelli encodes homologs of flagellar and chemotaxis genes conserved by other members of the Vibrionaceae, the regulatory network governing these genes have not been explored. We systematically deleted all 63 known flagellar and chemotaxis genes in V. campbellii and examined their effects on motility compared to their homologs in other Vibrios. We specifically focused on assessing the roles of the core flagellar regulators of the flagellar regulatory hierarchy established in other Vibrios: rpoN, flrA, flrC, and fliA. Although V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other Vibrios, we observed two critical differences: the σ54-dependent regulator FlrA is dispensable for motility, and Class II gene expression is independent of σ54 regulation. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae.
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: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. Three biological replicates of V. campbellii BAA-1116 (str) and hmgA mutants were grown to stationary phase (48 h, 200 rpm, 30M-BM-0C, in 50 mL LM 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.
Project description:Although many members of the genus Vibrio are known to inhabit the marine photic zone, an understanding of the influence of light on the molecular physiology of Vibrio spp. has largely been neglected. To begin to characterize the photophysiology of one such Vibrio sp. (Vibrio campbellii ATCC strain BAA-1116) we used microarray-based expression profiling to compare the transcriptomes of illuminated versus dark cell cultures. Specficially, we compared the transcriptomes of wild type V. campbellii (STR) cells that were cultured in M9 minimal salts medium plus glucose under two conditions: (i) after 24 hours of continuous dark and (ii) after a 12 hour dark:12 hour light cycle (white light illumination at 54 M-BM-5mol photons s-1 m-2). The results revealed a large photostimulon (differential expression of ~20% of the V. campbellii genome; adjusted p value < 0.0001) that surprisingly included ~75% of the type III secretion system (T3SS) genes which were found to be 1.6 M-bM-^@M-^S 5.4X more abundant in illuminated cultures. These findings, which were confirmed by quantitative reverse transcription PCR and quantitative membrane proteomics, strongly suggest that the photostimulon of strain BAA-1116 includes the T3SS. Five biological replicates of V. campbellii BAA-1116 (STR) were grown to log phase (200 rpm, 30M-BM-0C, 25 mL M9 minimal salts medium plus glucose in 125 mL baffled Erlenmeyer flasks) under continuous dark for 24 hours or under a 12 hour dark:12 hour light cycle (white light illumination at 54 M-BM-5mol photons s-1 m-2) 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:Small RNA and regulatory RNA discovery has been based on (i) computational predictions mainly in intergenic regions, (ii) microarray experiments, (iii) shotgun cloning of cDNA libraries, (iv) classical cloning of abundant small RNAs after size fractionation in polyacrylamide gels and (v) copurification with proteins like Hfq, CsrA and RNA polymerase. In this study, we attempted to experimentally validate in silico small and regulatory RNA predictions for V. campbellii BAA-1116 using a microarray expression profiling strategy. Specifically, we investigated transcripts from mid-log phase cultures grown in nutrient rich media and examined their hybridization to tiled probes targeting annotated V. campbellii BAA-1116 intergenic regions. Keywords: small RNA discovery Five replicates of wild type V. campbellii BAA-1116 were grown to Mid-log phase, 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 microarray (520694F, Affymetrix).