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: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: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: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: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:In marine Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome via homologous recombination. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in two Vibrio campbellii strains, DS40M4 and NBRC 15631, enables high frequencies of natural transformation. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4. This result is in contrast to Vibrio cholerae that requires the quorum-sensing regulator HapR to activate the competence regulator QstR. However, similar to V. cholerae, QstR is necessary for transformation in DS40M4. To investigate the difference in transformation frequencies between BB120 and DS40M4, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. BB120 encodes homologs of all known competence genes, but most of these genes were not induced by ectopic expression of TfoX, which likely accounts for the non-functional natural transformation in this strain. Comparison of transformation frequencies among Vibrio species indicates a wide disparity among even closely related strains, with Vibrio vulnificus having the lowest functional transformation frequency. We show that ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus.
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 µmol 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 – 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.