Project description:Transcriptional profiling of an El Tor biotype crp mutant The virulent V. cholerae El Tor Ogawa strain C7258 (Peru isolate 1991) and an isogenic deletion mutant (WL7258) lacking DNA sequences encoding the cAMP receptor protein were grown in LB medium to optical density at 600 nm of 1.5. The cultures were chilled in ice, cells quickly collected by centrifugation and total RNA imediately extracted. RNAwas extracted and purified using the Trizol plus RNA purification system (Invitrogen) followed RNEasy miniElute cleanup (Qiagen). RNA samples were conserved at - 80 C and used within a week. Keywords: Genetic modification
Project description:Transcriptional profiling of an El Tor biotype crp mutant The virulent V. cholerae El Tor Ogawa strain C7258 (Peru isolate 1991) and an isogenic deletion mutant (WL7258) lacking DNA sequences encoding the cAMP receptor protein were grown in LB medium to optical density at 600 nm of 1.5. The cultures were chilled in ice, cells quickly collected by centrifugation and total RNA imediately extracted. RNAwas extracted and purified using the Trizol plus RNA purification system (Invitrogen) followed RNEasy miniElute cleanup (Qiagen). RNA samples were conserved at - 80 C and used within a week. 4 replicates
Project description:Cholera is a diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139 that affects impoverished populations worldwide. The histone-like nucleoid structuring protein (H-NS) is a global regulator of environmentally-regulated gene expression that plays a fundamental role in V. cholerae adaptation to disparate ecological niches. We used RNA-seq to characterize the hns transcriptome of El Tor biotype V. cholerae. H-NS affected the expression of 18% of all predicted genes in a growth phase-dependent manner. It silenced the transcription of genes encoding virulence regulators and cytotoxic factors such as the VieSAB regulatory system, the repeat in toxin (RTX) and hemolysin. H-NS was 10 times more effective in silencing the vieSAB promoter in El Tor compared to classical biotype V. cholerae. In the El Tor biotype hns mutant, VieSAB significantly enhanced the expression of cholera toxin genes. For the RTX and hemolysin, we found that overexpression of the transcription activator HlyU diminished H-NS occupancy at the hlyA promoter but not at the rtxCA and rtxBDE promoters. H-NS had a significant impact on the cell envelope and the mutant expressed elevated rpoE encoding the extracytoplamic sigma factor E (sE), though this effect was indirect. A remarkable feature of the hns transcriptome was the down-regulation of numerous methyl-accepting chemotaxis proteins in early stationary phase that translated into diminished chemotaxis toward the amino acids glycine and serine. Our study suggests that H-NS transcriptional silencing can contribute to multiple phenotypic differences observed between V. cholerae biotypes, mainly by differentially repressing the VieSAB sensory pathway.
Project description:Cholera is a diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139 that affects impoverished populations worldwide. The histone-like nucleoid structuring protein (H-NS) is a global regulator of environmentally-regulated gene expression that plays a fundamental role in V. cholerae adaptation to disparate ecological niches. We used RNA-seq to characterize the hns transcriptome of El Tor biotype V. cholerae. H-NS affected the expression of 18% of all predicted genes in a growth phase-dependent manner. It silenced the transcription of genes encoding virulence regulators and cytotoxic factors such as the VieSAB regulatory system, the repeat in toxin (RTX) and hemolysin. H-NS was 10 times more effective in silencing the vieSAB promoter in El Tor compared to classical biotype V. cholerae. In the El Tor biotype hns mutant, VieSAB significantly enhanced the expression of cholera toxin genes. For the RTX and hemolysin, we found that overexpression of the transcription activator HlyU diminished H-NS occupancy at the hlyA promoter but not at the rtxCA and rtxBDE promoters. H-NS had a significant impact on the cell envelope and the mutant expressed elevated rpoE encoding the extracytoplamic sigma factor E (sE), though this effect was indirect. A remarkable feature of the hns transcriptome was the down-regulation of numerous methyl-accepting chemotaxis proteins in early stationary phase that translated into diminished chemotaxis toward the amino acids glycine and serine. Our study suggests that H-NS transcriptional silencing can contribute to multiple phenotypic differences observed between V. cholerae biotypes, mainly by differentially repressing the VieSAB sensory pathway. Transcriptome profiles of wild type and M-NM-^Thns mutant cells of the V. cholerae strain C7258 grown in LB medium were generated by Next Generation Sequencing using the Illumina HiSeq2000 platform. Samples from mid-exponential phase (OD600 0.5) and early stationary phase (OD600 2.0) were analyzed in duplicate
Project description:These experiments were performed to show a serogroup conversion of Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 El Tor (A1552) was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (4 ug each) from strain MO10, an O139 serogroup strain, was added after 24h and the cells were further grown for 24h. After detachment from the crab shell fragments, bacteria were poured into soft-agar and overlaid onto LB plates. Mukerjeee's El Tor phage V (a gift of Dr. M.S. Islam) was dropped onto the surface of the bacteria containing soft-agar. The plaques formed by killing non-transformed A1552 cells possessed resistant clones which were picked and further selected for opaque morphotype and agglutination by O139-specific antiserum. Four clones were selected from each independent experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone. Strain Names: ApO139#2 / ApO139#4 / ApO139#6 / ApO139#8 are four clones analyzed after the first experiment; AIIpO139#3 / AIIpO139#4 / AIIpO139#5 / AIIpO139#6 are four clones analyzed after the second independent experiment. Two MA replicates for each clone were done. CGHs of A1552 versus MO10 are provided as control.
Project description:In this study, we determined the TfoY regulon of V. cholerae using RNA-seq to better uderstand the protein's function. mRNA profiles of a WT V. cholerae O1 El Tor strain (A1552) and of a TfoY-producing derivative of the WT strain (A1552-TntfoY). 3 independent biological replicates are provided for each bacterial strain. The bacteria were grown to high cell density and in the presence of arabinose (to induce TfoY in strain A1552-TntfoY).
Project description:These experiments were performed to show a serogroup conversion of Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 El Tor (A1552) was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (4 ug each) from strain MO10, an O139 serogroup strain, was added after 24h and the cells were further grown for 24h. After detachment from the crab shell fragments, bacteria were poured into soft-agar and overlaid onto LB plates. Mukerjees El Tor phage V (a gift of Dr. M.S. Islam) was dropped onto the surface of the bacteria containing soft-agar. The plaques formed by killing non-transformed A1552 cells possessed resistant clones which were picked and further selected for opaque morphotype and agglutination by O139-specific antiserum. Four clones were selected from each independent experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone. Strain Names: AIIIpO139#1 / AIIIpO139#3 / AIIIpO139#4 / AIIIpO139#5 are four clones analyzed after the second experiment; AIVpO139#2 / AIVpO139#4 / AIVpO139#5 / AIVpO139#8 are four clones analyzed after the fourth independent experiment. Two MA replicates for each clone were done.
Project description:These experiments were performed to show serogroup conversion in Vibrio cholerae from O1 to O139 in a mixed communities / biofilms. For this purpose, V. cholerae O1 El Tor A1552 and VCO139-Kan strain (a MO10 derivative; O139 serogroup) were grown on crab shell fragments to induce natural competence for transformation. Transformants were selected on LB+Kan+Rif plates. O139 positive transformants have undergone a full exchange of the O1 region by the O139 region. This implies an exchange of an at least 32 kb spanning O1 genomic region by more than 42 kb of the O139 region. The transformation experiment was done at least five independent times; data from four experiments are shown; per experiment one to three clones were analysed by CGH with two experimental replicates each.
Project description:Investigation of whole genome gene expression level changes in a Vibrio cholerae O395N1 delta-nqrA-F mutant, compared to the wild-type strain. Total RNA recovered from wild-type cultures of VIbrio cholerae O395N1 and its nqrA-F mutant strain. Each chip measures the expression level of 3,835 genes from Vibrio cholerae O1 biovar eltor str. N16961 with twenty average probes/gene, with five-fold technical redundancy.