Project description:Rugose V. cholerae cells with an in-frame deletion of vpsT (VCA0952) were used in this study. Cells containing pBAD-Myc/His-B vector, or the vector containing a wild-type copy of vpsT, or vpsT with point mutations M17D, D60A, R134A, or I141E were grown to exponential phase and expression profiles compared.
Project description:We used RNA-seq to determine transcriptional profiles of whole guts or IPCs isolated from guts infected with wild type or type VI secretion system deficient Vibrio cholerae. We found significant differences between guts and progenitor cells infected wild type or type VI secretion system deficient Vibrio cholerae.
Project description:We identified the mutated gene locus in a pigment overproducing Vibrio cholerae mutant of strain A1552. The deduced gene product is suggested to be a oxidoreductase based on partial homology to putative homogentisate 1,2-dioxygenase in Pseudomonas aeruginosa and Mesorhizobium loti and we propose that the gene VC1345 in the V. cholerae genome be denoted hmgA in accordance with the nomenclature for other species. The hmgA::miniTn5 mutant showed a non-pigmented phenotype after complementation with a plasmid clone carrying the wild type hmgA+ locus. Microarray transcription analysis revealed that expression of hmgA, and the neighboring genes encoding a postulated two component sensor system, was growth phase dependent. Results from qRT-PCR analysis showed that hmgA operon expression was reduced in the rpoS mutant, but pigment production by the wild type V. cholerae or the hmgA mutant was not detectably influenced by the stationary phase regulator RpoS. The pigmented mutant showed increased UV resistance in comparison with the wild-type strain. Interestingly, the pigment producing mutant expressed more toxin co-regulated pili and cholera toxin in comparison with wild type V. cholerae. Moreover, the hmgA mutant showed a 5-fold increase in the ability to colonize the intestines of infant mice. A possible mechanism by which pigment production might cause induction of the ToxR regulon due to generation of hydrogen peroxide was supported by results from tests showing that externally supplied H2O2 led to higher TcpA levels. Taken together, our findings suggest that melanin pigment formation may play a role in V. cholerae virulence factor expression. Groups of assays that are related as part of a time series. Elapsed Time: Wild type V. cholerae strain A1552 bacteria were cultured statically for 4 h in AKI medium at 37C and then shifted to aerobic growth for 6 h using shaken culture flasks. Samples were taken at hourly during the this process. Keywords: time_series_design
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.
Project description:For expression analysis of wild-type V. cholerae, hapR, and rpoS deletion mutants in mid-exponential or stationary phase, the strains were grown to either OD600 of 0.3 or for 11 h in LB media at 37 0C, and bacteria from 2-ml culture were quickly pelleted, resuspended in Trizol reagent (GIBCO/BRL, San Diego, California, United States), and frozen on dry ice. RNA was isolated from the Trizol agent, treated with DNaseI (Ambion, Austin, Texas, United States), and cleaned by using the RNeasy kit (Qiagen, Valencia, California, United States). Labeling of cDNA and microarray hybridizations were performed as described [Yiliz et al. 2001, Mol. Micro. 53: 497-515]. Microarrays were scanned with a GenePix 400A instrument (Axon Instruments), using the GENEPIX 5.0 software. At least four microarray experiments were performed for each of two biological replicates for the tested strains. Gene expression of V. cholerae, rpoS, and hapR deletion mutants in stationary phase LB cultures was analyzed and compared to the wild-type parent under identical conditions. Gene expression of the wild-type parent during stationary phase after 11 h growth in LB was analyzed using RNA from an exponentially growing culture as a reference.
Project description:Lon protease is known to regulate various transcriptional regulators in other bacterial organisms. To understand whether lon protease is involved in transcriptional changes in Vibrio cholerae, wholel-genome level transcriptional profiling was performed using custom microarrays. Transcriptomes of lonA mutant and wild-type strains were compared in this study.
Project description:We identified the mutated gene locus in a pigment overproducing Vibrio cholerae mutant of strain A1552. The deduced gene product is suggested to be a oxidoreductase based on partial homology to putative homogentisate 1,2-dioxygenase in Pseudomonas aeruginosa and Mesorhizobium loti and we propose that the gene VC1345 in the V. cholerae genome be denoted hmgA in accordance with the nomenclature for other species. The hmgA::miniTn5 mutant showed a non-pigmented phenotype after complementation with a plasmid clone carrying the wild type hmgA+ locus. Microarray transcription analysis revealed that expression of hmgA, and the neighboring genes encoding a postulated two component sensor system, was growth phase dependent. Results from qRT-PCR analysis showed that hmgA operon expression was reduced in the rpoS mutant, but pigment production by the wild type V. cholerae or the hmgA mutant was not detectably influenced by the stationary phase regulator RpoS. The pigmented mutant showed increased UV resistance in comparison with the wild-type strain. Interestingly, the pigment producing mutant expressed more toxin co-regulated pili and cholera toxin in comparison with wild type V. cholerae. Moreover, the hmgA mutant showed a 5-fold increase in the ability to colonize the intestines of infant mice. A possible mechanism by which pigment production might cause induction of the ToxR regulon due to generation of hydrogen peroxide was supported by results from tests showing that externally supplied H2O2 led to higher TcpA levels. Taken together, our findings suggest that melanin pigment formation may play a role in V. cholerae virulence factor expression. Groups of assays that are related as part of a time series. Elapsed Time: Wild type V. cholerae strain A1552 bacteria were cultured statically for 4 h in AKI medium at 37C and then shifted to aerobic growth for 6 h using shaken culture flasks. Samples were taken at hourly during the this process. Keywords: time_series_design Using regression correlation
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.
Project description:Environmental isolates of Vibrio cholerae from California coastal water compared to reference strain N16961. A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's. Keywords: genotyping_design; array CGH