Transcriptional analysis of a Vibrio choleare rpoH mutant vs wild-type after a heat-shock
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ABSTRACT:
ORGANISM(S): Vibrio cholerae O1 biovar El Tor str. N16961 Vibrio cholerae
PROVIDER: GSE6097 | GEO | 2007/01/03
SECONDARY ACCESSION(S): PRJNA97659
REPOSITORIES: GEO
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Project description:The bacterial heat-shock response is regulated by the alternative sigma factor sigma 32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoterss for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden increase in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurs without arabinose-induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased fivefold within 15 min of arabinose addition. Whole genome transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many of the genes induced by RpoH are essential for P. aeruginosa growth. Other stress response genes induced by RpoH are involved in nucleic acid damage and repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also has increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside resistance of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes such as aminoglycoside acetyl transferases that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient limiting conditions. We used Affymetrix microarrays to characterize the RpoH regulon in P. aeruginosa. Using the P. aeruginosa ∆rpoH strain with rpoH under control of the PBAD promoter, we were able to perform transcriptomic analysis of genes induced by a sudden increase (15 min) in the cellular concentration of RpoH, independent from a sudden increase in temperature.
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Project description:Understanding gene expression by bacteria during the actual course of human infection may provide important insights into microbial pathogenesis. In this study, we evaluated the transcriptional profile of Vibrio cholerae, the causative agent of cholera, in clinical specimens from cholera patients. We collected samples of human stool and vomitus that were positive by dark-field microscopy for abundant vibrios and used a microarray to compare gene expression in organisms recovered directly from the early and late stages of human infection. Our results reveal that V. cholerae gene expression within the human host environment differs from patterns defined in in vitro models of pathogenesis. tcpA, the major subunit of the essential V. cholerae colonization factor, was significantly more highly expressed in early compared with late infection; however, the genes encoding cholera toxin were not highly expressed in either phase of human infection. Furthermore, expression of the virulence regulators, toxRS and tcpPH, was uncoupled. Interestingly, the pattern of gene expression indicates that the human upper intestine may be a uniquely suitable environment for the transfer of genetic elements that are important in the evolution of pathogenic strains of V. cholerae. These findings provide a more detailed assessment of the transcriptome of V. cholerae in the human host than previous studies of organisms in stool alone and have implications for cholera control and the design of improved vaccines. The V. cholerae microarray consists of 3,890 full-length PCR products representing the annotated open reading frames from the initial release of the V. cholerae N16961 genome. Each labeling and hybridization was performed in duplicate. Genomic DNA was used as a universal internal control for the quality of the microarray and to allow for the comparison of results across multiple experiments. Data were normalized using locally-weighted regression (Lowess) to obtain the relative abundance of each transcript as an intensity ratio with respect to that of genomic DNA. High correlation coefficients were observed between technical replicates (Pearsonâs correlation coefficient (r) > 0.80) and between results of separate clinical specimens of vomitus (r > 0.77) and of stool (r > 0.80). Hence, the results from the two clinical vomitus specimens and the five clinical stool specimens were pooled. Fold changes for the relative expression of a given gene between the two clinical specimens were calculated by dividing the normalized median intensity ratios with respect to genomic DNA.
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