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| Status |
Public on Nov 06, 2022 |
| Title |
Identification of genes regulated by rpoH in Pseudomonas aeruginosa |
| Organism |
Pseudomonas aeruginosa |
| Experiment type |
Expression profiling by array
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| Summary |
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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| Overall design |
For untreated control samples, P. aeruginosa ∆rpoH (PBAD-rpoH) cells were cultured plantonically for 8 hours in Biofilm Minimal Medium. For induced test samples, P. aeruginosa ∆rpoH (PBAD-rpoH) cells were cultured planktonically for 7.75 hours in Biofilm Minimal Medium, and then rpoH was induced for 15 minutes with 0.1% arabinose. For control and test samples, cells were collected at 8 hours and RNA was extracted for transcriptomic studies.
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| Contributor(s) |
Williamson KS, Dlakić M, Akiyama T, Franklin MJ |
| Citation(s) |
36675051 |
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| Submission date |
Nov 03, 2022 |
| Last update date |
Feb 05, 2023 |
| Contact name |
Michael J Franklin |
| Organization name |
Montana State University
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| Department |
Microbiology and Cell Biology
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| Lab |
Franklin Lab
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| Street address |
109 Lewis Hall
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| City |
Bozeman |
| State/province |
MT |
| ZIP/Postal code |
59717 |
| Country |
USA |
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| Platforms (1) |
| GPL84 |
[Pae_G1a] Affymetrix Pseudomonas aeruginosa Array |
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| Samples (6)
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| GSM6705792 |
Untreated control culture, biological replicate 1 |
| GSM6705793 |
Untreated control culture, biological replicate 2 |
| GSM6705794 |
Untreated control culture, biological replicate 3 |
| GSM6705795 |
Induced culture, biological replicate 1 |
| GSM6705796 |
Induced culture, biological replicate 2 |
| GSM6705797 |
Induced culture, biological replicate 3 |
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| Relations |
| BioProject |
PRJNA897807 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE217157_RAW.tar |
3.9 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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