Time course analysis of genes regulated by E. coli sigma 32 (rpoH) after overexpression
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE4321 | GEO | 2006/03/06
SECONDARY ACCESSION(S): PRJNA94741
REPOSITORIES: GEO
Similar Datasets
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.
2022-11-06 | GSE217157 | GEO
Project description:Time course analysis of genes regulated by E. coli sigma 32 (rpoH) after overexpression
| PRJNA94741 | ENA
Project description:Vibrio cholerae, the cause of cholera, can grow in a variety of environments outside of human hosts. During infection, the pathogen must adapt to significant environmental alterations, including the elevated temperature of the human gastrointestinal tract. σ32, an alternative sigma factor encoded by rpoH, activates transcription of genes involved in the heat-shock response in several bacterial species. We defined the V. cholerae RpoH regulon by comparing the whole genome transcription profiles of the wild-type and rpoH mutant strains after a temperature up-shift. Most of the V. cholerae genes expressed in an RpoH-dependent manner after heat-shock encode proteins that influence protein fate, such as proteases and chaperones, or are of unknown function. Keywords: heat-shock response, rpoH
2007-01-03 | GSE6097 | GEO
Project description:Sinorhizobium meliloti can live as a soil saprophyte, and can engage in a nitrogen fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma factors interacting with core RNA polymerase. The S. meliloti genome encodes 14 of these alternative sigmas, including two putative RpoH (heat shock) sigmas. We used custom Affymetrix Symbiosis Chips to characterize the global transcriptional response of S. meliloti rpoH1, rpoH2 and rpoH1 rpoH2 mutants during heat shock and stationary phase growth. Under these conditions, expression of over 300 genes is dependent on rpoH1 and rpoH2.
2012-10-02 | GSE36186 | GEO
Project description:Identification of genes regulated by rpoH in Pseudomonas aeruginosa
| PRJNA897807 | ENA
Project description:DNA microarray analysis of genes regulated by the alternative sigma factor, sigma E (rpoE). Sigma E-dependent genes were initially identified by comparing a wild type (wt) E. coli K-12 strain that has a low level of sigma E, with a strain over-expressing sigma E (following induction of rpoE from an inducible promoter by IPTG). We monitored changes in gene expression in 4 separate time-courses after induction and used SAM (Statistical Analysis of Microarrays) to identify 75 significantly induced and 8 significantly repressed genes. Some of these genes are part of operons in which other gene members were clearly induced but were not marked as significant in our strict selection criteria. Therefore, to fully describe the sigma E regulon we expanded this set by using the statistics from SAM to analyze the reproducibility and significance of the expression ratios of all the genes adjacent to and in the same orientation as the highly significant genes. This gave 96 genes organized in 50 sigma E-dependent transcription units (TUs), of which 42 were induced and 8 were repressed. This study is detailed in Rhodius et al 2006 (PLoS Biol 4(1): e2) Keywords: time course
2005-10-13 | GSE3437 | GEO
Project description:To characterize regulons of alternative sigma factor SigH, SigL, and SigC in Listeria monocytogenes, in-frame mutant strains were created in the 10403S background. Regulons controlled by these 3 alternative sigma factors were characterized by whole-genome microarrays. The L. monocytogenes 10403S wild type and sigma factor null mutation strains were grown at 37 °C to stationary phase (defined in this study as growth to OD600 = 1.0, followed by incubation for an additional 3 h) prior to RNA isolation. Transcriptional profiles of 10403S wild type were compared to those of null mutation strain. In addition to stationary phase condition, SigC regulon was further characterized using heat stress (cultures grown to log phase at OD600 = 0.4, 37 °C and then exposed to heat at 55 °C for 10 min) and a condition with IPTG-inducible expression of sigC (sigC gene is placed under Pspac promoter using pLIV2 vector in wild type 10403S background). Under these conditions, expression profiles were compared between (i) wild type and sigC null mutant for heat stress and (ii) IPTG-inducible sigC strain and sigC null mutant, respectively. Using adjusted P < 0.05 and ≥ 1.5 fold change as cutoff values, microarray analyses identified 169 SigH-dependent, 51 SigL-dependent, and 3 SigC-dependent genes. Keywords: Listeria monocytogenes, alternative sigma factor, SigH, SigL, SigC
2011-04-12 | GSE24339 | GEO
Project description:Sinorhizobium meliloti can live as a soil saprophyte, and can engage in a nitrogen fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma factors interacting with core RNA polymerase. The S. meliloti genome encodes 14 of these alternative sigmas, including two putative RpoH (heat shock) sigmas. We used custom Affymetrix Symbiosis Chips to characterize the global transcriptional response of S. meliloti rpoH1, rpoH2 and rpoH1 rpoH2 mutants during heat shock and stationary phase growth. Under these conditions, expression of over 300 genes is dependent on rpoH1 and rpoH2. Gene expression profiling of Sinorhizobium meliloti Rm1021 and its isogenic rpoH1, rpoH2, and rpoH1rpoH2 mutants, subjected to heat shock or stationary phase growth, was performed using custom Affymetrix GeneChips
2012-10-02 | E-GEOD-36186 | biostudies-arrayexpress
Project description:When performed at single bp resolution, the genome-wide location, occupancy level, and structural organization of DNA binding proteins provides mechanistic insights into genome regulation. Here we use ChIP-exo to provide the first high resolution view of the epigenomic organization of the E. coli transcription machinery and nucleoid structural proteins when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We suggest that indirect readout of DNA shape at the flanks of cognate motifs provide major contributions to site specificity at promoter positions -35/-24 and -10/-12. We examined the site specificity of three sigma factors (RpoD/70, RpoH/32, and RpoN/54), RNA polymerase (RNAP or RpoA, B, C) and two nucleoid proteins (Fis and IHF). Our results confirm and refine reports that RpoD binds most annotated promoters, whereas RpoH and RpoN bind a much smaller subset, each through their cognate motifs. However, only upon heat shock does RpoH becomes active for RNAP recruitment. In contrast, upon heat shock RpoD remains active at its cognate promoters (including at heat shock genes), whereas RpoN remains inactive at its cognate promoters. RpoN binds ~1,000 non-annotated RpoN motifs, which may reflect a large number of condition-specific transcription units. Occupancy patterns of sigma factors and RNAP suggest a common promoter recruitment mechanism that differs from the long-standing views that sigma and RNAP are co-recruited as a complex, and also simultaneously dissociate from promoters. Our findings suggest that sigma factors are recruited and/or maintained at most promoters via an RNAP-independent mechanism. When RNAP arrives, it dwells for a relatively short time before clearing the promoter, leaving sigma behind. Taken together these findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape and redefine mechanistic steps in regulated promoter assembly in E. coli.
2022-02-21 | GSE189206 | GEO
Project description:In this experiment two strains were compared. The control strain was the wild type strain containing the plasmid, pCD341. pCD341 is an inducible expression vector, a plasmid which can be used to make a bacterium produce a protein of interest. It is called inducible, because the protein of interest will only be produced if an inducer, in this case, IPTG, is present. In the control strain pCD341 is "empty", meaning the gene encoding the protein of interest has not been inserted into the vector, so when you add IPTG nothing will be produced. Because producing plasmid and being exposed to IPTG can stress cells, the wild type strain harboring the "empty" plasmid was used as the control. The experimental strain DL1/pCD341RpoN contained the a "full" vector into which the gene for the sigma factor RpoN was been inserted. Thus when IPTG is added to the growth medium, this strain will overproduce RpoN. RpoN is an alternative sigma factor, a subunit of the RNA polymerase which is required for binding of the polymerase to specific promoters and is therefore required for the transcription of genes with these promoter sequences. Most bacteria have multiple sigma factors, many of which have specialized functions. In many other bacteria, RpoN is required for the expression of genes involved in nitrogen fixation. However, in G.sulfurreducens and myxococcus xanthus, RpoN appears to essential for survival under all conditions tested and its function and promoter specificity are not known. This experiment was performed in order to gain insight into the physiological function of G. sulfurreducens RpoN. Since we could not knock out RpoN, we decided to try overproducing it, hoping that overproducing RpoN would increase expression of genes who's promoters are recognized by RpoN. We took the wild type strain + empty vector and and the wild type strain with the RpoN vector and transferred them both into medium containing IPTG. For this experiment, we used batch cultures, or closed culture containers (no media nutrients or cells flowing in and out). We harvested cells at mid log phase for RNA preparation. There were three biological replicates and two replicate hybridizations were carried out for each. Keywords: cell type comparison
2009-07-13 | GSE8022 | GEO