Diversity of HrpL-regulons across Pseudomonas syringae isolates
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ABSTRACT:
ORGANISM(S): Pseudomonas syringae
SUBMITTER: Emma Johnson
PROVIDER: E-GEOD-46930 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The effects of RyhB expression were examined by Ribo-seq and RNA-seq after 10 min to avoid indirect effects. Expression of RyhB was induced by arabinose from cells carrying pBAD-ryhB plasmid. The RyhB expression was confirmed by real-time PCR. As a control, cells with vector pNM12 were grown and induced. The cells were pulverized and total mRNAs were extracted from the pulverized cells and processed for Ribo-seq and RNA-seq.
2014-11-01 | E-MTAB-2903 | biostudies-arrayexpress
Project description:We implemented transcriptional analysis methods using cDNA and high-throughput sequencing data to identify HrpL-regulated genes for six strains of Pseudomonas syringae
2014-01-01 | GSE46930 | GEO
Project description:The bacterial Lon protease participates in a variety of biological processes. In Pseudomonas syringae, mutation of lon is known to activate hrpL and a few hrpL-regulated genes in the rich medium. The elevated expression of hrpL and hrpL-regulated genes results from of increased stability of HrpR, the transcriptional activator of hrpL, in the lon mutant. Here we conducted a microarray analysis to determine genes that are differently expressed in the lon- mutant of P. s. pv. tomato DC3000 grown in the rich medium KB. Most genes induced in the lon- mutant belong to the HrpL-regulon or are related to the transcription, protein synthesis, and energy metabolism. The major group of genes reduced in the lon- mutant is related to cell wall biogenesis. The HrpL-regulated genes exhibit different induction patterns in the lon- mutant, suggesting the involvement of regulators additional to HrpL in regulating these genes. Compared with the wild type bacteria, the lon- mutants exhibit elevated hrpL expression in KB medium but reduced hrpL expression in the minimal medium (MM). The reduced hrpL RNA is correlated with the reduced hrpR and hrpS RNAs, suggesting that the Lon-mediated regulation of hrpL involves different mechanisms in KB and MM. Consistent with the reduced expression of the hrpL in MM, the lon- mutants are less pathogenic on host plants. Keywords: Expression profiles of lonB mutant in KB three samples ( biological replicates) of mutation were analyzed
2007-10-14 | E-GEOD-6121 | biostudies-arrayexpress
Project description:To compare the transcriptional profiling of a FimZ-expressing strain or non-expressing strain in E. coli under stationary phase. For overexpression of fimZ gene was cloned into pBAD33 plasmid under control of the arabinose-inducible PBAD promoter, which was induced by addition of 0.2% arabinose. Goal was to determine the FimZ regulon in E. coli. Biological replicates: 2 replicates.
2021-08-05 | GSE181402 | GEO
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:To determine the targets of the small regulatory RNA DapZ in S. Typhimurium, we looked at the effect of a short pulse of DapZ over-expression on the Salmonella transcriptome, as well as a DapZ variant that lacks the GcvB-like R1 region. To achieve over-expression, the wild-type DapZ and its variant were cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control (also induced by L-arabinose). 2 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674-1688.
2015-12-01 | GSE38523 | GEO
Project description:The bacterial Lon protease participates in a variety of biological processes. In Pseudomonas syringae, mutation of lon is known to activate hrpL and a few hrpL-regulated genes in the rich medium. The elevated expression of hrpL and hrpL-regulated genes results from of increased stability of HrpR, the transcriptional activator of hrpL, in the lon mutant. Here we conducted a microarray analysis to determine genes that are differently expressed in the lon- mutant of P. s. pv. tomato DC3000 grown in the rich medium KB. Most genes induced in the lon- mutant belong to the HrpL-regulon or are related to the transcription, protein synthesis, and energy metabolism. The major group of genes reduced in the lon- mutant is related to cell wall biogenesis. The HrpL-regulated genes exhibit different induction patterns in the lon- mutant, suggesting the involvement of regulators additional to HrpL in regulating these genes. Compared with the wild type bacteria, the lon- mutants exhibit elevated hrpL expression in KB medium but reduced hrpL expression in the minimal medium (MM). The reduced hrpL RNA is correlated with the reduced hrpR and hrpS RNAs, suggesting that the Lon-mediated regulation of hrpL involves different mechanisms in KB and MM. Consistent with the reduced expression of the hrpL in MM, the lon- mutants are less pathogenic on host plants. Keywords: Expression profiles of lonB mutant in KB
2007-10-15 | GSE6121 | GEO
Project description:The type III secretion system (T3SS) is the main machinery for Pseudomonas syringae and other Gram-negative bacteria to invade plant cells. HrpR/HrpS heterodimer are key factors to activate HrpL which induces all T3SS genes by binding to a ‘hrp box’ in promoters. However, the molecular mechanisms of HrpRS on T3SS or non-T3SS genes have not been fully understood. Following by chromatin immunoprecipitation coupled to high-throughput DNA sequencing (ChIP-seq), we found that HrpRSL had 8, 38, and 36 targets on the genome, respectively. HrpS directly bound to the promoter regions of T3SS genes (hrpK1, hrpA2 and hopAJ1) as well as a group of non-T3SS genes (PSPPH_1496, PSPPH_1525, PSPPH_3494 and PSPPH_3495). Subsequent biochemical and genetic assays showed that HrpS independently regulated these genes in a hrpL deletion strain. In addition, a HrpS-binding motif (GTGCCAAA) in the hrpL promoter was identified by truncation, which was verified by EMSA in vitro and lux-reporter assay in vivo. On the contrary, HrpR alone couldn’t activate hrpL. Overall, our results demonstrated that HrpS directly regulated a group of T3SS and non-T3SS genes, which is independent on HrpL. HrpS functions as the center of the regulatory cascade in T3SS. This work deciphered the key HrpRSL-T3SS regulatory network, which provides insights to develop new strategies to control P. syringae infection in the future.
2018-06-30 | GSE103921 | GEO
Project description:Background Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. Methodology/Principal Findings To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. Conclusion/Significances We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens. Analysis used aerobic steady state RNA from wild-type as control samples for comparison to the experimental samples of hrpL mutant taken 6 h post-inoculation in T3SS inducible minimum media. The microarray results are based on the geomean of ten normalized expression values derived from three biological replicates and two dye-swap experiments with a technology replicate for each array.
2010-10-21 | E-GEOD-17185 | biostudies-arrayexpress
Project description:To screen the potential targets of Salmonella 3’ UTR derived sRNA FadZ, we compared global mRNA profiles in FadZ-deficient and -pulse expressed strains. The 40-nt FadZ sequences was cloned onto a plasmid downstream of the pBAD promoter and expression was induced by L-arabinose for 10-min in LB broth. Transcriptomic profiles were compared with strain carrying the empty control plasmid.
2021-12-27 | GSE192570 | GEO