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Chip-seq analysis of hrpRSL and Lon in Pseudomonas syringas

ABSTRACT: Pseudomonas syringae uses HrpRSL to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanism and the regulons of HrpRSL have yet to be fully elucidated. Here, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) on HrpRSL and Lon. The direct regulation of these genes by corresponding regulator has been confirmed by Electrophoretic mobility shift assays (EMSAs) and quantitative real-time polymerase chain reactions (qRT-PCR). Binding motifs are found by using MEME suite and verified by footprint assays in vitro. Collectively, this work provides new cues to better understand the detailed regulatory networks of T3SS systems in P. syringae. ChIP-seq analysis of HrpRSL and Lon in Pseudomonas syringas

ORGANISM(S): Pseudomonas syringae

SUBMITTER: Xin Deng

PROVIDER: E-GEOD-65437 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae

Project description:Pseudomonas syringae uses two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanism and the regulons of RhpRS have yet to be fully elucidated. Here we show that RhpS functions as an autokinase, an RhpR kinase, and a P-RhpR phosphatase. RhpR can also be phosphorylated by small phosphodonor acetyl phosphate. A specific RhpR-binding site containing an inverted repeat (IR) motif GTATC-N6-GATAC, was mapped to its own promoter by DNase I footprint analysis. Electrophoretic mobility shift assay (EMSA) indicated that P-RhpR has higher binding activity than RhpR to the IR motif. To identify additional targets of RhpR in P. syringae, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), which detected 167 enriched loci including the hrpR promoter, indicating the direct regulation of T3SS cascade genes by RhpR. Genome-wide microarray analysis showed that, besides the T3SS cascade genes, RhpR differentially regulates a large set of genes of various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes to coordinate the T3SS and many other biological processes. ChIP-seq analysis of RhpR

2014-10-01 | E-GEOD-58533 | biostudies-arrayexpress

Chip-seq analysis of hrpRSL and Lon in Pseudomonas syringas

Project description:Pseudomonas syringae uses HrpRSL to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanism and the regulons of HrpRSL have yet to be fully elucidated. Here, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) on HrpRSL and Lon. The direct regulation of these genes by corresponding regulator has been confirmed by Electrophoretic mobility shift assays (EMSAs) and quantitative real-time polymerase chain reactions (qRT-PCR). Binding motifs are found by using MEME suite and verified by footprint assays in vitro. Collectively, this work provides new cues to better understand the detailed regulatory networks of T3SS systems in P. syringae.

2015-12-31 | GSE65437 | GEO

RNA-seq and ChIP-seq for Lon

Project description:Lon protease plays vital roles in many biological processes in Pseudomonas syringae, including type III secretion systems (T3SS), transcription regulation, protein synthesis and energy metabolism. Lon also functions as a transcriptional regulator in other bacterial species (e.g., Escherichia coli and Brevibacillus thermoruber). Therefore, we hypothesise that Lon has dual functions in P. syringae. To reveal the molecular mechanisms of Lon as a transcriptional regulator and protease under different environmental conditions, we used a combination of transcriptome sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the genes or proteins regulated by Lon. As a transcriptional regulator, Lon bound to the promoter regions of PSPPH_4788, gacA, fur, gntR, clpS, lon and glyA and consequently regulated 1-dodecanol oxidation activity, motility, pyoverdine production, gluconokinase activity, N-end rule pathway, lon expression and serine hydroxymethyltransferase (SHMT) activity in King’s B medium (KB). In minimal medium (MM), Lon regulated SHMT activity and lon expression by binding to the promoter regions of glyA and lon, respectively. As a protease, Lon regulated the T3SS and metabolic pathways (e.g., amino acid metabolism). In MM, Lon regulated the polysaccharide metabolic process by controlling PSPPH_0514, AlgA, CysD and PSPPH_4991. Taken together, these data demonstrate that Lon acts as a transcriptional regulator or protease in different environments and tunes its virulence and metabolic functions accordingly.

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2023-06-02 | PXD040715 | Pride

Plant signals anticipate the induction of the type III secretion system in Pseudomonas syringae 2 pv. actinidiae facilitating efficient temperature-dependent effector translocation

Project description:Disease resistance in plants depends on a molecular dialogue with microbes that involves many known chemical effectors, but the time course of the interaction and the influence of the environment are largely unknown. The outcome of host–pathogen interactions is thought to reflect the offensive and defensive capabilities of both players. When plants interact with Pseudomonas syringae, several well-characterized virulence factors contribute to early bacterial pathogenicity, including the type III secretion system (T3SS), which must be activated by signals from the plant and environment to allow the secretion of virulence effectors. The manner in which these signals regulate T3SS activity is still unclear. Here, we strengthen the paradigm of the plant–pathogen molecular dialogue by addressing overlooked details concerning the timing of interactions, specifically the role of plant signals and temperature on the regulation of bacterial virulence during the first few hours of the interaction. Whole-genome expression profiling after 1 h revealed that the perception of plant signals from kiwifruit or tomato extracts anticipates T3SS expression in P. syringae pv. actinidiae compared to apoplast-like conditions, facilitating more efficient effector transport in planta, as revealed by the induction of a temperature-dependent hypersensitive response in the non-host plant Arabidopsis thaliana Col-0. Our results show that, in the arms race between plants and bacteria, the temperature-dependent timing of bacterial virulence versus the induction of plant defenses is probably one of the fundamental parameters governing the outcome of the interaction.

2022-10-12 | PXD036021 | Pride

Molecular Mechanisms of Master Regulator VqsM mediating Quorum-sensing and Antibiotic Resistance in Pseudomonas aeruginosa

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2014-08-04 | E-GEOD-57284 | biostudies-arrayexpress

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Project description:Pseudomonas syringae, a Gram-negative plant pathogen, infects more than 50 crops with its type III secretion system (T3SS) and causes severe economic losses around the world. Although the mechanisms of virulence-associated regulators of P. syringae T3SS have been studied for decades, the crosstalk and network underlying these regulators are still elusive. Previously, we have individually studied a group of T3SS regulators, including AefR, HrpS, and RhpRS. In the present study, we found 4 new T3SS regulator genes (envZ, ompR, tsiS and phoQ) via transposon-mediated mutagenesis. Two-component systems EnvZ and TsiS natively regulate T3SS. In order to uncover the crosstalk between 16 virulence-associated regulators, (including AefR, AlgU, CvsR, GacA, HrpL, HrpR, HrpS, MgrA, OmpR, PhoP, PilR, PsrA, RhpR, RpoN, TsiR and Vfr) in P. syringae, we mapped an intricate network named PSVnet (Pseudomonas syringae Virulence Regulatory Network) by combining differentially expression genes in RNA-seq and binding loci in ChIP-seq of all regulators.

2021-03-29 | GSE138204 | GEO

Whole-transcriptome microarray analysis of Pseudomonas syringae pv. actinidiae CRAFRU 8.43 and Pseudomonas syringae pv. tomato DC3000 grown in apoplast-like conditions supplemented or not with kiwifruit or tomato extract

Project description:Purpose: The outcome of host–pathogen interactions is thought to reflect the offensive and defensive capabilities of both players. When plants interact with Pseudomonas syringae, several well-characterized virulence factors contribute to early bacterial pathogenicity, including the type III secretion system (T3SS), which must be activated by signals from the plant and environment to allow the secretion of virulence effectors. The manner in which these signals regulate T3SS activity is still unclear. Conlusion: the analysis revealed that the perception of plant signals from kiwifruit or tomato extracts anticipates T3SS expression in P. syringae pv. actinidiae compared to apoplast-like conditions

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ChIP-seq analyses of 10 quorum sensing regulators in Pseudomonas aeruginosa

Project description:The Pseudomonas aeruginosa quorum-sensing (QS) systems contribute to bacterial homeostasis and pathogenicity. Although many regulators have been characterized to control the production of virulence factors and QS signaling molecules, its detailed regulatory mechanisms still remain elusive. Here, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) on 10 key QS regulators. The direct regulation of these genes by corresponding regulator has been confirmed by Electrophoretic mobility shift assays (EMSAs) and quantitative real-time polymerase chain reactions (qRT-PCR). Binding motifs are found by using MEME suite and verified by footprint assays in vitro. Collectively, this work provides new cues to better understand the detailed regulatory networks of QS systems. ChIP-seq of 10 QS regulators in Pseudomonas aeruginosa

2015-08-04 | E-GEOD-65356 | biostudies-arrayexpress

Vibrio cholerae lon protease mutant microarray analysis

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.

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