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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

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