A comprehensive analysis of pneumcoccal two-component system regulatory networks
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ABSTRACT:
ORGANISM(S): Streptococcus pneumoniae
PROVIDER: GSE225902 | GEO | 2024/04/02
REPOSITORIES: GEO
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Project description:To identify YehT-regulated genes, the transcriptome profiles of E. coli cells overproducing either the response regulator (RR) YehT or the RR KdpE (control) were comparatively analyzed. The expression level of 32 genes varied more than 8-fold.
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Project description:Identification of the direct target genes of a response regulators (RRs) of a bacterial two-component system (TCS) is critical to understand the roles of the TCS in bacterial environmental adaption and pathogenesis. A. pleuropneumoniae is an important respiratory bacterial pathogen causing great economic losses to swine industry worldwide. The triggering signal(s) and targets of the RR NarP belonging to the TCS NarQ/NarP of A. pleuropneumoniae are still unknown. In the present study, a DNA-affinity-purified sequencing (DAP-Seq) approach was established. The upstream regions of a total of 131 candidate genes from the genome of A. pleuropneumoniae were shown to be co-purified with the activated NarP protein. Electrophoretic mobility shift assay results confirmed the interactions of NarP with the promoter regions of five selected target genes, including dmsA, pgaA, ftpA, cstA and ushA. Then, the EMSA-confirmed target genes were shown to be significantly up-regulated in the narP deleted mutant in the presence of additional nitrate, while the transcriptional changes were restored in the complemented strain. The NarP binding motif in the upstream regions of the target gene dmsA and ftpA were further identified and confirmed by EMSA using the truncated binding motif. The NarP binding sites were present in a total of 25.2% of the DNA fragments captured by DAP-Seq. These results demonstrated that the DAP-Seq method established in this study was effective to explore the direct targets of RRs of bacterial TCSs, and indicated that the A. pleuropneumoniae NarP could be a repressor in response to nitrate.
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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.
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