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Identification of the genes regulated by the two-component system response regulator NarP of Actinobacillus pleuropneumoniae by DNA-affinity-purified (DAP) sequencing

ABSTRACT: 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.

ORGANISM(S): Actinobacillus pleuropneumoniae

PROVIDER: GSE107526 | GEO | 2019/11/30

REPOSITORIES: GEO

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Effects of two-component system NarQ/P deletion on gene expression of Actinobacillus pleuropneumoniae in nitrate-rich anaerobic growth

Project description:Nitrate metabolism is an adaptation mechanism used by many bacteria for survival under anaerobic environments. As a by-product of inflammation, nitrate is used by the intestinal bacterial pathogens to enable gut infection. However, the responses of bacterial respiratory pathogens to nitrate are less well understood. Actinobacillus pleuropneumoniae is an important bacterial respiratory pathogen of swine. Previous studies have suggested that adaptation of A. pleuropneumoniae to anaerobiosis is important for infection. In this work, A. pleuropneumoniae growth and pathogenesis in response to the nitrate were investigated. Nitrate significantly promoted A. pleuropneumoniae growth under anaerobic condition in vitro and lethality in mice. By using narQ and narP deletion mutants, and single-residue mutation complementary strains of ΔnarQ, the two-component system NarQ/P were confirmed to be critical for nitrate induced growth, with Arg50 in NarQ as an essential functional residue. Transcriptome analysis showed that nitrate up-regulated multiple energy-generating pathways, including nitrate metabolism, mannose and pentose metabolism and glycerolipid metabolism via the regulation of NarQ/P. Furthermore, narQ, narP and its target gene encoding the nitrate reductase Nap contributed to the pathogenicity of A. pleuropneumoniae. The Nap inhibitor tungstate significantly reduced the survival of A. pleuropneumoniae in vivo, suggesting that Nap is a potential drug target. These results give new insights into how the respiratory pathogen A. pleuropneumoniae utilizes alternative electron acceptor nitrate to overcome the hypoxia microenvironment, which can occur in the inflammatory or necrotic infected tissues.

2022-07-29 | GSE207497 | GEO

Mapping the DNA-binding response regulators of Desulfovibrio vulgaris Hildenborough

Project description:D. vulgaris is a model sulfate reducing bacteria whose genome encodes a high number of two component systems, including 29 DNA-binding response regulators (RRs) whose functions are unknown, but are very likley to be important to the environmental lifestyle of the organism. We determined the gene targets for 24 of these RRs using purified His-tagged RR and sheared genomic DNA in an in vitro DAP-chip (DNA-affinity-purified - chip) assay. For each RR, one target was identified first using gel shift assays, and qPCR was used to ensure that the target was enriched in the RR-bound DNA before the samples were hybridized to a tiling array. Based on the peaks generated by the array analysis, we determined that at least 200 genes are regulated by two component systems in this organism. We also predicted binding site motifs and validated them for 15 RRs using gel shift assays. In vitro DAP-chip for 28 response regulators where RR-bound enriched DNA is compared to input total genomic DNA

2011-05-31 | E-GEOD-25163 | biostudies-arrayexpress

Phylogenetic footprinting and transcriptome profiling reveal new roles for two Bacillus cereus two-component systems

Project description:Members of the Bacillus cereus group can adapt to a wide range of environmental challenges. In bacteria, these challenges are often translated into a transcriptional response via the cognate response regulators (RRs) of specialized two-component systems (TCSs). We have previously developed a phylogenetic footprinting approach that was successfully implemented to predict specific binding sites (operators) and target genes for the RRs of B. cereus and related species. In this study, this footprinting approach was integrated with transcriptome analyses of two B. cereus TCS deletion mutants, involving the TCSs YvrHG and YufLM. Comparison of mutant versus wild-type transcriptomes revealed that the respective TCSs were significantly active during the exponential growth phase in rich medium and that the footprinting-based predictions were accurate for the two TCSs. Moreover, the predicted specific operators were used in combination with the transcriptome data to guide the identification of more extended TCS regulons. This revealed new roles for the respective TCSs, including the participation in an intricate transcriptional network involved in antibiotic resistance, including the confirmed resistance to oxolinic acid (YvrHG) and the confirmed uptake and metabolism of fumarate and the repression of fermentative pathways (YufLM).

2014-12-31 | GSE18523 | GEO

Mapping the DNA-binding response regulators of Desulfovibrio vulgaris Hildenborough

2011-05-31 | GSE25163 | GEO

Genome wide transcriptional portrait of Actinobacillus pleuropneumoniae during invasive disease reveals new strategies for survival and persistence in the host

Project description:The transcriptional profile of the porcine lung pathogen, Actinobacillus pleuropneumoniae, was monitored during the acute phase of infection in its natural host. Bacterial expression profiles of A. pleuropneumoniae isolated from lung lesions of 25 infected pigs were compared in samples taken 6, 12, 24 and 48 hours post infection. A 84 chip study using total RNA recovered from A. pleuropneumoniae serotype 2 (4226) and serotype 6 (7712640) isolated from infected pig lung tissue during the first 48 of infection. Samples were taken 6, 12, 24 and 48 hours post infection, respectively. Before hybridization the samples were enriched for bacterial RNA and submitted to linear amplification. Each chip measures the expression level of 4,876 target genes from A. pleuropneumoniae with each gene covered by an average of 26.7 probes. Three biological replicates per sample. Microarray data from 3 pigs (no. 39, no. 43 and no. 72) were omitted from the final analysis due to poor signal intensity. A total of 75 microarrays were included in the final analysis.

2012-12-01 | E-GEOD-33999 | biostudies-arrayexpress

Genome wide transcriptional portrait of Actinobacillus pleuropneumoniae during invasive disease reveals new strategies for survival and persistence in the host

2012-12-01 | GSE33999 | GEO

Triclocarban vs Triclosan: A look into the protein targets of chlorinated biphenyl antimicrobials

Project description:Triclocarban (TCC), a formerly used disinfectant, kills bacteria via an unknown mechanism of action. A structural hallmark is its N,N'-diaryl urea motif which is also present in other antibiotics including the recently reported small molecule PK150. Like PK150, TCC exhibited a similar inhibitory effect on menaquinone metabolism via inhibition of the biosynthesis protein MenG, suggesting a contribution of this pathway to its mode of action. However, the activity spectrum (MIC90) of TCC across a broad range of resistant staphylococci and enterococci strains was much narrower compared to PK150. Accordingly, TCC did not cause an overactivation of signal peptidase SpsB, a hallmark of the PK150 scaffold. Furthermore, we were able to rule out inhibition of FabI, a confirmed target of the diaryl ether antibiotic triclosan (TCS). Differences in the target profile of TCC and TCS were further investigated by proteomic analysis, showing complex, but rather distinct changes in the protein expression profile of the bacteria. Additional evidence for an effect on bacterial energy metabolism was provided for TCC. Taken together, this study shows that N,N'-diaryl urea motifs exhibit shared and distinct molecular targets and for the first time highlights MenG as a target of TCC.

2020-06-04 | PXD018347 | Pride

Target-gene mediated activation of phosphatase activity of sensor kinase in the SaeRS two-component system of Staphylococcus aureus

Project description:In bacterial two-component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulator is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a ternary complex with the sensor kinase SaeS and activate the sensor kinase’s phosphatase activity. Efficient activation of the phosphatase activity of SaeS required the presence of both SaeP and SaeQ. When SaeP and SaeQ were expressed, the expression of coagulase, a sae target with low affinity to phosphorylated SaeR, was greatly reduced, while the expression of alpha-hemolysin, a sae target with high affinity to phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by SaeRS TCS, these results suggest that the SaeRS TCS returns to pre-activation state by a negative feedback mechanism. To examine the global effect of SaePQ on sae target gene expression, we treated the wild type strain of USA300-P23 and its P1 promoter mutant with HNP-1 and analyzed the transcription of sae target genes by microarray assays. WT and P1 cells were compared against a each other at the same time point and against a T=0 reference.

2012-03-09 | E-GEOD-36230 | biostudies-arrayexpress

Expression data of Actinobacillus pleuropneumoniae 4074 under aerobic and anaerobic environment

Project description:Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry worldwide. Oxygen deprivation is a stress that A. pleuropneumoniae will encounter during both early infection and the later, persistent stage. To understand modulation of A. pleuropneumoniae gene expression in response to the stress caused by anaerobic conditions, gene expression profiles under anaerobic and aerobic conditions were compared in this study. The microarray results showed that 631 genes (27.7% of the total ORFs) were differentially expressed in anaerobic conditions. Many genes encoding proteins involved in glycolysis, carbon source uptake systems, pyruvate metabolism, fermentation and the electron respiration transport chain were up-regulated. These changes led to an increased amount of pyruvate, lactate, ethanol and acetate in the bacterial cells as confirmed by metabolite detection. Genes encoding proteins involved in cell surface structures, especially biofilm formation, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis were up-regulated as well. Biofilm formation was significantly enhanced under anaerobic conditions. These results indicate that induction of central metabolism is important for basic survival of A. pleuropneumoniae after a shift to an anaerobic environment. Enhanced biofilm formation may contribute to the persistence of this pathogen in the damaged anaerobic host tissue and also in the early colonization stage. These discoveries give new insights into adaptation mechanisms of A. pleuropneumoniae in response to environmental stress.

2014-06-30 | GSE39801 | GEO

Target-gene mediated activation of phosphatase activity of sensor kinase in the SaeRS two-component system of Staphylococcus aureus

2012-03-09 | GSE36230 | GEO

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