Chemical inhibition of the Mycobacterium tuberculosis DosRST two-component regulatory system by targeting sensor kinase heme and response regulator DNA binding
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ABSTRACT:
PROVIDER: PRJNA476612 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7359820.fastq.gz | Fastqsanger.gz | |||
| SRR7359821.fastq.gz | Fastqsanger.gz | |||
| SRR7359812.fastq.gz | Fastqsanger.gz | |||
| SRR7359822.fastq.gz | Fastqsanger.gz | |||
| SRR7359813.fastq.gz | Fastqsanger.gz |
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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
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.
2012-03-09 | GSE36230 | GEO
Project description:In this experiment the transcriptional profile of the Pseudomonas aeruginosa PA14 two-component sensor kinase PA4398 was investigated under swarming conditions using DNA microarrays. To this aim three independent cultures of the PA14 wild-type and the PA4398 mutant were grown until mid-log phase in Luria-Bertani broth following an incubation on BM2-swarm plates containing 0.1 % (wt/vol) casaminoacids and 0.5 % (wt/vol) agar for 20 h at 37 °C. Subsequent total RNA was extracted from the leading edge of dendritic swarm colonies and analyzed by microarrays.
2015-01-02 | E-MTAB-2540 | biostudies-arrayexpress
Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum. Three biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown und FeSO4 or heme as iron source; Exp. 2: WT vs. hrrA deletion mutant grown on FeSO4; Exp. 3: WT vs. hrrA mutant grown on heme. For analysis via DNA microarraysose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 2.5 uM FeSO4 or 2.5 uM heme as iron source.
2010-12-17 | E-GEOD-26122 | biostudies-arrayexpress
Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum.
2010-12-17 | GSE26122 | GEO
Project description:ReeS, previously named as CPE1512, was originally annotated as the only hybrid sensor histidine kinase/response regulator in Clostridium perfringens. Further evidence suggests that ReeS is more likely to function as an orphan sensor histidine kinase. A reeS deletion mutant was constructed and the transcriptome analysed using microarrays.
2013-08-31 | GSE36786 | GEO
Project description:Heme is a cofactor with myriad roles and is essential to almost all living organisms. Accordingly, bacte-rial pathogens have developed numerous mechanisms to acquire heme from their hosts. Beyond classi-cal gas transport and catalytic functions, heme is increasingly appreciated as a tightly controlled signal-ling molecule regulating protein expression. However, heme acquisition, biosynthesis and regulation is poorly understood beyond a few model organisms, and the heme-binding proteome has yet to be fully characterised in bacteria. Yet as heme homeostasis is critical for bacterial survival, heme-binding pro-teins are promising drug targets. Herein we report a chemical proteomics method for global profiling of heme-binding proteins in live cells for the first time. Employing a panel of heme-based clickable and photoaffinity probes enabled the profiling of 32-54% of the known heme-binding proteomes in Gram-positive and Gram-negative bacteria, and revealed previously unknown potential heme interactors. This simple-to-implement profiling strategy could be interchangeably applied to different cell types and sys-tems and fuel future research into heme biology.
2023-12-05 | PXD036133 | Pride
Project description:We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study ChrSA was shown to be crucial for heme resistance of C. glutamicum by activation of the putative heme-detoxifying ABC-transporter HrtBA in the presence of heme. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards heme. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA provided evidence for ChrA being a repressor of the heme biosynthesis operon hemAC and hemH and an activator of the ctaE-qcrCAB operon, encoding subunits of the cytochrome bc1-aa3 supercomplex of the respiratory chain. The heme oxygenase gene, hmuO, showed a strongly decreased mRNA level in the ΔchrSA mutant, but no significant binding of ChrA was observed in vitro. Promoter fusion studies of PchrSA with eyfp indicated positive autoregulation of the chrSA operon in the presence of heme. Interestingly, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralog response regulator, suggesting a close link between HrrSA and ChrSA. Mutational analysis and in silico prediction resulted in the deduction of a 16-bp weakly conserved inverted repeat as consensus DNA-binding motif of ChrA. Altogether, the present study emphasizes ChrSA as a second TCS, besides HrrSA, involved in heme-dependent gene regulation in C. glutamicum.
2012-10-31 | GSE37327 | GEO
Project description:We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study ChrSA was shown to be crucial for heme resistance of C. glutamicum by activation of the putative heme-detoxifying ABC-transporter HrtBA in the presence of heme. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards heme. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA provided evidence for ChrA being a repressor of the heme biosynthesis operon hemAC and hemH and an activator of the ctaE-qcrCAB operon, encoding subunits of the cytochrome bc1-aa3 supercomplex of the respiratory chain. The heme oxygenase gene, hmuO, showed a strongly decreased mRNA level in the M-NM-^TchrSA mutant, but no significant binding of ChrA was observed in vitro. Promoter fusion studies of PchrSA with eyfp indicated positive autoregulation of the chrSA operon in the presence of heme. Interestingly, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralog response regulator, suggesting a close link between HrrSA and ChrSA. Mutational analysis and in silico prediction resulted in the deduction of a 16-bp weakly conserved inverted repeat as consensus DNA-binding motif of ChrA. Altogether, the present study emphasizes ChrSA as a second TCS, besides HrrSA, involved in heme-dependent gene regulation in C. glutamicum. To identify the influence of ChrSA on global gene expression , DNA microarray analyses were performed with the M-NM-^TchrSA mutant compared to C. glutamicum wild type. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII minimal medium with 4% glucose and either 2.5 M-BM-5M FeSO4 or 2.5 M-BM-5M hemin as iron source. Three biological replicates were performed.
2012-10-31 | E-GEOD-37327 | biostudies-arrayexpress
Project description:Natural mutations in the sensor kinase of the PhoPR two-component regulatory system modulate virulence of ancestor-like tuberculosis bacilli
| PRJNA955279 | ENA