Effect of small M. tuberculosis RNA MTS1338 on the transcriptome under various stress conditions
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ABSTRACT:
ORGANISM(S): Mycobacterium tuberculosis
PROVIDER: GSE218354 | GEO | 2023/05/17
REPOSITORIES: GEO
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Project description:We have performred RNA-seq analysis of WT, ∆phoP and ∆sigH Mtb H37Rv under normal and redox (Diamide) stress, to investigate the role of PhoP and SigH in maintaning the redox homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. Briefly, 25 ml of bacterial culture was grown to mid-log phase (OD600= 0.4 to 0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India. The main purpose of this study is to understand how mycobacteria can sense numerous stress conditions and mount an appropriate stress response. Although recent evidence suggests that at low pH M. tuberculosis encounters reductive stress, the mechanism of integrated regulation of stress response remains unknown. Unexpectedly, we find that PhoP contributes to mycothiol level and a PhoP-depleted bacilli shows enhanced susceptibility to redox stress. Because SigH is known to control expression of redox inducible genes, we probed whether previously-reported PhoP-SigH interaction accounts for mycobacterial redox stress response. Our results suggest that while PhoP controls pH homeostasis via its interaction with SigE, SigH-dependent PhoP expression, but not PhoP -SigH interaction, and direct recruitment of SigH, but not PhoP controls expression of mycobacterial thioredoxin genes. Together, these results uncover novel stress-specific interaction events of sigma factors and PhoP or lack thereof, as the underlying mechanisms of an adaptive programme, which couples low pH conditions and mycobacterial thiol homeostasis. The main purpose of this study is to understand how mycobacteria can sense numerous stress conditions and mount an appropriate stress response. Although recent evidence suggests that at low pH M. tuberculosis encounters reductive stress, the mechanism of integrated regulation of stress response remains unknown. Unexpectedly, we find that PhoP contributes to mycothiol level and a PhoP-depleted bacilli shows enhanced susceptibility to redox stress. Because SigH is known to control expression of redox inducible genes, we probed whether previously-reported PhoP-SigH interaction accounts for mycobacterial redox stress response. Our results suggest that while PhoP controls pH homeostasis via its interaction with SigE, SigH-dependent PhoP expression, but not PhoP -SigH interaction, and direct recruitment of SigH, but not PhoP controls expression of mycobacterial thioredoxin genes. Together, these results uncover novel stress-specific interaction events of sigma factors and PhoP or lack thereof, as the underlying mechanisms of an adaptive programme, which couples low pH conditions and mycobacterial thiol homeostasis.
2022-04-09 | GSE171775 | GEO
Project description:We have performred RNA-seq analysis of WT and ∆phoR Mtb H37Rv under normal and acidic stress, to investigate the role of PhoR in maintaning the pH homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. For acid stress, cells at OD600 of 0.6 were pelleted, washed twice with 7H9 medium buffered with 100 mM MOPS or 2N HCl for pH 7.0 or pH 4.5, respectively, re-suspended in media of indicated pH, and finally the cells were grown further for 2 hours at 37°C. After incubation cells were combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India. The main purpose of this study is to understand how mycobacteria can sense acidic stress conditions and mount an appropriate stress response.
2022-07-15 | GSE180161 | GEO
Project description:Transcriptional profile of Mycobacterium tuberculosis in in vitro acid-nitrosative multistress, comparing untreated control cells and bacteria under multi-stress.
2013-11-05 | GSE34623 | GEO
Project description:In other bacteria, arginine induces the expression of genes involved in arginine catabolism. This study obtained the identification of genes involved in the arginine metabolism of Mycobacterium tuberculosis.
2015-04-01 | GSE67445 | GEO
Project description:In other bacteria, arginine induces the expression of genes involved in arginine catabolism. This study obtained the identification of genes involved in the arginine metabolism of Mycobacterium tuberculosis. Mycobacterium tuberculosis was cultured with arginine or ammonium chloride as sole nitrogen source. In the log phase of growth, RNA was isolated and whole genome expression was determined. The study contains three biological replicates.
2015-04-01 | E-GEOD-67445 | biostudies-arrayexpress
Project description:This RNA-Seq study of gene expression in M. tuberculosis was done as part of the FLUTE project (NIH grant U19-AI107774, Eric Rubin, program director).
2016-03-30 | GSE67035 | GEO
Project description:We present the data obtained from high-resolution ribosome profiling analysis of Mycobacterium tuberculosis grown under standard conditions (log phase) and cells subjected to oxidative stress (50 µM cumene hydroperoxide) and pH stress (pH 4.5). Our data shows pervasive ribosome pausing in the M. tb translatome. A large number of genes show very high pause immediately downstream to the translation start site. Moreover, serines and alanines in the E site of the ribosome exhibit highest pause scores.
2021-06-02 | GSE151718 | GEO
Project description:Cellular respiration is a fundamental process undergone within energy-transducing membranes through the redox activity of multimeric protein assemblies, so-called respiratory complexes. In most cases, electron transport is ensured by lipophilic molecules, quinones, serving as electron shuttles. This redox activity is coupled to the net translocation of protons across the membrane thereby establishing an electrochemical gradient, the protonmotive force (pmf). Such a gradient powers the transport of molecules (such as proteins, ions or antibiotics) and ATP synthesis but was also shown to participate in protein localization in prokaryotes. Importantly, energy-transducing membranes show a high level of organization with heterogeneous distribution of respiratory complexes as observed across several bacterial lineages. Although electron transport in energy-transducing membranes is considered to be a kinetic process coupled to the diffusion of all reactants and in particular quinones, it is not clear to which extent subcellular distribution of respiratory complexes can have an influence on the overall kinetics. We previously evidenced polar clustering of nitrate reductase, NarGHI, an anaerobic respiratory complex in the gut bacterium Escherichia coli. Such spatial organization was shown to directly impact the electron flux of the associated respiratory chain. While dynamic localization of such complex plays an important role in controlling respiration, the mechanism by which it impacts the electron flux is not understood. Yet, under such conditions where nitrate is the sole terminal electron acceptor, functioning of the electron transport chain in enteric bacteria such as E. coli and Salmonella typhimurium is associated with the production of nitric oxide (NO) mainly resulting from the reduction of nitrite by nitrate reductase. As a consequence, both E. coli and S. typhimurium produce a multitude of enzymes controlling NO homeostasis, the primary source of nitrosative stress. Protein S-nitrosylation is a ubiquitous NO-dependent post-translational modification of cysteine that regulates protein structure and function. Recently, Seth et al demonstrated that, in absence of oxygen, NO oxidation is mainly catalyzed by the hybrid cluster protein Hcp allowing its further reaction with thiols of a broad spectrum of proteins. Among Hcp-dependent S-nitrosylated targets are the nitrate reductase complex, multiple metabolic enzymes and the transcription factor OxyR whose S-nitrosylation entails a distinct nitrosative stress regulon. Optimizing electron transport through clustering of nitrate reductase will lead to nitrite accumulation which may in turn be responsible for NO production. Here, the electron-donating respiratory complex, the formate dehydrogenase, FdnGHI was shown to cluster at the poles under nitrate respiring conditions. Its proximity with the nitrate reductase complex was confirmed by evaluating the interactome of the later under distinct metabolic conditions reported to impact its subcellular organization. All the identified partners were exclusively found when cells were grown under nitrate respiration. The proximity of two respiratory complexes provides mechanistic explanation on the importance of subcellular organization onto quinone pool turnover. Noteworthy is the identification of several components playing key roles in the protection against endogenous nitrosative stress.
2019-10-07 | PXD012320 | Pride
Project description:Mycobacterium tuberculosis has the ability to survive inside macrophages under acid-nitrosative stress. M. tuberculosis Rv1685c and its orthologue in M. smegmatis, MSMEG_3765, are induced on exposure to acid-nitrosative stress. Both genes are annotated as TetR transcriptional regulators, a family of proteins that regulate a wide range of cellular activities, including multidrug resistance, carbon catabolism and virulence. Here we demonstrate that MSMEG_3765 is co-transcribed with the upstream genes MSMEG_3762 and MSMEG_3763, encoding efflux pump components. RTq-PCR and GFP-reporter assays showed that the MSMEG_3762/63/65 gene cluster, and the orthologous region in M. tuberculosis (Rv1687c/86c/85c), was up-regulated in a MSMEG_3765 null mutant, suggesting that MSMEG_3765 acts as a repressor, typical of this family of regulators. We further defined the MSMEG_3765 regulon using genome-wide transcriptional profiling and confirmed that the MSMEG_3762/63/65 promoter is induced under acid-nitrosative stress. A putative 36 bp regulatory motif was identified upstream of the gene clusters in both M. smegmatis and M. tuberculosis and purified recombinant MSMEG_3765 protein was found to bind to DNA fragments containing this motif from both M. smegmatis and M. tuberculosis upstream regulatory regions. These results suggest that the TetR repressor MSMEG_3765/Rv1685c controls expression of an efflux pump with an, as yet, undefined role in the mycobacterial response to acid-nitrosative stress.
2017-10-24 | E-MTAB-5869 | biostudies-arrayexpress
Project description:Transcriptional profile of Mycobacterium tuberculosis in in vitro acid-nitrosative multistress, comparing untreated control cells and bacteria under multi-stress. Two-condition experiment, MTB-K vs. MTB-ACID-NO treated. Biological replicates: 3 controls, 3 Acid-NO treated, independently grown and harvested. In controls and Acid-NO treated cells, good quality RNA samples obtained from each pellet were pooled together and used for microarray experiments to get an average of three separate experiments, five replicates per array.
2013-11-05 | E-GEOD-34623 | biostudies-arrayexpress