scholarly journals The α10 helix of DevR, the Mycobacterium tuberculosis dormancy response regulator, regulates its DNA binding and activity

FEBS Journal ◽  
2016 ◽  
Vol 283 (7) ◽  
pp. 1286-1299 ◽  
Author(s):  
Atul Vashist ◽  
D. Prithvi Raj ◽  
Umesh Datta Gupta ◽  
Rajiv Bhat ◽  
Jaya Sivaswami Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Response Regulator

scholarly journals Expression, Autoregulation, and DNA Binding Properties of the Mycobacterium tuberculosis TrcR Response Regulator

2002 ◽  
Vol 184 (8) ◽  
pp. 2192-2203 ◽  
Author(s):  
Shelley E. Haydel ◽  
William H. Benjamin ◽  
Nancy E. Dunlap ◽  
Josephine E. Clark-Curtiss
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
Two Component System ◽  
Mobility Shift ◽  
Rich Region ◽  
Component System ◽  
Two Component ◽  
Gel Mobility Shift

ABSTRACT The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in β-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.

scholarly journals Inhibition of Mycobacterium tuberculosis DosRST two-component regulatory system signaling by targeting response regulator DNA binding and sensor kinase heme

2018 ◽  
Author(s):  
Huiqing Zheng ◽  
Bilal Aleiwi ◽  
Edmund Ellsworth ◽  
Robert B. Abramovitch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Treatment Time ◽  
Response Regulator ◽  
Transcriptional Profiling ◽  
Regulatory System ◽  
Dependent Manner ◽  
Triacylglycerol Synthesis ◽  
Two Component ◽  
Long Course

AbstractMycobacterium tuberculosis (Mtb) possesses a two-component regulatory system, DosRST, that enables Mtb to sense host immune cues and establish a state of non-replicating persistence (NRP). NRP bacteria are tolerant to several anti-mycobacterial drugs and are thought to play a role in the long course of tuberculosis (TB) therapy. Therefore, small molecules that inhibit Mtb from establishing or maintaining NRP could reduce the reservoir of drug tolerant bacteria and function as an adjunct therapy to reduce treatment time. Previously, we reported the discovery of six novel chemical inhibitors of DosRST, named HC101A-106A, from a whole cell, reporter-based phenotypic high throughput screen. Here, we report functional and mechanism of action studies of HC104A and HC106A. RNAseq transcriptional profiling shows that the compounds downregulate genes of the DosRST regulon. Both compounds reduce hypoxia-induced triacylglycerol synthesis by ~50%. HC106A inhibits Mtb survival during hypoxia-induced NRP, however, HC104A did not inhibit survival during NRP. An electrophoretic mobility assay shows that HC104A inhibits DosR DNA binding in a dose-dependent manner, indicating that HC104A may function by directly targeting DosR. In contrast, UV-visible spectroscopy studies suggest HC106A directly targets the histidine kinase heme, via a mechanism that is distinct from the oxidation and alkylation of heme previously observed with artemisinin (HC101A). Synergistic interactions were observed when DosRST inhibitors were examined in pair-wise combinations with the strongest potentiation observed between artemisinin paired with HC102A, HC103A, or HC106A. Our data collectively show that the DosRST pathway can be inhibited by multiple distinct mechanisms.

scholarly journals Inhibiting Mycobacterium tuberculosis DosRST Signaling by Targeting Response Regulator DNA Binding and Sensor Kinase Heme

2019 ◽  
Vol 15 (1) ◽  
pp. 52-62 ◽  
Author(s):  
Huiqing Zheng ◽  
John T. Williams ◽  
Bilal Aleiwi ◽  
Edmund Ellsworth ◽  
Robert B. Abramovitch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Response Regulator ◽  
Sensor Kinase

scholarly journals Abundance Changes of the Response Regulator RcaC Require Specific Aspartate and Histidine Residues and Are Necessary for Normal Light Color Responsiveness

2008 ◽  
Vol 190 (21) ◽  
pp. 7241-7250 ◽  
Author(s):  
Lina Li ◽  
David M. Kehoe
Keyword(s):  
Dna Binding ◽  
Dynamic Range ◽  
Response Regulator ◽  
Red Light ◽  
Green Light ◽  
Binding Activity ◽  
Transcriptional Responses ◽  
Dna Binding Activity ◽  
Normal Light ◽  
Light Color

ABSTRACT RcaC is a large, complex response regulator that controls transcriptional responses to changes in ambient light color in the cyanobacterium Fremyella diplosiphon. The regulation of RcaC activity has been shown previously to require aspartate 51 and histidine 316, which appear to be phosphorylation sites that control the DNA binding activity of RcaC. All available data suggest that during growth in red light, RcaC is phosphorylated and has relatively high DNA binding activity, while during growth in green light RcaC is not phosphorylated and has less DNA binding activity. RcaC has also been found to be approximately sixfold more abundant in red light than in green light. Here we demonstrate that the light-controlled abundance changes of RcaC are necessary, but not sufficient, to direct normal light color responses. RcaC abundance changes are regulated at both the RNA and protein levels. The RcaC protein is significantly less stable in green light than in red light, suggesting that the abundance of this response regulator is controlled at least in part by light color-dependent proteolysis. We provide evidence that the regulation of RcaC abundance does not depend on any RcaC-controlled process but rather depends on the presence of the aspartate 51 and histidine 316 residues that have previously been shown to control the activity of this protein. We propose that the combination of RcaC abundance changes and modification of RcaC by phosphorylation may be necessary to provide the dynamic range required for transcriptional control of RcaC-regulated genes.

scholarly journals Hanks-Type Serine/Threonine Protein Kinases and Phosphatases in Bacteria: Roles in Signaling and Adaptation to Various Environments

2018 ◽  
Vol 19 (10) ◽  
pp. 2872 ◽  
Author(s):  
Monika Janczarek ◽  
José-María Vinardell ◽  
Paulina Lipa ◽  
Magdalena Karaś
Keyword(s):  
Dna Binding ◽  
Essential Role ◽  
Current Knowledge ◽  
Response Regulator ◽  
Protein Targets ◽  
Regulatory Pathways ◽  
Translational Machinery ◽  
Signaling Systems ◽  
Cellular Processes ◽  
Division Cell

Reversible phosphorylation is a key mechanism that regulates many cellular processes in prokaryotes and eukaryotes. In prokaryotes, signal transduction includes two-component signaling systems, which involve a membrane sensor histidine kinase and a cognate DNA-binding response regulator. Several recent studies indicate that alternative regulatory pathways controlled by Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) also play an essential role in regulation of many different processes in bacteria, such as growth and cell division, cell wall biosynthesis, sporulation, biofilm formation, stress response, metabolic and developmental processes, as well as interactions (either pathogenic or symbiotic) with higher host organisms. Since these enzymes are not DNA-binding proteins, they exert the regulatory role via post-translational modifications of their protein targets. In this review, we summarize the current knowledge of STKs and STPs, and discuss how these enzymes mediate gene expression in prokaryotes. Many studies indicate that regulatory systems based on Hanks-type STKs and STPs play an essential role in the regulation of various cellular processes, by reversibly phosphorylating many protein targets, among them several regulatory proteins of other signaling cascades. These data show high complexity of bacterial regulatory network, in which the crosstalk between STK/STP signaling enzymes, components of TCSs, and the translational machinery occurs. In this regulation, the STK/STP systems have been proved to play important roles.

scholarly journals Cinnamaldehyde and cinnamaldehyde derivatives reduce virulence in Vibrio spp. by decreasing the DNA-binding activity of the quorum sensing response regulator LuxR

2008 ◽  
Vol 8 (1) ◽  
pp. 149 ◽  
Author(s):  
Gilles Brackman ◽  
Tom Defoirdt ◽  
Carol Miyamoto ◽  
Peter Bossier ◽  
Serge Van Calenbergh ◽  
...  
Keyword(s):  
Dna Binding ◽  
Quorum Sensing ◽  
Response Regulator ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Vibrio Spp

scholarly journals Real-time kinetic studies of Mycobacterium tuberculosis LexA-DNA interaction

2021 ◽  
Author(s):  
Chitral Chatterjee ◽  
Soneya Majumdar ◽  
Sachin Deshpande ◽  
Deepak Pant ◽  
Saravanan Matheshwaran
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Kinetic Parameters ◽  
Dna Sequences ◽  
Kinetic Studies ◽  
Dna Interaction ◽  
Damage Repair ◽  
Inducible Genes ◽  
Kinetics Of

Transcriptional repressor, LexA, regulates the “SOS” response, an indispensable bacterial DNA damage repair machinery.  Compared to its E.coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in “SOS” regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different “SOS” boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, this study provides insights into the kinetics of the interaction between Mtb LexA and its target “SOS” boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of “SOS” regulation and activation in Mtb.

scholarly journals Modulation of DNA-binding activity of Mycobacterium tuberculosis HspR by chaperones

Microbiology ◽  
2008 ◽  
Vol 154 (2) ◽  
pp. 484-490 ◽  
Author(s):  
Twishasri Das Gupta ◽  
Boudhayan Bandyopadhyay ◽  
Sujoy K. Das Gupta
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Binding Activity ◽  
Dna Binding Activity
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