scholarly journals Sequestration of histidine kinases by non-cognate response regulators establishes a threshold level of stimulation for bacterial two-component signaling

2021 ◽  
Author(s):  
Gaurav D Sankhe ◽  
Rubesh Raja ◽  
Narendra M Dixit ◽  
Deepak Kumar Saini
Keyword(s):  
Response Regulator ◽  
Intervention Strategy ◽  
Threshold Level ◽  
Model Systems ◽  
Adaptive Responses ◽  
Signaling Systems ◽  
Two Component ◽  
Bacterial Fitness

Two-component signaling systems (TCSs) in bacteria are often positively auto-regulated, where the histidine kinase (HK) and response regulator (RR) proteins comprising a TCS are expressed downstream of the signal they transduce. This auto-regulation improves the sensitivity of the TCS to stimuli and amplifies adaptive responses. The downside, however, is that the TCS may mount disproportionately large responses to weak or fleeting signals. How bacteria prevent such disproportionate responses is not known. Here, we show that sequestration of phosphorylated HKs by non-cognate RRs serves as a design to prevent such disproportionate responses. Using TCSs of M. tuberculosis as model systems, we found that with every one of the five HKs we studied, there was at least one non-cognate RR with higher affinity than that of the cognate RR for the HK. Phosphorylated HKs would thus preferentially bind the non-cognate RRs, suppressing signal transduction through the cognate pathways, which we demonstrated in vitro. Using mathematical modeling of TCS signaling in vivo, we predicted that this sequestration would introduce a threshold level of stimulation for a significant response, preventing responses to signals below this threshold. Finally, we showed in vivo using tunable expression systems in M. bovis that upregulation of a higher affinity non-cognate RR substantially suppressed the output from the cognate TCS pathway, presenting strong evidence of sequestration by non-cognate RRs as a design to regulate TCS signaling. Blocking this sequestration may be a novel intervention strategy, as it would compromise bacterial fitness by letting it respond unnecessarily to signals.

scholarly journals Quantitative Kinetic Analyses of Shutting Off a Two-Component System

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Rong Gao ◽  
Ann M. Stock
Keyword(s):  
Phosphatase Activity ◽  
Response Regulator ◽  
Two Component System ◽  
Component System ◽  
Signaling Systems ◽  
Cellular Environment ◽  
Two Component ◽  
The Impact

ABSTRACT Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo , which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro . We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression. IMPORTANCE Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.

scholarly journals Functional reconstitution of the Salmonella typhimurium PhoQ histidine kinase sensor in proteoliposomes

2005 ◽  
Vol 390 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Sarah Sanowar ◽  
Hervé Le Moual
Keyword(s):  
Salmonella Typhimurium ◽  
Histidine Kinase ◽  
Catalytic Domain ◽  
Response Regulator ◽  
Phosphoryl Group ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
High Concentrations

Two-component signal-transduction systems are widespread in bacteria. They are usually composed of a transmembrane histidine kinase sensor and a cytoplasmic response regulator. The PhoP/PhoQ two-component system of Salmonella typhimurium contributes to virulence by co-ordinating the adaptation to low concentrations of environmental Mg2+. Limiting concentrations of extracellular Mg2+ activate the PhoP/PhoQ phosphorylation cascade modulating the transcription of PhoP-regulated genes. In contrast, high concentrations of extracellular Mg2+ stimulate the dephosphorylation of the response regulator PhoP by the PhoQ kinase sensor. In the present study, we report the purification and functional reconstitution of PhoQHis, a PhoQ variant with a C-terminal His tag, into Escherichia coli liposomes. The functionality of PhoQHis was essentially similar to that of PhoQ as shown in vivo and in vitro. Purified PhoQHis was inserted into liposomes in a unidirectional orientation, with the sensory domain facing the lumen and the catalytic domain facing the extraluminal environment. Reconstituted PhoQHis exhibited all the catalytic activities that have been described for histidine kinase sensors. Reconstituted PhoQHis was capable of autokinase activity when incubated in the presence of Mg2+-ATP. The phosphoryl group could be transferred from reconstituted PhoQHis to PhoP. Reconstituted PhoQHis catalysed the dephosphorylation of phospho-PhoP and this activity was stimulated by the addition of extraluminal ADP.

scholarly journals Identification and Characterization of a Regulatory Sequence Recognized by Mycobacterium tuberculosis Persistence Regulator MprA

2005 ◽  
Vol 187 (1) ◽  
pp. 202-212 ◽  
Author(s):  
Hongjun He ◽  
Thomas C. Zahrt
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Response Regulator ◽  
Regulatory System ◽  
Direct Repeat ◽  
Regulatory Sequence ◽  
Spacer Length ◽  
Signaling Systems ◽  
Downstream Effector

ABSTRACT Establishment and maintenance of persistent, latent infection by Mycobacterium tuberculosis are dependent on expression of the mprA-mprB regulatory system. Previously, MprA and MprB were shown to participate in phosphotransfer reactions characteristic of two-component signaling systems. To begin identifying downstream effector genes regulated by mprA-mprB during persistent stages of infection, a search for the regulatory sequence(s) recognized by response regulator MprA was carried out. Here, evidence is presented demonstrating that MprA recognizes a 19-bp sequence comprising two loosely conserved 8-bp direct repeat subunits separated by 3 nucleotides. This motif, termed the MprA box, is found upstream of the mprA coding sequence and that of downstream gene pepD (Rv0983). Protein phosphorylation was not required for binding to this DNA sequence by MprA in vitro; however, phosphorylation enhanced DNA binding by MprA and was required for the regulation of mprA and pepD by MprA in vivo. Binding of MprA to the MprA box was dependent on conserved nucleotides contained within repeat subunits and on the spacer length separating these repeats. In addition, recognition of this sequence proceeded via tandem binding of two monomers of MprA. Identification of the genetic determinants regulated by MprA will ultimately enhance our understanding of the mechanisms utilized by M. tuberculosis to undergo latency.

scholarly journals Requirement of the Receiver and Phosphotransfer Domains of ArcB for Efficient Dephosphorylation of Phosphorylated ArcA In Vivo

2005 ◽  
Vol 187 (9) ◽  
pp. 3267-3272 ◽  
Author(s):  
Gabriela R. Peña-Sandoval ◽  
Ohsuk Kwon ◽  
Dimitris Georgellis
Keyword(s):  
Response Regulator ◽  
Growth Conditions ◽  
Sensor Kinase ◽  
Aerobic Growth ◽  
Two Component System ◽  
Two Component ◽  
Necessary And Sufficient ◽  
Respiratory Conditions

ABSTRACT The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to the respiratory conditions of growth. Under anoxic growth conditions, ArcB autophosphorylates and transphosphorylates ArcA, which in turn represses or activates its target operons. Under aerobic growth conditions, phosphorylated ArcA (ArcA-P) dephosphorylates and its transcriptional regulation is released. The dephosphorylation of ArcA-P has been shown to occur, at least in vitro, via an ArcAAsp54-P → ArcBHis717-P → ArcBAsp576-P → Pi reverse phosphorelay. In this study, the physiological significance of this pathway was assessed. The results demonstrate that the receiver and phosphotransfer domains of the tripartite sensor kinase ArcB are necessary and sufficient for efficient ArcA-P dephosphorylation in vivo.

scholarly journals Effect of d-Lactate on the Physiological Activity of the ArcB Sensor Kinase in Escherichia coli

2004 ◽  
Vol 186 (7) ◽  
pp. 2085-2090 ◽  
Author(s):  
Claudia Rodriguez ◽  
Ohsuk Kwon ◽  
Dimitris Georgellis
Keyword(s):  
Kinase Activity ◽  
Response Regulator ◽  
Physiological Activity ◽  
Growth Conditions ◽  
Sensor Kinase ◽  
Two Component System ◽  
Direct Signal ◽  
Two Component

ABSTRACT The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to the respiratory growth conditions. Under anoxic growth conditions ArcB autophosphorylates and transphosphorylates ArcA, which in turn represses or activates its target operons. The anaerobic metabolite d-lactate has been shown to stimulate the in vitro autophosphorylating activity of ArcB. In this study, the in vivo effect of d-lactate on the kinase activity of ArcB was assessed. The results demonstrate that d-lactate does not act as a direct signal for activation of ArcB, as previously proposed, but acts as a physiologically significant effector that amplifies ArcB kinase activity.

scholarly journals The ChvG–ChvI and NtrY–NtrX two-component systems coordinately regulate growth of Caulobacter crescentus

2021 ◽  
Author(s):  
Benjamin J. Stein ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Gene Expression ◽  
Phosphatase Activity ◽  
Environmental Changes ◽  
Caulobacter Crescentus ◽  
Genetic Interaction ◽  
Response Regulator ◽  
Cell Envelope ◽  
Signaling Systems ◽  
Two Component

Two-component signaling systems (TCSs) are comprised of a sensory histidine kinase and a response regulator protein. In response to environmental changes, sensor kinases directly phosphorylate their cognate response regulator to affect gene expression. Bacteria typically express multiple TCSs that are insulated from one another and regulate distinct physiological processes. There are certainly examples of cross-regulation between TCSs, but this phenomenon remains relatively unexplored. We have identified regulatory links between the ChvG–ChvI (ChvGI) and NtrY–NtrX (NtrYX) TCSs, which control important and often overlapping processes in α-proteobacteria, including maintenance of the cell envelope. Deletion of chvG and chvI in Caulobacter crescentus limited growth in defined medium and a selection for genetic suppressors of this growth phenotype uncovered interactions among chvGI , ntrYX , and ntrZ , which encodes a previously uncharacterized periplasmic protein. Significant overlap in the experimentally-defined ChvI and NtrX transcriptional regulons provided support for the observed genetic connections between ntrYX and chvGI . Moreover, we present evidence that the growth defect of strains lacking chvGI is influenced by the phosphorylation state of NtrX and, to some extent, by levels of the TonB-dependent receptor ChvT. Measurements of NtrX phosphorylation in vivo indicated that NtrZ is an upstream regulator of NtrY, and that NtrY primarily functions as an NtrX phosphatase. We propose a model in which NtrZ functions in the periplasm to inhibit NtrY phosphatase activity; regulation of phosphorylated NtrX levels by NtrZ and NtrY provides a mechanism to modulate and balance expression of the NtrX and ChvI regulons under different growth conditions. Importance Two-component signaling systems (TCSs) enable bacteria to regulate gene expression in response to physiochemical changes in their environment. The ChvGI and NtrYX TCSs regulate diverse pathways associated with pathogenesis, growth, and cell envelope function in many α-proteobacteria. We used Caulobacter crescentus as a model to investigate regulatory connections between ChvGI and NtrYX. Our work defined the ChvI transcriptional regulon in C. crescentus and revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. In addition, we identified NtrZ as a periplasmic inhibitor of NtrY phosphatase activity in vivo . Our work establishes C. crescentus as an excellent model to investigate multi-level regulatory connections between ChvGI and NtrYX in α-proteobacteria.

scholarly journals Construction and Characterization of Listeria monocytogenes Mutants with In-Frame Deletions in the Response Regulator Genes Identified in the Genome Sequence

2005 ◽  
Vol 73 (5) ◽  
pp. 3152-3159 ◽  
Author(s):  
Tatjana Williams ◽  
Susanne Bauer ◽  
Dagmar Beier ◽  
Michael Kuhn
Keyword(s):  
Listeria Monocytogenes ◽  
Response Regulator ◽  
Individual Response ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Regulator Genes ◽  
The Individual

ABSTRACT Two-component systems are widely distributed in prokaryotes where they control gene expression in response to diverse stimuli. To study the role of the sixteen putative two-component systems of Listeria monocytogenes systematically, in frame deletions were introduced into 15 out of the 16 response regulator genes and the resulting mutants were characterized. With one exception the deletion of the individual response regulator genes has only minor effects on in vitro and in vivo growth of the bacteria. The mutant carrying a deletion in the ortholog of the Bacillus subtilis response regulator gene degU showed a clearly reduced virulence in mice, indicating that DegU is involved in the regulation of virulence-associated genes.

scholarly journals Functional Analysis of theMycobacterium tuberculosis MprAB Two-Component SignalTransductionSystem

2003 ◽  
Vol 71 (12) ◽  
pp. 6962-6970 ◽  
Author(s):  
Thomas C. Zahrt ◽  
Christopher Wozniak ◽  
Denise Jones ◽  
Andrea Trevett
Keyword(s):  
Signal Transduction ◽  
Functional Analysis ◽  
Persistent Infection ◽  
Response Regulator ◽  
Sensor Kinase ◽  
Two Component System ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT The mechanisms utilized by Mycobacterium tuberculosis to establish, maintain, or reactivate from latent infection in the host are largely unknown but likely include genes that mediate adaptation to conditions encountered during persistence. Previously, a two-component signal transduction system, mprAB, was found to be required in M. tuberculosis for establishment and maintenance of persistent infection in a tissue- and stage-specific fashion. To begin to characterize the role of this system in M. tuberculosis physiology and virulence, a functional analysis of the mprA and mprB gene products was initiated. Here, evidence is presented demonstrating that sensor kinase MprB and response regulator MprA function as an intact signal-transducing pair in vitro and in vivo. Sensor kinase MprB can be autophosphorylated, can donate phosphate to MprA, and can act as a phospho-MprA phosphatase in vitro. Correspondingly, response regulator MprA can accept phosphate from MprB or from small phosphodonors including acetyl phosphate. Mutagenesis of residues His249 in MprB and Asp48 in MprA abolished the ability of these proteins to be phosphorylated in vitro. Introduction of these alleles into Mycobacterium bovis BCGattenuated virulence in macrophages in vivo. Together, these results support a role for the mprAB two-component system in M. tuberculosis physiology and pathogenesis. Characterization of two-component signal transduction systems will enhance our understanding of processes regulated by M. tuberculosis during acute and/or persistent infection in the host.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

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