TheEscherichia coliPII Signal Transduction Protein Regulates the Activities of the Two-Component System Transmitter Protein NRII by Direct Interaction with the Kinase Domain of the Transmitter Module

Biochemistry ◽  
2000 ◽  
Vol 39 (44) ◽  
pp. 13450-13461 ◽  
Author(s):  
Augen A. Pioszak ◽  
Peng Jiang ◽  
Alexander J. Ninfa
Keyword(s):  
Signal Transduction ◽  
Direct Interaction ◽  
Kinase Domain ◽  
Two Component System ◽  
Component System ◽  
Signal Transduction Protein ◽  
Two Component

scholarly journals CrdS and CrdA Comprise a Two-Component System That Is Cooperatively Regulated by the Che3 Chemosensory System in Myxococcus xanthus

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Jonathan W. Willett ◽  
John R. Kirby
Keyword(s):  
Signal Transduction ◽  
Myxococcus Xanthus ◽  
Complex Signal ◽  
Central Processor ◽  
Two Component System ◽  
Component System ◽  
Chemosensory System ◽  
Content Type ◽  
Phosphorylation State ◽  
Two Component

ABSTRACTMyxococcus xanthusserves as a model organism for development and complex signal transduction. Regulation of developmental aggregation and sporulation is controlled, in part, by the Che3 chemosensory system. The Che3 pathway consists of homologs to two methyl-accepting chemotaxis proteins (MCPs), CheA, CheW, CheB, and CheR but not CheY. Instead, the output for Che3 is the NtrC homolog CrdA, which functions to regulate developmental gene expression. In this paper we have identified an additional kinase, CrdS, which directly regulates the phosphorylation state of CrdA. Both epistasis andin vitrophosphotransfer assays indicate that CrdS functions as part of the Che3 pathway and, in addition to CheA3, serves to regulate CrdA phosphorylation inM. xanthus. We provide kinetic data for CrdS autophosphorylation and demonstrate specificity for phosphotransfer from CrdS to CrdA. We further demonstrate that CheA3 destabilizes phosphorylated CrdA (CrdA~P), indicating that CheA3 likely acts as a phosphatase. Both CrdS and CheA3 control developmental progression by regulating the phosphorylation state of CrdA~P in the cell. These results support a model in which a classical two-component system and a chemosensory system act synergistically to control the activity of the response regulator CrdA.IMPORTANCEWhile phosphorylation-mediated signal transduction is well understood in prototypical chemotaxis and two-component systems (TCS), chemosensory regulation of alternative cellular functions (ACF) has not been clearly defined. The Che3 system inMyxococcus xanthusis a member of the ACF class of chemosensory systems and regulates development via the transcription factor CrdA (chemosensoryregulator ofdevelopment) (K. Wuichet and I. B. Zhulin, Sci. Signal. 3:ra50, 2010; J. R. Kirby and D. R. Zusman, Proc. Natl. Acad. Sci. U. S. A. 100:2008–2013, 2003). We have identified and characterized a homolog of NtrB, designated CrdS, capable of specifically phosphorylating the NtrC homolog CrdA inM. xanthus. Additionally, we demonstrate that the CrdSA two-component system is negatively regulated by CheA3, the central processor within the Che3 system ofM. xanthus. To our knowledge, this study provides the first example of an ACF chemosensory system regulating a prototypical two-component system and extends our understanding of complex regulation of developmental signaling pathways.

scholarly journals Mycobacterium tuberculosis PhoY Proteins Promote Persister Formation by Mediating Pst/SenX3-RegX3 Phosphate Sensing

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Sarah B. Namugenyi ◽  
Alisha M. Aagesen ◽  
Sarah R. Elliott ◽  
Anna D. Tischler
Keyword(s):  
Signal Transduction ◽  
Mycobacterium Tuberculosis ◽  
System Component ◽  
Signal Transduction System ◽  
Two Component System ◽  
Component System ◽  
Phosphate Sensing ◽  
Two Component ◽  
Pst System

ABSTRACT The Mycobacterium tuberculosis phosphate-specific transport (Pst) system controls gene expression in response to phosphate availability by inhibiting the activation of the SenX3-RegX3 two-component system under phosphate-rich conditions, but the mechanism of communication between these systems is unknown. In Escherichia coli, inhibition of the two-component system PhoR-PhoB under phosphate-rich conditions requires both the Pst system and PhoU, a putative adaptor protein. E. coli PhoU is also involved in the formation of persisters, a subpopulation of phenotypically antibiotic-tolerant bacteria. M. tuberculosis encodes two PhoU orthologs, PhoY1 and PhoY2. We generated phoY single- and double-deletion mutants and examined the expression of RegX3-regulated genes by quantitative reverse transcription-PCR (qRT-PCR). Gene expression was increased only in the ΔphoY1 ΔphoY2 double mutant and could be restored to the wild-type level by complementation with either phoY1 or phoY2 or by deletion of regX3. These data suggest that the PhoY proteins function redundantly to inhibit SenX3-RegX3 activation. We analyzed the frequencies of antibiotic-tolerant persister variants in the phoY mutants using several antibiotic combinations. Persister frequency was decreased at least 40-fold in the ΔphoY1 ΔphoY2 mutant compared to the frequency in the wild type, and this phenotype was RegX3 dependent. A ΔpstA1 mutant lacking a Pst system transmembrane component exhibited a similar RegX3-dependent decrease in persister frequency. In aerosol-infected mice, the ΔphoY1 ΔphoY2 and ΔpstA1 mutants were more susceptible to treatment with rifampin but not isoniazid. Our data demonstrate that disrupting phosphate sensing mediated by the PhoY proteins and the Pst system enhances the susceptibility of M. tuberculosis to antibiotics both in vitro and during infection. IMPORTANCE Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to the lengthy treatment times required to cure Mycobacterium tuberculosis infection, but the molecular mechanisms governing their formation and maintenance are poorly characterized. Here, we demonstrate that a phosphate-sensing signal transduction system, comprising the Pst phosphate transporter, the two-component system SenX3-RegX3, and functionally redundant PhoY proteins that mediate signaling between Pst and SenX3-RegX3, influences persister formation. Activation of RegX3 by deletion of the phoY genes or a Pst system component resulted in decreased persister formation in vitro. Activated RegX3 also limited persister formation during growth under phosphate-limiting conditions. Importantly, increased susceptibility to the front-line drug rifampin was also observed in a mouse infection model. Thus, the M. tuberculosis phosphate-sensing signal transduction system contributes to antibiotic tolerance and is a potential target for the development of novel therapeutics that may shorten the duration of tuberculosis treatment. IMPORTANCE Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to the lengthy treatment times required to cure Mycobacterium tuberculosis infection, but the molecular mechanisms governing their formation and maintenance are poorly characterized. Here, we demonstrate that a phosphate-sensing signal transduction system, comprising the Pst phosphate transporter, the two-component system SenX3-RegX3, and functionally redundant PhoY proteins that mediate signaling between Pst and SenX3-RegX3, influences persister formation. Activation of RegX3 by deletion of the phoY genes or a Pst system component resulted in decreased persister formation in vitro. Activated RegX3 also limited persister formation during growth under phosphate-limiting conditions. Importantly, increased susceptibility to the front-line drug rifampin was also observed in a mouse infection model. Thus, the M. tuberculosis phosphate-sensing signal transduction system contributes to antibiotic tolerance and is a potential target for the development of novel therapeutics that may shorten the duration of tuberculosis treatment.

scholarly journals A Novel Two-Component System, GluR-GluK, Involved in Glutamate Sensing and Uptake in Streptomyces coelicolor

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Lei Li ◽  
Weihong Jiang ◽  
Yinhua Lu
Keyword(s):  
Signal Transduction ◽  
Glutamate Uptake ◽  
Streptomyces Coelicolor ◽  
Signal Transduction Pathway ◽  
Antibiotic Biosynthesis ◽  
Transduction Pathway ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Two Component

ABSTRACT Two-component systems (TCSs), the predominant signal transduction pathways employed by bacteria, play important roles in physiological metabolism in Streptomyces. Here, a novel TCS, GluR-GluK (encoded by SCO5778-SCO5779), which is located divergently from the gluABCD operon encoding a glutamate uptake system, was identified as being involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor. Under the condition of minimal medium (MM) supplemented with different concentrations of glutamate, deletion of the gluR-gluK operon (gluR-K) resulted in enhanced actinorhodin (ACT) but reduced undecylprodigiosin (RED) and yellow type I polyketide (yCPK) production, suggesting that GluR-GluK plays a differential role in antibiotic biosynthesis. Furthermore, we found that the response regulator GluR directly promotes the expression of gluABCD under the culture condition of MM with a high concentration of glutamate (75 mM). Using the biolayer interferometry assay, we demonstrated that glutamate acts as the direct signal of the histidine kinase GluK. It was therefore suggested that upon sensing high concentrations of glutamate, GluR-GluK would be activated and thereby facilitate glutamate uptake by increasing gluABCD expression. Finally, we demonstrated that the role of GluR-GluK in antibiotic biosynthesis is independent of its function in glutamate uptake. Considering the wide distribution of the glutamate-sensing (GluR-GluK) and uptake (GluABCD) module in actinobacteria, it could be concluded that the GluR-GluK signal transduction pathway involved in secondary metabolism and glutamate uptake should be highly conserved in this bacterial phylum. IMPORTANCE In this study, a novel two-component system (TCS), GluR-GluK, was identified to be involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor. A possible GluR-GluK working model was proposed. Upon sensing high glutamate concentrations (such as 75 mM), activated GluR-GluK could regulate both glutamate uptake and antibiotic biosynthesis. However, under a culture condition of MM supplemented with low concentrations of glutamate (such as 10 mM), although GluR-GluK is activated, its activity is sufficient only for the regulation of antibiotic biosynthesis. To the best of our knowledge, this is the first report describing a TCS signal transduction pathway for glutamate sensing and uptake in actinobacteria.

scholarly journals Genetic and Functional Characterization of the Escherichia coli BarA-UvrY Two-Component System: Point Mutations in the HAMP Linker of the BarA Sensor Give a Dominant-Negative Phenotype

2005 ◽  
Vol 187 (21) ◽  
pp. 7317-7324 ◽  
Author(s):  
Henrik Tomenius ◽  
Anna-Karin Pernestig ◽  
Claudia F. Méndez-Catalá ◽  
Dimitris Georgellis ◽  
Staffan Normark ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Functional Characterization ◽  
Point Mutations ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Two Component System ◽  
Component System ◽  
Two Component

ABSTRACT The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.

Spermidine triggering effect to the signal transduction through the AtoS–AtoC/Az two-component system in Escherichia coli

2007 ◽  
Vol 1770 (8) ◽  
pp. 1104-1114 ◽  
Author(s):  
Marina C. Theodorou ◽  
Evaggelos C. Theodorou ◽  
Christos A. Panagiotidis ◽  
Dimitrios A. Kyriakidis
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Two Component System ◽  
Component System ◽  
Two Component

scholarly journals Conformational Changes of an Interdomain Linker Mediate Mechanical Signal Transmission in Sensor Kinase BvgS

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Elodie Lesne ◽  
Elian Dupré ◽  
Camille Locht ◽  
Rudy Antoine ◽  
Françoise Jacob-Dubuisson
Keyword(s):  
Signal Transduction ◽  
Bordetella Pertussis ◽  
Coiled Coil ◽  
Protein Domain ◽  
Sensor Kinase ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Two Component ◽  
Sensor Kinases

ABSTRACT The whooping cough agent, Bordetella pertussis, controls the expression of its large virulence regulon in a coordinated manner through the two-component system BvgAS. BvgS is a dimeric, multidomain sensor kinase. Each monomer comprises, in succession, tandem periplasmic Venus flytrap (VFT) domains, a transmembrane segment, a cytoplasmic Per-Arnt-Sim (PAS) domain, a kinase module, and additional phosphorelay domains. BvgS shifts between kinase and phosphatase modes of activity in response to chemical modulators that modify the clamshell motions of the VFT domains. We have shown previously that this regulation involves a shift between distinct states of conformation and dynamics of the two-helix coiled-coil linker preceding the enzymatic module. In this work, we determined the mechanism of signal transduction across the membrane via a first linker, which connects the VFT and PAS domains of BvgS, using extensive cysteine cross-linking analyses and other approaches. Modulator perception by the periplasmic domains appears to trigger a small, symmetrical motion of the transmembrane segments toward the periplasm, causing rearrangements of the noncanonical cytoplasmic coiled coil that follows. As a consequence, the interface of the PAS domains is modified, which affects the second linker and eventually causes the shift of enzymatic activity. The major features of this first linker are well conserved among BvgS homologs, indicating that the mechanism of signal transduction unveiled here is likely to be generally relevant for this family of sensor kinases. IMPORTANCE Bordetella pertussis produces virulence factors coordinately regulated by the two-component system BvgAS. BvgS is a sensor kinase, and BvgA is a response regulator that activates gene transcription when phosphorylated by BvgS. Sensor kinases homologous to BvgS are also found in other pathogens. Our goal is to decipher the mechanisms of BvgS signaling, since these sensor kinases may represent new targets for antibacterial agents. Signal perception by the sensor domains of BvgS triggers small motions of the helical linker region underneath. The protein domain that follows this linker undergoes a large conformational change that amplifies the initial signal, causing a shift of activity from kinase to phosphatase. Because BvgS homologs harbor similar regions, these signaling mechanisms are likely to apply generally to that family of sensor kinases.

scholarly journals Regulation of Autophosphorylation ofEscherichia coli Nitrogen Regulator II by the PII Signal Transduction Protein

1999 ◽  
Vol 181 (6) ◽  
pp. 1906-1911 ◽  
Author(s):  
Peng Jiang ◽  
Alexander J. Ninfa
Keyword(s):  
Signal Transduction ◽  
Phosphatase Activity ◽  
Single Molecule ◽  
Kinase Activity ◽  
Two Component System ◽  
Phosphatase Activities ◽  
Signal Transduction Protein ◽  
Two Component ◽  
Additional Ligand ◽  
Two Component Signal Transduction

ABSTRACT The nitrogen regulator II (NRII or NtrB)-NRI (NtrC) two-component signal transduction system regulates the transcription of nitrogen-regulated genes in Escherichia coli. The NRII protein has both kinase and phosphatase activities and catalyzes the phosphorylation and dephosphorylation of NRI, which activates transcription when phosphorylated. The phosphatase activity of NRII is activated by the PII signal transduction protein. We showed that PII was also an inhibitor of the kinase activity of NRII. The data were consistent with the hypothesis that the kinase and phosphatase activities of two-component system kinase/phosphatase proteins are coordinately and reciprocally regulated. The ability of PII to regulate NRII is allosterically controlled by the small-molecule effector 2-ketoglutarate, which binds to PII. We studied the effect of 2-ketoglutarate on the regulation of the kinase and phosphatase activities of NRII by PII, using a coupled enzyme system to measure the rate of cleavage of ATP by NRII. The data were consistent with the following hypothesis: when not complexed with 2-ketoglutarate, PII cannot bind to NRII and has no effect on its competing NRI kinase and phosphatase activities. Under these conditions, the kinase activity of NRII is dominant. At low 2-ketoglutarate concentrations, PII trimers complexed with a single molecule of 2-ketoglutarate interact with NRII to inhibit its kinase activity and activate its phosphatase activity. However, at high 2-ketoglutarate concentrations, PII binds additional ligand molecules and is rendered incapable of binding to NRII, thereby releasing inhibition of NRII’s kinase activity and effectively inhibiting its phosphatase activity (by failing to stimulate it).

scholarly journals Diversity of two-component systems: insights into the signal transduction mechanism by the Staphylococcus aureus two-component system GraSR

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 252 ◽  
Author(s):  
Uzma Muzamal ◽  
Daniel Gomez ◽  
Fenika Kapadia ◽  
Dasantila Golemi-Kotra
Keyword(s):  
Signal Transduction ◽  
Staphylococcus Aureus ◽  
Abc Transporter ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Two Component System ◽  
Component System ◽  
Transduction Mechanism ◽  
Two Component ◽  
Auxiliary Protein

The response to cationic antimicrobial peptides (CAMPs) in Staphylococcus aureus relies on a two-component system (TCS), GraSR, an auxiliary protein GraX and an ATP-binding cassette (ABC) transporter, VraF/G. To understand the signal transduction mechanism by GraSR, we investigated the kinase activity of the cytoplasmic domain of histidine kinase GraS and the interaction with its cognate response regulator GraR. We also investigated interactions among the auxiliary protein GraX, GraS/R and the ATPase protein of the ABC transporter, VraF. We found that GraS lacks autophosphorylation activity, unlike a similar histidine kinase, BceS, of Bacillus subtilis. In addition, the interaction between GraS and GraR is very weak in comparison to the stronger interaction observed between BceS and its conjugated response regulator, BceR, suggesting that CAMP signaling may not flow directly from GraS to GraR. We found that the auxiliary protein GraX interacts with VraF and GraR, and requires the histidine phosphotransfer and dimerization domain of GraS to interact with this protein. Further, VraF requires the GraS region that connects the membrane-bound domain with the cytoplasmic domain of this protein for interaction with GraS. The interactions of GraX with GraS/R and VraF indicate that GraX may serve as a scaffold to bring these proteins in close proximity to GraS, plausibly to facilitate activation of GraS to ultimately transduce the signal to GraR.

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