scholarly journals Constitutive Activation of Two-Component Response Regulators: Characterization of VirG Activation in Agrobacterium tumefaciens

2006 ◽  
Vol 188 (14) ◽  
pp. 5204-5211 ◽  
Author(s):  
Rong Gao ◽  
Aindrila Mukhopadhyay ◽  
Fang Fang ◽  
David G. Lynn
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Conformational Changes ◽  
Response Regulator ◽  
Structural Features ◽  
Response Regulators ◽  
Two Component System ◽  
Plant Host ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.

scholarly journals Complex Regulation of Arsenite Oxidation in Agrobacterium tumefaciens

2006 ◽  
Vol 188 (3) ◽  
pp. 1081-1088 ◽  
Author(s):  
Des R. Kashyap ◽  
Lina M. Botero ◽  
William L. Franck ◽  
Daniel J. Hassett ◽  
Timothy R. McDermott
Keyword(s):  
Signal Transduction ◽  
Agrobacterium Tumefaciens ◽  
Quorum Sensing ◽  
Response Regulator ◽  
Structural Features ◽  
Rt Pcr ◽  
Two Component ◽  
In The Wild ◽  
Two Component Signal Transduction ◽  
Operon Expression

ABSTRACT Seminal regulatory controls of microbial arsenite [As(III)] oxidation are described in this study. Transposon mutagenesis of Agrobacterium tumefaciens identified genes essential for As(III) oxidation, including those coding for a two-component signal transduction pair. The transposon interrupted a response regulator gene (referred to as aoxR), which encodes an ntrC-like protein and is immediately downstream of a gene (aoxS) encoding a protein with primary structural features found in sensor histidine kinases. The structural genes for As(III) oxidase (aoxAB), a c-type cytochrome (cytc 2), and molybdopterin biosynthesis (chlE) were downstream of aoxR. The mutant could not be complemented by aoxSR in trans but was complemented by a clone containing aoxS-aoxR-aoxA-aoxB-cytc 2 and consistent with reverse transcriptase (RT) PCR experiments, which demonstrated these genes are cotranscribed as an operon. Expression of aoxAB was monitored by RT-PCR and found to be up-regulated by the addition of As(III) to cell cultures. Expression of aoxAB was also controlled in a fashion consistent with quorum sensing in that (i) expression of aoxAB was absent in As(III)-unexposed early-log-phase cells but was observed in As(III)-unexposed, late-log-phase cells and (ii) treating As(III)-unexposed, early-log-phase cells with ethyl acetate extracts of As(III)-unexposed, late-log-phase culture supernatants also resulted in aoxAB induction. Under inducing conditions, aoxS expression was readily observed in the wild-type strain but significantly reduced in the mutant, indicating that AoxR is autoregulatory and at least partially controls the expression of the aox operon. In summary, regulation of A. tumefaciens As(III) oxidation is complex, apparently being controlled by As(III) exposure, a two-component signal transduction system, and quorum sensing.

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.

Histidine kinases in signal transduction pathways of eukaryotes

1997 ◽  
Vol 110 (10) ◽  
pp. 1141-1145 ◽  
Author(s):  
W.F. Loomis ◽  
G. Shaulsky ◽  
N. Wang
Keyword(s):  
Signal Transduction ◽  
Response Regulator ◽  
Signal Transduction Pathways ◽  
Response Regulators ◽  
Histidine Kinases ◽  
Camp Phosphodiesterase ◽  
Wide Range ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Dependent Protein

Autophosphorylating histidine kinases are an ancient conserved family of enzymes that are found in eubacteria, archaebacteria and eukaryotes. They are activated by a wide range of extracellular signals and transfer phosphate moieties to aspartates found in response regulators. Recent studies have shown that such two-component signal transduction pathways mediate osmoregulation in Saccharomyces cerevisiae, Dictyostelium discoideum and Neurospora crassa. Moreover, they play pivotal roles in responses of Arabidopsis thaliana to ethylene and cytokinin. A transmembrane histidine kinase encoded by dhkA accumulates when Dictyostelium cells aggregate during development. Activation of DhkA results in the inhibition of its response regulator, RegA, which is a cAMP phosphodiesterase that regulates the cAMP dependent protein kinase PKA. When PKA is activated late in the differentiation of prespore cells, they encapsulate into spores. There is evidence that this two-component system participates in a feedback loop linked to PKA in prestalk cells such that the signal to initiate encapsulation is rapidly amplified. Such signal transduction pathways can be expected to be found in a variety of eukaryotic differentiations since they are rapidly reversible and can integrate disparate signals.

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 Crystal Structure of the Response Regulator 02 Receiver Domain, the Essential YycF Two-Component System of Streptococcus pneumoniae in both Complexed and Native States

2004 ◽  
Vol 186 (9) ◽  
pp. 2872-2879 ◽  
Author(s):  
Colin J. Bent ◽  
Neil W. Isaacs ◽  
Timothy J. Mitchell ◽  
Alan Riboldi-Tunnicliffe
Keyword(s):  
Streptococcus Pneumoniae ◽  
Active Site ◽  
Response Regulator ◽  
Thermotoga Maritima ◽  
Two Component System ◽  
Environmental Signals ◽  
Receiver Domain ◽  
Two Component ◽  
Protein Sequence Homology ◽  
Two Component Signal Transduction

ABSTRACT A variety of bacterial cellular responses to environmental signals are mediated by two-component signal transduction systems comprising a membrane-associated histidine protein kinase and a cytoplasmic response regulator (RR), which interpret specific stimuli and produce a measured physiological response. In RR activation, transient phosphorylation of a highly conserved aspartic acid residue drives the conformation changes needed for full activation of the protein. Sequence homology reveals that RR02 from Streptococcus pneumoniae belongs to the OmpR subfamily of RRs. The structures of the receiver domains from four members of this family, DrrB and DrrD from Thermotoga maritima, PhoB from Escherichia coli, and PhoP from Bacillus subtilis, have been elucidated. These domains are globally very similar in that they are composed of a doubly wound α5β5; however, they differ remarkably in the fine detail of the β4-α4 and α4 regions. The structures presented here reveal a further difference of the geometry in this region. RR02 is has been shown to be the essential RR in the gram-positive bacterium S. pneumoniae R. Lange, C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein, Gene 237:223-234, 1999; J. P. Throup, K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham, Mol. Microbiol. 35:566-576, 2000). RR02 functions as part of a phosphotransfer system that ultimately controls the levels of competence within the bacteria. Here we report the native structure of the receiver domain of RR02 from serotype 4 S. pneumoniae (as well as acetate- and phosphate-bound forms) at different pH levels. Two native structures at 2.3 Å, phased by single-wavelength anomalous diffraction (xenon SAD), and 1.85 Å and a third structure at pH 5.9 revealed the presence of a phosphate ion outside the active site. The fourth structure revealed the presence of an acetate molecule in the active site.

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 A Novel Three-Protein Two-Component System Provides a Regulatory Twist on an Established Circuit To Modulate Expression of the cbbI Region of Rhodopseudomonas palustris CGA010

2006 ◽  
Vol 188 (8) ◽  
pp. 2780-2791 ◽  
Author(s):  
Simona Romagnoli ◽  
F. Robert Tabita
Keyword(s):  
Response Regulator ◽  
Rhodopseudomonas Palustris ◽  
Sensor Kinase ◽  
Response Regulators ◽  
Transcription Activator ◽  
Two Component System ◽  
Bisphosphate Carboxylase ◽  
Control Of Gene Expression ◽  
Component System ◽  
Two Component

ABSTRACT A novel two-component system has been identified in the cbbI region of the nonsulfur purple photosynthetic bacterium Rhodopseudomonas palustris. Genes encoding this system, here designated cbbRRS, are juxtaposed between the divergently transcribed transcription activator gene, cbbR, and the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS. The three genes of the cbbRRS system represent a variation of the well-known two-component signal transduction systems, as there are a transmembrane hybrid sensor kinase and two response regulators, with no apparent DNA binding domain associated with any of the three proteins encoded by these genes. In this study, we showed that the membrane-bound full-length kinase undergoes autophosphorylation and transfers phosphate to both response regulators. A soluble, truncated version of the kinase was subsequently prepared and found to catalyze phosphorylation of response regulator 1 but not response regulator 2, implying that conformational changes and/or sequence-specific regions of the kinase are important for discriminating between the two response regulators. Analyses indicated that a complex network of control of gene expression must occur, with CbbR required for the expression of the cbbLS genes but dispensable for the synthesis of form II RubisCO (encoded by cbbM). The CbbRRS proteins specifically affected the activity and accumulation of form I RubisCO (CbbLS), as revealed by analyses of nonpolar, unmarked gene deletions. A tentative model of regulation suggested that changes in the phosphotransfer activity of the sensor kinase, possibly in response to a redox metabolic signal, cause modulation of the activity and synthesis of form I RubisCO.

scholarly journals Systematic Inactivation and Phenotypic Characterization of Two-Component Signal Transduction Systems of Enterococcus faecalis V583

2004 ◽  
Vol 186 (23) ◽  
pp. 7951-7958 ◽  
Author(s):  
Lynn E. Hancock ◽  
Marta Perego
Keyword(s):  
Signal Transduction ◽  
Antibiotic Resistance ◽  
Environmental Stress ◽  
Enterococcus Faecalis ◽  
Response Regulator ◽  
Phenotypic Characterization ◽  
Response Regulators ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Biofilm Development

ABSTRACT The ability of enterococci to adapt and respond to different environmental stimuli, including the host environment, led us to investigate the role of two-component signal transduction in the regulation of Enterococcus faecalis physiology. Using a bioinformatic approach, we previously identified 17 two-component systems (TCS), consisting of a sensory histidine kinase and the cognate response regulator, as well as an additional orphan response regulator (L. E. Hancock and M. Perego, J. Bacteriol. 184:5819-5825, 2002). In an effort to identify the potential function of each TCS in the biology of E. faecalis clinical isolate strain V583, we constructed insertion mutations in each of the response regulators. We were able to inactivate 17 of 18 response regulators, the exception being an ortholog of YycF, previously shown to be essential for viability in a variety of gram-positive microorganisms. The biological effects of the remaining mutations were assessed by using a number of assays, including antibiotic resistance, biofilm formation, and environmental stress. We identified TCS related to antibiotic resistance and environmental stress and found one system which controls the initiation of biofilm development by E. faecalis.

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 Cpx Two-Component Signal Transduction inEscherichia coli: Excessive CpxR-P Levels Underlie CpxA* Phenotypes

2000 ◽  
Vol 182 (5) ◽  
pp. 1423-1426 ◽  
Author(s):  
Peter De Wulf ◽  
E. C. C. Lin
Keyword(s):  
Signal Transduction ◽  
Response Regulator ◽  
Response Regulators ◽  
Signal Transduction System ◽  
Regulate Gene Expression ◽  
Adverse Conditions ◽  
Two Component ◽  
Sensor Protein ◽  
Two Component Signal Transduction ◽  
Cognate Response Regulator

ABSTRACT In Escherichia coli, the CpxA-CpxR two-component signal transduction system and the ςE and ς32response pathways jointly regulate gene expression in adaptation to adverse conditions. These include envelope protein distress, heat shock, oxidative stress, high pH, and entry into stationary phase. Certain mutant versions of the CpxA sensor protein (CpxA* proteins) exhibit an elevated ratio of kinase to phosphatase activity on CpxR, the cognate response regulator. As a result, CpxA* strains display numerous phenotypes, many of which cannot be easily related to currently known functions of the CpxA-CpxR pathway. It is unclear whether CpxA* phenotypes are caused solely by hyperphosphorylation of CpxR. We here report that all of the tested CpxA* phenotypes depend on elevated levels of CpxR-P and not on cross-signalling of CpxA* to noncognate response regulators.

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