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 ◽  
Caulobacter Crescentus ◽  
Genetic Interaction ◽  
Response Regulator ◽  
Cell Envelope ◽  
Defined Medium ◽  
Growth Defect ◽  
Phosphoryl Transfer ◽  
Signaling Systems ◽  
Two Component

AbstractTwo-component signaling systems (TCSs) regulate cellular homeostasis in response to changes in the environment. Typical TCSs comprise a sensor histidine kinase and a response regulator; the kinase senses environmental conditions and relays this information via phosphoryl transfer to its cognate response regulator, which controls gene expression. Bacteria often express many TCS gene pairs that control distinct physiological processes, but the regulatory connections between TCSs remain underexplored. 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. We found that NtrZ, a previously uncharacterized periplasmic protein, functions upstream of the NtrY sensor kinase. We observed significant overlap in the ChvI and NtrX transcriptional regulons, which provides support for the genetic connection between ntrYX and chvGI. Our analyses indicated that the growth defect of strains lacking ChvGI is determined by the phosphorylation state of NtrX and, to some extent, by the level of the TonB-dependent receptor ChvT. To explain the genetic interaction between these TCSs, we propose a model in which NtrZ functions in the periplasm to regulate the NtrY kinase, promoting phosphorylation of NtrX and modulating the regulatory overlap between NtrX and ChvI.ImportanceTwo-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 uncovered significant overlap with the NtrX regulon. We revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. Finally, we identified NtrZ as a novel NtrY regulator. Our work establishes C. crescentus as an excellent model to investigate multi-level regulatory connections between ChvGI and NtrYX in α-proteobacteria.

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 Molecular basis of unidirectional information transmission in two-component systems: lessons from the DesK-DesR thermosensor

2021 ◽  
Author(s):  
Sofia Lima ◽  
Juan Blanco ◽  
Federico Olivieri ◽  
Juan Andres Imelio ◽  
Federico Carrion ◽  
...  
Keyword(s):  
Histidine Kinase ◽  
Regulatory Networks ◽  
Response Regulator ◽  
Signal Transmission ◽  
Phosphoryl Transfer ◽  
Signaling Systems ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Histidine Phosphorylation

Cellular signaling systems transmit information over long distances using allosteric transitions and/or post-translational modifications. In two-component systems the sensor histidine kinase and response regulator are wired through phosphoryl-transfer reactions, using either a uni- or bi-directional transmission mode, allowing to build rich regulatory networks. Using the thermosensor DesK-DesR two-component system from Bacillus subtilis and combining crystal structures, QM/MM calculations and integrative kinetic modeling, we uncover that: i) longer or shorter distances between the phosphoryl-acceptor and -donor residues can shift the phosphoryl-transfer equilibrium; ii) the phosphorylation-dependent dimerization of the regulator acts as a sequestering mechanism by preventing the interaction with the histidine kinase; and iii) the kinase's intrinsic conformational equilibrium makes the phosphotransferase state unlikely in the absence of histidine phosphorylation, minimizing backwards transmission. These mechanisms allow the system to control the direction of signal transmission in a very efficient way, showcasing the key role that structure-encoded allostery plays in signaling proteins to store and transmit information.

Phosphorylation and DNA binding of the regulator DcuR of the fumarate-responsive two-component system DcuSR of Escherichia coli

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 877-883 ◽  
Author(s):  
Ingo G. Janausch ◽  
Inma Garcia-Moreno ◽  
Daniela Lehnen ◽  
Yvonne Zeuner ◽  
Gottfried Unden
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Response Regulator ◽  
Sensory System ◽  
Phosphoryl Transfer ◽  
Affinity Binding ◽  
High Affinity ◽  
Two Component ◽  
Target Promoters

The function of the response regulator DcuR of the DcuSR fumarate two-component sensory system of Escherichia coli was analysed in vitro. Isolated DcuR protein was phosphorylated by the sensory histidine kinase, DcuS, and ATP, or by acetyl phosphate. In gel retardation assays with target promoters (frdA, dcuB, dctA), phosphoryl DcuR (DcuR-P) formed a high-affinity complex, with an apparent K D (app. K D) of 0·2–0·3 μM DcuR-P, and a low-affinity (app. K D 0·8–2 μM) complex. The high-affinity complex was formed only with promoters transcriptionally-regulated by DcuSR, whereas low-affinity binding was seen also with some DcuSR-independent promoters. The binding site of DcuR-P at the dcuB promoter was determined by DNase I footprinting. One binding site of 42–52 nt (position −359 to −400/−410 nt upstream of the transcriptional start) was identified in the presence of low and high concentrations of DcuR-P. Non-phosphorylated DcuR, or DcuR-D56N mutated in the phosphoryl-accepting Asp56 residue, showed low-affinity binding to target promoters. DcuR-D56N was still able to interact with DcuS. DcuR-D56N increased the phosphorylation of DcuS and competitively inhibited phosphoryl transfer to wild-type DcuR.

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 Novel Role for an HPt Domain in Stabilizing the Phosphorylated State of a Response Regulator Domain

2000 ◽  
Vol 182 (23) ◽  
pp. 6673-6678 ◽  
Author(s):  
Fabiola Janiak-Spens ◽  
David P. Sparling ◽  
Ann H. West
Keyword(s):  
Response Regulator ◽  
Phosphoryl Transfer ◽  
Sensor Kinase ◽  
Signaling System ◽  
Phosphorylated Protein ◽  
Physiological Conditions ◽  
Regulatory Domains ◽  
Regulatory Systems ◽  
Two Component ◽  
Stabilization Effect

ABSTRACT Two-component regulatory systems that utilize a multistep phosphorelay mechanism often involve a histidine-containing phosphotransfer (HPt) domain. These HPt domains serve an essential role as histidine-phosphorylated protein intermediates during phosphoryl transfer from one response regulator domain to another. InSaccharomyces cerevisiae, the YPD1 protein facilitates phosphoryl transfer from a hybrid sensor kinase, SLN1, to two distinct response regulator proteins, SSK1 and SKN7. Because the phosphorylation state largely determines the functional state of response regulator proteins, we have carried out a comparative study of the phosphorylated lifetimes of the three response regulator domains associated with SLN1, SSK1, and SKN7 (R1, R2, and R3, respectively). The isolated regulatory domains exhibited phosphorylated lifetimes within the range previously observed for other response regulator domains (i.e., several minutes to several hours). However, in the presence of YPD1, we found that the half-life of phosphorylated SSK1-R2 was dramatically extended (almost 200-fold longer than in the absence of YPD1). This stabilization effect was specific for SSK1-R2 and was not observed for SLN1-R1 or SKN7-R3. Our findings suggest a mechanism by which SSK1 is maintained in its phosphorylated state under normal physiological conditions and demonstrate an unprecedented regulatory role for an HPt domain in a phosphorelay signaling system.

scholarly journals Regulation of Bacterial Cell Cycle Progression by Redundant Phosphatases

2020 ◽  
Vol 202 (17) ◽  
Author(s):  
Jérôme Coppine ◽  
Andreas Kaczmarczyk ◽  
Kenny Petit ◽  
Thomas Brochier ◽  
Urs Jenal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Environmental Changes ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Model Organism ◽  
Replication Initiation ◽  
Cycle Progression ◽  
Content Type ◽  
Two Component

ABSTRACT In the model organism Caulobacter crescentus, a network of two-component systems involving the response regulators CtrA, DivK, and PleD coordinates cell cycle progression with differentiation. Active phosphorylated CtrA prevents chromosome replication in G1 cells while simultaneously regulating expression of genes required for morphogenesis and development. At the G1-S transition, phosphorylated DivK (DivK∼P) and PleD (PleD∼P) accumulate to indirectly inactivate CtrA, which triggers DNA replication initiation and concomitant cellular differentiation. The phosphatase PleC plays a pivotal role in this developmental program by keeping DivK and PleD phosphorylation levels low during G1, thereby preventing premature CtrA inactivation. Here, we describe CckN as a second phosphatase akin to PleC that dephosphorylates DivK∼P and PleD∼P in G1 cells. However, in contrast to PleC, no kinase activity was detected with CckN. The effects of CckN inactivation are largely masked by PleC but become evident when PleC and DivJ, the major kinase for DivK and PleD, are absent. Accordingly, mild overexpression of cckN restores most phenotypic defects of a pleC null mutant. We also show that CckN and PleC are proteolytically degraded in a ClpXP-dependent way before the onset of the S phase. Surprisingly, known ClpX adaptors are dispensable for PleC and CckN proteolysis, raising the possibility that as yet unidentified proteolytic adaptors are required for the degradation of both phosphatases. Since cckN expression is induced in stationary phase, depending on the stress alarmone (p)ppGpp, we propose that CckN acts as an auxiliary factor responding to environmental stimuli to modulate CtrA activity under suboptimal conditions. IMPORTANCE Two-component signal transduction systems are widely used by bacteria to adequately respond to environmental changes by adjusting cellular parameters, including the cell cycle. In Caulobacter crescentus, PleC acts as a phosphatase that indirectly protects the response regulator CtrA from premature inactivation during the G1 phase of the cell cycle. Here, we provide genetic and biochemical evidence that PleC is seconded by another phosphatase, CckN. The activity of PleC and CckN phosphatases is restricted to the G1 phase since both proteins are degraded by ClpXP protease before the G1-S transition. Degradation is independent of any known proteolytic adaptors and relies, in the case of CckN, on an unsuspected N-terminal degron. Our work illustrates a typical example of redundant functions between two-component proteins.

The senX3–regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1423-1435 ◽  
Author(s):  
Tanya Parish ◽  
Debbie A. Smith ◽  
Gretta Roberts ◽  
Joanna Betts ◽  
Neil G. Stoker
Keyword(s):  
Gene Expression ◽  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Regulatory System ◽  
Normal Growth ◽  
Growth Defect ◽  
Stress Genes ◽  
Two Component ◽  
Whole Genome Microarray ◽  
Immunocompetent Mice

Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3–regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3–regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.

DevR–DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 865-875 ◽  
Author(s):  
Deepak Kumar Saini ◽  
Vandana Malhotra ◽  
Deepanwita Dey ◽  
Neha Pant ◽  
Taposh K. Das ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Regulatory System ◽  
Site Directed Mutagenesis ◽  
Phosphoryl Transfer ◽  
Dependent Manner ◽  
Two Component System ◽  
Component System ◽  
Two Component

Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS201) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His6-tagged proteins from Escherichia coli. DevS201 underwent autophosphorylation and participated in rapid phosphotransfer to DevR in a Mg2+-dependent manner. Chemical stability analysis and site-directed mutagenesis implicated the highly conserved residues His395 and Asp54 as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp8 and Asp9 residues, postulated to form the acidic Mg2+-binding pocket, and the invariant Lys104 of DevR, abrogated phosphoryl transfer from DevS201 to DevR. DevR–DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of the Rv3134c–devR–devS operon was induced at the RNA level in hypoxic cultures of M. tuberculosis H37Rv and was associated with an increase in the level of DevR protein. However, in a devR mutant strain expressing the N-terminal domain of DevR, induction was observed at the level of RNA expression but not at that of protein. DevS was translated independently of DevR and induction of devS transcripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia.

scholarly journals Convergence of PASTA Kinase and Two-Component Signaling in Response to Cell Wall Stress inEnterococcus faecalis

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Stephanie L. Kellogg ◽  
Christopher J. Kristich
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Antimicrobial Resistance ◽  
Enterococcus Faecalis ◽  
Opportunistic Pathogen ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Signaling Systems ◽  
Changing Environments ◽  
Two Component

ABSTRACTTwo common signal transduction mechanisms used by bacteria to sense and respond to changing environments are two-component systems (TCSs) and eukaryote-like Ser/Thr kinases and phosphatases (eSTK/Ps).Enterococcus faecalisis a Gram-positive bacterium and a serious opportunistic pathogen that relies on both a TCS and an eSTK/P pathway for intrinsic resistance to cell wall-targeting antibiotics. The TCS consists of a histidine kinase (CroS) and a response regulator (CroR) that become activated upon exposure of cells to cell wall-targeting antibiotics, leading to a modulation of gene expression. The eSTK/P pathway consists of a transmembrane kinase (IreK) and its cognate phosphatase (IreP), which act antagonistically to mediate antibiotic resistance through an unknown mechanism. Because both CroS/R and IreK/P contribute to enterococcal resistance toward cell wall-targeting antibiotics, we hypothesized that these signaling systems are intertwined. To test this hypothesis, we analyzed CroR phosphorylation and CroS/R-dependent gene expression to probe the influence of IreK and IreP on CroS/R signaling. In addition, we analyzed the phosphorylation state of CroS, which revealed the IreK-dependent phosphorylation of a Thr residue important for CroS function. Our results are consistent with a model in which IreK positively influences CroR-dependent gene expression through the phosphorylation of CroS to promote antimicrobial resistance inE. faecalis.IMPORTANCETwo-component signaling systems (TCSs) and eukaryote-like Ser/Thr kinases (eSTKs) are used by bacteria to sense and adapt to changing environments. Understanding how these pathways are regulated to promote bacterial survival is critical for a more complete understanding of bacterial stress responses and physiology. The opportunistic pathogenEnterococcus faecalisrelies on both a TCS (CroS/R) and an eSTK (IreK) for intrinsic resistance to cell wall-targeting antibiotics. We probed the relationship between CroS/R and IreK, revealing the convergence of IreK and the sensor kinase CroS to enhance signaling through CroS/R and increase antimicrobial resistance inE. faecalis. This newly described example of eSTK/TCS convergence adds to our understanding of the signaling networks mediating antimicrobial resistance inE. faecalis.

Sign in / Sign up

Export Citation Format

Share Document