scholarly journals Pneumococcal HtrA Protease Mediates Inhibition of Competence by the CiaRH Two-Component Signaling System

2005 ◽  
Vol 187 (12) ◽  
pp. 3969-3979 ◽  
Author(s):  
M. E. Sebert ◽  
K. P. Patel ◽  
M. Plotnick ◽  
J. N. Weiser
Keyword(s):  
Histidine Kinase ◽  
Transformation Efficiency ◽  
Signaling System ◽  
Inhibitory Effects ◽  
Genetic Competence ◽  
Constitutive Activation ◽  
Protein Target ◽  
Protein Levels ◽  
Unknown Protein ◽  
Two Component

ABSTRACT Activation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation in Streptococcus pneumoniae through a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression of htrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of a ciaRH + strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.

scholarly journals Feedback control of a two-component signaling system by an Fe-S-binding receiver domain

2019 ◽  
Author(s):  
Benjamin J. Stein ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Feedback Control ◽  
Histidine Kinase ◽  
Oxygen Sensor ◽  
Cell Physiology ◽  
Environmental Cues ◽  
Sensor Kinase ◽  
Signaling System ◽  
Receiver Domain ◽  
Two Component ◽  
Feedback Inhibitor

AbstractTwo-component signaling systems (TCSs) function to detect environmental cues and transduce this information into a change in transcription. In its simplest form, TCS-dependent regulation of transcription entails phosphoryl-transfer from a sensory histidine kinase to its cognate DNA-binding receiver protein. However, in certain cases, auxiliary proteins may modulate TCSs in response to secondary environmental cues. Caulobacter crescentus FixT is one such auxiliary regulator. FixT is composed of a single receiver domain and functions as a feedback inhibitor of the FixL-FixJ (FixLJ) TCS, which regulates the transcription of genes involved in adaptation to microaerobiosis. We sought to define the impact of fixT on Caulobacter cell physiology and to understand the molecular mechanism by which FixT represses FixLJ signaling. fixT deletion results in excess production of porphyrins and premature entry into stationary phase, demonstrating the importance of feedback inhibition of the FixLJ signaling system. Although FixT is a receiver domain, it does not affect dephosphorylation of the oxygen-sensor kinase FixL or phosphoryltransfer from FixL to its cognate receiver FixJ. Rather, FixT represses FixLJ signaling by inhibiting the FixL autophosphorylation reaction. We have further identified a 4-cysteine motif in Caulobacter FixT that binds an Fe-S cluster and protects the protein from degradation by the Lon protease. Our data support a model in which oxidation of this Fe-S cluster promotes degradation of FixT in vivo. This proteolytic mechanism facilitates clearance the of the FixT feedback inhibitor from the cell under normoxia and resets the FixLJ system for a future microaerobic signaling event.ImportanceTwo-component signal transduction systems (TCSs) are broadly conserved in the bacterial kingdom and generally contain two molecular components: a sensor histidine kinase and a receiver protein. Sensor histidine kinases alter their phosphorylation state in direct response to a physical or chemical cue, whereas receiver proteins “receive” the phosphoryl group from the kinase to regulate a change in cell physiology. We have discovered that a single-domain receiver protein, FixT, binds an Fe-S cluster and controls Caulobacter heme homeostasis though its function as a negative feedback regulator of the oxygen-sensor kinase, FixL. We provide evidence that the Fe-S cluster protects FixT from Lon-dependent proteolysis in the cell and endows FixT with the ability to function as a second, autonomous oxygen/redox sensor in the FixL-FixJ signaling pathway. This study introduces a novel mechanism of regulated TCS feedback control by an Fe-S-binding receiver domain.

scholarly journals Feedback Control of a Two-Component Signaling System by an Fe-S-Binding Receiver Domain

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Benjamin J. Stein ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Histidine Kinase ◽  
Oxygen Sensor ◽  
Cell Physiology ◽  
Phosphoryl Transfer ◽  
Environmental Cues ◽  
Sensor Kinase ◽  
Signaling System ◽  
Receiver Domain ◽  
Two Component ◽  
Feedback Inhibitor

ABSTRACT Two-component signaling systems (TCSs) function to detect environmental cues and transduce this information into a change in transcription. In its simplest form, TCS-dependent regulation of transcription entails phosphoryl-transfer from a sensory histidine kinase to its cognate DNA-binding receiver protein. However, in certain cases, auxiliary proteins may modulate TCSs in response to secondary environmental cues. Caulobacter crescentus FixT is one such auxiliary regulator. FixT is composed of a single receiver domain and functions as a feedback inhibitor of the FixL-FixJ (FixLJ) TCS, which regulates the transcription of genes involved in adaptation to microaerobiosis. We sought to define the impact of fixT on Caulobacter cell physiology and to understand the molecular mechanism by which FixT represses FixLJ signaling. fixT deletion results in excess production of porphyrins and premature entry into stationary phase, demonstrating the importance of feedback inhibition of the FixLJ signaling system. Although FixT is a receiver domain, it does not affect dephosphorylation of the oxygen sensor kinase FixL or phosphoryl-transfer from FixL to its cognate receiver FixJ. Rather, FixT represses FixLJ signaling by inhibiting the FixL autophosphorylation reaction. We have further identified a 4-cysteine motif in Caulobacter FixT that binds an Fe-S cluster and protects the protein from degradation by the Lon protease. Our data support a model in which the oxidation of this Fe-S cluster promotes the degradation of FixT in vivo. This proteolytic mechanism facilitates clearance of the FixT feedback inhibitor from the cell under normoxia and resets the FixLJ system for a future microaerobic signaling event. IMPORTANCE Two-component signal transduction systems (TCSs) are broadly conserved in the bacterial kingdom and generally contain two molecular components, a sensor histidine kinase and a receiver protein. Sensor histidine kinases alter their phosphorylation state in direct response to a physical or chemical cue, whereas receiver proteins “receive” the phosphoryl group from the kinase to regulate a change in cell physiology. We have discovered that a single-domain receiver protein, FixT, binds an Fe-S cluster and controls Caulobacter heme homeostasis though its function as a negative-feedback regulator of the oxygen sensor kinase FixL. We provide evidence that the Fe-S cluster protects FixT from Lon-dependent proteolysis in the cell and endows FixT with the ability to function as a second, autonomous oxygen/redox sensor in the FixL-FixJ signaling pathway. This study introduces a novel mechanism of regulated TCS feedback control by an Fe-S-binding receiver domain.

FOXD3 inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma through regulation of the PI3K–Akt pathway

2020 ◽  
Vol 98 (6) ◽  
pp. 653-660 ◽  
Author(s):  
Xiaoxing Xie ◽  
Gaoyun Xiong ◽  
Wenjun Chen ◽  
Hongdan Fu ◽  
Mingqian Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nasopharyngeal Carcinoma ◽  
Cell Apoptosis ◽  
Inhibitory Effect ◽  
Akt Pathway ◽  
Migration And Invasion ◽  
Inhibitory Effects ◽  
Protein Levels ◽  
Flow Cytometric ◽  
Phosphoinositide 3 Kinase

FOXD3 has been found previously to positively regulate miR-26b, a tumor inhibitor of nasopharyngeal carcinoma (NPC). However, FOXD3’s precise function and associated mechanism of action in NPC have not yet been investigated. In this study, the expression of FOXD3 mRNA and protein was evaluated using RT-qPCR, western blotting, and immunohistochemistry. Protein levels involved in the phosphoinositide 3-kinase – protein kinase B (PI3K–Akt) pathway were assessed by western blot, and cell proliferation was determined by MTT and colony forming assays. Additionally, cell apoptosis was assessed by flow cytometric assay. Finally, the migration and invasion capabilities of the NPC cells were determined using wound healing and Transwell assays. We found that FOXD3 levels were relatively low in NPC tissue and cells, while an increase caused the inhibition of the PI3K–Akt pathway. Functional experiments found that overexpression of FOXD3 suppressed cell proliferation, migration, and invasion and enhanced cell apoptosis in NPC C6661 cells. IGF-1, an activator of the PI3K–Akt pathway, reversed the inhibitory effect of FOXD3. Furthermore, we found upregulation of the PI3K–Akt pathway and upregulation of the inhibitory effects of FOXD3 on C6661 cellular activities. In conclusion, FOXD3 negatively affected the PI3K–Akt pathway to restrain the processes involved in C6661 cell pathology. These findings further exposed the function and downstream axis of FOXD3 in NPC and displayed a promising new target for NPC therapy.

scholarly journals The EnvZ-OmpR Two-Component Signaling System Is Inactivated in a Mutant Devoid of Osmoregulated Periplasmic Glucans in Dickeya dadantii

2018 ◽  
Vol 9 ◽  
Author(s):  
Marine Caby ◽  
Sébastien Bontemps-Gallo ◽  
Peggy Gruau ◽  
Brigitte Delrue ◽  
Edwige Madec ◽  
...  
Keyword(s):  
Signaling System ◽  
Dickeya Dadantii ◽  
Two Component

scholarly journals Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Reed M. Stubbendieck ◽  
Paul D. Straight
Keyword(s):  
Antibiotic Resistance ◽  
Abc Transporter ◽  
Bacterial Communities ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Bioactive Metabolites ◽  
Signaling System ◽  
Content Type ◽  
Two Component ◽  
Analytical Approaches

ABSTRACT Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilis. IMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that includes the gastrointestinal tract of animals and the rhizosphere of plants. Interactions between different species in bacterial communities have impacts on our health and industry. However, many approaches currently used to study whole bacterial communities do not resolve mechanistic details of interspecies interactions, including how bacteria sense and respond to their competitors. Using a competition model, we have uncovered dual functions for a previously uncharacterized two-component signaling system involved in specific antibiotic resistance and biofilm morphology. Insights gleaned from signaling within interspecies interaction models build a more complete understanding of gene functions important for bacterial communities and will enhance community-level analytical approaches.

scholarly journals The Interaction Network and Signaling Specificity of Two-Component System in Arabidopsis

2020 ◽  
Vol 21 (14) ◽  
pp. 4898
Author(s):  
Ruxue Huo ◽  
Zhenning Liu ◽  
Xiaolin Yu ◽  
Zongyun Li
Keyword(s):  
Signal Transduction ◽  
Molecular Mechanisms ◽  
Interaction Network ◽  
Response Regulators ◽  
Signaling Specificity ◽  
Protein Protein Interaction ◽  
Protein Levels ◽  
Two Component ◽  
Model Plant Arabidopsis Thaliana ◽  
External Signals

Two-component systems (TCS) in plants have evolved into a more complicated multi-step phosphorelay (MSP) pathway, which employs histidine kinases (HKs), histidine-containing phosphotransfer proteins (HPts), and response regulators (RRs) to regulate various aspects of plant growth and development. How plants perceive the external signals, then integrate and transduce the secondary signals specifically to the desired destination, is a fundamental characteristic of the MSP signaling network. The TCS elements involved in the MSP pathway and molecular mechanisms of signal transduction have been best understood in the model plant Arabidopsis thaliana. In this review, we focus on updated knowledge on TCS signal transduction in Arabidopsis. We first present a brief description of the TCS elements; then, the protein–protein interaction network is established. Finally, we discuss the possible molecular mechanisms involved in the specificity of the MSP signaling at the mRNA and protein levels.

Kinetic and mechanistic analyses of new classes of inhibitors of two-component signal transduction systems using a coupled assay containing HpkA–DrrA from Thermotoga maritima

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 885-896 ◽  
Author(s):  
J. Estelle Foster ◽  
Qin Sheng ◽  
Jonathan R. McClain ◽  
Mark Bures ◽  
Thalia I. Nicas ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Histidine Kinase ◽  
Competitive Inhibition ◽  
Thermotoga Maritima ◽  
Phosphorylation Site ◽  
Phosphoryl Group ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Coupled Assay ◽  
Signal Transduction Systems

Two-component signal transduction systems (TCSs) play fundamental roles in bacterial survival and pathogenesis and have been proposed as targets for the development of novel classes of antibiotics. A new coupled assay was developed and applied to analyse the kinetic mechanisms of three new kinds of inhibitors of TCS function. The assay exploits the biochemical properties of the cognate HpkA–DrrA histidine kinase–response regulator pair from Thermotoga maritima and allows multiple turnovers of HpkA, linear formation of phosphorylated DrrA, and Michaelis–Menten analysis of inhibitors. The assay was validated in several ways, including confirmation of competitive inhibition by adenosine 5′-β,γ-imidotriphosphate (AMP-PNP). The coupled assay, autophosphorylation and chemical cross-linking were used to determine the mechanisms by which several compounds inhibit TCS function. A cyanoacetoacetamide showed non-competitive inhibition with respect to ATP concentration in the coupled assay. The cyanoacetoacetamide also inhibited autophosphorylation of histidine kinases from other bacteria, indicating that the coupled assay could detect general inhibitors of histidine kinase function. Inhibition of HpkA autophosphorylation by this compound was probably caused by aggregation of HpkA, consistent with a previous model for other hydrophobic compounds. In contrast, ethodin was a potent inhibitor of the combined assay, did not inhibit HpkA autophosphorylation, but still led to aggregation of HpkA. These data suggest that ethodin bound to the HpkA kinase and inhibited transfer of the phosphoryl group to DrrA. A peptide corresponding to the phosphorylation site of DrrA appeared to inhibit TCS function by a mechanism similar to that of ethodin, except that autophosphorylation was inhibited at high peptide concentrations. The latter mechanism of inhibition of TCS function is unusual and its analysis demonstrates the utility of these approaches to the kinetic analyses of additional new classes of inhibitors of TCS function.

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