scholarly journals A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides in Geobacillus stearothermophilus

2006 ◽  
Vol 73 (3) ◽  
pp. 874-884 ◽  
Author(s):  
Smadar Shulami ◽  
Galia Zaide ◽  
Gennady Zolotnitsky ◽  
Yael Langut ◽  
Geoff Feld ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Transport System ◽  
Response Regulator ◽  
Transcriptional Unit ◽  
Geobacillus Stearothermophilus ◽  
Two Component System ◽  
Component System ◽  
Abc Transport ◽  
Two Component

ABSTRACT Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His6-XynC, bound to a 220-bp fragment corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the −53 and the +34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 μM XynC. The His6-tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (ΔC p) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system.

scholarly journals Identification of a novel two-component system SenS/SenR modulating the production of the catalase-peroxidase CpeB and the haem-binding protein HbpS in Streptomyces reticuli

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3603-3614 ◽  
Author(s):  
Darío Ortiz de Orué Lucana ◽  
Peijian Zou ◽  
Marc Nierhaus ◽  
Hildgund Schrempf
Keyword(s):  
Peroxidase Activity ◽  
Response Regulator ◽  
Binding Protein ◽  
Streptomyces Avermitilis ◽  
Binding Motif ◽  
Two Component System ◽  
Component System ◽  
Regulatory Cascade ◽  
Two Component

The Gram-positive soil bacterium and cellulose degrader Streptomyces reticuli synthesizes the mycelium-associated enzyme CpeB, which displays haem-dependent catalase and peroxidase activity, as well as haem-independent manganese-peroxidase activity. The expression of the furS–cpeB operon depends on the redox regulator FurS and the presence of the haem-binding protein HbpS. Upstream of hbpS, the neighbouring senS and senR genes were identified. SenS is a sensor histidine kinase with five predicted N-terminally located transmembrane domains. SenR is the corresponding response regulator with a C-terminal DNA-binding motif. Comparative transcriptional and biochemical studies with a designed S. reticuli senS/senR chromosomal disruption mutant and a set of constructed Streptomyces lividans transformants showed that the presence of the novel two-component system SenS/SenR negatively modulates the expression of the furS–cpeB operon and the hbpS gene. The presence of SenS/SenR enhances considerably the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting that this system could participate directly or indirectly in the sensing of redox changes. Epitope-tagged HbpS (obtained from an Escherichia coli transformant) as well as the native S. reticuli HbpS interact in vitro specifically with the purified SenS fusion protein. On the basis of these findings, together with data deduced from the S. reticuli hbpS mutant strain, HbpS is suggested to act as an accessory protein that communicates with the sensor protein to modulate the corresponding regulatory cascade. Interestingly, close and distant homologues, respectively, of the SenS/SenR system are encoded within the Streptomyces coelicolor A3(2) and Streptomyces avermitilis genomes, but not within other known bacterial genomes. Hence the SenS/SenR system appears to be confined to streptomycetes.

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 Characterization of an Alcohol Dehydrogenase from the Cyanobacterium Synechocystis sp. Strain PCC 6803 That Responds to Environmental Stress Conditions via the Hik34-Rre1 Two-Component System

2009 ◽  
Vol 191 (13) ◽  
pp. 4383-4391 ◽  
Author(s):  
Rebeca Vidal ◽  
Luis López-Maury ◽  
Miguel G. Guerrero ◽  
Francisco J. Florencio
Keyword(s):  
Alcohol Dehydrogenase ◽  
Interaction Analysis ◽  
Response Regulator ◽  
Stress Conditions ◽  
Primary Alcohols ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Pcc 6803

ABSTRACT The slr1192 (adhA) gene from Synechocystis sp. strain PCC 6803 encodes a member of the medium-chain alcohol dehydrogenase/reductase family. The gene product AdhA exhibits NADP-dependent alcohol dehydrogenase activity, acting on a broad variety of aromatic and aliphatic primary alcohols and aldehydes but not on secondary alcohols or ketones. It exhibits superior catalytic efficiency for aldehyde reduction compared to that for alcohol oxidation. The enzyme is a cytosolic protein present in photoautotrophically grown Synechocystis cells. The expression of AdhA is enhanced upon the exposure of cells to different environmental stresses, although it is not essential for survival even under such stress conditions. The induction of the expression of the adhA gene is dependent on the Hik34-Rre1 two-component system, as it is severely impaired in mutant strains lacking either the histidine kinase Hik34 or the response regulator Rre1. In vitro DNA-protein interaction analysis reveals that the response regulator Rre1 binds specifically to the promoter region of the adhA gene.

scholarly journals Regulation of osmC Gene Expression by the Two-Component System rcsB-rcsC inEscherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 5870-5876 ◽  
Author(s):  
Marcela Davalos-Garcia ◽  
Annie Conter ◽  
Isabelle Toesca ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Response Regulator ◽  
Regulatory Element ◽  
In Vitro Transcription ◽  
Proximal Promoter ◽  
Two Component System ◽  
Mobility Shift ◽  
Component System ◽  
Two Component ◽  
Gel Mobility Shift

ABSTRACT The Escherichia coli osmC gene encodes an envelope protein of unknown function whose expression depends on osmotic pressure and growth phase. The gene is transcribed from two overlapping promoters, osmCp 1 andosmCp 2. Several factors regulating these promoters have been reported. The leucine-responsive protein Lrp represses osmCp 1 and activatesosmCp 2, the nucleoid-associated protein H-NS represses both promoters, and the stationary-phase sigma factor ςs specifically recognizesosmCp 2. This work reports the identification of an additional regulatory element, the two-component systemrcsB-rcsC, affecting positively the distal promoter osmCp 1. The response regulator of the system, RcsB, does not affect expression of the proximal promoter osmCp 2. Deletion analysis located the site necessary for RcsB activation just upstream ofosmCp 1. In vitro transcription experiments and gel mobility shift assays demonstrated that RcsB stimulates RNA polymerase binding at osmCp 1.

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.

scholarly journals Characterization of the role of the RadS/RadR two-component system in the radiation resistance of Deinococcus radiodurans

Microbiology ◽  
2011 ◽  
Vol 157 (10) ◽  
pp. 2974-2982 ◽  
Author(s):  
Shruti S. Desai ◽  
Yogendra S. Rajpurohit ◽  
Hari S. Misra ◽  
Dileep N. Deobagkar
Keyword(s):  
Gamma Radiation ◽  
Radiation Resistance ◽  
Deinococcus Radiodurans ◽  
Response Regulator ◽  
Strand Break ◽  
Two Component System ◽  
Component System ◽  
Dna Damaging Agents ◽  
Two Component

Deinococcus radiodurans shows extraordinary tolerance to DNA damage, and exhibits differential gene expression and protein recycling. A putative response regulator, the DRB0091 (RadR) ORF, was identified from a pool of DNA-binding proteins induced in response to gamma radiation in this bacterium. radR is located upstream of drB0090, which encodes a putative sensor histidine kinase (RadS) on the megaplasmid. Deletion of these genes both individually and together resulted in hypersensitivity to DNA-damaging agents and a delayed or altered double-strand break repair. A ΔradRradS double mutant and a ΔradR single mutant showed nearly identical responses to gamma radiation and UVC. Wild-type RadR and RadS complemented the corresponding mutant strains, but also exhibited significant cross-complementation, albeit at lower doses of gamma radiation. The radS transcript was not detected in the ΔradR mutant, suggesting the existence of a radRS operon. Recombinant RadS was autophosphorylated and could catalyse the transfer of γ phosphate from ATP to RadR in vitro. These results indicated the functional interaction of RadS and RadR, and suggested a role for the RadS/RadR two-component system in the radiation resistance of this bacterium.

scholarly journals Phosphotransfer Reactions of the CbbRRS Three-Protein Two- Component System from Rhodopseudomonas palustris CGA010 Appear To Be Controlled by an Internal Molecular Switch on the Sensor Kinase

2006 ◽  
Vol 189 (2) ◽  
pp. 325-335 ◽  
Author(s):  
Simona Romagnoli ◽  
F. Robert Tabita
Keyword(s):  
Response Regulator ◽  
Rhodopseudomonas Palustris ◽  
Molecular Switch ◽  
Sensor Kinase ◽  
Two Component System ◽  
Conserved Residues ◽  
Component System ◽  
Receiver Domain ◽  
Two Component

ABSTRACT The CbbRRS system is an atypical three-protein two-component system that modulates the expression of the cbb I CO2 fixation operon of Rhodopseudomonas palustris, possibly in response to a redox signal. It consists of a membrane-bound hybrid sensor kinase, CbbSR, with a transmitter and receiver domain, and two response regulator proteins, CbbRR1 and CbbRR2. No detectable helix-turn-helix DNA binding domain is associated with either response regulator, but an HPt domain and a second receiver domain are predicted at the C-terminal region of CbbRR1 and CbbRR2, respectively. The abundance of conserved residues predicted to participate in a His-Asp phosphorelay raised the question of their de facto involvement. In this study, the role of the multiple receiver domains was elucidated in vitro by generating site-directed mutants of the putative conserved residues. Distinct phosphorylation patterns were obtained with two truncated versions of the hybrid sensor kinase, CbbSRT189 and CbbSRR96 (CbbSR beginning at residues T189 and R96, respectively). These constructs also exhibited substantially different affinities for ATP and phosphorylation stability, which was found to be dependent on a conserved Asp residue (Asp-696) within the kinase receiver domain. Asp-696 also played an important role in defining the specificity of phosphorylation for response regulators CbbRR1 or CbbRR2, and this residue appeared to act in conjunction with residues within the region from Arg-96 to Thr-189 at the N terminus of the sensor kinase. The net effect of concerted interactions at these distinct regions of CbbSR created an internal molecular switch that appears to coordinate a unique branched phosphorelay system.

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.

scholarly journals Identification of a Novel Two-Component System in Streptococcus gordonii V288 Involved in Biofilm Formation

2004 ◽  
Vol 72 (6) ◽  
pp. 3489-3494 ◽  
Author(s):  
Yongshu Zhang ◽  
Yu Lei ◽  
Ali Khammanivong ◽  
Mark C. Herzberg
Keyword(s):  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Streptococcus Gordonii ◽  
Wild Type ◽  
Two Component System ◽  
Component System ◽  
Two Component

ABSTRACT Streptococcus gordonii is a pioneer colonizer of the teeth, contributing to the initiation of the oral biofilm called dental plaque. To identify genes that may be important in biofilm formation, a plasmid integration library of S. gordonii V288 was used. After screening for in vitro biofilm formation on polystyrene, a putative biofilm-defective mutant was isolated. In this mutant, pAK36 was inserted into a locus encoding a novel two-component system (bfr [biofilm formation related]) with two cotranscribed genes that form an operon. bfrA encodes a putative response regulator, while bfrB encodes a receptor histidine kinase. The bfr mutant and wild-type strain V288 showed similar growth rates in Todd-Hewitt broth (THB). A bfr-cat fusion strain was constructed. During growth in THB, the reporter activity (chloramphenicol acetyltransferase) was first detected in mid-log phase and reached a maximum in stationary phase, suggesting that transcription of bfr was growth stage dependent. After being harvested from THB, the bfr mutant adhered less effectively than did wild-type strain V288 to saliva-coated hydroxyapatite (sHA). To simulate pioneer colonization of teeth, S. gordonii V288 was incubated with sHA for 4 h in THB with 10% saliva to develop biofilms. RNA was isolated, and expression of bfrAB was estimated. In comparison to that of cells grown in suspension (free-growing cells), bfr mRNA expression by sessile cells on sHA was 1.8-fold greater and that by surrounding planktonic cells was 3.5-fold greater. Therefore, bfrAB is a novel two-component system regulated in association with S. gordonii biofilm formation in vitro.

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