scholarly journals Glucose-Binding of Periplasmic Protein GltB Activates GtrS-GltR Two-Component System in Pseudomonas aeruginosa

2021 ◽  
Vol 9 (2) ◽  
pp. 447 ◽  
Author(s):  
Chenchen Xu ◽  
Qiao Cao ◽  
Lefu Lan
Keyword(s):  
Deletion Mutant ◽  
Binding Protein ◽  
Two Component System ◽  
Component System ◽  
Host Interactions ◽  
Regulatory Circuit ◽  
Glucose Binding ◽  
Two Component ◽  
Glucose Binding Protein

A two-component system GtrS-GltR is required for glucose transport activity in P. aeruginosa and plays a key role during P. aeruginosa-host interactions. However, the mechanism of action of GtrS-GltR has not been definitively established. Here, we show that gltB, which encodes a periplasmic glucose binding protein, is essential for the glucose-induced activation of GtrS-GltR in P. aeruginosa. We determined that GltB is capable of binding to membrane regulatory proteins including GtrS, the sensor kinase of the GtrS-GltR TCS. We observed that alanine substitution of glucose-binding residues abolishes the ability of GltB to promote the activation of GtrS-GltR. Importantly, like the gtrS deletion mutant, gltB deletion mutant showed attenuated virulence in both Drosophila melanogaster and mouse models of infection. In addition, using CHIP-seq experiments, we showed that the promoter of gltB is the major in vivo target of GltR. Collectively, these data suggest that periplasmic binding protein GltB and GtrS-GltR TCS form a complex regulatory circuit that regulates the virulence of P. aeruginosa in response to glucose.

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.

Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa

1993 ◽  
Vol 39 (7) ◽  
pp. 722-725 ◽  
Author(s):  
John L. Wylie ◽  
Elizabeth A. Worobec
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Glucose Transport ◽  
Transport System ◽  
Binding Protein ◽  
High Affinity ◽  
Glucose Binding ◽  
Glucose Transport System ◽  
Glucose Binding Protein

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.Key words: Pseudomonas aeruginosa, glucose transport, OprB, glucose binding protein.

scholarly journals Autoinducer-2 and QseC Control Biofilm Formation and In Vivo Virulence of Aggregatibacter actinomycetemcomitans

2010 ◽  
Vol 78 (7) ◽  
pp. 2919-2926 ◽  
Author(s):  
Elizabeth A. Novak ◽  
HanJuan Shao ◽  
Carlo Amorin Daep ◽  
Donald R. Demuth
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Biofilm Formation ◽  
Alveolar Bone ◽  
Wild Type ◽  
Two Component System ◽  
Component System ◽  
Autoinducer 2 ◽  
Two Component

ABSTRACT Biofilm formation by the periodontal pathogen Aggregatibacter actinomycetemcomitans is dependent upon autoinducer-2 (AI-2)-mediated quorum sensing. However, the components that link the detection of the AI-2 signal to downstream gene expression have not been determined. One potential regulator is the QseBC two-component system, which is part of the AI-2-dependent response pathway that controls biofilm formation in Escherichia coli. Here we show that the expression of QseBC in A. actinomycetemcomitans is induced by AI-2 and that induction requires the AI-2 receptors, LsrB and/or RbsB. Additionally, inactivation of qseC resulted in reduced biofilm growth. Since the ability to grow in biofilms is essential for A. actinomycetemcomitans virulence, strains that were deficient in QseC or the AI-2 receptors were examined in an in vivo mouse model of periodontitis. The ΔqseC mutant induced significantly less alveolar bone resorption than the wild-type strain (P < 0.02). Bone loss in animals infected with the ΔqseC strain was similar to that in sham-infected animals. The ΔlsrB, ΔrbsB, and ΔlsrB ΔrbsB strains also induced significantly less alveolar bone resorption than the wild type (P < 0.03, P < 0.02, and P < 0.01, respectively). However, bone loss induced by a ΔluxS strain was indistinguishable from that induced by the wild type, suggesting that AI-2 produced by indigenous microflora in the murine oral cavity may complement the ΔluxS mutation. Together, these results suggest that the QseBC two-component system is part of the AI-2 regulon and may link the detection of AI-2 to the regulation of downstream cellular processes that are involved in biofilm formation and virulence of A. actinomycetemcomitans.

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 Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

2021 ◽  
pp. mbc.E21-03-0151
Author(s):  
Pusparanee Hakim ◽  
Y Hoang ◽  
Anthony G. Vecchiarelli
Keyword(s):  
Active Site ◽  
Carbon Fixation ◽  
Binding Region ◽  
Two Component System ◽  
Component System ◽  
Ratchet Mechanism ◽  
Bacterial Microcompartment ◽  
Two Component ◽  
Bacterial Nucleoid

Carboxysomes, the most prevalent and well-studied anabolic bacterial microcompartment, play a central role in efficient carbon fixation by cyanobacteria and proteobacteria. In previous studies, we identified the two-component system called McdAB that spatially distributes carboxysomes across the bacterial nucleoid. McdA, a ParA-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to carboxysome-localized McdB. As McdB stimulates McdA ATPase activity, McdA is removed from the nucleoid in the vicinity of carboxysomes, propelling these proteinaceous cargos toward regions of highest McdA concentration via a Brownian-ratchet mechanism. How the ATPase cycle of McdA governs its in vivo dynamics and carboxysome positioning remains unresolved. Here, by strategically introducing amino acid substitutions in the ATP-binding region of McdA, we sequentially trap McdA at specific steps in its ATP cycle. We map out critical events in the ATPase cycle of McdA that allows the protein to bind ATP, dimerize, change its conformation into a DNA-binding state, interact with McdB-bound carboxysomes, hydrolyze ATP and release from the nucleoid. We also find that McdA is a member of a previously unstudied subset of ParA family ATPases, harboring unique interactions with ATP and the nucleoid for trafficking their cognate intracellular cargos. [Media: see text] [Media: see text] [Media: see text]

scholarly journals Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution

2021 ◽  
Author(s):  
Pusparanee Hakim ◽  
Anthony G. Vecchiarelli
Keyword(s):  
Active Site ◽  
Carbon Fixation ◽  
Binding Region ◽  
Two Component System ◽  
Component System ◽  
Ratchet Mechanism ◽  
Bacterial Microcompartment ◽  
Two Component ◽  
Bacterial Nucleoid

ABSTRACTCarboxysomes, the most prevalent and well-studied anabolic bacterial microcompartment, play a central role in efficient carbon fixation by cyanobacteria and proteobacteria. In previous studies, we identified the two-component system called McdAB that spatially distributes carboxysomes across the bacterial nucleoid. McdA, a ParA-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to carboxysome-localized McdB. As McdB stimulates McdA ATPase activity, McdA is removed from the nucleoid in the vicinity of carboxysomes, propelling these proteinaceous cargos toward regions of highest McdA concentration via a Brownian-ratchet mechanism. However, how the ATPase cycle of McdA governs its in vivo dynamics and carboxysome positioning remains unresolved. Here, by strategically introducing amino acid substitutions in the ATP-binding region of McdA, we sequentially trap McdA at specific steps in its ATP cycle. We map out critical events in the ATPase cycle of McdA that allows the protein to bind ATP, dimerize, change its conformation into a DNA-binding state, interact with McdB-bound carboxysomes, hydrolyze ATP and release from the nucleoid. We also find that McdA is a member of a previously unstudied subset of ParA family ATPases, harboring unique interactions with ATP and the nucleoid for trafficking their cognate intracellular cargos.

scholarly journals A hybrid two-component system protein of a prominent human gut symbiont couples glycan sensing in vivo to carbohydrate metabolism

2006 ◽  
Vol 103 (23) ◽  
pp. 8834-8839 ◽  
Author(s):  
E. D. Sonnenburg ◽  
J. L. Sonnenburg ◽  
J. K. Manchester ◽  
E. E. Hansen ◽  
H. C. Chiang ◽  
...  
Keyword(s):  
Carbohydrate Metabolism ◽  
Two Component System ◽  
Human Gut ◽  
Component System ◽  
Two Component

scholarly journals Two-Component System RgfA/C Activates the fbsB Gene Encoding Major Fibrinogen-Binding Protein in Highly Virulent CC17 Clone Group B Streptococcus

PLoS ONE ◽  
2011 ◽  
Vol 6 (2) ◽  
pp. e14658 ◽  
Author(s):  
Rim Al Safadi ◽  
Laurent Mereghetti ◽  
Mazen Salloum ◽  
Marie-Frédérique Lartigue ◽  
Isabelle Virlogeux-Payant ◽  
...  
Keyword(s):  
Binding Protein ◽  
Group B Streptococcus ◽  
Two Component System ◽  
Gene Encoding ◽  
Component System ◽  
Fibrinogen Binding ◽  
Two Component ◽  
Group B ◽  
Highly Virulent ◽  
Clone Group

scholarly journals Regulation of Galactose Metabolism through the HisK:GalR Two-Component System in Thermoanaerobacter tengcongensis

2010 ◽  
Vol 192 (17) ◽  
pp. 4311-4316 ◽  
Author(s):  
Zhong Qian ◽  
Quanhui Wang ◽  
Wei Tong ◽  
Chuanqi Zhou ◽  
Qian Wang ◽  
...  
Keyword(s):  
Phosphoryl Group ◽  
Regulation Mechanism ◽  
Two Component System ◽  
Galactose Metabolism ◽  
Component System ◽  
Two Dimensional Electrophoresis ◽  
Thermoanaerobacter Tengcongensis ◽  
Two Component ◽  
Dimensional Electrophoresis

ABSTRACT Thermoanaerobacter tengcongensis could utilize galactose as a carbon source via the enzymes encoded by a novel gal operon, whose regulation mechanism has yet to be elucidated. We propose here that the gal operon in T. tengcongensis is regulated through a HisK:GalR two-component system. By using radioactive isotope assay and genetic analysis, we found that the kinase of this system, HisK, is phosphorylated by ATP, and the regulator, GalR, accepts a phosphoryl group during phosphorelay, in which the phosphoryl group at HisK-His-259 is transferred to GalR-Asp-56. Two-dimensional electrophoresis, followed by Western blotting, revealed that phosphorylation status of GalR is uniquely dependent on the galactose stimulus in vivo. Furthermore, DNA pulldown assays demonstrated that the phosphorylated GalR prefers binding to the operator DNA O2 , whereas the unphosphorylated GalR to O1 . A model of HisK:GalR is proposed to explain how galactose mediates the expression of the gal operon in T. tengcongensis.

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.

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