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 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 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 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.

scholarly journals RegA, the Regulator of the Two-Component System RegB/RegA of Brucella suis, Is a Controller of Both Oxidative Respiration and Denitrification Required for Chronic Infection in Mice

2013 ◽  
Vol 81 (6) ◽  
pp. 2053-2061 ◽  
Author(s):  
Elias Abdou ◽  
Amélie Deredjian ◽  
María Pilar Jiménez de Bagüés ◽  
Stephan Köhler ◽  
Véronique Jubier-Maurin
Keyword(s):  
Oxygen Deficiency ◽  
Transcriptional Regulator ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Ubiquinol Oxidase ◽  
Two Component ◽  
Brucella Suis ◽  
Oxidative Respiration

ABSTRACTAdaptation to oxygen deficiency is essential for virulence and persistence ofBrucellainside the host. The flexibility of this bacterium with respect to oxygen depletion is remarkable, sinceBrucella suiscan use an oxygen-dependent transcriptional regulator of the FnrN family, two high-oxygen-affinity terminal oxidases, and a complete denitrification pathway to resist various conditions of oxygen deficiency. Moreover, our previous results suggested that oxidative respiration and denitrification can be simultaneously used byB. suisunder microaerobiosis. The requirement of a functional cytochromebdubiquinol oxidase for nitrite reductase expression evidenced the linkage of these two pathways, and the central role of the two-component system RegB/RegA in the coordinated control of both respiratory systems was demonstrated. We propose a scheme for global regulation ofB. suisrespiratory pathways by the transcriptional regulator RegA, which postulates a role for the cytochromebdubiquinol oxidase in redox signal transmission to the histidine sensor kinase RegB. More importantly, RegA was found to be essential forB. suispersistencein vivowithin oxygen-limited target organs. It is conceivable that RegA acts as a controller of numerous systems involved in the establishment of the persistent state, characteristic of chronic infections byBrucella.

scholarly journals The McdAB system positions α-carboxysomes in proteobacteria

2020 ◽  
Author(s):  
Joshua S. MacCready ◽  
Lisa Tran ◽  
Joseph L. Basalla ◽  
Pusparanee Hakim ◽  
Anthony G. Vecchiarelli
Keyword(s):  
Carbon Fixation ◽  
Synechococcus Elongatus ◽  
Bacterial Protein ◽  
Metabolic Processes ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Pcc 7942

SummaryCarboxysomes are protein-based organelles essential for carbon fixation in cyanobacteria and proteobacteria. Previously, we showed that the cyanobacterial nucleoid is utilized as a surface for the equidistant-spacing of β-carboxysomes across cell lengths by a two-component system (McdAB) in the model cyanobacterium Synechococcus elongatus PCC 7942. More recently, we found that McdAB systems are widespread among β-cyanobacteria, which possess β-carboxysomes, but are absent in α-cyanobacteria, which possess structurally distinct α-carboxysomes. Since cyanobacterial α-carboxysomes are thought to have arisen in proteobacteria and were subsequently horizontally transferred into cyanobacteria, this raised the question whether α-carboxysome containing proteobacteria possess a McdAB system for positioning α-carboxysomes. Here, using the model chemoautotrophic proteobacterium H. neapolitanus, we show that a McdAB system distinct from that of β-cyanobacteria operates to position α-carboxysomes across cell lengths. We further show that this system is widespread among α-carboxysome containing proteobacteria and that cyanobacteria likely inherited an α-carboxysome operon from a proteobacterium lacking the mcdAB locus. These results demonstrate that McdAB is a cross-phylum two-component system necessary for positioning α- and β-carboxysomes. The findings have further implications for understanding the positioning of other bacterial protein-based organelles involved in diverse metabolic processes.

scholarly journals Phosphorelay as the Sole Physiological Route of Signal Transmission by the Arc Two-Component System ofEscherichia coli

2000 ◽  
Vol 182 (13) ◽  
pp. 3858-3862 ◽  
Author(s):  
Ohsuk Kwon ◽  
Dimitris Georgellis ◽  
E. C. C. Lin
Keyword(s):  
Response Regulator ◽  
Signal Transmission ◽  
Phosphoryl Group ◽  
Sensor Kinase ◽  
Wild Type ◽  
Two Component System ◽  
Component System ◽  
Group Transfer ◽  
Two Component

ABSTRACT The Arc two-component system, comprising a tripartite sensor kinase (ArcB) and a response regulator (ArcA), modulates the expression of numerous genes involved in respiratory functions. In this study, the steps of phosphoryl group transfer from phosphorylated ArcB to ArcA were examined in vivo by using single copies of wild-type and mutantarcB alleles. The results indicate that the signal transmission occurs solely by His-Asp-His-Asp phosphorelay.

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