scholarly journals Phenotype MicroArray Analysis of Escherichia coli K-12 Mutants with Deletions of All Two-Component Systems

2003 ◽  
Vol 185 (16) ◽  
pp. 4956-4972 ◽  
Author(s):  
Lu Zhou ◽  
Xiang-He Lei ◽  
Barry R. Bochner ◽  
Barry L. Wanner
Keyword(s):  
Escherichia Coli ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Standard Technique ◽  
Common Mechanism ◽  
Phenotype Microarray ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
K 12

ABSTRACT Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in Escherichia coli K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.

scholarly journals Molecular Characterization of Two-Component Systems of Helicobacter pylori

2000 ◽  
Vol 182 (8) ◽  
pp. 2068-2076 ◽  
Author(s):  
Dagmar Beier ◽  
Rainer Frank
Keyword(s):  
Helicobacter Pylori ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Response Regulators ◽  
Reading Frame ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation. Here we report the characterization of members of the two-component systems of the gastric pathogenHelicobacter pylori deduced from the genome sequence of strain 26695. We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains. An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase-response regulator pairs. Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3′ end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165.

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.

scholarly journals A transcriptome study of the QseEF two-component system and the QseG membrane protein in enterohaemorrhagic Escherichia coli O157 : H7

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1167-1175 ◽  
Author(s):  
Nicola C. Reading ◽  
David Rasko ◽  
Alfredo G. Torres ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Response Regulator ◽  
Two Component System ◽  
E Coli ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Type Iii Secretion Effector

QseE is a sensor kinase that responds to epinephrine, sulfate and phosphate. QseE constitutes a two-component signalling system together with the QseF σ 54-dependent response regulator. Encoded within the same operon as qseEF is the qseG gene, which encodes a membrane protein involved in the translocation of a type III secretion effector protein of enterohaemorrhagic Escherichia coli (EHEC) into epithelial cells. The qseEGF genes also form an operon with the glnB gene, which encodes the E. coli nitrogen sensor PII protein. Here we report a transcriptome analysis comparing qseE, qseF andqseG single mutants with the wild-type strain. This study revealed that the proteins encoded by these genes play a modest but significant role in iron uptake. Although QseEFG regulate genes involved in nitrogen utilization, these proteins do not play a notable role in nitrogen metabolism. In addition, QseEFG regulate transcription of the rcsBC and phoPQ two-component systems, linking several signal transduction pathways. The similarity of the microarray profiles of these mutants also indicates that these proteins work together. These data indicate that QseEFG are involved in the regulation of virulence and metabolism in EHEC.

scholarly journals Regulation of Virulence by Two-Component Systems in Pathogenic Burkholderia

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Matthew M. Schaefers
Keyword(s):  
Histidine Kinase ◽  
Burkholderia Cepacia ◽  
Target Genes ◽  
Response Regulator ◽  
Pharmaceutical Products ◽  
Phosphoryl Group ◽  
Content Type ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT The regulation and timely expression of bacterial genes during infection is critical for a pathogen to cause an infection. Bacteria have multiple mechanisms to regulate gene expression in response to their environment, one of which is two-component systems (TCS). TCS have two components. One component is a sensory histidine kinase (HK) that autophosphorylates when activated by a signal. The activated sensory histidine kinase then transfers the phosphoryl group to the second component, the response regulator, which activates transcription of target genes. The genus Burkholderia contains members that cause human disease and are often extensively resistant to many antibiotics. The Burkholderia cepacia complex (BCC) can cause severe lung infections in patients with cystic fibrosis (CF) or chronic granulomatous disease (CGD). BCC members have also recently been associated with several outbreaks of bacteremia from contaminated pharmaceutical products. Separate from the BCC is Burkholderia pseudomallei, which is the causative agent of melioidosis, a serious disease that occurs in the tropics, and a potential bioterrorism weapon. Bioinformatic analysis of sequenced Burkholderia isolates predicts that most strains have at least 40 TCS. The vast majority of these TCS are uncharacterized both in terms of the signals that activate them and the genes that are regulated by them. This review will highlight TCS that have been described to play a role in virulence in either the BCC or B. pseudomallei. Since many of these TCS are involved in virulence, TCS are potential novel therapeutic targets, and elucidating their function is critical for understanding Burkholderia pathogenesis.

scholarly journals The Histidine Residue of QseC Is Required for Canonical Signaling between QseB and PmrB in Uropathogenic Escherichia coli

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Erin J. Breland ◽  
Ellisa W. Zhang ◽  
Tomas Bermudez ◽  
Charles R. Martinez ◽  
Maria Hadjifrangiskou
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Polymyxin B ◽  
Sensor Kinase ◽  
Histidine Residue ◽  
Content Type ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Tract Infections

ABSTRACT Two-component systems are prototypically comprised of a histidine kinase (sensor) and a response regulator (responder). The sensor kinases autophosphorylate at a conserved histidine residue, acting as a phosphodonor for subsequent phosphotransfer to and activation of a cognate response regulator. In rare cases, the histidine residue is also essential for response regulator dephosphorylation via a reverse-phosphotransfer reaction. In this work, we present an example of a kinase that relies on reverse phosphotransfer to catalyze the dephosphorylation of its cognate partner. The QseC sensor kinase is conserved across several Gram-negative pathogens; its interaction with its cognate partner QseB is critical for maintaining pathogenic potential. Here, we demonstrate that QseC-mediated dephosphorylation of QseB occurs via reverse phosphotransfer. In previous studies, we demonstrated that, in uropathogenic Escherichia coli, exposure to high concentrations of ferric iron (Fe3+) stimulates the PmrB sensor kinase. This stimulation, in turn, activates the cognate partner, PmrA, and noncognate QseB to enhance tolerance to polymyxin B. We demonstrate that in the absence of signal, kinase-inactive QseC variants, in which the H246 residue was changed to alanine (A) aspartate (D) or leucine (L), rescued a ΔqseC deletion mutant, suggesting that QseC can control QseB activation via a mechanism that is independent of reverse phosphotransfer. However, in the presence of Fe3+, the same QseC variants were unable to mediate a wild-type stimulus response, indicating that QseC-mediated dephosphorylation is required for maintaining proper QseB-PmrB-PmrA interactions. IMPORTANCE Two-component signaling networks constitute one of the predominant methods by which bacteria sense and respond to their changing environments. Two-component systems allow bacteria to thrive and survive in a number of different environments, including within a human host. Uropathogenic Escherichia coli, the causative agent of urinary tract infections, rely on two interacting two-component systems, QseBC and PmrAB, to induce intrinsic resistance to the colistin antibiotic polymyxin B, which is a last line of defense drug. The presence of one sensor kinase, QseC, is required to regulate the interaction between the other sensor kinase, PmrB and the response regulators from both systems, QseB and PmrA, effectively creating a “four-component” system required for virulence. Understanding the important role of the sensor kinase QseC will provide insight into additional ways to therapeutically target uropathogens that harbor these signaling systems.

scholarly journals Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Felipe Trajtenberg ◽  
Daniela Albanesi ◽  
Natalia Ruétalo ◽  
Horacio Botti ◽  
Ariel E. Mechaly ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Activation Mechanism ◽  
Response Regulators ◽  
Content Type ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. IMPORTANCE The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways.

scholarly journals Multiple Mechanisms of Action for Inhibitors of Histidine Protein Kinases from Bacterial Two-Component Systems

1999 ◽  
Vol 43 (7) ◽  
pp. 1693-1699 ◽  
Author(s):  
Jamese J. Hilliard ◽  
Raul M. Goldschmidt ◽  
Lisa Licata ◽  
Ellen Z. Baum ◽  
Karen Bush
Keyword(s):  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Membrane Integrity ◽  
Bacterial Killing ◽  
Gram Positive Bacteria ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Bacterial Cell Membrane ◽  
Cellular Incorporation

ABSTRACT Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC50s) ranging from 1.9 to >500 μM and MICs ranging from 0.5 to >16 μg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4× MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC50s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 μg/ml). Fifteen of 24 compounds also caused hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.

scholarly journals A survey of HK, HPt, and RR domains and their organization in two-component systems and phosphorelays proteins of organisms with fully sequenced genomes

2015 ◽  
Author(s):  
Baldiri Salvado ◽  
Ester Vilaprinyo ◽  
Albert Sorribas ◽  
Rui Alves
Keyword(s):  
Response Regulator ◽  
Signal Transmission ◽  
Pr Proteins ◽  
Component Systems ◽  
Two Component ◽  
Domains Of Life ◽  
Two Component Systems ◽  
Operon Organization ◽  
The Individual ◽  
Selection Of

Two Component Systems and Phosphorelays (TCS/PR) are environmental signal transduction cascades in prokaryotes and, less frequently, in eukaryotes. The internal domain organization of proteins and the topology of TCS/PR cascades play an important role in shaping the responses of the circuits. It is thus important to maintain updated censuses of TCS/PR proteins in order to identify the various topologies used by nature and enable a systematic study of the dynamics associated with those topologies. To create such a census, we analyzed the proteomes of 7609 organisms from all domains of life with fully sequenced and annotated genomes. To begin, we survey each proteome searching for proteins containing domains that are associated with internal signal transmission within TCS/PR: Histidine Kinase (HK), Response Regulator (RR) and Histidine Phosphotranfer (HPt) domains, and analyze how these domains are arranged in the individual proteins. Then, we find all types of operon organization and calculate how much more likely are proteins that contain TCS/PR domains to be coded by neighboring genes than one would expect from the genome background of each organism. Finally, we analyze if the fusion of domains into single TCS/PR proteins is more frequently observed than one might expect from the background of each proteome. We find 50 alternative ways in which the HK, HPt, and RR domains are observed to organize into single proteins. In prokaryotes, TCS/PR coding genes tend to be clustered in operons. 90% of all proteins identified in this study contain just one of the three domains, while 8% of the remaining proteins combine one copy of an HK, a RR, and/or an HPt domain. In eukaryotes, 25% of all TCS/PR proteins have more than one domain. These results might have implications for how signals are internally transmitted within TCS/PR cascades. These implications could explain the selection of the various designs in alternative circumstances.

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