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Blades, Gareth. "Re-engineering bacterial two-component signalling systems." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:2865c02d-c208-45fa-8108-d8ced9486c19.
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Bacteria use Two Component Systems (TCS) to sense and respond to changes in their external environment. TCS are used to navigate to nutrients or away from toxins (chemotaxis) and to adapt to changes in osmolarity (osomosensing). TCS are composed of a histidine protein kinase (HPK) which trans-autophosphorylates in response to environmental change, transferring the phosphoryl group to a cognate response regulator (RR). Phosphorylated RRs modulate an output response such as protein-protein interaction for chemotaxis, and transcription for osmosensing. RRs are composed of a conserved amino terminal REC domain, and where present a variable effector domain. CheY, the chemotaxis RR, contains only a REC domain, whilst OmpR, the osmosensing RR, also contains a DNA binding effector domain. Recently, TCS have been used in synthetic biology applications due to their modularity and conserved signalling mechanism. This thesis aimed to investigate whether it was possible to design a synthetic TCS composed of fused chemotaxis and osmosensing components. Synthetic RRs were designed, fusing the highly conserved REC domains of CheY and OmpR upstream of the OmpR effector domain. REC domains were fused across the α4-β5-α5 region, a region which transmits REC domain phosphorylation into effector domain activation. Synthetic RRs were designed to undergo phosphotransfer to their fused REC domains from the chemotaxis HPK, CheA, activate the attached OmpR effector domain and bind promoter DNA. Four chimeric RRs were created, although only three were structurally viable; F2, F3 and F4. Each fusion bound CheA, and F3 and F4 bound CheA with a significantly higher affinity than CheY. The chimeric RRs could all be phosphorylated byCheA-P; F4 and F3 were phosphorylated to wild-type levels. DNA binding affinitywas investigated with fluorescence anisotropy, hosphorylated and unphosphorylated F3 could not bind promoter DNA. F2 bound promoter DNA regardless of phosphorylation state. These data indicate that phosphorylation of the F2 REC domain does not lead to activation of the effector domain. F2 is likely to be constitutively active suggesting a previously unknown role for OmpR α5 as a mediator of effector domain activation. Furthermore, using a simple fusion approach to design RRs is not a viable method to create a synthetic TCS with a controllable output.
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Sheng, Xia. "Evolutionary and functional aspects of two-component signalling systems." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/17940.
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Two-component systems (TCSs) are critical for bacteria to interact with their extracellular environment. They define a type of signalling system that is composed of a transmembrane histidine kinase (HK) and a cytoplasmic response regulator (RR). In this thesis we have studied the evolutionary and functional aspects of these important signalling systems. By studying the distribution of the TCS orthologues of E.coli across more than 900 bacterial organisms, we have found that a pair of TCS proteins does not always coexist in one organism. The genomic localisation map of TCSs reveals a possible translocation mechanism for TCS evolution. The alignments of HKs and RRs have shown that HKs are genetically more divergent, probably due to their signal recognizing role. An analysis of the steady states of TCS dynamics has shown that the outputs of the TCSs are bistable if they have positive auto-regulation feedback loops in their transcriptional regulation. Our analysis has also shown that the phosphorylation process of a TCS is always monostable and the factors that affect steady states have been studied. For both orthodox and non-orthodox TCSs, autophosphorylation rates of the HKs are the most important factor to affect the steady states of the TCSs' outputs. To study the phosphorelay mechanism of the non-orthodox TCS ArcB/A, we constructed plasmids carrying different copy numbers of ArcB mutants with different phosphorylation sites ablated. By fitting our phosphorelay model to the data obtained from mutant ArcB constructs, we have found that ArcB most likely performs phosphorelay in an allosteric mechanism. Finally, Approximate Bayesian computation was used in order to evaluate the potential use of orthodox TCS and non-orthodox TCS architectures in synthetic biology contexts. Results show that neither of the orthodox TCS model or the nonorthodox TCS model are superior under all circumstances but that both models have advantages in some scenarios. In the appendix, we did some study on how the contact residue disorder would affect protein-ligand binding specificity.
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Puthiyaveetil, Sujith. "Two-component signalling systems of chloroplasts : function, distribution and evolution." Thesis, Queen Mary, University of London, 2008. http://qmro.qmul.ac.uk/xmlui/handle/123456789/121.
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Two-component signal transduction, comprising sensor kinases and response regulators, is the predominant signalling mechanism in prokaryotes. This signalling system originated in bacteria, and has spread to the eukaryotic domain of life through symbiotic, lateral gene transfer from the bacterial ancestors of chloroplasts and mitochondria. During the course of their evolution, chloroplasts, with the exception of a few instances in non-green algae, appear to have relinquished all genes encoding two-component systems to their eukaryotic host cell nuclei. In green algae and plants, chloroplast genes for two-component systems were neither known nor were chloroplast two-component proteins shown to exist as products of nuclear genes prior to the work described here. This thesis describes the identification and characterisation of a novel two-component sensor kinase in chloroplasts. This Chloroplast Sensor Kinase (CSK) is the product of a nuclear gene in algae and plants. CSK is synthesised in the cytosol of Arabidopsis thaliana and imported into the chloroplast as a protein precursor. CSK is autophosphorylated and couples photosynthetic electron transport to gene transcription in chloroplasts. The identity of the response regulator partner of CSK reveals an unexpected phylogenetic and functional relatedness of CSK with chloroplast two-component systems of non-green algae. Chloroplast two-component systems are likely to be universal in photosynthetic eukaryotes and they persist in chloroplasts as products of nuclear genes even where chloroplast genomes no longer encode them. Chloroplast twocomponent systems have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. The persistence of cyanobacterial two-component systems in chloroplasts and their function in coupling photosynthesis with chloroplast gene expression are central to the premise that chloroplasts retain genes whose expression is regulated by the activity of the photosynthetic electron transport chain, using a mechanism conserved from their cyanobacterial ancestors.
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Tomenius, Henrik. "Bacterial virulence and adaptation mediated by two-component system signalling /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-792-8/.
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Croxatto, Antony. "VanT, a central regulator of quorum sensing signalling in Vibrio anguillarum." Doctoral thesis, Umeå : Umeå University, Department of Molecular Biology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-702.
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Francis, Vanessa Ina. "Extensive communication between sensor kinases controlling virulence in the GacS network of Pseudomonas aeruginosa." Thesis, University of Exeter, 2015. http://hdl.handle.net/10871/17434.
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Two component systems (TCSs) are regulatory pathways in bacteria and lower eukaryotes that integrate multiple stimuli and bring about appropriate responses to promote adaptation of the bacteria to their niches. They are commonly insulated from cross-talk and form discrete regulatory systems where the sensor histidine kinase (SK) and the response regulator (RR) share high fidelity for one another. The GacS network controls the switch between acute and chronic virulence of P. aeruginosa. The network is unusual in having a 'core' SK, GacS, which is modulated directly by one other SK, RetS. Here the complex relationship between GacS and RetS is dissected to reveal three distinct mechanisms by which they interact. Two of these mechanisms involve the dephosphorylation of GacS-P by RetS and it is these mechanisms that are important in vivo for the regulation of biofilm formation, rsmY and rsmZ expression, swarming, and virulence in both Galleria mellonella and an acute model of infection in mice. This study reveals an unprecedented level of complexity in the ability of RetS to interact with GacS and suggests that RetS has a number of mechanisms by which it can downregulate the GacS network output. Furthermore, the interactions of additional SKs that have previously been linked to the GacS network were investigated. Here I demonstrate that many of these kinases can interact with one another but that RetS remained the only kinase tested that could directly interact with GacS. The interactions observed between kinases could be either stimulatory, having a synergistic impact on phosphorylation levels, or inhibitory. I also show that kinase-kinase interactions allow for the regulation of phosphorylation of downstream proteins. Finally, we searched for additional SKs that may be able to interact with the GacS network. Here I identify three new kinases, which show differing interactions with the kinases of the GacS network. The discovery of additional SKs in the GacS network indicates that the network is likely to respond to a far greater number of different signals than previously realised as it decides between acute and chronic virulence.
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Guan, Shuang. "A novel two-component signal transduction system in propionibacterium acnes and its association with a putative extracellular signalling peptide." Thesis, University of Leeds, 2011. http://etheses.whiterose.ac.uk/1752/.
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Propionibacterium acnes, a resident micro-organism of human skin, is thought to be involved in the development of inflammatory acne, which is an exclusive human disease affecting more than 80% of the whole population. Quorum sensing is the regulation of gene expression in response to cell density. As it is often involved in pathogenecity of bacteria, its signal transduction pathway has been suggested as potential target for new drug development. This project identified a putative unique quorum sensing system of P. acnes, consisting of a putative signalling peptide, and divergently transcribed histidine kinase and response regulator. The aim of this project is to investigate the relationship among these three components as being elements of a putative quorum sensing system. Using purified proteins and in vitro phosphorylation assays, the histidine kinase was demonstrated to phosphorylate the response regulator indicating they may constitute a legitimate pair of a two-component system, despite being encoded by divergently transcribed genes. By mapping transcriptional start site, it was found that the signalling peptide and histidine kinase were co-transcribed from the same start site, suggesting that the signalling peptide was associated with the two-component system. Gene expression analysis also revealed these three genes were co-regulated during the growth of P.acnes, which is consistent with these three genes functioning together as a part of quorum sensing system. The results of this project suggested that the signalling peptide, histidine kinase and response regulator are associated with each other and may constitute a quorum sensing system.
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Wojnowska, M. "Biochemical and structural characterisation of a two-component signalling system downstream of bacteriophytochrome photoreceptor 1 in Rhizobium NT-26." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1388782/.
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Prokaryotic signal transduction frequently involves two-component systems (TCSs), typically comprising a sensor histidine kinase (HK) and a response regulator (RR). Via a conserved phosphotransfer reaction, TCSs couple the detection of diverse stimuli with appropriate responses. The initial aim of this project was to characterise the two-component “signalome” of an arsenite oxidiser, Rhizobium NT-26, in the context of the environmental niche, and compare it to “signalomes” of other bacterial species. A light-sensing HK, bacteriophytochrome photoreceptor 1 (BphP1), was thus identified. Previous studies indicated that BphP1 and the members of its gene cluster - two single-domain RRs and a hybrid RR/HK protein, ExsG - may constitute a TCS. Functional characterisation revealed that BphP1 initiates a branched signalling pathway; however, the mediated output could not be identified. ExsG, a HWE-type HK, was shown to possess dual HK/RR activity and act as a novel type of signalling switch: phosphorylation of the N-terminal receiver domain downregulates the autokinase activity of the C-terminal kinase core. Ion mobility-mass spectrometry analysis indicated that while Asp62 phosphorylation stabilises the “closed” form of ExsG, nucleotide binding stabilises the “open” conformation. These observations led to a model in which phosphorylation of the receiver domain precludes ATP binding and thus inhibits autokinase activity. Furthermore, ExsG was demonstrated to hexamerise via the HWE core, which makes it the first non-dimeric HK. Notably, however, the basic unit of the hexameric assembly is a homodimer, and the HWE core shares homology with canonical HK cores. The results presented herein broaden the current knowledge on TCSs and identify previously unreported mechanisms involved in two-component signal transduction. The members of the BphP1 signalling cascade enrich the pool of modular communication units that can be exploited in engineering artificial signalling networks, biosensors and microorganisms with novel functionalities.
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Casali, Nicola. "Two component regulatory systems in Mycobacterium tuberculosis." Thesis, London School of Hygiene and Tropical Medicine (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322228.
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Goffri, Shalom. "Polymer FETs with crystalline two-component systems." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613082.
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Richard, Jessica. "The two-component signal transduction systems of Pseudomonas aeruginosa." Thesis, Montana State University, 2008. http://etd.lib.montana.edu/etd/2008/richard/RichardJ0508.pdf.
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Two-component signal transduction systems are important components for bacteria, because they allow the bacteria to sense environmental conditions and rapidly adapt to changes in the environment. Two-component systems generally contain a sensor histidine kinase, which detects an environmental signal and responds by autophosphorylation at a histidine residue using ATP as the phosphate donor. The phosphate group is then transferred to an aspartate residue in the receiver domain of the second component, the response regulator, which in its activated form responds by stimulating or repressing gene expression or motility, as needed for the physiological responses of the cell. The structural versatility of two-component systems reflects the wide range of signals to which bacteria respond. Despite this versatility, histidine kinases and response regulators show a conserved mechanism of signal transfer. Pseudomonas aeruginosa is a versatile organism that can use a variety of nutrient sources and is found in many different environments. It is a human pathogen in nosocomial infections as well as in pulmonary fluid of patients with cystic fibrosis. P. aeruginosa encodes genes for over 60 two-component regulatory systems. In this review, I discuss the structure and function of two-component systems in bacteria, and conduct a phylogenetic analysis of the P. aerugionsa two-component systems. Finally, I develop a model of the calcium-responsive two-component system of P. aeruginosa, PA2656/PA2657, which is closely related to the magnesium responsive PmrAB and phosphate responsive PhoPQ two-component systems. The results provide insight on how P. aeruginosa is able to detect and respond to changing environmental conditions.
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Lubin, Emma A. (Emma Alexandra). "Mechanism and specificity of bacterial two-component signaling systems." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/57799.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.Includes bibliographical references (p. 57-60).
Bacterial two component signaling (TCS) systems are the predominant means by which bacteria sense and respond to external signals. These systems represent a large family of paralogous proteins; often hundreds of the histidine kinase (HK) and response regulator (RR) pairs that make up a TCS system can be found in a single cell. How do these systems maintain faithful signal transmission and avoid cross-talk? To understand how specificity is determined, we examined co-evolving residues between HKs and RRs, and guided by this, aimed to rewire specificity of several activities of TCS systems. Previous work in the lab has successfully rewired specificity of histidine kinases for response regulators in the phosphotransfer reaction. By mutating different subsets of these co-evolving residues, we were able to rewire specificity of RRs in the phosphotransfer reaction, and partially rewire specificity of HKs and RRs in the phosphatase reaction. Additionally, we identified residues that may dictate specificity between two domains of the histidine kinase, and found that mutating them altered the rate of autophosphorylation. These analyses will allow rational rewiring of two component systems in vivo, and permit us to examine the fitness consequences of this altered specificity, providing insight into the evolutionary pressures on TCS systems.
by Emma A. Lubin.
S.M.
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Patel, Jenishkumar. "ENVIRONMENTAL RESPONSES OF TWO-COMPONENT SYSTEMS IN STREPTOCOCCUS SANGUINIS." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/2270.
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The gram-positive bacterium Streptococcus sanguinis is a member of human indigenous oral microbialflora and has long been recognized as a key player in the bacterial colonization of the mouth. S. sanguinis is also the most common viridians streptococcal species implicated in infective endocarditis. Although many studies have focused on two-component systems in closely related Streptococcus species such as S. mutans, S. pneumoniae and S. gordonii; the mechanism of the response regulator in S. sanguinis is still unknown. The ability of S. sanguinis to adapt and thrive in hostile environments suggests this bacterium is capable of sensing and responding to various environmental stimuli. The present study clearly demonstrates that a number of RR genes, SSA_0204, SSA_0217, SSA_1810, SSA_1794, and SSA_1842, in S. sanguinis are essential to the recognition and response to various environmental stresses. Results from this study also identified genes SSA_0260, SSA_0261, and SSA-0262, involved in acidic tolerance and suppressed by SSA_0204 response regulator.
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Salvadó, López Baldiri. "Design principles in two component systems and his-asp phosphorelays." Doctoral thesis, Universitat de Lleida, 2016. http://hdl.handle.net/10803/393740.
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L’objectiu d’aquesta tesi és trobar principis generals que permetin relacionar l’estructura i les propietats funcionals dels circuits moleculars de transducció de senyals two-component systems (TCS) i his-asp phosphorelays (PR).
La tesi s’inicia revisant els mètodes usats per a l’estudi de principis de disseny en sistemes moleculars i alguns dels resultats obtinguts fins ara, i discutint la importància de l’estudi dels principis de disseny.
A continuació, explorem els proteomes seqüenciats de més de 7000 organismes i fem un inventari dels diferents tipus d’organització en operons i proteïnes multidomini dels dominis proteics que intervenen en TCS i PR. A partir d’aquesta informació deduirem alternatives existents en la natura pel que fa al disseny d’aquests circuits moleculars.
Per acabar, comparem mitjançant modelització matemàtica el comportament dinàmic de 3 circuits diferents de TCS, i trobem que un tercer component que modula l’activitat de la histidina quinasa o bé el response regulator pot modificar l’espai paramètric on el sistema es comporta de forma biestable.El objectivo principal de esta tesis es la búsqueda de principios de diseño que relacionen la estructura y la función de redes bioquímicas de transducción de señales, concretamente en two-component systems (TCS) y phosphorelays (PR). La tesis se inicia con una revisión de los métodos usados para el estudio de principios de diseño en sistemas moleculares y algunos de los resultados obtenidos hasta ahora, seguida de una discusión sobre la importancia del estudio de dichos principios de diseño. A continuación, exploramos los proteomas secuenciados de más de 7000 organismos y hacemos un inventario de los distintos tipos de organización en operones o proteínas de los dominios proteicos implicados en TCS y PR, con el objetivo de deducir el repertorio de estructuras existentes en la naturaleza para estos circuitos moleculares. Para terminar, comparamos mediante modelización matemática las propiedades dinámicas de tres circuitos distintos de TCS, y observamos que una proteína adicional que interacciona con la histidina quinasa o con el response regulator modifica el espacio de valores de los parámetros del sistema en el cual existe biestabilidad.
The ultimate goal of this thesis is to set the stage for finding general design principles underlying the relationship between network design and network function in two-component (TCS) and His-Asp phosphorelay (PR) signal transduction systems. This thesis starts with a review of the methods for and results from the study of design principles in molecular systems, and a discussion about the importance of studying those design principles. Next, a survey of the fully sequenced and annotated genomes and proteomes of more than 7000 different organisms is performed in order to identify different types of organizations of the TCS/PR protein domains in operons and multidomain proteins. From this data, the existing diversity of TCS/PR circuit designs will be inferred. Finally, we compare through mathematical modeling the dynamic properties associated with three types of TCS circuit designs, and find that a third component that binds to and modulates the activity of either the sensor kinase or the response regulator can modify the parameter space in which bistability in the system’s response is possible.
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Shams, Eldeen Samir. "Instabilities and propagation properties in two-component reaction-diffusion systems." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12287/.
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This thesis deals with a detailed linear analysis for a two-component reaction-diffusion system with constant diffusion coefficients. A comprehensive linear stability analysis results in three types of instabilities: (1) stationary periodic instability, (2) oscillatory uniform and (3) stationary uniform. The first instability involves pattern formation and the other two do not. Precise parameter regimes are identified for each. Travelling wave analysis is performed for the system and a detailed and comprehensive analysis is undertaken of a linear mechanism governing the development and propagation of nonlinear patterns. This analysis focuses on a linear selection mechanism that gives some insights into the selected speed of invasion of an unstable state by a stable one, as described both by a fixed form of travelling wave and by a modulated travelling wave.
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Suggett, Jason Andrew. "An investigation of triboelectrification in two component pharmaceutical powder systems." Thesis, University of Sunderland, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263475.
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Reuter, Mark Andrew. "Intramolecular and intermolecular signal transduction within bacterial two component systems." Thesis, University of East Anglia, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390647.
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Capra, Emily Jordan. "Specificity and evolution of bacterial two-component signal transduction systems." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/83765.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references.
Cells possess a remarkable capacity to sense and process a diverse range of signals. Duplication and divergence of a relatively small number of gene families has provided the raw material enabling cells to quickly increase their signaling capacity. After duplication, however, all pathway components are identical in sequence and function. To evolve a new role, the pathways must become insulated at the level of signal transduction. Two-component signal transduction systems, consisting of a sensor histidine kinase and a cognate response regulator, are the main means by which bacteria sense and respond to their environment. These systems have undergone extensive duplication and lateral gene transfer such that most species encode dozens to hundreds of these pathways, yet there is little evidence of cross-talk at the level of signal transduction. Previous work has shown that interaction specificity is dictated by molecular recognition and determined by a small set of specificity residues. I begin by studying the evolutionary trajectories of specificity residues in a duplicated two-component system that lead to insulation of pathways while at the same time maintaining interaction between cognate kinases and regulators. I then examine specificity residues in orthologs of a single two-component system and show that specificity residues are typically under purifying selection, but, as a result of additions to the two-component signaling network, can undergo bursts of diversification followed by extended stasis. By reversing these mutations I demonstrate that avoidance of cross-talk is a major selective pressure. Finally, I show that covalent attachment of the response regulator to a kinase represents an alternative mechanism for enforcing specificity. In these cases, no changes are needed to accommodate a duplication; the high effective concentration of the covalently attached response regulator prevents cross-talk with other two component proteins in the cell. This may allow hybrid kinases to be duplicated or transferred between genomes more easily. This work sheds light on the apparent ease with which two-component systems have expanded to become the dominant signaling system in bacterial genomes and, more generally, how a small number of gene families can be responsible for signal transduction in all organisms.
by Emily Jordan Capra.
Ph.D.
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Cock, Peter J. A. "Two-component regulation : modelling, predicting & identifying protein-protein interactions & assessing signalling networks of bacteria." Thesis, University of Warwick, 2008. http://wrap.warwick.ac.uk/1939/.
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Two-component signalling systems (TCSs) are found in most prokaryotic genomes. They typically comprise of two proteins, a histidine (or sensor) kinase (HK) and an associated response regulator (RR), containing transmitter and receiver domains respectively, which interact to achieve transfer of a phosphoryl group from a histidine residue (of the transmitter domain in the HK) to an aspartate residue (of the partner RR’s receiver domain). An automated analysis pipeline using the NCBI’s RPS-BLAST tool was developed to identify and classify all TCS genes from completed prokaryotic genomes using the PFAM and CDD protein domain databases. A large proportion of TCS genes were found to be simple hybrid kinases (HYs) containing both a transmitter domain and a receiver domain within a single protein, presumably the result of the fusion or combination of separate HK and RR genes. This propensity to consolidate functionality into a single protein was found to be limited in the presence of either a transmembrane sensory/input domain or a DNA binding domain – two spatially separated functions. While HK and RR genes are usually found together in the genome, in some species a large proportion of TCS domains are found as part of complex hybrid kinases (genes containing multiple TCS domains), in isolated or orphaned genes, or in complex gene clusters. In such organisms the lack of paired HK and RR genes makes it difficult to define genome-encoded signalling networks. Identifying paired transmitter and receiver domains from a pan-genomic survey of prokaryotes gives a database of amino acid sequences for thousands of interacting protein-protein complexes. Covariation between columns of multiple sequence alignments (MSAs) identifies particular pairs of residues representing interactions within the docked complex. Using numerical scores, these amino acids pairs were successfully used as explanatory variables in a generalised linear model (GLM) to predict the probabilities of interaction between transmitter and receiver domains.
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Nishikawa, Yukihiro. "Interface Curvatures of Bicontinuous Phase-structures in Two-component Polymeric Systems." Kyoto University, 2000. http://hdl.handle.net/2433/181348.
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Ishchenko, Andrii [Verfasser]. "Molecular mechanisms of signal transduction in two-component signaling systems / Andrii Ishchenko." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2013. http://d-nb.info/1038680530/34.
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Hawkins, Peter F. "Investigation into the interactions of Pho-like two-component systems in Myxococcus xanthus." Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436648.
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Willett, Jonathan. "Bacterial two-component systems share a common mechanism to regulate signaling and specificity." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/4977.
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Despite years of intensive research, many of the fundamental aspects of two-component signal transduction pathways are not yet understood. Interestingly these systems are found throughout all domains of life including archaea, bacteria and eukaryotes and are known to regulate diverse cellular processes such as motility, pathogenesis, development, biofilm formation, and toxin production. Despite many groups working on two-component systems it is not yet appreciated whether these systems have conserved features, amino acid requirements, structures and specificity. By understanding the mechanisms by which signals propagate through these systems we could perhaps develop novel therapeutics targeting these pathways.
In order to address these questions my thesis has focused on studying the signaling pathways which regulate multicellular development in the model soil bacterium Myxococcus xanthus. The developmental process is highlighted by large changes in gene expression patterns and motility resulting in the production of large macroscopic fruiting bodies composed of metabolically dormant myxospores. My initial work focused on characterizing the Che3 chemosensory pathway known to regulate time of aggregation required for fruiting body formation. I discovered an additional kinase CrdS which works with the Che3 system to regulate phosphorylation of the important developmental regulator CrdA.
Additionally I performed mutagenesis on the kinase CrdS to demonstrate that specific residues in CrdS are required for both kinase and phosphatase activities. A conserved Thr/Asn was required for phosphatase activity while a conserved acidic residue was required for kinase activity. Importantly, these residues are highly conserved and when we made mutations in multiple other kinases, we saw similar requirements, indicating the importance of these residues.
Further analysis focused on 26 other CrdA homologs found within the M. xanthus genome. Using phosphotransfer profiling and a newly created phosphatase profiling method we were able to demonstrate signaling specificity whereby each kinase was able to phosphorylate and dephosphorylate a single response regulator. Since phosphotransfer and phosphatase activities are predicated upon protein-protein interactions, we also determined that cognate pairs exhibited preferential binding. Cumulatively this research highlights some of the conserved mechanism governing the signal transduction pathways regulating multicellular development in M. xanthus.
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Belzil-Lacasse, Christian. "Study of Dissipative Spots In Three-Component Reaction-Difussion Systems on Two-Dimensional Domains." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34257.
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Dissipative spots are found in physical experiments of many branches of natural science. In this thesis we use three-component reaction-diffusion systems on two-dimensional domains in order to generate these patterns. Using a dynamical system approach we proceed with a Fourier analysis on a linearized reaction-diffusion system in order to provide the bifurcation conditions for a given homogeneous state. We validate our results and establish it's limitations through numerical experiments. We report very interesting behavior during these simulations, notably hysteresis and multi-stability. We will then turn our attention to the relatively unexplored phenomenon of rotating spots. Based on previous work done for spiral waves, we investigate the effect of translational symmetry-breaking on a rotating spot mainly through careful numerical analysis.
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Fischer, Miriam [Verfasser], and Victor [Akademischer Betreuer] Sourjik. "Cross-talk between two-component systems in Escherichia coli / Miriam Fischer ; Betreuer: Victor Sourjik." Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/1180616820/34.
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Ann, Kevin. "Sudden death of entanglement and non-locality in two- and three-component quantum systems." Thesis, Boston University, 2011. https://hdl.handle.net/2144/34430.
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Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Quantum entanglement and non-locality are non-classical characteristics of quantum states with phase coherence that are of central importance to physics, and relevant to the foundations of quantum mechanics and quantum information science. This thesis examines quantum entanglement and non-locality in two- and three-component quantum states with phase coherence when they are subject to statistically independent, classical, Markovian, phase noise in various combinations at the local and collective level. Because this noise reduces phase coherence, it can also reduce quantum entanglement and Bell non-locality. After introducing and contextualizing the research, the results are presented in three broad areas. The first area characterizes the relative time scales of decoherence and disentanglement in 2 x 2 and 3 x 3 quantum states, as well as the various subsystems of the two classes of entangled tripartite two-level quantum states. In all cases, it was found that disentanglement time scales are less than or equal to decoherence time scales. The second area examines the finite-time loss of entanglement, even as quantum state coherence is lost only asymptotically in time due to local dephasing noise, a phenomenon entitled "Entanglement Sudden Death" (ESD). Extending the initial discovery in the simplest 2 x 2 case, ESD is shown to exist in all other systems where mixed-state entanglement measures exist, the 2 x 3 and d x d systems, for finite d > 2. The third area concerns non-locality, which is a physical phenomenon independent of quantum mechanics and related to, though fundamentally different from, entanglement. Non-locality, as quantified by classes of Bell inequalities, is shown to be lost in finite time, even when decoherence occurs only asymptotically. This phenomenon was named "Bell Non-locality Sudden Death" (BNSD).
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Taylor, Patrick. "The Role of Two-Component and Small RNA Regulatory Systems in Pseudomonas aeruginosa Biofilms." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39614.
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Biofilms are a crucial adaptation for bacterial survival against stresses from external environments. Biofilms are adherent colonies of sessile bacteria embedded within a self-produced matrix. Bacterial control over formation, maintenance, and response to external stresses are strictly regulated. However, complexities of intracellular signaling for biofilm regulation are still not fully understood. In this thesis, I report on two distinct regulatory systems important for biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. The first regulatory system I report on is the two-component system TctD-TctE. This system is involved in regulating the uptake of tricarboxylic acids such as citric acid and is involved in biofilm-specific susceptibility to aminoglycoside antibiotics. Here I describe work I performed characterizing the involvement of TctD-TctE in biofilm development when citric acid is present as a carbon source in nutrient media. In further characterizing a previously observed aminoglycoside susceptibility, I found that a strain with a deletion of TctD-TctE (ΔtctED) has a heightened accumulation of tobramycin in its biofilms when grown in the presence of citric acid. In ΔtctED, I determined that there was an inhibition of overall cell growth when citric acid was present in nutrient media. Additionally, in the presence of citric acid, ΔtctED displayed high levels of biofilm formation. This contrasted with normal biofilm development observed in the PA14 wild type strain where biofilm mass was reduced in the presence citric acid. The second project of this thesis reports on a novel regulatory small RNA, the Small RNA Regulator of Biofilms (SrbA). SrbA was found to be unique to P. aeruginosa and displayed no homology with any other sequenced bacterial species. I found that loss of SrbA resulted in a significant reduction in biofilm mass. Subsequently, loss of SrbA also leads to attenuation of P. aeruginosa pathogenicity in Caenorhabditis elegans nematodes. Bacterial biofilms possess specific regulatory programs that are still just being appreciated for their complexity. This thesis work adds to our understanding of biofilm regulation by studying roles of the two-component system TctD-TctE and the small RNA SrbA in P. aeruginosa.
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Amin, Munia. "Exploring and understanding signal-response relationships and response dynamics of microbial two-component signaling systems." Thesis, University of Exeter, 2013. http://hdl.handle.net/10871/15016.
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Two-component signaling systems are found in bacteria, fungi and plants. They mediate many of the physiological responses of these organisms to their environment and display several conserved biochemical and structural features. This thesis identifies a potential functional role for two commonly found architectures in two-component signaling system, the split kinases and phosphate sink, which suggests that by enabling switch-like behaviors they could underlie physiological decision making. I report that split histidine kinases, where autophosphorylation and phosphotransfer activities are segregated onto distinct proteins capable of complex formation, enable ultrasensitivity and bistability. By employing computer simulations and analytical approaches, I show that the specific biochemical features of split kinases “by design” enable higher nonlinearity in the system response compared to conventional two-component systems and those using bifunctional (but not split) kinases. I experimentally show that one of these requirements, namely segregation of the phosphatase activity only to the free form of one of the proteins making up the split kinase, is met in proteins isolated from Rhodobacter sphaeroides. While the split kinase I study from R. sphaeroides is specifically involved in chemotaxis, other split kinases are involved in diverse responses. Genomics studies suggest 2.3% of all chemotaxis kinases, and 2.8% of all kinases could be functioning as split kinases. Combining theoretical and experimental approaches, I show that the phosphate sink motif found in microbial and plant TCSs allows threshold behaviors. This motif involves a single histidine kinase that can phosphotransfer reversibly to two separate response regulators and examples are found in bacteria, yeast and plants. My results show that one of the response regulators can act as a “sink” or “buffer” that needs to be saturated before the system can generate significant responses. This sink, thereby allows the generation of a signal threshold that needs to be exceeded for there to be significant phosphoryl group flow to the other response regulator. Thus, this system can enable cells to display switch-like behavior to external signals. Using an analytical approach, I identify mathematical conditions on the system parameters that are necessary for threshold dynamics. I find these conditions to be satisfied in both of the natural systems where the system parameters have been measured. Further, by in vitro reconstitution of a sample system, I experimentally demonstrate threshold dynamics for a phosphate-sink containing two-component system. This study provides a link between these architectures of TCSs and signal-response relationship, thereby enabling experimentally testable hypotheses in these diverse two-component systems. These findings indicate split kinases and phosphate as a microbial alternative for enabling ultrasensitivity and bistability - known to be crucial for cellular decision making. By demonstrating ultrasensitivity, threshold dynamics and their mechanistic basis in a common class of two-component system, this study allows a better understanding of cellular signaling in a diverse range of organisms and will open the way to the design of novel threshold systems in synthetic biology. Thus, I believe that this study will have broad implications not only for microbiologists but also systems biologists who aim to decipher conserved dynamical features of cellular networks.
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Walker, Jennifer Nicole. "The two-component system, ArlRS, regulates agglutination and pathogenesis in Staphylococcus aureus." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/1414.
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Staphylococcus aureus is defined by its ability to agglutinate during exposure to human blood plasma. Although agglutination has long correlated with disease severity, the function of agglutination during infection remains unclear. Increasing evidence suggests the mechanisms of agglutination are highly complex and poorly understood. The goal of this dissertation was to characterize the mechanisms required for S. aureus agglutination in vitro and determine how these factors contribute to pathogenesis.
Chapter II focuses on the development of two in vitro agglutination assays, which allow the process to be measured quantitatively. Through these assays, we confirmed the major factors contributing to agglutination are human fibrinogen and the bacterial surface protein, ClfA. Productive interactions between these two factors are required for agglutination to proceed. Surprisingly, we also identified a novel regulatory system that significantly contributed to agglutination. Inactivation of the ArlRS two-component system (TCS) prevents agglutination in both of the developed assays.
Studies in Chapter III focused on characterizing the mechanism by which ArlRS inhibits agglutination. To examine regulation, quantitative PCR identified the major output of the ArlRS system as the gene ebh. Surprisingly, transcript levels of known extracellular matrix (ECM) binding proteins did not change. Characterization of ebh indicated that overexpression in an arlRS mutant is the major factor responsible for preventing agglutination. Deletion of ebh restores the ability of the arlRS mutant to agglutinate in both gravity and flow-based agglutination assays. Fluorescence microscopy of clumps indicates wildtype cells bind and incorporate fluorescently labeled human fibrinogen (Fg) displaying co-localization with the clumps. Surprisingly, arlRS mutants also bound human Fg, but these interactions were not productive for clumping, suggesting successful agglutination is more complex than binding ECM proteins. These studies indicate that ArlRS regulates agglutination through a unique mechanism that depends on the surface protein Ebh.
Studies in Chapter IV were performed to determine the role ArlRS played in pathogenesis. A rabbit model of infective endocarditis and sepsis was employed to assess ArlRS virulence because this model has been shown to require agglutination for disease progression. Mutants in arlRS displayed reduced virulence in the rabbit model of infective endocarditis, which correlated with the mutant's inability to form a vegetation of the heart valve. These studies provide further insight into the importance of S. aureus agglutination during infection and define a mechanism of regulation through a novel surface protein.
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Bent, Colin James. "Structural studies of the two-component signal transduction systems from the human pathogen Streptococcus pneumoniae." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400756.
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Revilla, Guarinos Ainhoa. "Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response." Doctoral thesis, Universitat Politècnica de València, 2014. http://hdl.handle.net/10251/43589.
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Lactobacillus casei es una bacteria del ácido láctico de interés aplicado por su uso como cultivo iniciador en la industria alimentaria y por el carácter probiótico de algunas cepas. Como probiótico, L. casei debe sobrevivir a las condiciones de producción industrial y a su paso por el tracto gastrointestinal manteniendo sus propiedades. Para ello, L. casei posee rutas de reconocimiento de señales ambientales específicas y convierten esta información en una respuesta fisiológica adecuada. Un mecanismo comúnmente encontrado en bacterias para la transducción de señal son los sistemas de dos componentes (Two Components Systems, TCS). Los TCS están formados por una proteína sensora con actividad histidina quinasa (HK) y un regulador de respuesta (RR). La detección de un estímulo específico por la proteína sensora induce su autofosforilación y la transferencia del fosfato al regulador de respuesta, produciéndose la activación del mismo. Los sistemas de dos componentes median la respuesta adaptativa a una amplia gama de estímulos ambientales en bacterias.
En el laboratorio de Bacterias Lácticas del Instituto de Agroquímica y Tecnología de Alimentos se ha iniciado el estudio de los TCS codificados por L. casei BL23 dentro del cual se incluye el presente proyecto de tesis doctoral.Revilla Guarinos, A. (2014). Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43589
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Daza, Oscar Eduardo. "Simulation and Validation of Two-Component Flow in a Void Recirculation System." DigitalCommons@CalPoly, 2011. https://digitalcommons.calpoly.edu/theses/504.
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Nuclear power plants rely on the Emergency Core Cooling System (ECCS) to cool down the reactor core in case of an accident. Occasionally, air is entrained into the suction piping of ECCS causing voids that decrease pumping efficiency, and consequently damage the pumps. In an attempt to minimize the amount of voids entering the suction side of the pump in ECCS, a Void Recirculation System (VRS) experiment was conducted for a proof of concept purpose. While many studies have been oriented in studying two-component flow behavior in ECCS, none of them propose a solution to minimize air entrainment. As a consequence, there are no simulation models that use computational fluid dynamics to address gas entrainment solutions in ECCS. The objectives of this thesis are to (1) simulate and investigate two-component air-water flow in a VRS that minimizes the amount of air in piping systems, using RELAP5/MOD3 as the computational tool, and (2) to validate the numerical results with respect to experimental results and observations.
A one-dimensional model of the VRS was built in RELAP5, in which eight different scenarios (replicating those from the VRS experiment) were simulated for a period of 150 seconds. Four Froude numbers of 0.8, 1.0, 1.3 and 1.6 were evaluated in two different pipe configurations, and the experimental data obtained from the VRS experiment was used to validate the numerical results obtained from these simulations. It was concluded that air recirculation occurs indefinitely throughout the entire 150 seconds of the simulation for Froude numbers up to 1.3; while for a Froude number of 1.6, air recirculation occurs for approximately 100 seconds and ceases after 125 seconds of the simulation. An average air reduction effectiveness of 90% was found for all simulation scenarios. The VRS model was successfully validated and can be used to investigate the effects of air entrainment in suction piping.
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Mioro, Miriam Kanyua. "Designing a Two Component System for Enzyme Immobilization Using a Modified Chitosan Support." Youngstown State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ysu15946615388307.
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Ramani, Anand. "Two-step Component Mode Synthesis with convergence for the eigensolution of large-degree-of-freedom systems." Diss., Virginia Tech, 1996. http://hdl.handle.net/10919/39162.
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Wang, Benjamin X. Ph D. Massachusetts Institute of Technology. "Investigation of two-component signaling systems in Pseudomonas aeruginosa and their roles in the mucus barrier." Thesis, Massachusetts Institute of Technology, 2021. https://hdl.handle.net/1721.1/130821.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February, 2021Cataloged from the official PDF of thesis.
Includes bibliographical references.
All living things must be able to sense and respond to signals in their environment in order to grow and survive. For bacteria, a common means through which this occurs is via two-component signaling systems, which are typically comprised of a membrane-bound histidine kinase that senses external cues signals, and a cognate cytoplasmic response regulator that triggers changes in gene expression. The importance of these systems is highlighted by the fact that they have been identified in the vast majority of sequenced bacteria. However, despite their prevalence, much remains to be discovered about these sensory systems. In particular, both the actual processes that these systems control, along with the signals that they sense and respond to, remain poorly characterized in many cases.
In this work, I further characterize two-component signaling systems in the opportunistic pathogen Pseudomonas aeruginosa, which is most well-known for chronically infecting the lungs of patients with cystic fibrosis. I begin by screening a collection of in-frame deletion mutants of each histidine kinase in the PA14 strain against a dozen virulence-associated phenotypes, including different types of motility, biofilm formation, virulence factor production, and antibiotic resistance. Through this approach, I identify nearly two dozen of these proteins as important regulators of virulence. As P. aeruginosa is a human-associated mucosal pathogen, I next search for host-derived signals in mucus that act through these sensory systems in P. aeruginosa. I identify mucins and their associated glycans as signals that act through the RetS histidine kinase and the GacS-GacA two-component signaling system.
One major output of this signaling is the downregulation of the type VI secretion system, which suggests that mucin glycans may serve as host-derived "safety signals" that suppress microbial competition under non-dysbiotic conditions. Finally, I characterize the molecular mechanisms by which RetS inhibits GacS activity to better understand the unusual interactions between these two histidine kinases. Overall, this work underscores the diverse and important roles that two-component signaling systems play in bacteria, and begins to shed light on how microbes like P. aeruginosa utilize these systems to sense and respond to signals in the host.
by Benjamin X. Wang.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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Blue, Clare Elizabeth. "Characterisation of the role of two two-component signal transduction systems and a putative zinc metalloprotease in the virulence of Streptococcus pneumoniae." Thesis, University of Glasgow, 2002. http://theses.gla.ac.uk/3575/.
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This thesis aimed to evaluate the contribution of several pneumococcal two-component systems to virulence by analysis of null mutants in a murine model of infection. In addition, a putative zinc metalloprotease, ZmpB, located immediately downstream of one of the TCS, was analysed for its role in virulence. Data indicated that one of the systems studied, TCS08, does not contribute significantly to virulence in serotype 2 pneumococcus, but may have a slightly more important role in a serotype 3 background. A second two-component system, TCS09, was found to be essential for virulence in a serotype 2. Despite the completely avirulent phenotype of the mutant, no difference in expression of many of the previously identified pneumococcal virulence-associated genes was detected in the mutant compared to its isogenic parental strain. Microarray analysis indicated that in serotype 2, TCS09 may be involved in nutrient perception in nutrient perception. TCS09 was found to be required for full virulence in a serotype 3 strain. In this strain, mutants appeared to be impaired in their ability to disseminate from the lungs to the blood in a pneumonia model of infection, but were not attenuated in virulence following direct inoculation into the systemic circulation. These data provide evidence that virulence determinants can behave differently based on the genetic background of the parental strain. ZmpB was found to contribute significantly to pneumococcal virulence in a serotype 3 strain. Further analysis of the contribution of this protein to infection found that ZmpB appears to have a role in promoting inflammation. Thus this work has identified ZmpB as being a novel pneumococcal virulence factor. The role of this protein in inflammation is being investigated further. This thesis has thus identified several genes important in the virulence of S. pneumoniae and work is currently ongoing to assess the potential of these genes as future vaccine or drug candidates. Data presented within this work also provides evidence that virulence determinants can behave differently based on the genetic background of the parent bacterial strain. This important observation could have significant implication for the future characterisation of pneumococcal virulence factors and may apply to other bacterial pathogens.
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Hentschel, Eva [Verfasser], and Julia [Akademischer Betreuer] Frunzke. "Interaction of the two-component systems HrrSA and ChrSA in Corynebacterium glutamicum / Eva Hentschel. Gutachter: Julia Frunzke." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2015. http://d-nb.info/107098129X/34.
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Lai, Tzung-Huei. "Two-Component Regulatory Systems of Anaplasma phagocytophilum and Outer Membrane Protein P44 Expression Locus of Anaplasma platys." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1276709646.
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Trinh, My. "Role of Two-Component System Response Regulators in Virulence of Streptococcus pneumoniae TIGR4 in Infective Endocarditis." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2374.
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Streptococci resident in the oral cavity have been linked to infective endocarditis (IE). While viridans streptococci are commonly studied and associated with IE, less research has been focused on Streptococcus pneumoniae. Two-component systems (TCSs), consisting of a histidine kinase (HK) protein and response regulator (RR) protein, are bacterial signaling systems that may mediate S. pneumoniae TIGR4 strain virulence in IE. To test this hypothesis, TCS RR mutants of TIGR4 were examined in vivo through use of rabbit models. There were 14 RR proteins identified and 13 RR mutants synthesized because SP_1227 was found to be essential. The requirement of the 13 RRs for S. pneumoniae growth in IE models was assessed by quantifying mutants after overnight inoculation in IE infected rabbits through use of real time PCR (qPCR), colony enumeration on antibiotic selection plates, and competitive index assays. Real time PCR pinpointed several candidate virulence factors. Candidate RR SP_0798 was selected to be further examined. In the in vivo model, mutant SP_0798 grew significantly less than our control mutant SP_1678, which encodes a hypothetical protein and grew at a comparable rate to wild-type TIGR4 strains. Literature and databases identified SP_0798 as the ciaR gene, which has roles in regulating many diverse cellular functions. Our data suggests that RR SP_0798 is a virulence factor of S. pneumoniae TIGR4 strain in IE. This research may place more emphasis on virulence factors and lead to novel methods to combat pneumococcal endocarditis.
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Yusuf, Rahmi. "Synecdoche, two-component systems : E. coli EnvZ/OmpR as a model of transmembrane communication and chimeric biosensor design." Thesis, University of Portsmouth, 2019. https://researchportal.port.ac.uk/portal/en/theses/synecdoche-twocomponent-systems-e-coli-envzompr-as-a-model-of-transmembrane-communication-and-chimeric-biosensor-design(8d99cbe9-a70f-4320-9615-c5f27b6e0d2a).html.
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Ma, Pikyee. "Structure-activity relationships of membrane proteins : the NCS1 family of transporters and sensor kinases of two component systems." Thesis, University of Leeds, 2010. http://etheses.whiterose.ac.uk/4689/.
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Membrane proteins perform in many critical physiological processes, constituting 25 – 30% of prokaryotic and eukaryotic genomes, and represent up to 70% of current drug targets. However, the number of three dimensional structures of membrane proteins elucidated has been modest in comparison with those of soluble proteins, and this is attributable to three major bottlenecks: expression, purification and structure determination of these hydrophobic proteins. A genomic approach was conducted to overcome these bottlenecks for two families of membrane proteins: (1) the NCS1 family of transporters and; (2) the histidine kinases of two-component signal transduction systems of E. faecalis. The genes encoding these membrane proteins were cloned separately into plasmid pTTQ18-His6 and subjected to expression trials in Escherichia coli. Purification of the recombinant intact proteins from E. coli membranes was undertaken using Ni-NTA chromatography, and for proteins that were produced in sufficient quantities crystallisation trials and investigations of structural characteristics were performed. The activities of membrane-bound and/or purified proteins were also tested, and in the case of the histidine kinases, these activity assays were expanded to successfully identify modulating signals. This is the first time that direct in vitro activity assays using intact proteins have proven successful for identification of environmental ‗signals‘ in enterococci. In this study, thirteen NCS1 transporters were cloned. Twelve of the thirteen cloned proteins were expressed in E. coli membranes, seven were successfully purified, and substrates transported by five of these were confirmed. Three of these transporters, were further characterised, including; (1) a cytosine transporter – CodB, (2) an allantoin transporter – PucI, and (3) a uracil transporter – PA0443, and their substrate specificities and kinetics were also determined. All sixteen membrane histidine kinases of E. faecalis were successfully cloned (six were assigned to me in a joint project with Dr Hayley Yuille). Fifteen of the sixteen histidine kinases were successfully expressed in E. coli, and thirteen were purified. Eleven of the fifteen membrane-bound histidine kinases and twelve of the thirteen purified proteins were active in autophosphorylation activity assays. Three histidine kinases were studied further, including; (1) EF3197, which responded to increasing concentrations of a reducing agent, suggesting a possible role in redox-sensing, (2) EF1051, which was further purified by size exclusion chromatography and showed promising results in crystallisation trials for future structural determination, and (3) EF1820 (FsrC), which was activated in in vitro assays by its pheromone signal, gelatinase biosynthesis-activating pheromone (GBAP). This study showed GBAP-induced activity of FsrC was inhibited by the anti-HIV inhibitor, siamycin I, identifying unequivocally and for the first time, the target protein for siamycin I inhibition in the Fsr pathway. This study presents a successful genomic approach for the production of milligram quantities of membrane proteins leading to characterisation and crystallisation studies. The methods can now be applied to further membrane proteins.
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Diomande, Sara Esther. "Adaptation au froid de la bactérie pathogène Bacillus cereus : étude de mécanismes impliqués et exploitation de la diversité génétique." Thesis, Avignon, 2014. http://www.theses.fr/2014AVIG0666/document.
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Bacillus cereus sensu stricto (ss) est un pathogène alimentaire majeur représentant la 2e cause de toxiinfectionalimentaire en France en 2012. Cette espèce fait partie du groupe Bacillus cereus sensu lato (sl)constitué d’espèces ubiquitaires génétiquement très proches et incluant d’autres pathogènes comme B.anthracis, B. thuringensis et B. cytotoxicus. Les souches de B. cereus sl sont d’autre part réparties en septgroupes phylogénétiques présentant des gammes de température de croissance variées et caractérisés partrois thermotypes principaux: thermotolérants, mésophiles, psychrotolérants. L’adaptation au froid dessouches B. cereus ss est un mécanisme clé car il conditionne sa capacité à se développer dans les alimentsréfrigéré pour atteindre des doses qui peuvent être dangereuse pour les consommateurs. Le but de cetteétude a été d’étudier les mécanismes moléculaires impliqués dans l’adaptation au froid de la diversité desouches représentant B. cereus sl.Nous avons mis en évidence que les gènes codant pour le système à deux composants CasK/R sontsurexprimés à basse température. CasK/R s’est révélé être un système générique d’adaptation de B. cereussl au froid, car son rôle a été mis en évidence lors de l’étude de quatre souches de thermotypes différents etleurs mutants isogéniques ΔcasK/R respectifs. Une étude transcriptomique réalisée sur une souche ATCC14579 et son mutant ΔcasK/R a révélé que seize des gènes différentiellement exprimés en début de phaseexponentielle et en phase stationnaire, à basse température, codent pour des protéines impliquées dans lemétabolisme des acides gras. Nous avons mis en évidence le rôle de CasK /R dans la modification de lacomposition en acides gras membranaires via une augmentation de la proportion en acides gras insaturéslors de la croissance de B. cereus au froid. Par ailleurs, le gène codant pour la désaturase DesA,principalement responsable des insaturations des acides gras à basse température est régulée positivementpar CasK/R au froid.Nous avons également démontré que les gènes casK/R sont organisés en opéron avec un gène codant pourun régulateur RpiR-like. De manière originale, cet opéron est négativement régulé par CasK/R à bassetempérature en phase stationnaire. Le promoteur individuel du rpiR est réprimé à basse température maisaussi à température optimale de croissance, ce qui suggère un rôle de CasK/R, même à températureoptimaleBacillus cereus sensu stricto (ss) is a major foodborne pathogen representing the second cause of foodpoisoning in France in 2012. This species belongs to Bacillus cereus sensu lato (sl) consisting of ubiquitousspecies genetically close-related and including other pathogens such as B. anthracis, B. thuringiensis and B.cytotoxicus. The strains of B. cereus sl are divided into seven phylogenetic groups with various growthtemperature ranges and characterized by three main thermotypes: thermotolerant, mesophilic,psychrotolerant. The B. cereus ss cold adaptation is a key mechanism because it determines B. cereusability to grow in refrigerated foods and achieve doses that can be dangerous to consumers. The aim of thisstudy was to study the molecular mechanisms involved in the cold adaptation of strains representing B.cereus sl diversity.We demonstrated that the genes encoding the two component system CasK/R are overexpressed at lowtemperature. CasK/R was found to be a generic mechanism for B. cereus sl cold adaptation as its role washighlighted in the study of four strains with different thermotypes and their respective isogenic mutantsΔcasK/R. A transcriptomic study on a B. cereus ATCC 14579 strain and its ΔcasK/R mutant strain revealedthat sixteen of the genes differentially expressed in both early log phase and stationary phase at lowtemperature encode proteins involved in the fatty acids metabolism. We showed the role of CasK/R in themodification of the membrane fatty acid composition via an increase of the proportion of unsaturated fattyacids during growth of B. cereus at low temperature. Furthermore, the gene encoding the desaturase DesA,mainly responsible of the fatty acids unsaturation at a low temperature is upregulated by CasK/R at lowtemperature.We also demonstrated that casK/R genes were organized in operon with a gene encoding a RpiR-likeregulator. Interstingly,, this operon is negatively regulated by CasK/R at low temperature in the stationaryphase. The individual rpiR promoter is repressed by CasK/R at low temperature but also optimal growthtemperature, suggesting also a role for CasK/R at optimal temperature
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Tatke, Gorakh Digambar. "Elucidating The Role of MifS-MifR Two-Component System in Regulating Pseudomonas aeruginosa Pathogenicity." FIU Digital Commons, 2016. http://digitalcommons.fiu.edu/etd/3002.
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Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections.
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Hayashi, Masaki. "Studies of time-evolution of domain structure and domain interface structure in phase-separation process of two-component polymer systems." 京都大学 (Kyoto University), 2003. http://hdl.handle.net/2433/148838.
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Tu, Nhan. "Characterization of Two Novel Gene Regulatory Systems in the Zoonotic Bacterium Bartonella henselae." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/6042.
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The genus Bartonella contains Gram-negative arthropod-borne bacteria that are found in many small animal reservoirs and are capable of causing human disease. Bacteria utilize a general stress response system to combat stresses from their surrounding environments. In α-proteobacteria, the general stress response system uses an alternate σ factor as the main regulator and incorporates it with a two-component system into a unique system. Our study identifies the general stress response system in the α-proteobacterium, Bartonella henselae, where the gene synteny is conserved and both the PhyR and alternate σ factor have similar sequence and domain structures with other α-proteobacteria. Furthermore, we showed that the general stress response genes are up-regulated under conditions that mimic the cat flea vector. We also showed that both RpoE and PhyR positively regulate this system and that RpoE also affects transcription of genes encoding heme-binding proteins and the BadA adhesin. Finally, we also identified a histidine kinase, annotated as BH13820 that can potentially phosphorylate PhyR. In addition, analysis of the transcriptome from the Houston-1 strain of B. henselae by RNA-Seq reveals a family of small RNAs (termed Brt1-Brt9 for Bartonella Regulatory Transcripts 1-9) that may rapidly adapt gene expression patterns to the diverse hosts of this bacterium. This family of RNAs consists of nine novel, highly expressed intergenic transcripts, ranging from 193-205 nucleotides with a high degree of homology (70-100%) and stable predicted secondary structures that are unique to the genus Bartonella. Northern blot analysis indicates that transcription of these sRNAs was highest under conditions mimicking those of the cat flea vector (low temperature, high hemin). The predicted promoters for Brt1-Brt9 have been cloned upstream of a β-galactosidase reporter gene in pNS2 to identify conditions altering transcription. Immediately downstream of each of the nine putative sRNAs is a helix-turn-helix DNA binding protein (termed Trp1-9 for Transcriptional Regulatory Protein 1-9) that is poorly transcribed as determined by RNA-Seq. This gene organization is suggestive of a potential cis-acting RNA mechanism or riboswitch with the RNA secondary structure controlling transcription of the cognate downstream trp.
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Draheim, Roger Russell. "The role of protein-membrane interactions in modulation of signaling by bacterial chemoreceptors." [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1336.
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Grob, Philipp. "Identification of two homologous two-component regulatory systems, NodV/NodW and NWsA/NwsB, in Bradyrhizobium japonicum and analysis of their role in nodulation and nod gene regulation /." [S.l.] : [s.n.], 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10399.
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Wang, Liang. "Structural characterisation of Histidine Kinase 2." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/33933.
Full textAbstract:
Two-component systems (TCS) are the predominant signal transduction pathways in prokaryotes, being present also in eukaryotic organisms, such as algae, fungi and yeast, and higher plants. TCSs play an important role in environmental signal perception and response, essentially implementing adaptation to the surrounding environment. Histidine Kinase 2 (Hik2) in cyanobacteria is a typical sensor histidine kinase, one component of a TCS, and has been identified to be a homologue protein of Arabidopsis Chloroplast Sensor Kinase (CSK). Previous research has elucidated Hik2 to regulate photosynthetic gene transcription with two response regulators, Rre1 and RppA via phosphorylation. A typical histidine kinase contains a variable sensor domain and a conserved kinase domain. It usually functions as a homodimer. This thesis describes the structural characterisation of Hik2, probing particularly its discovered oligomeric states. Results obtained from size exclusion chromatography, native-PAGE, chemical cross-linking analyses and mass spectrometry, amongst others, have shown a variety of Hik2 structural populations exist, further validated by negative stain transmission electron microscopy coupled to single particle analysis. Hik2 protein exists predominantly as a hexamer in low salt conditions, and adding NaCl dissociates hexamers into tetramers, critical for the autophosphorylation activity of Hik2. Thus, a model is proposed for the constitution change of Hik2 oligomers when salt concentration differs. In addition, the sensor domain is typically responsible for detecting environmental input, however, it is not yet clear how Hik2 and CSK sense signals. In this thesis, the structures of Hik2 and CSK sensor domains were analysed and discussed, to aid our understanding of their mechanism of signal perception and transduction.
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Faralla, Cristina <1986>. "Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6440/1/Faralla_Cristina_tesi.pdf.
Full textAbstract:
Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.
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Faralla, Cristina <1986>. "Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6440/.
Full textAbstract:
Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.