Dissertations / Theses on the topic 'Phosphorelays'
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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.
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.
Cochard, Clémence. "Régulation fine du système EnvZ/OmpR chez Dickeya dadantii : clef d'une infection réussie." Electronic Thesis or Diss., Université de Lille (2022-....), 2023. http://www.theses.fr/2023ULILS109.
Throughout their life, the bacteria must confront numerous environmental variations. They must adapt rapidly and effectively to ensure their survival. To accomplish this, they possess phosphorelays, or two-component systems, which are the major molecular tools enabling perception and adaptation to the environment in bacteria. These phosphorelays consist of a sensor and an associated regulator. Following the perception of a stimulus, the sensor autophosphorylates and transmits the phosphate group to the regulator, which then modulates the expression of the entire target gene set, known as a regulon. During the infection process, pathogenic bacteria must deal with multiple stresses. A significant number of these systems are found in various pathogenic bacteria, such as our study model Dickeya dadantii. Responsible for soft rot disease, D. dadantii is a wide-host-range phytopathogenic enterobacterium. It possesses a battery of 32 phosphorelays to deal with host defenses and the general stresses of nutritional deficiencies or physicochemical variations in the environment.First this study focuses on one of them, the EnvZ/OmpR system. My work initially shows that the pH in the plant remains acidic during infection. However, despite activation of the system by acidic pH, it is not activated during this process. To understand the reason for this inconsistency, the system's regulon was studied. It was then discovered that during the emergence of the Dickeya genus, the ompF gene, encoding the porin of the same name, was duplicated. Interestingly, the expression of ompF is constitutive, whereas that of ompF2, the duplicated gene, which is dependent on OmpR phosphorylation levels. The expression of this second porin is also detrimental to infection. Thus, during infection, the activation of EnvZ/OmpR is counteracted by the perception of host defense molecules to prevent the expression of ompF2 and enable proper virulence progression.In a second phase, a comprehensive study of the importance of each phosphorelay in D. dadantii's virulence was conducted in my work. The initial results show that only 6 systems are involved in virulence. The number and complexity of stresses encountered by pathogenic bacteria do not seem to align with this low number. Reducing the quantity of inoculated bacteria allowed for a more precise detection of the systems contributing to virulence, which now totals 12. Overall, these results indicate the significance of finely regulating the activity of a phosphorelay, as EnvZ/OmpR must be activated for infection, but this activation must be strongly controlled to avoid detrimental effects on virulence
Treffandier, Hélène. "Etude du phosphorelais RcsCDB/FA d'Escherichia coli." Toulouse 3, 2009. http://thesesups.ups-tlse.fr/797/.
Two-component systems, also called phosphorelays are the major signalling pathways in bacteria. They are widespread and mediate a large variety of adaptative cellular responses. The Rcs system of Escherichia coli is a complex phosphorelay composed of five proteins: RcsCDBFA. Exclusive to the Enterobacteriacae, the Rcs phosphorelay modulates biofilm formation and virulence in several pathogens. It is activated by membrane alterations and influences expression of 2 to 3% of the bacterial genome. However, the role of the Rcs system in adaptation to the environment remains elusive. The three objectives of my PhD were (i) to explore further the role of the Rcs system in adaptation to relevant environmental conditions (ii) to rationalize the complexity of the Rcs phosphorelay and, in particular, to explain the role of the accessory co-factor RcsA in the Rcs response (iii) to develop an integrated vision of the system through the identification of its protein partners. Within the framework of these objectives, my work proved that the Rcs system is essential for resistance to low pH, a situation encountered by E. Coli in mammals' stomach. My research also suggests that the accessory cofactor RcsA modulates the kinetics of expression of the whole RcsB regulon, in particular by prolonging the Rcs response after the phosphorelay's activating signal has stopped. To finish with, my work revealed a greater complexity in rcsA regulation than was previously thought to exist, by showing that it is twofold: transcriptional and post-transcriptional
Caby, Marine. "Rôle du phosphorelais EnvZ/OmpR chez la bactérie phytopathogène Dickeya dadantii." Thesis, Lille 1, 2020. http://www.theses.fr/2020LIL1S108.
During their lifetime, pathogenic bacteria are confronted with numerous environmental variations often referred to stress, particularly during infection. In order to survive and successfully colonize its host, the bacterium must perceive this new and dangerous environment to adapt quickly. This is the main role assigned to phosphorelays. These systems are composed of a sensor and a cognate regulator. Under the action of a stimulus, the sensor autophosphorylates and transmits the phosphate group to its regulator, which in turn modulates the activity of a set of target genes allowing adaptation to the new environment. Our experimental model Dickeya dadantii is a necrotrophic plant pathogen bacterium responsible for soft rot disease in a wide range of plant species. The variation of pH and osmolarity are two stresses often faced and fought by pathogenic bacteria. EnvZ/OmpR and RcsCDB phosphorelays are two major systems known to respond to these stresses. The laboratory had previously demonstrated that the level of activation of the RcsCDB system was dependent on the concentration of periplasmic osmoregulated glucans (OPG). Their concentration in the periplasm increases as the medium osmolarity decreases, making OPGs a major intermediate in the perception of osmolarity. This prompted us to decipher the relationship between EnvZ/OmpR and OPGs. I showed that, unlike for the activation of the RcsCDB system, the activation of EnvZ/OmpR doesn’t depend on the concentration of OPGs, but still requires its presence for proper activation of the phosphorelay. To go deeper into the EnvZ/OmpR system, activities of this system have been studied in vivo and in planta. While the EnvZ/OmpR system is activated in a medium with an acidic pH and a high osmolarity in E. coli, my work shows that only pH variation activates this phosphorelay in D. dadantii. In addition, only one major porin (versus two in E. coli) was previously detected in D. dadantii. My studies revealed the existence of a second porin expressed at acidic pH in vivo and in planta. These two OmpF-like porins are regulated by the pH via OmpR. After adaptation for a few hours in planta, the pattern of these two porines remains the same over the rest of the infection. However, the level of OmpR activation during the same period fluctuates indicating that at least one other environmental parameter modulates the activation of EnvZ/OmpR in planta. The steady state level of the porines in the envelope during this same period suggests that another regulatory system, perhaps RcsCDB may maintain their expression level
Huang, Ya-hui. "Genetic and functional analysis of the Rcs phosphorelay in the Enterobacteriaceae." Thesis, University of Bath, 2006. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436882.
Djeghdir, Inès. "Identification et caractérisation de facteurs de transcription appartenant à la famille des régulateurs de réponse de type B, impliqués dans la réponse à la sécheresse chez le peuplier." Thesis, Orléans, 2016. http://www.theses.fr/2016ORLE2056/document.
Plants are increasingly faced with a decrease in soil’s water availability, leading to a hydric and osmotic stress and impacting on their survival. Plant tolerance to this stress will be dependent on its perception. One of the signaling mechanisms related to this stress is called MultiStep Phosphorelay (MSP) and is composed by 3 partners: a histidine-aspartate receptor kinase (HK), histidine phosphotransfer proteins (HPt) and response regulators (RR), including the B-type RR transcription factors. In Arabidopsis, an MSP with AHK1, AHP2 and ARR18 has been identified for osmotic stress signaling. For poplar, HK1a and b, paralogous genes and homologous with AHK1, 10 HPt and 9 B-type RR genes have been isolated respectively. The osmosensor function of HK1a was proposed, and an osmosensing signaling pathway composed by HK1a, 3 HPt proteins, and 6 B-type RR has been suggested. The purpose of this work was focused on the identification and characterization of B-type RR transcription factors specifically linked to osmotic stress in poplar. The main results of this work are the highlight of the transcription factor function of two B-type RR, RR13 and RR19, through the study of their ability to dimerize and transactivate or not osmotic stress-responsive genes. The RR13 seems to be specific for cytokinins signaling pathway, whereas the RR19 seems to be specific for the osmosensing one. This work strongly supports the involvement of RR19 in the osmosensing MSP. Many studies have also been initiated during this work and will facilitate the characterization of the studied MSP
Borland, Stéphanie. "Rôle des systèmes à deux composants dans l’adaptation de la bactérie phytostimulatrice Azospirillum à la rhizosphère." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10037.
Bacterial two-component systems play an important role in the ability of bacteria to adapt to various environments. The aim of this thesis was to identify and characterize two-component systems involved in the adaptation of the phytostimulatory bacteria Azospirillum to its host plant. Analysis of the genomic distribution of genes encoding two-component systems across Azospirillum available genomes revealed the existence of a high number of genes encoding hybrid histidine kinases, and further analyses highlighted a complex multi-domain organization of this family of proteins. In order to understand their role in Azospirillum, as a first step we selected and inactivated four genes encoding complex hybrid histidines kinases. Using a multidisciplinary approach which combines genetics, biochemistry and phylogeny, we brought to light for the first time in Azospirillum, an atypical three-component system named PreSKR which controls a wide variety of processes involved in survival and rhizosphere colonization likely by modulating c-di-GMP levels. As a second step, we focused on a gene encoding a hybrid histidine kinase named RsiK which is induced in contact with its host plant. RsiK is involved in surface sensing and biofilm formation regulation. Transcriptomic analysis of rsiK regulon by RNA-seq showed that 78 genes were under the control of this system. The prevalence of genes encoding hybrid histidine kinase family in Azospirillum, coupled with the functional characterization of two of them, highlight the importance of phosphorelays, still largely unrecognized in rhizospheric bacteria
Fernandez, Marion. "Le rôle de systèmes à phosphorelais dans l’infection de la puce Xenopsylla cheopis par Yersinia pestis." Thesis, Lille, 2019. http://www.theses.fr/2019LILUS050.
Two-component systems are used by bacteria to sense and respond to environmental cues by modulating genetic expression. They are composed of an histidine kinase, a sensor which transmits the signal to a response regulator by a phosphotransfert mechanism. Yersinia pestis is the causative agent of plague, a zoonotic disease, and is transmitted by fleas. Biofilm formation in the flea proventriculus leads to flea blockage, which is an important step for Y. pestis transmission by its vector. During its life cycle, Y. pestis must sense and adapt to different environments. This is why two-component systems have been considered of interest for Y. pestis pathogenicity studies. Our work provided evidence that OmpR/EnvZ is activated in the flea’s digestive tract. Interestingly, neither osmolarity nor pH variation in the flea gut trigger OmpR-EnvZ. In contrast, nutrient depletion occurring after blood digestion could be responsible for the activation of the system. We further reported that OmpR/EnvZ is needed for flea blockage because it is needed for biofilm formation in the proventriculus, especially by activating ompF. In addition to OmpR-EnvZ, we also provided evidence that the activation of the GlrKR-YfhG regulatory system is also required for flea blockage. Strikingly, this system displays two distinct function. In the proventriculus, it promotes the production of c-di-GMP, a secondary messenger essential for biofilm formation, and thus flea blockage. In the midgut, it activates the transcription of small RNA glmY and glmZ genes to maintain the bacterial morphology. Overall, our data suggest that the flea’s digestive tract is a toxic environment for Y. pestis and that the proventriculus and the midgut are two distinct environments
Sexauer, Anne [Verfasser], and Nicole [Akademischer Betreuer] Frankenberg-Dinkel. "Characterization of a bacterial-like signal transduction phosphorelay in Methanosarcina acetivorans / Anne Sexauer ; Betreuer: Nicole Frankenberg-Dinkel." Kaiserslautern : Technische Universität Kaiserslautern, 2021. http://d-nb.info/1237268974/34.
Chefdor, Françoise. "Recherche d’un phosphorelais multiple impliqué dans la perception et la transduction du signal stress hydrique chez le peuplier." Orléans, 2006. http://www.theses.fr/2006ORLE2054.
Morgan, Jason Kyle. "Genetic basis for the virulence of enterohemorrhagic Escherichia coli strain TW14359." Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5277.
Francez-Charlot, Anne. "Le phosphorelais RcsCDB chez Escherichia coli : caractérisation du régulon et analyse de la régulation des gènes cibles flhDC et bdm." Toulouse 3, 2004. http://www.theses.fr/2004TOU30271.
To protect themselves against environmental stresses, bacteria use signal transduction systems, the so-called His-Asp phosphorelays. In Escherichia coli, the RcsCDB phosphorelay is essential under certain conditions of stress and is involved in biofilm development. This system controls capsule synthesis, cell division and sS production in response to envelope stresses. The objective of my thesis was to understand the biological role of the RcsCDB/A system and to identify the determinants of the regulation by the response regulator RcsB and its cofactor RcsA. Using a global approach, we characterized RcsB and RcsA regulons. This study suggests that one function of the Rcs system consists in envelope remodelling. A thorough study of the regulation of two targets of the Rcs system, flhDC and bdm, shows the first example of a negative regulation by RcsB and RcsA and strengthens the assumption that the Rcs system contributes to the effectiveness of biofilm formation
Bertheau, Lucie. "Caractérisation d'un phosphorelais multiple de type histidine-aspartate dans la transduction du signal de la contrainte osmotique chez le peuplier : mécanismes de régulation du fonctionnement d'un régulateur de réponse de type-B à l'échelle moléculaire." Thesis, Orléans, 2013. http://www.theses.fr/2013ORLE2076/document.
Multistep His-to-Asp phosphorelay systems are signaling pathways devoted to signal perception and transduction for establishment of specific responses. These systems are composed of three successive partners: Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). One of the best characterized corresponding systems is the osmo-responsive pathway in yeast. Such systems are also suspected in Arabidopsis. This work aimed to characterize the involvement of an osmosensing pathway in Populus by identifying HPt and RR elements downstream of HK1 and to reveal the underlying mechanisms for the activity of a RR-B. HK1, membrane osmosensor, is expected to be responsible for signal detection and propagation by triggering the activation of three preferential HPt. Furthermore, an interacting partnership between those HPts and particular B-type RRs was observed. Two of them appear to be regulated by an osmotic stress, suggesting their possible involvement in this pathway. The B-type RR members, the final output elements of the pathway, act as transcription factors, as shown for at least for one of them in planta. Taken together, the dimerization of the RR receiver domain and its interaction with its DNA binding domain (GARP), are likely key checkpoints in the regulation of RR-B activity. Besides, the ability of one RR-B to bind its cognate specific DNA sequences (AGAT boxes) was confirmed in vitro and those were found in promoters of osmotic response genes. This work opens up prospects for the involvement of RR-B in the osmotic stress signaling pathway and suggests mechanisms tuning induction of specific responses
Bernard, Rémi. "Résistance à la bacitracine chez Bacillus subtilis." Phd thesis, Université de la Méditerranée - Aix-Marseille II, 2007. http://tel.archives-ouvertes.fr/tel-00350345.
Le groupe des Firmicutes, dont la bactérie modèle est Bacillus subtilis, est un groupe ubiquitaire producteur d'antibiotiques et contenant de nombreux organismes pathogènes. Les analyses effectuées au laboratoire ont permis d'identifier chez les Firmicutes des systèmes associant un phosphorelais et un transporteur ABC. Dans chacun des cas, les gènes codant les différents partenaires du système sont à proximité sur le chromosome et le phosphorelais régule l'expression des gènes codant le transporteur ABC. On trouve trois de ces systèmes chez B. subtilis nommés BceRSAB (anciennement YtsABCD), YvcPQRS et YxdJKLM. L'objectif de notre étude était de tester l'hypothèse selon laquelle ces systèmes pouvaient être impliqués dans la résistance aux antibiotiques ciblant l'enveloppe bactérienne.
La bacitracine est un antibiotique qui se complexe à l'undécaprényl pyrophosphate (UPP) et inhibe la dernière étape de la biosynthèse du peptidoglycane : la régénération de l'undécaprényl phosphate (UP). Nos résultats indiquent que le système BceRSAB est le composant majeur de la résistance à la bacitracine chez B. subtilis. L'expression de l'opéron bceAB est activée, en présence de bacitracine, par le phosphorelais BceRS. Nous avons également identifié une protéine, nommée BcrC, qui participe à la résistance à la bacitracine chez B. subtilis. Nous avons démontré que cette dernière est une UPP phosphatase impliquée dans la régénération de l'UP et s'oppose ainsi à l'action de la bacitracine. L'expression du gène bcrC ne dépend pas du phosphorelais BceRS mais de trois facteurs sigma à fonction extracytoplasmique. En conclusion, B. subtilis possède deux types de systèmes de résistance à la bacitracine indépendants et complémentaires.
Nous avons par la suite analysé le mécanisme de régulation de l'induction du système BceRSAB par la bacitracine. Nos résultats sont surprenants et montrent clairement que le transporteur ABC BceAB participe à l'activation de l'expression de ses propres gènes de structure avec le phosphorelais BceRS. De plus, lorsque le pool cellulaire d'UPP diminue (lorsque la phosphatase BcrC est surproduite), et en présence de bacitracine, l'expression du gène bceA diminue également. Ceci montre que l'UPP participe, avec la bacitracine, au stimulus du système BceRSAB. Notre hypothèse de travail est que le transporteur ABC, prédit comme étant un exporteur, prend en charge le complexe UPP/bacitracine et agit comme une flippase pour créer une dissymétrie membranaire ressentie par le senseur BceS. Il n'est pas exclu, qu'en présence de bacitracine, le transporteur BceAB puisse interagir avec le senseur BceS pour permettre l'activation du système.
La majeure partie des bactéries du groupe des Firmicutes possède, d'une part, des protéines similaires à BcrC, et, d'autre part, des systèmes similaires au système BceRSAB. Toutes les protéines « BcrC-like » ont un motif caractéristique permettant de les classer dans la famille des phosphatases de type PAP2. Il est tentant de penser qu'elles sont toutes des UPP phosphatases assurant une des étapes clés de la synthèse du peptidoglycane.
Par ailleurs, les deux autres systèmes « Bce-like » de B. subtilis, YvcPQRS et YxdJKLM, sont également activés en présence d'antibiotiques ciblant l'enveloppe bactérienne et nous savons que le transporteur ABC YvcRS est aussi impliqué dans le mécanisme de régulation. Nous proposons donc que les systèmes « Bce-like » des Firmicutes sont tous des systèmes de détoxification dirigés contre des antibiotiques ciblant l'enveloppe bactérienne.
Prouvost, Anne-France. "Rôle du périplasme dans la perception par la bactérie de son environnement : utilisation des ß-galactanes par Erwinia chrysanthemi : voie de signalisation du système Rcs dans la virulence d'Erwinia chrysanthemi." Thesis, Lille 1, 2008. http://www.theses.fr/2008LIL10078/document.
Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plant species. The maceration of plant tissues is essentially caused by the secretion of a set of plant cell wall degrading enzymes (including pectinases and cellulases). The aim is to participate to the understanding of the role of the periplasm in environment perception by bacteria. We characterised genetically and biochemically the gan locus encoding 9 proteins involved in galactan transport and catabolism (galactan is a pectic component). Osmoregulated periplasmic glucans (OPGs) are essential for E. chrysanthemi pathogenicity. An opgG mutant lacks OPGs and shows a pleiotropic phenotype including nonvirulence. A rcsC point mutation (rcsC2) suppresses the phenotype induced by OPGs defect. We wanted to clarify the role of the Rcs system in pathogenicity. The rcsC2 mutation leads to the reduction of transcriptional activity of RcsB caused by an increase of phosphatase activity of RcsC. Mutations in RcsCDB proteins show that RcsB is not essential to virulence. We have shown that genes expression regulated by RcsCBD phosphorelay is influenced by the quantity of OPGs in the periplasm. An ectopic overexpression of a soluble version of RcsF leads to a RcsCDB phosphorelay activation, while an ectopic overexpression of DjlA and IgaA has no significatives effects. Finally, we have characterized RcsF structures by site-directed mutagenesis and deletion mutagenesis
Dautel, Rebecca [Verfasser], and Klaus [Akademischer Betreuer] Harter. "Molecular characterization of the Arabidopsis thaliana histidine kinase 1 and transitions from the multistep phosphorelay system to Ser/Thr/Tyr phosphorylation / Rebecca Dautel ; Betreuer: Klaus Harter." Tübingen : Universitätsbibliothek Tübingen, 2019. http://d-nb.info/1199615315/34.
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.
Sen, Shaunak. "Regulatory Consequences of Bandpass Feedback in a Bacterial Phosphorelay." Thesis, 2011. https://thesis.library.caltech.edu/6443/3/Abstract_ssen.pdf.
Chu, Pei-Hsuan, and 朱珮瑄. "Role of the HptB module in Peudomonas aeruginosa signaling phosphorelay." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/92rexy.
國立交通大學
生物科技系所
92
Pseudomonas aeruginosa PAO1 is an opportunistic pathogen, and owns great capability to adapt versatile environment. There are 123 genes encoding two-component system components in the bacteria, which serve as a stimulus-response coupling machinery allowing the organism sense and respond to the changes in environment. The system has been classified into three types: the classical system, the unorthodox system, and the hybrid system. We focus on the hybrid system which consists of a histidine kinase (HK) without output domain, a separated Histidine-containing phosphotransfer (Hpt) molecule, and a response regulator (RR). In order to demonstrate the role of Hpt modules in signaling phosphorelay, the gene fragments corresponding to each of the Hpt domain and one hybrid sensor (PA1611) and two RRs (PA3346 and PA0034) were amplified by PCR and subcloned respectively into pET30 expression vector. The recombinant proteins were expressed in E. coli and the proteins purified by His-Bind nickel column for the phosphorylation assays. The assay demonstrated a specific phosphoryl transfer from sensor kinase PA1611 to HptB and then to PA3346. However, using yeast two-hybrid screening failed to identify the protein interactions. Sequence comparisons of several Pseudomonas species revealed that the genome DNA contains the HptB homolog flanking by a flagella biosynthesis gene cluster. To identify the functional role of HptB, hptB mutant was constructed by homologous recombination. The hptB mutant showed a decrease of growth rate decreased in a nutrient limiting condition. The hptB mutation also affected the biofilm formation with a 2-fold higher activity than that of the wild type strain in a minimal medium supplemented with carbon and nitrogen source. A reducing swimming capability and an increase of twitching motility were also found in hptB mutant. Taken together, the HptB is likely mediated the signaling involved in flagella biosynthesis and regulation.
Hsu, Jye-Lin, and 徐婕琳. "Regulation of bacterial gene expression: elucidation of HptB-mediated phosphorelay signaling systems and an ATPase dependent transcriptional factor AcoK." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/35615304488824363872.
國立清華大學
分子醫學研究所
97
This thesis contains two major topics. The first one is “Characterization of the histidine-containing phosphotransfer (Hpt) protein B�{mediated multi-step phosphorelay system in Pseudomonas aeruginosa PAO1” and the second one is “The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43”. Pseudomonas aeruginosa is a gram-negative pathogen causing many acute and chronic infections, particularly in hospitalized individuals. It contains three genes that encode proteins with an Hpt domain but lack a kinase domain. Hpt proteins are signal mediators between hybrid sensors and response regulators. The proteins play a crucial role in directing signal transduction in bacteria, yeasts and plants. The study in first topic demonstrates that the Pseudomonas aeruginosa HptB-mediated signaling system consists of four orphan sensor kinases, HptB, and a specific response regulator, PA3346. We also present evidence of phosphatase activity of PA3346 on its neighboring gene product, PA3347. Finally, the swarming and biofilm forming activites of hptB, PA3346, and PA3347 knockout mutants are described. The second part of this thesis is to characterize a transcriptional factor AcoK in the regulation of acetoin catabolism. Many bacterial species utilize acetoin as a carbon source. The compound is oxidized by acetoin dehydrogenase encoded by acoABCD operon in Klebsiella pneumoniae. Previously, we have shown the expression of this operon is induced by acetoin through AcoK, the product of a gene located immediately upstream of acoABCD. AcoK contains a helix-turn-helix DNA binding domain of the LuxR transcription activator family at the C-terminal region and putative Walker A and B nucleotide binding motifs at N-terminal portion. The goal of the second study is to understand the contribution of different domains, in particular the nucleotide binding motif, in AcoK on its transcriptional activity. A number of truncations and site-directed mutations were constructed on AcoK and the biochemical and trans-activation activities of the resulting proteins were determined and reported herein. A mutation in the putative Walker A motif resulted in a significant reduction of ATP hydrolysis and trans-activation activity of AcoK on acoABCD expression, presumably was due to the loss of ATP-binding ability. The transcription factor bound specifically to a region comprising nucleotide -66 to -36 of the acoABCD promoter, although the DNA binding ability was not affected by the Walker A motif mutation. Together, this study provides an additional example in how a member of the Signal Transduction ATPases with Numerous Domains family activates its target gene expression.