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Studies on molecular recognition and degradation mechanism of plant cell wall polysaccharides-assimilating Clostridium cellulovorans using proteome analysis / プロテオーム解析を用いたクロストリジウムセルロボランスの植物細胞壁多糖分解と分子認識機構の解析Aburaya, Shunsuke 25 March 2019 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21808号 / 農博第2321号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5180(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 渡邊 隆司, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM
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Characterization of the Salmonella enterica Two-Component Regulatory System SsrA-SsrB and the SsrB Regulon / The Salmonella enterica Regulatory System SsrA-SsrBMulder, David January 2014 (has links)
Salmonella enterica is an intracellular bacterial pathogen of humans and the causative agent of the acute gastrointestinal disease, salmonellosis, and the chronic systemic infection, typhoid fever. Sensor proteins convert environmental signals, including signals detected within the host environment, into biochemical signals to control cellular responses. It has been previously established that the two component regulatory system SsrA-SsrB, consisting of the integral membrane sensor kinase protein SsrA and the cytoplasmic DNA-binding response regulator SsrB are essential for regulation of bacterial factors during systemic intracellular infection. The first chapter of this thesis describes characterization of the sensor kinase SsrA. The structure of the periplasmic sensor domain is modeled and evidence is presented that it is involved in enhancing signaling activity in response to environmental acidification encountered within the intracellular environment. A mechanism whereby protonation of histidine residues within this region in response to acidification drives conformational strain and thereby signaling is proposed. The second chapter describes identification of the DNA-binding motif of SsrB within regulated promoters as well as its regulon. Integration of experimental data with comparative genomics data resulted in identification of the palindromic heptameric DNA recognition motif of SsrB as well as identification of novel SsrB-regulated promoters. In addition, a DNA microarray analysis is described wherein the complete SsrB regulon is identified. Finally, the third chapter describes regulatory input of SsrB to the S. enterica type VI secretion system. This chapter also describes the contribution of this system to systemic dissemination of S. enterica during host infection. Altogether, these data advance understanding of how Salmonella controls factors essential for disease in response to the host environment during infection. / Thesis / Doctor of Philosophy (PhD)
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Transcriptional regulators of <i>Ehrlichia chaffeensis</i> during intracellular development and the roles of OmpA in the bacterial infection and survivalCheng, Zhihui 08 December 2008 (has links)
No description available.
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Bases moleculares de la especificidad en el mecanismo de transducción de señal en los sistemas de dos componentes bacterianosMideros Mora, Cristina 19 February 2021 (has links)
[ES] El contexto de esta Tesis se enmarca en los sistemas de dos componentes (TCS) para comprender el mecanismo de transducción de la señal. Se analizó la especificidad en el reconocimiento de los TCS abarcando estudios a nivel funcional, estructural y evolutivo. Primero se utilizó el sistema HK853-RR468, que al estar previamente caracterizado nos permitió analizar específicamente las regiones de reconocimiento (HK-RR) correspondientes a los Lß3α3 y Lß4α4 de RR468 mutando residuos que determinaran la influencia en la transferencia del grupo fosfato. Los mutantes se caracterizaron de manera bioquímica y se hicieron aproximaciones estructurales pudiendo asignar la reacción de fosfotransferencia a una estructura formada por un complejo entre HK853 y RR468 mutante. Esta estructura nos permitió observar el carácter disociativo de dicha reacción que ha sido descrito previamente y la nula participación del dominio CA. Al mismo tiempo, se analizó la influencia del pH en los residuos catalíticos de la HK y el RR (His y Asp), utilizando un rango de pH de 5 a 8. Los ensayos bioquímicos generados en este rango nos mostraron como la His catalítica perdía su carácter nucleofílico cuando el pH se acercaba y disminuía de 6. Esto se relaciona con el pKa del anillo de imidazol presente en el residuo de His, que se se encuentra en torno a 6 y la pérdida de protonación. También se cristalizó el complejo HK-RR a diferentes pHs donde observamos que la His adquiría un rotámero gauche- que se asignaba a un estado inactivo o de reposo. Por otra parte, se analizó la influencia de la mutación G63V en el RR OmpR, que fue descrita como una mutación relacionada con la resistencia al antibiótico ertapenem. Para esto se generaron mutantes en OmpR en la posición G63, tanto en el dominio REC aislado como en la proteína completa. Los estudios bioquímicos de estas mutaciones demostraron como la mutación en esta posición disminuía la capacidad del RR para fosforilarse e incluso a dimerizar. Esto afectaba a la afinidad de este RR para interaccionar con su ADN correspondiente, las cajas ompF y ompC. Estos efectos se lograron evidenciar con la estructura de OmpRRECG63V, donde se observó como la mutación generaba un cambio conformacional al reducir el tamaño del Lßα3 y generaba un bolsillo hidrofóbico donde quedaba atrapada la cadena lateral de la Val. Finalmente se analizó el aspecto evolutivo de la señalización, para lo que se buscaron organismos endosimbiontes que presentaran una HK y uno o varios RRs. Estas características nos sugerían que la menor presión selectiva nos iba a permitir encontrar organismos con TCSs menos evolucionados cuya especificidad se haya visto reducida. Se analizaron los sistemas de Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis y Methanobrevibacter sp. Abm4. Solo pudo evidenciarse reacción de fosfotransferencia en el sistema perteneciente a Methanobrevibacter, el cual presenta una HK y 4 RRs. Sin embargo, esta fosfotransferencia presentaba una eficiencia diferenciada, siendo más rápida en RRMet572 y RRMet589-1 mientras que era nula en RRMet589-2. Por su parte las HKs de C. trachomatis y S. negevensis, fueron capaces de fosfotransferir, de manera no selectiva, a RR468, probablemente debido a la alta similitud que presenta la hélice α1 de las HKs con HK853. La aproximación estructural de estos sistemas permitió obtener las estructuras de los RRsMet589-1 y RRMet572, ambos en estado no fosforilado. Las dos estructuras presentaron grandes diferencias conformacionales a partir del Lßα4. Esto sugiere que sus mecanismos de reconocimiento con HKMet y de regulación son diferentes lo que apoya la selectividad diferenciada entre los RRs de este sistema. / [CA] Esta Tesi s'emmarca en l'estudi dels sistemes de dos components (TCS) amb la finalitat d'entendre el seu mecanisme de transducció de senyal basat en l'especificitat de reconeixement a nivell funcional, estructural i evolutiu. Utilitzant el TCS HK853-RR468, analitzarem les regions Lß3α3 y Lß4α4 del regulador de la resposta (RR) RR468, que prèviament s'havien mostrat importants en el reconeixement, generant mutants i determinant la influència en la transferència del grup fosforil. Els mutants foren caracteritzats bioquímicament, observant que afectaven a una reacció específica i permetent-nos captar la reacció de fosfotransferència en una estructura formada per un complex entre HK853 i RR468 mutant. Aquesta estructura va mostrar el caràcter dissociatiu d'aquesta reacció i la nul·la participació del domini CA de la HK. Al mateix tems, s'analitzà l'efecte del pH sobre la transducció del senyal utilitzant els TCS K853-RR468 i EnvZ-OmpR. Els assajos bioquímics generats dins del rang de pH entre 5 i 8 ens mostraren com la His de la HK catalítica perdia el seu caràcter nucleofílic quan el pH s'aproximava i disminuïa de 6, valor del pKa de l'anell d'imidazole de la cadena lateral del residu d'His, indicant que aquesta disminució en l'activitat es correlacionava amb el canvi en la protonació de l'anell. Un exhaustiu estudi estructural del complex HK853-RR468 a diferents pHs mostrà que la His catalítica sempre adquiria un rotàmer gauche- independentment del valor del pH, invalidant el model que proposava que el pH regulava l'activitat de les HKs de la família HisKA induint un canvi en el rotàmer de la His catalítica. D'altra banda, s'analitzà la influència de la mutació G63V en el RR OmpR, que fou descrita com una mutació relacionada amb la resistència a l'antibiòtic ertapenem. Amb aquesta finalitat, es generaren mutants a OmpR a la posició G63 tant al domini REC aïllat com a la proteïna completa. Els estudis bioquímics demostraren com la mutació en aquesta posició disminuïa la capacitat de OmpR per fosforilar-se i per dimeritzar, afectant a la capacitat d'interaccionar amb les seqüències d'ADN palindròmiques diana, corresponents a les caixes ompF i ompC. Aquests efectes es visualitzaren a nivell molecular al resoldre l'estructura del mutant G63V d'OmpRREC, on s'observava com la mutació induïa un canvi conformacional al reduir la mida del Lßα3 generant una butxaca hidrofòbica degut a la presència de la nova Val en posició 63. Aquests canvis es transmeten a la resta de l'estructura d'OmpR produint canvis en Lßα4 i α4 que impedeixen la formació d'una superfície de dimerització competent i impedint la seua interacció amb l'ADN. Finalment, s'analitzà l'aspecte evolutiu de l'especificitat HK-RR. Buscaren organismes endosimbionts que presentaren TCS aïllats consistent en una HK i un o diversos RRs, suggerint que la menor pressió selectiva permetria trobar TCS menys evolucionats, on l'especificitat s'haguera vist reduïda. S'analitzaren HKs i RRs presents en Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis i Methanobrevibacter sp. Abm4. La reacció de fosfotransferència es va detectar en Methanobrevibacter, que presenta una sola HK i 4 RRs. Aquesta HK mostrà eficiència diferenciada per a la reacció de fosfotransferència, presentant major velocitat per als RRs RRMet572, RRMet589-1 i nul·la per a RRMet589-2. Per la seua banda, les HKs de C. trachomatis i S. negevensis, foren capaces de transferir, de manera no selectiva, a RR468 de Thermotoga maritima, probablement degut a l'alta similitud que presenta l'hèlix α1 de les HKs amb HK853. L'aproximació estructural d'aquests sistemes ens va permetre resoldre les estructures dels RRsMet589-1 i RRMet572 en estat no fosforilat. Les dues estructures presentaren grans diferències conformacionals a partir del Lßα4, els que ens suggereix que els seus mecanismes de reconeixement amb HKMet i de regulació són diferents, cosa que suporta la selectivitat diferenciada entre els RRs d’aquest sistema. / [EN] The context of the Thesis is framed in the two component systems (TCS) to understand the signal transduction mechanism. The specificity in the recognition of TCS was analyzed covering studies at the functional, structural, and evolutionary level. First, the previously characterized HK853-RR468 was used, this allowed us to analyze specific recognition regions corresponding to Lß3α3 and Lß4α4 of RR468 and induce mutations in these regions and understand the recognition between HK and RR and determine the phosphate group transfer's influence. The mutants were characterized biochemically, and structural approximations were prepared, thus assigning the phosphotransfer reaction in a formed structure by an HK8536 and a mutant RR468 complex. This structure allowed us to observe the dissociative character of this reaction that has been previously described and the null participation of the CA domain. Simultaneously, the influence of pH on the catalytic residues of HK and RR (His and Asp) was analyzed, using a pH range of 5 to 8. The biochemical assays generated in this range showed how HK's catalytic His lost the nucleophilic characteristic when pH reached six or below. This is related to the pKa of the imidazole ring present in the His residue that if found around 6 and the loss of protonation. The HK853-RR468 complex was also crystallized at different pHs where we observed that His acquired a gauche- rotamer that was assigned an inactive or resting state. In addition, the influence of mutation G63V in the RR OmpR was analyzed. This mutation was associated with resistance to the antibiotic ertapenem in E. coli. For this, mutants in OmpR were generated in position G63 in both the isolated REC domain and in the whole protein. Biochemical studies of this mutations showed how the mutation in this position reduced the capacity of RR to phosphorylate and even to form a dimer. This affected the affinity of the RR to interact with it's corresponding DNA, the boxes ompF and ompR. These effects were shown with the structure of the REC domain of the OmpR protein mutant G63V. This mutation generated a conformational change by reducing the Lßα3 and generating a hydrophobic pocket that trapped Val's lateral chain. Finally, the evolutive aspect of signaling was analyzed. For this, endosymbiotic organisms that had one HK or many RRs were identified. These characteristics suggested that lower selective pressure would allow us to find organisms with TCSs that showed lower KH-RR specificity. The systems of Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis, and Methanobrevibacter sp. Abm4 were analyzed. The phosphotransference reaction was only evident in the the Methanobrevigbacter system. This system presents only one KH and four RRs. This HK shows differentiated efficiency in the phosphotranference. It has a higher speed in RRMet572, RRMet589-1 and it is null in RRMet589-2. On the other hand, the HKs of C. trachomatis y S. negevensis were able to transfer in a nonselective manner the RR468 of T. maritima. This is due to the similarity between the α1 helix of the HKs with HK853. The structural approach of there systems allowed us to obtain the structure of RRMet589-1 y RRMet572, both in a non-phosphorylated state. The two structures presented large conformational differences from Lßα4. This suggests that the recognition mechanisms with KHMet and regulation are different. This supports differentiated selectivity between the RRs in this system. / Mideros Mora, C. (2021). Bases moleculares de la especificidad en el mecanismo de transducción de señal en los sistemas de dos componentes bacterianos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/161920 / TESIS
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Genes do metabolismo do nitrogênio e suas implicações na patogenicidade e virulência da Xanthomonas citri subsp. citri / Genes of nitrogen metabolism and its implications in the pathogenicity and virulence of Xanthomonas citri subsp. citriAmorim, Julie Anne Espíndola 27 April 2018 (has links)
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Previous issue date: 2018-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O cancro cítrico tipo A, causado pela bactéria Xanthomonas citri subsp. citri (XccA), é uma das doenças de citros mais importantes, afetando todas as cultivares comerciais, para a qual não existem ainda estratégias de controle eficientes. Os genes ntrB e ntrC codificam, respectivamente, a histidina quinase (HK) e o regulador de respostas (RR), pertencentes a um sistema de dois componentes (TCSs), que atuam no sistema regulador de nitrogênio (NTR). Porém, o possível papel desses genes na virulência da XccA e de outros fitopatógenos ainda não foi elucidado. Este estudo teve como objetivo investigar os efeitos dos genes ntrB e ntrC no desenvolvimento do cancro cítrico em limão-cravo (Citrus limonia Osbeck), bem como a possível relação desses genes com a regulação da expressão de genes do sistema de secreção tipo 3 (SST3), considerado um dos principais fatores de virulência da XccA. Os mutantes ΔntrB e ΔntrC foram obtidos pela técnica de mutagênese sítio-dirigida por reação em cadeia da polimerase de extensão por sobreposição. A mutação dos genes causou redução na sintomatologia do cancro cítrico e diminuição da população bacteriana no espaço intercelular do tecido foliar da planta. A análise das curvas de crescimento in vitro revelou que a ausência do gene ntrB não alterou a viabilidade da bactéria, enquanto a mutação do gene ntrC afetou o “fitness” bacteriano em meio de cultura NB. Análises in vitro indicaram que o mutante ΔntrC formou duas vezes mais biofilme e produziu cinco vezes mais goma xantana do que a XccA 306 in vitro. A expressão dos genes (hpa1, hrpG, hrpX, hrpE, hrpW e hrpD6) do SST3 avaliados foi significativamente maior (p < 0,05) no mutante ΔntrC do que na XccA 306 e no ΔntrB, indicando que ntrC possa atuar na regulação do SST3. Porém, o nível de expressão desses genes no mutante ΔntrB não apresentou diferença significativa (p > 0,05) em relação à XccA 306. A modelagem molecular revelou semelhança estrutural entre as regiões receptoras de NtrC e HrpG, sugerindo que a fosforilação de HrpG por NtrB possa ocorrer in vivo. Em síntese, os resultados obtidos neste estudo indicam que a mutação dos genes ntrB e ntrC afeta o desenvolvimento do cancro cítrico em limão-cravo e que o gene ntrC pode atuar na regulação dos mecanismos de formação de biofilme, produção de goma xantana e expressão de genes do SST3 e/ou que a ausência desse gene ocasione um desequilíbrio celular na XccA 306, resultando na alteração desses mecanismos, enquanto NtrB pode apresentar papel na regulação de genes do SST3 por meio da fosforilação de HrpG. / The citrus canker type A, provoked by the bacterium Xanthomonas citri subsp. citri (XccA), is one of themost important citrus diseases, affecting all the commercial cultivars, for which there are no effective control strategies. The ntrB and ntrC genes encode a histidine kinase (HK) and the response regulator (RR), respectively, belong to a two-component system (TCSs), related to the nitrogen regulatory system (NTR). However, the possible role of ntrB and ntrC genes in the virulence of XccA and other phytopathogens has not yet been elucidated. Therefore, the aim of this study was to investigate the impact of the ntrB and ntrC genes on the development of citrus canker in rangpur lime (Citrus limonia Osbeck), as well as the possible relation of ntrB and ntrC genes with the regulation of the type 3 secretion system (T3SS) gene expression, which is considered one of the main virulence factors of XccA. The ΔntrB and ΔntrC were obtained by site-directed mutagenesis through overlap extension polymerase chain reaction. The mutation of the ntrB and ntrC genes caused a reduction of the citrus canker symptoms, and decrease of the bacterial population in the intracellular space of the foliar tissue of the plant. In vitro growth curves analysis revealed that the ΔntrB did not affect the viability of the bacterium, whereas the ΔntrC affected the bacterial fitness in NB culture medium. In vitro analysis indicated that the ΔntrC formed 2x more biofilm, and produced 5x xanthan gum compared to the XccA 306. The T3SS related genes (hpa1, hrpG, hrpX, hrpE, hrpW and hrpD6) expression was significantly higher (p <0.05) in the ΔntrC than in the XccA 306 and the ΔntrB, indicating that ntrC can modulate the regulation of T3SS. However, the level of expression of these genes in the ΔntrB did not differ (p> 0.05) in relation to the XccA 306. Molecular modeling revealed structural similarity between NtrC and HrpG receptors motifs, suggesting that phosphorylation of HrpG by NtrB may occur in vivo. Overall, the results obtained in this study strongly suggest that the mutation of the ntrB and ntrC genes affect the development of rangpur lime citrus canker and that ntrC gene may play an important role in the regulation of the mechanisms of biofilm formation, xanthan gum production and T3SS gene expression and/or that the absence of this gene causes a cellular imbalance in XccA 306 resulting in the alteration of these mechanism, whereas the NtrB may have a role with the regulation of T3SS genes by phosphorylation of HrpG. / 3385/2013
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Contribution à la compréhension des mécanismes moléculaires impliqués dans la virulence de Staphylococcus lugdunensis. / Contribution to the understanding of the molecular mechanisms involved in the virulence of Staphylococcus lugdunensisDahyot, Sandrine 18 July 2019 (has links)
Nos travaux ont cherché à mieux comprendre les mécanismes à l’origine de la virulence de Staphylococcus lugdunensis, espèce au pouvoir pathogène proche de celui de Staphylococcus aureus. Nous avons procédé à la première caractérisation fonctionnelle chez cette espèce d’un système à deux composants, LytSR. Ce système s’est révélé impliqué dans le contrôle de processus métaboliques majeurs et dans la virulence. Il est en effet impliqué dans la formation de biofilm, probablement en lien avec le contrôle exercé sur la mort cellulaire. LytSR est de plus impliqué dans la pathogenèse des infections à S. lugdunensis, comme démontré dans le modèle d’infection du nématode Caenorhabditis elegans. Pour mieux caractériser et suivre la diffusion de clones prédominants chez cette espèce, nous avons dans un deuxième volet développé trois nouvelles méthodes de typage (MLVA, TRST et fbl-typing), reposant sur le polymorphisme de séquences répétées en tandem. Ces méthodes se sont révélées très discriminantes, permettant la définition de nouveaux génotypes chez cette espèce clonale. Ces outils sont à ce titre très prometteurs pour des études micro- comme macro-épidémiologiques chez S. lugdunensis, le fbl-typing apparaissant à bien des égards comme l’outil utilisable en première ligne (http://fbl-typing.univ-rouen.fr/). Enfin, nous avons montré que la variabilité de la liaison de S. lugdunensis au fibrinogène in vitro peut être en partie expliquée par des variations génétiques de fbl. / Our work has sought to better understand mechanisms involved in Staphylococcus lugdunensis virulence, a species whose pathogenicity is close to that of Staphylococcus aureus. We performed the first functional characterization of a two-component regulatory system, LytSR, in this species. This system has been shown to be involved in the control of major metabolic processes and in virulence. It is indeed involved in biofilm formation, probably in connection with the control of cell death. LytSR is furthermore implicated in the pathogenesis of S. lugdunensis infections, as demonstrated in the infection model of the nematode Caenorhabditis elegans. To better characterize and monitor the diffusion of predominant clones in this species, we have in a second part developed three new typing methods (MLVA, TRST and fbl-typing), based on the polymorphism of variable number of tandem repeats. These methods were highly discriminant, allowing the definition of new genotypes in this clonal species. These tools are very promising for micro- and macro-epidemiological studies in S. lugdunensis, fbl-typing appearing in many ways as the frontline tool (http://fbl-typing.univ-rouen.fr/). Finally, we have shown that the variability of the binding of S. lugdunensis to fibrinogen in vitro can be partly explained by some fbl genetic variations.
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Charakterisierung des Proteoms von Ralstonia eutropha H16 unter lithoautotrophen und anaeroben BedingungenKohlmann, Yvonne 18 June 2015 (has links)
Das Biopolymer-produzierende Knallgasbakterium Ralstonia eutropha H16 gilt mit seinem außergewöhnlichen Stoffwechsel als vielversprechender Produktionsstamm für die weiße Biotechnologie. Es wächst auf einer Vielzahl organischer Substrate sowie chemolithoautotroph mit H2 und CO2 als einzige Energie- bzw. Kohlenstoffquelle. Unter anaeroben Bedingungen ist es zudem zur Denitrifikation befähigt. In dieser Arbeit wurde das Proteinprofil von R. eutropha unter chemolithoautotrophen sowie anaeroben Bedingungen mittels GeLC-MS/MS untersucht. Beide Proteomstudien offenbarten, dass die Nutzung unterschiedlicher Elektronendonoren bzw. -akzeptoren mit zahlreichen Veränderungen im Proteinbestand der Zellen einherging. Hierbei waren neben Proteinen metabolischer und Transportprozesse auch jene der Zellbewegung betroffen. Die Ergebnisse stellen im Vergleich zu vorangegangenen Studien den bisher umfassendsten Überblick zum Proteinbestand beim H2-basierten sowie anaeroben Wachstum in R. eutropha dar. Von besonderer Bedeutung war dabei das Einbinden der Analyse der Membran als Ort wichtiger Energie- und Transportprozesse. Besonderes Interesse galt einem unter H2/CO2-Bedingungen abundanten Zweikomponentensystem. Sequenzvergleiche zeigten Ähnlichkeit zum Regulationssystem der Katabolitrepression des Biphenylabbaus in Acidovorax sp. KKS102. Die Deletion des Response-Regulator-Gens führte zu vielfältigen Wachstumseffekten auf Substraten wie Fructose, Glycerin sowie auf H2/CO2. Der pleiotrope Phänotyp sowie die Ergebnisse von Genexpressionsstudien und der Suche nach Regulator-Bindestellen lassen eine globale Rolle des Systems im Energie- und/oder Kohlenstoffmetabolismus von R. eutropha H16 annehmen. Histidin-Kinase und Response Regulator wurden in GloS bzw. GloR umbenannt. Die vorliegende Arbeit zeigt eindrucksvoll das Potential der Proteomik als Teil der funktionellen Genomik für den Anstoß neuer Forschungsansätze zur Evaluierung des biotechnologischen Potentials von Mikroorganismen. / Due to its remarkable metabolism the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16 is ranked as a promising production strain for white biotechnology. It grows on a wide range of organic substrates as well as lithoautotrophically on H2 and CO2 as sole energy and carbon source, respectively. Under anaerobic conditions it thrives by denitrification. This thesis focused on characterizing the protein profiles of lithoautotrophically and anaerobically grown R. eutropha cells. Proteome analyses revealed an extensive protein repertoire adapting the organism to alternative electron donors and acceptors, respectively. Changes concerned proteins involved in metabolic and transport processes as well as in cell movement. Compared to previous studies the results reported here offer the most comprehensive proteomic survey regarding the H2-based as well as anaerobic lifestyle of R. eutropha so far. In this context analyzing the cell membrane as a place for a number of energy, transport and signal transduction processes was of particular importance. Special interest aroused the identification of a two-component system upregulated on H2/CO2. Sequence analysis offered high similarity to the regulatory system for catabolite control of biphenyl degradation in Acidovorax sp. KKS102. Deletion of the response regulator gene led to versatile growth effects on substrates such as fructose and glycerol as well as H2/CO2. This pleiotrophic phenotype as well as the results of gene expression studies and the search for regulator binding sites suggests that the two-component system is a global player in energy and/or carbon metabolism in R. eutropha and possibly other bacteria. Thus, histidine kinase and response regulator have been renamed GloS/R. Since their characterization was initiated by proteomic data this study impressively elucidates the power of functional genomics in terms of revealing new research approaches to evaluate the biotechnological use of microbes.
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Searching for novel protein-protein specificities using a combined approach of sequence co-evolution and local structural equilibrationNordesjö, Olle January 2016 (has links)
Greater understanding of how we can use protein simulations and statistical characteristics of biomolecular interfaces as proxies for biological function will make manifest major advances in protein engineering. Here we show how to use calculated change in binding affinity and coevolutionary scores to predict the functional effect of mutations in the interface between a Histidine Kinase and a Response Regulator. These proteins participate in the Two-Component Regulatory system, a system for intracellular signalling found in bacteria. We find that both scores work as proxies for functional mutants and demonstrate a ~30 fold improvement in initial positive predictive value compared with choosing randomly from a sequence space of 160 000 variants in the top 20 mutants. We also demonstrate qualitative differences in the predictions of the two scores, primarily a tendency for the coevolutionary score to miss out on one class of functional mutants with enriched frequency of the amino acid threonine in one position.
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