Tesis sobre el tema "HrpA protein"

O Cancro Cítrico, um dos mais graves problemas fitossanitários da citricultura atual, é uma doença causada pelo fitopatógeno Xanthomonas axonopodis pv. citri (Xac). Um estudo funcional do genoma de Xac foi iniciado com o intuito de identificar interações proteína-proteína envolvidas em processos de patogenicidade de Xac. Através da utilização do sistema duplo-híbrido de levedura, baseado nos domínios de ligação ao DNA e ativação da transcrição do GAL4, nós analisamos os principais componentes dos mecanismos de patogenicidade de Xac, incluindo o Sistema de Secreção do Tipo III (TTSS), Sistema de Secreção do Tipo IV (TFSS) e Sistema de \"Quorum Sensing\" composto pelas proteínas Rpf. Componentes desses sistemas foram utilizados como iscas na triagem de uma biblioteca genômica de Xac. O TTSS é codificado pelos genes denominados hrp (\"hypersensitive response and pathogenicity\"), hrc (\"hrp conserved\") e hpa (\"hrp associated\") localizados no locus hrp do cromossomo de Xac. Esse sistema de secreção é capaz de translocar proteínas efetoras do citoplasma bacteriano para o interior da célula hospedeira. Nossos resultados mostraram novas interações proteínaproteína entre componentes do próprio TTSS além de associações específicas com uma proteína hipotética: 1) HrpG, um regulador de resposta de um sistema de dois componentes responsável pela expressão dos genes hrp, e XAC0095, uma proteína hipotética encontrada apenas em Xanthomonas spp; 2) HpaA, uma proteína secretada pelo TTSS, HpaB e o domínio C-terminal da HrcV; 3) HrpB1, HrpD6 e HrpW, 4) HrpB2 e HrcU e 5) interações homotrópicas envolvendo a ATPase HrcN. Em Xac, foram encontrados dois loci vir que codificam proteínas que possuem similaridade com componentes do TFSS envolvido em processos de conjugação/secreção bacteriana: TFSS-plasmídeo localizado no plasmídeo pXAC64 e TFSS-cromossomo localizado no cromossomo de Xac. O TFSS-plasmídeo, o qual possui maior similaridade com sistemas de conjugação, mostrou interações envolvendo proteínas cujos genes estão localizados na mesma região do plasmídeo pXAC64: 1) interação homotrópica da TrwA; 2) XACb0032 e XACb0033; 3) interações homotrópicas da proteína XACb0035; 4) VirB1 e VirB9; 5) XACb0042 e VirB6; 6) XACb0043 e XACb0021b. O TFSS-cromossomo apresentou interações envolvendo as proteínas: 1) VirD4 e um grupo de 12 proteínas que contém similaridade entre si, incluindo XAC2609 cujo gene encontra-se no locus vir, 2) XAC2609 e XAC2610; 3) Interações homotrópicas da VirB11; 4) XAC2622 e VirB9. A análise do sistema de \"Quorum-Sensing\" composto pelas proteínas Rpf mostrou interações envolvendo componentes do próprio sistema: 1) RpfC e RpfF; 2) RpfC e RpfG; 3) interações homotrópicas da RpfF; 4) RpfC e CmfA, uma proteína similar a Cmf de Dictyostelium discoideum que, neste organismo, é fundamental para processos de \"quorum-sensing\". As interações proteína-proteína encontradas permitiram-nos entender melhor a composição, organização e regulação dos fatores envolvidos na patogenicidade de Xac.
Citrus Canker, caused by the bacterial plant pathogen Xanthomonas axonopodis pv. citri (Xac) presents one of the most serious problems to Brazilian citriculture. We have initiated a project to identify protein-protein interactions involved in pathogenicity of Xac. Using a yeast two-hybrid system based on GAL4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators and substrates of: Type Three Secretion System (TTSS), Type Four Secretion System (TFSS) and Quorum Sensing/Rpf System. Components of these systems were used as baits to screening a random Xac genomic library. The TTSS is coded by the hrp (hypersensitive response and pathogenicity), hrc (hrp conserved) and hpa (hrp associated) genes in the chromosomal hrp locus. This secretion system can translocate efector proteins from the bacterial cytoplasm into the host cells. We have identified several previously uncharacterized interactions involving: 1) HrpG, a two-component system response regulator responsible for the expression of Xac hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp; 2) HpaA, a protein secreted by the TTSS, HpaB and the C-terminal domain HrcV; 3) HrpB1, HrpD6 and HrpW; 4) HrpB2 and HrcU; 5) Homotropic interactions were also identified for the ATPase HrcN. Xac contains two virB gene clusters, one on the chromosome and one on the pXAC64 plasmid, each of which codes for a unique and previously uncharacterized TFSS. Components of the TFSS of pXAC64, which is most similar to conjugation systems, showed interactions involving proteins coded by the same locus: 1) Homotropic interactions of TrwA; 2) XACb0032 and XACb0033; 3) XAC0035 homotropic interactions; 4) VirB1 and VirB9; 5) XACb0042 and VirB6; 6) XACb0043 and XACb0021 b. Components of the chromosomal TFSS exhibited interactions involving: 1) VirD4 and a group of 12 uncharacterized proteins with a common C-terminal domain motif, include XAC2609 whose gene resides within the vir locus; 2) XAC2609 and XAC261 O; 3) Homotropic interactions of VirB11; 4) XAC2622 and VirB9. Analysis of Quorum Sensing/Rpf System components revealed interactions between the principal Rpf proteins which control Xanthomonas quorum sensing: 1) RpfC and RpfF; 2) RpfC and RpfG; 3) RpfF homotropic interactions; 4) RpfC and CmfA, a protein that presents similarity with Cmf (conditioned medium factor) of Dictyostelium discoideum, which contrais quorum sensing in this organism. The protein-protein interactions that we have detected reveal insights into the composition, organization and regulation of these important mechanisms involved in Xanthomonas pathogenicity.

Orientador: Marcos Antonio Machado
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A bactéria Xanthomonas axonopodis pv. citri (Xac) é o agente causador do cancro cítrico, doença que leva a severas perdas econômicas devido à contaminação e à erradicação de plantas de citros. Com o seqüenciamento completo do genoma de Xac, vários genes supostamente envolvidos com a patogenicidade de Xac foram identificados. Os genes ligados à resposta de hipersensibilidade (hrp) e avirulência (avr) em geral estão relacionados à patogenicidade de Xanthomonas, entretanto, poucos estudos funcionais destes genes de Xac foram feitos. Foram construídas linhagens mutantes de Xac para a perda de função dos genes hrpF e avrXacE2 e, nas análises in vivo, foi verificado que hrpF está envolvido na patogenicidade de Xac e é essencial para a manifestação dos sintomas primários da doença. A mutação de avrXacE2 não provocou alterações na capacidade de Xac em provocar os sintomas do cancro, portanto, este gene não parece ser essencial para a patogenicidade da bactéria, podendo não estar envolvido diretamente na patogenicidade de Xac. Os genes hrpF e avrXacE2 foram clonados em vetores de expressão e foram realizados testes de indução da expressão destas proteínas em sistemas heterólogos. Somente a proteína AvrXacE2 foi expressa, purificada e submetida a teste de interação com as proteínas citoplasmáticas da linhagem mutante de Xac para o gene avrXacE2. Os testes de interações não confirmaram a identificação de proteínas com afinidade específica pela proteína recombinante AvrXacE2. A proteína HrpF não foi super-expressa em sistema heterólogo. Nas análises de proteoma comparativo da linhagem de Xac selvagem versus linhagem mutante para o gene hrpF, foram detectadas alterações na expressão de proteínas citoplasmáticas e "pericelulares". Com base nas observações pode-se supor que HrpF possa influenciar processos celulares relacionados à respostas à situações de estresse e não somente atuar na translocação de moléculas efetoras via T3SS. A partir do que foi exposto neste trabalho, sugere-se que as técnicas de estudos funcionais de genes e análises proteômicas podem conjuntamente permitir que novos mecanismos relacionados a patogenicidade de Xac sejam interpretados. Com os mutantes produzidos neste estudo, espera-se criar condições para novos ensaios funcionais visando a melhor compreensão da patogenicidade de Xac e buscar novas formas de combate ao cancro cítrico
Abstract: The bacterium Xanthomonas axonopodis pv. citri (Xac) is the causative agent of the citrus canker disease, which leads to economic losses due the contamination and erradication of citrus plants. The complete sequencing of its genome identified a number of genes supposedly involved with pathogenicity. Genes that code for hipersensitivity response (hrp) and avirulence (avr), in general, are related to the pathogenicity of Xanthomonas, however, only a few functional studies of these genes in Xac have been made. Here we report findings based on genomics and proteomics methods for Xac. Mutant strains of Xac for genes hrpF and avrXacE2 and in vivo assays demonstrated that hrpF is strongly involved in the pathogenicity of Xac and is essential for the manifestation of the primary symptoms of the citrus canker. On the other hand, the lack of avrXacE2 expression did not result in modifications in the capacity of Xac to elicite the symptoms of canker, therefore, this gene does not seem to be essential for the pathogenicity of the bacterium. The genes hrpF and avrXacE2 were cloned in expression vectors and tests of induction of the expression of these proteins in heterologous systems were carried out. The protein AvrXacE2 was expressed, purified and tested on interaction assays with cytoplasmic proteins of the mutant of Xac for the gene avrXacE2. The tests of interactions had not confirmed the identification of proteins with specific affinity for the recombinant protein AvrXacE2. The protein HrpF was not overexpressed in heterologous system. In the comparative proteome of the wild versus mutant strains for hrpF, modifications in the cytoplasmic protein expression and "pericellular" expression levels were detected. We postulate that, besides acting as a translocator of molecules through T3SS, HrpF may influence stress-related cellular responses. Thus, it is an opportune time to highlight the new and different ways in which HrpF serves Xac function. Moreover, we can assume that the techniques of functional genomics and proteomics analyses will clarify the mechanisms of pathogenicity used by Xac to cause citrus canker and, thus, enable the search for additional information to control the disease
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

Both mammalian and bacterial peroxidases contain novel covalent linkages. In the former a sulfonium linkage to a critical Met residue is thought to modify the normal planarity of the haem affecting the functional properties. Following on from the work of Metcalfe et al., 2004 which showed that such links could be engineered in ascorbate peroxidase, horseradish peroxidase (HRP) has been used as convenient model system to try and understand the structural and electronic effects of engineered covalent linkages. Previous work in the group (Cali, 2008) had shown that mutation of Ser167 to Met in HRP-C* resulted in autocatalytic cross-linking on incubation with hydrogen peroxide. In this thesis, two additional HRP variants, S167Y and S167W were studied and a new novel structural linkage discovered. The UV/Vis spectrum of the S167Y variant suggested a more 6-coordinate high spin character. The molar extinction coefficients were markedly increased, 180 mM-1 cm-1 for S167Y and 135 mM-1 cm-1 for S167W, compared to 100 mM-1 cm-1 for the WT enzyme, consistent with a more 6 coordinate high spin character normally seen in lignin peroxidase. In contrast, the dissociation constant (Kd) of the S167W variant mutant for the aromatic donor BHA was hardly affected, whilst that of the S167Y variant increased two-fold relative to the WT, implying a significant perturbation of the aromatic donor binding site and / or the associated haem-linked hydrogen bonding network. After peroxide treatment the haem group of the S167Y variant could not be extracted into acid butanone in contrast to the WT. Only a proportion of the haem could be extracted even from the untreated S167Y variant, implying that a substantial fraction of the protein had formed the haem-protein linkage during folding and purification. These results were confirmed during reverse phase HPLC and MALDI-TOF / ESI mass spectroscopy measurements. The haem and protein completely co-eluted in the case of peroxide treated S167Y, while only ~50% of the haem was linked to the protein in the untreated as isolated enzyme. The MALDI-TOF and ESI mass spectrum showed that there was a large increase (614 Da) in the mass of the linked S167Y protein, compared to that of the unlinked enzyme. Unlike the sulfonium linkage obtained earlier, treatment with hydrogen peroxide was unnecessary to observe this increase. Interestingly, the 100% unlinked S167Y protein could only be isolated if enzyme was prepared in the presence of an efficient peroxidase substrate as an antioxidant scavenger. It appears that a Tyr residue at position 167 is highly reactive with respect of the haem vinyl side chain forming a spontaneous covalent link not otherwise seen in nature. Pre steady-state comparison of the intermediates has shown that Compound I was formed essentially normally at near WT rates, however its stability was greatly affected in the S167Y variant (linked or unlinked), the life time being decreased to ~0.04 s, compared to of the WT enzyme, where it was ~80 s. The substrate preference of the cross-linked S167Y variant was also altered. Stopped-flow measurements of the individual rate constants for the partial reactions of the catalytic cycle with luminol as reducing substrate revealed an increase in the rate of reduction of Compound I to Compound II (k2). The X-ray crystal structure of S167Y variant was solved to 1.7 Å resolution and the structure has been modelled and determined by x-ray crystallography. The x-ray structure reveals an unanticipated linkage containing an additional ring structure bonded to the engineered Tyr. ESI mass measurements supported this structure.

O gene hrcA é encontrado em quase todos os ramos da árvore filogenética das eubactérias, e seu produto, a proteína HrcA, funciona como repressor da expressão dos operons de choque térmico groESL e dnaKJ, ligando-se à seqüência repetida invertida denominada CIRCE (controlling inverted repeat of chaperonin expression) presente na região regulatória destes operons. O sistema HrcA-CIRCE está, portanto, amplamente representado nas eubactérias. Particularmente, em Caulobacter crescentus, uma α-proteobactéria, este sistema está envolvido no controle da expressão do operon groESL durante o ciclo celular da bactéria. Conhecer a estrutura e as interações de HrcA é importante para entender este processo. Neste trabalho são apresentadas as análises de seqüência das HrcA\'s de C. crescentus e de Xylella fastidiosa, uma proteobactéria do grupo γ, as quais são muito similares. Este estudo levou à proposta de um modelo estrutural com a delimitação dos domínios da proteína, os dobramentos de cada domínio, com base nas interações da HrcA de C. crescentus com o elemento CIRCE e ATP, que estão sendo caracterizadas em nosso laboratório, assim como a atribuição de aminoácidos e motivos conservados funcionais. Adicionalmente, embora a expressão da HrcA recombinante de X. fastidiosa não tenha tido sucesso, a HrcA recombinante de C. crescentus purificada tem se prestado aos ensaios espectroscópicos, ainda que tenha sido detectada uma microagregação que está sendo enfrentada com um protocolo de purificação baseado no uso de α ciclodextrina. Os estudos espectroscópicos preliminares da HrcA C. crescentus dão suporte ao modelo estrutural proposto.
The hrcA gene is found in almost all branches of the filogenetic tree of eubacteria, and its product, the protein HrcA, functions as a repressor regulating the expression of the heat shock operons groESL and dnaKJ, by binding to the inverted repeat sequence called CIRCE (controlling inverted repeat of chaperonin expression). The system HrcA-CIRCE, therefore, is widely represented in eubacteria. Specifically in Caulobacter crescentus, an α-proteobacterium, this system is involved in the cell-cycle control of groESL expression (Baldini et al, 1998). Knowledge of the structure of HrcA and its interactions is important to understand this process. This work presents the analysis of the sequences of HrcA from C. crescentus and Xylella fastidiosa, a proteobacterium of the γ group, which are very similar. A structural model has been proposed, with protein domain delimitation, specific domain folding, based on known interactions of C. crescentus HrcA with the CIRCE element and ATP, obtained in our laboratory, as well as assignment of functional residues and conserved motifs. Additionally, even though no sucess was obtained the expression of recombinant HrcA from X. fastidiosa, purified recombinant HrcA from C. crescentus has been shown to be suitable for spectroscopic studies, in spite of microagregation observed, which is being faced with a purification protocol based on the use of α cyclodextrin. The preliminary spectroscopic studies of HrcA from C. crescentus support the proposed structural model.

In biosensors, printing involves the transfer of materials, proteins or cells to a substrate. It offers many capabilities thatcan be utilized in many applications, including rapid deposition and patterning of proteins or other biomolecules.However, issues such as stability when using biomaterials are very common. Using proteins, enzymes, as biomaterialink require immobilizations and modifications due to changing in the structural conformation of the enzymes, whichleads to changes in the properties of the enzyme such as enzymatic activity, during the printing procedures andrequirements such as solvent solutions. In this project, an innovative approach for the fabrication of proteinmicroparticles based on cross-linking interchange reaction is presented to increase the stability in different solvents.The idea is to decrease the contact area between the enzymes and the surrounding environment and also preventconformation changes by using protein microparticles as an immobilization technique for the enzymes. The theory isbased on using a cross-linking reagent trigging the formation of intermolecular bonds between adjacent proteinmolecules leading to assembly of protein molecules within a CaCO3 template into a microparticle structure. TheCaCO3 template is removed by changing the solution pH to 5.0, leaving behind pure highly homogenous proteinmicroparticles with a size of 2.4 ± 0.2 μm, according to SEM images, regardless of the incubation solvents. Theenzyme model used is Horse Radish Peroxidase (HRP) with Bovine Serum Albumin (BSA) and Glutaraldehyde (GL)as a cross-linking reagent. Furthermore, a comparison between the enzymatic activity of the free HRP and the BSAHRPprotein microparticles in buffer and different solvents are obtained using Michaelis-Menten Kinetics bymeasuring the absorption of the blue product produced by the enzyme-substrate interaction using a multichannelspectrophotometer with a wavelength of 355 nm. 3,3’,5,5’-tetramethylbenzidine (TMB) was used as substrate. As aresult, the free HRP show an enzymatic activity variation up to ± 50 % after the incubation in the different solventswhile the protein microparticles show much less variation which indicate a stability improvement. Moreover, printingthe microparticles require high microparticle concentration due to contact area decreasing. However, usingmicroparticles as a bioink material prevent leakage/diffusion problem that occurs when using free protein instead.

O operon groESL de C. crescentus apresenta dupla regulação. A indução deste operon por choque térmico é dependente do fator sigma de choque térmico σ32. A temperaturas fisiológicas, a expressão de groESL apresenta regulação temporal durante o ciclo celular da bactéria e o controle envolve a proteína repressora HrcA e o elemento CIRCE (controlling inverted repeat of chaperonin expression). Para estudar a atividade da proteína repressora in vitro, produzimos e purificamos de E. coli a HrcA de C. creseentus contendo uma cauda de histidinas e a ligação especifica ao elemento CIRCE foi analisada em ensaios de migração retardada em gel de poliacrilamida (EMRGP). A quantidade de DNA retardada pela ligação a HrcA aumentou significativamente na presença de GroES/GroEL, sugerindo que estas proteínas modulam a atividade de HrcA. Corroboração desta modulação foi obtida analisando fusões de transcrição da região regulatória de groESL com o gene lacZ, em células de C. crescentus produzindo diferentes quantidades de GroES/EL. HrcA contendo as substituições Pro81 AJa e Arg87Ala, aminoácidos que se localizam no domínio putativo de ligação ao DNA da proteína, mostraram ser deficientes na ligação a CIRCE, tanto in vitro como in vivo. Em adição, HrcA Ser56Ala expressa na mesma célula juntamente com a proteína selvagem produziu um fenótipo dominante-negativo, indicando que a HrcA de C. crescentus liga-se a CIRCE como um oligômero, provavelmente um dímero. As tentativas de obtenção de mutantes nulos para os genes groESL ou dnaKJ falharam, indicando que as proteínas GroES/GroEL e DnaK/DnaJ são essenciais em C. crescentus, mesmo a temperaturas normais. Foram então construídas no laboratório as linhagens mutantes condicionais SG300 e SG400 de C. crescentus, onde a expressão de groESL e de dnaKJ, respectivamente, está sob controle de um promotor induzido por xilose (PxyIX). Estas linhagens foram caracterizadas quanto á sua morfologia em condições permissivas ou restritivas, assim como quanto à capacidade de sobrevivência frente a vários tipos de estresse. As células da linhagem SG300, exauridas de GroES/GroEL, são resistentes ao choque térmico a 42°C e são capazes de adquirir alguma termotolerância. Entretanto, estas células são sensíveis aos estresses oxidativo, salino e osmótico. As células da linhagem SG400, exauridas de DnaKlJ, são sensíveis ao choque térmico, à exposição a etanol e ao congelamento, e são incapazes de adquirir termotolerância. Além disso, tanto as células exauridas de GroES/GroEL quanto as exauridas de DnaK/DnaJ apresentam problemas na sua morfologia. As células de SG300 exauridas de GroES/GroEL formam filamentos longos que possuem constrições fundas e irregulares. As células de SG400 exauridas de DnaK/DnaJ são apenas um pouco mais alongadas que as células pré-divisionais selvagens e a maioria das células não possuem septo. Estas observações indicam bloqueio da divisão celular, que deve ocorrer em diferentes estágios em cada linhagem.
In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction of the operon is dependent on the heat shock sigma factor σ-32. At physiological temperatures, groESL expression is cell cycle regulated and the control involves the repressor protein HrcA and the element CIRCE (controlling inverted repeat of chaperonin ~xpression). To study the activity of HrcA in vitro, we produced and purified from E. coli a histidine-tagged version of the protein, and specific binding to the CIRCE element was analyzed in electrophoretic mobility shift assays (EMSA). The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that these proteins modulate HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells producing different amounts of GroES/GroEL. The mutants proteins HrcA Pro81Ala and HrcA Arg87Ala, that contain amino acid substitutions in the putative DNA-bindíng domain of the protein, were found to be deficient in binding to CIRCE in vitro and in vivo. Furthermore, HrcA Ser56Ala expressed together with the wild type protein within the same cell, produced a dominant-negative phenotype, indicating that C. crescentus HrcA binds to CIRCE in an oligomeric form, most likely as a dimer. Attempts to obtain null mutants for groESL or dnaKJ were unsuccessful indicating the importance of GroES/GroEL and DnaK/lDnaJ to the survival of C. crescentus cells. Conditional mutants were then constructed in our laboratory in which groESL and dnaKJ expression is under the control ofaxylose inducible promoter (PxyIX) , giving rise to strains SG300 and SG400, respectively. These strains were characterized in regard to their morphology under permissive and restrictive conditions, as well as their viability under different types of environmental stresses. SG300 cells depleted of GroES/GroEL are resistant to heat shock at 42°C and can acquire some thermotolerance, but they are sensitive to oxidative, saline and osmotic stresses. SG400 cells depleted of DnaKlJ are quite sensitive to heat shock, ethanol and freezing, and are unable of acquiring thermotolerance. Cells depleted of either GroES/EL or DnaKlJ also present morphological problems. SG300 cells depleted of GroES/EL form long and pinched filaments. SG400 cells depleted of DnaKlJ are only somewhat more elongated than wild-type predivisional cells and most cells do not present septum. These observations indicate a cell division arrest, which should occur at different stages in each strain.

碩士
國立中興大學
生物科技學研究所
101
Acidovorax avenae subsp. avenae (Aaa) CH12 causes bacterial leaf stripe disease on corn. The bacterial proteins secreted via type III secretion system (T3SS) which is encoded by a hrp/hrc cluster are the major virulence factors to cause diseases in host plants or elicit the hypersensitive response (HR) on nonhost plants. The genes residing the region between hrcT and GALA genes in hrp/hrc cluster of A. avenae isolated from different host are variable. To elucidate whether the diversity is involved in virulence or not, the two ORFs annotated in this region from AaaCH12 were characterized in this study. These two ORFs were named as hrpY and hrpW based on their amino acid sequence shared 99% and 98% identities with those from AaaN1141, respectively. Moreover, HrpY and HrpW from AaaN1141 strain were predicted to be harpin proteins, which are glycine rich and thermal stable, could be an HR elicitor, have no N-terminal signal peptide and secreted via T3SS. In this study, HrpY and HrpW proteins from AaaCH12 are glycine rich and have no N-terminal signal peptide. The crude extracted or purified HrpY-His6 and HrpW-His6 proteins which were overexpressed by T7 RNA polymerase system could elicit the HR on Nicotiana tabacum with or without heat treatment, suggesting that HrpY-His6 and HrpW-His6 proteins are thermal stable. A pectate lyase domain residing in C-terminus of HrpW from AaaCH12 has pectate lyase activity based on the pectate lyase assay using polyglacturonic acid as a substrate. Gel filtration assay showed both purified HrpY-His6 and HrpW-His6 could be detected as about 2000 kDa in size. HrpY-His6 protein was applied to raise a polyclonal antibody in rabbit for Western blot analysis. Under T3SS inducing condition by cultured in modified XVM2 medium, both HrpY and HrpW could be secreted in wild type but not in T3SS-deficient hrcV mutant of AaaCH12, suggesting that HrpY and HrpW are secreted via T3SS. The results strongly suggest that HrpY and HrpW are harpin-like proteins. In addition, hrpY, hrpW and hrpYW deletion mutants were generated by using unmarked gene deletion mutagenesis. The hrpY, hrpW and hrpYW mutants elicited the delay HR on N. tabacum. The hrpY and hrpYW deletion mutants also reduced the virulence of Aaa CH12 by decreasing disease lesion length and bacterial growth in corn leaves, but the hrpW deletion mutant did not. Additionally, the hrpYW double mutant is significantly less virulent than hrpY, suggesting that HrpYW proteins have an additive effect in virulence on corns.

The market globalisation and the climate change are contributing substantially to the possible and rapid spread of alien and invasive plant pathogens in areas where they were previously absent, or are intensifying the incidence and severity of endemic pathogens, thus contributing significantly to increase the possible threats to the agricultural sector. Moreover, the lack of effective alternative molecules to copper compounds in plant protection, whose negative effects on both human health and environmental protection, have been neglected for far too long, and the need to adapt to European legislation, have led the operators in plant protection sector to reflect about the urgent need to implement a cultural revolution in which major innovation efforts are required. The study was carried out in order to achieve the following main aims: I) analysing pathogenic and virulence systems of phytopathogenic Gram-negative bacteria such as the Type Three Secretion System (TTSS), and in particular the main structural protein of TTSS pilus, i.e. “HrpA”, in order to design molecules able to block the pathogenicity and virulence of these bacteria without undermining their viability; II) verifying the in vitro and in vivo efficacy of anti-infective molecules, such as small oligopeptides and polyphenolic extracts obtained in a circular economy framework, to reduce or to block symptoms development caused by plant pathogenic bacteria; finally, as a future objective to analyse a possible correlation among virulence systems, fitness and efflux pumps related to xenobiotic compounds extrusion in phytopathogenic bacteria, in order to identify underdeveloped targets, against which innovative molecules can be designed.

碩士
國立中興大學
生物科技學研究所
96
Bacterial fruit blotch (including watermelon, muskmelon and bitter gourd) is a bacterial plant disease caused by Acidovorax avenae subsp. citrulli (Aac) . HrpW protein in Pseudomonas syringae pv. tomato is secreted by type III secretion system (TTSS). In previous study, a hrpW gene was cloned from Aac156 isolated from bitter gourd. The feature of this gene product similar to the HrpW in Pseudomonas syringae pv. tomato is a harpin-like protein with the amino sequence consisting of the harpin domain in N-termini and the pectate lyase domain in C-termini, and can induce hypersensitive response on nonhost leaves. However, hrpW gene doesn’t exist in every strain of A. avenae subsp. citrulli. HrcV is a component of TTSS. In plant pathogenic bacteria ,the TTSS and its secreting proteins are the major virulence factors. So the expression of the TTSS in the medium will benefit for the studies on these proteins and their pathogenic mechanism. The gene composition encoding TTSS in Aac is similar to those in Xanthomonas campestris and Ralstonia solanacearum, but the XVM2 medium which was commonly used for inducing the TTSS in Xanthomonas campestris was not suitable for inducing the TTSS in Aac. In previous study, it was found that the XVM2 medium containing 1mM Tween80 could activate the TTSS promoter of Aac by using a plasmid carrying a TTSS promoter from Aac148 fused to luxA-luxB reporter gene. In this study, the distribution and homology of hrpW in Aac was conferred by Southern blotting with probes prepared from the two domains of hrpW. HrpW protein was overexpressed and was applied to raise polyclonal antibody in rabbit. Based on western blot analysis with HrpW antibody, expression of HrpW in Aac 156 was observed in XVM2 medium and XVM2 medium containing 1mM Tween80 and the protein was detected in supernatant fraction. By construction of hrcV mutant, the secretion of proteins can be investigated whether it is TTSS dependent.

碩士
國立中山大學
生物科學系研究所
94
HarpinPss, a proteinaceous elicitor from Pseudomonas syringae pv. syringae, is a glycine-rich, cysteine-lacking, heat-stable protein. It can elicit the hypersensitive response (HR) when delivered to the surface of plant cells. HRAP (hypersensitive response assisting protein) is an amphipathic protein purified from sweet pepper and could intensify harpinPss–mediated HR in sweet pepper. In the previous research, harpinPss was present as monomer, dimer, trimer, tetramer, and ocatamer forms in neutral pH buffer. Only monomer and dimer forms of harpinPss induced hypersensitive response in nonhost plants. HRAP could cause multimeric forms of harpinPss dissociation into monomer forms. The interaction between HRAP and harpinPss is an important issue. HRAP contained three positively charged regions, a typical signal peptide and a cAMP-dependent phosphorylation site. In this study, these regions of HRAP would be truncated and identified whether these truncated HRAP fragments could promote harpinPss dissociation. Different combinations of truncated HRAP and harpinPss were used to identify the protein-interaction regions between two proteins. HarpinPss triggers HR via interaction with cAMP phosphorated region of HRAP and MAPK pathway transduction. When cAMP region of HRAP was truncated, harpinPss still triggers HR via polymerization and anchor on lipid bilayers to form an ion-conducting pore.

碩士
國立中興大學
生物化學研究所
102
The study contains two parts: the first part is determing the structure of protein XccFhrR, which regulates causative hrp genes, in Xanthomonas campestris pv. campestris (Xcc). FhrR transcription factor belongs to the TetR family, and regulates the expression of genes that encode flagella, Hrp and ribosomal proteins. We have obtained the native crystals of XccFhrR that diffracted to a resolution of 2.9 A. We tried to get the phase by the molecular replacement approach using the most similar protein Sco0520, but were not able to successfully extract the phase information. To solve the problem, we have prepared the Se-Met labeled protein. We have obtained preliminary Se-Met labeled crystals, but they are too small for X-ray diffrection. More efforts are required to grow bigger crystals. The second part is to study the complex structure of the dsRNA receptor protein DDX1 and DDX21 in the mammalian innate immune system. The full-lenth DDX1 and DDX21 are very difficult to obtain in soluble form, and we finally found the DDX11-440 and DDX211-571 fragements are very soluble. We have tried to obtain their co-crystal by using a variety of crystallization conditions, yet no crystal could be obtained so far.

碩士
國立中興大學
農業生物科技學研究所
90
Protein secretion is required for numerous aspects of the bacterial life cycle, including organelle biogenesis, nutrient acquistion, and virulence-factor expression. Plant pathogenic bacteria translocate proteins into plant cells, which cause diseases on the host plants and elicit the hypersensitive response (HR) in nonhost plants, by type III secretion (TTS) system. In Pseudomonas syringae pv. syringae 61(Pss61), a 25 kb hrp/hrc gene cluster encodes a TTS system. The aim of this study is to understand the interactions among Hrp/Hrc proteins of the Pss61 TTS apparatus. In yeast two-hybrid system, hrcN or hrpE gene constructed either in pGADT7 or pGBKT7 resulted in high levels of reporter gene activation, and HrpE could interact to itself. For convenience to detect target protein, a FLAG peptide was fused to the C-terminus of HrcN, HrpE, or HrcU proteins. Also, rabbit antisera against HrcN and HrpE were generated. In order to confirm the interactions between HrcN and HrpE, the protein affinity blotting was performed using HrcN and HrpE as probes. The results suggest that HrcN specifically recognizes and binds to HrpE. In addition, we performed protein affinity blotting using FLAG-tagged HrcU as a probe, which can be detected by the commercial monoclonal antibody M2. These data showed that HrcU specifically recognizes and binds to HrpE and HrcN. In column binding experiments, either HrcN-FLAG or HrcU-FLAG could be copurified with HrpE-His. Besides, base on sequence analysis, HrpE exhibits amino acid similarity with subunit b of ATPase and FliH of flagellar. It suggested that HrpE may form homodimer similar to its homologs subunit b of ATPase and FliH of flagellar, and may play an important role to regulate ATPase activity of HrcN and protect HrcN. HrcU seems to play a dual role: a direct one in secretion and a probable indirect one on transcription. According to these results and previous data, we propose that the cytoplasmic HrcN and HrpE together form a (HrpE)2HrcN ATPase-like complex and then bind to HrcU, an inner membrane protein of the TTS apparatus, to energize secretion or to provide the energy for assembly of the secretion apparatus.

碩士
國立中興大學
分子生物學研究所
90
Heat shock protein HrcA acts as a repressor interacting with the CIRCE element at the transcriptional level in the majority of microorganisms which do not include E. coli. The hrcA in Xanthomonas campestris pv. campestris 17 ( Xc17 ) has been cloned and sequenced. To understand the interaction between HrcA and CIRCE elements, the Xc17 hrcA was cloned into pET-21a vector for over-expression in E. coli. Since the over-expressed HrcA protein was present as insoluble form, dialysis was performed to renature the HrcA. The DNA fragments carrying the CIRCE elements upstream of groES of X. campestris pv. phaseoli and A. tumefaciens were amplified by PCR. However, when the PCR fragments were incubated with the His-bind purified HrcA for gel mobility shift assays, no interaction was observed. To analyze the effects of HrcA on CIRCE element in vivo, the PCR fragments were cloned in the promoter-proving vector pFY13-9, which use the promoter-less lacZ as the reporter, and transformed into the wild-type Xc17 and hrcA mutant. The levels of b-galactosidase in hrcA mutant were higher than that in Xc17, indicating that the expression is repressed by HrcA. After heat shock treatments, the promoter activity increased by 30%, implicating that HrcA interacts with CIRCE elements in vivo, although the effect is not strong. It was previously found that following heat shock treatments, Xc17 de novo synthesizes a protein of about 18 kDa. N-terminal sequencing data suggested this protein to be a Hsp20 homologue of bacteria. In this study, the Xc17 hsp20 gene was cloned and sequenced. The results indicated that it is 477-bp long able to code for a polypeptide of 158 amino acids with a calculated molecular weight of 17.8 kDa. The deduced amino acid sequence has 87.8 % similarity to the low molecular weight heat shock protein in Xylella fastidiosa. Primer extension analysis showed that the transcription start site is located 64 bp upstream of the hsp20 start codon. Consensus —10 and —35 sequences similar to that recognized by s32 in many other bacteria are present upstream of the transcription start site. Presence of promoter activity at the hsp20 upstream region was demonstrated by transcriptional fusion assay. In addition, the promoter activity was found to increase by 5.8 folds after heat shock. An open reading frame homologous to peroxidase gene which transcribed divergently is present upstream of the Xc17 hsp20.

碩士
國立中興大學
生物化學研究所
98
Xanthomonas campestris pv. campestris (Xcc) is a gram-negative plant- pathogenic bacterium that hosts mainly in crucifers. When infected by this bacterium, hosts show syndrome of marginal leaf chlorosis, or rotten leafs. In Xcc, the hrp (hypersensitive reaction and pathogenicity) gene cluster are involved in the pathogenicity of host plants and induction of HRs in nonhost plant. When Xcc infects plants, its hrp gene cluster are activated. The structure and function associated with some of the hrp gene products were studied. In Xcc, HrpG is a key master regulator necessary for activating other hrp genes. It cpmprises 266 amino acids and belongs to a response regulator. The full-length HrpG was found to precipitate after cell lysis. To solve this problem, a hrpG (1-201) were cloned and purified successfully. A variety of crystallization condition were screened to obtain a crystal. However, the quality of the crystal was not good enough to obtain good X-ray diffraction data. The crystallization condition is being fine tuned. hrpB, the largest operon in the Xcc, could encode eight proteins (HrpB1-8), seven of which were soluble when expressed in E.coli. The HrpB5 were purified and the crystallization condition were screened for X-ray diffraction. A crystal was obtained and diffracted to a resolution of 2.5 Å. To solve the phase problem, its Se-Met labeled derivative is being preparing. XAC3683 is a histidine kinase and was found to interact with HrpG in the XAC from a prior yeast two-hybrid experiment. Based on sequence comparison, XC3070 is a XAC3683 homologue in Xcc. Three different clones : the full-length, the N-terminus deleted XC3070 (115-630) and XC3070 (250-630) were constructed. The soluble form of XC3070 (115-630) was obtained. After purification, a crystal was obtained via in situ proteolysis. The Gel filtration, biacore or ITC experiments to determine the binding strength between XC3070 and XcHrpG will be tested.

碩士
國立中興大學
生物科技學研究所
98
Acidovorax avenae subsp. cattleyae (Aaca) OAC1 causes a brown spot disease on Cattleyae, while A. a. subsp. citrulli (Aac) causes Bacterial fruit blotch disease (BFB) on cucurbitaceae. These bacteria causing diseases in host plants or eliciting hypersensitive response (HR) on nonhost plants depend on type III secretion system (T3SS), encoded by hrp/hrc gene cluster. In this study, we analyzed the biological function of hrpWAaca of Aaca by loss-of-function strategy. The hrpWAaca mutant elicited a delayed HR on Nicotiana tabacum, but still maintained the same virulence as its wild type on its host Oncidium and Phalaenopsis. Hence, HrpWAaca might be not play an essential role in pathogenicity. The purified or crude extracted HrpWAaca-His6 proteins could elicit HR with or without heat treatment. Moreover, HrpWAaca-His6 protein contains 17.58% glycine but no cysteine, and HrpWAaca could be secreted into bacterial milieu under T3SS inducing condition. Therefore, HrpWAaca is a harpin-like protein. The Pel domain in C-terminus of HrpWAaca has the pectate lyase activity based on the pel assay with polyglacturonic acid as a substrate. Besides, Gel filtration assay showed the purified HrpWAaca-His6 could be detected as dimer (120 kDa)、hexamer (400 kDa) and oligomer (>2000 kDa) in size. To control the tomato bacterial spot caused by Xanthomonas vesicatoria, HrpWAaca-His6 solution was sprayed onto tomato leaves 1 or 4 days before inoculated with X. vesicatoria. The result showed that HrpWAaca-His6 could reduce bacterial spot severity after 4 days harpin treatment. To improve the T3SS induction medium for Aac, extracts of watermelon suspension cell and loofah were total using gfp as a reporter gene under the control of hrcC promoter. The results indicated that both extracts significantly improved induction efficiency of the XVM2 medium.

Neisseria meningitidis, Auslöser der Meningokokken-Meningitis und Sepsis, trägt auch heute noch zur hohen Kindersterblichkeit in Entwicklungsländern bei und sorgt, vor allem im afrikanischen Meningitis-Gürtel, immer wieder für Epidemien mit gravierenden Folgen für die Betroffenen. Im Rahmen dieser Arbeit wurden zwei an der Pathogenität von N. meningitidis beteiligte Proteine, der Transkriptionsregulator FarR und der Transportkanal HrpB, näher charakterisiert, um weitere Einblicke in die immer noch nicht vollständig entschlüsselte Pathogenese der Meningokokken-Meningitis zu erhalten. Das Neisseria adhesin A NadA ist Bestandteil der sich aktuell in der Entwicklung befindenden Impfung gegen Meningokokken der Serogruppe B. Im dem bekapselten B-Stamm MC58 wurde gezeigt, dass nadA unter der negativen Kontrolle des Transkriptionsregulators FarR steht (Schielke et al., 2009). In den ebenfalls zur Gattung Neisseria gehörenden Neisseria gonorrhoeae (Ng) wurde bereits 2001 ein FarR-Homolog beschrieben (Shafer et al., 2001). NgFarR ist an der Resistenz gegenüber antimikrobiellen, langkettigen Fettsäuren beteiligt, indem es die Expression des FarABEffluxpumpen-Systems reguliert, welches eingedrungene Fettsäuren wieder nach extrazellulär befördert. Dagegen zeigten Palmitinsäure-Resistenztests, dass FarR nicht an der intrinsischen Fettsäure-Resistenz der Meningokokken beteiligt ist. Die Deletion und die Komplementierung von farR hatten weder in bekapselten noch in unbekapselten Meningokokken Einfluss auf das normale Wachstumsverhalten. Ein Western Blot- Nachweis des FarR-Proteins in der frühen, mittleren und späten exponentiellen Wachstumsphase von Wildtyp, Kapsel-Deletionsmutante und farR-Komplementante zeigte, dass die Menge an FarR im zeitlichen Verlauf kontinuierlich zunimmt und FarR damit Wachstumsphasen-abhängig exprimiert wird. Dabei scheint es einer posttranskriptionalen oder posttranslationalen Regulation zu unterliegen, da auch in dem farRkomplementierten Stamm unabhängig vom farR-Promotor eine entsprechende Hochregulation stattfindet. In Infektionsversuchen wurde die Interaktion zwischen Meningokokken und humanen polymorphkernigen Granulozyten untersucht. In den Infektionsassays wurde die farRDeletionsmutante innerhalb des dreistündigen Versuchsrahmens deutlich stärker durch die Granulozyten abgetötet als der Serogruppe B-Wildtyp. Als Mitglied der in Bakterien und Archaeen weit verbreiteten Familie der MarR-Transkriptionsregulatoren (Multiple antibiotic resistance Regulator, MarR) bindet FarR mit hoher Wahrscheinlichkeit auch als Homodimer an seine Bindesequenz auf der DNA. FarR erkennt eine 16 bp lange, palindromische Sequenz in der Promotorregion von nadA (NMB1994), wodurch die nadA-Expression verhindert wird. Außerdem erkennt FarR eine ähnliche Bindesequenz im Promotorbereich von farAB (NMB0318/0319), wobei es aber keinen regulatorischen Einfluss ausübt. Mit einer aus diesen beiden Bindestellen berechneten minimalen Bindesequenz wurde im Genom von MC58 weitere mögliche Bindepartner detektiert. Eine Auswahl dieser möglichen Bindestellen wurde in Electrophoretic Mobility Shift Assays auf eine direkte Interaktion mit dem FarR-Protein hin untersucht, wobei sich allerdings keine direkte Bindung nachweisen ließ. Diese Ergebnisse darauf hin, dass der Transkriptionsregulator FarR hoch spezifisch bestimmte DNA-Bindesequenzen erkennt und die entsprechenden Gene reguliert. In der Promotorregion des TpsB-Proteins HrpB wurde in den sequenzierten Referenzstämmen Z2491, MC58, FAM18 und α14 eine mit der minimalen FarR-Bindesequenz kompatible Sequenz gefunden. In Electrophoretic Mobility Shift Assays konnte allerdings gezeigt werden, dass FarR nicht direkt daran bindet. Um das Transport-Protein HrpB näher zu charakterisieren, wurde das entsprechende Gen in 22 N. meningitidis-Isolaten sequenziert. Dabei zeigte sich, dass das Transportprotein hrpB in allen untersuchten invasiven und nicht-invasiven Stämmen vorhanden ist. Dieses äußerst konservierte Protein weist nur im seinem C-terminalen Bereich eine relativ variable Region auf, was vermutlich auf Rekombinationsereignisse zurückzuführen ist. Ein Alignment der Aminosäure-Sequenz des Serogruppe C-Stamms FAM18 mit der des homologen Bordetella pertussis TpsB-Proteins FhaC zeigte, dass die dreidimensionale Struktur des HrpB ebenfalls eine α-Helix, eine transmembranöse Domäne und variable extrazelluläre Loops enthält. Zusammengenommen erfüllt HrpB somit wichtige Bedingungen, um als Vakzine-Bestandteil in Betracht gezogen zu werden
Function of the transcriptional regulator FarR in Neisseria meningitidis