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Garin, Tiffany. "Impact des compétitions inter-microbiennes médiées par le Système de Sécrétion de Type VI (T6SS) sur la dynamique de transmission du microbiote des graines aux plantules". Electronic Thesis or Diss., Angers, 2024. https://dune.univ-angers.fr/documents/dune18434.
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Les graines constituent la source d’inoculum initiale pour le microbiote des plantes. Lors de la germination, le relargage d’exsudats à proximité des graines déclenche une intense compétition microbienne qui joue un rôle moteur dans l’assemblage du microbiote des plantes. Ce travail de thèse vise à explorer le rôle de la compétition d'interférence médiée par le système de sécrétion de type VI (T6SS). Le T6SS d’une souche de Stenotrophomonas rhizophila, une espèce bactérienne transmise efficacement aux plantules, a été utilisé comme modèle d’étude. Ce T6SS est impliqué dans l’inhibition de la croissance de nombreuses espèces bactériennes in vitro, dont la souche phytopathogène Xanthomonas campestris pv. campestris 8004 (Xcc8004). Cette inhibition de croissance bactérienne est corrélée à la proximité phylogénétique et métabolique entre S. rhizophilaet les espèces bactériennes testées. L’activité antimicrobienne du T6SS de S. rhizophila limite la transmission de Xcc8004 de la graine de radis à la plantule en conditions gnotobiotiques. Cette activité module également la dynamique d’assemblage des communautés bactériennes lors de la transition graine-plantule en sol non stérile. La composition du consortium microbien co-inoculé sur graine influence l’avantage compétitif conféré par le T6SS à S. rhizophila. La compétition d’interférence médiée par le T6SS intervient donc bien dans la dynamique d’assemblage du microbiote des plantes et l'inoculation de consortiums bactériens composés de bactéries porteuses de T6SS apparait comme une solution potentielle de réduction de la transmission des agents phytopathogènes aux plantulesSeeds represent the initial inoculum source for plant microbiota. During germination, the release of exudates near the seeds triggers intense microbial competitions, which play a pivotal role in the assembly of the plant microbiota. This thesis work aims to explore the role of interference competition mediated by the Type VI secretion system (T6SS). The T6SS of a strain of Stenotrophomonas rhizophila, a bacterium efficiently transmitted to seedlings, was used as a study model. This T6SS inhibits the growth of many bacterial species in vitro, including the phytopathogenic strain Xanthomonas campestris pv. campestris 8004 (Xcc8004). This inhibition of bacterial growth is correlated with the phylogenetic and metabolic proximity between S. rhizophila and the tested bacterial species. The antimicrobial activity of S. rhizophila T6SS limits the transmission of Xcc8004 from radish seed to seedling under gnotobiotic conditions. Its activity also influences the dynamics of bacterial community assembly during the seed-to-seedling transition in non sterile soil. The composition of the co-inoculated microbial consortium on the seed affects the competitive advantage conferred by the T6SS to S. rhizophila. Therefore, T6SS-mediated interference competition plays a significant role in the assembly dynamics of the plant microbiota, and the inoculation of bacterial consortia composed of T6SS-carrying bacteria appears as a potential solution for reducing the transmission of phytopathogenic agents to seedlings
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DE, CRECY-LAGARD VALERIE. "Transport et metabolisme du fructose chez xanthomonas campestris pv. Campestris". Paris 7, 1991. http://www.theses.fr/1991PA077025.
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Xanthomonas campestris pv. Campestris est une bacterie phytopathogene qui secrete un polysaccharide, le xanthane. Le but de ce travail etait l'etude de la regulation du transport des sourcs de carbone chez x. Campestris. Les regulations trouvees chez e. Coli etaient-elles conservees ou existait-il d'autres mecanismes de regulation dans cette bacterie? chez e. Coli, deux systemes de regulation du transport des sources de carbone ont ete etudies en detail: le controle transcriptionnel par l'amp-cyclique associe a sa proteine receptrice cap; la regulation par le systeme des phosphotransferases dependantes du phosphoenolpyruvate (pts). Une proteine de x. Campestris similaire a la proteine cap d'e. Coli a ete isolee. Cette proteine cap, a la difference de celle d'e. Coli, ne regule pas chez x. Campestris l'expression des operons cataboliques. Nous avons montre que cette proteine est impliquee dans la regulation des facteurs de phytopathogenicite. Le systeme pts de x. Campestris a ete caracterise. Ce pts est specifique du fructose. Des mutants pts ont ete isoles, les genes du pts fructose clones et sequences. L'operon fructose de x. Campestris contient au moins trois genes: le premier, frub, code pour une proteine multifonctionnelle portant les trois activites eiii, pseudo-hpr et ei; le deuxieme, fruk, code pour la 1-phosphofructokinase et le troisieme, frua, code pour l'eii#f#r#u. Bien qu'il soit specifique du glucose, le pts de x. Campestris joue comme le pts plus general d'e. Coli un role essentiel dans l'utilisation des sources de carbone
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Drouin, François. "Caractérisation de l'intégron de Xanthomonas campestris pv badrii". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0019/MQ56399.pdf.
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Monteiro, Leila. "Produção de Substaâncias biotivas de Bacillus spp. contra Xanthomonas campestris pv. campestris". Universidade Federal de Pernambuco, 2002. https://repositorio.ufpe.br/handle/123456789/1304.
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Previous issue date: 2002O gênero Bacillus é um dos mais utilizados no biocontrole de doenças de plantas. São microrganismos encontrados facilmente em solos e plantas, que formam esporos tolerantes ao calor e à dessecação, o que facilita sua comercialização e estoque. O principal mecanismo de ação desses organismos no controle de fitopatógenos é a produção de substâncias antimicrobianas, entre as quais os lipopeptídeos, que apresentam também atividade hemolítica. Dentre os fitopatógenos que sofrem inibição por Bacillus spp., está a bactéria Xanthomonas campestris pv. campestris, causadora da podridão negra das crucíferas, doença de abrangência mundial, responsável por grandes perdas nas plantações de couve, repolho, nabo, rabanete, entre outras. Pesquisas anteriores mostraram que Bacillus spp. epifíticos, isolados de rabanete e couve, apresentam alta eficiência no controle da podridão negra em couve e repolho no campo. O presente estudo teve como objetivos a investigação de mecanismo de antibiose de oito isolados de Bacillus: B. subtilis R14, B. megaterium pv. cerealis RAB7, B. megaterium pv. cerealis C211, B. megaterium C116, Bacillus sp. RAB9, B. cereus C240, Bacillus sp. C11 e B. cereus C210, contra nove linhagens de X. campestris pv. campestris e a participação de lipopeptídeos neste mecanismo. Além disso, para os Bacillus que apresentaram resultados positivos de antibiose, foram realizados estudos de fermentação para acompanhar o crescimento e a produção de substâncias bioativas e tensoativas. Para o estudo de antibiose, foram realizados testes de atividade dos oito isolados contra as linhagens fitopatógenas de X. campestris pv. campestris, pelo método de difusão em ágar. Para verificar a produção de lipopeptídeos pelos Bacillus, foram realizados testes de hemólise em meio ágar sangue a 30o C e a 37o C. As fermentações foram realizadas em frascos de Fernbach, contendo 500 mL de meio de cultura a base de glicose e (NH4)2SO4, em mesa agitadora, a 150 rpm e 30°C. Os testes de atividade antimicrobiana se apresentaram positivos para todas as linhagens de X. campestris pv. campestris frente a quatro dos isolados de Bacillus testados: B. subtilis R14, B. megaterium pv. cerealis RAB7, B. megaterium C116 e B. cereus C210, os quais foram também os que apresentaram halos de hemólise, principalmente a 37o C. Os isolados, B. subtilis R14 e B. megaterium pv. cerealis RAB7 se mostraram os mais eficientes no antagonismo contra as linhagens de X. campestris pv. campestris. A correlação observada entre a atividade antimicrobiana e a atividade hemolítica indica que lipopeptídeos estão envolvidos no mecanismo de antibiose dos isolados investigados. Nos estudos de fermentação, observou-se a produção de substâncias bioativas e surfactantes durante a fase de crescimento de B. subtilis R14, B. megaterium pv. cerealis RAB7 e B. megaterium C116. Assim como nos testes em meio sólido, maiores atividades antimicrobianas foram observadas nos cultivos de B. subtilis R14 e B. megaterium pv. cerealis RAB7
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Silva, Rafael Salomão da. "Bactérias de solos supressivos com atividade antimicrobiana sobre Xanthomonas campestris pv. campestris". Universidade Federal de Sergipe, 2016. https://ri.ufs.br/handle/riufs/3021.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESXanthomonas campestris pv. campestris is a phytopathogenic bacterium, the causative agent of black rot in crucifers. For the control of plant pathogens diseases, there is the use of bacteria with activity antagonistic to the pathogen. Recent studies show that Bacillus species have on X. campestris a strong biological control. One of the mechanisms of this control is the production of secondary metabolites by these species. The objective of this work was to select bacteria X. campestris and antagonists to evaluate the antimicrobial activity of extracellular filtered bacteria (FEB) antagonist activity. To this, 257 bacteria isolated from a suppressive soil. They were evaluated in vitro antagonist activity by the technique of double layer. Ninety-two isolates (44.6%) were able to inhibit growth of the target pathogen (X.campestris). Of the 92 isolates selected on double layer of the test, 51 (55.43%) showed inhibition of growth of X. campestris on the inhibition assays with FEB in liquid medium. Thirteen of 50% or more inhibited the growth of the target pathogen, and the FEB-8, FEB-31-FEB 68, FEB 74-FEB-87 and were able to inhibit 100% growth of X. campestris. The FEB isolated TC-DT08, belonging to the genus Paenibacillus, it was used for in vivo tests in plant farming kale. The artificial inoculation kale with X. campestris pretreated with FEB-08 showed that the bacterium loses the ability to colonize and cause the cabbage black rot, indicating the potential use of this isolate to protect kale butter infection by X. campestris.
Xanthomonas Campestris. pv campestris é uma bactéria fitopatogênica, agente causal da podridão negra em crucíferas. Dentre os mecanismos para o controle de doenças de fitopatógenos, destaca-se o uso de bactérias com atividade antagonista ao patógeno. Estudos recentes mostram que espécies de Bacillus exercem sobre X. Campestris um forte controle biológico. Um dos mecanismos deste controle é a produção de metabólitos secundários por essas espécies. O objetivo deste trabalho foi selecionar bactérias antagonistas a X. campestris e avaliar a atividade antimicrobiana dos filtrados extracelulares das bactérias (FEB) com atividade antagonista. Para isso, 257 bactérias isoladas de solos supressivos foram avaliadas quanto a atividade antagonista in vitro pela técnica da dupla camada. Noventa e dois isolados (44,6%) foram capazes de inibir o crescimento do fitopatógeno alvo (X.campestris). Dentre os 92 isolados selecionados no teste da dupla-camada, 51 (55,43%) apresentaram inibição do crescimento da X. campestris nos ensaios de inibição com os FEB em meio líquido. Treze destes inibiram 50% ou mais do crescimento do fitopatógeno-alvo, sendo que os FEB-08, FEB-31, FEB-68, FEB-74 e FEB-87 foram capazes de inibir 100% do crescimento de Xanthomonas campestris. O FEB do isolado TC-DT08, pertencente ao gênero Paenibacillus, foi utilizado para testes in vivo em plantas de couve-manteiga, em condições de casa de vegetação. A inoculação artificial de couve-manteiga com X. campestris pré-tratada com o FEB-8 demonstrou que a bactéria perde a habilidade de colonizar a couve e causar a podridão negra, o que indica o potencial do uso deste isolado para proteger a couve-manteiga da infecção por X. campestris.
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Sanders, Gina Mercia. "Detection of Xanthomonas Campestris PV. magniferaeindicae in mango plants". Diss., University of Pretoria, 1993. http://hdl.handle.net/2263/39792.
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The yearly losses incurred by bacterial blackspot disease are high. Often trees are
asymptomatic, with the pathogen either in the resident phase or latent stage of infection.
Detection of the pathogen in these asymptomatic trees is one of the most important
means of controlling the disease. Isolates which consistently differed in virulence were
isolated from symptomatic mango plants. These isolates could be categorised into four
groups based upon differences in virulence. Monoclonal antibodies (mAbs) were
successfully raised using separate and pooled isolates for immunisation. MAbsraised were
of the lgG class and reacted with a proteinaceous epitope. These monoclonal antibodies
could distinguish between different virulence groups of Xanthomonas campestris pv.
mangiferaeindicae by means of Western Blot analysis. These antibodies were used along
with a selective medium, BVGA for detection of epiphytic populations as well as latent
infections in mango. An enrichment step prior to the enzyme- linked immunosorbent assay
(ELISA) is important, since bacterial counts on trees with latent infections are too low to
result in a positive signal. These techniques in combination are thus useful for detection
and monitoring of the pathogen, which may play an important role in controlling the
spread of the disease.Dissertation (MSc Agric)--University of Pretoria, 1993.
gm2014
Microbiology and Plant Pathology
Unrestricted
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Pigatto, Gisele [UNESP]. "Irradiação UV em Xanthomonas campestris pv. campestris visando a produção da goma xantana". Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/94867.
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pigatto_g_me_sjrp.pdf: 376138 bytes, checksum: 0d3df3ba13a94adf6610c9416261a91c (MD5)Xanthomonas campestris é uma bactéria fitopatogênica que causa a podridão negra no sistema vascular das plantas da família das cruciferaceaes. Produz um exopolissacarídeo denominado goma xantana, que possui propriedades reológicas únicas sendo utilizada amplamente como agente de suspensão, espessante, emulsionante e estabilizante. É aplicados em indústrias petrolíferas, alimentícias, farmacêuticas, mineração, têxtil, termoquímicas, tintas, cosméticos e produtos agropecuários. O Brasil é um grande produtor mundial de cana de açúcar e álcool etílico. Produtos estes utilizados para a produção de xantana; o primeiro como substrato da fermentação e o segundo para a separação da goma. Apesar de todo esse potencial, o Brasil importa grande quantidade de goma xantana que poderá ser produzida com grande competitividade internacional. Portanto, este trabalho objetivou a utilização da técnica de irradiação ultravioleta, em uma linhagem específica de Xanthomonas campestris, para a obtenção de mutantes estáveis que possam melhorar o rendimento e/ou qualidade de goma obtida. A quantificação foi realizada através da determinação da biomassa, viscosidade, cálculos do rendimento da biomassa e goma. A irradiação UV por 600 segundos causou uma redução de 92,2% na população irradiada e as linhagens sobreviventes foram isoladas e analisadas nos testes de produção e viscosidade da goma xantana. As linhagens I6, I7, I9 e I10 apresentaram um aumento de 102% na produção de goma comparando com a linhagem não irradiada. Em relação à viscosidade do caldo, as linhagens irradiadas obtiveram um aumento de 48% comparadas com as não irradiadas de 20 e 30 rpm. A viscosidade da solução de goma xantana 1%, também foram superiores quando comparadas com a não irradiada. O aumento de...
Xanthomonas campestris is fitopatogenic bacterium that causes the black rotten in the vascular system of the plants of the family of the cruciferaceaes. It produces an exopolysaccharides that forms the xanthan gum, which is used in ample variety as agent of suspension, thicker, emulsifier and stabilizing, and singular rheological properties. It is applied in petroliferous, nourishing, pharmaceutical industries, of mining, textile, thermo chemistries, inks, cosmetics and farming products. Brazil is the worldwide producing greater of sugar cane of sugar and ethyl alcohol. Products theses used for the xanthan production; the first one as substratum of the fermentation, and the second as for the separation of the gum. Despite all this potential, the Brazil imports lot of xanthan gum that could be produced with great international competitiveness. This aimming work the used of the ultraviolet technique of irradiation in a specific strain of Xanthomonas campestris to obtain the mutants that can improve the income and/or quality of produced gum, through the determination of the biomass, viscosity, calculations of the income of the biomass and gum. The UV irradiation during 60 seconds caused a reduce of the 92.2% in the irradiated strains and the survived strains were isolated and analysed in the tests of production and viscosity of xanthan gum. The strains I6, I7, I9 e I10 showed increased in the xanthan production of 102% comparing with the non-irradiated strain. In relation the viscosity of the broth the irradiated strains the increase of 48% in shear rate of 20 and 30 rpm compared with the no irrdiated. The viscosity of the xanthan solution 1% irradiated were higher also whwn compared with no irradiated in both shear rate (20 and 30 rpm. The increase of viscosity was of 17% to rotational speed of 20 rpm and 16% to 30 rpm. The ...(Complete abstract click electronic access below)
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Pigatto, Gisele. "Irradiação UV em Xanthomonas campestris pv. campestris visando a produção da goma xantana /". São José do Rio Preto : [s.n.], 2008. http://hdl.handle.net/11449/94867.
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Orientador: Pedro de Oliva NetoBanca: Valéria Marta Gomes de Lima
Banca: Ivanise Guilherme Branco
Resumo: Xanthomonas campestris é uma bactéria fitopatogênica que causa a podridão negra no sistema vascular das plantas da família das cruciferaceaes. Produz um exopolissacarídeo denominado goma xantana, que possui propriedades reológicas únicas sendo utilizada amplamente como agente de suspensão, espessante, emulsionante e estabilizante. É aplicados em indústrias petrolíferas, alimentícias, farmacêuticas, mineração, têxtil, termoquímicas, tintas, cosméticos e produtos agropecuários. O Brasil é um grande produtor mundial de cana de açúcar e álcool etílico. Produtos estes utilizados para a produção de xantana; o primeiro como substrato da fermentação e o segundo para a separação da goma. Apesar de todo esse potencial, o Brasil importa grande quantidade de goma xantana que poderá ser produzida com grande competitividade internacional. Portanto, este trabalho objetivou a utilização da técnica de irradiação ultravioleta, em uma linhagem específica de Xanthomonas campestris, para a obtenção de mutantes estáveis que possam melhorar o rendimento e/ou qualidade de goma obtida. A quantificação foi realizada através da determinação da biomassa, viscosidade, cálculos do rendimento da biomassa e goma. A irradiação UV por 600 segundos causou uma redução de 92,2% na população irradiada e as linhagens sobreviventes foram isoladas e analisadas nos testes de produção e viscosidade da goma xantana. As linhagens I6, I7, I9 e I10 apresentaram um aumento de 102% na produção de goma comparando com a linhagem não irradiada. Em relação à viscosidade do caldo, as linhagens irradiadas obtiveram um aumento de 48% comparadas com as não irradiadas de 20 e 30 rpm. A viscosidade da solução de goma xantana 1%, também foram superiores quando comparadas com a não irradiada. O aumento de...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Xanthomonas campestris is fitopatogenic bacterium that causes the black rotten in the vascular system of the plants of the family of the cruciferaceaes. It produces an exopolysaccharides that forms the xanthan gum, which is used in ample variety as agent of suspension, thicker, emulsifier and stabilizing, and singular rheological properties. It is applied in petroliferous, nourishing, pharmaceutical industries, of mining, textile, thermo chemistries, inks, cosmetics and farming products. Brazil is the worldwide producing greater of sugar cane of sugar and ethyl alcohol. Products theses used for the xanthan production; the first one as substratum of the fermentation, and the second as for the separation of the gum. Despite all this potential, the Brazil imports lot of xanthan gum that could be produced with great international competitiveness. This aimming work the used of the ultraviolet technique of irradiation in a specific strain of Xanthomonas campestris to obtain the mutants that can improve the income and/or quality of produced gum, through the determination of the biomass, viscosity, calculations of the income of the biomass and gum. The UV irradiation during 60 seconds caused a reduce of the 92.2% in the irradiated strains and the survived strains were isolated and analysed in the tests of production and viscosity of xanthan gum. The strains I6, I7, I9 e I10 showed increased in the xanthan production of 102% comparing with the non-irradiated strain. In relation the viscosity of the broth the irradiated strains the increase of 48% in shear rate of 20 and 30 rpm compared with the no irrdiated. The viscosity of the xanthan solution 1% irradiated were higher also whwn compared with no irradiated in both shear rate (20 and 30 rpm. The increase of viscosity was of 17% to rotational speed of 20 rpm and 16% to 30 rpm. The ...(Complete abstract click electronic access below)
Mestre
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Silva, Fernanda Pereira da. "Caracterização biológica e molecular de um bacteriófago específico para Xanthomonas campestris pv. campestris". Universidade Federal de Viçosa, 2015. http://www.locus.ufv.br/handle/123456789/6502.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Os vírus que infectam bactérias são denominados bacteriófagos, sendo também referidos como “fagos”. Os bacteriófagos que infectam bactérias fitopatogênicas, tem despertado crescente interesse devido ao seu potencial para o biocontrole. A bactéria Gram negativa Xanthomonas campestris pv. campestris, é o agente causal da podridão negra das brássicas (Brassicaceae), sendo responsável por perdas econômicas, resultantes do difícil controle. Assim, este trabalho teve como objetivo realizar o isolamento e a caracterização biológica e molecular de bacteriófagos infectando Xanthomonas campestris pv. campestris. Para isso, plantas da família Brassicaceae apresentando sintomas da podridão negra e solo rizosférico, foram coletados em campos de cultura em Coimbra-MG e triados para a presença de bacteriófagos adotando a técnica de formação de placas de lise por meio da sobrecamada de ágar. Das nove amostras analisadas, uma amostra apresentou placas de lise persistente nos quatro ciclos de propagação. O fago foi denominado Xacp1 e apresentou cabeça icosaédrica de aproximadamente 30 ± 5 nm de diâmetro e uma cauda curta com 6 ± 0,2 nm de comprimento e 7 ± 0,2 nm de diâmetro. O bacteriófago possui ácido nucleico composto por uma única molécula de DNA fita dupla (dsDNA) com tamanho estimado em 65 Kpb. De acordo com a morfologia e tipo de genoma, o bacteriófago Xacp1 foi classificado na família Podoviridae (Ordem Caudovirales). A sequencia do genoma completo do fago está sendo analisada e será utilizada para estudar o relacionamento filogenético com outros bacteriófagos que infectam Xanthomonas campestris. O bacteriófago mostrou capacidade em infectar apenas isolados de Xanthomonas vcampestris pv. campestris no teste de susceptibilidade, apresentando placas de lise com coloração, tamanho e formato padrão. Isolados bacterianos relacionados e não relacionados a Xanthomonas campestris pv. campestres não foram suscetíveis à infecção. Em conjunto esses resultados demostram que Xacp1 apresentou características ideais de especificidade e virulência que motivam futuros estudos para sua utilização como uma ferramenta biotecnológica para a utilização no biocontrole da podridão negra em brássicas.
Viruses that infect bacteria are termed bacteriophages, and also referred to as “phage”, or “bacteriovírus”. The bacteriophages that infect bactéria plant pathogens, has attracted increasing interest due to its potential for biocontrol. The gram-negative bacterium Xanthomonas campestris pv. campestris is causal agent of black rot of crucifers (Brassicaceae), accounting for economic losses resulting from difficult to control. This study had objective perform the isolation and molecular characterization of biological and bacteriophage infecting Xanthomonas campestris pv. campestris. For this, plants of the family Brassicaceae showing symptoms of black rot and rhizosphere soil were collected in crop fields in Coimbra-MG and screened for the presence bacteriophage adopting the technique of forming plaques by agar overlay. The analyzed nine samples, one sample showed signs of Persistent lysis in four cycles of the spread. The phage was named φXacp1 and icosahedral head showed approximately 30 ± 5 nm in diameter and a tail short-± 5 nm in length and 0.7 ± 0.3 4 nm diameter. The bacteriophage has nucleic acid consists of a single double stranded DNA molecule (dsDNA) with size estimated at 65 kbp. According to the morphology and type of genome, bacteriophage φXacp1 scored in Podoviridae family (Order Caudovirales). The sequence the complete genome of the phage is being analyzed and will be used to study the phylogenetic relationships with other bacteriophages that infect Xanthomonas campestris. The bacteriophage showed ability to infect only Xanthomonas campestris pv. campestris in susceptibility testing, presenting plaques staining, size and pattern shape. Isolated bacterial related and unrelated to Xanthomonas campestris. Country were not susceptible to infection. viiTogether these results demonstrate that φXacp1 presented ideal characteristics of specificity and virulence that motivate future studies for its use as a biotechnological tool for use in biocontrol of black rot in crucifers.
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Wruck, Dulândula Silva Miguel. "Análises bioquímica, patogênica, sorológica e molecular de isolados de Xanthomonas campestris pv. campestris". Universidade Federal de Viçosa, 2001. http://www.locus.ufv.br/handle/123456789/11176.
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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Trinta e três isolados de Xanthomonas campestris pv. campestris, obtidos de oito diferentes brássicas, provenientes de diferentes regiões do Brasil e do exterior, foram analisados com base em caracterizações bioquímica, patogênica, sorológica e genética, objetivando verificar a existência ou não de relacionamentos quanto à origem geográfica e ao hospedeiro. No estudo bioquímico, foram realizados 22 testes, além da avaliação da atividade de esterase, em que com base no tamanho do halo e utilizando o teste de Scott Knott no nível de 5% de probabilidade, obtiveram-se cinco grupos de similaridade. Avaliando a capacidade de produção de bacteriocina, verificou-se que três isolados apresentaram antibiose contra sete outros. Avaliou-se, também, a sensibilidade dos isolados a alguns antibióticos e, com base na análise de agrupamento, foi possível a observação de três grupos distintos: o primeiro constituído por quatro isolados resistentes a 11 ou mais antibióticos; o segundo grupo composto por um isolado resistente somente a três antibióticos; e o terceiro grupo composto pelos demais isolados. Na caracterização patogênica, foi realizada a inoculação cruzada, de forma que os 33 isolados foram inoculados artificialmente em sete diferentes brássicas. Desses, 14 não mostraram especificidade quanto aos hospedeiros, enquanto os 19 isolados restantes apresentaram relativo grau de especificidade, uma vez que não causaram doença em uma ou mais das brássicas inoculadas. Para a análise sorológica, foram obtidos sete antissoros, pelos quais se reconheceram, no teste de imunodifusão dupla, 32 dos 33 isolados estudados, além de mostrarem especificidade quanto ao reconhecimento dos isolados de X. campestris pv. campestris. Na caracterização molecular, realizou-se a extração do DNA total de todos os isolados, seguida de amplificação pela técnica do RAPD. Procedeu-se, também, à extração do DNA plasmidial. Para avaliação do comportamento dos 33 isolados de X. campestris pv. campestris de diferentes origens e hospedeiros, com relação às diferentes análises realizadas, foram efetuadas análises estatísticas univariadas, para a avaliação da atividade de esterase, e multivariadas, para as demais, bem como a análise conjunta de todos os resultados. Com base nos resultados das caracterizações bioquímica, patogênica e sorológica, não se observou correlação da variabilidade dos isolados estudados com a origem geográfica e o hospedeiro do qual foram obtidos. Isso pode ter sido devido à constante introdução de novos isolados de X. campestris pv. campestris nas áreas de plantio, por meio de sementes contaminadas.
Thirty-three isolates of Xanthomonas campestris pv. campestris, obtained from eight different Brassica species and originated from distinct geographic regions in Brazil and other countries, were analyzed in terms of their biochemical, pathogenic, serological and molecular properties, in order to determine whether there is a relationship between isolate variability and host or geographic origin. Twenty-two biochemical tests, besides the determination of sterase activity, were carried out for the biochemical characterization. Five similarity groups were identified using Scott- Knot s test at 5% probability. The analysis of bacteriocin production indicated that three isolates displayed antibiosis against seven other isolates. Sensitivity to several antibiotics was also evaluated. Using cluster analysis, three distinct groups were identified: the first group consisted of four isolates which were resistant to 11 or more antibiotics; the second group consisted of one isolate which was resistant to only one antibiotic; the third group consisted of all the other remaining isolates. For the pathogenic characterization, all 33 isolates were cross-inoculated onto seven different Brassica species. Fourteen isolates did not display host specificity. The remaining 19 isolates displayed a relative degree of host specificity, since they did not induce disease in one or more of the inoculated species. Seven polyclonal antisera were raised for the serological analysis. In immunodiffusion tests, 32 out of the 33 isolates tested were recognized by these antisera. The antisera specifically recognized isolates of X. campestris pv. campestris. For the molecular characterization, total DNA was extracted from each isolate and used as a template for RAPD reactions. Plasmid DNA was also extracted. Together, the data from the biochemical, pathogenic, serological and molecular analyses failed to indicate a relationship among the isolates, hosts and geographic origin. This could be due to the repeated introduction of new isolates of X. campestris pv. campestris into different geographic regions, via contaminated seeds.
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Rosseto, Flávio Rodolfo. "Caracterização bioquímica, biofísica e estrutural da principal endoglucanase secretada por Xanthomonas campestris pv. campestris ATCC33913". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-20092011-101408/.
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O esgotamento das fontes de combustíveis fósseis e questões ambientais têm gerado grandes preocupações para a sociedade, e alternativas sustentáveis e eficientes para solucionar e trazer inovações na produção de biocombustíveis estão cada vez mais próximas. O uso de biomassa pode ser uma vantajosa opção, mas para isso, é necessária a degradação das moléculas constituintes de sua parede celular a açúcares fermentáveis. A biotecnologia tem melhorado e aumentado as opções para suprir fontes sustentáveis e preservação do meio ambiente. A transformação de biomassa na escala industrial para produção de bioetanol depende da capacidade de otimizar o processo de hidrólise enzimática da celulose utilizando enzimas de complexo celulolítico, principalmente de fontes bacterianas e fungos filamentosos. Dessa forma, estudamos a principal endoglucanase de Xanthomonas campestris pv. Campestris ATCC33913, que foi inicialmente identificada através de zimogramas e espectrometria de massas que mostrou boa cobertura de sequência e purificada utilizando passos de precipitação com sulfato de amônio seguida do uso de uma coluna de exclusão molecular. Esta enzima teve seus parâmetros cinéticos determinados, mostrando-se ativa para diversos substratos testados e também grande estabilidade para variações de pH e temperatura, com condições ótimas de pHótimo = 7.0 e Tótima = 45°C. Além da caracterização enzimática, a posição relativa entre o domínio catalítico e de ligação da celulose (CBM) da Endoglucanase por SAXS também foram determinadas, e ainda, para finalizar os estudos estruturais, a estrutura cristalográfica do domínio catalítico da enzima foi determinada com resolução de 2.8Å, contendo quatro moléculas por unidade assimétrica.The exhaustion of fossil fuels and the environment issues related have been generated many concerns for the society and searching for sustainable and effective alternative are being done in order to solve and bring innovations for biofuel production. Biomass can be a good alternative, but, for the success, it will be necessary degrading the cellular wall molecules into fermentable sugar. Biotechnology has improved and increased options in order to supply sustainable sources and environment preservation. The biomass transformation for bioethanol production, at the industrial scale, depends on the capacity of optimize the enzymatic hydrolysis of cellulose using enzymes of the cellulolytic complex, mainly produced by bacteria and filamentous fungi. We have studied the main endoglucanase of Xanthomonas campestris pv. Campestris ATCC33913 which has been initially identified by zymograms and mass spectrometry with high coverage sequence, and purified using precipitation with ammonium sulfate followed by size exclusion column. This enzyme had its kinetic parameters determined showing activity for the substrates tested and also has showed stability for pH and temperature changes, with optimal conditions of pHopt = 7.0 and Topt = 45°C. Following this enzymatic characterization, the relative position of the catalytic domain and cellulose binding domain (CBM) was determined by SAXS. In order to complete the structural studies, the crystallographic structure of the catalytic domain has been determined with 2.8Å of resolution, showing four molecules by asymmetric unity.
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Li, Pei-Zhen, e 李佩真. "Application of Xanthomonas campestris pv. campestris bla promoter". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/32617569531060590271.
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碩士國立中興大學
分子生物學研究所
103
Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium that causes black rot in crucifers. Many strains of Xcc are β-lactam resistant via expressing β-lactamase (AmpC/Bla) constitutively. Previous studies showed that transcription from bla promoter is regulated by a global transcriptional regulator, Clp (cAMP receptor protein-like protein). Mutation in Clp changes constitutive expression of bla to an ampicillin-inducible manner. This study aimed to explore the possibility in application of Ap-inducibility in the clp-deficient mutant strain, TC817, derived from Xc17. For the testing, several plasmids were constructed, which carried genes encoding lysis protein from the Xcc phage phiL7 or antimicrobial peptide (AMPs) under control of the bla promoter. These plasmids were introduced into TC817, and the resultant transformants were incubated in ampicillin (Ap)-containing medium. Results showed that growtn rate of the cells was reduced, suggesting that Ap can induce bla promoter to express lysis protein and AMPs. Thus cell growth was inhibited by Ap. Growth inhibition was also observed with P20H, a mutant derived from Xc11A. The results also indicated that lysis protein p27 (hollin) can effectively inhibit the growth of P20H. In the second part of this thesis, the filamentous phage phiLf of Xcc was used to construct a new vector system for expression of p27 gene driven by the plant-inducible promoters (PIP) of hrpB, hrpE and hrpF. The recombinant RF DNA were then introduced into Xcc. However, no lysis was observed for the recombinant phiLf-infected P20H in XVM2 medium in which PIP would activate the PIP promoters. Then we replay PIP by bla promter and incubate cell on Ap-containing medium. Results showed that cell growth was inhibited after recombinant phage infection.
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王旭川. "Gene prediction and annotation in Xanthomonas campestris pv. campestris". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/53320871199889246880.
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碩士國立清華大學
生命科學系
90
Abstract Xanthomonas campestris pv. campestis is a Gram-negative bacterium and one of the most important plant pathogens. It attacks cruciferous plants and causes worldwide agricultural loss. We annotate 575 contigs of X. campestris pv. campestis, which were sequenced by Genome Research Center at NYMU. We got 6327 and 3811 ORFs (open reading frame) predicted by Glimmer 2.0 and ORPHEUS. Then we performed BLAST search against Genbank nr (non-redundant) protein database and there were 5854 ORFs having similarity with protein sequences in the database ( sequence identity > 30% ). We did the sequence search with COG (clusters of orthologous groups) database for protein function assignment, and we got 2819 hits in our ORFs (E-value cut-off = e-20). The pathogenesis mechanism is one of the most important issues for plant pathogen study. 487 candidate genes are found responsible for pathogenesis after comparing our sequences with the annotations of the other two plant pathogens, Xylella fastidiosa and Ralstonia solanacearum. This result could apply to study the pathogenesis mechanisms of different plant pathogens and those genes could be good targets for structure determination. We collected all annotation results to create a X. campestris pv. campestis gene annotation database ( http://xcc.life.nthu.edu.tw ). This database can help the research related to X. campestris pv. campestis and our final goal is to select the valuable target proteins for structure genomics .
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HUANG, PEI-YI, e 黃佩儀. "Characterization of icd Gene of Xanthomonas Campestris pv. Campestris". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/638686.
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碩士中臺科技大學
醫學檢驗生物技術系碩士班
105
The Gram-negative plant pathogen Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease that causes tremendous agricultural loss. The isocitrate dehydrogenase (IDH) is an enzyme that catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and CO2 and NADPH or NADH, using NADP+ or NAD+ as a cofactor. In Xcc genome, the coding product of icd gene was annotated as a NADP+-dependent IDH. To characterize the function of Xcc icd gene, the icd mutant and its complementary strain were constructed previously. Here, the phenotypic changes were evaluated among these strains, and the biochemical properties of the coding product of icd gene were studied. Virulence assay revealed that the lesion length caused by icd mutant was reduced than the wild type. Inactivation of icd gene caused reduction of IDH activity in Xcc. Complementation of full length icd can restore these phenotypic changes of the mutant strain. The purified recombinant ICD (rICD) protein possessed IDH activity, and was shown to have a pH optimum of about pH 8.0 and a temperature optimum of about 37 ℃. Irreversible denaturation of rICD mainly occurred above 50 ℃. In addition, divalent cations Mg2+ and Mn2+ were found to be required for IDH activity of rICD. Keyword：Xanthomonas, black rot , isocitrate dehydrogenase, purification
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Chen, Chih-Hua, e 陳智華. "Transcriptional Regulation by Clp in Xanthomonas campestris pv. campestris". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/56300758274356683357.
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博士國立中興大學
分子生物學研究所
98
The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. It is capable of producing large amounts of an exopolysaccharide, xanthan gum, and secreting an array of extracellular enzymes, including proteases, pectinases, and endoglucanases, which have long been considered important virulence determinants. Production of these extracellular products is regulated by the global transcription factor Clp (cyclic AMP receptor protein-like protein, a homolog of the Escherichia coli CRP) and DSF whose biosynthesis involves RpfF protein. It is also known that Xcc encodes no adenylate cyclase required for the production of cyclic AMP, an effector that is involved in activation of CRP for binding to CRP-regulated promoters. Furthermore, it has previously been demonstrated in our laboratory that Clp 1) in vitro can bind to the regulated promoters in the absence of cyclic AMP, and 2) exerts transcriptional activation by binding to the promoter of some regulated genes (i.e., engA, pehA, and pelA1), but without direct binding to others (i.e., fliC, groESL, prt1, and manA). Despite these findings, prediction of the sequences for Clp binding (Clp-binding site, CBS) was merely based on similarity to the consensus E. coli CRP-binding site, which is 22-bp long (5’-AAATGTGATCTAGATCACATTT-3’) exhibiting perfect twofold sequence symmetry, because Clp has been shown to have the same DNA binding specificity as CRP at positions 5, 6, and 7 (GTG motif) of the DNA half site. However, close examination of the above CBSs that have been characterized revealed that some of them possess sequences of arms and a central region deviated from the CRP consensus sequence. Therefore, this study was aimed to deduce consensus CBS sequence(s) on the basis of a larger number of characterized Xcc CBSs than what was already available. To achieve this end, PromScan program was used to search in Xcc genome. Several putative CBSs were identified, among which promoters of clp, gumB, and xpsE were then confirmed to be up-regulated by Clp, via deletion mapping in conjunction with electrophoretic mobility shift assay (EMSA) and transcriptional fusion assay. The results suggested that 1) clp is auto-regulated positively by Clp via binding to a typical CBS, 2) gum promoter possesses two atypical CBSs, in which a more conserved right arm compensates for the lack of conservation in the left arm, a high GC content in the central region (6 bp) is important for binding, and binding is enhanced by the palindromic GC-rich central region, and 3) two typical CBSs are present upstream of xpsE and both could bind to Clp in EMSA; however, only the one in proximity to the xpsE transcription initiation site is required for transcription activation. The three atypical CBSs (gumB CBS I, gumB CBS II, and xpsF CBS) were combined to create a frequency matrix which was then used in PromScan to perform genome-wide search. Twenty-one possible atypical CBSs were identified and five of them were confirmed by competitive EMSA. These CBS-containing genes have previously been shown to be up-regulated by Clp in microarray assay; however, most of the CBSs confirmed here are situated at different positions from those predicted by the microarray assay. Based on these eight atypical CBSs, a consensus atypical CBS, 5’-AnAGGCGAACGCnGTCACAnAA-3’, was compiled. In the meantime, by aligning 8 known typical CBSs, a more informative consensus typical CBS (5’-TnGTGTGnnnnAnnTCnCATCG-3’) was deduced. Using a frequency matrix based on this consensus sequence for search with PromScan, 30 Xcc genes, which have been previously identified by microarray assay to be regulated by Clp, were identified to possess typical CBS. Further EMSA is needed for confirmation of these typical CBSs. In conclusion, these results suggest that both typical and atypical CBSs exist in Xcc genome and that Clp has evolved to gain the capability to bind both types of CBSs. In addition to the above results, two important findings were also made here: 1) xpsE promoter is also up-regulated by RpfF, and 2) binding of Clp in vitro is inhibited by cyclic di-GMP, as demonstrated by using the Xcc engA promoter region as the probe in EMSA.
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Shiau, Shuh-Lian, e 蕭澍濂. "Classification of Insertion Sequences in Xanthomonas campestris pv. campestris". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/66048286784555202974.
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Lo, Ta-Chun, e 羅大鈞. "Analysis of insertion sequences in Xanthomonas campestris pv. campestris". Thesis, 1999. http://ndltd.ncl.edu.tw/handle/00898840262981581923.
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碩士國立中興大學
分子生物學研究所
87
Xanthomonas campestris pv. campestris (Xcc) is the causing agent of black rot of crucifies. Using a sucrose-sensitive plasmid as a baiting plasmid, our laboratory previously isolated 34 plasmids containing IS insertions. Among these plasmids, thirty were characterized and were found to have IS476, IS1478, IS1479 and IS1480 insertion in the plasmids. All but one IS476 insertion mutant has target duplications of 4-6 bases at the insertion sites. An IS476 insertion mutant does not have any target site duplication. This study demonstrates that pXC11-11 containing IS476 insertion carries a 952-bp duplication at the insertion site. Based on presence of both monomers and multimers of plasmids in Xcc11 from which the pXC11-11 mutant plasmid was originally isolated, a model is prepared herein to explain how this long direct repents are generated. The other four insertion sequence-containing plasmids were found to have IS1477, IS1481 and IS1482. Southern blot hybridization of twenty related bacteria indicates that six IS elements (IS476, IS1477, IS1478, IS1479, IS1480, IS1481) are present in multiple copies in the seven Xcc strains tested and two or three other pathovars of Xanthomonas campestris; IS1482 is present in a single copy in four of the seven Xcc strains tested and two other pathovars of Xanthomonas campestris. A spontaneous avirulent mutant (Xcc11A) of Xcc11 carries an extra 352-bp DNA fragment located upstream from an IS1478 in the chromosome. This study demonstrates that the same 352-bp DNA fragment is located six bases downstream from the same IS1478 in Xcc11. It is speculated that an IS1478 is precise-excised from the Xcc11 chromosome and inserted into a site 352-bp downstream from the excision site, thus generating Xcc11A. An Xcc11 2.6-kb EcoRI-BamHI DNA fragment including the 352-bp fragment was found capable of transforming Xcc11A into a strain pathogenic to turnip seedling. An orf (orfF) encoding a protein of 113 amino acid residues is found in the 2.6-kb sequence, which is interrupted by an IS1478 in Xcc11A. This indicates that the protein is pathogenicity-related. Its amino acid sequence shows low but significant homology with the amino acid sequence of the transcripation intiation factor IIF beta subunit (RAP30) of Xenous laevis.
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Wang, Ching-Hua, e 王清華. "Characterization of hns Gene of Xanthomonas campestris pv. campestris". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/56591956311878025919.
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碩士國立中興大學
分子生物學研究所
95
The gram-negative plant pathogenic Xanthomonas campestris pv. campestris causes black rot in crucifers. The virulence of this bacterium is mainly depending on its ability to produce large mounts of an exopolysaccharide (EPS, also called xanthan gum) and several extracellular enzymes, the virulence factors. It is known that the global transcription factor Clp (cAMP receptor protein-like protein) is involved in the production and secretion of these virulence factors. Using the consensus Clp-binding sequences (CBS) predicted in our laboratory for genome-wide search, 46 genes with at least one CBS located in the upstream region, suggesting possible regulation by Clp, were found in X. campestris pv. campestris strain 17. One of them was XCC0637, which was predicted to be a DNA-binding protein genes belonging to functional category Ⅲ possibly involved in macromolecule metabolism. It was designated as an X. campestris pv. campestris H-NS, because it possesses an H-NS conserved domain and shares 72, 62, 46 and 35% identities with the global regulator H-NS from Stenotrophomonas maltophilia, Xylella fastidiosa, Rhodopseudomonas palustris and Escherichia coli, respectively. In enterobacteria, H-NS regulates gene expression primarily as a transcriptional repressor, with only a few examples required for a direct positive control. In addition, H-NS and CRP in E. coli regulate gene expression independently. In this study, gel retardation and Phns-lacZ transcriptional fusion assays show that Clp positively regulates expression of hns by direct binding to the promoter. An hns mutan was constructed by insertional mutation. Testing with this mutant indicated that, relative to the wild-type, i) production of EPS and extracellular enzymes was slightly reduced, ii) appearance of the black rot symptom was slightly delayed, by about one day slower, iii) swarming motility was drastically reduced. Finally, transcriptional fusion assays also demonstrated that the X. campestris pv. campestris H-NS exerts negative control on the expression of hns, pelB and xpsE and positive control on the expression of engA.
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Wu, Hsung-Chi, e 吳順吉. "Transcriptional regulation of Xanthomonas campestris pv. campestris engXCA gene". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/83810288653140348308.
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碩士國立中興大學
分子生物學研究所
90
The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris causes black rot in crucifers, which results in large damage in agriculture. X. campestris. pv. campestris secretes a large mount of exopolysaccharide (EPS), xanthan gum, which is widely used as a stabilizing, viscosifying, emulsifying, thickening, and suspending agent. Therefore, it is of importance to study the biosynthesis of xanthan gum. Previously, by using Tn5(pfm)CmKm transposon mutagenesis, the gene in locus eps8 involved in xanthan synthesis and pathogenicity was cloned, sequenced and found to encode Clp, homologous to the global transcription factor CRP (cyclic AMP receptor protein) in Escherichia coli. It was reported that in clp mutant of X. campestris pv. campestris, cellulase production is decreases by 85% compared to its wild-type cells. Therefore, it was interesting to know whether transcription of engXCA gene is regulated by Clp. In this study, two fragments in the upstream region of engXCA were amplified by PCR and cloned into the promoter-less promoter-probing vector pFY13-9 with lacZ as the reporter gene. The resultant plasmids were introduced into the wild type Xc17 and the clp mutant AU56E, followed by measuring the b-galactosidase activities. It was found that expression of the engXCA promoter was much lower in AU56E than Xc17, ranging from 22 to 40 versus 1793 to 4166 Miller Units. These data indicate that Clp is essential for the transcription of engXCA gene. It was also interesting to understand whether proteins other than Clp are involved in the regulation of engXCA expression. To achieve this end, the promoter-less xylE reporter gene (encoding catechol 2,3-dioxygenase) was inserted into engXCA gene of Xc17 resulting in mutant HC117(engXCA::xylE-GmW), a strain deficient in cellulase and showing XylE activity. HC117(engXCA::xylE-GmW) was then used as the recipient for Mini-Tn5-Tc mutagenesis for the selection of mutants that exhibited lower or none XylE activity, with the rational that mutation of a gene required for the transcription of engXCA would turn off the xylE expression in HC117(engXCA::xylE-GmW). Six such mutants were obtained and two of them were further studied. Sequence analysis of the responsible DNA regions from these two mutants revealed that one has a mutated ppsA gene (coding for phosphoenolpyruvate synthase) and the other has a mutated gpdA gene (coding for glycerol-3-phosphate dehydrogenase). Although further studies are needed to assure the involvement of these genes in transcription of the engXCA, the pathogenicity tests have indicated that gpdA gene is required for pathogenicity.
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Chou, Fan-Li, e 周芳莉. "Characterization of the gumD mutant from Xanthomonas campestris pv. campestris". Thesis, 1995. http://ndltd.ncl.edu.tw/handle/67481305091835184718.
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碩士國立中興大學
分子生物研究所
83
X. campestris pv. campestris 會引起十字花科蔬菜的黑腐病, 且會產 生大量的胞外多醣 (exopoly-saccharide, 簡稱 EPS), 又稱黃原膠 ( xanthan gum)。 為了研究黃原膠合成的基因, 選擇 p3DA01B 片段上的 pSR3, pRR4 與其下游片段的 pHN4, 利用標誌基因互換, 分離乾菌落突變 株。結果只有送進 pSR3 片段所得到的菌落為乾菌落, 此突變株命名為 XKSR。在 pSR3 片段上帶有一 484 個胺基酸 之 ORF, 相似於 GumD, 但 與 GumD 有 5 個胺基酸序列不同的差異, 推測其功能為轉移第一個 glucose 至 lipid carrier 之 glucosyl transferase。此外, 在 pSR3 片段上帶有 gumE 基因產物 399 個胺基酸中的 304 個胺基酸。 ORF484 與 Rhizobium legusarum 胞外多醣調節基因產物 Pss2 有 42.2％ 的同 質性。XKSR 之 EPS產量 為野生型菌株 Xc17 的 3.7％, 而 XKSR( pP2201) 僅達 Xc17 之 27％, 此可能是因 gumD (orf484) 基因之破壞, 影響其下游基因的表現所造成 polar effect 的影響。而相同 gumD基因 被破壞的突變株之互補株 P22(pP2201), 其 EPS 產量可達野生型菌株 Xc11 之 64％, 可見 P22 突變株 gumD 基因之破壞,影響其下游基因的表 現所造成 polar effect 的影響較小。純化突變株 XKSR 之 EPS 進行 HPLC 分析, 發現與野生型株 Xc17 之 EPS 組成不同, 可能為其它種類 之 EPS。關於突變株 XKSR 與 XKSR(pP2201) 對小白菜、青花菜與蕪菁之 致病力測試, 結果顯示突變株 XKSR 與互補株 XKSR(pP2201) 之 EPS 產 量減少則毒性降低, 但其致病力仍在。 The gram-negative bacterium Xanthomas campestris pv. campestris (Xc) is phytopathogenic causing black rot in cruciferous plants. It produces extracellular polysaccharide called xanthan. Some n p3DA01B have been studied, which ionvolved in xanthan gum biosynthesis.Two plasmids,pSR3 and pRR4,were constructed which contained inserts from the downsteram of p3DA01B,and a plasmid,pHN4, which contained insert from the region downstream to 3DA01B. A Km-resistance gene was inserted into the SacI site of these fragments and isolated non-mucoid mutants by marker exchange.A non-mucoid mutant,XKSR, was thus obtained by disruption of the pSR3 insert.Sequence analysis and computer search revealed a ORF484 similar to gumD, an incomplete ORF contain 304 amino acids which is 100% identity to GumE.The ORF484 have 5 amino acids residue different from that of GumD. GumD has been proposed to be a glucosyl transferase which transfer a glucose residue from UDP-glucose to a lipid carrier. Homology comparision of the amino acids sequence of ORF- 484 showed 42.2 % identity to that of the R.leguminosarum Pss2 which is a regulatory gene for polysaccharide synthesis and required for nodulation in peas. The amount of EPS produced by XKSR and XKSR(pP2201) were about 3.7%,27% of the amount produced by Xc17.These suggest that insertion of the cartidge might have a polar effect on the genes downstream to ORF484. The amount of EPS produced by gumD mutant P22 containing pP2201 is about 64% of the amount produced by Xc11, show the polar effect is not as strong as that in XKSR.Analysis of the EPS produced by XKSR using HPLC, indicated that the EPS composition is different from that from the wild type Xc17,suggesting that maybe there is an- other kind of EPS produced by XKSR. About the pathogenicity test for XKSR and XKSR(pP2201), showed that both XKSR and XKSR(pP2201) are pathogenic, but the mutant possessed reduced virulence.
21
Chang, Yu-Chia, e 詹于佳. "Charaterization of dsbA and dsbC of Xanthomonas campestris pv. campestris". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/d9rq39.
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碩士中臺科技大學
醫學生物科技研究所
96
Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in cruciferous plants. Xcc produces exopolysaccharide (EPS) and various extracellular enzymes, such as a-amylase, pectate lyase, endoglucanase and proteases, which are collectively essential for pathogenesis. Pathogenicity of Xcc is regulated by rpf gene cluster (for regulation of pathogenicity factors). Previous studies have shown that production of these virulence factors is positive regulated by rpf gene cluster. The amino acid sequence of RpfF has highly identity to enoyl-CoA hydratase and is demonstrated directly leading DSF synthesis. DSF regulate various virulence factors synthesis by signal transduction. Twelve protein spots difference was found by 2D-PAGE which was compared between wild type and P20H(rpfF::Gm), rpfF mutant obtained in previous study. The number 5 protein spot was identified as XCC3400 (DsbA) (ATCC33913 strain) which is periplasmic thiol: disulfide oxidoreducatase by LC/MS/MS. XCC3399, upstream gene of XCC3400, also belong to DsbA family. We named those two genes as dsbA1 and dsbA2 to make XCC3400 and XCC3399 could be distinguishable. Serial experiments were carried out for understanding how RpfF regulate dsbA1 and dsbA2, and whether DsbA1 and DsbA2 influence the Xcc virulence by catalyses folding of various factors. The results of above-mentioned study: (1) Mutation in dsbA1 or dsbC abolish ability of virulence, but mutation in dsbA2 shows no difference with wild type. (2) Secretion of protease slightly decreases while mutate in dsbA1 or dsbC, but decreases while mutate of dsbA1 and dsbC. (3) The deletion of dsbA1 and dsbC brings filamentous phage fLf resistibility to host, while mutation in dsbA2 is no effect. (4) Swimming motility abolish when mutation in 5 dsbA1 and dsbC. (5)We suppose possible inferences about the assay of transcription expression: the transcription of dsbA1 is upregulated by RpfF and fliC is indirectly upregulated by DsbA1 and DsbA2; transcription of pilA1 is not regulated by DsbA1 and DsbA2, these two genes probably effect on protein folding.
22
Chen, Jyu-Wei, e 陳巨威. "Regulation of β-lactamase of Xanthomonas campestris pv. campestris 17". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/65305099154396541785.
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碩士國立中興大學
分子生物學研究所
97
Most of Xanthomonas camprstris pv. campestris(Xcc) have ability that they resisted beta-latam antibiotic. Most of Xcc destroyed beta-latam antibiotic by beta-lactamase. In genome of many germ negative bacteria, they existed ampR-bla system genes. When ampicillin existed in environment, the system is only induced and produced beta-lactamase. Although, Genome of Xc17 exist the system, but the system expressed beta-lactamase(XCC3039) continuously. Clp(cAMP receptor protein-like protein) is global transcription factor. Extracellular protein between P20H and P20H clp mutant analyzed by protein 2-D electrophoresis. The result express more Bla(XCC3039)protein of P20h than Bla protein of P20h clp mutant. However, in 2007 He. published the Bla protein(XCC2873) was regulated positively by Clp in XC1. But He’s result is different with result of predecessor’s experiment. In beta-lactamase activity, TC817 (Xc17 clp mutant) loss activity in medium without ampicillin. however, when medium include ampicillin, the beta-lactamse activity is induced in TC817.The activity is enhance 30 folds approximately when medium is added ampicillin. In MIC test, resistance of beta-lactam antibiotic of TC817 reduced. Therefore, Clp effected continuous expression of Bla protein in Xc17.Promoter region between bla and ampR excite 60 percents of Clp-Binding Site(CBS).In gel retardation, tre result that Clp can bind this region is confirmed. In promoter activity assay, when medium included ampicillin, the activity of promoter region of ampR in TC817 was enhanced slightly. But the activity of promoter region of bla in TC817 was not different. In Western blotting assay, when medium included ampicillin in Xc17, Bla protein was still monitored and growth rate was also not different. However, when medium only included ampicillin in TC817, Bla protein was just monitored and growth rate was suppressed.
23
Lu, Chun-Da, e 盧俊達. "Characterization of the fleN gene in Xanthomonas campestris pv. campestris". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/84854581535403713972.
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碩士臺中健康暨管理學院
生物科技學系碩士班
93
Abstract Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot disease in cruciferous plants. This bacterium synthesizes great amounts of xanthan gum rendering the colonies mucoid. Xanthan gum has many industrial applications, such as oil drilling, food, agriculture and industry. Production of xanthan gum is also refuired for the virulence of this bacterium. To adapt to the diverse environmental changes, bacteria develop various responsive mechanisms. Bacteria are able to activate or switch off specific sets of genes as they face changing environmental conditions. This can be achieved through the activities of RNA polymerases containing alternate sigma factors and their cognate regulatory proteins. Biogenesis of flagellum is very complicate and is delicately regulated by several sigma factors and transcription regulators. Xcc fleN mutants were constructed by insertional mutagenesis. Mutation in fleN does not interfere with the pathogenicity and exocellular enzymes production and growth of the cell. Transcriptional fusion assay indicated that fleN Electron microscopy photography revealed that fleN mutants are hyper-flagellated but immotile, fleN regulates expression of six flagellar genes, including fliE, fliL, fliQ, flgB, flgG and flhF, in a negative manner. However, fleN is not necessery for the expression of the major flagellin gene fliC, nor for the type IV pilin gene pilA1. .
24
黃培瑄. "Transcriptional analysis of pilBA2A1CD cluster in xanthomonas campestris pv. campestris". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/56470531185800389731.
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碩士國立中興大學
分子生物學研究所
90
AU56E, a clp mutant derived from Xanthomonas campestris pv. campestris strain 17, synthesizes no significant amounts of xanthan gum (an exopolysaccharide). In addition, this mutant is resistant to filamentous phage fLf due to the failure in phage adsorption. Furthermore, studies carried out in our laboratory have indicated that 1) fLf infection is through attachment to the type IV pilus, the primary receptor, 2) pilRSBA1A2CD gene cluster is essential for the normal biogenesis of pilus, and 3) biogenesis of the type IV pilus is regulated by Clp, a global transcription factor. In order to understand the expression and regulation of genes pilA1, pilA2, pilB, pilC, and pilD, the promoter activities of pilA1, pilB and pilC were measured in two types of transcriptional fusions: one with the promoter-less xylE-GmW cartridge and the other with the promoter-less lacZ as the reporter. In the former assays, the cartridge was inserted into the coding regions of the genes to be studied while in the latter assays a promoter region was cloned upstream of the lacZ gene. The results of xylE-GmW reporter assays showed that pilA1 promoter is drastically reduced in clp mutant, indicating that it is positively regulated by Clp. No effects were observed in the expression of pilB and pilC promoters. In lacZ fusion assays, the result indicated that 1) neither growth of each strains nor the activities of the promoters were affected by fLf infection, 2) expression of the pilA1 and pilC promoter was higher in the wild-type Xc17 than those in AU56E, indicating that that transcription of pilA and pilC gene requires Clp, and 3) the promoter activities of pllA2, pilB and pilD were not affected by the mutation of clp gene. Primer extension analysis showed that the transcription start site of pilB is the T located at 15 nt upstream from the ATG start codon, and that of pilC is the G located at 21 nt upstream from the ATG start codon. In addition, Northern blot analysis results suggest that pilA1, pilA2 and pilC are transcribed from different promoters.
25
WU, CHENG-YU, e 吳承宇. "Molecular characterization of secretion genes in xanthomonas campestris pv. campestris". Thesis, 1990. http://ndltd.ncl.edu.tw/handle/77875113105871899036.
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26
Choy, Ka-Tim, e 蔡家添. "Physical Mapping and Gene Localization of Xanthomonas campestris pv. campestris". Thesis, 1994. http://ndltd.ncl.edu.tw/handle/84563355524873210594.
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碩士國立中興大學
分子生物研究所
82
Xanthomonas campestris pv. campestris is a Gram (-) bacterium which has a G+C content of about 65%. It is important in causing black rot in crucifers. In addition, it produces xanthan gum which has high viscosity and is widely used in industry. The purpose of this study was to construct a physical map of strain Xc17 and localize some genes in the physical map. Intact chromosomal DNA was prepared by embedding method, subjected to enzyme digestions with PacI, SpeI and SwaI, respectively, followed by pulsed-field gel electrophoresis running at different parameters to get optimum resolution. The sizes of the fragments were calculated by using lambda II, lambda ladder and yeast chromosomes as markers. During this study, several multi-bands with similar fragment sizes were found in the SpeI-digested samples. These problems are solved by two dimensional PFGE. By calculating the sum of the length of the fragments released by enzyme digestions, the Xc17 genome size was estimated to be 4725 kb. A Tn5 derivative, pUT-Tn5(pfm) CmKm, which carries sites for rare-cut restriction enzymes, including PacI, SpeI and SwaI, was used to mutagenize the Xc17 genes. Different type of mutants were thus obtained, which included Gum-, pigmentation, auxotrophic, protease and secretion mutants. The chromosomal DNAs of these mutant have one more rare cut restriction sites than that of the wild type at sites of Tn5 insertion. After PFGE and hybridization with appropriate probes, the site of Tn5 insertion in a mutant was determined. This in turn represents the location of that gene in the physical map. A combination of partial digestion, two dimensional PFGE, and Tn5 mutant analysis were the strategies employed for SwaI physical mapping. The results thus showed a fragment order WA-WC-WD-WF-WB-WE in the SwaI circular map. In addition, locations for the genes involved in uracil synthesis, a gene cluster required for the synthesis of xanthan gum and the dif site were assigned in physical map of SwaI.
27
Chen, Day-Yu, e 陳戴瑜. "Mutational studies of xpsD gene of Xanthomonas campestris pv. campestris". Thesis, 1994. http://ndltd.ncl.edu.tw/handle/56936596280293823056.
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碩士國立中興大學
分子生物研究所
82
XpsD 為一外胞膜酯蛋白，是十字花科黑腐病菌分泌胞外酵素所必需的蛋 白質之一。本研究利用一系列 xpsD 變異基因，探討 XpsD蛋白在胞外酵 素分泌過程中可能的功能。以西方墨點法檢查 XpsD在細胞內的分佈位置 ，結果顯示 XpsD變異蛋白之分佈與野生株相同，免疫螢光標定實驗亦顯 示，各變異蛋白均嵌入外胞膜，並有部分裸露於菌體外。除 pCD103及 pKU2 合成的 XpsD變異蛋白具分泌功能外，其餘皆喪失分泌功能。將變異 基因以廣寄主性載體送入野生株 XC1701，利用固體培養基檢查澱粉及 蛋白的分泌時發現在失去分泌功能的變異蛋白中,有部分呈現干擾分泌 的現象，包括 pCD105，pKD2，pKdA6 及 pYL4 所合成的 XpsD 變異蛋白 ；pMH7，pKdPs 及 pKD20 所表現者，則無顯著干擾。利用澱粉抗體檢 查XC1701轉接合菌株之澱粉分泌情形時， pMH7，pKdPs 及 pKD20表現 的 XpsD 變異蛋白亦呈現微弱的干擾現象。以 XpsD 抗體偵測 xpsD基因 表現時，除成熟型之 77kDa XpsD 蛋白產物外，尚有一分子量約為55kDa 之XpsD55 的產物出現，為探討 XpsD55是否為 xpsD基因中連續 6 個 A 經 translational frameshift 後，提早終止所產生。本研究第二部分將 連續6個 A 序列改換為 CAAGAA序列，並進行 6 個 A 序列的刪除，發現 對 XpsD55 的產生沒有影響；同時於6個 A 之上、下游構築 xpsD-phoA 融合基因的實驗結果，亦證實 XpsD55的出現，可能與 6 個 A 造成 frameshift 的關聯性不大。 XpsD is an outer membrane lipoprotein, required for the secretion of extracellular enzymes by Xanthomonas campestris pv. campestris. A series of mutant xpsD genes were constructed. Immunodetection of fractionated cell extracts using antibody against XpsD showed that the mutant XpsD proteins were, similar to the wild type XpsD, mainly located in membrane. Immunofluo- rescence labeling in intact cells further revealed that some regions of the proteins were exposed to the cell surface. Results from complementation experiments indicated that all mutants, except two (encoded in pCD103 and pKU2), were defective in protein secretion. Secretion interference was caused by introducing into XC1701 some (encoded in pCD105, pKD2, pKdA6, pYL4), but not the other (encoded in pMH7, pKdPs and pKD20) nonfunctional, mutant xpsD genes. Cell fractionation followed by immunodetection using antibody against α-amylase revealed that slight interference in secretion was also caused by mutant XpsD made from pMH7, pKdPs, and pKD20. The molecular weight of mature XpsD is 77kDa. In addition to the 77kDa product, a 55kDa protein, XpsD55, could also be detected on immunoblot. In order to study the significance of the A6T sequence in xpsD coding region in the production of XpsD55, base substitutions (CAAGAA), A6T deletion and XpsD-PhoA fusions were constructed. The results suggested that XpsD55 was probably not produced as a result of frameshifting downstream of A6T.
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Yu, Cheng-Tai, e 蔚承太. "Purification and Characterization of Xanthomonas campestris pv. campestris RNA Ploymerase". Thesis, 1994. http://ndltd.ncl.edu.tw/handle/24965549804306715399.
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碩士國立中興大學
分子生物研究所
82
In this study, we applied column chromatography and purified the RNA polymerase from the wild type strain of Xc.11. To achieve this purpose, 70.19 g of wet cell were collected and then broken by sonication, fractionation by 38% to 65% ammonium sulfate cut . The sample were applied into Heparin-Sepharose CL-6B column, and collected from bufferA with 0.2M, 0.35M and 0.5M KCl. The samples with enzyme activity eluted from 0.35M KCl were applied into Bio- Gel A 1.5 column and DEAE-Sephadex A 25 column and obstain 5.31mg purified RNA polymerase holoenzyme. The samples with enzyme activ- ity eluted from 0.5 M KCl buffer A were applied into Bio - Rex 70 column and Bio-Gel A 1.5 column and finally obtained 3.47mg holo- enzyme . In order to check the purity of the purified RNA polymerase,a two dimentional gel electrophoresis was performed and only four spots( beta'', beta,sigma and alpha) were found after staining. In the mean time,the 81.51kDa sigma factor of Xc11 was isolated from the SDS-PAGE and used to immunize the rabbit. Antiserum against the σsubunit of Xc.11 was then prepared and the title was deter- mined by Western-blot analyses. In order to examine the enzymatic activity of the Xc. 11 RNA polymerase several DNA templates were used in the transcription reactions in vitro found that (1) Xc 11 RNA polymerase can recog- nize E. coli lac promoter and produced a 198 nt transcript . ( 2) The purified Xc. 11 RNA polymerase can recognize either the E. coli thr promoter or terminator and produced a 164 nt terminated transcript and a 300 nt read - through transcript as E. coli RNA polymerase . (3) The purified Xc11 RNA polymerase also can recog- nized T7A1 promoter and produce 110 nt RNA product . (4) The pro- moter of xpsA is recognized well by the purified RNA polymerase and E. coli RNA polymerase.
29
Lee, Wen-Hung, e 李文弘. "Characterization of the ppk mutant of Xanthomonas campestris pv. campestris". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/63585401359034153133.
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碩士亞洲大學
生物科技學系碩士班
93
Xanthomonas campestris pv. campestris (Xcc) is an important plant pathogen which causes black rot disease in cruciferous plants. The ppk gene encodes a polyphosphate kinase (PPK) responsible for the synthesis of inorganic polyphosphate (poly P). Poly P is a polymer of tens to hundreds of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds , as in ATP. Poly P is ubiquitous , having been found in all organisms (archaea, bacteria, fungi, plants, insects, and animal cell). It is well known that poly P plays a role in the physiological adaptation of microbial cells during growth and development and is required for the adaptation to stress. Xcc ppk mutant is motile and is normal in the secretion of virulence factors, such as exopolysaccharide and exocellular enzymes which are essential for Xcc to invade and establish infections in host cells , production. Neither ppk mutants nor their parental strains can survive after a prolonged incubation at stationary phase. Both ppk mutants and wild-type strains are sensitive to acidified media (pH below 4.0). No homolog of rpoS is present in Xcc genome , that might explain the fragility of Xcc in stringency. In rich medium, ppk mutants grow as rapid as their parental strains. Nevertheless, ppk mutants present a serious delay in growth in poor medium especially after a downshift of nutrition. This suggests that ppk mutants need more time to adapt to an environmental change.
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Chang, Wen-Hui, e 張文輝. "Proteomic analysis of Clp-regulated Xanthomonas campestris pv. campestris genes". Thesis, 2003. http://ndltd.ncl.edu.tw/handle/53777619398096985145.
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碩士國立中興大學
分子生物學研究所
91
Xanthomonas campestris pv. campestris is a Gram-negative bacterium causing black-rot disease in crucifers. TC820 was a mutant derived from strain P20H by mini-Tn5 transposition at clp gene, encoding a protein homologous to cyclic AMP receptor protein (CRP), Clp (CRP-like protein). In this study, crude extracts prepared from P20H and TC820 were subjected to two-dimensional gel electrophoresis. In P20H and TC820, 201 and 148 spots were labeled, respectively, with 125 of the spots matched. Six of the proteins showing differences in amounts were analyzed by mass spectrometry. One of them, upregulated by Clp, was identified as GroEL encoded by groEL, a gene forming an operon with the upstream groES gene. To understand whether transcription of groESL gene is regulated by Clp, transcriptional fusion constructs using LacZ as the reporter were assayed for β-galactosidase levels. One construct (pWHP552) carried the region from —32 to —583 relative to groESL initiation codon, containing a putative Clp-binding site（-243 to -264）, a CIRCE element（-51 to -77）and a σ32 binding region（-10/-35 at -118~-127 and -142~-148, respectively）. The other (pWHP150) contained a 150 bp fragment（-29 to -178）without Clp-binding site. The results indicated that the activity of groESL promoter is about 3-fold lower in clp mutant TC820 than that in the parental P20H. Using antibodies specific to GroES for Western blotting revealed the amounts of GroES were found to be reduced in clp mutant TC820, confirming that Clp upregulates the expression of groESL operon. This is the first report identifying the stress-responsive groESL as Clp dependent.
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Huang, Yi-ling, e 黃伊伶. "Transcriptional regulation of pmeA gene in Xanthomonas campestris pv. campestris". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/10700874467252550548.
Texto completo da fonteResumo:
碩士中臺科技大學
醫學生物科技研究所
98
Abstract Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative plant-pathogenic bacterium that causes black rot in crucifers, resulting in tremendous losses in agriculture. This organism is capable of producing large amounts of exopolysaccharides and secreting several extracellular enzymes (such as protease, endoglucanase, mannanase, and pectinase), which have been considered to be important virulence determinants. The production of these extracellular products is coordinated by a cell-cell communication mechanism through diffusible signal factor (DSF), which is dependent on RpfF, a putative enoyl-CoA hydratase, for synthesis and the RpfC/RpfG two component system for perception and signal transduction. A recent study also indicated that global transcription factor cAMP receptor protein-like protein (Clp) is essential for DSF regulation of virulence factor production. The aim of this study was to characterize the pmeA gene encoding one of the pectinases, pectin methyl esterase (PME), in Xcc. First, the pmeA mutant YL17 and its complementary strain YL17(pRKpmeA) were constructed and the extracellular PME activity was compared with different Xcc strains. The result showed that the PME activity of AU56E (clp mutant), RM17F (rpfF mutant), and YL17 were slightly reduced, whereas the activity was restored in YL17(pRKpmeA). It was suggested that Clp and RpfF are involved in PME expression and pmeA gene might code for a secondary PME in Xcc. In pathogenicity test, the rate of symptoms development in YL17 was slightly slower than Xc17. These findings deduced that PmeA of Xcc plays a minor role in pathogenesis. Second, nucleotide G at 54 nt upstream of the pmeA start codon was mapped as the pmeA transcriptional initiation site by using the 5’-RACE (rapid amplication of cDNA ends) technique. Third, seven PpmeA-lacZ transcriptional fusion constructs were generated by cloning PCR fragments into the broad-host-range-promoter-probing vector pFY13-9, which uses lacZ as the reporter. The resultant PpmeA-lacZ transcriptional fusion constructs were introduced into Xc17, AU56E and RM17F by electroporation, respectively. Reporter assay indicated that the -359/+46 region contains the complete promoter and is capable of maximal-level expression. Transcriptional fusion analysis also revealed that the promoter activity of pmeA was reduced in either AU56E or RM17F, suggested that Clp and RpfF positively regulate transcription of the pmeA gene. In addition, the pmeA expression was affected by catabolite repression, osmolarity, oxygen limitation, and nitrogen starvation.
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Shen, Jheng-Syun, e 沈政勳. "Functional study of fliF gene in Xanthomonas campestris pv. campestris". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/13818431905553598846.
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碩士亞洲大學
生物科技學系碩士班
100
Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium. It can infect cruciferous plants and cause black rot disease. Xcc secrets various type of enzymes and polysaccharides which are essential for the bacterial virulence. Previous data from our lab have indicated that Xcc has a single polar flagellum. Biogenesis of the flagellum is regulated by the three-tier regulation of sigma factors RpoD, RpoN and FilA. A basal body composed of a flagellar type-III secretion system is essential for the secretion of the flagellin protein FliC. In other Gram-negative bacteria, FliF protein is the first element constituting the type III secretion system. Homolog of fliF was identified in the complete sequenced genome of Xcc. The aim of this study is to characterize the biological function of fliF gene in Xcc. Mutant of fliF was constructed by insertional mutagenesis. Phenotypes of the fliF mutant were analyzed by motility assay and pathogenic analysis. Regulatory function of fliF was analyzed by western blot. Interactions between FliF and other flagellar proteins were studied with the yeast-two hybrid system. The results demonstrated that 1) FliF is essential for the motility; 2) FliF is not involved in the pathogenicity; 3) FliF positively regulates the production of FliC, and 4) FliF interacts with FlhF protein.
33
Chen, Sho-Yi, e 陳脩易. "Characterization of the typA gene in Xanthomonas campestris pv. campestris". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/77286188697345000967.
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34
CHEN, DONG-TAI, e 陳東泰. "Nine filamentous phagesfrom Xanthomonas campestris pv. citri". Thesis, 1991. http://ndltd.ncl.edu.tw/handle/31235591119072936691.
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碩士國立中興大學
植物病理學研究所
79
Cf26、Cf29、Cf32、Cf86、Cf87、Cf89、Cf99、Cf117、Cf134等九個噬菌體分別從柑 桔潰瘍病菌的XW26、XW29、XW32、XW86、XW87、XW89、XW99、XW117、XW134等潛溶菌 株分離而得。此等噬菌體皆為絲狀, 大小在1250∼1550×8∼10nm 之間; 在XW47指示 菌株的頂層培養基上, 形成直徑0.2∼1.5毫米的稍為混濁或混濁的溶菌斑; 每一溶菌 斑含有約10 ∼10 PFU 的噬菌體顆粒; 以環狀的單股去氧核醣核酸組成它們的基因組 ,Cf117及其它8 個噬菌體基因組的分子量分別約為8.9kb 及7.9kb。 此等噬菌體在70 ∼80℃ 10分鐘, pH值為2,15瓦的紫外燈距60公分下照射90∼150 秒, 或懸浮在50mM 的硫酸亞鐵溶液內1 小時, 以及丙酮、氯仿、乙醇、正丁醇、二甲苯等有機溶劑內5 分鐘, 則接近完全或完全不活化; 當懸浮於蒸餾水、Nutrient broth、GY broth、磷 酸緩衝液、Tris緩衝液、或硫酸銨、醋酸鉛、硫酸鎂、氯化鉀、二鹽基磷酸鉀、氯化 鈉等50mM溶液中時, 有些噬菌體的感染率, 降低至大約1∼14%。此等噬菌體在XW47菌 株上吸附良好, 當感染比率為0.1,反應10分鐘, 吸附率可達到96.68∼99.95%。 當感 染比率為20, 感染24小時, 此等噬菌體皆能使XW47菌株潛溶化, 其百分率分別為93、 80、85、86、84、35、69、100、80%。此等噬菌體能感染柑桔潰瘍病菌的70個菌株中 之56個菌株, 但不能感染所供試的其它植物病原細菌及土壤、臨床、環境等細菌。
35
DONG, GUO-XIN, e 董國鑫. "Monoclonal antibody to xanthomonas campestris pv. cittri". Thesis, 1990. http://ndltd.ncl.edu.tw/handle/64646671354209534191.
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36
Wu, Cheng-Der, e 吳承德. "Cloning and Characterization of hrcA from Xanthomonas campestris pv. campestris 17". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/81769534272027949086.
Texto completo da fonteResumo:
碩士國立中興大學
分子生物學研究所
88
The hrcA and its flanking sequences of Xanthomonas campestris pv. campestris 17 were cloned from genomic library using the DNA fragment carrying the 3'' end of hrcA sequence as a probe. After subcloning and sequencing the X. campestris hrcA and its upstream sequence were determined. Sequence analysis showed that the 1,053 bp hrcA can code for a protein of 350 amino acids with a predicted molecular mass of 38.3 kDa and PI of 5.87. Comparison of the amino acid sequences with those of other HrcA showed 36% identity to that of the HrcA of Caulobacter crescentus and 34% identity to that of the HrcA of Bradyrhizobium japonicum. The putative ribosomal binding site is located 6 to 13 bp upstream from the translational start codon ATG of hrcA, and the putative terminator is located 7 to 23 bp downstream from the translational stop codon TAA. Evolutionary analysis of the relationship of various HrcA sequences appeared that X. campestris is most closely related to C. crescentus, B. japonicum and Agrobacterium tumefaciens. The first 100 amino acids of HrcA are most consensus suggesting that this region maybe important in function. Northern blotting analysis detected an mRNA with a size similar to that of the X. campestris hrcA coding region suggesting the hrcA gene-containing transcript may be monocistronic. Primer extension showed that the nucleotide cytosine locating at 20 nt upstream from the translational start codon of the X. campestris hrcA is the possible transcriptional initiation site. The putative promoter region was cloned into promoter-proving vector pFY7 for analysis. The hrcA promoter activity was detectable under nonstressed condition but was not inducible by heat shock treatment. In addition, the DNA fragment containing the full length hrcA and the N-terminal deleted hrcA were cloned for protein expression. Based on sequences available in database, hrcA present in many eubacteria but not in Escherichia coli, Haemophilus influenzae, Aquifex aeolicus and archaebacteria. Therefore, it is suggested that hrcA genes evolved in eubacteria after diverging from archaebacteria evolution.
37
Wang, Szu-Wen, e 王仕文. "Construction and Characterization of Xanthomonas campestris pv. campestris 17 hrcA Mutant". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/76465066687145192425.
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碩士國立中興大學
分子生物學研究所
88
Plasmid pOKhrca carrying a DNA fragment span within the heat shock regulatory gene hrcA was constructed. After electroporation of pOKhrca into Xanthomonas campestris pv. campestris 17, several Kmr transformants were obtained, and one of them was verified to be a Xc17 hrcA mutant by Southern blotting. The growth rate of the hrcA mutant was slightly faster than that of Xc17 at 37℃. Northern blot analysis suggested that the expression of groESL in Xc17was mainly regulated by sigma factor s32, not by HrcA. Western blot analysis showed high levels of GroEL in hrcA mutant and Xc17; after heat shock, only slight increase of GroEL was observed in both strains. 2-D gel electrophoresis results showed no obvious differences between protein profiles of Xc17 and hrcA mutant grown at 28℃. However, after heat shock at 37℃ for 20 min, a novel protein of 20 kDa, with a pI of 5.8 was observed on the 2-D map of the Xc17 or hrcA mutant, which was named Hsp20. The N-terminal 15 amino acids determined chemically share identity with the corresponding regions of small heat shock proteins from different organisms. According to the N terminal sequence of Hsp20 and the consensus sequence in the a-crystalline domain of the small heat shock proteins, two degenerated primers were designed. Using these primers, a 194 bp PCR fragment was amplified from Xc17 chromosome and sequenced. The amino acid sequence encoded by the 194 bp DNA fragment showed 86% identity to a low molecular weight heat shock protein of Xylella fastidiosa. The effect of heat shock response on the level of hsp20 expression was analyzed by Northern blot analysis. Results showed that the expression of hsp20 is greatly increased after heat shock at 37℃ for 15 min.
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Chang, Chun-Yan, e 張君妍. "The study of regulatory protein, AlgR in Xanthomonas campestris pv. campestris". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/48546956713086174625.
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碩士臺中健康暨管理學院
生物資訊研究所
94
Abstract Lf is a filamentous bacteriophage which specifically infects Xanthomonas campestris pv. campestris. A Lf resistant X. campestris strain was isolated by mini-Tn5 transposon insertional mutagenesis. The insertion site was mapped within algR, gene encoding for a transcriptional activator belonging to the microbial two component systems. Mutants of algR and algZ, the cognate sensor gene of algR, were reconstructed by insertional mutagenesis and the phenotypes of mutants were analyzed and summarized: (1) algR mutant is resistant to Lf infection and algZ mutant is still sensitive to Lf. (2) algR and algZ mutants showed no difference in colony morphology, growth rate and extracellular enzymes secretion. And (3) algR mutant, but not algZ mutant, moved slower than their parental strain on a semisolid plant. The phage sensitivity and mobility deficiency of the algR mutant were recovered after introducing an algR gene containing plasmid, pBBADalgR. A DNA-binding domain LytTR (FhRhH[RK][SNQ]hhVN) which binds specifically to [TA][AC][CA]GTTN[AG][TG] sequence, was identified in the c-terminal of AlgR. To identify putative AlgR-regulated gene, RSAT consensus program was used to search in X. campestris genome for AlgR-binding consensus sequence. Five genes (fabA, gltD, plcN, phoR and slp) were selected for promoter activity assay. The results indicated that expression of fabA, gltD, plcN and phoR genes were not dependent on AlgR or AlgZ. slp gene promoter activity mutant was significantly lower in an algR mutant than in an algZ mutant or in their parental strain Xc17. This suggests that slp gene is positively regulated by algR. The early step of Lf infection depends on type IV pilus. Gene pilA1 encodes for the mayor pilin of type IV pilus. The promoter assay demonstrated that the expression of pilA1 gene is positively regulated by AlgR.
39
Deng, Fu-Sheng, e 鄧福勝. "Translocation and expression of β-lactamase in Xanthomonas campestris pv. campestris". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/fjtjed.
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40
Chi-Kuo, Hu, e 胡智國. "The locational analysis of endogeneous IS1404s in Xanthomonas campestris pv. campestris". Thesis, 1999. http://ndltd.ncl.edu.tw/handle/13455349563604571453.
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碩士輔仁大學
生物學系
87
Three DNA fragments containing iso-IS1404s, named IS1404a, IS1404c and IS1404d, from Xanthomonas campestris pv. campestris Xcc1-1 genomic DNA were cloned and sequenced. Based on nucleotide sequence analysis of the flanking sequences of these iso-IS1404s, IS1404a is located in the ORFb of transposase gene of a new IS3 family member and IS476-like insertion sequence, ISA2. In addition to IS1404a, another new IS3 family member and IS1400-like insertion sequence, ISA1, also integrate the same ISA2. IS1404c is located in the transposase gene of an IS5 family insertion sequence, IS1478. IS1404d is transposed in an open reading frame (ORF) whose deduced amino acid sequence shows homology to virD4 of Agrobacterium tumefaciens, traG and taxB of Escherichia coli, and cag5 pathogenicity island gene of Helicobacter pylori. The ORF was named vag1 for virD4-taxB--traG-like gene. Further analysis of the flanking sequences of four iso-IS1404s, including IS1404b from Xcc14-1, revealed that there is not any conserved sequence or hot spot for IS1404 transposition, but the sequences are GC-rich and contain a 5-bp inverted-repeat consensus sequence In addition to vag1, another two DNA fragments of Xcc1-1 genome, 3.9-kb and 8.3-kb EcoRI DNA fragments, contained genes homologous to vag1 determined by Southern hybridization. The 3.9-kb EcoRI DNA fragment were cloned and sequenced. Sequence analysis revealed that the fragment contains an operon (named vag2AB) with two open reading frames, named vag2A and vag2B, respectively. There is 92.34% identity between vag1 and vag2A nucleotide sequence. The amino-terminus region of Vag2A has a signal peptide and two hydrophobic regions, which may function for membrane-spanning. Vag2A also contains two NTP-binding Walker motifs, which are highly conserved in VirD4, TraG, TaxB, and Cag5. In 8.3-kb EcoRI DNA fragment, there is another complete and vag2A-like gene, named vag3, determined by polymerase chain reaction (PCR). The results indicated that vag genes in the genome of Xcc1-1 exist in three different forms and can be expressed as vag1::IS1404, vag2AB, vag3. When an internal fragment of vag2A was used as a probe, three heterogeneous groups (A, B, and C) of X. campestris pv. campestris can be easily distinguished by hybridization patterns. Strains in each group have two or three EcoRI fragments hybridized to vag2A, indicating that strains contain at least two vag genes. Further PCR and Southern hybridization analyses revealed (1) that all tested strains in group A and some strains in group B have a vag gene integrated by IS1404, but no IS1404-integrated vag gene was found in group C strains; (2) that strains in group A, B, and C contain at least one complete and vag2A-like gene. For example, Xcc1-1 of group A contains vag2A and vag3; (3) that vag2B and vag2AB operon only exist in strains of group A, but not in group B and C. The results suggested that vag3 of Xcc1-1 might be a common vag gene existing in X. campestris pv. campestris, and vag2AB was acquired by horizontal gene transfer. A Xcc1-1 vag2A knock-out mutant strain (vag1::IS1404, vag2A::WB, vag3) was obtained by a marker-exchange method. No difference was found between vag2A knock-out mutant and wild type of Xcc1-1 in the capacities to cause disease in cabbage (host plants) and to induce hypersensitive reaction in tobacco (non-host plants). The vag gene is widely distributed in pathovars of Xanthomonas spp. but do not exist in other phytopathogenic bacteria such as Erwinia, Pseudomonas, and Ralstonia solanacearum. According, vag gene is a Xanthomonas-specific gene.
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Wang, Tsai-Yuh, e 王彩諭. "Characterization of the Reconstituted RNA Polymerase from Xanthomonas campestris pv. campestris". Thesis, 1999. http://ndltd.ncl.edu.tw/handle/42836205073532390667.
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碩士國立中興大學
分子生物學研究所
87
Abstract After identified and characterized each subunits of Xc 11 RNA polymerase, the in vitro function of Xc 11 RNA polymerase by means of reconstituted RNA polymerase was subjected to be studied. The DNA fragment of each subunit were amplified by PCR with proof-reading Pfu or UlTma polymerase, and subcloned into protein expression vector pET-21. Concerning with the possibility of introducing mutation into DNA fragment after PCR amplification, we have sequenced the full length DNA of each RNA polymerase subunit genes in the protein expression plasmids. Two mutations (G139S and P717L) were found in the deduced amino acid sequence of RpoB, one mutation (P488A) in RpoD and none in other subunits. Cell cultures were induced with IPTG to overexpress the cloned DNA fragment and after passing the cell extract through Ni2+-NTA agarose column the purified subunits were reconstituted into RNA polymerase. However, no transcript could be detected when in vitro transcription assay was performed with the reconstituted RNA polymerase. The reasons for losing activity of reconstituted RNA polymerase are remained to establish. However, it could be either from deactivation of RpoB and RpoC proteins by long purification time, or mutation in RpoB and RopD. In order to distinguish these two possibilities, we have constructed Xc 11 mutant strain, Xc TY109, which has a truncated rpoC gene in its chromosome and the functional RpoC has His-tag in its C terminus after translation. The b￠His RNA polymerase holoenzymeof Xc TY109 could be partially purified through Ni2+-NTA agarose column and the yield was about 6 mg RNA polymerase per liter of cell culture. The b￠His RNA polymerase was denatured by 6M Guanidine-HCl, and the enzyme was reconstituted following the same procedures. The reconstituted b￠His RNA polymerase regain its transcription activity was identified by in vitro transcription assay.
42
Shao, Yuan-Yuan, e 邵源源. "Expression and Regulation of Xanthomonas campestris pv. campestris α-Amylase Gene". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/40911903765128769440.
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碩士國立中興大學
分子生物研究所
84
Xanthomonas campestris pv. campestris (Xc)可分泌產生胞外酵素 α- amylase，將可溶性 starch 水解成 maltose、部分 maltotriose 以及微 量之 glucose。為探討 Xc α-amy gene 啟動子之表現與調控，首先進行 Xc α-amy gene 上游核甘酸之定序。得到之核甘酸序列包含 α-amy gene coding region 之 255th bp 起至 5'' 端，全長共 1091 bps。分析 結果顯示，α-amy 基因之調節區域可能包含 putative upstream activator-binding site ΩUAS, potential promoter -10/-35 cononical sequences, 2 段 inverted repeats ΩI 及 ΩII, 與 Shine-Dalgarno sequence (SD)。本研究中，利用報導基因 luxAB 反應 Xc α-amy gene 啟動子表現，選殖了調節區域並作進一步分析。實驗結 果顯示，在 maltose medium 中 Xc α-amy 啟動子及調控區域因受到誘 發而表現最高；在 starch、glucose 及 sucrose medium 中，亦有良好 之螢光表現；在 lactose、galactose、fructose 或 glycerol medium 中之螢光表現則非常低。推測 maltose 為一重要之 inducer 直接或間接 誘發 positive regulator 調控α-amy 之表現。 Xc17(AMY800) 在 glucose 中生長時，其螢光表現較 α-amylase 酵素活性為高，故在轉錄 層面，其對α-amy 有誘發 之功能，但在轉錄後或轉譯後，則可能有 glucose inactivation 之作用。因此 glucose 對 Xc α-amy gene 之調 控則可能是多層面的。利用刪短與突變等方式測試 α-amy 調節區域之核 甘酸序列之功能及重要性後，結果顯示 α-amy 轉錄起始點 (+1) 上游 -152th 至 -117th bp 處，可能為啟動子之邊界區域。當除去 putative -35 region，仍可有極弱之表現。此外， putative -35 region 內之 -158th 及 -147th bp 位置的 G，對啟動子之效率亦十分重要。可能由於 這是 -35 region ，核甘酸序列之改變影響轉錄因子與 DNA 的接合能力 。當 ΩUAS 之 inverted repeat 上游一段序列被刪除時，maltose 誘發 之現象即開始減弱。以 site-directed mutagenesis 將 ΩUAS 之對稱性 破壞後，α-amy 受 maltose 及 glucose 誘發之現象與原來相同，但在 galactose medium 中表現受 repression 之現象則被解除。故 ΩUAS 之 對稱性可能與 galactose 有關之 negative regulator 認辨所須。將 pAMY800 送入 E. coli 中，螢光表現測試之結果顯示，Xc α-amy 在 E. coli 不表現。
43
Du, Shin-Chiao, e 杜歆喬. "Characterization of galE and galU Genes in Xanthomonas Campestris pv. Campestris". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/27039360000747718760.
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碩士中臺科技大學
醫學檢驗生物技術系碩士班
101
UDP-galactose 4-epimerase (encoded by galE gene) and UTP-glucose-1-phosphate uridylyltransferase (encoded by galU gene) are enzymes responsible for the synthesis of UDP-galactose and UDP-glucose, substrates involved in the synthesis of different surface structures (lipopolysaccharide, exopolysaccharide, and capsular polysaccharide) in many bacteria. In a number of Gram-negative pathogens, galE and galU are known as important virulence factors. Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative plant-pathogenic bacterium causing black rot in crucifers. The virulence of Xcc depends on a number of factors, including exopolysaccharide and extracellular enzymes. The expression of these virulence determinants is upregulated by RpfF and Clp. Although putative galE and galU genes have been annotated in the fully sequenced Xcc genome, the biological functions are unclear. The aims of this study were to functionally characterize the galE and galU genes in Xcc. Seqeunce and mutational analysis indicated that the Xcc galE encodes a UDP-galactose 4-epimerase and the putative catalytic residues (S123, Y147 and K151) in GalE are essential for epimerase activity. The galE transcription initiation site was located at nucleotide G, 201 nt upstream of the start coden. Reporter assay indicated that galE transcription is positively regulated by RpfF and Clp. Phenotypic evaluation and genetic complementation showed that inactivation of galE caused reduction of bacterial attachment and GalE activity. In addition, it was also found that galU is involved in extracellular protease activity, exopolysaccharide and lipopolysaccharide production, bacterial attachment, cell motililty, and pathogenicity of Xcc. This is the first time that galE and galU genes have been characterized in the crucifer pathogen Xcc.
44
Chien, Hui-Chen Chang, e 張簡惠甄. "Characterization of the fliA, flgM, flhF genesin Xanthomonas campestris pv. campestris". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/40096826737703368468.
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碩士亞洲大學
生物科技學系碩士班
95
Xanthomonas campestris pv. campestris (XCC) is a Gram negative, rod-shaped bacterium. It is the causative agent of black rot disease in cruciferous plants. The disease results in heavy loss in agriculture worldwide. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive and necrosis. Full leaf yellowing, wilting, and necrosis occur as the disease advances. XCC has a single unipolar flagellum. From the completed genome sequence of XCC, more than 40 genes are predicted to be involved in the flagellar biogenesis. Research results in our laboratory showed that FleQ and RpoN2 positively regulate and FleN negatively regulates the XCC flagellar biosynthesis by up- or downregulating the promoter activity of seven flagellar operons, fliQ, flhF, flgG, fliL, fliC, flgB and fliE, respectively. The purpose of we were to elucidate the role of fliA, encoding a putative σ28 factor, flgM, encoding a putative anti-σ28 factor and flhF which was reported to be a positive regulator of flagellar biogenesis in Pseudomonas. Mutants of fliA, flgM or flhF were constructed by insertional mutagenesis and the phenotypes of the mutant were analyzed. The results showed that 1) fliA, flgM and flhF mutants have a decreased motility, 2) deficiency in motility of flgM mutant can be restored by a plasmid-born flgM, 3) as shown by transmission electron microscopy (TEM) fliA mutants have no flagellum; flgM mutants have a polar but shorter flagellum and flhF mutants have one to two lateral flagella, 4) mutations in any of these genes affect the growth rate in a poor medium, 5) mutations in fliA, flgM or flhF also decrease the pathogenicity. 6) all the three mutants are still sensitive to the infection of bacteriophage φLf and φL7, and 7) none of the mutation changed activity of extracellular enzymes. 6 The results of transcriptional fusion assays demonstrated that 1) FliA positively regulates the expression of fliC and negatively regulated the expression of the fliQ, flhF and flgG operons, 2) FlgM positively regulates the expression of flgB, fliQ, flhF and fliL operons and negatively regulate the expression of the fliC and flgG operons, and 3) FlhF positively regulates the expression of fliE and pilA1 and negatively regulates the expression of flgB, fliQ and fliL. The results of Western blot demonstrated that 1) RpoN2 positively regulated the expression of FliA, and 2) expression of fliC is positively regulated by FliA and RpoN2, but is negatively regulated by FlgM.
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Chien, Hsu-Ling, e 簡旭伶. "Regulation of pglA Gene of Plant Pathogenic Xanthomonas campestris pv. campestris". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/62414802313961724839.
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碩士國立中興大學
分子生物學研究所
95
Abstract The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) causes black rot in crucifers. This bacterium produces large mounts of an exopolysaccharide (EPS), xanthan gum, and an array of extracellular enzymes, including proteases, pectinases and cellulases. These substances are collectively required for pathogenicity. In addition to these substances, Avr (avirulence) proteins and the hrp genes encoded Type III secretory pathway are also essential for virulence. Regulation of the virulence factors in Xcc is not fully understood, but it is known that rpf (regulation of pathogenicity factors) gene cluster and the global transcription factor Clp（cAMP receptor protein-like protein）are involved in regulation of EPS and the extracellular enzymes. In Xcc, pglA is a pectinase gene encoding one of the polygalacturonases. Sequence analysis performed previously indicated that the upstream region of this gene has a region conserved to a Clp-binding site and a putative HrpX-binding site (plant-inducible promoter, also called PIP box). The purposes of this study were to investigate the regulation of the pglA gene by Clp, rpf system and HrpX protein. My sequence analysis revealed that PglA belongs to family 28 of glycosyl hydrolases, which typically have four conserved domains (NTD, DD, HG, and RIK). In plate assays, the pglA mutant constructed by marker exchange exhibited extracellular polygalacturonase at a level similar that of the wild-type, indicating that pglA is not the major pectinase in Xcc. In pathogenicity tests, the symptoms caused by the pglA mutant appeared slightly slower than Xc17. Using 5’RACE technique, nucleotide A at 102 nt upstream of the pglA translation initiation codon GTG was mapped as the 5’ end of pglA mRNA, the transcription star site. Gel retardation assays revealed that Clp can directly bind to the pglA promoter. Nested fragments of the pglA upstream region were separately cloned into the broad host range promoter-probing vector pFY13-9 to form transcriptional fusion constructs, each of which was then introduced by electroporation into the wild-type Xc17, clp mutant Xc17(clp::Gm), rpfF mutant Xc17(rpfF::Gm), and hrp mutant Xc17(hrpX::Gm). Results of reporter assays indicated that when the cells were grown in the modified XVM2 medium supplemented with 0.5% polygalacturonic acid, the pglA promoter activity was greatly reduced in hrpX mutant compared to that in than Xc17, but increased by 2.27 and 1.76 times in clp and rpfF mutant, respectively.
46
Wang, Yun-Ching, e 王雲慶. "Characterization of the Xanthomonas campestris pv. campestris lexA1 and lexA2 Regulons". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/68391760802020700583.
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碩士國立中興大學
分子生物學研究所
94
Two lexA genes, named lexA1 and lexA2, had been identified in Xanthomonas campestris pv. campestris strain 17 (Xc17) in our previously studies. Computational search for genes possibly regulated by LexA1 and LexA2 proteins in the available genomic sequence of the X. campestris showed that the sequence in the upstream region of umuDC gene has an identical LexA1 binding motif. Southern blot analysis revealed that umuDC gene only exists in X. campestris ATCC33913 among the ten Xanthomonas strains studied. UmuD gene was demonstrated to be positively regulated by LexA1 protein using UV sensitivity test and promoter activity assay. To elucidate the regulatory mechanisms of the two lexA genes and recA in SOS response in the ten Xanthomonas strains, a currently constructed lexA1 mutant strain (XDA1) and previous constructed lexA2 mutant strain (XDA2) and recA mutant strain (NT3) were used in this study. Growth curves showed no difference among mutants and wild type strains. The XDA1, XDA2 and NT3 mutant strains were found to be UV sensitive because an exposure to 300 J/m2 UV irradiation resulted in respective 220, 200 and 3370-fold decrease in survival rate. LexA1, lexA2 and recA genes should have their importance when DNA was damaged. Because the recA and recX genes are located just downstream of lexA1 in Xc17, the promoter activities of lexA1, recA, and recX were determined. The results indicated that the lexA1 and recA promoters were induced by MMC but recX was not. On the other hand, it has been shown that sulA2, dinP2, and dnaE2 genes were located just downstream of the lexA2 gene. Promoter activity assay indicated that the lexA2 was induced by MMC and dinP2 was slightly induced but sulA2 and dnaE2 were not. Northern blot analysis and RT-PCR indicated that the two lexA genes could be regulated to form operons lexA1-recA-recX and lexA2-sulA2-dinP2-dnaE2 for downstream gene expression. By using the lacZ as a reporter gene to measure the promoter activity of lexA1, lexA2 and recA, the transcriptional activities of these promoters were enhanced by MMC and the induction was time-dependent. After inducing for 3 hours, the recA increased 3.5-fold but the ratio of lexA1 and lexA2 was 1.1~1.4-fold, indicating that the recA promoter was the first target among these three promoters. Because recA is the most important gene in SOS response, the genes affecting recA expression were investigated by Tn5 mutagenesis. To date, recD, recC and piuB mutants were confirmed to be three of the mutagenesis colonies and waited for further studies of the relationship between piuB, recD, recC and recA.
47
Chen, Ching-Jiun, e 陳俊磬. "Study on SOS response related genes in Xanthomonas campestris pv. campestris". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/74375043032962647410.
Texto completo da fonteResumo:
碩士國立中興大學
分子生物學研究所
94
It has been demonstrated the Xanthomonas campestris pv. campestris strain 17 (Xc17) contains two function and independent lexA genes. One of them (lexA1) constitutes a lexA-recA-recX operon and the second lexA (lexA2) belongs to a multiple gene cassette with the following gene order: lexA2-sulA2-dinP2-dnaE2. In the previous work, the Xc17 lexA1 regulon has been showed to be different form that E.coli and an analysis of the sequence of the recA upstream region revealed that the sequences required for LexA1 and LexA2 binding was not present. The aim of this study was to explore the regulatory mechanism of SOS response in Xc17. A total of 29 SOS response related genes in Xc17 were selected and investigated their expression levels in wild-type or LexA1 deficient (XDA1) and LexA2 deficient (XDA2) strains by RT-PCR. Results revealed that only genes in the lexA1 and lexA2 operons were affected by the corresponding mutant of the operon. Additionally, except genes in the lexA1 and lexA2 operons, the expression levels of the six tested genes, dinG, dinP, recN, icfG, rfaY and algU were found to be induced by mitomycin C (MMC) treatment. To measure of the transcriptional responses induced by DNA damage, the promoters of these genes were each cloned and fused to the promoterless lacZ reporter present within plasmid pFY13-9 and then introduced into wild-type, recA-(NT3) and lexA deficient Xc17 strains. Results revealed that the transcriptional activities of the dinG, dinP, recN and algU were DNA damage inducible. No significant different in the transcriptional activities of the dinG, dinP and recN promoters in the wild-type and lexA deficient Xc17 strains were observed. However, the promoter activity of algU was relatively higher in XDA1 and lower in NT3 than that of the wild-type strain. These results were further confirmed by real-time PCR analyses. Moreover, DNA damage induced by exposure of Xc17 strain to UV radiation was carried out and the promoter activities of these genes were elucidated by real-time PCR analyses. Results showed that the expression levels of most of these genes were relatively higher by UV treatment as compared to MMC induction. Finally, the transcription start sites of the dinG, dinP and recN were identified by primer-extension analyses and no typical -35 and -10 promoter consensus sequences of these genes were observed. Deficient of the LexA1 and LexA2 binding sequences in the upstream regions of these genes was also confirmed by gel retardation alalysis.
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Yang, Shu-hui, e 楊淑惠. "The study of regulatory protein, FleQ in Xanthomonas campestris pv. campestris". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/50995752607181959016.
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碩士臺中健康暨管理學院
生物資訊研究所
92
To adapt to the diverse environmental changes, bacteria develop various methods. One of the methods is to use the alternative sigma factors to regulate gene expression. The 54 factor, encoded by rpoN, regulates genes involved in utilization of nitrogen and carbon sources, synthesis of flagellum and pilus, pathogenicity, RNA modification, and chemotaxis. Two functionally independent rpoN genes have been identified in Xanthomonas campestris pv. campestris (XCC). An interaction with an NtrC-like activator (also called enhancer-binding protein, EBP) is essential for activation of the transcription initiation by a 54-containing RNA polymerase. Activation of EBP is mediated by a series of eukaryotic-like phosphorylation-dephosphorylation pathways. Based on sequence homology to the 54-activating domains present in other bacteria (Pfam PF00158), eight and nine EBPs were identified in the genome of XCC strain 33913 and XC17, respectively, including among others the fleQ gene downstream of rpoN2 essential for flagellum biogenesis. In Pseudomonas aeruginosa, FleQ works in concert with RpoN in activating flagellar genes. To investigate the role fleQ plays in XCC, a fleQ mutant from XC17 was constructed by insertional mutation. This mutant was found to be immotile and does not possess flagellum. However, the mutation does not interfere with the pathogenicity and growth of the cell. Promoter-probing assays were carried out to elucidate the role fleQ plays in regulation of expression of the 54-dependent promoters. The results showed that FleQ is required for the expression of flagellar genes (fliE, fliL, fliQ, flgB, flgG and flhF) which are RpoN2-dependent. Nevertheless, mutation in fleQ does not interfere with the expression of RpoN1-dependent genes, such as pilF (encoding fimbrial biogenesis protein), pilA1 (encoding pilin), nasA (encoding nitrate transporter), prpB (encoding carboxyphosphonoenolpyruvate phosphonomutase) and glnA (encoding glutamine synthetase). Putative FleQ binding sites were predicted by bioinformatics tools. A consensus sequence containing a GC-rich region flanked by an upstream oligo-A and a downstream oligo-T regions was identified immediately upstream of the RpoN-binding site in each of the analyzed flagellar promoters. Destruction of this motif in fliE gene was shown to eliminate the normal promoter function. This data suggests that this region may play an essential role in the interaction with FleQ.
49
Chen, Po-hsuan, e 陳柏軒. "Functional analysis and application of vagA of Xanthomonas campestris pv. campestris". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/24932032101804625138.
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碩士輔仁大學
生物學系
89
The study of the insertion sequence IS1404 of Xanthomonas campestris pv. campestris (XCC) leads to a discovery of a new gene, whose deduced amino acid sequence shows homology to VirD4 of Agrobacterium tumefaciens, TraG and TaxB of Escherichia coli, Cag5 of Helicobacter pylori, and RP293 of Rickettesia prowazekii. Those proteins contain NTP-binding Walker motif A and B, and the identity and similarity of the amino acid sequences range from 17% to 29% and 31% to 48%, respectively. The G+C content of the nucleotide sequences is from 33.3% to 63.6%. The gene knock-out mutant strain of XCC33 was obtained by a marker-exchange method. It was found that the appearance of symptoms was later than that of wild-type about three days after inoculation of the knock-out mutant and wild-type strains into cabbage, respectively. However, after symptom appeared, the rate of symptom progress was not significantly different between mutant and wild type strains. The results indicated that the gene was involved in initiation step of symptom development. Since the gene is related to the virulence of X. campestris pv. campestris, it was named vagA (virulence-associated gene; GenBank accession no. AF360374). The 64.3-kD VagA protein was produced in E. coli pET system. Although most of VagA were insoluble and formed an inclusion body, a few soluble VagA still existed in soluble portion and can be purified. The purified soluble VagA has an NTPase activity, and prefers dATP and dGTP to dTTP and dCTP as substrates. The specific activity is 48.92 unit/mg for dATP, 74.71 unit/mg for dGTP, 22.43 unit/mg for dTTP, and 10.03 unit/mg for dCTP. Based on Southern hybridization, it was found that vagA gene is widely distributed in pathovars of Xanthomonas spp. but does not exist in other phytopathogenic bacteria, such as Erwinia, Pseudomonas, and Ralstonia solanacearum. Comparison of nucleotide sequences of ten vagA partial fragments cloned from other pathovars of Xanthomonas spp. revealed that the identity of nucleotide sequences ranges from 71% to 100%, indicating that vagA gene is conserved in Xanthomonas spp. A PCR primer set was designed based on conserved regions of vagA, and can amplified a 0.4-kb PCR product from all pathovars of Xanthomonas spp. tested, but not from other plant pathogenic bacteria in a PCR reaction. Therefore, the primer set is Xanthomonas-specific and can be used to identify pathogenic Xanthomonas spp. Analysis of a DNA fragment containing vagA from X. campestris pv. citri XCI3-1 revealed that there is an ORF, vagX, in the down stream of vagA, and its deduced amino acid sequence shows similarity to VirB8 of A. tumefaciens VirB8, TraE of E. coli, and Cag10 of H. pylori, which are also involved in type IV secretion/transfer system. In conclusion, plant pathogenic Xanthomonas spp. contain a vagA, which affects symptom development and has NTPase activity, and the genes clustered with vagA and vagX are related to type IV secretion/transfer system.
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Hsia, Tai Wei, e 夏代威. "Cloning and analysis of lon gene in Xanthomonas campestris pv campestris". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/25435732332034877119.
Texto completo da fonteResumo:
碩士國立中興大學
分子生物學研究所
89
Xanthomonas campestris pv. campestris is a gram-negative, rod-shaped and monotrichously flagellated plant pathogenic bacterium. The chromosome of X. campestris pv. campestris has a G + C content more then 60%. It produces copious amounts of xanthan gum, which has been implicated to associate with the virulence of the bacterium. Biosynthesis of xanthan gum involves multiple steps and a multitude of enzymes. Recently, our laboratory has localized the genes involved in xanthan synthesis at eight loci on the X. campestris pv. campestris 17 (Xc17) chromosome map. In E. coli, lon gene mutations made cell mucoid, UV sensitive and long form cell. In this study, I employed a homologous recombination strategy to clone the of lon gene from the Xc17 chromosome . pOKRV carrying the DNA fragment of complete lon gene as thus obtained. Sequence analysis revealed 3955 bp containing two ORFs. The lon gene, nt 721 to nt 3189; encodes a protein that has 823 amino acids with a molecule weight of 90,631.27. The Lon protease deduced from the Xc17 possesses 65% similarity to that of E. coli and X. fastidiosa. Two motifs include the gene are ATP/GTP binding motif and ATP-dependent serine protease active site. The hupb, nt 3307 to 3680; encodes a protein 90 amino acids with a molecule weight of 9327.13, whose product is named Histone-like DNA binding protein. The sequence similarity of hupb is more then 65% with E. coli and P. fluorescens. A motif, the bacterial histone-like DNA-binding proteins signature motif, is present in the deduced HupB protein. Mutants of lon were constructed by insertional mutagenesis. No differences were found in colony morphology, pathogenicity, growth rate and UV-sensitivity between the mutant and the wild type cells