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Mouville, Clémence. "Interaction entre les pili de type IV et le phage filamenteux MDA : impact potentiel sur la virulence de Neisseria meningitidis". Electronic Thesis or Diss., Université Paris Cité, 2024. http://www.theses.fr/2024UNIP5235.
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Neisseria meningitidis (Nm) est une bactérie commensale du nasopharynx humain qui traverse quelques fois la barrière nasopharyngée et se propage dans la circulation sanguine jusqu'à atteindre les méninges. Un bactériophage filamenteux appelé MDA (Meningococcal Disease Associated) est associé aux infections invasives à méningocoques chez les jeunes adultes. Le MDA semble augmenter l'incidence de la maladie en augmentant la colonisation bactérienne au point d'entrée. L'objectif de ce travail a été de comprendre le mécanisme moléculaire précis de l'infection de Nm par le MDA. L'étude des mutants des gènes impliqués dans la machinerie des pili de type IV (PT4) a montré que l'entrée du phage nécessite un PT4 rétractable. Ce résultat est cohérent avec la littérature sur les phages Ff ou CTX qui interagissent directement avec l'extrémité du pilus. Mais aucune preuve de l'interaction du MDA avec l'extrémité du pilus n'a été trouvée. L'interaction possible entre la fibre du pilus et la capside du phage a été examinée. Comme PilE, la piline majeure, est sujette à variation antigénique, des variants de PilE qui diminuent l'entrée du phage ont été identifiés. L'infection par le phage se produit dans des populations de bactéries exprimant des séquences de PilE spécifiques. Par imagerie, nous avons montré que les pili et les MDA s'associaient ensemble. Une analyse de la charge des acides aminés de PilE et celle de la capside soutient l'hypothèse d'une interaction variable selon le variant de PilE. Finalement, il a été montré que les P4T avec un potentiel électrostatique positif favorisaient l'infection par le phage et permettaient, de plus, une forte adhésion des Nm aux cellules humaines. L'inverse est observé pour les P4T chargés négativement. Ce travail présente également les premières caractérisations de la machinerie de sécrétion du phage. Des études préliminaires avaient montré que dans un biofilm formé sur cellules épithéliales, Nm produisaient soit des pili, soit des phages, mais pas les deux en même temps. Nous avons montré que la sécrétion du phage nécessitait PilQ, PilW et TsaP de la machinerie des P4T. Les analyses bio-informatiques suggèrent que l'ORF8 du phage, qui aurait une activité ATPase, s'associerait avec l'ORF11 pour former un complexe qui interagirait avec la sécrétine bactérienne PilQ. Ce modèle est soutenu par des tests d'interaction par double-hybride. Cette interaction mobiliserait les PilQ de la bactérie, les rendant inaccessible à la machinerie de piliation. La surexpression d'ORF8 entraine une inhibition de la piliation. Ces données permettent d'établir un nouveau modèle d'interaction entre les phages filamenteux et les P4T, ce qui pourrait participer à la sélection des souches pathogènes de NmNeisseria meningitidis (Nm) is a commensal bacterium of the human nasopharynx that sometimes crosses the nasopharyngeal barrier and spreads through the bloodstream to reach the meninges. A filamentous bacteriophage called MDA (Meningococcal Disease Associated) is associated with invasive meningococcal disease in young adults. MDA appears to increase the incidence of the disease by increasing bacterial colonization at the point of entry. The aim of this work was to understand the precise molecular mechanism of infection of Nm by MDA. The study of mutants of genes involved in the type IV pili (T4P) machinery showed that phage entry requires a retractable T4P. This result is consistent with the literature on Ff or CTX phages, which interact directly with the pilus tip. However, no evidence was found for MDA interacting with the T4P tip. The possible interaction between the pilus fiber and the phage capsid was investigated. Since PilE, the major pilin, is subject to antigenic variation, variants of PilE that reduce phage entry were identified. Phage infection occurs in populations of bacteria that express specific PilE sequences. Using imaging, we showed that pili and MDA associate with each other. An analysis of the amino acid charge of the pilin and that of the capsid supports the hypothesis of a variable interaction depending on the PilE variant. Finally, it was shown that T4P with a positive electrostatic potential favored phage infection and allowed the bacteria to adhere strongly to human cells. The opposite is observed for negatively charged T4P. This work also presents the first characterizations of the phage secretion machinery. Previous studies had shown that in a biofilm formed on epithelial cells, Nm produce either pili or phages, but not both at the same time. We showed that phage secretion requires PilQ, PilW and TsaP of the pili machinery. Bioinformatic analyses suggest that phage ORF8, which has ATPase activity, associates with ORF11 to form a complex that interacts with the bacterial secretin PilQ. This model is supported by two-hybrid interaction assays. This interaction would mobilize the bacterial PilQ, making them inaccessible to the piliation machinery. Overexpression of ORF8 inhibits piliation. These data provide a new model for the interaction between filamentous phages and T4P, which could be involved in the selection of pathogenic strains of Nm
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Weber, Patric. "Display of trypanosomal antigens on the surface of filamentous phage /". [S.l.] : [s.n.], 1994. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Deng, L. W. "Infection mechanism of filamentous bacteriophage fd : interaction between E. coli F-pilus and phage protein pIII". Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598493.
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Gene 3 protein (pIII), a minor coat protein located at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. An introduction and the experimental materials and methods are described in Chapters 1 and 2, respectively. Chapter 3 describes the construction and expression of the N-terminal di-domain and individual domains of pIII protein. The plaque-forming assay in vivo was used to investigate the ability of the isolated pIII domains to interact with F-pilus. Chapter 4 presents the investigation of the structure of F pilus, including displaying fragments of F pilus subunit (pilin) by means of phage display technology, expression of pilin subunit and X-ray fibre-diffraction of F pilus. Site-directed alanine mutagenesis of the second N-terminal domain of pIII in the phage as well as the evaluation of their infectivity are described in Chapter 5. Chapter 6 describes the establishment of a competitive ELISA assay in vitro in order to analyse all the mutated phages. Combining the results from two assays and mapping out the affected residues on the 3D structure, a region located at the outer rim of pIII-D2 domain was implicated as the F pilus binding epitope. Second generation mutagenesis and evaluation of these mutants were performed in order to define the interface region more closely (Chapter 7). Furthermore, several pIII proteins each containing an alanine mutation were isolated to investigate whether any major disruption of protein integrity occurs. Finally, Chapter 8 places this work in perspective and offers ideas for further work.
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Lima, Mayara Ingrid Sousa. "Seleção e caracterização de peptídeos recombinantes do Mycobacterium leprae ligantes à IgG por meio da tecnologia de phage display". reponame:Repositório Institucional da FIOCRUZ, 2011. https://www.arca.fiocruz.br/handle/icict/4253.
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Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia,Brasil
A hanseníase é uma doença infecciosa crônica, causada pelo Mycobacterium leprae, que apresenta manifestações clínicas variadas. Essas variações refletem em diferenças que vão de uma forte resposta imune celular com controle do crescimento do bacilo, no pólo tuberculóide, a uma anergia em resposta celular, no pólo virchoviano. A caracterização do perfil antigênico do M. leprae frente a esse quadro de múltiplos aspectos clínicos representa uma ferramenta fundamental para o desenvolvimento de novas plataformas para um diagnóstico diferencial mais sensível e/ou desenvolvimento de unidades vacinais. Dessa forma, o objetivo desse trabalho foi selecionar e caracterizar peptídeos miméticos de antígenos do M. leprae reativos contra IgGs totais purificadas de pacientes com hanseníase. Para a seleção foi utilizada a tecnologia de phage display, usando bibliotecas randômicas de peptídeos expressos em fagos filamentosos. Foi realizada uma seleção com IgGs de pacientes Tuberculóides e outra com IgGs de pacientes Virchovianos. A validação dos peptídeos foi realizada utilizando o imunoensaio ELISA, o teste de redução de colônias e análise de bioinformática. Após a pré-validação e sequenciamento foram encontradas 17 mimotopos para o pólo Vichorviano e 12 no pólo Tuberculóide. Foram validados 4 peptídeos, sendo 2 do pólo Tuberculóide (T03, T04) e 2 do pólo Virchoviano (V06 e V13). Os peptídeos TALFPWL (T03) e YSTTLSY (T04) foram imunorreativos em soros de pacientes paucibacilares, bem como em pacientes Virchovianos, além de terem alinhado com proteínas de membrana do M. leprae com potencial antigênico. O peptídeo V06 apresentou especificidade de 100% e sensibilidade de 94,74%, o que se complementa com os dados do teste de redução da pIII, o qual obteve uma taxa de redução de 82% em soros Virchovianos. O peptídeo V13 também foi reativo e apresentou similaridades com chaperonas e proteínas de membrana. Este estudo aponta perspectivas para a identificação de novos antígenos, propiciando a descoberta de novos alvos biológicos com potencial diagnóstico e/ou terapêutico.
Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which has varied clinical manifestations. These variations reflect differences that spans from a strong cellular mediated immunity and bacili growth control the tuberculoid pole to a poor T cell immunity at the lepromatous pole. The antigenic profile characterization in both clinical forms represents a fundamental tool for the development of new platforms for a differential diagnosis more sensitive and/or development of vaccine units. Thus, the objective was to select and characterize mimetics peptides antigens of M. leprae reactive against total IgG purified from leprosy patients. The phage display technology was used for selection using random peptides libraries expressed on filamentous phages. A selection was performed with IgGs from tuberculoid patients and other IgGs of lepromatous patients. Peptides validation was performed using the ELISA immunoassay, the plaque reduction test and bioinformatics analysis. After the pre-validation and sequencing were found 17 valid sequences for the lepromatous pole and 12 tuberculoid pole. Four peptides were validated, two of tuberculoid pole (T03, T04) and two lepromatous pole (V06 and V13). The peptides TALFPWL (T03) and YSTTLSY (T04) were imunoreatives in sera from paucibacillary patients and in lepromatous patients. They had alignment with membrane proteins of M. leprae antigenic potential. The V06 peptide showed 100% specificity and 94.74% sensitivity, which is supplemented with the plaque reduction test, who obtained a reduction rate of 82% in lepromatous sera. The V13 peptide was also reactive and showed similarities with chaperones and membrane proteins. This study presents insights for new antigens identification, leading to discovery of new biological targets with potential diagnostic or therapeutic
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Pommier, Stéphanie. "Le système Tol-Pal : l'intégrité membranaire et les interactions entre TolA, colicine A et G3p, protéine de capside des phages filamenteux". Aix-Marseille 2, 2005. http://theses.univ-amu.fr.lama.univ-amu.fr/2005AIX22070.pdf.
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Lößner, Holger. "Autolytische Salmonellen als Vektoren für die orale genetische Vakzinierung". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2003. http://dx.doi.org/10.18452/15000.
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Die Entwicklung einer mukosal verabreichbaren, effektiven DNA-Vakzine gegen Infektionskrankheiten oder Tumorerkrankungen auf der Basis invasiver attenuierter Bakterien ist eine vielversprechende Alternative zu bisherigen parenteralen Strategien der genetischen Vakzinierung. Innerhalb dieser Arbeit wurden Salmonellen-Impfstämme für die orale Übertragung eines eukaryontischen Expressionsplasmids mit dem kleinen Oberflächenantigen des Hepatitis-B-Virus (HBsAg) als Modellantigen optimiert. Die kontinuierliche Sezernierung von Plasmiden als filamentöse Phagenpartikel wurde als ein erster Ansatz getestet, um mit lebenden Bakterien eine DNA-Vakzine innerhalb infizierter Zellen freizusetzen. Die Salmonellen-vermittelte Phagensekretion in der Wirtszelle ist jedoch nicht effizient genug, die Expression des Transgens zu vermitteln. Alternativ wurde ein Ansatz gewählt, durch eine spontan induzierte Lyse der Impfbakterien, Plasmid-DNA in die Wirtszelle zu übertragen. Dazu wurde ein neuartiges bakterielles Autolysesystem etabliert, basierend auf einem Zwei-Phasen-Expressionssystem und von Bakteriophagen abgeleiteten Lysedeterminanten. Dieses System ermöglicht erstmals die kontinuierliche Freisetzung von Plasmid-DNA und Proteinen aus einzelnen, lysierenden Salmonellen innerhalb einer sonst gesunden bakteriellen Gesamtpopulation. Innerhalb infizierter COS7-Zellen führt die Freisetzung des porenformierenden Proteins Listeriolysin O durch autolytische Salmonellen zur Zerstörung der Vakuole, in der die Impfbakterien replizieren, und erleichtert somit den Transfer der Plasmid-DNA aus den Bakterien in das Zytoplasma der Wirtszelle. Die Lysedeterminante und die eukaryontische Expressionskassette für HBsAg wurden auf einem Plasmid kombiniert, sowie eine Kassette zur konstitutiven Expression des Histon-ähnlichen Proteins aus Thermotoga maritima (TmHU) in ein solches Konstrukt integriert. TmHU stabilisiert die Plasmiderhaltung unter nicht selektiven Bedingungen und besitzt das Potential, die Effizienz der DNA-Translokation innerhalb der Wirtszelle zu erhöhen. Durch die orale Gabe optimierter autolytischer Impfbakterien konnte eine potente HBsAg-spezifische Antikörperantwort sowie eine zytotoxische zelluläre Antwort induziert werden. Bereits die einmalige Gabe der autolytischen Bakterien induzierte eine höhere antigenspezifische Antikörperantwort, als die herkömmliche intramuskuläre DNA-Vakzine. Das im Rahmen dieser Arbeit entwickelte Konzept autolytischer Salmonellen stellt also eine neuartige, effiziente Strategie für den mukosalen DNA-Transfer dar. Die Übertragung des Konzeptes der Autolyse auf andere bakterielle Trägersysteme ist möglich und kann zur Erweiterung des Anwendungspektrums bakterieller Vektoren beitragen.The development of an effective mucosal DNA vaccine against infectious diseases or tumors based on invasive attenuated bacteria is a very promising alternative to common parenteral routes of genetic vaccination. This work aimed at the optimization of Salmonella vaccine strains for the oral delivery of an eukaryotic expression plasmid encoding the small Hepatitis B Virus surface antigen (HBsAg), here used as model antigen. The continuous secretion of plasmids as filamentous phage particles was first tested as a mean for the delivery of the DNA vaccine by living bacteria inside infected host cells. However, Salmonella-mediated phage secretion inside cells did not suffice for the induction of transgene expression. As alternative approach, inducible spontanous lysis of bacteria was used to mediate the release of plasmid DNA into host cells. For this purpose a novel bacterial autolytic system was established on the basis of a two-phase expression system and lysis determinants derived from bacteriophages. This system allows for the first time the continuous release of plasmid DNA and proteins from only few lysing Salmonella within an otherwise healthy bacterial population. Inside COS7 cells the release of the pore-forming protein listeriolysin O by autolytic Salmonella mediates the destruction of the Salmonella-harbouring vacuole, thereby facilitating the transfer of plasmid DNA from bacteria into the host cell cytoplasm. The lysis determinant was combined with the eukaryotic expression cassette for HBsAg on one plasmid. In addition, a cassette for the constitutive expression of TmHU, a histon-like protein derived from Thermotoga maritima, was integrated in such vector. TmHU stabilizes the plasmid propagation in the absence of selective pressure and has the potential to increase the efficiency of plasmid translocation inside the host cell. The oral administration of the optimized autolytic bacteria stimulated a potent HBsAg-specific antibody response as well as a cytotoxic cellular response. Already a single inoculation of the oral vaccine induced a higher specific antibody response than the conventional intramuscular DNA vaccine. Therefore the concept of autolytic Salmonella carrier strains developed in this work constitutes a novel efficient strategy for mucosal DNA delivery. The transfer of this concept to other bacterial carriers is possible and may widen the application field for bacterial vectors.
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CHEN, SONG-YUN, i 陳松芸. "Characterization of filamentous phage Cf1t imm -". Thesis, 1992. http://ndltd.ncl.edu.tw/handle/79599876065467010926.
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LIN, JING-HUI, i 林靜慧. "Identification of DNA region involved in phage integration of filamentous phage Cflt". Thesis, 1991. http://ndltd.ncl.edu.tw/handle/03339821352724891199.
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SU, WEI-ZHI, i 蘇偉誌. "Localization of the replication origin of filamentous phage Cf". Thesis, 1990. http://ndltd.ncl.edu.tw/handle/54165762240788842565.
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Yen, Ming-Ren, i 嚴明仁. "Comparative Genomics of Filamentous Phages from Xanthomonas". Thesis, 2003. http://ndltd.ncl.edu.tw/handle/42138177638249238083.
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博士國立中興大學
分子生物學研究所
91
Several filamentous phages from Xanthomonas, including Lf, Xv, Xo, and Cflc, have previously been reported. In this study, two novel filamentous phages, Xv2 and Xo2, were isolated from Xanthomonas axonopodis pv. vesicatoria and Xanthomonas oryzae pv. oryzae, respectively. They are similar to other filamentous phages of Xanthomonas in having (i) restrictive host specificity, (ii) a single-stranded DNA genome, (iii) a replicative form (RF) as the intermediate during propagation, and (iv) a life cycle without lysis of host cells. Sequence determination revealed that the genome of Xv2 and Xo2 are 6,293 and 8,341 nt in size, respectively, containing seven genes on the viral strand which are corresponding to those of Lf, gII, gV, gVII, gVIII, gIII, gVI, and gI, which are defined as the core genes herein. In order to elucidate the phylogenetic relatedness, the six phages were analyzed with programs BLAST, CLUSTAL-X, TREEVIEW, TMHMM, Dot plot, and SignalP. Results indicated that the genome of filamentous phages of Xanthomonas consist of three different modules i.e., DNA replication module, structure module, and morphogenesis module. Each of the modules has its own high degree of conservation. Combination of different modules thus provides diversities and results in different phages. Data of phylogenetic analyses suggested that structure module and morphogenesis module are linked during the process of evolution, implicating that there is a direct contact between coat proteins and assembly proteins during morphogenesis. The ratio of size of the region containing core genes to that of the whole genome is almost the same among different phages, but the sizes of intergenic region (IR) vary. Several open reading frames are present in the IRs and two of them, encoding a 13-kDa and a 16-kDa protein, are found in all the filamentous phages. Based on sequence analysis, these two proteins are proposed to play important roles in phage replication. A novel mini plasmid pXV1.3, with a 1,313-bp genome consisting of a single gene (rep) encoding a replication protein, was discovered in X. axonopodis pv. vesicatoria Xvt146. In the presence of a filamentous Xanthomonas phage, pXV1.3 can be packaged and released into medium becoming transducing particles, which can infect the susceptible host cells.
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CHEN, YUAN-QUAN, i 陳淵銓. "Functional analysis of the replication origin of filamentous phage Cf". Thesis, 1992. http://ndltd.ncl.edu.tw/handle/99825415880337360802.
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Wang, Chen-Nai, i 王貞乃. "Identification of the 5' ends of filamentous phage cf transcripts". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/18113013508412437764.
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碩士國立臺灣大學
植物學系
84
Filamentous phage cf was isolated from Xanthomonas campestri pv. citri .The nucleotide sequence of the cf genome has been determined. Base on the result of Northern blot hybridization, a rough transcription map of the cf could be established. To construct a more detail cf genetic map, primer extention was used to map the 5 ends of the cf transcripts. Our data indicated that eight cf-specific transcripts have been found, and at least three of them are primary products. It suggested that there were three promoters in the cf genome. The transcriptional initiation sites of the primary transcripts were G or A.
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Hsiao, Miao-Yi, i 蕭妙宜. "The Host pilA Gene Required for Infection of Filamentous Phage phiLf". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/93081880382931854122.
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碩士國立中興大學
分子生物學研究所
88
The gram negative Xanthomonas campestris pv. campestris is the causative agent of black rot in cruciferous plants. This bacterium, short rod in shape with one polar flagellum, can synthesize great amounts of exoplysaccharide that has many applications in industry, cosmetics, and food industry. A filamentous phage phiLf specifically infect X. campestris pv. campestris is isolated in our laboratory. This phage is similar to other filamentous phages such as the Ff and IKe phages in morphology and genome organization. In Ff phages which infection mechanism has been studied very clear absorbs the F pili of host and infects. The further examination of morphological surface by electron microscopy in wild type in X. campestris pv. campestris showed no visible pili structure. The infection pathway of phiLf was unknown. To understand the phiLf infection pathway, our laboratory used the transposon mini-Tn5 to mutagenesis P20H. Three phiLf-resistance mutants of Xanthomonas campestris pv. campestris obtained. They are named FR9, FR26 and T5. The mutant T5 electroporated the phiLf RF DNA can release the bacteriophage particle. Using the strategy, chromosome walking obtain the fragments adjust mini-Tn5 on the T5 chromosome. Sequencing analysis found three ORF adjust the mini-Tn5 on the T5 chromosome. Three ORFs was named pilC, pilA and pilE. The pilC can’t translate a complete ORF highly identity with P. aeruginosa pilC gene. Two complete ORFs pilA and pilE translate 147 a.a. and 144 a.a., respectively, which highly identical to type IV fimbriae on amino acid level. Type IV fimbriae gene encode the protein which is the major compound of type IV fimbriae（or pili）. Using E. coli S30 expression system express pilA gene obtain a 14.7 kDa protein product. The molecular weight of product is equal with predict of sequence. The mini-Tn5 breaks the predicted promoter of pilA so that pilA unexpressed. The hosts lack receptor so that can’t be infected by phiLf.
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Yang, Ming-Haw, i 楊明浩. "Cloning and Analysis of the Promoter Regions of filamentous Phage phiLf". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/31449772070749412701.
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碩士國立中興大學
植物學系
85
Abstract Xanthomonas campestris pv. campestris is the phytopathogenic bacterium causing black rot in crucifer. In industry, this bacterium is the producer of xanthan gum, an exopolysaccharide that has a variety of application. phiLf is a filamentous bacteriophage that specifically infects Xanthomonas campestris pv. campestris. It has a circular, single-stranded DNA genome of 6008 nt. Like most Ff phages, it also has similar genomic organization in the following order: the intergenic region - gene II/X - V - VII - IX - VIII - III -VI - I, and a rho-independend transcription terminator distal to gene VIII. In this study, we use a promoter-proving vector, pFY9, containing a promoterless-lacZ gene to select the promoter regions of phiLf. Five clones have b-galactosidase activity with inserts ranging from 300-700 bp were obtained. The transcription direction of four inserts among them revealed that there are promoters on the complementary strand of pliLf viral strand. Slot blotting analysis using strand-specific DNA probes hybridized with total RNA isolated from P20H infected with phiLf also showed that there are RNA transcribed from the complementarystrand of phiLf viral strand. The transcription of phiLf viral strand was studied by using Northern blotting analysis with strand-specific DNA probes. Our results suggested that there are promoter regions upstream to gene II, gene V, gene VII and gene III, and further, the transcripts depend on them are 1200 nt, 950 nt, 420 nt and 1500 nt, respectively.Primer extension analysis of gene V indicated the transcription initiation site is 121 nt upstream to the start codon of gene V, correspond to nt 2262 of phiLf viral strand. Primer extension analysis of gene IX indicated the transcription initiation site is G at nt 2743 of phiLf viral strand. Primer extension analysis of gene VII indicated there are two transcription initiation sites, correspond to nt 2676 and nt 2687 of phiLf viral strand. Primer extension analysis of gene III indicated there are two transcription initiation sites upstream to the start codon of gene III, correspond to nt 3204 and nt 3209 of phiLf viral strand. Primer extension analysis of gene II also indicated there are two transcription initiation sites distal to the 5''-end of gene II.
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方鈺森. "Sequence Analysis of the Filamentous phage fXo of Xanthomonas oryzae pv. oryzae". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/29443668606349610279.
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碩士國立中興大學
分子生物學研究所
88
fXo, isolated in our laboratory, is a filamentous bacteriophage specifically infecting Xanthomonas oryzae pv. oryzae, a gram-negative pathogen causing bacterial blight in rice. Similar to other filamentous phages, fXo possesses a circular, single-stranded DNA genome (7.6 kb), replicates using a replicative form (RF) as an intermediate, and propagates without lysis of the host cells. The nucleotide sequence of fXo has previously been determined for intergenic region (IR), genes III, VIII and VI (encoding capsid proteins pIII, pVIII and pVI, respectively) and genes I and XI (encoding pI and pXI, respectively, that are presumably required for morphogenesis). In order to understand the genome organization, the rest regions of the fXo RF DNA were sequenced and analyzed. Putting all the sequences determined together, a total of 7,613 bp was obtained. Open reading frame (ORF) analysis indicated the presence of 10 putative genes. These genes are arranged in the same order as that in Ff phages (f1, M13 and fd), IR-gII/X-gV-gVII-gIX-gVIII-gIII-gVI-gI/XI. In this study, the origin for fXo replication was located in a 1.5-kb MluI DNA fragment within the coding region of gII, the gene encoding the replication initiation protein. This 1.5-kb DNA fragment was inserted into the HindIII-SmaI sites of pUC19G to generate plasmid pGII1561G. After electroporation, pGII1561G was found to be maintained in X. oryzae pv. pryzae; however, no deletion clones containing an insert smaller than that of pGII1561G were maintained in complementation tests with the gII being provided in trans.
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Wei, Chiu Chien, i 邱健維. "Identification and Characterization of the Filamentous Phage phi Lf Gene VIII Promoter". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/07755766093269514691.
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碩士國立東華大學
生物技術研究所
89
A filamentous phage Lf which infects Xanthomonas campestris pv. campestris, an important plant pathogen, has a circular single-stranded DNA genome of 6,008 nt. It is similar to the Ff phages that infect E. coli in terms of morphology and gene organization. Ten genes arranged as gII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI-gXI have been found in Lf. The most abundant structural protein is the major coat protein encoded by gVIII. To isolate the promoter region of this gene, the 2 kb SacI fragment of Lf was partially digested with Sau3AI, or specific fragments within this region were amplified by PCR, and each individual fragment was subcloned into two promoterless vectors, pFY12-8MD1 and/or pFY13-9, respectively. The clones expressing lacZ in Xanthomonas campestris pv. campestris were sequenced. The results demonstrated that the promoter region encoded gVIII is mapped between Lf nt2,886-2,966. In an effort to examine the gene products of Lf, Northern analysis was performed using end-labeled oligonucleotides or double-stranded DNA probes specific to gV, gVII, gIX, and gVIII, respectively. Three species of mRNAs of size 870 nt, 320 nt, and 250 nt were detected with probes specific to gVIII, two species of mRNAs of size 870 nt and 320 nt were observed with either gVII or gIX and only one message of 870 nt was detected with a probe specific to gV. To identify the transcription initiation site, primer extension analysis was carried out. The results show that the transcripts start at the positions of Lf nt2,780, 2,807, 2,840, 2,880, and 2,922. The first three bands contribute to the 320 nt of mRNAs and the nt2,880, and nt2,922 bands denote the 250 nt of mRNAs shown on Northern analysis. Construct pG8PS containing nt2,886-2,966 still shows promoter activity, suggesting that the transcription initiation site of gVIII is at nt2,922, and the promoter for gVIII could be narrowed down at nt2,886-2,921. Within this region, however, no E. coli-type -35 and -10 consensus sequences of promoter were found. The most abundant transcript shown on primer extension analysis was the transcript initiated from nt2,880. However, no promoter activity could be identified near upstream of this region suggesting that this transcript could be a degradation product of larger mRNAs. The abundant transcript and the best fit of Shine-Dalgarno sequence for gVIII demonstrate that gene VIII has the advantage to generate a great amount of major coat proteins.
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Liao, Tsai-Ling, i 廖采苓. "The genes required for packaging and export of the filamentous phage φLf". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/74851909388582844731.
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碩士國立中興大學
分子生物研究所
84
Abstract Xanthomonas campestris pv. campestris is the phytopathogenic bacterium causing black rot in crucifers. In industry, this bacterium is the producer of xanthan gum, a substance that finds a variety of applications. fLf is a filamentous bacteriophage that specifically infects X. campestris. It has a circular, single-stranded DNA genome of 6008 bp. It is similar to other filamentous phages in most parts of genome organization and biological properties. However, properties different from other filamentous phageshave also been observed. For example, the region between genes II and I in the fLf genome is only 1481 bp, comparing to 1786 bp containing intergenic region (508 bp) and gene IV (1278 bp) in Ff phages (f1, fd and M13). In this study, the function of the 1481-bp region was investigated. The ability of fLf to propagate was not affected by insertion of a kanamycin cartridge into the PstI site or gentamycin cartridge into the SmaI site, or replacing the 141-bp EcoRI fragment with kanamycin cartridge. These results indicate that these regions are not essential and the homologue of the filamentous phage gIV, required for phage export, may be absent from the fLf genome. ORF137 is an open reading frame in the complementary strand, locating in the 863-bp EcoRI-PstI fragment between gII and gI of fLf genome, which is able to encode a 13 kDa product. Deletion of ORF137 from the fLf genome was found to result in the loss of phage assembly and export. The amino acid sequence of ORF137 shows 30% identity to that of the thioredoxin of E. coli, which is required for Ff phage assembly and export. Therefore, it is possible that ORF137 may play a role similar to that of thioredoxin fophage assembly and export. XpsD is a multimeric outer membrane lipoprotein required for the secretion of extracellular enzymes by X. campestris. It shares strong homology with the widespread homologues in Gram-negative bacteria involved in the secretion pathways, including pIV of the Ff phages. The phage titer produced by xpsD mutant A92 was about 1.2 x 107 PFU/ml versus 3.7 x 109 PFU/ml in the fLf- infected parental strain XcP20H, a 300 fold decrease was observed. Similar differences were also observed in phage production when the flf RF DNA was electroporated into the cells, about 9.5 x 106 PFU/ml for XcP20H versus 3.6 x 104 PFU/ml for A92 after 5 hours.
18
Lu, Shao-Tzu, i 呂紹慈. "Study of infection of Xanthomonas campestris pv. campestris by filamentous phage phiLf". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/23248360919541445820.
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碩士國立中興大學
分子生物學研究所
103
Filamentous phage φLf specifically infects Xanthomonas campestris pv. campestris (Xcc). It has a single-stranded circular DNA with a genome site of 6,008 nucleotides. The φLf viral strand encodes nine genes organized into the order of gII-gX-gV-gVII-gIX-gVIII- gIII-gVI-gI, with gII, gX and gV shown to be required for replication, gVII, gIX, gVIII, gIII, and gVI encoding coat proteins, and gI responsible for assembly and morphogenesis. φLf-homologous region (fhr) is present on the chromosome of Xcc strain Xc17, next to the dif (deletion induced filamentation) site that is the end of chromosome replication. This region has been shown to site specific integration between dif site (attB) and the dif-homologous attP site in φLf, and homologous recombination via the fhr region outside dif. φLf can infect Xcc strain P20H, but can’t infect Xc17. Xc17fhrΔ8361 was constructed from Xc17 containing gVIII, gIII, gVI and gI of fhr region replaced by Gmr cartridge.φLf can infect Xc17fhrΔ8361. The purpose of this study was to understand why φLf can’t infect Xc17 ? Two experimental strategies were used for study: (I) Xc17 mutants with Gmr cartridge replaced the gVIII,gIII,gVI or gI gene in the fhr region were contracted and infected by φLf. (II) Xc17fhrΔ8361 transformants complemented with DNA fragments carrying Xc17fhr gVIII,gIII,gVI and gI were obtained and infected by φLf. Results showed that mutation of gVIII,gIII,gVI or gI gene in the fhr region of Xc17 causes no effects on φLf infection. Adsorption test results appeared that Xc17 mutants is no significant difference by φLf. Therefore, mutation in the gVIII~gIII, gVIII~gVI,gVIII~gI and gVIII~gI gene of Xc17 causes no effects on φLf infection. PCR amplification analysis results, showed that fhr region is absent in the genome of Xc17fhrΔ8361 and P20H. Based on the results, φLf infection isn’t associated with gVIII,gIII,gVI,gI gene but pilA1. Expression levels of pilA1 are various among Xc17, Xc17fhrΔ8361 and P20H by real-time PCR. Higher expression level of pilA1 in P20H, φLf can efficiency infect host, and lower expression level of pilA1 in Xc17fhrΔ8361, φLf also can efficiency infect host, but lower expression level of pilA1 in Xc17, φLf can’t infect host. Based on the results, presumably there is other factor more influence than pilA1 efficiency when φLf infected host.
19
徐晨圃. "analysis of protein-protein and protein-DNA interactions during filamentous phage assembly". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/21416762171222116744.
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碩士國立交通大學
生物科技系所
95
Filamentous phages are small , highly evolved parasites that can reproduce and disseminate without killing their host . The genome of filamentous phage is single stranded circular DNA . The process by which filamentous phage are concomitantly assembled and secreted across the cell membranes is likely to involve a series of protein – protein and protein – DNA interactions .The previous findings that deletion of the filamentous phage packaging signal can be compensated for by mutations in g7p , g9p , and g1p , strongly suggest a role for these proteins in the initiation of assembly . In addition , thioredoxin is required for efficient filamentous phage production and may interact to g1p to drive an engine for filamentous phage assembly . This report tests the gel shift assay for examining previous proposal about protein- packaging signal interaction . We also investigate the possible interactions between these proteins by various assays. These results would show information on the structure and assembly process of filamentous phage .
20
Hao, Peng Chi, i 彭啟豪. "Characterization of gene IX and its protein in the filamentous phage phiLf". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/89830508627658897096.
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碩士國立中興大學
分子生物學研究所
92
Containing a single-stranded circular genomic DNA, Lf is a filamentous bacteriophage which specifically infects Xanthomonas campestris pv. campestris. Like other filamentous phages, it infects the host, replicating using replicative form (RF DNA) as the intermediate,and produce phage progeny without killing the host cells. The Lf genome has 10 predicted genes on the viral strand which are organized into an order of gII-gX-gV-gVII (a 43-codon open reading, orf43)-gIX (a 38-codon open reading, orf38)-gVIII-gIII-gVI—gI-gXI, whereas three possible genes are present between gII and gI on the complementary strand (orf155-orf137-orf102). gII and gX are required for DNA replication, gVIII encodes the major coat protein, gIII, gVII, gIX possibly encode the minor coat proteins, gI and gXI are possibly involved in morphogenesis. Among these, presence of the gene products from gII, gV, gVI, gVIII, gIII, and gI has been demonstrated. However, the previously proposed orf43 and orf38 have not been verified. ORF search using FramePlot revealed that an orf of 83 codons (orf83) is present in the region encompassing orf43 and orf38, which has the same reading frames as orf38 in its C-terminus. In other words, should this prediction be true, the presence of orf43 becomes questionable. The purpose of this study was to detect the possible protein products from these orfs. To detect the possible protein, orf83 was cloned into pET expression vector and expressed in E. coli. The recombinant protein was used to raise antibodies by immunize a rabbit. No protein band corresponding to the size of orf83 (9,130 dal) was detected in the Lf particles using this antiserum for Western blotting. Instead, a band of about 4,000 dal which is similar to the size of the orf38 protein (pIX) was observed. These data indicate that, while whether orf43 is a true gene remains unknown, orf38 (gIX) indeed encodes a minor coat protein of Lf, pIX. In Western blottings with fractionated subcellular components, pIX was found be located in the inner membrane of X. campestris pv. campestris.
21
Wang, Yu-Ru, i 王玉如. "Characterization of five filamentous phages from xanthomonas campestris pv. citri". Thesis, 1995. http://ndltd.ncl.edu.tw/handle/74962702204266617801.
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HU, JIAN-GUO, i 胡建國. "Lysogenicity of filamentous phages isolated from xanthomonas campestris pv. citri". Thesis, 1992. http://ndltd.ncl.edu.tw/handle/36873360053346338739.
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碩士國立中興大學
植物病理學研究所
80
Cf26，Cf29，Cf32，Cf86， Cf87，Cf99，Cf117 及 Cf134 等九種噬菌體分別從柑 桔潰瘍病菌 XW26，XW29，XW86， XW87，XW99，XW117 and XW134 等潛溶菌株分離 而得。此等噬菌體分別以 35-100 ％的頻率將 XW47 菌株的細胞潛溶化，而所獲得 的所有潛溶菌株，經五年來的長期更新培養，或經ㄚ定橙、紫外線照射、軟瓊脂培 養基五年穿刺保存，或接種至柳橙葉片二星期後，皆仍具有潛溶性。所有潛溶菌株 在牛肉液培養過程中皆自然地釋放噬菌體， 0 小時測得 10-10 P.F.U./mL，而 48 小時後達 10-10 P.F.U./mL 的噬菌體顆粒。XW47 （ Cf26 ） -12、XW47 （ Cf29 ） -69、XW47 （ Cf32 ） -16、 XW47 （ Cf86 ） -58、 XW47 （ Cf87 ） -6、 XW47 （ Cf89 ） -31、 XW47 （ Cf99 ） -22、 XW47 （ Cf117 ） -13 及 XW47 （ Cf13 ） -25 等潛溶菌株對於潛溶化自己本身的 Cf26、 Cf29、Cf32、 Cf86、 Cf89Cf99、Cf117 及 Cf134 等噬菌體皆具有免疫性。 潛溶化後，此等九種噬菌體 的 DNA 存在於其寄主細胞內，但其位置還未確定。 （關鍵字：柑桔潰瘍病，Xanthomonascampestris pv. citri，絲狀溫和噬菌體，噬 菌） #9201882 #9201882
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Chen, Chin-Huai, i 陳治淮. "The interaction between thioredoxin and Gene I protein of the filamentous phage f1". Thesis, 1998. http://ndltd.ncl.edu.tw/handle/02863666200090294347.
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碩士國立交通大學
生物科技研究所
86
Thioredoxin是一個任何型態細胞中皆有，且具有許多功能的熱穩定蛋 白。常見的功能是在細胞內，扮演氧化還原蛋白的角色，例如 thioredoxin參與核糖核甘酸二磷酸(ribonucleotide diphosphate)的還 原、動物細胞中的胰島素還原劑、高等植物細胞葉綠體中某些酵素的活化 。噬菌體T7去氧核糖核酸聚合酵素(T7 DNA polymerase)之次單元、及線 形噬菌體f1(filamentous bacteriophage f1)所須之蛋白。對於大腸桿菌 Thioredoxin影響線形噬菌體f1生長的機制，目前並不是非常清楚，只知 其影響的步驟可能發生在線形噬菌體f1組裝子代移出宿主細胞時。而一些 遺傳實驗證據顯示，thioredoxin可能與線形噬菌體f1的Gene I蛋白有交 互作用。線形噬菌體f1為單股去氧核糖核酸病毒(single-stranded DNA virus)，對於帶有F質體(F+)的大腸桿菌有專一性。f1構型與遺傳物質為 核糖核酸(RNA)的煙草鑲嵌病毒(TMV)類似，遺傳物質外由許多相同單元之 殼蛋白包裹；另外，f1的感染也與許多動物病毒相似，皆不會造成宿主死 亡。一些動物病毒感染細胞皆與Thioredoxin有關聯，但作用機制現仍不 甚了解。本研究的目的是希望藉由研究Thioredoxin及Gene I蛋白是否能 夠交互作用以了解thioredoxin在線形病毒生長的作用機轉及所扮演的角 色。經由活體內酵母菌雙雜交系統實驗結果呈現藍色可知，Thioredoxin 及Gene I蛋白確實有交互作用。 Thioredoxin is a multifunctional heat stable protein found in all cell types. It appears to play a variety of roles in different cell types. In E. coli, it is a general protein disulfide reductant. It serves as the reductant for systhesis of deoxyribonucleotides and is an effective reductant of insulin in animal cells. The protein also acts as an activator of certain enzymes of carbon dioxide fixation in the chloroplasts of higher plants. It may also be involved in activation of the glucocorticoid receptor, phage T7 growth, and filamentous phage assembly. Filamentous phage f1 is a single- stranded circular DNA virus. It is specific for the F+ strains of Escherichia coli. In common with other filamentous bacteriophages, these viruses exhibit unusual mechanisms of replication and maturation and in many respects resemble endosymbiotic animal viruses rather than typical lytic bacteriophages. Thioredoxin is in fact related to the infection of animal cells by some animal viruses. We are interested in studying the relationship between thioredoxin and virus infection. In this study, we start with filamentous phage f1. Genetic studies have indicated that thioredoxin can interact with Gene I protein of filamentous phage f1. In this study we investigate whether thioredoxin can interact with Gene I protein of the phage by using yeast two-hybrid system. The results show that thioredoxin indeed interacts with filamentous phage f1 Gene I protein.
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Liu, Tzu-Jun, i 劉祖君. "Purification, Expression and Functional Study of gene III Protein from Filamentous phage fLf". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/44366043448568620492.
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碩士國立中興大學
分子生物研究所
85
Filamentous phages fLf, fXv and fXo were isolated from Xanthomonas campestris pv. campestris, X. campestris pv. vesicartoria and X. oryzae pv. oryzae, respectively. These phages have restrictive host specificity. However, they are highly homologous in nucleotide sequences as shown by DNA hybridization. The sequence of the single-stranded DNA genome has been determined for fLf and fXv. The sizes and organization of the fLf and fXv genes are similar to those of the Escherichia coli Ff phages (M13, f1,fd), except that the gIV homologs are absent from the genomes. The homology between the corresponding gene products of fLf and fXv range from 60.4% to 98 %, with the adsorption protein, pIII, showing the lowest degree of homology, and the major coat protein having the highest dagree of the homology.In Ff phages, infection of the host is a two-step event. First, the phage adsorbs to conjugative pili via pIII. When the phage particle reaches the cell surface, the phage DNA is released into the cell in a process dependent on the host Tol system. Interaction with the Tol system also depends on the adsorption protein. In this study, I have purified the adsorption protein of fLf , fXv and fXo. The open reading frame of fLf gIII was determined on the basis of (i) the amino terminal sequence of the fLf pIII which showed that the fLf pIII, like the cases in Ff phages, has a cleavable signal peptide, (ii) computer analysis of the probable region of signal peptide in front of the cleavage site, (iii) the position of the Shine-Dalgarno sequence which is complementary to the 3' end of 16S rRNA ( anti-Shine-Dalgarno sequenc), and (iv) the transcriptional initiation site of the fLf gIII mRNA determined by primer extension. Based on the information obtained by analyzing the fLf gIII , the ORF of fXv and fXo gIII have also been deduced. I also found that after several times of chloroform extraction, most of the fLf pVIII can be removed. This treatment rendered the pIII and pVI readily visible on SDS- PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).fXv carrying disrupted gIII could not infective to X. c. pv. vesicartoria but maintained as a RF DNA, after being electroporated into X. c. pv. campestris. With a cloned fLf gIII being provided in trans, recombinat fXv particles were released which could infect X. c. pv. campestris but not X. c. pv. vesicartoria. These results indicate that pIII adsorption protein of the Xanthomonas filamentous phages is responsible for host specificity.
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Chang, Yu-Feng, i 張瑜鳳. "The Xanthomonas campestris pv. campestris pilL gene involved in filamentous phage phiLf infection". Thesis, 2003. http://ndltd.ncl.edu.tw/handle/45384275939133772897.
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碩士國立中興大學
分子生物學研究所
91
phiLf is a filamentous phage of Xanthomonas campestris pv. campestris. To investigate the genes involved in phage infection, phiLf-resistant mutants were isolated from 8000 transconjugants after Tn5 mutagenesis of strain P20H. One of the mutants was found to have Tn inserted within pilL gene, resulting in reduced sensitivity to phiLf. The pilL gene, 7,044-nt long able to encode 2,347 amino acid resides, is clustered with four other pil genes, pilGHIJL. Using anti-PilLD12 antibodies raised against truncated PilL for Western blotting has shown that a protein of 251 kDa, similar to the size of PilL, is present in P20H. Sequence comparison revealed that the P20H PilL is a CheA homolog (chemotaxis genes) having six functional domains, H-box domain 1 (D1), H-box domain 2 (D2), H-box domain 3 (D3), histidine kinase domain (D4), CheW-like domain (adapter) (D5), and CheY-like domain (response regulator) (D6). Sequence conservation and genome organization of these pil genes are similar to those of pilGHIJKL in P. aeruginosa required for pili synthesis and twitching motility, which is likely functioning through interaction with a signal transduction system. The similarities suggest that these X. campestris pv. campestris pil genes may play similar roles in pilus production and cell motility, and probably constitute a chemotaxis-like signal transduction pathway. Mutation in X. campestris pv. campestris pilL gene does not affect virulence, colony morphology, extracellular enzymes secretion and PilA1 expression, but slightly reduces swarming mobility and the ability to adsorb phiLf. In addition, several other mutants were created by inserting Gmr or Gmr cartridge at different sites of pilL gene. The results of complementation tests on these mutants carried out with plasmids carrying different regions of pilL indicated that regions PilLD3, PilLD4, and PilLD5 are essential for function of PilL.
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You, Bih-Yuh, i 游碧堉. "Comparison of Adsorption Protein Genes (gene III) of Filamentous Xanthomonas phages". Thesis, 1993. http://ndltd.ncl.edu.tw/handle/07148639368967598242.
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碩士國立中興大學
分子生物研究所
81
Filamentous phages phiLf, phiXo and phiXv were isolated from Xanthomonas campestris pv. campestris, X. oryzae pv. oryzae and X. campestris pv. vesicartoria, respectively. To understand the evolutionary relationships of Xanthomonas filamentous phages and the mechanism of host-specificity, the adsorption protein genes (gIII) of phages phiXo and phiXv were cloned and sequenced for comparison. Gene III of phiXo was located in a 1.4 kb HindIII-KpnI fragment, phiXv gIII was in a 1.25 kb HindIII-HincII fragment. Both gIII-containing fragments were cloned and sequenced. Comparison of gIII sequences of phiLf, phiXo and phiXv revealed several features. (1) They all used GTG as start codon and contained weak promoter sequences which were overlapped with the terminator pf gVIII.(2) The gIII products contained conserved N-terminal signal peptide and C- terminal anchor domains. (3) The constitution of glycine-rich domains varied between phages.
27
陳政男. "Analysis of ATPase Activity between Thioredoxin and Gene I Protein of the Filamentous Phage f1". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/99534527330175429261.
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28
Chiang, Ying-Chuan, i 江瑩娟. "Filamentous phage fLf infection of Xanthomonas campestris pv. campestris are regulated by RpfF/DSF system". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/85595367238859799133.
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碩士中臺科技大學
醫學生物科技研究所
95
Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in cruciferous plants. Xcc produces a range of extracellular enzymes (including alpha-amylase, cellulases proteases and pectinases) and an exopolysaccharide (EPS), which are collectively essential for pathogenesis. Previous studies have shown that production of these virulence factors is positively regulated by Clp (cyclic AMP receptor protein–like protein) and rpf gene cluster (for regulation of pathogenicity factors). Synthesis of diffusible signal factor (DSF) is completely dependent on RpfF. As well as being implicated in the perception of DSF and signal transduction leading to virulence factors synthesis. P20H(rpfF::Gm), a mutant derived from Xcc P20H, which plaque is more clear than wild-type when filamentous phage (fLf) infected., transposon mutagenesis was used in this study with mini-Tn5 to induce P20H(rpfF::Gm), to obtain rpfF and clp double mutant, M67(rpfF::Gm;clp::Tc), while plaque return to as clear as wild-type when filamentous phage (fLf) infected. The experiments were designed for understanding how RpfF/DSF system and Clp influence the
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Yu, Chiang-Pei, i 江珮榆. "Involvement of type II secretin in export of filamentous Xanthomonas campestris pv. campestris phage phiLf". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/68107802407790677007.
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碩士慈濟大學
微生物學免疫學暨生物化學碩士班
100
phiLf is a filamentous phage that specifically infects Xanthomonas campestris pv. campestris (Xcc), a gram-negative plant pathogenic bacterium causing black rot disease in crucifers. Xcc produces large amounts of an exopolysaccharide (called xanthan gum) and an array of extracellular enzymes including amylase, pectinases, cellulases and proteases. These extracellular substances have been implicated as virulence factors involved in pathogenesis. Secretion of extracellular enzymes in this bacterium requires the type II secretion machinery, with the xps cluster being the major genes participating in the secretion. It is known that XpsD, encoded by xpsD, is an outer membrane protein that forms a channel (secretin) containing at least 12 mature XpsD polypeptides to facilitate the secretion of extracellular enzymes across the outer membrane. Interestingly, XpsD shares homology with pIV, the outer membrane protein encoded by filamentous phage Ff of Escherichia coli (f1, fd and M13), required for exporting Ff phage particles. The strongest homology is found at their COOH termini of approximately 200 amino acids residues. Although phiLf has an almost identical genome organization as Ff, this Xcc phage lacks the gene analogous to the Ff gIV. These findings suggest that XpsD may play a role analogous to that of pIV in phage export. Based on this prediction, the aims of this study are to test whether 1) XpsD is required for phage export by testing with xpsD mutant, 2) the wild-type phenotype can be restored by complementation with cloned wild-type xpsD gene, and 3) cloned gIV can substitute for the function of XpsD in phage export, should XpsD be shown to have the desired function.
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Hung, Chih-Hsin, i 洪志勳. "Xanthomonas campestris pv. campestris Genes Required for Phage phiL7 Infection and Determination of Filamentous Phage phiLf pIII Region Responsible for Host Recognition". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/88611798013119627447.
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博士國立中興大學
分子生物學研究所
90
The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris is the causal agent of black rot in crucifers. The exopolysaccharide (EPS) has a variety of applications in agriculture, petroleum production, cosmetics, and food industry as a stabilizing, viscosifying, emulsifying, thickening, and suspending agent. phiL7 and phiLf are phages that specifically infect X. campestris pv. campestris. phiL7, a virulent phage with a double-stranded DNA genome of 37 kb, has a hexagonal head that measures 60 nm in diameter and a noncontractile tail that is about 10 ´ 180 nm. fLf, a filamentous phage of 1000±200 ´ 8 nm in size with a single-stranded, circular DNA genome of 6,008 nt, propagates without lysis of the host cells. To understand phage-host interactions, two phiL7-resistant mutants isolated from strain X. campestris pv. campestris 17 by Tn5 mutagenesis were studied. One was mutated in xanA gene and the other in tonB gene. The xanA gene has previously been shown to code for the bifunctional phosphoglucomutase/phosphomannomutase required for the synthesis of precursors, Glu-1-P and Man-1-P, for the biosynthesis of both exopolysaccharide and lipopolysaccharide (LPS). Therefore, the xanA mutant is non-mucoid and rough in morphology. In spot tests with phiL7, lysis zones can be formed in 1.5 hr on the lawn of the wild-type cells. In contrast, it took 36 hr for turbid lysis zones to appear on the lawns of the xanA mutant cells. The ability to form the wild-type clear lysis zones was restored by complementation with the wild-type xanA gene. The adsorption efficiency of xanA mutant is decreased by 1,500-fold compared with that of the wild-type cells. EPS mutants are still sensitive to phiL7, indicating that EPS is not required for phiL7 infection. These data together suggest LPS is one of the components forming the complex receptors for fL7. The tonB is the gene previously shown to code for an inner membrane protein required for iron uptake and pathogenesis. Sequencing of the DNA fragment containing tonB, revealed three downstream genes, exbB, exbD1 and exbD2. Insertional mutation showed that exbB and exbD1 are also required for phiL7 infection. Northern hybridization detected a transcript of 2.4 kb, equivalent to the sum of the four genes, with a transcription initiation site located 79 nt upstream of tonB as determined by primer extension. Promoter-proving assay showed only one promoter located upstream of tonB. These data together indicate that they are organized into an operon, tonB-exbB-exbD1-exbD2. It is suggested that the tonB, exbB, and exbD1 genes are required for penetration instead of adsorption, based on the observations that 1) no differences in the efficiency of phiL7 adsorption were found between the wild-type and mutant cells, and 2) the mutant cells were able to release infective phage particles upon electroporation with the phiL7 genomic DNA. The third part of this study was to determine the region of pIII (the adsorption protein) of phiLf and phiXv (a filamentous phage of X. campestris pv. vesicatoria) that is responsible for host recognition. For this purpose, the N-terminal 154 amino acid residues of the phiXv pIII were exchanged with the N-terminal 164 residues of phiLf pIII. The resultant recombinant phage, phiXvfIII, is able to infect X. campestris pv. campestris, but not X. campestris pv. vesicatoria. These data indicate that the region controlling host recognition locates in the N-terminal third of the pIIIs.
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ZHENG, JUN-MING, i 鄭俊明. "Chromatographic analysis and identification of phycobiliproteins from filamentous phase of some red algae". Thesis, 1992. http://ndltd.ncl.edu.tw/handle/45650515485954428460.
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32
FAN, JIN-HAI, i 范金海. "The structure involved in the integration of the DNA of filamentous phage Cflt into hosthromosomal DNA". Thesis, 1989. http://ndltd.ncl.edu.tw/handle/10023446518817587744.
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ZHANG, RUI-YI, i 張瑞宜. "Integration of cloned DNA into chromosome of Xanthomonas campestris mediated by the filamentous phage 0Lf DNA". Thesis, 1989. http://ndltd.ncl.edu.tw/handle/14441664171983920106.
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XIE, GUO-ZHEN, i 謝國珍. "Studies on the plaque turbidity gene and the integration of phage DNA into host chromosome of filamentous phage cf1 from xanthomonas campestris pv. citri". Thesis, 1992. http://ndltd.ncl.edu.tw/handle/76754906706644707538.
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Chuang, Ming-Fen, i 莊明芬. "Construction of recombinant filamentous phages for expression of heterologous genes using plant-inducible promoter". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/86712322411262665832.
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碩士國立中興大學
分子生物學研究所
100
Xanthomonas campestris pv. campestris (Xcc), a gram-negative plant pathogen, causes black rot in crucifers. Currently, no effective method is available for controlling this disease. Phage therapy is a form of biological control. Mixtures of lytic phages have been used to treat diseases caused by bacteria, based on their high specificity to and lysis effect on the hosts. However, lytic phages usually have complex structure and are large in genome size, rendering the phages not easy to manipulate. In contrast, genomes of filamentous phages are stable and small in size. Replicative form of phage genomic DNA can be transformed into bacteria as a plasmid. Most gram-negative plant pathogens carry hrp (hypersensitive response and pathogenicity) gene clusters, regulated by HrpG and HrpX. Promoter regions of hrpX regulated genes usually have a conserved sequence known as PIP (plant-inducible promoter) box. Levels of gene expression from the PIP box are high in bacteria grown in planta or in poor media (such as XVM2), but very low in rich media (such as NYG). Xc17 hpaR gene (MarR family transcriptional regulator) promoter region contains a typical PIP box (TTCGC-N15-TTCGC), a Clp-binding site, and three inverted repeat (IR) sequences. In this study, filamentous phage phiLf of Xcc was used as a backbone to construct recombinant phages for expression of lactoferricin and lysis genes (p27p28 and p28 from phage phiL7) using the PIP-containing promoters of hpaR and hrpF. Recombinant phage particles were recovered after electroporation of the constructed recombinant phages into X. campestris pv. campestris P20H (P20H). However, these recombinant phages caused no lytic effect on P20H in the XVM2 medium, because the Xc17 hpaR and hrpF promoters exhibited no plant-inducible effect in P20H, Xc11 and Xc11A; furthermore, the cloned lactoferricin might not be able to kill Xanthomonas. RT-PCR analysis was then used to evaluate the expression of several hrpX related genes (hrpG, hrpX, hrcQ, hrcV, zur and clp) in various Xcc strains. When cultured in XVM2, the hrpX expression was almost not detectable in P20H, Xc11 and Xc11A, consistent with the data showing weak activities of hpaR and hrpF promoters in these strains. In addition, Xc11, Xc11A and P20H displayed low or no virulence in the pathogenicity test, which might be due to the low expression of hrpX and its related genes. Recombinant plasmid expressing hrpX was constructed and transformed into Xc11. Results showed that expression levels of hrpX and the downstream genes, hrcV and hrcQ, were low in the transformants. Extracellular enzyme levels among the virulent strain Xc17 and nonvirulent strains P20H, Xc11 and Xc11A were similar, suggesting that the loss of pathogenicity was due to factors other than synthesis of the extracellular enzymes.
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Chang, Kaung-Huey, i 張光慧. "Expression of Filamentous Phage ψLf Gene I Encodingn a 48-kDa Protein Associated with Host Cell Membrane". Thesis, 1998. http://ndltd.ncl.edu.tw/handle/55952036363135393567.
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碩士國立中興大學
分子生物學研究所
86
Filamenous phage ΦLf specfically infects xanthomonas py campestris ,the causative agent of black rot in crucifers ΦLf contains a citcular single-stranded DNA genome of 6,008bp ,uses a replicative form RF as the intermediate of replication, and manifests a non-Lytic life cycle . Like Ff phages (fl,fd and M13), ΦLf has similar biological proerties and genomic organization i,e,in the order gII-gX-gV-gVII-gIX-gVIII-gIII-gVI-. AII the open reading frames for ΦLf have already been identified in our laboratory ,except for orf440 which is located downstream from gVI . The orf440 may be required for phage assembly ,because i) similar to the assembly protein of fl ,a membrane-spanning domain was found between aa 221 and 236 and ii) an ATP -birdngmotif is present in the N-terminal 97-aa region implying that the phage is assembled by ATP hydrolysis . This gene codes for pI (48kDa) detectable in the crude extract of the ΦLf-infected cells, Xcp20H(ΦLf), but not in the Xcp20h and mature phage particle by Western blot analysis using the antibody raised against the pI protein expressed in Escherichia coli. To understand more about the cellular location of pI,the cell envelope of the ΦLf -iffected Xcp20H was fractionjaed by sarkosyl solubilization followed by western blot analysis .Results from these .Results from these experiments showed that the pI the Kf- infected host ,Xcp20h f ,is an inner membrane protein . Deletioe of the 3,-terminal part of I the intergenic region (IR) from the ΦLf genome reulting in murant phage H2785m,was found to cause loss of phage export . Therefore ,it is possobe that the ΦLf PI plays a role similar to that of the Ff pI required for phage assembly and export The transcription of ΦLf gI was studiedd by Northern blkot analysis using a strand -speific DNA probe for gI mRNA ,but no signal mas detecred, In this study, a hairpin structure nt 4,627 and 4,657 in the downstream from g1 was found. Therefore, it is possible that φLf gI is co-transcribed with gIII and gVI till this putative transcription, similar to the case of Ff gI.
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Su, Wei-Chih, i 蘇偉誌. "Identification of the Filamentous Phage cf A Protein Gene and the Host Pilus Gene Required for the Infection". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/21010165954000642965.
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博士國立臺灣大學
植物學系
85
Filamentous bacteriophage cf was isolated from Xanthomonas campestris pv. citri. It was confirmed to consist of two coat proteins, major coat protein B and minor coat protein A. Those were encoded separately from the gene VIII and gene III within the 2.0 kb EcoRI/HincII fragment of cf genome. In order to investigate the function of A protein in the infection of phage cf, the A protein gene of cf was disrupted by introducing the kanamycin resistant gene from plasmid pUC4-KIXX in the HindIII site within the2.7 kb EcoRI/BamHI fragment of cf RF DNA. The RF DNA mutant (RF:Km) of cf was used to trasform X. c. pv. citri, and then the kanamycin resistant trasformant was further trasformed by a recombinant plasmid containing a intact A protein gene of cf by electroporation. The result of plaque assay showed that the infectivity of the non-infectious phage mutant (cf1k) could be restored by the trans-complement of A protein from the chimeric plasmid. It indicated that the function of A protein of cf is essential forphage infection. X. c. pv. citri was further mutated by Tn5tac1 mutagenesis for the search of host factor(s) which is responsible for the infection of cf. A mutant, XT501, was found to be resistant to cf infection and confirmed to contain only one copy of the trasposon inserting in the bacterial chromosome by Southern hybridization. The mutant could, however, still produce progeny virion of cf by the transfection of cf RF DNA into the mutant. It indicated that the mutated gene of XT501 was required only forthe infection, neither for the replication nor for the morphogenesis of phage cf. One of the flanking end aside the end of the trasposon was cloned directly by a procedure termed marker-rescue. A 0.4 kb EcoRI fragment flanking the transposon was used to probe genomic library of wild-type X. c. pv. citri constructed in cosmid pHC79 by colony hybridization. Four cosmid clones were selected and confirmed by Southern hybredization. The subclones of screened cosmid clone were used to perform complementation test. The results showed that a 1.7 kb fragement was required for the phage cf infection. The 1.7 kb fragment was then subcloned and subjected to determine the DNA sequence. A new gene, fimA, was found in the region of trasposon insertion site. The DNA sequence and the N-terminus of the predicted amino acid sequence of the fimA are homologous to type IV fimbriae gene (pilA). The predicted product of the fimA is a protein of 146 amino acid which contains a N-terminal leader peptide (Met- Lys-Lys-Gln-Gln-Gly)similar to type IV fimbriae of other bacteria.
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CHEN, YU-LI, i 陳玉麗. "Utilization of transfection of replicative form DNA from filamentous phage Cf to establish transformation system in xanthomonas campestris PV. citri". Thesis, 1987. http://ndltd.ncl.edu.tw/handle/75054334428979502878.
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Lin, Nien-Tsung, i 林念璁. "DNA regions of the filamentous phage φLf required for autonomous replication and integration into the chromosome of Xanthomonas campestris pv. campestris". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/52354368683585702067.
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博士國立中興大學
植物學系
84
Abstract Different regions of the filamentous bacteriophage φLf were previously found to integrate into the host Xanthomonas campestris pv. campestris chromosome. In this study, efforts were made to investigate the mechanisms involved in the integration of φLf. The results indicated that himA, the gene encoding the α subunit of integration host factor (IHF), was not required for integration. The recA gene was required for homologous recombination, but not for site-specific integration. In order to localize the sites for φLf integration, a 4,445-bp φ Lf-homologous region was cloned from the host chromosome. Sequence comparison showed high homology between the fragment and φLf, including the gene encoding the major coat protein, the attB carrying a 15-bp AT-rich core for φLf integration, and the putative IHF binding site. Deletion of this 4,445-bp region caused filamentation of the cells, a consequence similar to dif deletion in E. coli which loses normal partitioning of chromosome. In addition, after deletion of the 4,445-bp fragment, φLf could no longer integrate, indicating the φLf-homologous region to be required for homologous recombination of φLf. Replacement of the 4,445-bp fragment with a 59-bp fragment, containing the attB core sequence of Xc17, restore to the deletion mutant, a 72-bp segment including the attP core sequence simultaneously the wild-type phenotype and the ability to facilitate site-specific integration. These data indicate that the integrase required for site-specific integration of φLf is not located in the 4,445-bp fragment of Xc17 chromosome or included in the φLf genome. This situation is different from the site-specific integration systems for phages and plasmids which have integrase genes located near attP. A 1,013-bp fragment (nt 1386-2398) of the φLf RF DNA containing the gene II coding region was found to be maintained autonomously as a minireplicon, and the replication origin (ori) was localized in a segment of 121-bp (nt 1,591 to 1,711) within the gene II coding region, as assayed by providing gene II function in trans. Gene II was able to provide function for the kanamycin resistance gene from Tn903 to replicate in Xc17. Finally, by cloning the autonomous replicating fragment (nt 1,235-2,400) of φLf into pOK12, a shuttle cloning vector, p2GP, was constructed which could maintain in X. campestris and E. coli.
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Lin, Shu-Chin, i 林書進. "Effects of active site amino acid substitution of Thioredoxin on the growth of bacteriophage T7 and filamentous phages". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/77884192449220348752.
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碩士國立交通大學
生物科技研究所
85
abstract Thioredoxin appears to play a variety of roles in different cell types. It was isolated initially as a co-factor for the reduction of ribonucleotide diphosphatesby ribonucleotide reductase, has recently been shown to be required for the assemblyof the filamentous Escherichia coli phages f1, fd, M13, and required for phage T7growth. T7 DNA polymerase is composed of the gene 5 protein of the phage and Thioredoxin. Interaction has been predicted as an engine for phage assembly between the geneI protein of filamentous phage and thioredoxin system. However, little is known of of the role of thioredoxin in different" protein-protein interaction" types. In order to understand the influence of the different amino acid substitutionsin E.coli thioredoxin on the growth of bacteriophages T7 and filamentous phages , we have constructed a mutant in the thioredoxin gene changing its catalytic core.PCR site-directed-mutagenesis was crrried out to replace conservative Gly33 by Asp. The mutant was characterized using an in vivo assay based on the ability of cellto support growth of T7 and filamentous phages. The results indicated that the charged group side-chain in the position 33 of amino acid residues have great effecton the growth of filamentous phages, and little effect on the growth of T7 phages.
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Stolle, Tim Oliver [Verfasser]. "Development of bispecific filamentous bacteriophages for the generation of a novel automated screening system based on phage display technology / vorgelegt von Tim Oliver Stolle". 2005. http://d-nb.info/976116758/34.
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Wang, Wen-Horng, i 王文宏. "Sequence Analysis of the Filamentous phage f Xv of Xanthomonas campestris pv. vesicatoria. Roles of the two conserved isoleucine residues in the promoter -10 binding region on the structural and func". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/10130479351969448482.
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碩士國立中興大學
植物學系
84
fXv是Xanthomonas campestris pv. vesitoria線狀噬菌體，其基因體可 以穿插入寄主細胞染色體上。此外，此噬菌體顆粒和fLf線狀噬菌體顆粒 之抗血清具有交互反應之現象。為探討fXv和fLf線狀噬菌體在演化親緣上 的相關性，本研究乃繼續從事fXv基因體核甘酸序列之分析，並和fLf之核 甘酸序列加以比對。實驗結果顯示，fXv基因體由6479個核甘酸所組成， 比含6008個核甘酸之fLf基因體為大，但是兩者DNA序列間之相似性高達74 %。綜合本研究DNA序列分析資料、及先前同一噬菌體基因體之部份DNA序 列分析結果，整體上來說，fXv噬菌體含有九個可能的開放式解譯區，及 一個基因間區；這些區域以基因II/X- V- VII- IX- VIII- III- VI- I之 順序於噬菌體環狀基因體上排列，這種排列方式正如發現於fLf線狀噬菌 體上者。進一步有關fXv和fLf相對應解譯區蛋白之胺基酸序列排列分析結 果顯示，此兩噬菌體之各個可能蛋白間均有相當高之相同性，其相同性百 分率分布在98 %至78 %之間。其間以主要外套蛋白和單股DNA結合蛋白之 相同性最高；而以吸附蛋白（orf 362蛋白產物）之58 %相同性最低。 綜合上述結果，我們認為fXv和fLf兩種噬菌體在演化上具有相當接近之親 緣關係。 fXv is a filamentous bacteriophage of Xanthomonas cam- pestris pv. vesicatoria, which can integrate its genome into the host chromosome. This phage particle has a high cross reactivity to antisera of fLf, a filamentous bacteriophage of Xanthomonas campestris pv. campestris. In order to understand the evolutionary relationship between fXv and fLf, the circlular and single-stranded DNA genome of fXv was sequenced and analyzed. The genome size of fXv de-termined by DNA sequencing is 6479 nucleotides, larger than that of fLf which comprises6008 nucleotides. Comparison of DNA sequences revealed that there was a 74 % similarity be-tween these two phage genomes. In combination with previ-ously data, fXv totally contains nine open reading frames (orfs) and one intergenic region which are organized in the order of in- tergenic region -gene II/ X -V -VII -IX -VIII -III -VI -I similar to that reported for fLf. Amino acid sequence alignment of corre-sponding orfs of fXv and fLf always revealed very high per-centages of sequence identity between proteins of these two phages. The highest identity ( 98 %) was observed for both the putative capsid and single-stranded DNA binding proteins and the lowest ( 58 %) was for the adsorption protein of the phages. The high similarity in DNA sequences as well as the high identity of amino acid sequences between proteins of these two filamentous phages suggest that fXv and fLf are evolutionarilly closely related.