Dissertations / Theses on the topic 'Pectate lyase'

Cell wall loosening and degradation are important processes in major stages of plant development including fruit ripening. Three main mechanisms have been proposed to contribute towards cell wall polysaccharide degradation in vivo: enzymic hydrolysis by endopolygalacturonase (EPG), enzymic elimination by pectate lyase (PL), and non-enzymic scission by hydroxyl radicals (•OH). However, little idea as to which of these three mechanisms predominates in homogalacturonan degradation especially during fruit ripening. This study presents an attempt to discover the respective contribution of those three mechanisms of attack. The strategy used to achieve the objective of this study was to identify and measure homogalacturonan molecules that exhibit symptoms of each mechanism of attack. A method that was developed in this study is a fluorescent labelling method mainly to study the •OH attack on pectic polysaccharides. This labelling method is based on the ability of 2-aminoacridone (2-AMAC) to reductively aminate oxo groups of sugar moieties followed by exhaustive digestion with Driselase. In a model in-vitro experiment, the developed novel fluorescent labelling method, when applied to homogalacturonan, that had been attacked by •OH (Fenton reagent), produced at least three fluorescent ‘fingerprint’ compounds, separable by high-voltage paper electrophoresis (HVPE) based on their charge/mass properties at pH 6.5 and also by high pressure liquid chromatography (HPLC) on a C18 column with a fluorescence detector at λem= 520 nm. These fingerprint compounds include: a monomer, 1A*; a dimer, 2A*; and an unidentified compound, X*. In-vivo application with alcoholinsoluble residue (AIR) of seven species of fruit (pear, mango, banana, apple, avocado, strawberry and strawberry tree fruit) at three stages of softening produced at least two fluorescent fingerprint compounds: a monomer, 1AF and a dimer, 2AF. XF, an interesting compound found in a few samples in in-vivo experiments, showed electrophoretic mobility similar to X*; however, the retention time of this compound on HPLC did not agree with that of X*. 2AF was suggested to be exclusive evidence for •OH attack in vivo while 1AF was suggested to be a useful evidence not only to reveal •OH attack but also to reveal EPG and PL attack on pectic polysaccharides during fruit softening. HVPE and HPLC results showed an increasing pattern of 2AF in mango, banana, avocado and strawberry tree fruit, which indicated progressive •OH attack on pectic polysaccharides during the softening process. There was no clear evidence of 2AF at any stage of softening in apple and strawberry, which may suggest that fruit softening in apple and strawberry was not associated with •OH attack. On the other hand, HVPE analysis of 1AF showed and increasing pattern in pear, mango, banana, avocado and strawberry tree fruit, which may indicate EPG, PL and/or •OH attack during fruit softening. Production of these fluorescent fingerprint compounds provides good evidence for •OH attack on pectic polysaccharides, and has the potential to give useful information for EPG and PL attack in vivo.

Erwinia chrysanthemi est une enterobacterie phytopathogene responsable de la pourriture molle de nombreux vegetaux. Son pouvoir pathogene est du essentiellement a la production de pectinases et cellulases parmi lesquelles 8 pectate lyases (pels) sont des determinants majeurs. La plupart des pels et la cellulase celz sont exportees dans le periplasme ou elles acquierent une structure repliee grace a des catalyseurs de repliement tels que les disulfure isomerases et, probablement, des peptidyl prolyl cis-trans isomerases. L'etude de la region adjacente au locus pelb-pelc, codant deux pels, nous a permis d'identifier deux genes convergents, pelz et rota. L'expression du gene pelz est soumise a de nombreuses regulations qui affectent aussi la transcription des autres genes pel d'e. Chrysanthemi. Unite transcriptionnelle independante en conditions non induites, le gene pelz est co-transcrit avec pelc en presence de derives pectiques ; la synthese d'un arnm polycistronique demontre l'existence du premier operon de genes de la pectinolyse d'e. Chrysanthemi. Pelz est une endo-pectate lyase secondaire appartenant a une nouvelle famille de pels. Bien que presentant des caracteristiques enzymologiques proches de celles des autres pels d'e. Chrysanthemi, et notamment pelb et pelc, elle est la seule pel utilisant l'ion manganese comme cofacteur. Une synergie d'action a ete mise en evidence entre pele et pelz. Cette derniere est un determinant mineur de la pathogenicite d'e. Chrysanthemi, qui semble implique dans une certaine specificite d'hote. Le gene rota code une peptidyl prolyl cis-trans isomerase periplasmique de la famille des cyclophilines. Son expression, independante de celle des genes pel, est peu regulee. Aucune fonction de la proteine rota n'a pu etre determinee in vivo. Contrairement aux disulfure isomerases, rota ne semble pas etre essentielle pour le repliement des pels et de celz.

Die Pektat-Lyasen gehören zu einer Proteinfamilie, die meistens von pflanzenpathogenen Mikroorganismen sekretiert werden. Die Enzyme katalysieren den Abbau von Polygalakturonsäure, einem Hauptbestandteil in <br /> pflanzlichen Mittellamellen und Primärzellwänden. Der Abbau der alpha-1,4-verbrückten Galakturonsäurereste erfogt durch eine beta-Eliminierungsreaktion, dabei entsteht ein Produkt mit einer ungesättigten C4-C5 Bindung am nicht reduzierenden Ende, das durch spektroskopische Messungen beobachtet werden kann. Für die enzymatische Reaktion der Pektat-Lyasen ist Calcium nötig und das pH-Optimum der Reaktion liegt bei pH 8.5. Alle bis jetzt bekannten Strukturen der Pektat- und Pektin-Lyasen haben das gleiche Strukturmotiv - eine rechtsgängige parallele beta-Helix. Die Struktur der Pektat-Lyase aus Bacillus subtilis (BsPel) ist im Komplex mit Calcium gelöst worden. BsPel ist ein monomeres Protein mit einer ungefähren Molekularmasse von 43 kDa, das keine Disulfidbrücken enthält. Dies erlaubte sowohl eine effiziente rekombinante Expression des Wildtypproteins, als auch von destabilisierten Mutanten im Cytoplasma von E. coli. Parallele beta-Helices sind relativ große, jedoch verhältnismäßig einfach aufgebaute Proteine. Um detailliertere Informationen über die kritischen Schritte bei der in vitro-Faltung von parallelen beta-Helices zu erhalten, sollte in der vorliegenden Arbeit versucht werden, den Faltungsmechanismus dieses Proteins näher zu charakterisieren. Dabei sollte vor allem die Frage geklärt werden, welche Wechselwirkungen für die Stabilität dieses Proteins einerseits und für die Stabilität von essentiellen Faltungsintermediaten andererseits besonders wichtig sind.<BR><br>Rückfaltung von BsPel, ausgehend vom guanidiniumchlorid-denaturierten Zustand, war bei kleinen Proteinkonzentrationen und niedrigen Temperaturen vollständig möglich. GdmCl-induzierte Faltungsübergänge waren aber nicht reversibel und zeigten eine apparente Hysterese. Kinetische Messungen des Fluoreszenz- und CD-Signals im fernen UV ergaben eine extreme Denaturierungsmittelabhängigkeit der Rückfaltungsrate im Bereich des Übergangmittelpunktes. Der extreme Abfall der Rückfaltungsraten mit steigender Denaturierungsmittelkonzentration kann als kooperative <br /> Entfaltung eines essentiellen Faltungsintermediats verstanden werden. Dieses Faltungsintermediat ist temperaturlabil und kann durch den Zusatz Glycerin im Renaturierungspuffer stabilisiert werden, wobei sich die Hysterese verringert, jedoch nicht vollständig aufgehoben wird. Durch reverse Doppelsprungexperimente konnten zwei transiente Faltungsintermediate nachgewiesen werden, die auf zwei parallelen Faltungswegen liegen und beide zum nativen Zustand weiterreagieren können. Fluoreszenzemissionsspektren der beiden Intermediate zeigten, daß beide schon nativähnliche Struktur aufweisen. Kinetische Daten von Prolin-Doppelsprungexperimenten zeigten, daß Prolinisomerisierung den geschwindigkeitsbestimmenden Schritt in der Reaktivierung des denaturierten Enzyms darstellt. Desweiteren konnte durch Prolin-Doppelsprungexperimenten an Mutanten mit Substitutionen im Prolinrest 281 gezeigt werden, daß die langsame Renaturierung von BsPel nicht durch die Isomerisierung der einzigen cis-Peptidbindung an Prolin 281 verursacht wird, sondern durch die Isomerisierung mehrerer trans-Proline. Die beiden beobachteten transienten Faltungsintermediate sind somit wahrscheinlich zwei Populationen von Faltungsintermediaten mit nicht-nativen X-Pro-Peptidbindungen, wobei sich die Populationen durch mindestens eine nicht-native X-Pro-Peptidbindung unterscheiden.<BR><br>Der Austausch des Prolinrestes 281 gegen verschiedene Aminosäuren (Ala, Ile, Leu, Phe, Gly) führte zu einer starken Destabilisierung des nativen Proteins und daneben auch zu einer Reduktion in der Aktivität, da die Mutationsstelle in der Nähe der putativen Substratbindetasche liegt. Die Rückfaltungskinetiken der Prolinmutanten war bei 10&#176;C annähernd gleich zum Wildtyp und die geschwindigkeitsbestimmenden Schritte der Faltung waren durch die Mutation nicht verändert. Die durch die Mutation verursachte drastische Destabilisierung des nativen Zustands führte zu einem reversiblen Entfaltungsgleichgewicht bei pH 7 und 10&#176;C. GdmCl-induzierte Faltungsübergänge der Mutante P281A zeigten bei Messungen der Tryptophanfluoreszenzemission und der Aktivität einen kooperativen Phasenübergang mit einem Übergangsmittelpunkt bei 1.1 M GdmCl. Durch die Übereinstimmung der Faltungsübergänge bei beiden Messparametern konnten die Faltungsübergänge nach dem Zwei-Zustandsmodell ausgewertet werden. Dabei wurde eine freie Sabilisierungsenthalpie der Faltung für die Mutante von <nobr>-&nbsp;64.2&nbsp;&#177;&nbsp;0.4&nbsp;kJ/mol</nobr> und eine Kooperativität des Übergangs <br /> von <nobr>-&nbsp;58.2&nbsp;&#177;&nbsp;0.3&nbsp;kJ/(mol&#183;M)</nobr> bestimmt.<BR> <br /> <br>BsPel enthält, wie die meisten monomeren rechtsgängigen parallelen beta-Helix-Proteine, einen internen Stapel wasserstoffverbrückter Asparagin-Seitenketten. Die Mehrheit der erzeugten Mutanten mit Substitutionen im Zentrum der Asn-Leiter (N271X) waren als enzymatisch aktives Protein zugänglich. Die Auswirkung der Mutation auf die Stabilität und Rückfaltung wurde an den Proteinen BsPel-N271T und BsPel-N271A näher analysiert. Dabei führte die Unterbrechung des Asparaginstapels im Inneren der beta-Helix zu keiner drastischen Destabilisierung des nativen Proteins. Allerdings führten diese Mutationen zu einem temperatur-sensitiven Faltungsphänotyp und die Hysterese im Denaturierungsübergang wurde verstärkt. Offenbar wird durch die Unterbrechung des Asparaginstapel ein essentielles, thermolabiles Faltungsintermediat destabilisiert. Der Asparaginstapel wird somit bei der Faltung sehr früh ausgebildet und ist wahrscheinlich schon im Übergangszustand vorhanden.<br>Pectate lyases belong to a family of proteins secreted by plant pathogenic microbes. The enzymes cleave alpha-1,4 linked galacturonic acid by a beta-elimination that results in an unsaturated product, which can be quantified spectrophotometrically. Calcium is essential for the activity and the pH-optimum is near 8.5. All known structures of pectate and pectin lyases have the same structural motif - a right handed parallel beta-helix. The structure of pectate lyase from Bacillus subtilis (BsPel) has been solved in complex with calcium. It is a monomeric protein, with a molecular mass of about 43 kDa and without disulfide bonds. This allows its high-yield recombinant expression in the cytoplasm of Escherichia coli. Parallel beta-helices are relative large proteins, however with a simple folding topology. The objective of this work was to characterize the folding mechanism of BsPel. In particular we investigated the role of the interactions of certain residues in the parallel beta-helix for the stability of the native protein and the stability of essential folding intermediates.<br /> <br /> Refolding of BsPel was possible at low protein concentrations and low temperature. However, denaturation of BsPel was not freely reversible. De- and renaturation curves showed a large apparent hysteresis. Furthermore, the folding rate constant deduced from fluorescence and circulardichroism measurements showed a very strong dependence on denaturant concentrations near the midpoint of the renaturation transition. This can be explained with a cooperative unfolding of an essential folding intermediate. Upon stabilisation of the temperature-sensitive intermediate by addition of glycerol in the renaturation buffer, the hysteresis is reduced, but does not disappear. Reverse double mixing kinetic experiments have shown that two transient folding intermediates are on the folding pathway. These intermediates are on parallel pathways and both can fold to the native state. Fluorescence emission spectra have shown the native-like structure of both intermediates. Furthermore, data from proline double mixing kinetic experiments revealed that isomerization of peptidyl-prolyl bonds was responsible for the slow kinetics in the reactivation of the enzyme. However, the isomerization of the single cis-peptidyl-prolyl bond at Pro281 was not responsible for the slowest folding phase observed, but rather the isomerization of other trans-peptidyl-prolyl bonds. Thus, both transient folding intermediates observed probably represent two populations of folding intermediates with non-native X-Pro-peptide bonds. The difference of the two populations is at least one non-native X-Pro-peptide bond.<br /> <br /> Mutations of the proline 281 against various residues (Ala, Ile, Leu, Phe, Gly) resulted in a strong destabilization of the native protein. Also, the activity of the mutant proteins was strong reduced due to the position of the mutation site near the putative active center of the protein. At 10&#176;C the kinetic folding behavior of the proline mutants was not significant changed. However, the strong destabilization of the native state in the proline mutants resulted in a reversible folding equilibrium at pH 7 and 10&#176;C. The unfolding of the P281A mutant was reversible as determined by fluorescence emission and enzyme activity measurements. The coincidence of these detected transitions is consistent with a two-state equilibrium transition. At pH 7 and 10&#176;C the delta G&#176;(H₂O) for folding of P281A was <nobr>-&nbsp;64.2&nbsp;&#177;&nbsp;0.4&nbsp;kJ/mol,</nobr> with a midpoint of the transition at 1.1 M GdmCl and a cooperativity of <nobr>-&nbsp;58.2&nbsp;&#177;&nbsp;0.3&nbsp;kJ/(mol&#183;M).</nobr><br /> <br /> BsPel has an asparagine ladder in turn 2 of the parallel beta-helix with extensive network of side-chain hydrogen bonds between the Asn residues. Such an Asn-ladder is a conserved feature of many monomeric beta-helices crystallized so far. The middle Asn residue (271) was selected and exchanged for various residues. Most of the mutants were expressed at 25&#176;C as soluble and active proteins but with a significant reduction in yield. Mutants N271T and N271A were selected to study the stability and refolding of these proteins in comparison with the wild-type protein. The substitution in the Asn-ladder did not drastically destabilize the native protein, but caused a temperature-sensitive-folding (tsf) phenotype with an increased hysteresis in the de- and renaturation transition curves. In addition, the disruption of the Asn-ladder resulted in destabilization of an essential, thermosensitive folding intermediate. Thus, the Asn-ladder is formed very early during the folding, probably well before the transition state of folding.

L'objectif de cette thèse est, d'une part, axée sur les glycosidases modifiées pour la synthèse d'analogues de l'antigène H-1 et, d'autre part, de créer une activité hydratase à partir d'une pectate lyase. A cet effet, nous avons évalué trois glycosynthases et des transglycosidases. Nos résultats montrent que les glycosynthases permettent d'obtenir des disaccharides avec des rendements quasi quantitatifs. De plus, les transglycosidases permettent la synthèse de composés de transglycosylation avec des rendements de 60 à 75% selon l'accepteur utilisé. La modification de la pectate lyase de Thermotoga maritima (TmPel) aux travers d'approches complémentaires a été tentée. Nos efforts pour l'évolution dirigée de TmPel nous ont permis de mettre au point un système de sélection in vivo dans Escherichia coli pour la recherche de nouvelles activités enzymatiques. Nous avons également construit un modèle de TmPel permettant de discuter des possibilités d'ingénierie rationnelle<br>The aim of this PhD thesis was first to use modified glycosidases for the synthesis of antigen H-1 analogs and second to generate an hydratase activity from a pectate lyase. In this end, we evaluated three glycosynthases and transglycosidases. We showed that glycosynthases can synthesize disaccharides with nearly quantitative yields. Furthermore, transglycosidases can synthesize transglycosylation compounds with 60 to 75% yields depending of the acceptor. Modification of the Thermotoga maritima pectate lyase (TmPel) was attempted with complementary approaches. Our efforts for the directed evolution of TmPel led us to the development of an in vivo selection system in Escherichia coli allowing the seak of new enzymatic activities. We also constructed a model of TmPel to discuss about its rationnal engineering

Grewia optiva är en utav många outnyttjade stjälkfibrer som skulle kunna bidra till ökandet utav de naturliga fiberalternativen. Fibern har idag inte så många användningsområden på grund utav dess hårda och styva uppbyggnad, vilket gör den svår att spinna till garn. På uppdrag av organisationen Bhartiya Gramotthan Sanstha (BGS) har i detta projekt en redan befintlig metod utvecklats för att förädla fibern. Vad som främst eftersöktes var nedbrytandet av pektin som är en av de faktorer som bidrar till fiberns hårda och styva struktur. I metoden användes biologiskt nedbrytbara enzym som katalysatorer. En fungerande metod skulle kunna öka användningsområdet hos stjälkfibrer generellt och öka möjligheten till användandet utav fler naturliga fibrer. Enzymet som har använts i metoden är ett pektatlyas EC 4.2.2.2 som katalyserar reaktionen som sker då pektinmolekyler klyvs. För att effektivisera processen adderades en komplexbildare, EDTA, som tidigare visat goda resultat för lin. Efter enzymbehandlingen skedde en viktreduktion av fibrerna samt förändring av deras utseende. I svepelektronmikroskop observerades förändring av ytstruktur samt separation mellan fiberbuntarna. Dessa parametrar är viktiga och har stor inverkan på spinnbarheten hos fibrer. I projektet har försök att spinna fibern gjorts men inte lyckats helt. Förändringen på ytstruktur och separation mellan fibrerna tyder dock på att behandlingen är ett steg i rätt riktning.<br>Grewia optiva is one of many unused bast fibres that could contribute to an increase of natural textile fibres on the industrial market. This fibre has to-day not as many applications due to its stiff and hard structure that makes the fibre difficult to spin into yarn. On behalf of the organisation Bhartiya Gramotthan Sanstha (BGS) has an existing method been developed to process the Grewia optiva fibre. The method is developed to break down substances like pectin that is responsi-ble for the hard and stiff structure of the fibre. Degradable biological en-zymes were used as catalyser in the method. With a functioning method like this the applications of bast fibres could increase and contribute to the use of more natural fibres. The enzyme used to catalyse the chemical reaction and the cleavage of pec-tin molecules in this method was a pectate lyase EC 4.2.2.2. In this method EDTA was used as a chelator to efficient the chemical process. EDTA has been used as a chelator in earlier reports and showed good results. After the enzymatic treatment a weight reduction of the fibre was notable. In SEM-analysis separation between fibres and changes on the fibre surfaces was observed. These parameters are important and affect the spinning capability of the fibre. To test the spinning capability of the enzyme treated fibre they were spun in a ring spinning system, unfortunately not successfully. The surface changes and the separation shows that the enzymatic treatment had occurred and indicates that the method has developed in the right direction.

La nucléoline est une protéine nucléolaire servant de marqueur des cellules cancéreuses et qui interagit avec des acides nucléiques. Le protocole de purification de divers fragments de cette protéine a été optimisé afin de l’adapter aux exigences d’une étude cristallographique. Une analyse de l’interaction de ces fragments avec diverses séquences d’ADN structurées en G-quartet a été réalisée par des méthodes biophysiques. Parallèlement, des essais de cristallisation ont été menés sur ces fragments seuls et en complexe avec des acides nucléiques. La structure d’un ADN structuré en G-quartet a été obtenue. La pectate lyase PelI d’E. Chrysanthemi catalyse la dégradation de la pectine par un mécanisme de beta-élimination. Les structures de l’enzyme seule et en complexe avec un substrat, un acide tétragalacturonique, ont été déterminées. Divers mutants ont été produits, caractérisés et cristallisés, nous permettant de mieux comprendre le mécanisme réactionnel<br>Nucleolin is a classical marker of cancerous cells and which interacts with nucleic acids. The purification protocol of various fragments of the protein was optimized in order to meet requirements for crystallographic studies. The protein-G-quartet structured DNA complex was subjected to a detailed characterization using biophysical methods. At the same time, crystallization trials were performed on the protein alone and in complex with nucleic acids. The structure of a DNA structured in G-quartet has been solved. E. Chrysanthemi pectate lyase PelI catalyzes the degradation of pectin by a beta-elimination chemistry. The structure of the protein has been solved in its native state and in complex with a substrate, a tetragalacturonic acid. Different mutants have been produced, characterized and crystallized, leading us to a better understanding of the reaction mechanism

Made available in DSpace on 2015-03-26T13:51:58Z (GMT). No. of bitstreams: 1 texto completo.pdf: 952076 bytes, checksum: 7fbcb43414ecacd3439620826b6172cd (MD5) Previous issue date: 2012-07-20<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico<br>Colletotrichum lindemuthianum is the causal agent of common bean anthracnose. Genes that encode cell wall-degrading enzymes are essential for the development of this disease. The pectinases are characterized as the most important group of cell wall- degrading enzymes produced by phytopathogen fungi. The gene coding for pectate lyase, pecCl1, was previously identified in a suppressive subtractive library of bean infected with C. lindemuthianum. Isolation of the gene pecCl1 made it possible to obtain mutants and to analyze the regulation of this gene during development of anthracnose, determining whether the pectate lyase is a pathogenic factor. Thus, the aim of our study was structurally and functionally characterize the gene encoding pectate lyase in C. lindemuthianum. Initially, was performed the structural analysis of the gene pecCl1. The complete nucleotide sequence of the gene pecCl1 was deposited in Genbank with accession number JX270683. The analysis of the promoter region revealed some putative cis-elements and potential binding motifs of transcription factors involved in the regulation of pectate lyase gene expression. The deduced amino acid sequence of pecCl1 showed sequence identity with the pectate lyase F of Colletotrichum higginsianum and the pectate lyase C of Glomerella graminicola M1.001. Furthermore, it was found putative conserved domain pfam03211 of the pectate lyases superfamily. The gene pecCl1 is represented by a single copy in the C. lindemuthianum genome. However, into the genome of Colletotrichum graminicola, three sequences encoding pectate lyase showed sequence identity with the gene pecCl1 of C. lindemuthianum, and into the genome of C. higginsianum seven sequences encoding pectate lyase showed sequence identity with the gene pecCl1 of C. lindemuthianum, indicating that the C. lindemuthianum genome can possess other genes encoding pectate lyase. Phylogenetic analysis of pectate lyase amino acid sequences of filamentous fungi exhibited the formation of two distinct groups which are grouped on the basis of members of the pectate lyases multigene family. The Split-Marker technique was effective in C. lindemuthianum pecCl1 gene inactivation, allowing the study of pecCl1 function in a mutant by specific integrations and without ectopic integrations. The pecCl1 gene inactivation did not lead to complete loss of the pectate lyase activity, and consequently only decreased anthracnose symptoms in its host, which is consistent with the presence of other genes coding pectate lyase, allowing greater flexibility in pathogen aggressiveness. The analysis of differential expression of gene pecCl1 by qPCR was performed at different stages of bean infection and were observed expression levels of pecCl1 at all stages of development of the fungus in the plant, but a significant increase was observed five days after infection, in the onset of necrotrophic stage. At this stage, secondary hyphae cause extensive degradation of plant cell wall through the secretion of wide range of depolymerases, among these, the pectate lyase. Thus, the pectate lyase encoded by the gene pecCl1 is important to aggressiveness of C. lindemuthianum. The analysis of pectate lyases in C. lindemuthianum can not only assist in understanding the disease, but may also lead to discovery of one more target for disease control.<br>Colletotrichum lindemuthianum é o agente causal da antracnose do feijoeiro comum. Genes que codificam enzimas que degradam a parede celular são essenciais para o desenvolvimento dessa doença. As pectinases são caracterizadas como o grupo de enzimas que hidrolisam a parede celular mais importante produzidas por fungos fitopatogênicos. O gene pecCl1, que codifica pectato liase, foi previamente identificado em uma biblioteca subtrativa supressiva de feijoeiro infectado com C. lindemuthianum. O isolamento do gene tornou possível a obtenção de mutantes e análise da regulação deste gene durante o desenvolvimento da antracnose, visando determinar se a pectato liase é um fator de patogenicidade. Desta forma, o objetivo do nosso trabalho foi caracterizar estruturalmente e funcionalmente o gene que codifica pectato liase em C. lindemuthianum. Inicialmente, foi realizada a análise estrutural do gene pecCl1. A sequência completa de nucleotídeos do gene pecCl1 foi deposita no Genbank com número de acesso JX270683. A análise da região promotora revelou alguns possíveis cis-elementos e sítios de ligação a fatores de transcrição envolvidos na regulação da expressão gênica da pectato liase. A sequência de aminoácidos deduzida de pecCl1 apresentou identidade de sequências com a pectato liase F de Colletotrichum higginsianum e a pectato liase C de Glomerella graminicola M1.001. Além disso, detectou-se um possível domínio conservado pfam03211 da superfamília de pectato liases. O gene pecCl1 encontra-se representado por uma cópia única no genoma de C. lindemuthianum. No entanto, no genoma de Colletotrichum graminicola, três sequências que codificam pectato liase apresentaram identidade de sequências com o gene pecCl1 de C. lindemuthianum, e no genoma de C. higginsianum sete sequências que codificam pectato liase apresentaram identidade de sequências com o gene pecCl1 de C. lindemuthianum, indicando que o genoma de C. lindemuthianum pode possuir além do gene pecCl1 outros genes que codificam pectato liase. A análise filogenética de sequências de aminoácidos de pectato liases de fungos filamentosos mostrou a formação de dois grupos distintos, que se agruparam com base nos membros da família multigênica de pectato liases. A técnica de Split-Marker mostrou-se eficiente na inativação do gene pecCl1 de C. lindemuthianum, possibilitando o estudo da função do gene pecCl1, em um mutante com integração específica e livre de integrações ectópicas. A inativação do gene pecCl1 não levou a perda completa da atividade de pectato liase, e consequentemente, somente diminuiu os sintomas de antracnose em seu hospedeiro, o que é consistente com a presença de outros genes que codificam pectato liase no fungo, permitindo ao patógeno uma maior flexibilidade em sua agressividade. Foi realizada a análise da expressão diferencial do gene pecCl1 por qPCR nos diferentes estágios de infecção no feijoeiro e foram observados transcritos de pecCl1 em todas as fases de desenvolvimento do fungo na planta, mas houve um aumento significativo destes transcritos cinco dias após a infecção, no início da fase necrotrófica do fungo. Nesta fase, as hifas secundárias causam degradação extensiva da parede celular vegetal por meio da secreção de vasta gama de despolimerases, dentre estas, a pectato liase. Portanto, a pectato liase codificada pelo gene pecCl1 é importante para agressividade de C. lindemuthianum. A análise de pectato liases poderá não somente auxiliar na compreensão da antracnose em feijoeiro comum, mas também poderá levar a descoberta de mais um alvo para o controle dessa doença.

The aim of this research was to study the effect of pre-treatment and modification processes on the properties of hemp cellulose fibre for biopolymer composites application. Hemp fibres have been modified by various extraction, swelling, chemical and enzymatic treatments. The morphology and mechanical properties of the modified fibres have been measured. Biopolymer composites have been prepared using the modified fibres and matrices of cellulose acetate butyrate and cellulose solutions derived from hemp. The first fibre treatment employed was acetone extraction and mercerization. A low pressure acrylonitrile grafting initiated by azo-bis-isobutylonitrile was performed using alkali treated fibre. The AN grafted fibres had no transformation of crystalline structure as observed after mercerization. The mechanical properties performed by a single fibre test method were strongly influenced by the cellulose structure, lateral index of crystallinity, and fraction of grafting. Bioscouring of hemp using pectate lyase (EC 4.2.2.2), Scourzyme L, was performed. Greater enzyme concentration and a longer treatment improved the removal of the low methoxy pectin component. Removal of pectate caused no crystalline transformation in the fibres, except for a slight decline in the X-ray crystalline order index. Smooth surfaces and separated fibres were evidence of successful treatment. The shortening of fibre by grinding and ball-milling was introduced to achieve a desired fibre size. An increase in the milling duration gradual ly destroyed the crystalline structure of the cellulose fibres. An increase in solvent polarity, solvent-fibre ratio, agitation speed and drying rate resulted in the rearrangement of the ball-milled cellulose crystalline structure to a greater order. The thermal degradation behaviour of hemp fibres was investigated by using TGA. The greater activation energy of treated hemp fibre compared with untreated fibre represented an increase in purity and improvement of structural order. The all hemp cellulose composites were prepared by an introduction of fibres into 12% cellulose N-methyl-morpholine N-oxide (NMMO) solution and water-ethanol regeneration. A broadening of the scattering of the main crystalline plane, (002) and a depression of the maximum degradation temperature of the fibres were observed. These revealed a structural change in the fibres arising from the preparation. The mechanical properties of composites depended on size, surface area, crystallinity and the structural swelling of the fibres. Composites of cellulose acetate butyrate (CAB) and modified hemp fibres were prepared. Composites containing pectate lyase enzyme treated fibres showed better mechanical property improvement than untreated and alkali treated fibres respectively.

Pektatlyase (Pel-15) aus dem alkalophilen Bodenbakterium Bacillus spec. KSM-P15 ist mit 197 Aminosäuren eines der kleinsten, bekannten β-3-Solenoidproteine. Sie spaltet Polygalakturonsäurederivate in einem Ca2+-abhängigen β-Eliminierungsprozess. Wie bei allen Proteinen dieser Enzymfamilie ist auch die Polypeptidkette von Pel-15 zu einer einsträngigen, rechtsgängigen, parallelen β-Helix aufgewunden. In diesem Strukturmotiv enthält jede Windung drei β-Stränge, die jeweils durch flexible Schleifenbereiche miteinander verbunden sind. Insgesamt acht Windungen stapeln sich in Pel-15 übereinander und bilden entlang der Helixachse flächige, parallele β-Faltblätter aus. Im Bereich dieser β-Faltblätter existiert ein ausgedehntes Netzwerk von Wasserstoffbrückenbindungen, durch das der hydrophobe Kern, der sich im Inneren der β-Helix befindet, vom umgebenden Lösungsmittel abgeschirmt wird. Besondere Abschlussstrukturen an beiden Enden der β-Helix, wie sie typischerweise bei anderen Ver-tretern dieser Strukturklasse ausgeprägt werden, sind in Pel-15 nicht zu beobachten. Stattdessen sind die terminalen Bereiche der β-Helix über Salzbrücken und hydrophobe Seitenkettenkontakte stabilisiert. In der vorliegenden Dissertation wurde die Pektatlyase Pel-15 hinsichtlich ihres Faltungsgleichgewichtes, ihrer enzymatischen Aktivität und der Kinetik ihrer Strukturbildung charakterisiert. In eine evolutionär konservierte Helixwindung wurden destabilisierende Mutationen eingeführt, und deren Auswirkungen mittels spektroskopischer Methoden analysiert. Die Ergebnisse zeigen, dass Pel-15 in Gegenwart des Denaturierungsmittels Guanidiniumhydrochlorid einen hyperfluoreszenten Gleichgewichtsustand (HF) populiert, der nach Messungen von Faltungs- und Entfaltungskinetiken ein konformationelles Ensemble aus den Zuständen HFslow und HFfast darstellt. Diese HF-Zustände sind durch eine hohe Aktivierungsbarriere voneinander getrennt. In Rückfaltungsexperimenten populieren nur etwa 80 % der faltenden Moleküle den Zwischenzustand HFslow, der mit einer Zeitkonstante von ca. 100 s zu HFfast weiterreagiert. Die Denaturierungsmittelabhängigkeit dieser Reaktion ist sehr gering, was eine trans-/cis-Prolylisomerisierung als geschwindigkeitslimitierenden Schritt nahelegt. Die Existenz eines cis-Peptides in der nativen Struktur macht es erforderlich, den denaturierten Zustand als ein Ensemble kinetisch separierter Konformationen, kurz: DSE, zu betrachten, das durch die Spezies Ufast und Uslow populiert wird. Nach dem in dieser Arbeit aufgestellten „Minimalmodell der Pel-15 Faltung“ stehen die HF-Spezies (HFslow, HFfast) mit den Konformationen des DSE in einem thermodynamischen Kreisprozess. Das Modell positioniert HFfast und die native Konformation N auf die „native Seite“ der Aktivierungsbarriere und trägt damit der Tatsache Rechnung, dass die Gleichgewichtseinstellung zwischen diesen Spezies zu schnell ist, um mit manuellen Techniken erfasst zu werden. Die hochaffine Bindung von Ca2+ (Kd = 10 μM) verschiebt sich das Faltungsgleichgewicht bereits in Gegenwart von 1 mM CaCl2 soweit auf die Seite des nativen Zustandes, das HFfast nicht länger nachweisbar ist. Entgegen anfänglicher Vermutungen kommt einer lokalen, evolutionär konservierten Disulfidbrücke im Zentrum der β-Helix eine wichtige Stabilisierungsfunktion zu. Die Disulfidbrücke befindet sich in einem kurzen Schleifenbereich der β-Helix nahe dem aktiven Zentrum. Obwohl ihr Austausch gegen die Reste Val und Ala die freie Stabilisierungsenthalpie des Proteins um ca. 10 kJ/mol reduziert, lässt die Struktur im Bereich der Mutationsstelle keine gravierende Veränderung erkennen. Auch die katalytisch relevante Ca2+-Bindungsaffinität bleibt unbeeinflusst; dennoch zeigen Enzymaktivitätstests für VA-Mutanten eine Reduktion der enzymatischen Aktivität um fast 50 % an. Die evolutionär konservierte Helixwindung im Allgemeinen und die in ihr enthaltene Disulfidbrücke im Besonderen müssen nach den vorliegenden Ergebnissen also eine zentrale Funktion sowohl für die Struktur des katalytischen Zentrums als auch für die Strukturbildung der β-Helix während der Faltungsreaktion besitzen. Die Ergebnisse dieser Arbeit finden in mehreren Punkten Anklang an Faltungseigenschaften, die für andere β -Helixproteine beschrieben wurden. Vor allem aber prädestinieren sie Pel-15 als ein neues, β-helikales Modellprotein. Aufgrund seiner einfachen Topologie, seiner niedrigen Windungszahl und seiner hohen thermodynamischen Stabilität ist Pel-15 sehr gut geeignet, die Determinanten von Stabilität und Strukturbildung des parallelen β-Helix-Motivs in einer Auflösung zu studieren, die aufgrund der Komplexität bestehender β-helikaler Modellsysteme bislang nicht zur Verfügung stand.<br>Pectate lyase Pel-15 was isolated from alcaliphlic Bacillus spec. strain KSM-P15. Like all pectate lyases Pel-15 binds and subsequently cleaves polygalacturonic acid, the main pectic compound in plant cell walls and middle lamellae, in a Ca2+ dependent beta-elimination reaction. With 197 amino acids and a molecular mass of only 21 kDa the protein is one of the smallest right-handed parallel beta-helical proteins known today. Polypeptide chains that are classified into this structural family adopt super-helical folds in which each “solenoid stack” consists of three beta-structured regions that are connected by flexible turn segments. Along its longitudinal axis the right-handed parallel beta-helix thus comprises three elongated parallel beta-sheets that are stabilized by an extensive network of hydrogen bonds wrapping around the densely packed hydrophobic core. Together with the shield-like arrangement of hydrogen bonds this hydrophobic core is considered as the main contributor to an exceptionally high stability that is a common feature of all beta-helical proteins. In contrast to most right-handed parallel beta-helices, Pel-15 is devoid of any terminal capping domains and laterally associated secondary structure. Therefore, this protein is considered to be a promising model protein of a pure beta-helix which will help to understand the determinants of both parallel beta-sheet formation and stability. In the dissertation at hand optical spectroscopic methods were used to assess the enzymatic activity, the folding/unfolding equilibrium and the kinetic mechanism of structure formation in neutral buffered solutions. Results indicate that Pel-15 populates a hyper-fluorescent equilibrium intermediate (HF) that is effectively populated in presence of the denaturing agent guanidinium hydrochloride (GdmCl). According to kinetic folding and unfolding experiments HF is not only an essential on-pathway intermediate but has to be considered as a conformational ensemble in which several hyperfluorescent states are in thermodynamic equilibrium with each other. According to their existence in kinetic folding trajectories these different HF-species were termed HFslow and HFfast. The activation energy between both states is remarkably high leading to a time constant of about 100 seconds for the reaction HFslow ⇆ HFfast. Since native Pel-15 contains an energetically disfavoured cis-prolyl peptide between A59 and P60 it is proposed that HFslow and HFfast differ in their prolyl peptide conformations. Two main results emerge from this dissertation. First, an extensive study of the Pel-15 folding- and unfolding behaviour facilitated the proposal of a “minimal folding model”. According to this model the HF-states and the according denatured species Uslow and Ufast are aligned into a thermodynamic circle. This implies that unfolded polypeptide chains reach the HF-ensemble via parallel folding trajectories. Since the native conformation N together with HFfast are on the same side of the activation barrier, it is the reaction HFslow ⇆ HFfast that is the rate limiting step in the folding reaction of Pel-15. Second, the importance of an evolutionarily conserved disulfide bond in the central region of Pel-15 was tested by site directed mutagenesis and subsequent spectroscopic characterization. The exchange of the disulfide against a hydrophobic pair of alanine and valine decreases the folding free energy by about 10 kJ/mol. Although this value is unexpectedly high, structural perturbations around both mutational positions are small as was deduced from X-Ray crystallography. Interestingly, the stability decrease is accompanied by a major loss of enzymatic activity while the Ca2+ binding affinity is not significantly affected. It is therefore concluded that the allosterically relevant disulfide bond stabilizes long-range interactions that stabilize several adjacent solenoid turns near the N-terminus of the protein. Indeed, planar stacking interactions are perturbed and flexibility of N-terminal loops is increased once the disulfide bond is removed. This dissertation establishes Pel-15 as a novel beta-helical model protein and – even more important – smoothes the way for a generally accepted perspective on the formation and stability of parallel beta-sheet proteins.

Les oligosaccharides acides possèdent un grand intérêt biologique et présentent de nombreuses applications thérapeutiques. La synthèse in vitro de ces composés est indispensable car ceux-ci sont présents en faible quantité dans la nature et leur purification par extraction du milieu naturel est délicate. Leur synthèse chimique régiosélective est fastidieuse du fait du grand nombre d'étapes de protection-déprotection nécessaires et de la formation de structures furaniques et pyraniques. Les enzymes, par leur sélectivité, peuvent réduire considérablement l'ensemble de ces manipulations. Les lyases et les glycuronidases sont susceptibles de répondre à cette problématique. L'objectif du travail présenté dans cette thèse est d'évaluer les capacités des pectine- et pectate-lyases et des glycuronidases à produire des oligomères d'acides uroniques de façon régio- et stéréosélective. . .

Erwinia chrysanthemi est une bacterie phytopathogene responsable de la pourriture molle de nombreux vegetaux. La pathogenicite de cette bacterie est essentiellement due a la production et a la secretion de pectate lyases, codees par les genes pel. Nous nous sommes interesses a une de ces pectate lyases, pela, codee par le gene pe/a. Nous avons determine la sequence du gene pe/a et etudier l'expression de ce gene grace a une fusion pela::uida. Les genes impliques dans les relations plante-bacterie sont frequemment induits par des molecules vegetales. Nous avons demontre que les genes pel d'e. Chrysanthemi sont induits en presence d'extraits de plantes, mais seulement en synergie avec des derives pectiques inducteurs connus de la synthese des pectate lyases. Une purification partielle du compose inducteur a ete entreprise

La porriture molle de végétaux provoquée par Erwinia cluysanthemi est due à la sécrétion dans le milieu extracellulaire d'enzymes lytiques : pectinases, cellulases, protéases. Huit mutants (outJ) dans lesquels les pectate-lyases et les cellulases ne sont plus sécrétées dans le milieu extérieur mais retenues dans le périplasme ont été isolés, par mutagenèse chimique ou par insertion de phage Mu-lac. L'étude cinétique de la sécrétion de pectate-lyasés montre que le périplasme semble être un compartiment obligatoire pour la sécrétion. Les mutations outJ sont localisées dans un même locus sur le chromosome d'E. Chrysanthemi. Dans plusieurs mutants, une protéine péri plasmique de 65 kd est absente. Par étude de fusions outJ-lac, l'expression du gène outJ est apparue constitutive. Le clonage du gène outJ a été entrepris par utilisation du plasmide RP4::mini Mu. La sélection directe de ce gène étant difficile, le transposon Tn5 a été inséré près du gène out. ! par mutagenèse avec un plasmide suicide. La sélection de clones portant le Tn5 chez E. Coli puis la réintroduction chez un mutant outJ d'E. Chrysanthemi ont permis d'obtenir un fragment d'environ 30 kb portant l'insertion du Tn5 et le gène outJ. Le plasmide R551 complémente nos 8 mutations outJ. Le sous-clonage a permis de réduire cette région à un fragment ClaI/SspI de 4 kb. Le sens de transcription de la région codante a été établi à J'aide de fusions au gène lacZ réalisées par insertion de phages mini Mu-lac. Le gène outJ semble s'étaler sur environ 2,5 kb. Les fusions de protéines obtenues avec le transposon Tn5::phoA montrent que la protéine codée possède une séquence signal et donc qu'il s'agit d'une protéine exportée.

Le but poursuivi dans ce travail était de préciser au niveau moléculaire les déterminants du pouvoir phytopathogène de l'entérobactérie Erwinia chrysanthemi et d'aborder l'analyse des mécanismes de régulation intervenant dans l'expression de cette pathogénicité. E. Chrysanthemi produit des enzymes pectinolytiques qui sont les principales responsables de l'activité macératrice sur les végétaux. Une banque génomique de cette bactérie a d'abord été construite dans laquelle nous avons identifié une région pe1 composée de trois gènes distincts pe1A, pe1D, pe1E codant pour trois pectinases. La caractérisation physique de cette région nous a conduit à la connaissance de l'organisation des gènes~ les uns par rapport aux autres et a montré qu'il s'agit de trois unités de transcription indépendantes. Chaque gène a été isolé~par des expériences de sous-clonage,sur des fragments d'ADN de 217 à 516 kb. L'un de ces gènes pe1EJa été fusionné à l'opéron lac d'Escherichia coli ; l'étude de ces fusions de gènes a abouti à la détermination du sens de transcription du gène pe1E. Enfin, l'introduction de ces fusions dans E. Chrysanthemi par une méthode de transformation que nous avons mise au point, a permis d'aborder la régulation du gène pe1E. Par ailleurs, ce travail peut s'inscrire en préliminaire à la production industrielle de pectinases.

Erwinia carotovora subsp. Carotovora MFCL0 est une souche bactérienne phytopathogène isolée de la surface de céleris-raves conservés en chambre froide. Psychotrophe, elle est capable de se développer dans une large gamme de température (0°C à 37°C). Il a été montré que des pectate lyases (Pels), enzymes sécrétées par cette bactérie et impliquées dans la dégradation du tissu végétal, sont produites de façon maximale à une température de croissance inférieure à sa température optimale de croissance (28°C). Dans ce travail, l’implication de l’expression des gènes spécifiques des endo-pectate lyases (PelI, PelII, PelIII et PelZ) et de l’exo-pectate lyase (PelX) a été étudiée. Cela à la fois dans l’activité Pel globale (soit exo-cellulaire, soirpériplasmique) et le pouvoir pathogène de la bactérie. Les différents gènes pels ont été d’abord caractérisés. A l’instar de ceux d’Eca C18, les gènes des endo-Pels d’Ecc MFCL0 sont organisés à un même locus en tandem pel123Z. Des expériences de mutagénèse intégratives ciblées ont ensuite été entreprises. Des 5 souches mutées obtenues et spécifiquement affectées chacune dans les gènes pels, 2 sont significativement modifiées dans les phénotypes étudiés. Ainsi, l’inactivation des gènes pel1 ou pelZ entraîne des conséquences remarquables sur les activités enzymatiques testées à différentes températures de croissance et sur le pouvoir de macération du végétal par la souche. Les gènes pel1 et pelZ joueraient un rôle primordial dans l’activité Pel globale et dans la virulence de la souche Ecc MFCL0 vis-à-vis du céleri-rave. Enfin, grâce à la technique de RT-PCR relative, il a été montré que les effets de la température de croissance sur l’expression des gènes pels de la souche Ecc MFCL0 ne s’exerce pas à un niveau transcriptionnel<br>Erwinia carotovora subsp. Carotovora MFCL0, isolated from the surface of spoiled celeriac stored at low temperature, is a psychotropic and phytopathogenic bacteria able to grow in a wide growth temperatures range (0°C to 37°C). Several exo-cellular enzymes are produced by this strain, at a temperature lower than the optimal growth temperature (28°C), particularly pectate lyases enzymes (Pels). The characterization of the corresponding genes, for endo-Pels (PelI, PelII, PelIII and PelZ) and exo-Pel (PelX), shows that, like Eca C18 pels, endo-Pels genes are located in the same locus pel123Z on Ecc MFCL0 genome. The study of Ecc MFCL0 mutants, obtained by integrative mutagenesis and individually affected in pels genes, shows that PelI and PelZ have a main role in Pel activity and in pathogenesis against the host plant. Using relative RT-PCR experiments, it has been shown that the growth temperature effect on Ecc MFCL0 pels genes expression, doesn’t act at a transcriptional level

L'enterobacterie erwinia chrysanthemi souche 3937 provoque le symptome de pourriture molle sur son hote saintpaulia ionantha. Le developpement de la maladie requiert la production d'enzymes pectinolytiques, notamment cinq pectate lyases (pla a ple), et un systeme d'assimilation du fer a haute affinite base sur la production d'un siderophore de type catechol, la chrysobactine. L'operon fct-cbsceba code le recepteur de membrane externe de la ferrichrysobactine (fct) et des enzymes de biosynthese de la chrysobactine. Cette bacterie produit un second siderophore, l'achromobactine. Les mutants de transport de la ferriachromobactine (cbr) ont la particularite de produire la chrysobactine en conditions normalement repressives. Nous avons mis en evidence, par des tests de pouvoir pathogene, que des bacteries cultivees en carence en fer sont plus agressives. En revanche, l'apparition et la propagation des symptomes sont retardees avec les mutants cbr. L'etude in vitro de fusions pel::gus nous a permis de montrer que l'expression des genes pelb, pelc et pele est stimulee en conditions pauvres en fer, mais pas dans les mutants cbr. Nous nous sommes interesses ensuite a l'operon fct-cbsceba. La sequence de la region promotrice et du gene fct a ete realisee. La region promotrice presente une forte ressemblance avec les promoteurs divergents des operons fepa-entd et fes-entf d'e. Coli, impliques dans l'assimilation du fer via l'enterobactine. Nous avons montre qu'un gene similaire au gene fes d'e. Coli se trouve en orientation inverse de fct. Un site consensus du represseur fur chevauche la region promotrice et une fusion fct::lacz est regulee par fur chez e. Coli. Fct presente 36% d'identite avec le recepteur du ferrichrome fhua d'e. Coli et le recepteur de la ferrioxamine foxa de yersinia enterocolitica. Des predictions de structure secondaires et l'alignement de fct, fhua et foxa nous ont permis de proposer un modele de l'arrangement de fct dans la membrane externe

Le gene pela de la souche 3937 d'erwinia chrysanthemi qui code une pectate lyase de point isoelectrique acide, a ete sequence et caracterise. Le gene de structure est constitue d'une phase ouverte de lecture de 1179 bp codant un polypeptide de 41555 daltons, qui possede une sequence signal. Le clonage du gene pela derriere le promoteur de lacz du vecteur ptz19r a permis la surproduction de pla dans une souche deletee de ces autres genes pel construite a partir de la souche 3937. La proteine mature a ete purifiee et obtenue en grande quantite a partir du surnageant de cette souche. Ses proprietes biochimiques ont ete determinees et des anticoprs polyclonaux diriges contre la pla purifiee ont ete obtenus. La production des pl est stimulee dans les tissus de plante, en anaerobiose et en carence en fer, mais dans tous les cas, l'activite pla ne represente que 3 a 5% de l'activite pl totale produite par la souche sauvage. Bien que necessaire, cette enzyme n'est pas suffisante pour obtenir une virulence comparable a la souche 3937. Une mutagenese a l'aide du mini-mu mudiipr13 (cm#,lac), dans la region du gene pela, a permis de mettre en evidence un gene en amont de pela qui semblait etre son regulateur. La sequence de ce gene, pecx a ete realisee et fait apparaitre une phase ouverte de lecture de 1185 pb codant une proteine de 44230 daltons. La sequence peptidique de pecx est fortement homologue a celle de la proteine d'escherichia coli, codee par le gene hmp et qui possede une activite dihydropteridine reductase. L'analyse de la sequence a mis en evidence que pecx etait constitue d'au moins deux domaines, l'un etant homologue aux globines et l'autre correspondant a une activite reductase. Une fusion pecx-lac, a ete introduite par recombinaison homologue dans le chromosome de la souche 3937 d'erwinia chrysanthemi. En condition de culture micro-oxygenee, ce mutant pecx# produit une quantite reduite de pectate lyase et autres enzymes depolymerisantes et inocule a des plants de saint-paulia, il presente une agressivite tres reduite par rapport a la souche 3937. Le role possible de pecx dans une regulation dependante de la concentration en oxygene est discute

碩士<br>國立中興大學<br>分子生物學研究所<br>93<br>Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium causing black rot in cruciferous plants. The virulence of Xcc depends on a number of pathogenic genes and virulent factors including the exopolysaccharide and extracellular enzymes such as proteases, endoglucannases, and pectinases. It is known that extracellular enzymes in various Gram-negative bacteria are secreted by type II secretion pathway using PilD as the signal pepetidase for N-terminus processing. A pilD mutant, MC1220 (pilD::pSMP104), has previously been constructed from Xcc strain P20H in our laboratory. Plate assays for the extracellualr enzymes showed that pilD mutant exhibited the same levels of activity as those in the wild-type cells. To compare the N-terminal sequence, extracellular proteins from the culture supernatants of P20H and MC1220 (pilD::pSMP104) were separated in SDS-polyacrylamide gel electrophoresis, transblotted onto Nylon membrane, then three of the proteins (24, 28 and 35-kDa) were subjected to chemical determination of the N-terminal sequences. The results showed that each of the protein pairs has the same terminal ends. N-terminal sequencing also identified the 24, 28, and 35-kDa proteins as the conserved hypothetical protein (XCC0694 in the genome of Xcc strain ATCC33913), cellulose S (XCC3381), and pectate lyase II (XCC2815, designated as PelB herein), respectively. These results indicate that PilD is not the signal peptidase responsible for the N-terminus processing during secretion of these extracellular proteins. In a separate experiment, SDS-PAGE showed that the amounts of extracellular PelB were significantly reduced in a mutant deficient in Clp, a transcription factor homologous to CRP (cyclic AMP-receptor protein), suggesting that expression of pelB is regulated by Clp. Because of the latter finding, pelB was further studied. The results indicate that 1) the pelB mutant of Xcc acquired by insertional mutation showed no change in plate assays for pectinolytic activity, indicating that PelB is not the major pectate lyase in Xcc, 2) in pathogenicity testing using pelB mutant, appearance of symptoms was delayed compared with that of the wild-type virulent strain, and 3) in transcriptional fusion (pelB-lacZ) assays, the promoter activities were found to be greatly reduced in clp and rpfF mutant; suggesting that transcription of the pelB gene is positively regulated by Clp as well as RpfF.

碩士<br>國立高雄師範大學<br>生物科技系<br>106<br>Plants can't move freely like animals, When the environment is not suitable for survive, plants must start various mechanisms to protect itself from the stresses. Plant stress mainly divided into biological stress, such as herbivores and pathogens attack, and abiotic stress, such as high/low temperature, sunlight, flooding and drought. Salicylic acid, jasmonic acid and ethylene are plant hormones which activate, and promote defense system to achieve disease- and insect-resistant ability. Pectate lyase was first discovered in the cultures of Erwinia carotovora in 1962, and the function of pectate lyase was involved in maceration and degradation of cell wall. Pectate lyase was wildly exist in plants, including tomato. Pectate lyase participate in plant grow, ageing, fruit ripening, and softening. Pectate lyase gene was found to be induced by ethylene in bananas, and by salicylic acid in Arabidopsis. However, it is not clear about how to regulate the gene expression of pectate lyase in tomato. Therefore, this study analyze the promoter sequence of pectate lyase in tomato, and the result shows that there are many cis-acting elements which were regulated by abiotic and biotic stress present on the promoter DNA. We also found that the gene expression of pectate lyase was up-regulated by wounding, jasmonate and salicylic acid, not regulated by ethylene. Regarding the effect of abiotic stress, the gene expression of pectate lyase was induced by drought, flooding, and low temperature, but not regulated by constant light.

碩士<br>輔仁大學<br>生命科學系碩士班<br>95<br>Phalaenopsis soft rot bacterium, Erwinia chrysanthemi produces several pectic enzymes. Pectate lyases (Pels) the main E. chrysanthemi enzyme, depolymerize pectic substances of plant cell walls. The degradation products, unsaturated oligogalacturonates, have been proposed to induce plant defence reactions. If healthy plants are treated with Pels or oligosaccharides to make them produce defence reactions or resist soft rot pathogen. Constructed E. coli BL21 (DE3) and E. coli Rosetta (DE3) pLysS E.coli strains could express PelE and PelZ. Purified 47.4 kDa PelE and 48.5 kDa PelZ were used to produce PelE and PelZ antibodies. Soft rot leaves filtering solution had Pel activity. PelE and PelZ antibodies were used in Western blot for detecting soft rot leaves filtering solution. There had detected PelE and PelZ expression, the results indicated that PelE and PelZ express in soft rot pathogenesis. E. coli BL21 (DE3) (pET29a-pelE) and E. coli Rosetta (DE3) pLysS (pET29a-pelE) could secrete PelE protein out of the cell and was also able to degrade polygalacturonic acid. Its molecular weight of 40 kDa was smaller than we calculated. The N-terminal amino acid sequence of PelE had a signal peptide and its cleavage site based on sequence analysis and N-terminal amino acid sequencing of PelE. Signal peptide DNA sequence amplification by PCR was used to construct a new expression vector for other protein expression. PelE enzyme was purified from culture supernatant. PelE enzyme activity decreased significantly after seven days at room temperature. A complete removal of PelE enzyme activity required 100 ℃ for 30 minutes. The Km of PelE enzyme was 0.46 and the Vmax of PelE enzyme was 1.75. Analysis the products of PelE degraded polygalacturonic acid by HPLC. A maximum absorption was found at 230 nm, and the HPLC separation test showed a clear peak at 5.3 minute after sample injection. The time was similar to digalacturonic acid showed a clear peak at 5.2 minute after sample injection. The E.coli strains which could secrete PelE protein were inoculated into tobacco and triggered hypersensitive reaction but not seen in phalaenopsis. Purified active PelE enzyme was inoculated into tobacco and phalaenopsis both triggered hypersensitive reaction. The products of PelE degraded polygalacturonic acid were inoculated into tobacco and phalaenopsis both could not trigger hypersensitive reaction.

碩士<br>輔仁大學<br>生命科學系碩士班<br>92<br>Soft rot symptoms on white flowered calla lily (Zantedeschia aethiopica) were found in some nurseries in the Yang Ming Shan area, Taipei, Taiwan. Isolations from diseased flower stems consistently yielded bacterial colonies. Koch's postulates were completed by injecting bacterial suspensions (108 CFU/ml) into stems of white flowered calla lily. Control plants were inoculated with sterile distilled water. Symptoms developed 1-2 days in all inoculated plants and appeared to be identical to those observed on diseased material in nurseries. All of the control plants did not become rotten. Ten representative isolates were chosen for further characterization. All isolates were gram-negative rods, facultatively anaerobic, sensitive to erythromycin (25 g/ml), negative for oxidase, and positive for tryptophanase (indole production), and lecithinase. The isolates cannot produce acid from trehalose. This result revealed that this pathogen was identified as Erwinia chrysanthemi. Primers designed from 16S rRNA gene sequence were further utilized to perform PCR. PCR product was cloned and sequenced. Sequence analysis of 16S rRNA gene (AY360397) of this pathogen, revealed 98% of homology to 16S rRNA gene of E. chrysanthemi. By using indC and tryptophanase genes as probes in Southern hybridization, it was found that E. chrysanthemi showed positive result, but not with E. carotovora subsp. carotovora. A PCR primer set (pelZ exp) was designed based on conserved regions of pelZ gene, and a 1.2 kb PCR product can be amplified from E. chrysanthemi, but not from E. carotovora subsp. carotovora. Therefore, indC, tryptophanase probes, and the pelZ exp primer set are E. chrysanthemi-specific and can be used to identify E. chrysanthemi. Base on the results of physiological and biochemical test, sequence analysis of 16S rDNA gene, and molecular detection of specific genes, confirmed that this pathogen which infected white flowered calla lily and caused soft rot disease is E. chrysanthemi and named this strain as S3-1. By inoculating pedicels of white flowered calla lily with E. chrysanthemi isolated from different hosts, it was found that E. chrysanthemi which was isolated from white flowered calla lily showed the most serious symptom, pathogenicity of E. chrysanthemi isolated from phalaenopsis is the second, and those isolated from green onions and celery, their pathogenicity is weaker. In the other hand, E. carotovora subsp. carotovora isolated from the Chinese cabbage and potato also showed weak pathogenicity after inoculated to the pedicels of white flowered calla lily. By inoculating E. chrysanthemi strain S3-1 to different plants, we found that this pathogen can cause soft rot disease in potato, sweet potato, onion, carrot, phalaenopsis, sweet pepper, and celery. E. chrysanthemi S3-1 and E. carotovora subsp. carotovora CS3-2 were cultured in 28℃and 37℃ water respectively. We found that E. chrysanthemi S3-1 survive longer than E. carotovora subsp. carotovora CS3-2 either in 28℃ or 37℃ water. In order to understand whether the soft rot disease of the white flowered calla lily was caused by E. chrysanthemi of other plant hosts in Taiwan, molecular typing methods including Restriction Fragment Length Polymorphism （RFLP）, PCR-RFLP, and pulsed field gel electrophoresis （PFGE）were used. In the experiment of RFLP, indC, tryptophanase, pel AED, and pelZ genes were used as probes to perform Southern hybridization, it was found that E. chrysanthemi from white flowered calla lily and other plants host are two different patterns. A PCR primer set was designed based on the conserved regions of pelZ gene. PCR-RFLP study was undertaken by digesting the amplified fragment with Ahd I and Pst I enzymes. The digestion pattern revealed that E. chrysanthemi of white flowered calla lily is different from E. chrysanthemi of other hosts. In the PFGE experiment, the banding patterns obtained with enzyme Xba I digestion revealed significant differences among E. chrysanthemi and E. carotovora subsp. carotovora strains from different hosts. Results from the above-mentioned molecular typing methods, E. chrysanthemi isolated from the white flowered calla lily can be distinguished from those isolated from other plants as different molecule types. Besides, the molecule typing differences between E. chrysanthemi and E. carotovora subsp. carotovora are greater. Via pathogenesis test, and molecular typing, the E. chrysanthemi strain that caused soft rot diseases among white flowered calla lily is different from E. chrysanthemi strain found in Taiwan before, and due to the soft rot disease has not been found among the white flowered calla lily previously, so we suggested that the E. chrysanthemi strain which isolated from white flowered calla lily is newly introduced from abroad. Soft rot Erwinia spp. causes the soft rot disease of plants by producing pectate lyase that able to degrade plant cell wall. The pectate lyase will lead to the realease of the oligosaccharides, these plant cell wall fragments function as elicitors to stimulate plant defense responses. We reasoned that the introduction of pectate lyase gene into plants would alter the situation to the benefit of the plant host by triggering pectate lyase synthesis, which is already at low bacterial cell densities. Due to lack of complete tissue cultivation and transgenic technology for white flowered calla lily (Zantedeschia aethiopica), while there are complete tissue culture and transgenic technique for phalaenopsis, and the soft rot disease pathogen of phalaenopsis is also E. chrysanthemi, therefore this study is to introduce pelE and pelZ of the pectate lyase gene of E. chrysanthemi into phalaenopsis separately, in order to cultivate the phalaenopsis which can resist the soft rot disease. The pelE and pelZ genes from E. chrysanthemi were cloned and the 47.13-kDa PelE enzyme and 48.38-kDa PelZ enzyme was produced in E. coli pET system separately. Although most of PelE and PelZ are insoluble and formed an inclusion body, a few soluble PelE and PelZ still existed in soluble portion and can be purified. The purified soluble PelE and PelZ have the pectate lyase activity. Phalaenopsis leaves were inoculated with these enzymes. We found that PelE and PelZ will induce phalaenopsis's defense responses to E. chrysanthemi. At this stage the pelE gene and pelZ genes have been cloned to pBI121 vector, and in the process of being transgened to phalaenopsis.

Yes<br>Dickeya chrysanthemi is well known as a plant pathogen that caused major blackleg in the European potato industry in the 1990s. D. chrysanthemi strain L11 was discovered in a recreational lake in Malaysia. Here, we present its draft genome sequence.<br>University of Malaya High Impact Research (HIR) Grants UM C/625/1/HIR/MOHE/CHAN/01 (grant no. A-000001-50001) and UM C/625/1/HIR/MOHE/CHAN/14/1 (grant no. H-50001-A000027)

L’une des particularités fondamentales caractérisant les cellules végétales des cellules animales est la présence de la paroi cellulaire entourant le protoplaste. La paroi cellulaire joue un rôle primordial dans (1) la protection du protoplaste, (2) est impliquée dans les mécanismes de filtration et (3) est le lieu de maintes réactions biochimiques nécessaires à la régulation du métabolisme et des propriétés mécaniques de la cellule. Les propriétés locales d’élasticité, d’extensibilité, de plasticité et de dureté des composants pariétaux déterminent la géométrie et la forme des cellules lors des processus de différentiation et de morphogenèse. Le but de ma thèse est de comprendre les rôles que jouent les différents composants pariétaux dans le modelage de la géométrie et le contrôle de la croissance des cellules végétales. Pour atteindre cet objectif, le modèle cellulaire sur lequel je me suis basé est le tube pollinique ou gamétophyte mâle. Le tube pollinique est une protubérance cellulaire qui se forme à partir du grain de pollen à la suite de son contact avec le stigmate. Sa fonction est la livraison des cellules spermatiques à l’ovaire pour effectuer la double fécondation. Le tube pollinique est une cellule à croissance apicale, caractérisée par la simple composition de sa paroi et par sa vitesse de croissance qui est la plus rapide du règne végétal. Ces propriétés uniques font du tube pollinique le modèle idéal pour l’étude des effets à courts termes du stress sur la croissance et le métabolisme cellulaire ainsi que sur les propriétés mécaniques de la paroi. La paroi du tube pollinique est composée de trois composantes polysaccharidiques : pectines, cellulose et callose et d’une multitude de protéines. Pour comprendre les effets que jouent ces différents composants dans la régulation de la croissance du tube pollinique, j’ai étudié les effets de mutations, de traitements enzymatiques, de l’hyper-gravité et de la gravité omni-directionnelle sur la paroi du tube pollinique. En utilisant des méthodes de modélisation mathématiques combinées à de la biologie moléculaire et de la microscopie à fluorescence et électronique à haute résolution, j’ai montré que (1) la régulation de la chimie des pectines est primordiale pour le contrôle du taux de croissance et de la forme du tube et que (2) la cellulose détermine le diamètre du tube pollinique en partie sub-apicale. De plus, j’ai examiné le rôle d’un groupe d’enzymes digestives de pectines exprimées durant le développement du tube pollinique : les pectate lyases. J’ai montré que ces enzymes sont requises lors de l’initiation de la germination du pollen. J’ai notamment directement prouvé que les pectate lyases sont sécrétées par le tube pollinique dans le but de faciliter sa pénétration au travers du style.<br>One of the most important features characterizing plant cells and differentiating them from animal cells is the cell wall that surrounds them. The cell wall plays a critical role in providing protection to the protoplast; it acts as a filtering mechanism and is the location of many biochemical reactions implicated in the regulation of the cell metabolism and the mechanical properties of the cell. The local stiffness, extensibility, plasticity and elasticity of the different cell wall components determine the shape and geometry of the cell during differentiation and morphogenesis. The goal of my thesis is to understand the role played by the different cell wall components in shaping the plant cell and controlling its growth behaviour. To achieve this goal, I studied the pollen tube, or male gametophyte, as a cellular model system. The pollen tube is a cellular protuberance formed by the pollen grain upon its contact with the stigma. Its main purpose is to deliver the sperm cells to the female gametophyte to ensure double fertilization. The pollen tube is a tip-growing cell characterized by its simple cell wall composition and by the fact that it is the fastest growing cell of the plant kingdom. This makes it the ideal model to study the effects of drugs, mutations or stresses on cellular growth behaviour, metabolism and cell wall mechanics. The pollen tube cell wall consists mainly of proteins and three major polysaccharidic components: pectins, cellulose and callose. To understand the role played by these components in regulating pollen tube growth, I investigated the effects of mutations, enzymatic treatments, hyper-gravity and omni-directional gravity on the pollen tube cell wall. Using mathematical modeling combined with molecular biology and high-resolution electron and fluorescent microscopy I was able to show that the regulation of pectin chemistry is required for the regulation of the growth rate and pollen tube shape and that cellulose is crucial for determining the pollen tube diameter in the sup-apical region. Moreover, I investigated the role of the pectate lyases, a group of pectin digesting enzymes expressed during pollen tube development, and I showed that this enzyme activity is required for the initiation of pollen germination. More importantly, I directly showed for the first time that the pollen tube secretes cell wall loosening enzymes to facilitate its penetration through the style.