Chapter 1 Hydrocarbon autoxidation, a free radical chain reaction, is believed to play a key role in the onset and developments of most degenerative diseases and disorders. The two propagating steps: 1) H-atom abstraction from the hydrocarbon by a hydrocarbon-derived peroxyl radical, and 2) addition of oxygen to the resultant alkyl radical to form a new peroxyl, play a role in determining the rate of hydrocarbon autoxidation, as well as the regio- and stereochemistry of the product hydroperoxides. In the current study, we carried out a set of calculations to provide a detailed framework for understanding the mechanism of the first two steps of autoxidation. Chapter 2 Radical-trapping chain-breaking antioxidants inhibit hydrocarbon autoxidation. Phenols are the prototypical radical-trapping antioxidants and are employed in nature, as well as in industry, to inhibit the autoxidation of hydrocarbons. The mechanism of inhibiting radical chain propagation has recently been suggested to be a PCET on the basis of theoretical calculations. It has been demonstrated that the antioxidant activitiy of phenols is increased in the presence of either protic acids or alcohols, but the basis of this acceleration is not well understood. In the current study, we used computational methods to investigate the effects of acids and alcohols on the PCET pathway for the reaction of phenol with a peroxyl radical. Chapter 3 The antibiotic pyrrolnitrin [3-chloro-4-(2’-nitro-3’-chlorophenyl) pyrrole] (PRN) is biosynthesized from L-tryptophan in four steps, catalyzed by the enzymes PrnA, B, C and D encoded by the prn operon. Two of the four gene products, PrnA and PrnC, are flavin-dependent halogenases, a recently discovered and highly interesting class of enzymatic halogenation catalysts. Their activities have never been unequivocally demonstrated by reconstitution of the activity from a recombinant protein. Herein, we report the results of our efforts to clone the genes encoding PrnA and PrnC, and overexpress, isolate and purify the proteins from E. coli. We were able to successfully reconsistute halogenation activity of both and have obtained the first kinetic data for PrnC, which shows kinetics similar to other flavin-dependent halogenases, along with substrate inhibition. / Thesis (Master, Chemistry) -- Queen's University, 2010-02-03 15:42:39.67

This thesis details a straightforward synthetic route to the antifungal compound pyrrolnitrin 1.2, along with several analogous halogenated phenylpyrroles. The proposed synthetic protocol involved the Suzuki-Miyaura cross-coupling of appropriately halogenated pyrrole pinacolboronate esters and aryl compounds. In the efforts towards preparing the cross-coupling partners, we report a regiospecific and high yielding synthesis of a 3-chloro pyrrole compound 2.14, its brominated analog 2.16, an iodinated analog 2.17, and the corresponding pinacolboronate ester 2.18. We also report a generalized reaction sequence (lithiation/carboxylation/Schmidt reaction/oxidation) for the preparation of halogenated benzoic acids, anilines and nitrobenzenes. In particular, we synthesized the desired halogenated nitrobenzene coupling partner 3.27 in excellent yield. We were also able to show that the conditions employed in this sequence were mild enough to allow preparation of the 2-bromo-6-iodo compound 3.33. Once the coupling partners were prepared, we developed the optimal conditions for our Suzuki-Miyaura cross-coupling reactions. In doing so, we were able to prepare our target compound 1.2 and several halogenated analogs in good yields. We also prepared brominated and deuterated arylpyrroles 4.27 and 4.28, respectively, for future use in mechanistic studies of the pyrrolnitrin biosynthetic enzymes, PrnB, Prn C and PrnD. This required preparation of the corresponding brominated and deuterated pyrrole pinacolboronate esters 4.24 and 4.26. / Thesis (Master, Chemistry) -- Queen's University, 2009-09-29 13:58:35.186

A Terra Preta Antropogênica (TPA) é considerada um dos solos mais férteis do mundo e recebe essa denominação por ser originada da ação antrópica, provavelmente de populações pré-colombianas que viveram nestes sítios arqueológicos. O crescente aumento por agricultura sustentável torna a utilização de bactérias produtoras de antibióticos uma alternativa de controle para doenças de plantas. Pirrolnitrina (PRN) é um antibiótico que tem ampla atividade antimicrobiana produzida por várias estirpes de Burkholderia e Pseudomonas que foram isoladas de diferentes solos. Entretanto, não se tem conhecimento de isolados bacterianos de TPA que produzem PRN, assim com, sobre a ecologia e freqüência do gene para este antibiótico. A PRN é codificada por um operon composto por 4 genes sendo o gene prnD responsável por catalisar a oxidação que forma a pirrolnitrina. Neste trabalho, foi estudado o gene prnD através de métodos dependente e independente de cultivo. Foram utilizados isolados bacterianos para detectar o gene prnD através de PCR convencional e a identificação feita pelo seqüenciamento. As bactérias com amplificação positiva tiveram suas sequências do gene analisadas no programa MOTHUR o qual reuniu as em 10 grupos. Um representante de cada grupo foi empregado no teste de antagonismo contra o fitopatógeno Fusarium oxysporum. Amostras de solo de TPA e seus solos adjacentes foram coletados de dois sítios: Caldeirão Capoeira (floresta secundária por mais de 20 anos) e Cultivado (cultivado com mandioca por pelo menos 30 anos). Os DNAs totais das amostras de solo extraído foram usados como molde nas reações de PCR quantitativo em tempo real para verificar a abundância do gene prnD nas amostras de solo. Em amostras de solo também foi quantificado o gene 16S rRNA. No estudo dependente de cultivo, do total de 219 isolados (175 Burkholderia e 44 Pseudomonas), 60 isolados do gênero Burkholderia e 3 de Pseudomonas exibiram amplificação positiva. A análise filogenética do gene prnD mostrou que a maioria das seqüências obtidas deste estudo não agruparam com as seqüências do banco de dados do GenBank indicando que há diversidade do gene prnD nos isolados de solos amazônicos e que podem ser distintos dos descritos anteriormente. O teste de antagonismo demonstrou que isolados com potencial genético para produção de pirrolnitrina também são bioativos contra Fusarium oxysporum, apresentando forte atividade antimicrobiana. No estudo independente de cultivo, no sítio Caldeirão Cultivado, solo de TPA apresentou maior número de cópias do gene prnD e do gene 16S rRNA em relação ao solo ADJ. No sítio Caldeirão Capoeira, o solo adjacente apresentou maior quantidade do gene prnD (1,74x105 cópias/g solo) que o solo de TPA (2,48x104 cópias/ g de solo), no entanto, na TPA as bactérias totais foram mais abundante. Estes resultados evidenciam que a metodologia de PCR quantitativo em tempo real desenvolvida foi altamente sensível e específica permitindo a detecção de diferenças sensíveis e significativas entre os solos na quantificação do gene prnD. A abundância do gene prnD correlacionou significativamente com parâmetros químicos do solo tais como pH, fósforo, cálcio, magnésio e micronutrientes / Anthropogenic Dark Earth (ADE) is considered one of the world\'s most fertile soils and receives this name because it originated from human action, probably by pre-Columbian populations who lived in these archaeological sites. Because of the common trend in agriculture towards sustainability antibiotic-producing bacteria is an alternative for biocontrol to plant diseases. Pyrrolnitrin (PRN) is a broad-spectrum antibiotic produced by various strains of Burkholderia and Pseudomonas that have been isolated from different soils. However, little is known about PRN-producing bacteria screened from ADE, even as the ecology and frequency of pyrrolnitrin gene. PNR is encoded by an operon comprised by four genes and prnD gene is responsible for catalyze the oxidation to form pyrrolnitrin. In this work, we studied the gene prnD through culture dependent and independent methods. Conventional PCR were established to detect prnD gene in bacterial isolates screened in ADE and their adjacent soils (ADJ) and identification were done by sequencing. Based on the results generated by MOTHUR prnD sequences from isolates were grouped into 10 groups. A representative of each group was used in the test of antagonism against the plant pathogen Fusarium oxysporum. Soil samples of ADE and its adjacent soils were collected from two sites: Caldeirão Capoeira (secondary forest for over 20 years) and Caldeirão Cultivado (cultivated with cassava for at least 30 years). The total DNA extracted from soil samples was used as template in quantitative PCR reactions to determine the abundance of prnD gene. In soil samples was also quantified the 16S rRNA. In the study culture-dependent, in a total of 219 isolates (175 Burkholderia and 44 Pseudomonas), 60 isolates of Burkholderia and 3 Pseudomonas exhibited positive amplification for prnD gene. Phylogenetic analysis of prnD gene showed that most of the sequences obtained in this study grouped distinctly from sequences of the GenBank database. It indicates that there is diversity in prnD gene of isolates from amazonian soils and they may differ from those previous described. The antagonism test showed that isolates with genetic potential for production of pyrrolnitrin are also bioactive against Fusarium oxysporum, exhibiting strong antimicrobial activity. In culture-independent study, the site Caldeirão Cultivado, ADE soil showed higher copies number of prnD gene and 16S rRNA gene than in ADJ soil. At the site Caldeirão Capoeira, surrounding soil had a higher amount of prnD gene (1.74 x105 copies / g soil) than ADE soil (2.48 x104 copies / g soil), however, in ADE total bacteria was more abundant. These results show that the real-time PCR assay developed in this study was highly sensitive and specific enabling the detection of sensitive and significant differences between soils in the quantification of prnD gene. Soil variables such as pH, phosphorus, calcium, magnesium and micronutrients significantly correlated with abundance of prnD gene

A Terra Preta Antropogênica (TPA) é considerada um dos solos mais férteis do mundo e recebe essa denominação por ser originada da ação antrópica, provavelmente de populações pré-colombianas que viveram nestes sítios arqueológicos. O crescente aumento por agricultura sustentável torna a utilização de bactérias produtoras de antibióticos uma alternativa de controle para doenças de plantas. Pirrolnitrina (PRN) é um antibiótico que tem ampla atividade antimicrobiana produzida por várias estirpes de Burkholderia e Pseudomonas que foram isoladas de diferentes solos. Entretanto, não se tem conhecimento de isolados bacterianos de TPA que produzem PRN, assim com, sobre a ecologia e freqüência do gene para este antibiótico. A PRN é codificada por um operon composto por 4 genes sendo o gene prnD responsável por catalisar a oxidação que forma a pirrolnitrina. Neste trabalho, foi estudado o gene prnD através de métodos dependente e independente de cultivo. Foram utilizados isolados bacterianos para detectar o gene prnD através de PCR convencional e a identificação feita pelo seqüenciamento. As bactérias com amplificação positiva tiveram suas sequências do gene analisadas no programa MOTHUR o qual reuniu as em 10 grupos. Um representante de cada grupo foi empregado no teste de antagonismo contra o fitopatógeno Fusarium oxysporum. Amostras de solo de TPA e seus solos adjacentes foram coletados de dois sítios: Caldeirão Capoeira (floresta secundária por mais de 20 anos) e Cultivado (cultivado com mandioca por pelo menos 30 anos). Os DNAs totais das amostras de solo extraído foram usados como molde nas reações de PCR quantitativo em tempo real para verificar a abundância do gene prnD nas amostras de solo. Em amostras de solo também foi quantificado o gene 16S rRNA. No estudo dependente de cultivo, do total de 219 isolados (175 Burkholderia e 44 Pseudomonas), 60 isolados do gênero Burkholderia e 3 de Pseudomonas exibiram amplificação positiva. A análise filogenética do gene prnD mostrou que a maioria das seqüências obtidas deste estudo não agruparam com as seqüências do banco de dados do GenBank indicando que há diversidade do gene prnD nos isolados de solos amazônicos e que podem ser distintos dos descritos anteriormente. O teste de antagonismo demonstrou que isolados com potencial genético para produção de pirrolnitrina também são bioativos contra Fusarium oxysporum, apresentando forte atividade antimicrobiana. No estudo independente de cultivo, no sítio Caldeirão Cultivado, solo de TPA apresentou maior número de cópias do gene prnD e do gene 16S rRNA em relação ao solo ADJ. No sítio Caldeirão Capoeira, o solo adjacente apresentou maior quantidade do gene prnD (1,74x105 cópias/g solo) que o solo de TPA (2,48x104 cópias/ g de solo), no entanto, na TPA as bactérias totais foram mais abundante. Estes resultados evidenciam que a metodologia de PCR quantitativo em tempo real desenvolvida foi altamente sensível e específica permitindo a detecção de diferenças sensíveis e significativas entre os solos na quantificação do gene prnD. A abundância do gene prnD correlacionou significativamente com parâmetros químicos do solo tais como pH, fósforo, cálcio, magnésio e micronutrientes / Anthropogenic Dark Earth (ADE) is considered one of the world\'s most fertile soils and receives this name because it originated from human action, probably by pre-Columbian populations who lived in these archaeological sites. Because of the common trend in agriculture towards sustainability antibiotic-producing bacteria is an alternative for biocontrol to plant diseases. Pyrrolnitrin (PRN) is a broad-spectrum antibiotic produced by various strains of Burkholderia and Pseudomonas that have been isolated from different soils. However, little is known about PRN-producing bacteria screened from ADE, even as the ecology and frequency of pyrrolnitrin gene. PNR is encoded by an operon comprised by four genes and prnD gene is responsible for catalyze the oxidation to form pyrrolnitrin. In this work, we studied the gene prnD through culture dependent and independent methods. Conventional PCR were established to detect prnD gene in bacterial isolates screened in ADE and their adjacent soils (ADJ) and identification were done by sequencing. Based on the results generated by MOTHUR prnD sequences from isolates were grouped into 10 groups. A representative of each group was used in the test of antagonism against the plant pathogen Fusarium oxysporum. Soil samples of ADE and its adjacent soils were collected from two sites: Caldeirão Capoeira (secondary forest for over 20 years) and Caldeirão Cultivado (cultivated with cassava for at least 30 years). The total DNA extracted from soil samples was used as template in quantitative PCR reactions to determine the abundance of prnD gene. In soil samples was also quantified the 16S rRNA. In the study culture-dependent, in a total of 219 isolates (175 Burkholderia and 44 Pseudomonas), 60 isolates of Burkholderia and 3 Pseudomonas exhibited positive amplification for prnD gene. Phylogenetic analysis of prnD gene showed that most of the sequences obtained in this study grouped distinctly from sequences of the GenBank database. It indicates that there is diversity in prnD gene of isolates from amazonian soils and they may differ from those previous described. The antagonism test showed that isolates with genetic potential for production of pyrrolnitrin are also bioactive against Fusarium oxysporum, exhibiting strong antimicrobial activity. In culture-independent study, the site Caldeirão Cultivado, ADE soil showed higher copies number of prnD gene and 16S rRNA gene than in ADJ soil. At the site Caldeirão Capoeira, surrounding soil had a higher amount of prnD gene (1.74 x105 copies / g soil) than ADE soil (2.48 x104 copies / g soil), however, in ADE total bacteria was more abundant. These results show that the real-time PCR assay developed in this study was highly sensitive and specific enabling the detection of sensitive and significant differences between soils in the quantification of prnD gene. Soil variables such as pH, phosphorus, calcium, magnesium and micronutrients significantly correlated with abundance of prnD gene

La pourriture grise, causée par le champignon Botrytis cinerea, est l'une des principales maladies aériennes fongiques sur diverses cultures d’importance agronomique. La diversité génétique de B. cinerea est très forte et la capacité rapide d’adaptation de ce champignon à une pression sélective est également avérée. Ce champignon est ainsi capable de développer des résistances à une grande variété de composés fongicides de synthèse ou d'origine naturelle. Des méthodes alternatives de lutte ont de ce fait été développées ces dernières années : divers agents de lutte biologique (ALB) présentant différents modes d’actions ont été identifiés et pour certains d’entre eux commercialisés pour contrôler B. cinerea. Cependant la durabilité de la lutte biologique est un domaine encore très peu étudié. La perte d'efficacité d'un ALB pourrait résulter de la préexistence d’isolats moins sensibles de pathogènes dans les populations naturelles et/ou de la capacité de l’agent pathogène à produire, sous une pression de sélection continue exercée par l’ALB, des mutants ayant une sensibilité réduite. L'objectif global de la présente étude est d'évaluer le risque potentiel de perte d'efficacité de la lutte biologique vis-à-vis de B. cinerea. Dans cette étude, les efforts ont été concentrés sur la pyrrolnitrine, un antibiotique produit par divers ALBs, dont certains sont efficaces contre B. cinerea. Les objectifs spécifiques de l'étude étaient (i) d’évaluer la diversité de la sensibilité à la pyrrolnitrine au sein de la population naturelle de B. cinerea, (ii) d'estimer le risque de perte d'efficacité des ALBs produisant la pyrrolnitrine due à la pression de sélection exercée par la pyrrolnitrine et (iii) d'étudier le mécanisme de résistance à la pyrrolnitrine chez B. cinerea. Parmi 204 isolats de B. cinerea, une gamme importante de sensibilité à la pyrrolnitrine a été observée, avec un facteur de résistance de 8,4 entre l’isolat le plus sensible et l'isolat le moins sensible. La production de 20 générations successives pour 4 isolats de B. cinerea, sur des doses croissantes de pyrrolnitrine, a abouti au développement de mutants avec des niveaux élevés de résistance à l'antibiotique, et à une réduction in vitro de la sensibilité à la bactérie productrice de pyrrolnitrine Pseudomonas chlororaphis PhZ24. La comparaison entre les mutants résistants à la pyrrolnitrine et leurs parents sensibles pour la croissance mycélienne, la sporulation et l'agressivité sur plantes a révélé que la résistance à la pyrrolnitrine est associée à un fort coût adaptatif. Des observations cytohistologiques sur tomates ont confirmé que l’isolat sensible à la pyrrolnitrine attaque le pétiole rapidement et envahit la tige, alors que le mutant résistant à la pyrrolnitrine ne s'étend pas au-delà du pétiole. De plus, ce dernier mutant forme un mycélium anormal et des cellules ressemblant à des chlamydospores. Les résultats ont d'autre part révélé que les mutants de B. cinerea résistants à la pyrrolnitrine sont résistants au fongicide iprodione, suggérant ainsi qu'une pression exercée par la pyrrolnitrine sur le champignon conduit à une résistance au fongicide. Réciproquement, la production de générations successives sur iprodione conduit à une résistance à l'antibiotique. Afin d'étudier les déterminants moléculaires de la résistance de B. cinerea à la pyrrolnitrine, le gène histidine kinase Bos1, impliqué entre autres dans la résistance aux fongicides chez B. cinerea a été séquencé chez les souches sensibles et les mutants résistants. La comparaison des séquences a mis en évidence des mutations ponctuelles différentes chez les mutants de B. cinerea obtenus sur la pyrrolnitrine et ceux obtenus sur l'iprodione. De plus, les résistances à la pyrrolnitrine et à l'iprodione ne sont pas systématiquement associées à une mutation ponctuelle dans le gène Bos1. Enfin, aucune modification n'a été détectée dans la taille des allèles de neuf locus microsatellites quelle que soit la pression sélective exercée et quelle que soit le phénotype du mutant produit. Cette étude montre qu'un champignon pathogène des plantes est capable de développer progressivement une moindre sensibilité à un agent de lutte biologique mais que cette moindre sensibilité est associée à une forte perte de fitness / Gray mould, caused by Botrytis cinerea, is a severe disease on a wide range of crops. Disease control generally relies on chemicals, although biological control strategies have been intensively studied over the last decades. This pathogen can withstand a wide variety of fungitoxic compounds including fungicides and natural molecules. This capacity to adapt to different stress might, potentially, compromise the durability of biological control methods. The global purpose of that work was to estimate the potential of B. cinerea to overcome the efficacy of biological control agents. Knowledge on the potential development of resistance to biological control agents can help to devise or improve resistance management strategies. In this work, efforts have been focused on the antibiotic pyrrolnitrin produced by various bacteria described as potential biological control agents against B. cinerea. The specific objectives of the study were (i) to evaluate the diversity in susceptibility to pyrrolnitrin among natural population of B. cinerea, (ii) to estimate the risk of loss of efficacy of pyrrolnitrinproducing biological control agent due to selection pressure exerted by pyrrolnitrin and (iii) to study the mechanism of resistance to pyrrolnitrin in B. cinerea. An important range of sensitivity to pyrrolnitrin with an 8.4-fold difference in EC50 values between the most sensitive and the least sensitive isolates was observed within the 204 isolates tested. The production of 20 generations, for 4 isolates of B. cinerea, on increasing doses of pyrrolnitrin, resulted in the development of mutants of B. cinerea with high levels of resistance to the antibiotic and a reduced sensitivity in vitro to the pyrrolnitrin-producing Pseudomonas chlororaphis PhZ24. Comparison of the pyrrolnitrin-resistant mutants and their sensitive parent isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin has resulted in a high fitness cost. Additional cytohistological investigations revealed that while the sensitive isolate spread throughout the petiole and rapidly invaded the stem via the abscission zone, the pyrrolnitrinresistant mutant failed to extend beyond petiole to invade the stem. Moreover, the pyrrolnitrin-resistant mutant formed abnormal mycelium and chlamydospore-like cells. The comparison of resistance to pyrrolnitrin and to the iprodione fungicide in B. cinerea revealed that fungicide pressure exerted on the fungus is able to build-up resistance to pyrrolnitrin. Comparison of sequences of the osmosensing class III histidine kinase encoding gene bos1 revealed different mutations in pyrrolnitrin- and iprodione-resistant mutants. However, resistance to pyrrolnitrin and to iprodione was not systematically associated with a point mutation in the Bos1 gene. Finally, no changes were observed in the allele size at nine microsatellite loci whatever the four selective pressure endured by the fungus despite their phenotypic changes. This study provides evidence that a fungal plant pathogen is able to gradually build-up resistance to an antibiotic produced by a biocontrol agent