The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway and virulence factor production, motility tests and biofilm assays were conducted using pyrC- mutant. Even though no significant difference in growth rates was observed, there were significant differences between the wild type and mutant in the production of biofilm and virulence factors. This study will help us to understand the structure and regulation of ATCase holoenzyme with DHOase, and facilitate the use of B. cepacia as an applicable bio-tool. Additionally, we can potentially pursue more efficient drug targets for B. cepacia.

This diploma thesis focuses on an optimalization of simple laboratory model systems which serve as an innovative tool for an experimental study on the adsorption of bacterial cells on solid surfaces. In the description of living biological systems, an adsorption is labelled as an adhesion. Designed model systems were validated with a physical-chemical analysis. Various techniques were used to determine bacteria properties, more specifically Burkholderia cepacia and Bacillus megaterium. The solid surfaces after sorption of bacterial cells of Bacillus megaterium were subjected to a structural and visual analysis. Applying the theoretical approach (e.g. using different physical-chemical models) to study the adhesion of microorganisms to a particular surface allows a prediction of the conditions for a successful adhesion. The results will give us a better understanding of a formation and development of a biofilm.

No / To investigate whether there are differences between members of the Burkholderia cepacia complex in their ability to invade human respiratory epithelial cells, 11 strains belonging to genomovars I-V were studied in an antibiotic protection assay using the A549 cell line. Strains belonging to genomovars II and III were more invasive than those of genomovars I, IV and V. There was also intra-genomovar variation in invasiveness. No correlation between invasiveness and other putative virulence factors of importance in B. cepacia infection in individuals with cystic fibrosis, cable pilus and B. cepacia epidemic strain marker was identified.

Cystic Fibrosis (CF) is a common autosomal genetic disorder in Caucasian populations. CF is caused by mutations in the cftr gene, which encodes the CF transmembrane conductance regulator (CFTR). CFTR regulates chloride and sodium ion transport across the epithelial cells lining the exocrine organs. Mutations in the cftr result in a failure to mediate chloride transport, which leads to dehydration of the mucus layer surrounding the epithelial cells. The mucus coating in the lung epithelia provides a favorable environment for invasion and growth of several opportunistic bacterial pathogens resulting in life threatening respiratory infections in CF patients. Pseudomonas aeruginosa(Pa) and Burkholderia cepacia complex (Bcc) are associated with chronic lung infections and are responsible for much of the mortality in CF. Little is known about interactions between these two, often co-infecting, species. When in competition, it is not known whether Bcc replaces the resident Pa or if the two species co-exist in the CF lung. Bacteriocins are potent toxins produced by bacteria. They have a quite narrow killing range in comparison to antibiotics and have been implicated in intra-specific and inter-specific bacterial competition brought on by limited nutrients or niche space. Both Pa and Bcc produce bacteriocins known as pyocins and cepaciacins, respectively. More than 90% of Pa strains examined to date produce one or more of three pyocin types: R, F, and S. A limited number of phenotypic surveys suggest that approximately 30% of Bcc also produce bacteriocins. The goals of my thesis study were to determine if clinical strains of Pa and Bcc produce bacteriocins and to determine whether these toxins play a role in mediating intra- and inter-specific bacterial interactions in the CF lung. The final goal was to identify novel bacteriocins from clinical Pa and Bcc strains. First, I designed a phenotypic bacteriocin survey to evaluate bacteriocin production in 66 clinical Pa (38) and Bcc (28) strains procured from CF patients. This study revealed that 97% of Pa strains and 68% of Bcc strains produce bacteriocin-like inhibitory activity. Further phenotypic and molecular based assays showed that the source of inhibition is different for Pa and Bcc. In Pa, much of the inhibitory activity is due to the well known S- and RF-type pyocins. S-and RF pyocins were the source of within species inhibitory activity while RF pyocins were primarily implicated in the between species inhibitory activity of Pa strains. In contrast, Bcc inhibition appeared to be due to novel inhibitory agents. Finally, I constructed genome libraries of B. multivorans, B. dolosa, and B. cenocepacia to screen for genes responsible for the inhibitory activity previously described in Bcc. ~10,000 clones/genome were screened, resulting in fifteen clones with the anticipated inhibition phenotype. Of these fifteen, only five clones had stable inhibitory activity. These clones encoded proteins involved in various metabolic pathways including bacterial apoptosis, amino acid biosynthesis, sugar metabolism, and degradation of aromatic compounds. Surprisingly, none of Bcc clones possessed typical bacteriocin-like genes. These data suggest that, in contrast to all bacterial species examined in a similar fashion to date, Bcc may not produce bacteriocins. Instead, Bcc may be using novel molecular strategies to mediate intra- and inter-specific bacterial interactions.

No description available.

Cette étude a porté sur l’analyse de l’énantiosélectivité de la lipase de Burkholderia cepacia (BCL) pour les acides 2-substitués, synthons chiraux d’intérêt pharmaceutique, avec pour objectif d’examiner le rôle de l’accès au site actif enfoui de BCL sur l’énantiosélectivité et de développer une procédure d’ingénierie permettant de créer des mutants d’énantiosélectivité améliorée. Pour traiter le problème, une nouvelle approche de calcul, basée sur des algorithmes de planification de mouvements issus de la robotique a été développée. Elle permet l’exploration conformationnelle des espaces multi-dimensionnels contraints et a été appliquée au calcul des trajectoires de plusieurs racémiques dans le site actif de BCL et à l’identification de résidus pouvant potentiellement gêner le déplacement du substrat le long du site actif. Les résultats obtenus in silico ont révélé une corrélation qualitative avec les valeurs d’énantiosélectivité et ont permis de proposer des cibles de mutagénèse. Sur cette base, l’ingénierie du site actif de BCL a été entreprise pour moduler sélectivement l’accès des énantiomères R et S à la triade catalytique. Un système d’expression hétérologue de BCL chez E. coli compatible avec une expression en microplaque, a été développé. Une librairie de 57 (3x19) mono-mutants sur les positions : Leu17, Val266 et Leu287 a été construite par iPCR puis criblée en utilisant une procédure à moyen débit pour identifier les variants actifs pour l’hydrolyse du pNPB. L’énantiosélectivité de ces mutants a ensuite été évaluée pour l’hydrolyse du racémique (R,S)-2 bromophényl acétate de 2-chloro-éthyle, par utilisation d’une nouvelle procédure de criblage en deep-wells. Ce crible a permis de mettre en évidence plusieurs mutants dont les plus prometteurs ont été caractérisés. Ainsi les mutants Leu17Ser et Leu17Met présentent une augmentation de l’énantiosélectivité d’un facteur 10 accompagnée d’une augmentation de leur activité d’un facteur 4 à 5. Le mutant Val266Gly présente, quant à lui, une inversion de l’énantiosélectivité pour le substrat d’intérêt. L’étude des trajectoires par les techniques de planification combinée à une représentation sous la forme de carte de voxels a été réalisée en parallèle. Pour les mutants sélectionnés, une bonne corrélation a été observée entre les résultats obtenus in silico et expérimentalement. De plus, cela a permis de proposer de nouvelles combinaisons de mutations ayant conduit à l’identification de deux double-mutants Leu17Met/Val266Met et Leu17Ser/Leu287Ile d’énantiosélectivité supérieure à 150 pour le substrat modèle, révélant ainsi l’intérêt de l’approche semi-rationnelle proposée / This work has been focused on the understanding of the Burkholderia cepacia lipase (BCL) enantioselectivity towards 2-substituted acids which are chiral building blocks of pharmaceutical interest. The main objective of this work was the investigation of the potential role of substrate accessibility toward the buried active site of BCL on enantioselectivity and the development of an engineering procedure for the design of enantioselective mutants. To study further this hypothesis, a novel computational approach, based on motion-planning algorithms, originally used in robotics, was developed. It allows the conformational exploration of constrained high-dimensional spaces and was applied to the computation of trajectories for a set of racemates within the catalytic site. This methodology also enables the identification of residues potentially hindering substrates displacement along the active site. Results obtained in silico were correlated qualitatively with experimental values of enantioselectivity. On the basis of these results, engineering of the narrow active site of BCL has been undertaken to modulate selectively the access of R and S enantiomers to the catalytic triade. An heterologous expression system of BCL in E. coli compatible with production at microplate scale was developed. A library of 57 (3x19) variants targeted at positions Leu17, Val266 and Leu287 was built by iPCR and subsequently screened using a medium-throughput procedure to identify active variants against pNPB hydrolysis. Next, the enantioselectivity of these mutants was evaluated towards a given racemate, the (R,S)-2-chloro ethyl 2-bromophenylacetate, using a novel screening procedure developed in deep wells. Such screening enabled the identification of several variants amongst which the most promising were characterized. Mutants Leu17Ser and Leu17Met showed a remarkable 10-fold increase of their enantioselectivity and a 4- and 5-fold improvement of their specific activity. Compared to the wild-type enzyme, mutant Val266Gly displayed a reversed enantioselectivity for the substrate of interest. Investigation of the trajectories using motion-planning techniques combined to a voxel map representation was carried out. For selected variants, a fair correlation was observed between in silico and experimental results. Moreover, this enabled us to suggest novel combinations of mutations that led to the identification of two double-mutants Leu17Met/Val266Met and Leu17Ser/Leu287Ile showing an enantioselectivity value higher than 150 for the racemic substrate, revealing thus the effiency of the semi-rational strategy

Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado. / The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.

Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado. / The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.

A cana-de-açúcar é uma das principais culturas do Brasil e nos últimos anos está recebendo especial atenção devido ao crescente aumento da área cultivada e produção de etanol para uso como biocombustível. Considerando-se sua importância econômica e a possibilidade do uso de plantas geneticamente modificadas, essa cultura tem se tornado foco de pesquisas relacionadas à produtividade e sustentabilidade. Neste contexto, o estudo de comunidades microbianas associadas à cana-de-açúcar é de fundamental importância, pois além dessas comunidades desempenharem importante papel funcional na interação com a planta, os estudos realizados nas condições tropicais são limitados. Comunidades de fungos e bactérias associadas à cana-de-açúcar geneticamente modificada IMI-1 e sua isolinha convencional SP80-1842 foram sistematicamente isoladas de plantas cultivadas em área experimental em Piracicaba, SP, Brasil. Por meio de isolamento e PCR-denaturing gradient gel electrophoresis (PCR-DGGE) foi verificado que a transgenia não afeta a diversidade da comunidade fúngica associada à cana-deaçúcar. A diversidade dessas comunidades foi descrita e caracterizada molecularmente. O fungo Fusarium moniliforme apresentou alta freqüência de isolamento e também foi observado que o gene da endopoligalacturonase, pgIII, desempenha um importante papel no tipo de associação, endofítica ou patogênica, do F. moniliforme e a planta. O complexo Burkholderia cepacia constitui uma importante fração da comunidade de bactérias associada à cana-de-açúcar no Brasil e isolados deste complexo são capazes de inibir o crescimento do patógeno F. moniliforme. Análises filogenéticas indicaram que os isolados de bactérias endofíticas de Burkholderia são proximamente relacionados com linhagens-tipo do complexo B. cepacia isoladas de pacientes de fibrose cística. / In Brazil, the sugarcane is one of the most important cultivated crops. The sugarcane has received increased interest in the last years because of increase of the cultivated area and ethanol production to be used as biofuel. Considering its economical importance and the possibility of the use of the genetically modified plants; this crop has become the aim of current research for productivity and sustainability. In this context, the work on microbial communities associated with sugarcane is remarkable, because both, these communities play important functional role in the interaction with the plant, and studies performed in tropical conditions are limited as well. Fungal and bacterial communities associated with genetically modified sugarcane IMI-1 and its conventional isoline SP80-1842 were systematically isolated from plants cultivated in an experimental area in Piracicaba, SP, Brazil. The fungal communities associated with sugarcane were accessed by means of cultivation approach and PCR-denaturing gradient gel electrophoresis; the results revealed that these communities are not affected by transgeny. The microbial communities\' diversity was characterized and identified by using molecular tools. The fungus Fusarium moniliforme showed high frequency in association with the plant and it was observed that the endopolygalacturonase gene, pgIII, plays important role in order to determine the sort of association, either endophytic or pathogenic, between F. moniliforme and the host. The Burkholderia cepacia complex is an integral part of the endophytic bacterial community of sugarcane in Brazil and isolates of this complex are able to control F. moniliforme growth. Phylogenetic analyses indicated that the endophytic Burkholderia are closely related to clinical isolates of the B. cepacia complex isolated from cystic fibrosis patients.

A cana-de-açúcar é uma das principais culturas do Brasil e nos últimos anos está recebendo especial atenção devido ao crescente aumento da área cultivada e produção de etanol para uso como biocombustível. Considerando-se sua importância econômica e a possibilidade do uso de plantas geneticamente modificadas, essa cultura tem se tornado foco de pesquisas relacionadas à produtividade e sustentabilidade. Neste contexto, o estudo de comunidades microbianas associadas à cana-de-açúcar é de fundamental importância, pois além dessas comunidades desempenharem importante papel funcional na interação com a planta, os estudos realizados nas condições tropicais são limitados. Comunidades de fungos e bactérias associadas à cana-de-açúcar geneticamente modificada IMI-1 e sua isolinha convencional SP80-1842 foram sistematicamente isoladas de plantas cultivadas em área experimental em Piracicaba, SP, Brasil. Por meio de isolamento e PCR-denaturing gradient gel electrophoresis (PCR-DGGE) foi verificado que a transgenia não afeta a diversidade da comunidade fúngica associada à cana-deaçúcar. A diversidade dessas comunidades foi descrita e caracterizada molecularmente. O fungo Fusarium moniliforme apresentou alta freqüência de isolamento e também foi observado que o gene da endopoligalacturonase, pgIII, desempenha um importante papel no tipo de associação, endofítica ou patogênica, do F. moniliforme e a planta. O complexo Burkholderia cepacia constitui uma importante fração da comunidade de bactérias associada à cana-de-açúcar no Brasil e isolados deste complexo são capazes de inibir o crescimento do patógeno F. moniliforme. Análises filogenéticas indicaram que os isolados de bactérias endofíticas de Burkholderia são proximamente relacionados com linhagens-tipo do complexo B. cepacia isoladas de pacientes de fibrose cística. / In Brazil, the sugarcane is one of the most important cultivated crops. The sugarcane has received increased interest in the last years because of increase of the cultivated area and ethanol production to be used as biofuel. Considering its economical importance and the possibility of the use of the genetically modified plants; this crop has become the aim of current research for productivity and sustainability. In this context, the work on microbial communities associated with sugarcane is remarkable, because both, these communities play important functional role in the interaction with the plant, and studies performed in tropical conditions are limited as well. Fungal and bacterial communities associated with genetically modified sugarcane IMI-1 and its conventional isoline SP80-1842 were systematically isolated from plants cultivated in an experimental area in Piracicaba, SP, Brazil. The fungal communities associated with sugarcane were accessed by means of cultivation approach and PCR-denaturing gradient gel electrophoresis; the results revealed that these communities are not affected by transgeny. The microbial communities\' diversity was characterized and identified by using molecular tools. The fungus Fusarium moniliforme showed high frequency in association with the plant and it was observed that the endopolygalacturonase gene, pgIII, plays important role in order to determine the sort of association, either endophytic or pathogenic, between F. moniliforme and the host. The Burkholderia cepacia complex is an integral part of the endophytic bacterial community of sugarcane in Brazil and isolates of this complex are able to control F. moniliforme growth. Phylogenetic analyses indicated that the endophytic Burkholderia are closely related to clinical isolates of the B. cepacia complex isolated from cystic fibrosis patients.