Dans la rhizosphère du riz (Oryza sativa), certaines bactéries qualifiées de PGPR (Plant Growth Promoting Rhizobacteria), dont Azospirillum, sont capables de stimuler la croissance de cette céréale. Les effets bénéfiques de l’interaction PGPR-plante semblent conditionnés par la capacité de PGPR à interagir de façon précoce avec son hôte et d’induire chez ce dernier une réponse physiologique spécifique. Pour autant, peu d’informations sont disponibles quant à la nature et le degré de spécificité de la réponse physiologique de la plante à son interaction avec une PGPR. L’objectif de cette étude a donc été d’analyser, par profilage métabolique, la réponse physiologique du riz à son interaction avec Azospirillum et d’évaluer le degré de spécificité de cette réponse en fonction des partenaires de l’interaction. Dans ce but, nous avons comparé les profils chromatographiques des métabolites secondaires de deux cultivars de riz, Cigalon et Nipponbare, après inoculation ou non par les PGPR Azospirillum lipoferum 4B et Azospirillum sp. B510. Les résultats ont été comparés à ceux obtenus en présence d’une bactérie pathogène Burkholderia glumae AU6208 et d’une bactérie commensale Escherichia coli B6. En parallèle, la croissance végétale et l’architecture racinaire des cultivars ont été comparées, et la colonisation des systèmes racinaires par les PGPR a été suivie. Nous avons montré que chaque souche d’Azospirillum est capable de coloniser les deux cultivars, et qu’elle augmente préférentiellement la croissance du cultivar dont elle a été isolée. Le métabolisme secondaire du riz est profondément modifié en réponse à chacune des interactions biotiques testées. Dans ce cas des interactions riz-PGPR, la réponse est souche-cultivar dépendante. L’ensemble des résultats suggèrent l’existence d’une spécificité d’interaction entre la souche d’Azospirillum et son cultivar d’origine / In rice (Oryza sativa) rhizosphere, some bacteria designed as PGPR (Plant Growth Promoting Rhizobacteria) like Azospirillum, are able to enhance the growth of this crop. The beneficial effects of PGPR-plant interactions seem to be influenced by the ability of PGPR to interact at an early stage with its host and induce a specific physiological response. However, little information is available about the nature and degree of specificity of the physiological response of the plant to its interaction with PGPR. Thus, the aim of this study was to analyse, by using a metabolic profiling approach, the physiological response of rice to its interaction with Azospirillum and to assess the degree of specificity of this response according to interaction partners. To this purpose, chromatographic profiles of secondary metabolites of two rice cultivars, Nipponbare and Cigalon, were compared in non-inoculated plants and in plants inoculated by the PGPR Azospirillum lipoferum 4B and Azospirillum sp. B510. The results were compared with those obtained in the presence of pathogenic bacteria Burkholderia glumae AU6208 and a commensal bacteria Escherichia coli B6. In parallel, plant growth and root architecture of rice cultivars were compared, and root system colonization by PGPR was followed. We showed that each strain of Azospirillum is able to colonize both cultivars, and to preferentially increase the growth of the cultivar from which it was isolated. In addition, rice secondary metabolism is profoundly altered in response to each tested biotic interaction. In the case of rice-PGPR interactions, the response is strain-cultivar dependent. The overall results of this work suggest the existence of interaction specificity between Azospirillum strain and its cultivar of origin

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.

In under a century, melioidosis, the infection due to Burkholderia pseudomallei, has emerged from Whitmore’s series of glanders-like infections amongst the morphia addicts in Burma to a major cause of mortality in northeastern Thailand and northern Australia. Also endemic in other parts of south-east Asia, melioidosis may have varied presentations ranging from severe, overwhelming infection to chronic, low grade disease. Observational evidence had suggested that granulocyte colony stimulating factor (G-CSF), a naturally occurring substance produced by the body in response to infection, may have been useful in reducing the high mortality associated with the more severe forms of this infection. Other observations linked the occurrence of this disease to various environmental factors, such as contamination of drinking water and the annual rainfall. This thesis explores and attempts to quantify these associations. There are three parts to this thesis. In the first part, I reviewed the epidemiology and management of patients with melioidosis. The use of G-CSF and meropenem was associated with a fall in mortality, although other factors may have at least partially contributed to this effect. In the second part, I progressed towards a clinical trial of G-CSF. There was no other evidence supporting the use of G-CSF in severe sepsis and ethical issues precluded a trial in Darwin. There was not evidence from laboratory models of G-CSF action in melioidosis to support the use of G-CSF in patients, although there remained some doubt regarding the applicability of such models to human disease. I examined clinical methods to identify patients at high risk of death from melioidosis. A simple scoring system based on clinical and laboratory parameters was developed and externally validated. However, clinical definitions of severe sepsis appeared to be better predictors of mortality. A clinical trial based on clinical definitions was commenced in Thailand. In the final part, I explored the question of whether different strains or B. pseudomallei or different environmental conditions caused different patterns of infection. There was no evidence that strain types of this bacterium determine the pattern or severity of disease, but weather conditions appeared to influence the distribution of disease in northern Australia.

Le complexe Burkholderia cepacia (Bcc) englobe 17 espèces retrouvées dans les infections pulmonaires d'individus atteints de mucoviscidose. Les bactéries de ce complexe sont présentes dans les sols, la rhizosphère de grandes cultures, les eaux usées et peuvent également être rencontrées dans le cadre d'infections nosocomiales. En France, les espèces B. multivorans et B. cenocepacia (Bcen) sont les espèces majoritaires au niveau des infections de patients atteints de mucoviscidose. Divers clones épidémiques ont été décrits au sein de l'espèce Bcen dont le clone ET12 associé au "syndrome cepacia". En 2004, une épidémie nosocomiale impliquant un clone du Bcc est survenue dans un hôpital de l'Ain. Durant ce travail, l'origine de ce clone (B&B), sa classification au sein du Bcc et certains phénomènes génétiques liés à son émergence ont été étudiés. Cela a permis d'identifier ce clone comme appartenant à l'espèce Bcen et une forte proximité de celui-ci avec la lignée ET12. L'étude des facteurs transcriptionnels de la famille σ70 au sein du Bcc a mis en évidence une structure génétique similaire entre la lignée ET12 et ce clone, mais différente de celle observée chez les autres espèces du Bcc. L'analyse d'éléments génétiques répétés de la famille des séquences d'insertion (IS) a cependant permis d'observer une organisation génomique distincte de la lignée ET12. Celle-ci a été reliée à des phénomènes d'instabilité génétique notamment à des phénomènes d'acquisition d'éléments génétiques mobiles de type îlot génomique. L'ensemble de ce travail a permis de caractériser un ensemble de phénomènes génétiques pouvant expliquer l'émergence de clones épidémiques tels que le clone B&B.

Aromatic compounds comprise approximately one quarter of the Earth's biomass and thus play a critical role in the biogeochemical carbon cycle. These compounds are degraded almost exclusively by specialized microbial enzymes that are part of complex metabolic pathways. Detailed characterization of these enzymes is both a gateway to understanding a biological process fundamental to nature and a platform for bioengineering applications in bioremediation. Recently, a novel pathway was shown to metabolize two key aromatic intermediates: Benzoate and Benzoyl-Coenzyme A. Designated as the box pathway (benzoate oxidation), this metabolic conduit incorporates in succession; CoA-ligation, oxygenation, ring cleavage and neutralization of the aldehydic ring cleavage product, catalyzed by a Benzoate Coenzyme A Ligase (BCL), BoxAB, BoxC and an Aldehyde Dehydrogenase (ALDH) respectively. Collectively, these steps define the initial and unique segment of the box pathway. The objective of the research described here was to establish a molecular blueprint of the substrate binding pocket of the initial BCL and elucidate mechanistic details for both BoxC and ALDH enzymes from Burkholderia xenovorans LB400 through in-depth structural and functional characterizations. An intriguing feature of the box pathway in LB400 is a paralogous genetic organization. Functional studies on the BCL paralogs (BCLM and BCLC) show that BCLM is more active towards benzoate than BCLC. Structural analysis of the 1.84 Å resolution co- crystal structure of BCLM with benzoate reveals that the substrate binding pocket is closely contoured to bind benzoate, leaving little room to accommodate substituted benzoates, especially in the para position owing to a histidine (H339) residue that renders the pocket particularly shallow. Overall, while corroborative, the structural data provides a molecular rationale to our functional data where both the BCLs were seen to be highly specific for benzoate. Structural analysis of the 1.5 Å resolution crystal structure of the novel ring cleaving BoxC reveals an intriguing structural demarcation consistent with the primary sequence based divergence of BoxC within the crotonase superfamily. A highly divergent region in the C-terminus likely serves as a structural scaffold for the conserved N-terminus that harbors the active site. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site resulting in a compelling mechanistic model involving a pair of conserved glutamates (E146 and E168) and a novel cysteine (C111). Lastly, the 1.6 Å resolution co-crystal structure of ALDHC with NADPH and PEG allows identification of residues that are involved in rendering ALDHC selective for NADP+ and linear, medium to long chain aldehydes, as observed in our initial kinetic analyses. Functional and structural characterization of strategic ALDHC mutants enables us to propose a detailed reaction mechanism which involves the essential roles for C296 as the nucleophile, E257 as the general base and a proton relay network anchored by E496 and supported by E167 and K168. Overall, this research provides a molecular blueprint for three key box enzymes, thereby enhancing our understanding of central aromatic metabolism. / Graduate

Four type II toxin-antitoxin (TA) systems were previously identified in Burkholderia pseudomallei K96243. Type II TA toxins are able to induce cell growth arrest or death by interfering with key processes within the organism. BPSS0390-0391 is one of the TA systems previously identified and has homology to hicBA system in Acinetobacter baumannii. B. pseudomallei HicA is able to cause a reduction in the number of culturable cells after expression in E. coli. This study aimed to characterise B. pseudomallei HicA in three ways: by inducing expression of HicA in bacterial species other than E. coli, by identifying amino acids in HicA involved in toxicity and neutralisation by the antitoxin HicB and by examining the interaction of HicA with other TA antitoxins identified within B. pseudomallei genome. A broad host range plasmid encoding BPSS0390 was transformed into a range of Gram negative bacteria including Yersinia pseudotuberculosis IP32953, Vibrio vulnificus E64MW, Salmonella enterica serovar Typhimurium SL1344 and Burkholderia thailandensis E264. Expression of BPSS0390 was toxic in all bacterial species tested, despite the presence of antitoxin BPSS0391 homologues in some species. Unregulated expression in E. coli resulted in the appearance of escape mutants encoding non-toxic variants of HicA. An alanine scanning mutagenesis study of HicA identified 20 mutants where toxicity was abolished despite high levels of expression, but identified no mutants that affected TA complex formation. Finally an existing co-expression assay was modified to examine interactions between HicA and other type II TA antitoxins in B. pseudomallei. The assay revealed no interaction between HicA and non-cognate antitoxins and clarified the role of IPTG as an inhibitor of PBAD promoter on the arabinose operon.

Submitted by GUILHERME DEOMEDESSE MINARI null (gdminari@gmail.com) on 2016-03-02T21:34:04Z No. of bitstreams: 1 Dissertação Guilherme D. Minari.pdf: 1275061 bytes, checksum: b7c17ca0d2c40870526f019d55c9b421 (MD5) / Rejected by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: A versão do arquivo submetida é considerada a versão final do trabalho, não sendo possível realizar alterações posteriormente. Por favor verifique as páginas que contém a ficha catalográfica e o certificado de aprovação e realize uma nova submissão certificando-se de que o arquivo PDF contém a versão final do trabalho. Agradecemos a compreensão. on 2016-03-04T13:14:40Z (GMT) / Submitted by GUILHERME DEOMEDESSE MINARI null (gdminari@gmail.com) on 2016-03-04T18:31:39Z No. of bitstreams: 2 Dissertação Guilherme D. Minari.pdf: 1275061 bytes, checksum: b7c17ca0d2c40870526f019d55c9b421 (MD5) Dissertação_Guilherme_Deomedesse_Minari.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-04T19:56:27Z (GMT) No. of bitstreams: 1 minari_gd_me_jabo.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) / Made available in DSpace on 2016-03-04T19:56:27Z (GMT). No. of bitstreams: 1 minari_gd_me_jabo.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) Previous issue date: 2016-02-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A contaminação do solo pode ser decorrente do descarte inadequado de substâncias com potencial poluente ou do uso agrícola prolongado com aplicação de materiais corretivos de acidez, fertilizantes e defensivos. O objetivo deste estudo foi avaliar a distribuição espacial de cádmio (Cd), cromo (Cr) e níquel (Ni) em Latossolo sob plantio direto, convencional e convencional com aplicação de lodo de esgoto, em áreas com exploração agrícola há mais de 80 anos. As concentrações dos metais em 422 amostras de solo foram mensuradas por espectroscopia de absorção atômica com chama ar-acetileno, sendo os extratos obtidos por extração com HNO3, H2O2 e HCl em sistema aberto (USEPA-3050B). As variáveis pH e matéria orgânica também foram determinadas. Os mapas de distribuição espacial foram elaborados a partir do software Spring v. 5.2.7 e auxiliaram na visualização da dispersão dos metais na área de estudo, indicando níveis de contaminação. A análise estatística multivariada por componentes principais diferenciou as amostras de solo de mata nativa das que estão sob atividade agrícola, apontando a uma mesma fonte de contaminação para Cr e Ni, como também para a correlação da matéria orgânica com Cd. Conclui-se que a área estudada apresenta contaminação, em larga escala, por Cd e Cr, e contaminações pontuais por Ni, estando alguns valores dentro da faixa de intervenção. / Soil contamination may result from inappropriate use of substances with pollution potential or from the prolonged agricultural use with application of acidity corrective materials, fertilizers and pesticides. The aim of this work was to evaluate the spatial distribution of cadmium (Cd), chromium (Cr) and nickel (Ni) in latosol under no tillage, conventional tillage and conventional with sewage sludge application, in agricultural exploration areas for over 80 years. Metals concentration in 422 soil samples were measured by atomic absorption spectroscopy with acetylene-air flame, obtaining the extracts by extraction with HNO3, H2O2 e HCl in open system (USEPA-3050B). The variables pH and organic matter were obtained. Maps of spacial distribution were elaborated by software Spring v. 5.2.7 and assisted in the visualization of the metals dispersion in the study area, indicating contamination levels. The statistical multivariate by main components differentiated the native forest soil samples from those under agricultural activity, pointing to a same contamination source to Cr and Ni, and to the organic matter correlation to Cd as well. It can be concluded that the studied area presents contamination in large scale by Cd and Cr and pontual contamination by Ni, being some of the values into the intervention group.

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.

Mit einer miniaturisierten Schnellmethode (experimentelles Taxa Profile-Plattensystem, Merlin, Bornheim-Hersel, Deutschland) wurden 570 biochemische Stoffwechselleistungen der 36 Spezies des Genus Burkholderia (B.) untersucht. Die drei verwendeten 384-Loch-Mikrotitratons-Platten sind mit vakuumgetrockneten Substraten beschichtet: Profile A-Platte mit 191 Aminosäuresubstraten, Profile C-Platte mit 191 Kohlenhydratsubstraten und Profile E-Platte mit 188 enzymatischen Substraten. In die Studie wurden 160 Stämme aus insgesamt 36 Spezies, einschliesslich der beiden S 3 Erreger, B. mallei, Verursacher des Rotz und B. pseudomallei, Auslöser des Pseudorotzes (Melioidose), einbezogen. Insgesamt wurden 44 Reaktionsgruppen (Taxons) dargestellt. In fünf Spezies, einschließlich B. mallei und B. pseudomallei, konnten interne homologe Taxons festgestellt werden. Es erfolgte die Erstellung von Prozentwerttabellen der Reaktivität der untersuchten Isolate. Jedes Taxon wurde dann separat ausgewertet. Alle Taxons können eindeutig Spezies zugewiesen werden und ermöglichen so eine Diagnose. Aus den biochemischen Reaktionen wurden 88 Substrate, die eine Differenzierung aller 44 Taxons erlaubt, ausgewählt. Für 14 Taxons konnte eine einzige Schlüsselreaktion beschrieben werden. Mit diesen Substraten wird eine 96-Loch-Mikrotitratons-Platte belegt werden. In Zukunft wird mit diesem optimierten, kostengünstigen Testsystem in jedem Routinelabor innerhalb von 24h die exakte Identifizierung eines unbekannten Burkholderia-Isolates möglich sein. Mit den 160 phänotypisierten Stämmen wurde eine in vitro-Resistenztestung mit 32 antibakteriellen Wirkstoffen mittels einer Mikrodilutionsmethode (GENARS GN4-Platte, Merlin, Bornheim-Hersel, Deutschland) durchgeführt. Die Auswertung erlaubt erstmalig einen vollständigen Überblick über die Resistenzlage des gesamten Genus.

Burkholderia cenocepcia is one of the serious infectious agents of respiratory tract among cystic fibrosis patients. There are problems mainly with strains which are capable of epidemic spread. The known epidemic in the Czech Republic was caused by ST32 strain in the past. In this work, there was completed whole genome sequence of referential isolate 1232 of B. cenocepacia ST32 in cooperation with bioinformatics by new generation sequencing techniques and by determining the problematic areas by a combination of Sanger sequencing bioinformatics approaches and manual assembling of sequence reads localized in these areas. The final version of the genome sequence was annotated by PGAAP and at the present time it is finalized. Second part of this work is dedicated to looking for a prognostic marker of infection caused by ST32 strain in patients with cystic fibrosis. We analysed the results of ST32 trancriptomic experiment and chose genes possibly connected with the cepacia syndrome - serious, mostly fatal state of infection. By quantitative PCR we compared their expression in isolates from 4 patients from time of cepacia syndrome and month before that. We checked the possibility of direct detection of the expression of these genes in clinical material. We identified genes for type III secretion system as...