The initiation of transcription by the Bacillus subtilis sigma-M3 RNA polymerase at two Bacillus phage ɸ29 promoters and the effect of the delta subunit on initiation have been investigated by an in vitro kinetic analysis. The templates for the analysis were plasmids which carried the ɸ29 A2 or G2 promoter. The cloning and localization of the A2 promoter are reported here. The kinetics of RNA synthesis initiation were examined using a single-round run-off transcription assay in which multiple initiation events at a single promoter were inhibited with heparin. It was observed that the formation of heparin-resistant complexes at the A2 promoter required the presence of the initiating nucleotides, while the RNA polymerase alone was able to form heparin-resistant, non-initiated complexes at the G2 promoter. The G2 promoter was also shown by a competition assay to be a stronger promoter than A2. The effect of the delta subunit on complex formation at the two promoters was investigated with the single-round transcription assay. Delta had no effect on the formation of initiation complexes at the G2 promoter but lowered the rate and extent of complex formation at the A2 promoter. The effect of delta on the kinetic parameters of complex formation at the A2 promoter was also investigated. The data suggested that delta affects the efficiency with which the enzyme/promoter complexes undergo the transition(s) to a complex from which RNA synthesis can be initiated, although other interpretations were possible. A model for the effect of delta is proposed, in which it is postulated that the release of delta from the enzyme/promoter complex is essential for initiation. Enzyme which is associated with delta can interact with both the A2 and G2 promoters but complexes at the weaker A2 promoter do not efficiently release delta, thus slowing the formation of initiation complexes. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Les nanoparticules métalliques sont utilisées dans une large gamme de produits, ce qui a pour conséquence un rejet croissant de ces nanoparticules et de leurs produits secondaires dans l’environnement. Il est donc nécessaire d’évaluer leur devenir et leurs impacts dans l'environnement. Les bactéries constituent l’une des premières cibles des nanoparticules. La plupart des études se limitent à des analyses de mortalité et ne prennent pas en compte les transformations des nanoparticules dans l’environnement. Cette étude se concentre sur un modèle bactérien, Bacillus subtilis, dont les biotopes principaux incluent la rhizosphère du sol et le tractus gastro intestinal, et sur trois nanoparticules : Ag-NPs, ZnO-NPs et TiO2-NPs. Nous évaluons d’une part l’impact des nanoparticules sur le métabolisme de Bacillus subtilis, et d’autre part celui de l’activité bactérienne et en particulier des molécules sécrétées par Bacillus subtilis sur les nanoparticules, les deux étant interdépendants. / Metallic nanoparticles are used in variety of consumer products (solar screen, paint or medicine), which results in an increasing release of nanoparticles in the environment. There is a need of better evaluating their fate and impacts in the environment. Microorganisms are one of the first targets of nanoparticles in the environment. Most studies on microorganisms and bacteria have focused on cellular mortality, and did not take into account possible transformations of NPs in the environment, which modify their toxicity. This study is focused on model bacteria, Bacillus subtilis and three nanoparticles: Ag-NPs, ZnO-NPs and TiO2-NPs. We evaluate on one hand the impact of nanoparticles on the metabolism on the metabolism of Bacillus subtilis, and on the other hand the impact of Bacillus subtilis and of its secretome on the nanoparticles, both being mutually dependent.

L'acquisition et le maintien de la forme bactérienne ont été consciencieusement étudiés pendant une très longue période. Néanmoins, il reste encore beaucoup de questions sans réponse. Les bactéries Gram-positives présentent une couche externe rigide (la paroi cellulaire) qui permet de préserver la pression osmotique interne et la morphologie cellulaire. La paroi cellulaire (CW) est principalement formée par un maillage de polymères de sucres, le peptidoglycane (PG), sur lequel sont accrochés des acides téichoïques. L'absence de cette barrière essentielle provoque la perte de forme et, finalement, la lyse de la cellule. L’intégrité du CW est par conséquent d'une importance vitale pour les bactéries. La structure ainsi que la synthèse correcte du CW dépendent de supposées machineries d'élongation du peptidoglycane (PGEM) chargées d’assembler le réseau du PG. Le fonctionnement et la composition des PGEMs restent incertains, mais on sait qu’ils dépendent d’une protéine essentielle : MreB, une protéine procaryote similaire à l'actine. MreB est suspectée de contrôler l’activité et/ou l’assemblage des PGEMs, mais sa fonction exacte comme son mode de régulation sont actuellement inconnus. J’utilise Bacillus subtilis, le modèle des bactéries Gram-positives, pour mieux comprendre les fonctions de MreB via i- le développement et l’utilisation d’un criblage génétique pour l’identification de mutants de mreB non fonctionnels et ii- l'étude d'un effecteur potentiel de MreB.(i) MreB a été étudié pendant près de deux décennies et pourtant, sa (ses) fonction(s) reste(nt) mal comprise(s). Comme les approches biochimiques se sont révélées particulièrement difficiles jusqu'à présent, la plupart des études se sont concentrées sur la localisation cellulaire et la dynamique de la protéine. Au cours de mes travaux, j’ai conçu un criblage génétique au moyen duquel j’ai obtenu une collection de mutants de mreB fonctionnellement déficients, chez B. subtilis. La caractérisation de ces mutants a révélé de nombreux résidus importants pour le fonctionnement de la protéine. De façon intéressante, mes résultats indiquent que certains mutants ont conservé leurs propriétés dynamiques (suggérant une association fonctionnelle aux PGEMs) en plus d'une morphologie de type sauvage, tout en étant fortement affectés pour la croissance. Des résultats préliminaires indiquent que ces mutants sont compromis dans leur capacité à utiliser certaines sources de carbone, reliant MreB au métabolisme cellulaire. Ceci suggère l'existence soit d'un point de contrôle, soit d'un couplage entre le métabolisme du carbone et l'expansion du CW chez B. subtilis.(ii) Des résultats non publiés de notre groupe ont révélé l'existence d'un opéron non caractérisé (ydcFGH) dont l'expression est fortement induite en absence de MreB, par comparaison à la souche sauvage. J’ai 1- mis en évidence la cause probable de l’induction de cet opéron en l’absence de MreB, révélant ainsi l’existence de nombreuses mutations dans la souche MreB et 2- réalisé une caractérisation poussée de chaque gène de l'opéron ydcFGH. Bien que le lien exact entre MreB et ydcFGH soit encore inconnu, nos résultats suggèrent un rôle potentiel d’YdcH dans le contrôle du métabolisme du carbone et l'adaptation à la phase stationnaire. À la lumière de mes données issues du criblage génétique (i), ces résultats indiquent un lien fort entre MreB et le métabolisme du carbone. / Acquisition and maintenance of the bacterial shape has been conscientiously studied for a long time. Nevertheless, there are still many unanswered questions. Gram-positive bacteria present a rigid external coating (cell wall) that allows them to preserve internal osmotic pressure and cell morphology. The cell wall (CW) is mainly formed by the peptidoglycan meshwork (PG), that confers its structure to the CW, to which are connected teichoic acids. The absence of this essential barrier causes the loss of shape and, ultimately, lysis of the cells. Integrity of the CW is, therefore, a matter of vital importance for bacteria. Proper CW synthesis and structure depends on the so-called peptidoglycan elongation machineries (PGEM) in charge of building the PG meshwork. The precise composition and functioning of the PGEM is not completely understood but they rely on a key player: MreB, a conserved prokaryotic actin-like protein. MreB is suspected to control PGEM activity and/or assembly but its precise function and mode of regulation are currently unknown. I used Bacillus subtilis, the model for Gram-positive bacteria, to gain a better understanding of MreB functions via i- the development and use of a genetic screen for loss-of-function mutants of mreB and ii- the study of a potential effector of MreB.(i) MreB has been studied for almost two decades now and still, little is known about its function(s). Since biochemical approaches proved to be difficult so far, most of the studies have focused on cellular localization and dynamics of the protein. Here, I have designed a genetic screen by means of which I have obtained a collection of functionally impaired mreB mutants in B. subtilis. Characterization of these mutants revealed numerous key residues for the functioning of the protein. Interestingly, my results indicate that some mutants have kept their dynamic properties (suggesting functional association to the PGEM) together with a wild type shape, while being strongly affected for growth. Preliminary results indicate an impaired ability to use certain carbon sources linking MreB to cellular metabolism. This suggests the existence of either a checkpoint or a coupling between carbon metabolism and CW expansion in B. subtilis.(ii) Unpublished results from our group revealed the existence of an uncharacterized operon (ydcFGH), whose expression is highly induced in the absence of MreB by comparison to the wild type. I have 1- deciphered the cause of ydcFGH induction in the absence of MreB, revealing the existence of multiple mutations in the MreB strain and 2- realized a thorough characterization of each gene of the ydcFGH operon. Although the exact link between MreB and ydcFGH is yet unknown, my results suggest a potential role of YdcH in the control of carbon metabolism and adaptation to stationary phase. In light of my mutagenesis screen data (i), these results are pointing towards a strong link between MreB and carbon metabolism.

This work explores bacterial motility from the mechanisms of propulsion of an individual cell to the complex behavior of collective motility. The shear modulus of bacterial flagella was measured by stretching isolated flagella with an optical trap and by measuring force extension curves of the stretched flagella shedding light onto the me-chanics involved in the motility of single micro-organisms. Experiments in concentrated suspensions of bacteria show collective behavior with large scale mixing on a time and length scale greater than can be understood from the standard model of "run and tumble" motility of a single organism are reported. To further understand the transition from individual to collective motility a novel form of motility where an individual bacterium can reverse direction without changing cell orientation is reported here. These experiments further the understanding of bacterial motility.

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Orientador: Wagner Bettiol / Banca: Antonio Carlos Maringoni / Banca: Eduardo Alves / Banca: Renate Krause Sakate / Banca: Zayame Vegette Pinto / Resumo: O presente trabalho teve por objetivos avaliar o produto comercial Kalegreen®, à base de bicarbonato de potássio, e óleos fixos de café para o controle do oídio da soja; e Bacillus subtilis e Bacillus licheniformes, em tratamento de sementes, como promotores de crescimento de plantas, associado ou não a Bradyrhizobium elkanii. Para avaliar a eficiência de bicarbonato de potássio (Kaligreen®) no controle do oídio da soja cultivar MGBR-46 (Conquista), as pulverizações do bicarbonato, nas concentrações de 0; 0,25%, 0,5%, 0,75% e 1,0%, e do fungicida (piraclostrobina e epoxiconazole) e as avaliações foram semanais. Folhas foram coletadas para análise ultraestrutural através da microscopia eletrônica de varredura. O bicarbonato de potássio controlou a doença em todas as concentrações, mas causou fitotoxicidade a partir de 0,5%. O produto apresentou ação direta sobre o patógeno ocasionando murchamento e redução na germinação dos conídios. O produto apresenta potencial para controlar a doença, porém deve ser adequada a concentração a ser recomenda, bem como a frequência de aplicação para evitar os problemas de fitotoxicidade. Nos estudos com óleos extraídos de grãos de café torrado e cru no controle do oídio da soja, as metodologias e os materiais foram semelhantes, sendo que os óleos foram pulverizados semanalmente nas concentrações de 0%, 0,5%, 1% e 2%. Os óleos controlaram a doença na faixa de 84,8% a 99,8%, sendo superior ao fungicida padrão. Nas observações em MEV, constatou-se a presença de uma camada protetora na superfície das folhas, impedindo a ação do patógeno. Com isso, pode-se inferir que os produtos utilizados neste trabalho apresentam potencial para proteção de plantas de soja contra oídio. Os experimentos para avaliar o efeito de Bacillus subtilis e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to evaluate the commercial product Kalegreen ®, based on potassium bicarbonate, and fixed oils of coffee for the control of powdery mildew of soybeans, and Bacillus subtilis and Bacillus licheniformes in seed treatment, as growth promoters in plants, with or without Bradyrhizobium elkanii. To evaluate the efficiency of potassium bicarbonate to control powdery mildew of soybean cultivar MGBR-46 (Conquest), the bicarbonate sprays, at concentrations of 0, 0.25%, 0.5%, 0.75% and 1.0%, and the fungicide (pyraclostrobin and epoxiconazole) and ratings were weekly. Leaves were collected for ultrastructural analysis by scanning electron microscopy. The potassium bicarbonate controlled the disease in all concentrations, but caused phytotoxicity from 0.5%. The product has a direct effect on the pathogen causing wilting and reduction in spore germination. The product has the potential to control the disease, but should be adequate concentration to be recommended, and the frequency of application to avoid phytotoxicity problems. With the same methodology we evaluate the potential of coffee oils to control soybean powdery mildew. The oils were weekly sprayed at concentrations of 0%, 0.5%, 1% and 2%. For four weeks we evaluated the severity of the disease in a pair of clover in the middle third of the plants, using diagrammatic scale of notes. Oils controlled the disease in the range of 84.8% to... (Complete abstract click electronic access below) / Doutor

Microbes live in diverse, challenging and competitive environments. To survive and propagate microbes must be able to sense and respond to environmental fluctuations, such as changes in pH, nutrient status or temperature. As such, bacteria have a number of signal transduction mechanisms at their disposal that allow them to detect a range of different stimuli, integrate different signals and react to them appropriately. The work presented in this thesis aimed to understand more about the signalling cascades that the Gram positive soil-dwelling bacterium <em>Bacillus subtilis</em> uses to mediate its transition from a motile lifestyle that requires rotating helical flagella, to a sessile lifestyle called the biofilm, where cells adhere to a surface and are encased in a self-produced extracellular polymeric matrix. Bacterial tyrosine phosphorylation is required for <em>B. subtilis</em> biofilm formation and has been suggested to also play a role in regulating the putative motility protein, YvyG. This led to the hypothesis that tyrosine phosphorylation might play a role in both motility and biofilm formation. The first part of this thesis investigates this hypothesis and successfully ascribes a function to YvyG as an orthologue of a flagellar type 3 secretion system chaperone that is essential for flagellar assembly. Crucially this work provides further evidence that the <em>B. subtilis</em> flagellum is regulated by both conserved and species-specific means. These experiments led to YvyG being re-named as FlgN. Despite previous work suggesting that phosphorylation of YvyG was important for protein function and localisation, the data presented here found no evidence of this, and therefore indicate that the impact of bacterial tyrosine phosphorylation must be assessed in vivo before any significance can be drawn from the identification of such modifications by in vitro approaches. The second part of this study examines the role of the DegS-DegU two component signal transduction system in mediating the transition from motility to biofilm formation. DegS-DegU is required for both motility and biofilm formation, and previous work indicated that DegS-DegU may sense flagellar assembly. The data presented show that upon an inhibition of flagellar rotation DegU~P levels are increased, as inferred from accepted proxies. This could conceivably be the first step in biofilm formation to allow cells to sense and respond to a surface and change their gene expression profile. The <em>B. subtilis </em>flagellum therefore acts as a mechanosensor to control the DegS-DegU two component system. Collectively, the work presented here contributes to our understanding of how <em>B. subtilis</em> regulates flagellar assembly, and further enhances our knowledge of how bacteria are able to use their flagella not only as devices for propulsion, but also to sample changes in the extracellular environment.

Orientador: Antonio Olavo Cardoso Jorge / Banca: Juliana Campos Junqueira / Banca: Martha Simões Ribeiro / Resumo: Os esporos de Bacillus spp. são encontrados amplamente distribuídos na natureza, podendo ocasionar contaminação do meio ambiente e, eventualmente, doenças ao homem e animais. Em resposta a crescente resistência microbiana, a terapia fotodinâmica (PDT) surge para atuar como um tratamento alternativo e eficaz. O presente estudo teve como objetivo comparar e avaliar a ação exercida pelo LASER de baixa intensidade (vermelho visível) e pelo diodo emissor de luz verde (LED) em esporos de Bacillus atrophaeus e Bacillus subtilis na PDT, com o uso dos fotossensibilizadores azul de metileno (37,5 M), rosa bengala (12,5 M) e verde malaquita (300 M). Utilizou-se cepa padrão de Bacillus atrophaeus (ATCC 9372) e Bacillus subtilis (ATCC 19659). As cepas foram cultivadas, durante 7 dias, em Ágar Nutriente acrescidas de 0,003% de sulfato de manganês e analisadas quanto a formação de esporos (coloração de Wirtz-Conklin). Os esporos foram suspensos em água destilada esterilizada e centrifugados por 10 min a 653 Xg. As suspensões receberam choque térmico de 70 °C por 30 min. As suspensões foram padronizadas com 106 células/mL. Em placas de 96 poços adicionou-se 0,1 mL de suspensão dos esporos de Bacillus atrophaeus ou de Bacillus subtilis e 0,1 mL do fotossensibilizador ou de solução de NaCl a 0,9%, sendo agitadas durante 5, 10 e 30 minutos e irradiadas. Realizaram-se diluições seriadas. Alíquotas de 0,1 mL das diluições foram semeadas em placas com Ágar Infusão Cérebro-Coração e incubadas a 37 °C por 48 horas. Os resultados foram analisados estatisticamente (ANOVA, teste de Tukey, p<0,05). As maiores reduções observadas em UFC/mL (Log10) para os esporos de Bacillus atrophaeus foram 0,71 Log10 para azul de metileno (10 min); 2,49 Log10 para rosa bengala (30 min) e 0,42 Log10 para verde... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The spores of Bacillus spp. are found widely distributed in nature, which may cause contamination of the environment and eventually diseases to humans and animals. In response to increasing microbial resistance, photodynamic therapy (PDT) appears to act as an alternative treatment and effective. The present study aimed to compare and evaluate the action exerted by low intensity laser and green light emitting diode (LED) in spores of Bacillus atrophaeus and Bacillus subtilis in PDT, with the use of photosensitizers blue methylene (37.5 M), rose bengal (12.5 M) and malachite green (300 M). It was used a standard strain of Bacillus atrophaeus (ATCC 9372) and Bacillus subtilis (ATCC 19659). The strains were cultivated for 7 days in Nutrient Agar added to 0.003% of manganese sulphate and analyzed for the formation of spores (Wirtz-Conklin staining). The spores were suspended in sterile distilled water and centrifuged for 10 min at 653 Xg. The suspensions were heat shock at 70 °C for 30 min. The suspensions were standardized to 106 cells / mL. In 96-well plates were added 0.1 ml of Bacillus subtilis or Bacillus atrophaeus and 0.1 ml of the photosensitizer or NaCl 0.9%, stirred for 5, 10 and 30 minutes and irradiated. Serial dilutions were performed. Aliquots of 0.1 mL of the dilutions were plated on Brain Heart Infusion Agar and incubated at 37 °C for 48 hours. The results were analyzed statistically (ANOVA, Tukey test, p<0.05). The highest observed reductions in CFU/ml (Log10) for Bacillus atrophaeus were 0.71 Log10 for methylene blue (10 minutes), 2.49 Log10 for rose bengal (30 min) and 0.42 Log10 for malachite green (10 min). In relation to Bacillus subtilis the largest reductions were 0.82 Log10 for methylene blue (10 minutes), 3.86 Log10 for rose bengal (30 min) and 0.63 Log10 for malachite green (10 min). The conclusion... (Complete abstract click electronic access below) / Mestre

Thèse doctorat : Enzymologie Moléculaire : Nancy 1 : 2005. / Titre provenant de l'écran-titre.