Die Besiedlung der Pflanzenwurzel und das Vorhandensein aktiver Zellen sind grundlegende Voraussetzungen für das Zustandekommen phytoeffektiver und Pathogen unterdrückender Effekte durch Applikation des Nutzbakteriums Bacillus subtilis. Der Einfluss ökologischer und edaphischer Faktoren auf das Besiedlungsverhalten der introduzierten Bakterien wurde im Rahmen umfangreicher populations- und aktivitätsdynamischer Untersuchungen ergründet. Die Versuche wurden in drei verschiedenen Substrattypen an der Modellpflanze Erbse (Pisum sativum L. cv. ''Bördi'') unter kontrollierten Bedingungen in der Klimakammer durchgeführt. Die Populationsdichte von Bacillus subtilis war in starkem Maße abhängig von der angewendeten Dosis. Die Verwendung von Kaliumnitrat, Sand oder Maisstärke als Trägerstoff für die Bacillus subtilis-Formulierung war für das Besiedlungsverhalten des Nutzbakteriums unerheblich. Die Entwicklung von Bacillus subtilis-Populationen an Pflanzenwurzel und im Substrat war temperaturabhängig. Höhere Versuchstemperaturen hatten größere Populationsdichten zur Folge. Der Temperatureinfluss war in Quarzsand wesentlich stärker als in Feldboden. Die peripheren Wurzelteile wiesen in Quarzsand zumeist größere Populationsdichten auf als die in Samennähe befindlichen Wurzelteile. Der Zusatz des Neem-Präparates Rhakshak Gold führte in vivo weder zu einer signifikanten Erhöhung der Populationsdichte von Bacillus subtilis noch zu einer Aktivitätserhöhung. Die Gesamtkeimzahlen in Rhizosphäre und Substrat wurden durch die Anwendung von Bacillus subtilis nicht beeinflusst. Die Aktivität von Bacillus subtilis in Rhizosphäre, Rhizoplane und Substrat war gering, der größte Teil der Keime lag versport vor. Die geringsten Versporungsgrade wurden in Quarzsand in der Rhizoplane sowie in Feldboden im Substrat festgestellt. Die Applikation des Nutzbakteriums führte zu einem reduzierten Krankheitsbefall der Erbsenpflanzen mit Phoma pinodella und Rhizoctonia solani. Die antifungale Wirkung kam in Feldboden und Aussaaterde wesentlich stärker zum Tragen als in Quarzsand. Es war kein direkter Zusammenhang zwischen Populationsdichte der introduzierten Bakterien in Rhizosphäre und Substrat und deren phytoeffektiver sowie antifungaler Leistung erkennbar. Als Ursache dafür wird der hohe Anteil versporter Zellen in der Bacillus subtilis-Population angesehen. Die Ergebnisse werden hinsichtlich der Bedeutung des Besiedlungsverhaltens und der Aktivität von Bacillus subtilis für dessen phytoeffektive und antifungale Wirkung diskutiert. / The colonization of the plant root and the presence of physiologically active bacterial cells are basic conditions to obtain plant growth promotion and disease suppression by application of Bacillus subtilis. Studies of the population- and activity dynamics of the introduced bacteria were carried out to determine the influence of different soil types and ecological factors on colonization of plant roots by Bacillus subtilis. Pea plants (Pisum sativum L. cv. ''Boerdi'') which were treated with these bacteria have been grown in three different types of substrate under controlled conditions in the climate chamber. The population density of Bacillus subtilis was strongly dependend on the applied number of cfu (colony forming units). The use of potassium nitrate, sand or maize starche as carrier for the bacterial formulation did not affect the colonization behaviour. The development of Bacillus subtilis-populations at the plant roots and in the substrate was significantly influenced by temperature. Higher temperatures during the trials resulted in larger population sizes of the introduced bacteria. The influence of temperature was much stronger in quartz sand compared with field soil. In most cases the colonization of the outer parts of the root near the root tip was better than the colonization of the root parts near the seed. The addition of the neem-extract Rakshak Gold did not have a significant influence on population density and activity of Bacillus subtilis in vivo. Nearly no effects of the bacterial treatments on the indigenous microflora could be found. The physiological activity of Bacillus subtilis in the rhizosphere, on the root surface and in the substrate was low. The bacterial population consisted largely of spores. The lowest percentage of spores was determined directly on the root surface (in quartz sand) and in the substrate (in field soil) respectively. The bacterial treatments led to a reduced disease severity on pea plants caused by Phoma pinodella and Rhizoctonia solani. The antifungal activity was higher in field soil and horticultural substrate compared with quartz sand. There was no correlation between the population density of the introduced bacteria in rhizosphere and substrate and their plant growth promoting or antifungal effects. A possible reason for that is the high percentage of non-active cells within the population of Bacillus subtilis. The results would be discussed towards the importance of population dynamics and activity of the rhizobacterium Bacillus subtilis for its plant growth and health promoting effects.

Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile.

Resumo: As técnicas mais usadas atualmente para alterar o fluxo metabólico por uma determinada via são a deleção e/ou inserção de sequências regulatórias ou genes no cromossomo bacteriano. Estes são métodos que alteram permanentemente a via metabólica em questão, perturbando o balanço metabólico durante a fase lag de crescimento, o que muitas vezes leva a um excessivo prolongamento desta fase além de diminuir a viabilidade celular. O metabolismo de purinas em Bacillus subtilis tem grande potencial para manipulação genética e produz metabólitos com valor biotecnológico. Cinco riboswitches (purE, xpt, nupG, pbuE e pbuG) controlam o metabolismo de purina em B. subtilis promovendo a terminação precoce da transcrição em ligação com guanina ou adenina. O GTP gerado nesta via é utilizado na biossíntese da vitamina B2 (riboflavina), apresentando o mesmo mecanismo de regulação com riboswitch de flavina-monofosfato (ribDG). Neste estudo, um novo modelo de controle de metabolismo de purina foi construído visando o aumento reversível do fluxo de carbono pela via em B. subtilis utilizando pequenos RNAs não-codificadores sintéticos (sRNAs). Estes foram desenhados e sintetizados para interferir na regulação da expressão gênica realizada pelos riboswitches, mantendo assim a via biossintética ativa. Os riboswitches foram caracterizados por uma nova metodologia in vitro na presença dos ligantes guanina, adenina ou FMN, e nas células de B. subtilis, sendo possível constatar o papel regulador sobre o op... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The currently employed tools to redirect metabolic flux through a specific pathway are deletion and/or insertion of regulatory sequences or genes in the bacterial chromosome. These methods permanently modify the pathway perturbing the metabolic balance during the lag growth phase, which generally extends this process and also decreases cell viability. The purine metabolism in Bacillus subtilis has great potential for genetic manipulation and produces value-added biotechnological products. Five riboswitches (purE, xpt, nupG, pbuE and pbuG) control the purine metabolism in B. subtilis by promoting the premature transcription termination guided by guanine or adenine levels in the cell. The GTP generated in this pathway is used in the biosynthesis of vitamin B2 (riboflavin), which is regulated by a flavin-monophosphate riboswitch (ribDG). In this study, a new metabolism control model was developed aiming to reversibly increase the carbon flux through selected pathways employing small synthetic non-coding RNAs (sRNAs). These sRNAs were designed and synthesized to interfere with the regulation of gene expression performed by the riboswitches, thus maintaining the biosynthetic pathways active. The riboswitches were characterized by a new in vitro methodology in the presence of the ligands: guanine, adenine or FMN. The riboswitches were characterized in B. subtilis cells using the luxABCDE bioluminescence operon as reporter. Synthetic sRNAs were designed in the Ribomaker software to ... (Complete abstract click electronic access below) / Doutor

O aumento populacional e a elevação no consumo de alimentos tem aumentado a necessidade de intensificação da aquicultura objetivando maiores produtividades para atendimento à elevação na demanda. Consequentemente, aumentam as chances de ocorrência de surtos epizoóticos dentro dos sistemas de produção, devido ao maior estresse imposto aos animais densidade elevada, manejo constante, transporte, baixa qualidade da água, etc. Alterações no estado fisiológico dos animais fazem com que seu sistema imunológico não consiga combater agentes inócuos em situações normais, mas que se tornam patogênicos nos casos de imunodepressão, levando a grandes perdas econômicas, seja com mão de obra, mortandade, medicamentos, etc. Assim, este estudo avaliou o efeito da administração individual ou conjunta de -glucano e Bacillus subtilis na dieta de juvenis de pacu, Piaractus mesopotamicus. Em um primeiro ensaio, quantidades crescentes de -glucano (60; 120; 180; 240 e 300 mg kg-1 de ração) e de B. subtilis (109 UFC g-1 de B. subtilis; 0,025%, 0,050%, 0,075%, 0,100%, 0,125%) foram adicionados a uma dieta basal extrudada (32% de proteína bruta PB; 3200 kcal kg-1 de energia digestível ED) e administrados a juvenis de pacu (8,91 ± 0,30 g) até a saciedade aparente por 75 dias, em um delineamento inteiramente aleatorizado. Ao final do período experimental, os animais foram anestesiados, pesados, submetidos à coleta de sangue e tecidos, para determinação do ganho de peso, conversão alimentar, taxa de crescimento específico, proteínas totais, albumina e globulina séricas, índice albumina:globulina (A:G), atividade da lisozima sérica, explosão respiratória de leucócitos e morfometria intestinais. Em um segundo ensaio conduzido nas mesmas condições ambientais, juvenis de pacu (2,0 ± 0,1 g; 15 peixes/aquário) foram alimentados por 59 dias com rações suplementadas pelos aditivos em questão, mais um grupo controle: -glucano de levedura 240 mg kg-1 de ração; B. subtilis comercial (109 UFC B. subtilis g-1) 0,100% de inclusão; tratamento simbiótico 240 mg de -glucano kg-1 + 0,100% de B. subtilis. Ao final do período experimental os peixes foram amostrados para determinação dos mesmos parâmetros anteriormente descritos. A inclusão dos níveis de -glucano e de Bacillus subtilis na dieta não influenciou o desempenho (P>0,05) de juvenis de pacu, mas peixes alimentados com a dieta contendo o B. subtilis consumiram mais ração (P<0,05). Não houve alterações nos parâmetros de morfologia intestinal (P>0,05), nem nos valores de explosão respiratória, atividade de lisozima, proteínas totais séricas e globulinas séricas. Quando os dois imunoestimulantes foram adicionados às dietas em combinação, também não foi observado efeito sobre os parâmetros de desempenho, explosão respiratória e atividade de lisozima, mas as proteinas totais séricas e as globulinas séricas foram positivamente influenciadas pelos tratamentos (P<0,05). As alturas de microvilosidades da porção cranial do intestino dos pacus alimentados com o tratamento simbiótico (-glucano + B. subtilis) foram maiores se comparadas ao grupo controle (P<0,05). Apesar de algumas respostas terem indicado efeito positivo sobre alguns parâmetros imunológicos e morfologia intestinal de pacus alimentados por uma combinação de -glucano + B. subtilis, não houve clara evidência que a administração simbiótica é superior à administração individual de cada imunoestimulante. / Stimulation of immune response by dietary supplements has already been proven effective in aquaculture. This work evaluates effects of dietary supplementation with the prebiotic -glucan, the probiotic Bacillus subtilis and their synbiotic combination on growth performance, immune responses, and intestinal morphology of pacu Piaractus mesopotamicus. In a first trial, a basal diet (32% crude protein; 3200 kcal kg-1) was supplemented with either -glucan (60; 120; 180; 240 e 300 mg kg-1) and B. subtilis (109 UFC g-1 of B. subtilis; 0.025%, 0.050%, 0.075%, 0.100%, 0.125%) levels and fed to groups of juvenile pacu (eight fish; 8.91 ± 0.3 g) stocked in 33 glass aquariums (70 L) for 75 days, setting up a totally randomized design trial (n=3). Fish were then weighed and had their blood drawn for determination of leucocytes respiratory burst activity, serum lysozyme activity and serum immunoglobulin concentration; intestinal tissue was sampled from two fish from each unit for micromorphology analysis. The inclusion of -glucan and Bacillus subtilis in the diet did not affect growth (P>0.05) of pacu juvenile, but fish fed diet containing levels of B. subtilis showed higher feed consumption (P<0.05). Intestinal morphology, respiratory burst activity, lysozyme activity, total serum proteins and total globulins were not affected in either inclusion of -glucan and Bacillus subtilis (P>0.05). In a second trial, a basal diet (34% crude protein; 3400 kcal kg-1) was supplemented with either -glucan (240 mg kg-1), B. subtilis (0.1 %) and -glucan + B. subtilis mix and fed to groups of juvenile pacu (15 fish; 2.0 ± 0.1 g) stocked in 16 glass aquariums (70 L) for 59 days, setting up a totally randomized design trial (n=4). Fish fed diets supplemented with -glucan + B. subtilis mix presented better growth rate, but it was not significantly different from other feeding groups. Serum total proteins and serum globulins (serum total proteins serum albumin) were affected by dietary supplements (P < 0.05); fish receiving the synbiotic treatment had higher serum globulin contents, but respiratory burst and lysozyme activity were not significantly affected. Dietary synbiotic mix altered the micromorphology of anterior intestine (P < 0.05), but the same effect was not observed in the mid and posterior intestines (P > 0.05). Dietary -glucan + B. subtilis synbiotic mix affected the intestinal ultrastructure of pacu, improved responses of immune system but did not significantly affect growth rate.

Arsenic contamination of groundwater remains a serious health concern in many areas of the world. Developing countries such as Bangladesh and Nepal are particularly affected because access to high quality water infrastructure is low. Since the 1970s, most water in these countries is sourced from shallow tube wells installed to reduce the spread of diseases associated with poor water hygiene. In this goal they were successful, however by the mid 1990s it became apparent that many of these wells were contaminated by arsenic and that these countries’ rural poor were being slowly poisoned. No simple, cheap, and reliable test for arsenic exists, and efforts to mitigate arsenic contamination have been severely limited by this over the past two decades. Government backed well-testing efforts using commercially available field kits have many issues with reliability, safety, rigour, and transparency, and have lost their urgency over the past decade, while the expensive field test kits remain out of the reach of most ordinary people in these areas. Synthetic Biology offers the technology to develop a new class of biosensor by exploiting bacteria’s natural ability to sense and respond to levels of arsenic considerably lower than commercially available kits which are based on analytical chemistry. In order to reach this goal, we must first develop our understanding of the natural response to arsenic in our chosen host, B. subtilis. Although we have a reasonably good qualitative understanding of the operon responsible for arsenic sensing, very little quantitative analysis has been carried out, and a robust system for ratiometric characterisation has not been established in the bacteria. In this work, a robust platform for rapid ratiometric characterisation is established in B. subtilis. A rigorous mathematical model of the ars operon is developed and analysed before being verified experimentally. This new knowledge is then used to explore synthetic permutations to the natural system aimed at improving the sensor properties of the system. Finally, a biological architecture for an easily tunable biosensor with good characteristics is recommended.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-13T13:52:09Z No. of bitstreams: 1 Flavia Maria Maciel Carneiro de Souza.pdf: 432222 bytes, checksum: 47380c92c63248a05ae6b56a5134cf13 (MD5) / Made available in DSpace on 2017-02-13T13:52:09Z (GMT). No. of bitstreams: 1 Flavia Maria Maciel Carneiro de Souza.pdf: 432222 bytes, checksum: 47380c92c63248a05ae6b56a5134cf13 (MD5) Previous issue date: 2007-02-15 / Marine shrimp Farfantepenaeus subtilis is found along the Brazilian northeast coast. It tolerates salinity variations and grows quickly under culture conditions. However, it has a feeding habit predominantly carnivorous, using mainly polichaets as your main feeding source. The present work aimed at to induce the natural food in the experimental culture of F. subtilis through different fertilization strategies. A randomized entirely design with three treatments and three replicates was applied, being one with inorganic fertilizer: Control (CT) - 3 mg/L of urea and 0.3 mg/L of mono ammonium phosphate; and two with organic fertilizers: Wheat bran (FT) - 25 g/m2; Soybean meal (FS) – 18.75 g/m2. Nine 500L fiber glass tanks were used, with a stocking density of 16 shrimps/m2 (≈ 2.0 g), that were fed with a 35% crude protein commercial ration at 8:00, 12:00 and 16:00 hrs. The water and plankton samples were accomplished biweekly, and benthos was monthly. During the culture the water quality maintained it self adequate to shrimp culture. The growth data showed no significant difference (P≤0.05) among the treatments, where it was verified a growth rate of 0.44 g/week and a mean survival of 75%. With relation to the natural food, the phytoplankton (921 cells/mL) was predominated by the diatoms with 86, 49 and 83%, respectively, for thetreatments CT, FT and FS. The phytobenthos was also represented by Bacilariofíceae with 88%, 66% and 78%, respectively, for CT, FT and FS. The zooplankton (9.220 individuals/L) it was represented mainly by rotifers in the treatments CT (45%), FT (38%) and FS (65%). In zoobenthos the predominance was copepods in the treatments CT (97%), FT (91%) and FS (88%). The study showed similarity among the organic and inorganic fertilizers, indicating that the fertilization process was not efficient to supply enough natural food organisms, but it was observed that the three protocols applied were similar in terms of the F. subtilis shrimps growth and maintenance of water quality. / O camarão marinho Farfantepenaeus subtilis é encontrado por toda a costa do Nordeste do Brasil, tolera variações de salinidade e se desenvolve rapidamente sob condições de cultivo. Porém, tem um hábito alimentar predominantemente carnívoro, destacando-se os poliquetas como seu principal item alimentar. O presente trabalho objetivou induzir o alimento natural no cultivo experimental de F. subtilis, através de diferentes estratégias de fertilização. Foi adotado um delineamento inteiramente casualizado com três tratamentos, sendo um com fertilizante inorgânico: Controle (CT) - 3 mg/L de uréia e 0,3 mg/L de monoamônio fosfato; e dois com fertilizantes orgânicos: Farelo de Trigo (FT) - 25 g/m2 e Farelo de Soja (FS) – 18,75 g/m2, com três réplicas para cada tratamento. Foram utilizados nove tanques circulares em fibra de vidro, com capacidade de 500 L, os quais foram estocados com 16 camarões/m2 (≈2,0g). A alimentação artificial constou de ração comercial com 35% de proteína bruta e foi ofertada em bandejas, às 8:00, 12:00 e 16:00 h. As coletas de água e de plâncton foram realizadas quinzenalmente, e as coletas de bentos mensalmente.Durante o cultivo a qualidade de água se manteve adequada ao cultivo da espécie. Os dados de crescimento demonstraram não haver diferença estatística (P≤0,05) entre os tratamentos, onde foi constatado um crescimento de 0,44 g/semana e sobrevivência média de 75%. Quanto ao alimento natural, no fitoplâncton a média geral observada foi de 921 cél./mL e predominaram as diatomáceas com 86%, 49% e 83%, respectivamente, nos tratamentos CT, FT e FS. O fitobentos também foi representado pelas bacilariofíceas com 88%, 66% e 78,%, respectivamente, para os tratamentos CT, FT e FS. O zooplâncton teve uma média geral de 9.220 ind./L e esteve representado principalmente por rotíferos 45%, 38% e 65%, respectivamente, para CT, FT e FS. O zoobentos foi representado por copépodos com 97%, 91% e 88,%, respectivamente, para CT, FT e FS. O estudo demonstrou similaridade entre os fertilizantes orgânicos e inorgânicos, sugerindo que a fertilização não foi eficiente quanto à indução do alimento natural. Porém, demonstrou-se que os três protocolos testados foram igualmente eficientes para o crescimento do F. subtilis e para manutenção da qualidade da água.

Proteins performing catalytic roles predominantly occur in a few protein folds. Functional diversity within a common structural scaffold has been attributed to conformational features that enable exploration of reaction space. In this study, we examined specific aspects of functional diversity in the Double Stranded β-helix(cupin) fold. The cupin domain is a hyper-stable protein fold that can support a variety of functions. Variation in function using a conserved active site in the cupin fold is achieved by changes in the residues that line the active site cavity as well as by the choice of a metal cofactor. Although this appears to be a likely basis for functional diversification, a few exceptions exist. It is thus interesting to examine how enzymes with the same structure, metal cofactor and ligand coordination catalyze a diverse range of reactions. This thesis describes two bi-cupins, BacB (also known as bacilysin synthase, YwfC) and Quercetinase (YxaG). BacB is a part of the protein machinery involved in the synthesis of a di-peptide antibiotic bacilysin. The case of the bicupin protein BacB illustrates the problem of functional annotation of proteins with the cupin fold. None of the predicted functions for this enzyme could be experimentally validated in vitro. The crystal structure, determined by Single-wavelength Anomalous Dispersion (SAD) based on the bound metal-ion at the active site provided a basis to evaluate the catalytic role of this protein. Eventually, the function of this protein could be determined based on characterizing the gene product of bacA, the gene preceding bacB in the B. subtilis bac operon. The crystal structure determination of BacB also led to an analysis of multiple crystal forms, with implications for the role of molecular symmetry in forming protein crystals. The stability of the cupin domain was examined using B. subtilis quercetinase as a model system. The availability of the crystal structure and a robust activity assay enabled us to examine the role of fragment complementation in the stability of the cupin scaffold and its implications for the function of this enzyme. This thesis also has a section on the use of structural homology for function annotation for cupin proteins. The results presented here thus provide a frame-work to understand the structural basis for functional diversity in the cupin family. This thesis is organized as follows: Chapter 1: This chapter provides an introduction to the Double Stranded β-Helix-Helix (DSBH or cupin) fold. Proteins with a cupin scaffold are remarkably diverse - spanning both enzymatic and non-enzymatic functions. This chapter presents a compilation of previous reports encompassing eighteen different functional classes. These functions include seed storage, transcription factors and a host of various enzymatic activities. Cupin proteins can be monocupins, bicupins or multi-domain cupins based on the number of DSBH domains in a single polypeptide chain. Very few multi-domain cupin proteins have been identified and this is generally not considered to be a significant sub-group. The inference that cupin proteins with more than one domain are products of gene duplication events is also examined in detail. The latter part of this chapter aims to provide an introduction to the two model proteins B. subtilis BacB and Quercetinase. Chapter 2: This chapter describes studies on a bi-cupin protein BacB involved in bacilysin synthesis. Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. The production of this antibiotic is regulated via a stringent response and branches off the pathway for aromatic amino-acid biosynthesis at prephenate. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of L-anticapsin. This data, however, could not be reconciled with the putative functional assignments for these proteins whereby BacA, a prephenate hydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize L-anticapsin. Here, based on the characterization of the reaction products of BacA and BacB as well as the crystal structure of BacB, we demonstrate that B. subtilis BacB catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to L-anticapsin. The mass and NMR spectra of the reaction product of BacA suggest that BacA is a decarboxylase that acts on prephenate. BacB is an oxidase. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvicacid (PPY). A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis. Chapter 3 is based on the crystal packing analysis of three different crystal forms of B. subtilis BacB. BacB is an oxidase that catalyzes the production of the di-peptide antibiotic bacilysin. This protein is a bi-cupin with two double stranded β-helix domains fused in a compact arrangement. BacB crystallizes in three crystal forms, belonging to the triclinic, monoclinic and tetragonal space groups. These different crystal forms could be obtained in similar crystallization conditions. We also note that a slight disturbance to the crystallization droplet results in nucleation events, eventually resulting in a different crystal form. All three crystal forms of BacB diffract to high resolution, thus enabling the structure determination and analysis of the packing arrangements of BacB in different space groups. Metal ions at the lattice interface dominate the different packing arrangements. The crystal packing reveals that a dimer of BacB serves as the template on which higher order symmetrical arrangements are formed. BacB, however, is a monomer in solution. The different crystal forms of BacB thus provide experimental evidence to the hypothesis that molecular symmetry could aid crystallization. Chapter 4 provides a conformational analysis of the cupin fold using B. subtilis quercetinase as a model system to understand the conformational determinants of functional diversity. Controlled proteolysis experiments revealed that this enzyme is active, thermo-stable and maintains its quaternary arrangement even after substantial (ca 33 %) cleavage of the protein. The results presented in this chapter thus show that the cupin scaffold offers a balance between protein stability and function by locating the active site and substrate recognition features in the most stable region of the protein. Chapter 5 is based on the phylogenetic analysis of cupin domains. The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary association and the nature of bound metal ion despite having a conserved β-barrel structural scaffold. Here, an attempt was made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which structures are available through world-wide structural genomics initiatives but characterized as “hypothetical”. We have explored the feasibility of obtaining clues to functions of such proteins by means of comparative analysis with cupins of known structure and function. This phylogenetic strategy was applied to BacB leading to clustering with oxidoreductases. BacB was experimentally demonstrated to be an oxidase. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises additional experimental details, methodology and aspects of the techniques used in this study. Appendix I contains a description of a methodology for Molecular Replacement (MR) calculations in obtaining phase information for protein crystallography. Appendix II provides additional details of experimental protocols.

Faithful chromosome segregation is essential for all living organisms. Bacterial chromosome segregation utilizes highly conserved directional SpoIIIE/FtsK translocases to move large DNA molecules between spatially separated compartments. These translocases employ an accessory DNA-interacting domain (gamma) that dictates the direction of DNA transport by recognizing specific DNA sequences. To date it remains unclear how these translocases use DNA sequence information as a trigger to expend chemical energy (ATP turnover) and thereby power mechanical work (DNA movement). In this thesis, I undertook a mechanistic study of directional DNA movement by SpoIIIE from the Gram-positive model bacterium Bacillus subtilis. Specifically, I was interested in understanding the information transfer within the protein from sequence recognition, to ATP turnover, and ultimately to chromosome translocation. How do DNA sequences trigger directional chromosome movement?

No description available.

Chez Bacillus subtilis l'expression de l'opéron mtl pour l'utilisation du mannitol est contrôlé par MtlR. MtlR est un activateur transcriptionnel qui appartient à la famille des régulateurs DeoR composé d'un domaine HTH suivi de deux PRDs, un domaine EIIBGat et un domaine EIIAMtl-like.Le mécanisme général de la régulation de l'activité de MtlR est basé sur sa phosphorylation par des composants du PTS. La phosphorylation sur la Cystéine 419 du domaine EIIBGat par P~EIIAMtl a un effet négatif majeur sur l'activité de MtlR. Par conséquent, dans un mutant mtlF où EIIAMtl est délétée MtlR est constitutivement actif.Dans cette étude nous avons mis en évidence un nouveau phénomène de régulation de MtlR impliquant la protéine du PTS, EIIBMtl.Nous avons observé que lorsque on déléte l'opéron mtl ou EIIBMtl et EIIAMtl, l'activité constitutive de MtlR dans un mutant mtlF déjà observée est abolie d'où notre hypothèse que EIIBMtl aura un effet sur l'activité de MtlR. Par des expériences de double hybride nous avons montré une interaction directe, spécifique et bidirectionnelle entre les deux protéines EIIBMtl et EIIBGatEIIAMtl-like de MtlR. D'une manière comparable à la cellule où EIIBMtl est fusionnée à la protéine EIICMtl nous avons démontré que seulement la forme EIIBMtl fusionné à la perméase EIICMtl est capable d'activer MtlR mais nous avons également démontré que ce n'est pas EIICMtl qui est essentielle à l'interaction entre EIIBMtl et MtlR mais c'est le voisinage de la membrane qui est essentielle pour l'établissement de cette interaction et l'activation de MtlRUn modèle de régulation de l'activité du régulateur MtlR est proposé. Dans ce modèle l'induction de l'opéron mtl via l'activation de MtlR requiert la phosphorylation de PRDII de MtlR par P~His-HPr, la déphosphorylation de EIIBGat de MtlR par EIIAMtl et la présence de la forme non-phosphorylée de l'EIIBMtl qui est dominante en présence du substrat inducteur, le mannitol. Ainsi, l'EIIBMtl non-phosphorylée séquestre MtlR déphosphorylé sur sa cystéine 419 à la membrane, l'active et induit l'expression de l'opéron mtl.