Penicillin-binding proteins (PBP's) are membrane-associated enzymes involved in the polymerization of peptidoglycan. PBP's are divided into three classes based upon their molecular weights and functional domains. Gene expression is regulated in the two differentiated cells in Bacillus subtilis, the mother cell and the forespore, by coordinated expression of different sigma factors that recognize specific promoters in each compartment. The functional and compartmental specificity of individual penicillin-binding proteins from the different classes of PBP's were examined during sporulation in B. subtilis. Analyses of three class A high molecular weight PBP's indicated that pbpF and pbpG must be expressed in the forespore to carry out their specific role during spore peptidoglycan synthesis. Expressing pbpD in either the forespore or the mother cell could not complement for the loss of pbpF and pbpG, suggesting that there must be additional sequence information in PBP2c and PBP2d that allows them to carry out their specific role during germ cell wall synthesis. Analyses of a low molecular weight PBP, PBP5*, suggested that expressing dacB in either the mother cell or in the forespore could regulate the level of spore peptidoglycan cross-linking to what is typical of wild type spore peptidoglycan. / Master of Science

Reducing herbicide carryover in soybean production is imperative to sustain future soybean cropping systems in Mississippi and the rest of the world. This research was conducted to determine the response of inoculated soybean to corn (Zea mays L.) residual herbicides. The herbicide treatments included aatrex (atrazine), callisto (mesotrione), lexar (atrazine+mesotrione+s-metolachlor), and steadfast (nicosulfuron+rimsulfuron) at 100%, 75%, 50%, and 25% of the total label rate. Inoculant treatments were Bradyrhizobium japonicum, Bacillus subtilis, and no inoculant. Bacterial inoculants positively impacted most shoot and root growth parameters, significantly increasing depending on the herbicide x inoculant treatment combination and its rate. B.japonicum alone and in combination with B.subtilis can be potentially used in soybean production to alleviate corn residual herbicide carryover effects. However, due to the complex interaction between soybean, rhizobia, and herbicides, further long-term evaluations are needed to develop a more robust technology and make recommendations for use by farmers.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2007-2008. / La découverte d’oxyde nitrique synthases (NOS) chez les bactéries a imposé un questionnement quant à la fonction de l’oxyde nitrique (NO) chez les microorganismes. Chez les mammifères, le NO remplit des fonctions de signalisation et de stress pour lesquelles il n’y a aucun équivalent chez les bactéries. Des études ont démontré que in vitro, comme chez les NOS animales (mNOS), les NOS bactériennes (bNOS) peuvent catalyser la conversion du L-arginine en L-citrulline et libérer du NO. Cependant, il a été découvert que les bNOS de Deinococcus radiodurans et Streptomyces turgidiscabies pouvaient catalyser la nitrosation de groupements trytophanyles. Jusqu’à maintenant ces études soutiennent l’hypothèse que les bNOS synthétisent du NO lors d’un processus de catalyse semblable à celui des mNOS. Cependant, elles indiquent que sa fonction pourrait être de modifier une molécule au site du cofacteur. Nous avons proposé l’étude des propriétés cinétiques et structurales des bNOS de Bacillus subtilis (BsNOS) et Staphylococcus aureus (SaNOS) afin d’investiguer le mécanisme catalytique des bNOS, d’approfondir les connaissances de la catalyse chez les NOS en général et d’explorer les particularités de ces enzymes susceptibles de révéler leur fonction. Nos études ont porté sur les caractéristiques structurales du site actif de SaNOS et BsNOS, sur les cinétiques de formation et de disparition du complexe oxygéné chez SaNOS, sur les interactions des substrats avec les ligands de l’hème et sur le rôle du cofacteur chez les bNOS. Nos résultats montrent que SaNOS et BsNOS peuvent catalyser les mêmes réactions biochimiques que les mNOS et apportent des éléments nouveaux permettant de mieux comprendre le mécanisme d’activation du complexe oxygéné, une étape cruciale à la catalyse. Par ailleurs, certaines ressemblances observées entre SaNOS, BsNOS et les mNOS ont révélé des propriétés conservées chez ces enzymes qui sont importantes pour la production et la fonction du produit synthétisé. Notamment la spécificité des interactions des différents substrats avec les ligands de l’hème et la déformation de la molécule d’hème. Cependant des différences reliées aux effets structuraux du cofacteur ont été observées et s’ajoutent à celles découvertes chez les autres bNOS qui indiquent que la fonction de ces enzymes pourrait se distinguer de celle des mNOS. / The discovery of nitric oxide synthases (NOS) in bacteria has raised many questions regarding the function nitric oxide (NO) might fulfill in prokaryotes. In mammals, NO is implicated in signal and stress events for which no equivalent functions exist in bacteria. In vitro studies have revealed that, like mammalian NOS (mNOS), bacterial NOS (bNOS) catalyze the hydroxylation of L-arginine to L-citrulline and release NO. In addition, it has been shown that the bNOS of Deinococcus radiodurans and Streptomyces turgidiscabies catalyse the nitrosation of tryptophanyl compounds. As of now these studies support the hypothesis that bNOS synthesize NO in a catalytic process similar to the one described for mNOS. However, these studies also indicated that the newly synthesized NO might be used to modify a molecule bound to the cofactor binding site. We proposed the investigation of the kinetic and structural properties of the bNOS of Bacillus subtilis (BsNOS) and Staphylococcus aureus (SaNOS) to probe the catalytic mechanism of bNOS, deepen our understanding of catalysis in NOS and explore the distinctive features of these new enzymes that might reveal their function. Our studies targeted the structure of the active site of SaNOS and BsNOS, the kinetics of formation and decay of the oxygenated complex of SaNOS, the interactions of the substrates with heme-bound ligands and the function of the cofactor in bNOS. Our results support the proposal that bNOS are able to catalyze the same biochemical reactions than those carried out by mNOS and bring new information that provide us with a better understanding of the mechanism of oxygen activation, a crucial step during catalysis. In addition the observation of some similarities between SaNOS, BsNOS and mNOS revealed conserved features in these enzymes that are important for the synthesis and function of the product. Notably the specificity of the interactions the two substrates make with the heme-bound ligands and the deformation of the heme molecule. Meanwhile structural differences related to the cofactor have been observed, and in addition to those observed in the other bNOS, point to a novel function for these enzymes.

La bactérie Bacillus subtilis est considérée comme une solution alternative aux pesticides conventionnels pour la gestion des agents phytopathogènes. Les mécanismes de biocontrôle de cette bactérie sont multiples et reposent fortement sur la production de lipopeptides. A l'heure actuelle, l'influence des mycètes/oomycètes phytopathogènes sur la physiologie et les mécanismes de biocontrôle de B. subtilis est peu connue. L'objectif de ce projet de doctorat était d'étudier chez B. subtilis l'influence de différents mycètes/oomycètes phytopathogènes sur les mécanismes de biocontrôle et plus particulièrement sur la production de lipopeptides. Dans un premier temps, les lipopeptides produits par B. subtilis PTB185 ont été caractérisés par spectrométrie de masse et une méthode permettant leur quantification relative a été développée à l'aide d'un MALDI-TOF. Cette première étape a permis de montrer que la souche PTB185 produit de la surfactine, de l'iturine et de la fengycine. Les essais de confrontation sur gélose ont montré que l'activité antagoniste et la production de lipopeptides par B. subtilis variaient significativement (P ≤ 0.05) selon l'agent pathogène testé. Aucune corrélation entre la production de lipopeptides et l'activité antimicrobienne de B. subtilis n'a toutefois été observée. Le mycélium autoclavé des agents phytopathogènes a également influencé significativement les quantités de lipopeptides produites par B. subtilis en milieu liquide. La production de fengycine et/ou d'iturine a augmenté significativement en présence du mycélium de Botrytis cinerea, Mucor sp., Pythium ultimum ou Rhizoctonia solani, alors que l'addition de mycélium n'a pas affecté de façon significative la production de surfactine, en comparaison avec le traitement témoin. Les bactéries cultivées en milieu liquide en présence du mycélium de Mucor sp. ont causé une réduction significativement plus importante de la croissance de B. cinerea, R. solani et S. sclerotiorum, comparativement aux bactéries cultivées en absence de mycélium. Ces résultats montrent pour la première fois l'influence du mycélium autoclavé des agents phytopathogènes sur l'activité antagoniste et la production de fengycine/iturine chez B. subtilis. Une étude plus approfondie de l'influence du mycélium autoclavé et des composés extracellulaires des mycètes/oomycètes phytopathogènes sur B. subtilis PTB185 a par la suite été réalisée en protéomique. Le mycélium autoclavé de tous les agents phytopathogènes testés a fortement inhibé les protéines associées au métabolisme et à la biosynthèse de la thiamine chez la bactérie. Le mycélium autoclavé de Mucor sp. a fortement stimulé les protéines associées au « phage-like element PBSX » et fortement diminué celles du métabolisme et de la biosynthèse de la biotine. Les composés extracellulaires de P. ultimum ont diminué les protéines associées à la formation des flagelles et stimulé celles associées à la production de subtilosine. Le mycélium autoclavé et les composés extracellulaires de R. solani ont augmenté de façon importante les protéines associées à la synthèse de sidérophores. Les composés extracellulaires de S. sclerotiorum ont pour leur part très faiblement impacté le protéome de la bactérie. Les résultats de cette étude, en plus de témoigner de l'influence des interactions biotiques sur la production des lipopeptides, apportent des informations supplémentaires quant à l'influence de ces dernières sur la physiologie/les mécanismes de biocontrôle de B. subtilis. Enfin, cette étude offre de nouvelles perspectives pour optimiser le milieu de culture de B. subtilis à des fins biotechnologiques. / The bacterium Bacillus subtilis is considered as a promising alternative to conventional pesticides for plant disease management. The mechanisms underlying the biocontrol properties of this bacterium are multiple and are closely related to the production of lipopeptides. Currently, little is known about the influence of plant pathogenic fungi/oomycetes on B. subtilis physiology and biocontrol mechanisms. The objective of this doctoral research project was to study the influence of fungi/oomycetes on B. subtilis biocontrol mechanisms, especially on the production of lipopeptides. In the first instance, the lipopeptides produced by B. subtilis PTB185 were characterised by mass spectrometry and a method allowing the relative quantification of these compounds was developed using MALDI-TOF instrumentation. This first step displayed the capacity of strain PTB185 to produce surfactin, iturin, and fengycin. Confrontation assays conducted on agar showed that B. subtilis antagonistic activity and production of lipopeptides vary significantly (P ≤ 0.05) according to the pathogen tested. However, no correlation between lipopeptides production and B. subtilis antimicrobial activity was observed. Autoclaved mycelia of plant pathogens were also shown to significantly influence the quantities of lipopeptides produced by B. subtilis in liquid culture. Fengycin and/or iturin were produced in significantly higher amounts in presence of mycelium of Botrytis cinerea, Mucor sp., Pythium ultimum, or Rhizoctonia solani, while addition of mycelium in the medium did not significantly affect surfactin production as compared to the control. Bacteria grown in liquid medium amended with Mucor sp. mycelium caused a significantly higher reduction of mycelial growth of B. cinerea, R. solani, and Sclerotinia sclerotiorum as compared to the bacteria grown with no mycelium. These results show for the first time the influence of autoclaved plant pathogen mycelium on B. subtilis antagonistic ability and production of fengycin/iturin. The influence of autoclaved mycelia and extracellular compounds of plant pathogenic fungi/oomycetes on B. subtilis was further investigated by proteomics. Autoclaved mycelium of all tested pathogens strongly inhibited the proteins associated with B. subtilis thiamine metabolism and biosynthesis. Autoclaved mycelium of Mucor sp. strongly increased proteins associated with the phage-like element PBSX and strongly decreased those related to biotin metabolism and biosynthesis. Extracellular compounds of P. ultimum reduced proteins associated with flagellar assembly and stimulated those related to the production of subtilosin. Autoclaved mycelium and extracellular compounds of R. solani strongly increased proteins associated with the production of siderophores. Extracellular compounds of S. sclerotiorum barely affected the proteome of the bacterium. The results of this study, besides providing additional evidence of the influence of biotic interactions on lipopeptides production, give further information on the influence of the latter on B. subtilis physiology and biocontrol mechanisms. Finally, this study provides new insights into the optimisation of the culture medium to grow B. subtilis for biotechnological applications.

L’objectif de ce projet de recherche était de déterminer les effets d’une supplémentation de probiotiques du genre Bacillus sur les performances de production de vaches de race holstein en mi-lactation et sur le profil en acides gras de leur lait. Pour ce faire, six vaches multipares porteuses d’une canule ruminale ont été utilisées selon un dispositif en chassé croisé répliqué et randomisé. Les vaches ont ainsi reçu soit 200 g/j de poudre de lactosérum comme traitement témoin ou 200 g/j de BioPlus 2B (Chr Hansen, Milwaukee, WI), un probiotique commercial de Bacillus subtilis et de Bacillus licheniformis, représentant un apport quotidien de 6,4 × 10¹¹ UFC et utilisant la poudre de lactosérum comme diluant. La production laitière, la composition du lait et son profil en acides gras, les paramètres de fermentation ruminale et le microbiote ruminal ont été évalués. Les traitements n’ont pas affecté les performances laitières. L’utilisation du probiotique a cependant augmenté la concentration relative d’anteiso 13:0 et d’anteiso 15:0 et tendait à augmenter la concentration totale d’acides gras à chaîne ramifiée dans les matières grasses du lait lorsque comparé au traitement témoin. Les traitements n’ont pas modifié le pH, l’azote ammoniacal ou les concentrations en acétate, propionate et butyrate du rumen. Cependant,le probiotique a augmenté la concentration ruminale d’isovalérate et tendait à augmenter celle de l’isobutyrate. Cette expérience a permis d’observer la sensibilité du profil en acides gras du lait face aux modifications du microbiote ruminal. Les bactéries probiotiques du genre Bacillus pourraient donc être utilisées dans un protocole de maximisation des acides gras à chaîne ramifiée du lait pour permettre une augmentation du niveau de ces acides gras dans les produits laitiers. / The aim of the study was to determine the effect of a Bacillus-based direct-fed microbial on dairy performance of mid-lactating Holstein dairy cows and on their milk fatty acid composition. To this end, six multiparous cows fitted with a rumen cannula were used in a randomized replicated crossover design. Cows received 200 g/d of either whey powder, as a control, or BioPlus 2B (Chr Hansen, Milwaukee, WI), a commercial direct-fed microbial providing Bacillus subtilis and Bacillus licheniformis, representing a daily dose of 6.4 ×10¹¹ cfu, and using whey powder as a carrier. Milk production, composition and fatty acid profile, as well as ruminal parameters and microbiota were evaluated. Bacillus concentrationin the rumen increased with the addition of the direct-fed microbial supplement. Treatmentdid not affect ruminal pH, NH3-N and concentrations of acetate, propionate, and butyrate.However, direct-fed Bacillus increased ruminal concentration of isovalerate and tended to increase the concentration of isobutyrate. Treatments did not affect milk performance. The direct-fed Bacillus increased the relative concentration of anteiso 13:0 and anteiso 15:0 in milk fat, and tended to increase total concentration of branched-chain fatty acids as compared with control. This trial indicates that milk fatty acid composition is sensitive to ruminal microbiota modifications. Direct-fed Bacillus could be used as part of a standard protocol aiming to produce milk enriched in branched-chain fatty acids.

No description available.

Estudos envolvendo o emprego de novas técnicas de esterilização têm apresentado nítido crescimento nos últimos anos como alternativa aos processos convencionais. A grande possibilidade consiste no emprego do plasma como agente de esterilização. Essa tecnologia tem considerável potencial para desenvolver o meio mais eficiente e seguro de esterilização de artigos termossensíveis com ênfase para a indústria farmacêutica e médica e até mesmo em outras áreas industriais. O objetivo deste trabalho foi investigar a influência de alguns parâmetros de processos por plasma e correlacionar sua eficácia com processos por óxido de etileno. Neste trabalho, estudos foram realizados empregando tecnologia de esterilização por plasma usando reator Reactive Ion Etching (RIE). Os valores aplicados de potências de rádio-frequência, a 13,56 MHz foram 25 W, 50 W, 100 W e 150 W. Os gases testados foram oxigênio puro e misturas de oxigênio e peróxido de hidrogênio (190/10, 180/20 e 160/40 sccm) e fluxo constante de 200 sccm, pressão de 0,100 torr e temperatura abaixo de 60°C. Esterilizador por óxido de etileno foi empregado a 450 mg/L (Oxyfume 2002®), 55°C, 60% de umidade e -0,65 e 0,60 kgf/cm2 de pressão. Os indicadores biológicos empregados foram constituídos de esporos de Bacillus subtilis var. niger ATCC 9372, inoculados em lamínulas de vidro de 18 x 18 mm e discos de papel de 13 mm de diâmetro em uma carga de 2,0 x 107 UFC/suporte. Os tempos de exposição aos processos por plasma foram de 3 a 120 minutos. Reduções progressivas da contagem inicial de microrganismos foram observadas nos valores D: 215,91,55,55,9,19 e 2,91 minutos para processos por plasma com oxigênio puro, a 25 W, 50 W, 100 W e 150 W, respectivamente. Misturas de oxigênio e peróxido de hidrogênio apresentaram os seguintes valores D: 190/10 sccm (6,41 min), 180/20 sccm (6,47 min) e 160/40 sccm (4,02 min), a 100 W e 190/10 sccm (1,47 min), 180/20 sccm (3,11 min) e 160/40 sccm (1,94 min), a 150 W. Processos empregando óxido de etileno apresentaram valor D de 2,86 minutos. Análises por Microscopia Eletrônica de Varredura demonstraram danos causados ao córtex dos esporos. Sistema empregando plasma como principal agente de esterilização apresentou-se efetivo em desafios com indicadores biológicos. Os processos por plasma provaram ser a mais apropriada tecnologia de esterilização de materias termossensíveis e com grande potencial para substituir os métodos convencionais de esterilização em futuro próximo. / Studies involving new sterilization techniques have increased in the past few years as alternatives to conventional processes. The great possibility consists in the use of plasma as sterilization agent. This technology has the enormous potential to develop a more efficient and safer means of sterilization at thermo-sensitive matters, focusing the pharmaceutical and medical industry - even though it can be applied to other industrial areas. The aim of the present study was to investigate the influence at some parameters of plasma processes and correlate the effectiveness plasma with ethylene oxide sterilizer. In this work, studies were performed taking into account a plasma sterilization technology using a Reactive Ion Etching (RIE) reactor. Power was applied at 13.56 MHz using a 6 inch diameter electrode. The gases tested were pure oxygen and oxygen-hydrogen peroxide mixtures (190/10, 180/20, and 160/40 sccm), and gas flow held constant 200 sccm, pressure at 0.100 torr and radio-frequency power at 25 W, 50 W, 100 W, and 150 W and temperature below 60°C. Ethylene oxide sterilizer were performed using 450 mg/L (Oxyfume 2002®) at 55°C, 60% humidity and -0.65 and 0.60 kgf/cm2 pressure. The biological indicator used was Bacillus subtilis var. niger ATCC 9372, witch was inoculated in glass carries (18 x 18 mm) and paper discs (13 mm diameter) in a load of 2.0 x 107 CFU/support. The exposition times were 3 to 120 minutes. Progressive reductions of the initial microbial count could be observed in the D values witch were 215.91, 55.55, 9.19, and 2.91 minutes for pure oxygen plasma at 25 W, 50 W, 100 W and 150 W, respectively. Oxygen-hydrogen peroxide mixtures plasma showed D values: 190/10 sccm (6.41 min), 180/20 sccm (6.47 min) and 160/40 sccm (4.02 min) at 100 W and 190/10 sccm (1.47 min), 180/20 sccm (3.11 min) and 160/40 sccm (1.94 min) at 150 W. Ethylene oxide processes showed D value to 2.86 minutes. Scanning Electron Microscopy analyses showed some damage on the spore cortex. Processes using plasma as main sterilization agent are presented effective in challenge with biological indicators. The plasma proved to be the most appropriate sterilization technology in thermosensitive matters and to have a great potential to replace conventional sterilization methods in the near future.

A cárie dental é uma doença bacteriana infecciosa considerada como um dos principais problemas de saúde pública. O principal agente etiológico para o desenvolvimento da doença é o Streptococcus mutans. A proteína P1, também conhecida como antígeno I/II, do S. mutans é fundamental para a etapa inicial de adesão à superfície dental (sacarose-independente), sendo, portanto, considerada essencial para o processo de colonização deste patógeno. Algumas regiões dessa proteína vêm sendo empregadas como antígenos em estratégias vacinais contra a cárie, entre elas a região A, localizada na porção N-terminal, conhecida como região de ligação à saliva Saliva Binding Region (SBR). No entanto, apesar dos avanços, não existe vacina para a prevenção da cárie licenciada para uso em humanos. Diante disso, o presente trabalho tem como objetivo o desenvolvimento e caracterização de uma nova estratégia vacinal contra o S. mutans baseada em esquema de dose-reforço heterólogo, utilizando o fragmento P139-512, na forma de proteína purificada, expressa a partir de linhagens recombinantes de Bacillus subtilis. A proteína P139-512 compreende os aminoácidos 39-512 da proteína nativa, o que corresponde a toda região A e uma pequena porção da região variável da proteína P1. Utilizamos esporos de B. subtilis 1012, modificados para expressar o antígeno P139-512 (LDV702). A linhagem LDV704, além de expressar o antígeno, foi modificada para expressar uma invasina (InvA) com capacidade de se ligar a epitélios de mucosa. Camundongos da linhagem BALB/c foram imunizados por via sublingual com uma dose de esporos de B. subtilis (linhagens 1012, LDV702, LDV704 ou PBS) seguidos por dois reforços com a proteína P139-512, associada ou não com o adjuvante LTK63. Níveis significativos de anticorpos séricos foram induzidos pelas formulações em associação com o adjuvante após a terceira dose, e mostraram-se capazes de reconhecer os epítopos em diferentes linhagens de S. mutans. No entanto, nenhuma das formulações mostrou-se capaz de ativar respostas de mucosa (S-IgA). Porém, observamos que o adjuvante LTK63 empregado na estratégia dose-reforço heterólogo potencializou a resposta sérica de anticorpos IgG, sendo capaz de modular e melhorar qualitativamente as respostas induzidas. Assim, a administração das formulações na presença do adjuvante representa uma alternativa promissora para o controle do S. mutans. / Dental caries is an infectious bacterial disease considered as one of the main public health problems. The main etiological agent for the development of the disease is the Streptococcus mutans. The P1 protein, also known as S. mutans Ag I / II antigen, is essential for the initial stages of adhesion to the dental surface (sucrose-independent) and is, therefore, considered essential for the colonization process of this pathogen. Some regions of this protein have been used as antigens in vaccine strategies against caries, among them the A region, located at the amino terminal region also known as the Saliva Binding Region (SBR). However, despite the advances, there is no licensed anti-caries vaccine for human use. Therefore, the present work aims to develop and characterize a new vaccine strategy against S. mutans based on a heterologous priming/boost immunization regimen using the recombinant P139-512 fragment, expressed and purified from a Bacillus subtilis strain. The P139-512 protein comprises amino acids that encompasses the entire A region and a small portion of the variable region of the P1 protein. We used spores of B. subtilis 1012 (wild-strain) and recombinants that were modified to express the antigen P139-512 (LDV702). In addition to express the antigen, the LDV704 strain was modified to express a surface-exposed bacterial invasin (InvA) capable of binding to the mucosal epithelia. BALB/c mice were primed via the sublingual route with a dose of B. subtilis spores (1012, LDV702, or LDV704 strains) followed by two boosting doses with the purified protein P139-512, associated or not with the LTK63 adjuvant, by the same administration route. Significant serum antibody levels were induced by the formulations with the adjuvants after the third dose and the antibodies were shown to recognize epitopes exposed on the surface of different S. mutans strains. However, none of the formulations were capable to activate mucosal responses (S-IgA). Nevertheless, we observed that the LTK63 enhanced the serum IgG responses and qualitatively improved the induced antibody response. Thus, the administration of the formulations in the presence of the adjuvant represents a promising alternative for the control of S. mutans.

No description available.

No description available.