L'enveloppe cellulaire bactérienne joue plus qu'un rôle de barrière d'échange. Elle est au coeur des processus cellulaires essentiels comme la morphogenèse et la division. Cette structure abrite environ un quart des protéines codées par le génome. Le but de mon travail a été de mieux comprendre le rôle de deux protéines membranaires dans la construction et la dynamique de l'enveloppe chez Bacillus subtilis. GluP est une protéase intramembranaire rhomboïde. Ces protéases clivent des segments transmembranaires dans la membrane afin de moduler l'activité de diverses protéines. Elles participent à de nombreux processus cellulaires chez les eucaryotes. Cependant, les fonctions biologiques des rhomboïdes procaryotes sont pour l'heure presque totalement inconnues. Nos résultats suggèrent que GluP participe au contrôle qualité des protéines membranaires à la manière des pseudo-rhomboïdes associées au système ERAD eucaryote. Elle forme un complexe avec FtsH, une protéase majeure du contrôle qualité des protéines. Ce complexe est impliqué dans la dégradation d'un substrat de rhomboïde. Le rôle de GluP serait de permettre la dislocation du segment transmembranaire et faciliter la prise en charge du substrat par FtsH. Le second projet auquel j'ai participé a consisté à comprendre le rôle de la protéine CmmB dans la morphogenèse. Son absence conduit à une morphologie cellulaire élargie. CmmB semble faire partie de la machinerie de synthèse du peptidoglycane au cours de l'élongation de la paroi. Elle serait nécessaire au bon fonctionnement d'une ou de plusieurs penicillin-binding proteins (PBPs). En particulier, nous proposons que CmmB est un cofacteur de la transpeptidase PBP2a. / The bacterial cell envelope is an obligatory barrier. It is a fundamental component in essential cellular processes such as morphogenesis and cell division. It hosts about a quarter of the proteins encoded in the genome. My work was aimed at understanding the function of two membrane proteins in the building and the dynamics of the cell envelope in the model bacterium Bacillus subtilis.GluP is a rhomboid intramembrane protease. Usually, rhomboids cleave transmembrane segments within the membrane to modulate protein functions. In eukaryotes, they participate in many cellular processes and their dysfunction lead to several pathologies. However, prokaryotic rhomboid functions remain almost totally unknown. Our results suggest that GluP is involved in bacterial membrane protein quality control, in a process akin to pseudo-rhomboid dependent endoplasmic reticulum associated protein degradation in eukaryotes. GluP forms a complex with FtsH, a major protease in protein quality control. That complex is not involved in the cleavage of a membrane substrate but in its degradation. We propose that GluP is required for the dislocation of the transmembrane segment, thus facilitating full-length substrate degradation by FtsH in the cytoplasm. My thesis second objective was to understand the role of the CmmB protein in morphogenesis. The absence of CmmB leads to slightly enlarged cells. CmmB seems to belong to the peptidoglycan synthesis machinery for cell-wall elongation. Our data support the idea that it is required for the proper activity of one or several penicillin-binding proteins (PBPs). In particular, we propose that CmmB is a cofactor of the PBP2a transpeptidase.

Made available in DSpace on 2017-06-01T18:20:28Z (GMT). No. of bitstreams: 1 dissertacao_alicia_jara.pdf: 8514825 bytes, checksum: e7811d2022af27360b50bd77f223f4ba (MD5) Previous issue date: 2007-12-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In recent years, the consumption of the poly ethylene terephtalate plastic - PET is used in the manufacture of bottles, comes increasing in Brazil. PET is a polar thermoplastic, with raised dimensional stability and temperature of fusion, high impermeability the acid gases and chemical resistance to solvents. The biodegradation has been described as a possible methodology to reduce the accumulation of plastics. In this work it was carried through the evaluation by Bacillus subtilis and Phanerochaete chrysosporium performance on the biodegradation of the polyethylene terephtalate. In this direction, particles of polymer were submitted to the treatments: exposition to ultra violet light (6 and 36 hours) and temperatures (35ºC and 50ºC), followed incubation with the microorganisms during 30 and 60 days. The polymer degradation process was accompanied by determination of pH, biofilm formation and the cells viability, enzymes detection (amylase, protease, esterase, and polyphenoloxidase), as well as the scanning electron microscopy of biofilm and toxicity tests. The results obtained observed the biofilm formation by Bacillus subtilis on polyethylene terephtalate surface particles. The treatment using the temperature of 50ºC demonstrated a higher alteration in the surface of the polymer, supported the colonization of the microorganisms followed of the hydrolytic enzymes production. It was observed that Bacillus subtilis does not produced polyphenoloxidase. The results indicated the temperature (50º.C), induces the esterase production and it is related to degradation process. The P. chrysosporium produced esterases and polyphenoloxidase, whose enzymes had demonstrated to be involved with the polyethylene terephtalate degradation process, and were formed products with higher toxicities to Artemia salina / O Tereftalato de Polietileno PET é um termoplástico polar,com elevada estabilidade dimensional e temperatura de fusão, alta impermeabilidade a gases e resistência química a ácidos e solventes, empregado na fabricação de garrafas no Brasil. A biodegradação tem sido descrita como uma possível metodologia para reduzir o acúmulo de plásticos. Neste trabalho foi avaliado o desempenho das linhagens de Bacillus subtilis e Phanerochaete chrysosporium isoladamente na biodegradação do tereftalato de polietileno. Neste sentido, foram preparadas partículas do polímero sendo submetidas aos tratamentos com luz ultra violeta (6 e 36 horas) e temperaturas (35ºC e 50ºC) em seguida, foram colocadas nos meios caldo nutriente (B.subtilis) e Sabouraud (P. chrysosporium), incubados por 30 e 60 dias, incubados a 35ºC e 28ºC, respectivamente. Com a degradação das partículas observou-se que o pH passou de 5 para >8, com formação de biofilmes e indução da produção de enzimas (amilase, protease, esterase e polifenoloxidases). A formação do biofilme foi evidenciada por microscopia eletrônica de varredura. Os produtos metabólicos formados no meio de cultura foram avaliados pelo teste de toxicidade utilizando Artemia salina. A microscopia eletrônica demonstrou que B. subtilis colonizou completamente a superfície das partículas do PET, tanto nas condições controle (sem tratamento), como tratados. Os melhores resultados foram observados com o tratamento à temperatura de 50ºC, onde ocorreu alteração na superfície do polímero, perda da massa polimérica, permitindo maior colonização de ambos os microrganismos. As enzimas hidrolíticas foram produzidas pelos microrganismos em todos os tratamentos, em especial, à temperatura de 50ºC. Contudo, observou-se que B. subtilis não produziu polifenoloxidases. Os subprodutos da degradação do PET nas condições estudadas apresentaram alta toxicidade para Artemia salina no caso do P. chrysosporium e baixa toxicidade para B. subtilis. Os resultados obtidos sugerem o tratamento o prévio com a temperatura de 50ºC como importante para o processo de biorremediação

A divisão celular nas bactérias requer a formação do divisomo, um complexo protéico que tem como o primeira etapa a polimerização da proteína FtsZ, seguida pela associação de 15 outras proteínas conhecidas. Os mecanismos envolvidos na regulação espacial do divisomo são bem caracterizados, mas o controle temporal da divisão celular em relação a outros eventos do ciclo, como a replicação do cromossomo, segue controversa. Neste trabalho, aplicamos a metodologia de microscopia de série temporal para estudar duas questões fundamentais do processo de divisão: a montagem do complexo que executa a divisão e a possibilidade da oscilação periódica na expressão de um ou mais genes envolvidos em divisão possa participar do controle temporal da montagem do divisomo. Para investigar se há oscilação da expressão gênica, construímos inicialmente variantes instáveis GFP através da adição de sequências peptídicas C-terminais que encaminham para a degradação em B. subtilis e utilizamos estes repórteres para criar fusões transcricionais sob o controle de promotores de genes centrais do processo de divisão. Depois de otimizar as condições de microscopia de série temporal com fusões transcricionais usando a variante instável GFPAISV, observamos que a autofluorescência de B. subtilis interferia nas nossas quantificações. Como forma de contornar a autofluorescência, construímos então fusões transcricionais com duas variantes de YFP (proteína fluorescente amarela) e optamos por trabalhar com Ypet-AISV. A análise de filmes de células individuais, tanto com fusões a GFPAISV como a Ypet-AISV, indicou que apenas o promotor do operon ftsL-pbpB apresentava um padrão de oscilação significativamente diferente de um promotor artificial usado como controle negativo. Esta hipótese, no entanto, não foi confirmada por medidas estáticas de populações de células nas quais correlacionamos intensidade de fluorescência com posição no ciclo celular. Portanto, nossos dados não foram capazes de evidenciar flutuações na expressão dos genes ftsL-pbpB, minCD, ftsZ, ftsA e zapA ao longo do ciclo celular. Para estudar a cinética de montagem divisomo foram realizados experimentos de microscopia de série temporal de FtsZ-mCherry e Pbp2B-GFP, onde observamos que a associação de Pbp2B ao divisomo ocorre 3 minutos após a formação do anel de FtsZ em meio rico e 4 minutos em meio mínimo. Também realizamos experimentos de microscopia de série temporal com uma cepa contendo FtsZ-YFP e DivIVA-CFP, determinando que DivIVA é incorporado ao divisomo 16 minutos após a formação do anel de FtsZ em meio rico e 20 minutos em meio mínimo. Estes dados confirmam que a montagem do divisomo ocorre em três etapas, e não duas, como anteriormente proposto. / Cell division in bacteria requires the formation of the divisome, a protein complex that has as the first step polymerization of FtsZ, followed by the assembly of 15 other known proteins. The mechanisms that underlie spatial regulation of divisome assembly have been largely elucidated, but the temporal control that ties the timing of cell division to other cell cycle events, such as chromosomal replication, remains surrounded by controversy. In this work, we use time-lapse microscopy to address two issues in B. subtilis cell division: the timing of divisome assembly, and the possibility that a periodic oscillation in expression of one or more genes essential for divisome assembly may play a role in defining the timing of cell division. To study the possibility of oscilation in gene expression, we have first built unstable variants of GFP by adding to its C-terminus peptide sequences that target the protein for degradation and used those variants to build transcriptional fusions to access the promoter activity of core cell division genes. After optimizing time-lapse conditions with transcriptional fusions to cell divison genes with the unstable GFPAISV, we observed that B. subtilis autofluorescence was an issue to our quantifications. To improve our signal-to-noise ratio, we built transcriptional fusions with two variants of YFP (Yellow Fluorescent Protein), and decided to work with Ypet. In our single-cell analysis for GFPAISV and for Ypet-AISV, only the ftsL operon promoter presented an oscilating pattern different from our negative control. This was not confirmed, however, when we attempted to correlate fluorescence signal with cell cycle position in static single-cell measurements. Thus, we conclude that that there are no fluctuations in ftsL, pbpB, minCD, ftsZ, ftsA or zapA gene expression throughout the cell cycle. To study divisome assembly we performed time-lapse microscopy of FtsZ-mCherry and Pbp2B-GFP, and determined that the association of Pbp2B occurs 3 minutes after FtsZ polymerization in rich medium and 4 minutes in minimal medium. We also performed time-lapse microscopy with FtsZ-YFP and DivIVA-CFP, determining that DivIVA is incorporated to the divisome in 16 minutes after FtsZ polymerization in rich medium and 20 minutes in minimal medium. This data confirms the assembly of the divisome in three steps rather than two, as previously proposed.

Les théories actuelles suggèrent que l'efficacité de traduction (nombre de protéines produitespar ARN messager ; notion spécifique à chaque à gène) reste constante lorsque le taux decroissance varie. Néanmoins, une efficacité de traduction constante est incompatible avec lafaible corrélation observée, à l’échelle du génome, entre l’évolution en fonction du taux decroissance de la concentration des ARN messagers et des protéines pour lesquelles ils codent.Pour faire face à ce paradoxe, nous avons développé un modèle mathématique de la traductionbasé sur les connaissances actuelles de ce processus au niveau moléculaire. L’exploration despropriétés du modèle nous a amené à conduire des expériences de transcriptomique, de PCRquantitative et de Live Cell Array (LCA) dont l’analyse a montré que l'efficacité de traductiondiminue jusqu'à 4 fois entre faible et fort taux de croissance chez la bactérie modèle Bacillussubtilis. Notre modèle a révélé que la chute de l’efficacité de traduction repose sur une chutede la concentration des ribosomes libres. Pour étudier les conséquences de la chute desribosomes libres sur la production des protéines, nous avons rationnellement défini à partir dumodèle mathématique des constructions génétiques combinant des promoteurs, naturel ousynthétique, et différentes régions d'initiation de la traduction (TIRs) contrôlant l’expressiondu gène gfp. En utilisant ces constructions génétiques, nous avons montré que la productiondes protéines est non-linéaire en fonction de la concentration en ribosomes libres. Uneproduction non-linéaire entraine des efficacités de traduction différentielles des ARNmessagers en fonction de leurs TIRs. Ce mécanisme de régulation général des protéinesparticipe à la perte de corrélation entre la concentration des ARN messagers et des protéinescorrespondantes et nous a amené à revisiter la Physiologie Moléculaire Bactérienne. / Current theories suggest that translation efficiency (i.e. number of proteins produced permRNA) remains invariant with increasing growth rate, which is inconsistent with the scanty correlation between mRNAs and cognate proteins abundances at the genome-scale level. We tackled this apparent paradox using a systems biology approach. We developed a knowledgebased, nonlinear mathematical model of translation. The in-depth analysis of the model led us to reassess experimentally, using high-throughput and genome-wide technologies, each measurable RNA entity at different growth rates. In contrast to the current knowledge, the total mRNA abundance was not constant but linearly increased with respect to the growth rate. A model-driven integration of genome-wide and molecular experimental datasets demonstrated that the drop in abundance of a constitutively expressed protein with increasing growth rate is not only due to the dilution but also to an unexpected up to 4-fold decrease of translation efficiency. Our model revealed that this drop relies on a drastic decrease in free (untranslating) ribosomes, a non-measurable entity. Using a set of 18 Bacillus subtilis strains combining 9 synthetic translation initiation regions (TIRs) and 2 constitutive promoters, we show that TIRs together with free ribosome abundance strongly contribute to a nonlinear modulation of single proteins as a function of the growth rate. The nonlinearity accounted for the loss of correlation between mRNAs and cognate proteins abundances. Altogether, our results evidenced a unique, hard-coded and global growth-rate-dependent regulation of single bacterial proteins without dedicated regulators.

Bacillus subtilis ist einer der bislang am besten untersuchten Organismen. Es gilt als Modellbakterium, an welchem stoffwechselphysiologische und genetische Phänomene grundlegend aufgeklärt wurden. Daneben gehört B. subtilis zu den generell unbedenklichen Mikroorganismen und wird von der U.S. Food and Drug Administration (FDA) als ‚GRAS‘ (Generally Regarded As Safe) eingestuft. Dementsprechend wird B. subtilis in der biotechnologischen Industrie zur Herstellung vielfältiger Produkte, z.B. in der Lebensmittelindustrie, eingesetzt. Als Gram-positives Bakterium eignet sich B. subtilis vor allem zur Produktion extrazellulärer Enzyme wie Proteasen und Amylasen. Die Untersuchung der Sekretion dieser Proteine ist sowohl für die grundlagen- als auch für die anwendungsorientierte Forschung von wichtiger Bedeutung. Vorarbeiten der Arbeitsgruppe von Prof. Wiegert haben gezeigt, dass eine Deletionsmutante im Gen der Protease RasP einen vollständigen Defekt der Sekretion einer α-Amylase verursacht. In meiner Promotionsarbeit soll untersucht werden, welche Rolle RasP bei der Sekretion dieser α-Amylase spielt, und welchen Einfluss diese Protease auf die Proteinsekretion allgemein ausübt. Zudem soll, u.a. am Beispiel von Signalpeptiden, der Mechanismus der Substraterkennung durch RasP untersucht werden. Ich erhoffe, mit dieser Arbeit einen entscheidenden Beitrag zum Verständnis der Funktion der Protease RasP zu leisten und damit eine weitere gezieltere Optimierung von B. subtilis Produktionsstämmen in der biotechnologischen Industrie zu ermöglichen.

We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...

La synthèse de protéines recombinantes chez les microorganismes est d'un intérêt majeur pour la production de produits biopharmaceutiques, thérapeutiques et enzymatiques industriels. Cependant, la surproduction de protéines a un effet néfaste sur la physiologie cellulaire. Les ressources cellulaires (métabolites, énergie, machinerie moléculaire, espace cytosolique, etc.) sont en effet partagées entre les protéines de l'hôte et la protéine "gratuite". Cette surcharge non naturelle entraîne une croissance plus lente et des rendements en protéines plus faibles, un phénomène connu sous le nom de "burden". Dans mon projet de doctorat, il s'agissait (1) de déchiffrer les conséquences de la surproduction de protéines gratuites sur la physiologie cellulaire, (2) d'identifier le type de ressources limitantes, et (3) de surmonter cette limitation pour améliorer la production de protéines. Afin de déchiffrer les conséquences de la surproduction de protéines (1), nous avons analysé le taux de croissance, la production de protéines d'intérêt et le protéome de souches de Bacillus subtilis surproduisant divers niveaux de protéines rapportrices. Les protéines rapportrices ont été choisies de manière à être facilement quantifiables par fluorescence et par des tests d'activité (i.e. GFP, mKate2, LacZ, etc.). Pour obtenir les différents niveaux d'expression, nous avons construit des séquences synthétiques par assemblage de promoteurs constitutifs et inductibles et de régions d'initiation de traduction (TIR, RBS) variés. Nous avons ainsi montré que plus la quantité (et la taille) de la protéine produite était élevée, plus les taux de croissance étaient faibles et plus la taille des cellules était élevée. Par exemple, le taux de croissance a diminué de plus de 20 % lorsque la GFP était surproduite à plus de 5 % de la quantité totale de protéines solubles, selon des quantifications biochimiques et de fluorescence. Pour identifier le type de ressources limitantes (2), nous avons effectué une quantification relative des protéines sur les souches surproductrices de GFP et montré que certaines protéines non essentielles étaient moins abondantes dans ces souches. Nous avons ensuite dégradé spécifiquement les protéines rapportrices à l'aide d'un outil de biologie de synthèse précédemment mis au point pour B. subtilis, afin que les acides aminés puissent être recyclés dans le pool de ressources cellulaires. Avec une dégradation de 50-60% de GFP et mKate2, nous avons observé une restauration de 50% du taux de croissance. Ces résultats suggèrent que la quantité d'acides aminés (et par conséquent leur utilisation dans la synthèse des protéines) est le principal type de ressources limitantes. Pour améliorer la production de protéines (3), nous avons cherché à développer un système synthétique de recyclage des acides aminés basé sur le système de dégradation mentionné ci-dessus en surproduisant les protéases d'E. coli et B. subtilis (ClpXP) avec une protéine adaptatrice (SspB) d'E. coli. Cet outil pourrait permettre de dégrader spécifiquement des protéines non essentielles pour économiser des ressources cellulaires. Nous avons montré que la surproduction de ClpXP ou de SspB/ClpXP était suffisante pour permettre une dégradation complète des protéines produites à des niveaux bas et intermédiaires, et jusqu'à 50% des protéines fortement produites. Comme ClpXP est une protéase impliquée dans la réponse au stress, nous avons cherché à savoir si la surproduction de ClpXP pouvait avoir des conséquences négatives sur la physiologie cellulaire. Une quantification relative des protéines sur une souche surproductrice de ClpXP a montré que la surproduction de ClpXP provoque une réorganisation globale du protéome sans toutefois affecter le taux de croissance de la cellule. / Recombinant protein production in microorganisms is of great interest for the production of biopharmaceuticals, therapeutics and industrial enzymes. However, recombinant protein production has always shown a harmful effect on the microorganism cell physiology when excessively produced. Cell resources (i.e. metabolites, energy, molecular machinery, cytosolic space, etc.) are used to produce the host's proteins and the overproduced gratuitous protein. As a result, this unnatural extra load typically leads to slower growth and lower protein yields, a phenomenon known as ʻburdenʼ. This burden comes from the fact that the recombinant protein has no benefit for the microorganism, and that it only uses cell resources at the expense of the production of the endogenous essential proteins. In my PhD project, the issues were (1) to decipher the consequences of gratuitous protein overproduction on the cell physiology, (2) to identify the limiting type of resources, and (3) to overcome this limitation to improve protein production. To address the first issue (1), we analyzed growth rates, production of several proteins of interest, and genome-wide proteomes of Bacillus subtilis strains overproducing various levels of reporter proteins. The reporter proteins were chosen so that they were easily quantifiable by fluorescence and β-galactosidase activity assays (i.e. GFP, mKate2, LacZ, etc.). To obtain the various levels of expression, we built synthetic sequences made of the assembly of various constitutive and inducible promoters and translation initiation regions (TIR, RBS). Hence, we showed that higher was the amount (and size) of the protein produced, lower were the rates of growth and higher were the cell sizes. For instance, the growth rate decreased down by over 20% when GFP was overproduced above 5% of the total soluble protein amount according to both biochemical and fluorescence assays. To further identify the limiting type of resources (2), we performed a relative protein quantification on the strains overproducing GFP at different levels. Hence, we showed that some non-essential proteins were less abundant in the strains overproducing GFP. We next targeted the reporter proteins for degradation using a synthetic tool previously engineered in B. subtilis, so that amino acids can be recycled back to the pool of cell resources. Degrading the reporter gratuitous protein should also relieve the constraint on the cytosolic density by liberating intracellular space. With a degradation of 50-60% of GFP and mKate2, we observed a 50% restoration of the growth rate. This result together with the proteome analysis suggested that the amount of amino acids (and consequently their utilization in protein synthesis) was the main limiting type of resources. To overcome this limitation and improve protein production (3), we aimed at exploring a synthetic, amino acid recycling system based on the above mentioned degradation system. We decided to improve the targeted degradation system by overproducing the E. coli and B. subtilis ClpXP proteases together with an E. coli adaptor protein SspB. This tool may allow to target proteins for degradation in order to save resources and improve the production of a protein of interest. We showed that the overproduction of either ClpXP or SspB/ ClpXP were sufficient to allow a complete degradation of the proteins produced low and intermediate levels, and up to 50% of degradation of the proteins highly produced. As ClpXP is a protease involved in stress responses, we aimed to know whether the overproduction of ClpXP may have negative consequences on the cell physiology. We therefore performed relative protein quantification on a strain overproducing ClpXP. The results showed that ClpXP overproduction causes a global reorganisation on the proteome without affecting the growth rate of the cell.

Okra (Abelmuschus esculentus (L) Moench.) is one of the most popular crops within the Malvaceae family of plants. It is a common vegetable eminently cultivated in regions experiencing constraints to manage climate change. In South Africa climate change coupled with aluminium-enriched soils are responsible to drawbacks crop performance. Therefore, it is worthwhile to whether okra will thrive as an alternative crop in the country. Many studies have identified potential of okra to improve yields of resource poor farmers in Africa. The physiological responses of okra seed to variations in aluminium ions and temperature were not determined. Therefore, a study with okra, cv. Clemson Spineless, seed coated and uncoated with B. subtilis, was initiated to assess germination on moist filter paper in 90mm diameter Petri plates. Germination medium consisted of various concentrations of aluminium chloride (AlCl3), 0M, 0.001M, 0.01M, 0.05M and 0.1M. Each aluminium treatment was allocated into incubators adjusted to 22°C, 25°C and 37°C temperatures. This resulted into a 5 x 3 x 2 factorial experiment with five replicates and was conducted in three cycles. Daily scores of germinated seeds were assessed from the second to the fifth day after initiation of germination. During termination, five days after the initiation of the experiment 10 seeds with the longest coleoptiles had their coleoptiles measured using a digital caliper. At the fifth day after initiation of the experiment, coleoptile lengths from 10 seeds per treatment were measured using digital caliper. A total of 50 plates (10 from 37°C in Cycle 1; 30 from 22°C, 25°C and 37°C from Cycle 2; 10 from 37°C in Cycle 3), were selected and germinated were ground and stored at - 20°C before 1H NMR analysis. Metabolites were extracted from 50mg ground seed material with 750 μL methanol-D4 and 750 μL buffer (deuterium oxide + potassium dihydrogen phosphate). The mixture was vortexed for three minutes, sonicated for 20 minutes, centrifuged at 18000 rpms for 20 minutes and the supernatant filtered through cotton wool. Then the supernatant was dispensed into NMR tubes for further 1H NMR spectroscopic processing using a 600 MHz NMR xiii Varian spectrometer to generate magnetic spectra of the fifty samples. Results of this study demonstrated that in all the experimental cycles, regardless of aluminium concentration and bacterial seed coating, 37°C inhibited germination percentages and coleoptile lengths in okra seed germination. Germination percentages and coleoptile lengths of bacteria-coated seeds growing in 25°C were most stimulated at all aluminium concentrations, but not at 0.1M. In this temperature germination percentages and coleoptile lengths were highly influenced by the interaction of aluminium concentrations and bacterial coating, respectively. 1H NMR metabolomic association showed no distinct grouping, but clusters across treatments showed to be linked through a subset of metabolites amongst aluminium concentrations, bacterial seed coating and temperatures, respectively. This infers that treatment variations in both seed and bacterial physiological responses were associated through shared metabolic pathways. In conclusion, the study proved that 25°C provide temperature environment within which B. subtilis can be able to stimulate growth and remediate physiological constraints from aluminium ions during okra seed germination. / Agriculture, Animal Health and Human Ecology / M. Sc. (Agriculture)

No description available.

No description available.