Dissertations / Theses on the topic 'Bacillus genera'

Дисертація присвячена науково-технічним основам технології ферментації сільськогосподарських відходів з отриманням водню. В дисертаційній роботі було виділено ефективну асоціацію мікроорганізмів-деструкторів целюлози та продуцентів водню. За основу взято природну асоціацію мікроорганізмів з ґрунту, що зменшує вміст в асоціації консументів водню – сульфатредукуючих мікроорганізмів, в порівнянні з природними асоціаціями з мулу та проточних водойм. В роботі експериментально визначено, що для знешкодження метаноутворюючих мікроорганізмів необхідно проводити температурну обробку інокуляту при t = 90ºС протягом 1 години. За даної обробки мікроорганізми родів Clostridium та Bacillus утворюють спори, що проростають вже за 2 дні за сприятливих умов. Визначено, що додаткове збагачення асоціації мікроорганізмами родів Clostridiuт та Bacillus призводить до збільшення виходу водню в порівнянні з вихідною асоціацією. При чому вміст водню в біогазі залежить від кількості та співвідношення мікроорганізмів, що додаються. Встановлено, що збагачення асоціації культурами мікроорганізмів родів Clostridium та Bacillus у співвідношенні 1:2,5 дає можливість збільшити вихід водню в 2,3 рази. Визначено раціональний метод попередньої обробки сировини (3 год, 20% NaOH) що дозволяє підвищити вихід водню у 3 рази. Луг ефективно видаляє лігнін з біомаси, покращує доступність целюлози та збільшує площу адсорбція субстрату для мікроорганізмів. В дисертаційній роботі наведено математичний опис продукування біогазу залежно від значення рН та концентрації субстрату, що дозволяє моделювати довільний характер процесу і визначати оптимальні умови продукування водню в залежності від змінних параметрів процесу. Визначено основні технологічні параметри процесу ферментації целюлозовмісної сировини з продукуванням водню: температура процесу – 35ºC, рН - 7 – 7,5, концентрація сировини - 50 ± 5 г/дм3, співвідношення інокуляту до субстрату 1:4, постійне відведення водню з зони ферментації, що дозволяє одержати біогаз з вмістом водню 87,5±4,2%.

Bacterial endospores are 10 to 20 times more resistant to ultraviolet radiation than their vegetative counterparts, due to two interlocking mechanisms: the DNA photochemistry in spores, and the presence of two DNA repair systems. Spore DNA is closely associated with small acid soluble spore proteins (SASP) which change the conformation of DNA from the B form to an A-like form. When spores are subjected to UV radiation, SASP-bound DNA accumulates the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, informally referred to as spore photoproduct (SP). Spores possess two DNA repair pathways that repair SP, the general nucleotide excision repair (NER) pathway encoded by the uvr genes and the SP-specific SP lyase repair system encoded by the splB gene. Most of the information regarding spore UV resistance has traditionally been obtained using commercial UV lamps that emit predominantly 254-nm UV (UV-C). In contrast, solar UV radiation that reaches the Earth's surface spans 290 to 400-nm wavelengths, the so-called UV-B and UV-A portions of the UV spectrum, whereas the UV-C portion of solar UV is mainly filtered by the stratospheric ozone layer. Ten percent of bacterial spore dry mass consists of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA). DPA has been implicated in triggering germination in germination-deficient mutant B. subtilis spores. DPA has also been shown to photosensitize spore DNA to UV radiation. In this dissertation the SP lyase repair system, spore DNA damage cause by environmental UV, and the role of DPA in the survival of spores to UV radiation were investigated. SplB protein containing an N-terminal 10-Histidine tag [(10His) SplB] was over-expressed and purified from Escherichia coli. The purified (10 His) SplB was used for characterizing the binding of the enzyme to its substrate, SP. A 35-bp oligonucleotide (oligo) was constructed with a single pair of adjacent thymidines on one strand. The oligo was irradiated with 254-nm UV under conditions to produce either SP or cyclobutane pyrimidine dimers (Py<>Py). By DNase I protection, (10His) SplB was shown to bind specifically to the SP-containing oligo and not to the oligo containing Py<>Py or the unirradiated oligo. (10His) SplB bound to the oligo containing SP exhibited a 9-bp DNase I footprint with two hypersensitive sites within the footprint. Bacillus subtilis spores were exposed to UV-C, UV-B, solar UV-A and full spectrum sunlight. Chromosomal DNA was then extracted and probed for the presence of damage using a combination of enzymatic and electrophoretic treatments. Spores were shown to accumulate Py<>Py, single stranded breaks and double stranded breaks in addition to SP. No apurinic/apyrimidinic sites were detected under any irradiation conditions used. Mutant spores that make DPA (DPA⁻) or that were DPA-deficient (DPA⁻) were exposed to UV-C, UV-B, solar UV-A, and full spectrum sunlight as a dried-film or in suspension. When irradiated as a dried-film DPA⁻ spores were the most sensitive followed by the DPA⁻ spores and wild-type spores under all irradiation conditions except for solar UV-A where the DPA⁻ spores were the most resistant. On the other hand, DPA⁻ spores irradiated with UV-C in suspension were 8 times more resistant in comparison to the same spore irradiated as a dried-film.

Two selective media which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form were developed. Using these media it was conclusively proved for the first time that B. thuringiensis can reside on the phylloplane in a metabolically active form. This was corroborated by evidence, also for the first time, that conjugation can take place on the phylloplane between such endemic strains. A new bacteriophage, infecting one of the endemic stains, which was activated by the process of genetic recombination, was isolated and characterized. The appearance of naturally occurring strains of B. thuringiensis in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. These could not be correlated with weather conditions. A genetically stable population of strains seemed to be maintained throughout the growing season. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers, a considerably diversity of toxin types was found with the majority of isolates possessing multiple d-endotoxin genes. Bioassays against a lepidopteran insect (Pieris brassicae) of purified d-endotoxins showed that they were not more potent than those from a laboratory-adapted strain. A high percentage, however, of the endemic isolates (82%) possessed the ‘Vegetative insecticidal protein’ (Vip) gene, vip3. This might indicate an involvement of Vips in the establishment of these strains on the phylloplane. A mechanism for colonization of annual plants by B. thuringiensis was demonstrated for the first time. It was shown that spores added to seeds, even in non-sterile soil, can germinate and replicate on the resulting seedlings. The level of colonization achieved did not have a consistent influence on the feeding behaviour of third instar larvae of P. brassicae which were present on the plants for three days. Nevertheless, the fact that the number of CFU of B. thuringiensis recovered per gram of insect increased with time is evidence of proliferation of the bacterium inside the insects. Four isolates of B. thuringiensis that had been recovered in the vegetative phase from the phylloplane of T. hybridum were grown for 500 generations in a rich medium. Changes were observed in all of the strains but one isolate changed remarkably in all of the characteristics assessed which included: structure; plasmid profile; fatty acid composition; and d-endotoxin production. Moreover, the sequence of the Vip3 protein harboured by the evolved strain showed changes when compared with that of the parental strain.

Thesis (Ph. D.)--Georgia State University, 2008.
Title from title page (Digital Archive@GSU, viewed June 24, 2010) Sidney Crow Jr., committee chair; Kuk-Jeong Chen, Robert Simmons, George Pierce, committee members. Includes bibliographical references (p. 66-68).

La mosca mediterránea de la fruta, Ceratitis capitata, es la principal plaga de la fruticultura en el mundo. El desarrollo de métodos ambientalmente seguros de control de plagas, como Bacillus thuringiensis (Bt), adquiere cada vez mayor importancia. Esta tesis doctoral evalúa la validez de Bt y sus delta-endotoxinas como agentes de control de C. capitata. El análisis de la biodiversidad de Bt ha reflejado la gran riqueza presente en el agroecosistema de los cítricos. Sin embargo, ninguna de las cepas ensayadas (905) muestra alta toxicidad sobre C. capitata cuando esporas/cristales o sobrenadantes de su cultivo son ensayados. En cambio, la solubilización de los cristales de una selección de 42 cepas de Bacillus sp. ha demostrado que, para las cepas de la subespecie israelensis (Bti), este tratamiento produce una ganancia de función biológica. Adicionalmente, la predigestión de dichas protoxinas con proteasas de otro díptero, Culex pipiens, aumenta su actividad larvicida (CL50 31.26 µg/cm2). Por otro lado, se ha demostrado que, entre las delta-endotoxinas producidas por Bti, la protoxina Cyt1Aa es el factor determinante, con una CL50 sobre larvas de 4.93 µg/cm2. Sobre adultos, Cyt1Aa produce efectos subletales. Complementariamente, esta tesis propone un nuevo método de control basado en el desarrollo de toxinas recombinantes de fusión Cry-anticuerpo. Se ha puesto en práctica un sistema modelo para evaluar esta estrategia: se han desarrollado 4 variantes proteicas por la fusión entre partes de la protoxina Cry1Ab y un anticuerpo específico contra GFP (VHH anti-GFP) y éstas se han ensayado sobre larvas transgénicas de Drosophila melanogaster que expresan GFP en su intestino. Deficiencias en la unión de las toxinas de fusión a GFP, han impedido demostrar, por el momento, la estrategia propuesta. Por último, se han detectado al menos 160 proteínas distintas en las membranas intestinales de C. capitata y se han identificado las siguientes: V-ATPasa subunidades A y B, y alfa-tubulina.
Vidal Quist, JC. (2010). Estrategias para la utilización de la bacteria entomopatógena Bacillus thuringiensis (Berliner) en el control de Ceratitis capitata (Wiedemann) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8336
Palancia

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...

The heme-based aerotaxis transducer (HemAT) from B. subtilis is a heme-containing protein and functions as an oxygen sensor. It can detect oxygen and transmit the signal generated from oxygen binding to regulatory proteins through its putative methyl-accepting chemotactic domain. Through other components, the signaling information is transferred to motor proteins, which control the direction of rotation of flagella and in turn lead to changes in the swimming behavior of bacteria. There is a great deal of information known about chemotaxis signaling transduction for Escherichia coli and Salmonella typhimurium. However, the detailed molecular mechanism of chemotaxis of Bacillus subtilis is in a sense reversed, because attractant binding to chemotactic receptors strengthens the activity of the downstream histidine kinase, instead of inhibiting reaction in Escherichia coli and Salmonella typhimurium. Multiple-wavelength anomalous dispersion (MAD) data were collected from crystals of HemAT using the intrinsic anomalous scatterer, iron, with synchrotron radiation. Three wavelength iron MAD data were collected to 2.8A resolution. The native data set was collected to 2.15A resolution. The crystallographic analysis reveals that the crystal belongs to P21212 1 space group with the cell dimension a = 50.00A, b = 80.12A, c = 85.95A. There are two molecules in one asymmetric unit with 40% solvent content. I have determined the crystal structures of the HemAT sensor domain in liganded and unliganded forms at resolutions of 2.15A and 2.7A. The structures show that the HemAT sensor domain is a dimeric protein with one heme group in each subunit. The structure of liganded form of HemAT sensor domain reveals a more symmetrical organization than that of the unliganded form. Tyrosine70 in one subunit shows distinct conformations in the liganded and unliganded structures. Our study suggests that disruption of HemAT symmetry plays an important role in initiating the chemotaxis signaling transduction pathway. Our kinetic and thermodynamic studies of ligand binding suggest that HemAT may employ negative cooperativity for detecting external ligand in the signal transduction. The sensor domain provides the structural evidence for such a molecular mechanism.

ROLE OF THE YXKO GENE OF BACILLUS SUBTILIS IN RESPONCE TO ENVIRONMENTAL STRESS Abstract Mutation of the yxkO gene, which encodes a putative ribokinase and belongs to the σB general stress response regulon, leads to reduced salt tolerance under potassium limitation in Bacillus subtilis. The biological function of the yxkO gene has not been determined yet, but it may be involved in the high affinity potassium uptake system, which has been described in Escherichia coli in contrast to Bacillus subtilis. Our goal was to describe another features of a mutant in the yxkO gene and to try to propose the role of this gene. Using the integration vector pMutin4, we prepared a Bacillus subtilis strain MP2 with a yxkO gene inactivation. The MP2 strain displays limited growth in a rich medium and it is a sensitive strain to tetracycline. Furthermore, this strain is unable to form endospores and the cells are longer, which indicates a septum formation defect. We accomplished a 2-D protein gel analysis to compare expression profiles of the MP2 strain and the 1A680 standard strain after salt and ethanol stress. The MP2 strain shows changes in productions of some energy metabolism enzymes and flagellin protein. We conclude that yxkO is a regulatory gene, whose product has a pleiotropic effect on many of cell functions.

In a bacterium's environment, life conditions are subject to constant changes. One of the proposed mechanisms of adaptation to these changes is the increase in mutation rate. Bacterial mutability is generally kept very low by action of various mechanisms of control and repair, one of the most important ones being the Mismatch Repair, which is the master regulator of genetic stability of organisms. When its function is impaired, larger amounts of mutations occur in cells. In adverse conditions, these might be beneficial for cells' adaptation. The role of these repair mechanisms in adaptive processes in Bacillus subtilis has not yet been definitely resolved. The previous work in our lab focused on establishing an experimental system to measure the extent of mutagenesis in B. subtilis, and the influence of several stresses on mutation rate was assessed. No significant increase in mutability was found to be triggered by nutrient limitation in stationary growth phase, hyperosmotic stress or increased cultivation temperature. Furthermore, a system to monitor the expression of mismatch repair proteins was constructed, which has not revealed significant differences between stressed and nonstressed growth conditions. This thesis follows the results of previous experiments, expanding the range of stresses...

Cry6Aa1, une nouvelle toxine produite par Bacillus thuringiensis (Bt), agit comme insecticide sur la chrysomèle du maïs (WCRW). Dans cette étude, on démontre que Cry6Aa1 est une toxine formeuse de pores (TFP) en bicouches lipidiques planes (BLP). Contrairement aux autres toxines de Bt étudiées jusqu’à présent, la formation de pores par Cry6Aa1 ne requiert pas de prétraitement par protéases et se produit à des doses de toxine deux à trois ordres de grandeur plus faibles que celles nécessaires pour les autres toxines de Bt dans les mêmes conditions. La formation de pores par la forme non traitée de Cry6Aa1 dépend du pH; les pores obtenus ont des conductances comprises entre 31 et 689 pS en conditions symétriques de 150 mM de KCl; ils sont cationiques avec un comportement cinétique complexe. Les propriétés biophysiques des pores ne changent pas lorsque la toxine est traitée avec le suc du mésenthéron de l’insecte (Cry6Aa1 WCR1). Par contre, un traitement à la trypsine (Cry6Aa1 TT) modifie la conductance et la sélectivité des pores à pH 5,5 (le pH physiologique de l’intestin de WCRW). La reconstitution en BLP de fraction de membrane native du mésenthéron de WCRW affecte les propriétés des pores formés par Cy6Aa1. Les déterminants moléculaires du mode d’action de cette nouvelle toxine formeuse de pores semblent donc différer de ceux décrits précédemment pour d’autres toxines de Bt. La structure atomique tridimensionnelle de Cry6Aa1 vient tout juste d’être élucidée. Elle montre que la toxine adopte une conformation riche en hélices α qui ressemble fortement à celle de la TFP ClyA produite par E. coli. En se fondant sur les données disponibles pour ClyA, on a étudié l’effet de divers changements dans les régions N et C terminales de Cry6Aa1 sur sa capacité de former des pores en BLP.
Cry6Aa1 is a new toxin produced by Bacillus thuringiensis (Bt), which displays insecticidal activity against the Western corn rootworm (WCRW). The present work demonstrates that Cry6Aa1 is a pore-forming toxin (PFT) in planar lipid bilayers (PLBs). Contrary to other Bt toxins tested so far, pore formation by Cry6Aa1 does not require protease pretreatment and takes place at doses that are two to three orders of magnitude lower than those required for other Bt toxins under similar conditions. Pore formation by Cry6Aa1 is pH-dependent; the conductances of the pores range between 31 and 689 pS under symmetrical 150 mM KCl conditions; they are cationic and display a complex kinetic behaviour. The treatment of the toxin with midgut juice (Cry6Aa1 WCR1) does not change the biophysical properties of the pores. However, the treatment with trypsin (Cry6Aa1 TT) affects their conductance and selectivity at pH 5.5 (the WCRW gut physiological pH). The incorporation in PLBs of native membrane material from WCRW midgut affects the behaviour of the Cry6Aa1 pores. The molecular determinants of the mode of action of this new PFT appear therefore to differ from those reported before for other Bt toxins. The three-dimensional (3-D) atomic structure of Cry6Aa1 has just been elucidated. It shows that the toxin assumes an α-helix-rich configuration, which is quite similar to that of the ClyA PFT produced by E. coli. Based on the data available for ClyA, we have studied how different changes in the N- and C-terminal regions of Cry6Aa1 affect its pore formation ability in PLBs.

Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (prtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (lgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and lgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the lgt190-685 mutant did still exhibit signs of disease. In contrast, only the prtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the lgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

La plupart des toxines de Bacillus thuringiensis perméabilisent la membrane intestinale des insectes sensibles en formant des pores qui abolissent le potentiel électrique et les gradients ioniques. Plusieurs toxines ont été étudiées avec des vésicules purifiées de la bordure en brosse intestinale des insectes. Malheureusement, la membrane intestinale de beaucoup d’insectes ne forme pas des vésicules suffisamment étanches pour les expériences de perméabilisation. Une nouvelle technique utilisant des liposomes géants et une sonde de perméabilité membranaire a été développée pour caractériser deux nouvelles toxines particulièrement prometteuses pour le biocontrôle d’un des principaux ravageurs du maïs, la chrysomèle des racines du maïs (Diabrotica virgifera virgifera LeConte), Cry6Aa1 et la toxine binaire DS10/DS11. Les deux toxines perméabilisent efficacement les liposomes. La toxine binaire forme des pores qui sont légèrement sélectifs pour les cations, comme la plupart des toxines de B. thuringiensis. Bien que la Cry6Aa1 puisse former des pores sélectifs pour les anions, les résultats suggèrent aussi qu’elle pourrait, contrairement aux autres toxines de cette bactérie, ne former des pores qu’en présence d’une force ionique élevée. La formation des pores par ces deux toxines semble être sensible à la courbure de la membrane cible étant donné qu’elle est beaucoup plus efficace dans des liposomes géants que dans des liposomes de même composition, mais plus petits. Ce travail jette les bases de la mise au point d’une technique qui permettrait l’étude des toxines dans des liposomes géants enrichis avec des protéines et des lipides provenant de la membrane intestinale des insectes cibles.
Most Bacillus thuringiensis toxins permeabilize the intestinal membrane of susceptible insects by forming pores that abolish transmembrane electrical potentials and ionic gradients. Several toxins have been studied using brush border membrane vesicles purified from the insect midgut. Unfortunately, the intestinal membrane from many insects does not form vesicles that are tight enough to be used in permeabilisation experiments. A new technique using giant liposomes and a membrane permeability probe was developed to evaluate the pore-forming ability of two particularly promising toxins for the biocontrol of a major corn pest, the Western corn rootworm (Diabrotica virgifera virgifera LeConte), Cry6Aa1 and the binary toxin DS10/DS11. Both toxins permeabilized the liposomes efficiently. However, analysis of the permeabilisation rates under different experimental conditions indicates that these toxins differ in their biophysical properties. The binary toxin forms pores which are slightly selective for cations, like most B. thuringiensis toxins. On the other hand, although the results suggest that Cry6Aa1 could form anion-selective pores, they could also indicate that, in contrast with other toxins produced by this bacterium, it could form pores only under high ionic strength conditions. Pore formation by both toxins appears to be sensitive to membrane curvature since it is much more efficient in giant liposomes than in liposomes with identical composition, but smaller in size. This study sets the bases for the development of a technique that would allow the toxins to be studied in giant liposomes enriched with proteins and lipids from the intestinal membrane of target insects.

Les toxines Cry sont des protéines synthétisées sous forme de cristaux par la bactérie bacille de Thuringe pendant la sporulation. Elles sont largement utilisées comme agents de lutte biologique, car elles sont toxiques envers plusieurs espèces d’invertébrées, y compris les nématodes. Les toxines Cry5B sont actives contre certaines espèces de nématodes parasites, y compris Ankylostoma ceylanicum un parasite qui infeste le système gastro-intestinal des humains. Jusqu’au présent, le mode d’action des toxines Cry nématicides reste grandement inconnu, sauf que leurs récepteurs spécifiques sont des glycolipides et qu’elles causent des dommages importants aux cellules intestinales. Dans cette étude, on démontre pour la première fois que la toxine nématicide Cry5Ba, membre de la famille des toxines à trois domaines et produite par la bactérie bacille de Thuringe, forme des pores dans les bicouches lipidiques planes en absence de récepteurs. Les pores formés par cette toxine sont de sélectivité cationique, à pH acide ou alcalin. Les conductances des pores formés sous conditions symétriques de 150 mM de KCl varient entre 17 et 330 pS, à pH 6.0 et 9.0. Les niveaux des conductances les plus fréquemment observés diffèrent les uns des autres par environ 17 à 18 pS, ce qui est compatible avec l’existence d’arrangement d’un nombre différent de pores élémentaires similaires, activés de façon synchronisée, ou avec la présence d’oligomères de tailles variables et de différents diamètres de pores.
Cry toxins are proteins synthetized as crystal inclusions by the Bacillus thuringiensis bacterium upon sporulation. They are used widely as biological control agents, as they exhibit toxicity to a range of invertebrates, including nematodes. The Cry5B toxins are active against a number of parasitic nematode species, such as Ancylostoma ceylanicum a human gastro-intestinal parasite. So far, the mode of action of nematicidal Cry toxins is largely unknown, except for the facts that their specific receptors are glycolipids and that they cause prominent damage to nematode intestinal cells. In this study, we show for the first time that the nematicidal Cry5Ba toxin, a member of the three domain family of toxins produced by the Bacillus thuringiensis forms pores in receptor-free planar lipid bilayers. The pores formed by the toxin were cation selective, both under acid and alkaline pH conditions. Under symmetrical 150 mM KCl conditions, pore activity was characterized by conductances ranging from 17 to 330 pS, at both pH 6.0 and 9.0. The most frequently observed conductance levels differed from each other by approximately 17 to 18 pS consistent with the existence of clusters of different number of elementary, similar, co-operatively gated pores, or with the presence of variable size oligomers with different pore diameters.

碩士
國立中興大學
食品暨應用生物科技學系所
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Part I: Recently, the minimally-processed food products have been gradually popular in Taiwan. Fresh cut products can be purchased easily from retailers, convenience stores, hypermarkets, supermarkets, or instant restaurants. The characteristics of fresh-cut products are natural nutrition, handiness, savoriness and freshness. However, during processing, the minimally-processed fruits and vegetables are easily contaminated by bacteria. Therefore, investigating the amount of microbes especially pathogens in RTE food products becomes necessary. The study sampled a total of 165 minimally-processed fruits or vegetables from 20 companies to investigate their total plate count (TBC), coliform, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella spp., Clostridium perfringens, Escherichia coli, and Bacillus cereus. In this study, the most contaminated issue in RTE products were coliform since 40% of samples are unqualified. Moreover, there were 7 samples including papaya (Company D), guava (Company H), beef tomato (Company L), lettuce (Company M), and cabbage (Company S) which were contaminated by Salmonella spp. In addition, there was one sample, rape (Company T) which was contaminated by L. monocytogenes. Conclusively, the survey of this study shows that the companies need to improve their sanitary quality or the process of RTE and RTC products. Part II The liposomal nanovesicles and silica nanoparticles were used as fluorescent reporters for the detection of Bacillus cereus ces gene in a sandwich-type hybridization assay and their assay performance was compared in order to develop a sensitive assay with a lower detection limit. Oligonucleotide capturer pobes was conjugated to the surface of magnetic beads through an avidin–biotin linkage. The reporter DNA-tagged liposomes encapsulating SRB or silica nanoparticles were used as the label reagent to hybridize to DNA-tagged magnetic beads-ces sequence complex. Through a sandwich-type DNA hybridization procedure, the target DNA was captured and quantified by the fluorescent signals from liposomal nanovesicles or silica nanoparticles. The results of this study show that liposomal nanovesicle was a better detection reagent than silica nanoparticles and the assay with liposomal nanovesicles could have a dynamic range as 3.3-100 nM of target DNA with a detection limit of around 0.9 nM. Hence, this study has developed a probe tagged fluorescent nanovesicle method to detect B. cereus ces gene.

Une fois ingérées par un insecte sensible, les toxines insecticides du bacille de Thuringe doivent être activées par les protéases intestinales de cet insecte. Leur premier domaine, un ensemble de sept hélices-α amphipathiques, est responsable de leur insertion dans la membrane luminale de certaines cellules de l’intestin médian, ce qui crée des pores peu sélectifs. La toxicité et la capacité à former des pores d’une telle toxine, la Cry9Ca, de ses mutants simples R164A et R164K et d’un fragment de 55 kDa résultant d’un clivage protéolytique au niveau de son résidu 164 ont été étudiées à l’aide d’une combinaison de modélisation par homologie, de bioessais, d’expériences de gonflement osmotique avec des vésicules de membrane en bordure en brosse de larves de sphinx du tabac et de mesures électrophysiologiques sur des intestins isolés. Ni les mutations simples ni le clivage protéolytique n’ont altéré la toxicité de la Cry9Ca. Dans une solution à faible force ionique, toutefois, la formation des pores dépend fortement du pH : une augmentation de celui-ci de 6,5 à 10,5 a entraîné une baisse irrégulière et par étapes successives de la perméabilité membranaire. Les quatre préparations de toxine ont néanmoins dépolarisé la membrane apicale d’intestins médians fraîchement isolés baignant dans une solution contenant 122 mM de KCl à pH 10,5. L’activité de la Cry9Ca, et des mutants R164A et R164K, a été grandement stimulée lorsque les expériences ont été effectuées en présence de suc intestinal, de lipides extraits d’un volume équivalent de suc intestinal ou d’un cocktail d’inhibiteurs de protéases solubles dans l’eau. De plus, le rôle des boucles inter-hélicales du Domaine I lors de l’insertion dans la membrane a été étudié avec des mutants doubles de la Cry9Ca dont les mutations introduisaient, neutralisaient ou renversaient une charge électrique. À l’exception de trois d’entres eux, tous ces mutants ont conservé une toxicité et une capacité à former des pores comparables à celles de la toxine parentale. L’ensemble de ces résultats suggère que le micro-environnement de l’intestin médian contribue à minimiser l’influence des charges de surface portées par les résidus des boucles inter-hélicales du Domaine I sur la capacité des toxines du bacille de Thuringe à former des pores. Il indique aussi que, d’une part, selon le site de clivage et les conditions expérimentales utilisées, des protéolyses supplémentaires de la toxine Cry9Ca activée peuvent soit stimuler, soit nuire à son activité et que, d’autre part, le suc intestinal du sphinx du tabac contient probablement un inhibiteur de protéases qui pourrait jouer un rôle important dans l’activité des toxines du bacille de Thuringe.
Once ingested by susceptible insects, Bacillus thuringiensis insecticidal toxins must be activated by the insect’s intestinal proteases. Their first domain, a bundle of seven amphipathic -helices, is responsible for their insertion into the luminal membrane of midgut cells, thereby creating poorly selective pores. The toxicity and pore-forming ability of one such toxin, Cry9Ca, its single-site mutants, R164A and R164K, and of the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using a combination of homology modeling, bioassays, osmotic swelling experiments with Manduca sexta larval midgut brush border membrane vesicles and electrophysiological measurements on isolated midguts. Neither the single mutations nor the proteolytic cleavage altered Cry9Ca toxicity. In low ionic strength solutions however, pore formation was highly dependent on pH: increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization. All four toxin preparations nevertheless depolarized the apical membrane of freshly isolated midguts bathing in a solution containing 122 mM KCl at pH 10.5. The activity of Cry9Ca, R164A and R164K was greatly enhanced when the experiments were conducted in the presence of midgut juice, the lipids extracted from an equivalent volume of midgut juice or a cocktail of water-soluble protease inhibitors. Additionally, the role of the interhelical loops of Domain I in membrane insertion was investigated with Cry9Ca double mutants with mutations that either introduced, neutralized or reversed an electrical charge. All but three mutants retained a toxicity and a pore-forming ability that were comparable to those of their parental toxin. Overall, the results suggest that the midgut microenvironment contributes to minimizing the influence of surface charges carried by Domain I interhelical loop residues on B. thuringiensis toxins pore-forming ability. They also indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.