Bacillus popilliae and B. lentimorbus, causative agents of milky disease in Japanese beetles and related scarab larvae, have been differentiated based upon a small number of phenotypic characteristics, but they have not previously been examined at the molecular level. Thirty-four isolates of these bacteria were examined for DNA similarity. Three distinct but related similarity groups were identified; the first contained strains of B. popilliae, the second contained strains of B. lentimorbus, and the third contained two strains distinct from but related to B. popilliae. Some strains received as B. popilliae were found to be most closely related to B. lentimorbus and some received as B. lentimorbus were found to be most closely related to B. popilliae." Geographically distinct strains of B. popilliae and B. lentimorbus were analyzed using RAPD. Eight decamer primers were tested against nineteen new and seventeen isolates previously described by randomly amplified polymorphic DNA (RAPD) analysis (M. Tran). Of the new isolates, ten were found to be B. popilliae while nine isolates were more related to the B. lentimorbus species. Paraspore formation, believed to be a characteristic unique to B. popilliae, was found to occur among a subgroup of B. lentimorbus strains. Using a combination of two PCR primer pairs, the cry18Aa1 gene was detected in 31 of 35 B. popilliae isolates and in 1 of 18 B. lentimorbus isolates. When hemolymph smears were examined microscopically, a parasporal crystal was seen in three of the four B. popilliae strains where the PCR primers could not amplify the paraspore gene. The fourth strain was not tested due to the unavailability of infected hemolymph. A paraspore was also detected by microscopic examination in a subgroup of 14 B. lentimorbus strains. In combination, the primer pairs CryBp1 and CryBp2 are effective at detecting the paraspore gene in B. popilliae isolates, but not in the B. lentimorbus isolates. Growth in media supplemented with 2% NaCl was found to be less reliable in distinguishing the species than was vancomycin resistance, the latter present only in B. popilliae. The basis for vancomycin resistance in all isolates was investigated using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci. An amplicon was identified and sequenced. The amplified portion of the putative ligase gene in B. popilliae had 77% and 68-69% nucleotide identity to the sequences of the vanA gene and the vanB genes, respectively. There was 75% and 69-70% identity between the deduced amino acid sequence of the putative ligase gene in B. popilliae and the deduced amino acid sequence of the vanA gene and the vanB genes, respectively. It has been determined that the vanE gene is located either on a plasmid greater than 16 kb in size or on the chromosome. The gene in B. popilliae may have had an ancestral gene in common with vancomycin resistance genes in enterococci. Bacillus sphaericus strains isolated on the basis of pathogenicity for mosquito larvae and strains isolated on the basis of a reaction with a B. sphaericus DNA homology group IIA 16S rRNA probe were analyzed for DNA similarity. All of the pathogens belonged to homology group IIA, but this group also contained nonpathogens. It appears inappropriate to designate this homology group a species based solely upon pathogenicity. / Ph. D.

Media for the selective recovery of the mosquito pathogens Bacillus sphaericus 1593 and B. thuringiensis serovar. israelensis were developed. Streptomycin at 100 µg/ml and 500 µg/ml in NY agar (nutrient agar with 0.05% yeast extract) successfully selected for B. sphaericus, and allowed counts equivalent to those obtained on the nutrient medium NY without antibiotics. The medium containing 100 µg/ml of streptomycin (NYST) was used to recover B. sphaericus 1593 from a mixed microbial population in pond water. Sodium chloride, penicillin G, and pH adjustment of the medium were found to be unsatisfactory selective agents. Two selective media for the recovery of B. thuringiensis serovar. israelensis gave counts equivalent to those obtained on the nonselective NY medium. One medium contained 100 µg/ml of polymyxin with 1.0 µg/ml chloramphenicol (NYPC), and the other contained 500 µg/ml of polymyxin alone. The use of the higher level of polymyxin with chloramphenicol reduced the number of viable B. thuringiensis serovar. israelensis. NYPC was used to recover B. thuringiensis serovar. israelensis from a mixed microbial population in pond water. The selective media reduced the number of pond water microorganisms on plates by 90 to 99%. A heat treatment of 50ºC for 10 minutes also reduced pond water microbiota by approximately one log. The use of heat treatment plus either NYST or NYPC reduced the pond water microbiota further. The heat treatment had little effect on sediment microbiota. A selective-differential medium for B. thuringiensis serovar. israelensis was developed for use when heat treatment of samples would be undesirable. This medium, PEMBAC, permitted the observation of peptone deamination and hydrolysis of lecithin, which are characteristic of B. thuringiensis serovar. israelensis. The medium contained 50 µg/ml of polymyxin and 1.0 µg/ml chloramphenicol as selective agents. The parasporal crystals of B. thuringiensis serovar. israelensis are the site of the mosquito larval toxin. Because the crystals are not viable, another method for their enumeration was examined. Antisera to whole crystals and to solubilized crystal antigens were prepared in rabbits for use in the indirect fluorescent antibody technique. Because of the small size and irregular shape of the parasporal crystals of B. thuringiensis serovar. israelensis, the crystals were difficult to distinguish from other small fluorescing particles. The antisera prepared precipitated several antigens in solubilized crystals, but did not adsorb to the majority of the antigens in whole crystals. / Master of Science

<p>To make bacterial-based self-healing concrete, alkaline-resistant bacterial spores, nutrient sources, and a calcium source are incorporated into a concrete matrix. Two ureolytic spore-forming bacteria, <i>Sporosarcina pasteurii</i>, <i>Lysinibacillus sphaericus</i>, and two non-ureolytic spore-forming bacteria, <i>Bacillus cohnii</i>, and <i>Bacillus pseudofirmus</i>, which have been used in previous studies as bacterial concrete healing agents, were compared in this study. The four bacteria were compared for their (1) sporulation rates on different sporulation agar plates, (2) growth in five liquid media, (3) survival rates in light weight aggregates (LWA) and in mortar samples, and (4) calcium carbonate precipitation rates from either calcium lactate or calcium nitrate. Sporulation was successfully induced after three-day incubation at 30°C on an appropriate sporulation medium. High sporulation rates of <i>B. cohnii</i>, and <i>B. pseudofirmus</i>(93% and 99% respectively) were found on alkaline R2A medium (AR2A). A sporulation rate (89%) of <i>S. pasteruii</i>was observed on tryptic soy agar supplemented with 2% urea (TSAU)<i>.</i>The highest sporulation rate (60%) of <i>L. sphaericus</i>was found on R2A medium supplemented with 2% urea (R2AU). In the growth study, tryptic soy broth supplemented with 2% urea (TSBU) was a positive control which supported rapid growth of all four bacteria. <i>Sporosarcina pasteurii </i>and <i>L. pasteurii</i>showed rapid growth rates in alkaline yeast extract broth (AYE) and yeast extract with 2% urea broth (YEU) respectively. In contrast, <i>B. cohnii</i>, and <i>B. pseudofirmus</i>grew poorly in all media except in the positive control. Viable counts of the four bacterial spores reduced (1.8–3.3 logs) during the first 24 h in mortar samples and then remained stable for next 27 days testing period. Among the four, <i>S. pasteurii</i>showed the smallest reduction of viable counts (1.8–2.5 logs) in mortar after one day of incubation. Both <i>S. pasteurii</i>and <i>L. sphaericus</i>showed high CaCO₃productions (>80%) after 24 h incubation at 30°C in YEU containing either calcium nitrate or calcium lactate. However, <i>B. pseudofirmus</i>and <i>B. cohnii </i>showed<i></i>low calcite recovery rates (<11%) in AYE containing either<i></i>calcium nitrate or calcium lactate under the same incubation condition. Overall, <i>S. pasteurii</i>was the best bacterial concrete healing agent of the four. This bacterium had (1) rapid growth rate in AYE, (2) about 90% sporulation rate within 3 days, (3) highest survival rates after 24 h in mortar samples and, (4) high CaCO₃precipitation rates, 82 or 98%, in broth containing calcium nitrate or calcium lactate respectively.</p><p>In addition, two different lightweight aggregates (LWA), expanded shale (ES) and expanded clay (EC), which were used as bacterial carriers and protective materials, were compared in this study. Each type of LWA was separated into three sizes (<0.85 mm, 0.85– 2.0 mm, and >2.0 mm) and immobilized with spores of <i>B. cohnii</i>or <i>B. pseudofirmus.</i>Viable counts recovered from EC and ES reduced <1.0 log after the immobilization process and remained stable during the 150 days testing period. Neither the type nor the particle sizes of the two LWA significantly affected the survival rates of the bacterial spores. This result showed that both EC and ES could be used as carriers for bacterial healing agents. It was also found that when the spores were immobilized with nutrients in LWA, their survival rates in mortar samples can be improved slightly (<1.0 log).</p><p><br></p>

Bakterielle Zellhüllenproteine (S-Layer) können als formgebende Muster für die bottom-up Materialsynthese Verwendung finden. Auf S-Layern von Bacillus sphaericus und Sporosarcina ureae lassen sich nasschemisch Platin- bzw. Palladiumcluster abscheiden, die sich durch ihren gleichmäßig geringen Durchmesser und ihre hohe laterale Dichte auszeichnen. Am Beginn der vorliegenden Arbeit steht die Charakterisierung des Proteintemplates, welches grundlegenden Einfluss auf die sich bildenden Edelmetallcluster hat. Die Topographie der S-Layeroberfläche wird atomkraftmikroskopisch untersucht. Durch Photoemissions- und NEXAFS-Spektroskopie werden Aussagen zur elektronischen Struktur des Proteins gewonnen, die nach entsprechender Interpretation Erklärungen für das Verhalten des Proteintemplates liefern. Daneben sind Syntheseparameter ausschlaggebend für das Erscheinungsbild des dispersen Metalls. Insbesondere der Einfluss des Reduktionsmittels auf die Clustergröße wird elektronenmikroskopisch und durch Kleinwinkelstreuung untersucht. Die katalytische Aktivität von auf gamma-Al2O3 und SiC immobilisierten metallisierten S-Layern für die Oxidation ausgewählter Kohlenwasserstoffe und Kohlenmonoxid wird bestimmt. Außerdem werden Verfahren zur Erzeugung von Gold- und Silberclustern auf S-Layern vorgestellt.

Bakterielle Zellhüllenproteine (S-Layer) können als formgebende Muster für die bottom-up Materialsynthese Verwendung finden. Auf S-Layern von Bacillus sphaericus und Sporosarcina ureae lassen sich nasschemisch Platin- bzw. Palladiumcluster abscheiden, die sich durch ihren gleichmäßig geringen Durchmesser und ihre hohe laterale Dichte auszeichnen. Am Beginn der vorliegenden Arbeit steht die Charakterisierung des Proteintemplates, welches grundlegenden Einfluss auf die sich bildenden Edelmetallcluster hat. Die Topographie der S-Layeroberfläche wird atomkraftmikroskopisch untersucht. Durch Photoemissions- und NEXAFS-Spektroskopie werden Aussagen zur elektronischen Struktur des Proteins gewonnen, die nach entsprechender Interpretation Erklärungen für das Verhalten des Proteintemplates liefern. Daneben sind Syntheseparameter ausschlaggebend für das Erscheinungsbild des dispersen Metalls. Insbesondere der Einfluss des Reduktionsmittels auf die Clustergröße wird elektronenmikroskopisch und durch Kleinwinkelstreuung untersucht. Die katalytische Aktivität von auf gamma-Al2O3 und SiC immobilisierten metallisierten S-Layern für die Oxidation ausgewählter Kohlenwasserstoffe und Kohlenmonoxid wird bestimmt. Außerdem werden Verfahren zur Erzeugung von Gold- und Silberclustern auf S-Layern vorgestellt.

The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of the cell growth characteristics and bioassy results, TBL medium designed with 2:1 C:N ratio was chosen as the best for further steps. In addition, running time of the culture determined as 60 hours as was also determined in the previous experiment. As the last step of this study, the pre-determined optimal conditions were applied to a 30L batch type fermentor for toxin production by using Bacillus thuringiensis subsp. israelensis ONR60. Unfortunately, the toxicity was not satisfactory, being much below the level of that expected as based on the results of the laboratory scale studies.

Made available in DSpace on 2016-06-23T12:13:54Z (GMT). No. of bitstreams: 2 811.pdf: 3335097 bytes, checksum: 1e0ff860185019e7337aefd1db19cb05 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Made available in DSpace on 2016-07-05T22:00:08Z (GMT). No. of bitstreams: 3 811.pdf.txt: 201518 bytes, checksum: 5c6a06ebfc70a1c9165e7d65ca4a2ac9 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 811.pdf: 3335097 bytes, checksum: 1e0ff860185019e7337aefd1db19cb05 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / Devido a suas características epidemiológicas a filariose linfática é uma das doenças potencialmente erradicáveis. Por esse aspecto, a Organização Mundial de Saúde propôs em 1997 sua eliminação mundial prevista para 2020. O principal objetivo do Programa de eliminação da Filariose Linfática é reduzir o reservatório humano com microfilárias circulantes, para menos de 1 por cento, somado a redução para o índice 0.1 por cento nas crianças nascidas após o início do tratamento em massa, ambos diagnosticados pelas técnicas de gota espessa e pesquisa de antígeno circulante filarial (cartão ICT e Og4C3) resultando na interrupção da transmissão. Entre os objetivos do Plano de Controle e Eliminação da filariose linfática está à utilização da pesquisa de antígeno circulante como indicador da monitorização da eficácia do tratamento específico realizado nas comunidades endêmicas. Pesquisas demonstram resultados inconsistente nos padrões de clareamento da circulação sangüínea do antígeno da W.bancrofti após o tratamento, pois ainda não se pode assegurar a cura parasitológica e correlacionar esse fato concreto com a cronologia do desaparecimento do antígeno circulante filarial. O presente estudo comparou o efeito do tratamento seletivo com dietilcarbamazina (dose única e tradicional) e a intervenção cirúrgica sobre a microfilaremia e o marcador sorológico de infecção ativa (antigenemia), diagnosticados pelas técnicas parasitológicas da filtração (microfilaremia), utrason (vermes adultos filarias) e a pesquisa de antígeno circulante filarial (cartão ICT e Og4C3) frente aos 31 indivíduos de ambos os sexos, micro e amicrofilarêmicos, no período pré, 1, 6, 12, 96 e 120 meses. Observou-se uma correlação positiva (r=0,3118, P0,0001) crescente da densidade de MF/mL com o antígeno circulante filarial diagnosticado de forma quantitativa pela ferramenta diagnostica do Og4C3. O tratamento que observamos um menor número de indivíduos positivos foi através da medicação dietilcarbamazinna dose tradicional seguido de cirurgia e dose única. No presente estudo observamos que o tempo eleito pela OMS de 48 a 82 meses é insuficiente para assegurar a cura da infecção, visto quem em 96 meses ainda encontra-se vestígios de antígeno circulante. Sugere-se que exames paralelos sejam realizados para confirmar e assegurar a cura do indivíduo

Bacterial cell wall proteins (S-layer) are - due to both the capability to self-assemble into two-dimensional crystals and their distinct chemical and structural properties - suitable for the deposition of metallic particles at their surface . The cluster growth is subject of this thesis. The binding of metal complexes to S-layers of Bacillus sphaericus and Sporosarcina ureae and their subsequent reduction leads to the formation of regularly arranged platinum or palladium cluster arrays on the biomolecular template. A heterogeneous nucleation mechanism is proposed for this process consisting of the binding of metal complexes and their subsequent reduction. The kinetics of the process and the binding of the complexes to the protein are characterized by UV/VIS spectroscopy. This thesis focuses on structural investigations by means of transmission electron microscopy, electron holography, scanning force microscopy, image analysis, and image processing. Preferred cluster-deposition sites are determined by correlation averaging. A more precise determination and quantification is obtained by Multivariate Statistical Analysis. Furthermore a method for the electron beam induced formation of highly-ordered metallic cluster arrays in the transmission electron microscope and a fast screening method for surface layers of Gram-positive bacteria are presented. / Bakterielle Zellhüllenproteine (S-Layer) eignen sich durch ihre Fähigkeit zur Selbstassemblierung zu zweidimensionalen Kristallen und durch ihre besonderen chemischen und strukturellen Eigenschaften zur Abscheidung regelmäßiger metallischer Partikel auf ihrer Oberfläche. In dieser Arbeit wird das Clusterwachstum auf S-Layern untersucht. Die Anbindung von Metallkomplexen an S-Layer von Bacillus sphaericus und Sporosarcina ureae und deren Reduktion führt zur Abscheidung periodisch angeordneter metallischer Platin- bzw. Palladiumcluster auf dem Biotemplat. Für diese Clusterbildung wird ein heterogener Keimbildungsmechanismus vorgeschlagen, bestehend aus Komplexanbindung und Reduktion. Die Bestimmung der Prozeßkinetik und die Charakterisierung der Anbindung der Komplexe an das Protein erfolgt mittels UV/VIS-Spektroskopie. Den Schwerpunkt dieser Arbeit bilden strukturelle Untersuchungen mit Hilfe der Transmissionselektronenmikroskopie, der Elektronenholographie, der Rasterkraftmikroskopie und der Bildanalyse und Bildverarbeitung. Durch Korrelationsmittelung werden Strukturinformationen gewonnen, die eine Bestimmung der lateral bevorzugten Clusterpositionen ermöglichen. Für die auf S-Layern erzeugten Clusterarrays wird die Belegung der einzelnen Positionen mittels Multivariater Statistischer Analyse genauer quantifiziert. Außerdem werden eine Methode zur Erzeugung hochgeordneter metallischer Partikelarrays unter dem Einfluß des Elektronenstrahles im Transmissionselektronenmikroskop und eine Methode zum schnellen Test Gram-positiver Bakterienstämme auf die Existenz von S-Layern vorgestellt.

Bacterial cell wall proteins (S-layer) are - due to both the capability to self-assemble into two-dimensional crystals and their distinct chemical and structural properties - suitable for the deposition of metallic particles at their surface . The cluster growth is subject of this thesis. The binding of metal complexes to S-layers of Bacillus sphaericus and Sporosarcina ureae and their subsequent reduction leads to the formation of regularly arranged platinum or palladium cluster arrays on the biomolecular template. A heterogeneous nucleation mechanism is proposed for this process consisting of the binding of metal complexes and their subsequent reduction. The kinetics of the process and the binding of the complexes to the protein are characterized by UV/VIS spectroscopy. This thesis focuses on structural investigations by means of transmission electron microscopy, electron holography, scanning force microscopy, image analysis, and image processing. Preferred cluster-deposition sites are determined by correlation averaging. A more precise determination and quantification is obtained by Multivariate Statistical Analysis. Furthermore a method for the electron beam induced formation of highly-ordered metallic cluster arrays in the transmission electron microscope and a fast screening method for surface layers of Gram-positive bacteria are presented. / Bakterielle Zellhüllenproteine (S-Layer) eignen sich durch ihre Fähigkeit zur Selbstassemblierung zu zweidimensionalen Kristallen und durch ihre besonderen chemischen und strukturellen Eigenschaften zur Abscheidung regelmäßiger metallischer Partikel auf ihrer Oberfläche. In dieser Arbeit wird das Clusterwachstum auf S-Layern untersucht. Die Anbindung von Metallkomplexen an S-Layer von Bacillus sphaericus und Sporosarcina ureae und deren Reduktion führt zur Abscheidung periodisch angeordneter metallischer Platin- bzw. Palladiumcluster auf dem Biotemplat. Für diese Clusterbildung wird ein heterogener Keimbildungsmechanismus vorgeschlagen, bestehend aus Komplexanbindung und Reduktion. Die Bestimmung der Prozeßkinetik und die Charakterisierung der Anbindung der Komplexe an das Protein erfolgt mittels UV/VIS-Spektroskopie. Den Schwerpunkt dieser Arbeit bilden strukturelle Untersuchungen mit Hilfe der Transmissionselektronenmikroskopie, der Elektronenholographie, der Rasterkraftmikroskopie und der Bildanalyse und Bildverarbeitung. Durch Korrelationsmittelung werden Strukturinformationen gewonnen, die eine Bestimmung der lateral bevorzugten Clusterpositionen ermöglichen. Für die auf S-Layern erzeugten Clusterarrays wird die Belegung der einzelnen Positionen mittels Multivariater Statistischer Analyse genauer quantifiziert. Außerdem werden eine Methode zur Erzeugung hochgeordneter metallischer Partikelarrays unter dem Einfluß des Elektronenstrahles im Transmissionselektronenmikroskop und eine Methode zum schnellen Test Gram-positiver Bakterienstämme auf die Existenz von S-Layern vorgestellt.