Isolation of bacillus thuringiensis and investigation of crystal protein genes

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2002Includes bibliographical references (leaves: 43-55)Text in English; Abstract: Turkish and Englishvi, 55 leavesBacillus thuringiensis is a ubiquitous, gram-positive and spore-forming bacterium. During sporulation, it produces intracellular crystal proteins (cry proteins), which are toxic to insects. Because of its insecticidal activity, it has been used for nearly fifty years to control certain insect species among the orders Lepidoptera, Coloeptera, and Diptera. However, it is still necessary to search for more toxins to control other insect orders and to provide alternatives for coping with the problem of insect resistance. The genetic diversity of B. thuringiensis strains shows differences according to the regions where they were isolated. Thus, each habitat may contain novel B. thuringiensis strains, which have some toxic effects on target spectra of insects. The aim of this study was to isolate B. thuringiensis strains from different environments and to identify the crystalline protein gene content of the isolates. Sixty five samples including soil, stored product dust, insect cadavers, and dry leaf residues were collected from Akhisar/Manisa, İzmir, and Ereğli/Konya. Three approaches were applied for the isolation of B. thuringiensis: sodium acetate selection, heat treatment, and endospore staining. Polymerase Chain Reaction (PCR) method was used for the characterization of cry gene content of B. thuringiensis strains. The universal primers specific to cry 1, cry2, cry 3, and cry 9 genes were used to detect the type of cry gene carried by each environmental isolate of B. thuringiensis strains. In addition, 16S rRNA based PCR-restriction fragment length polymorphism (RFLP) was carried out to confirm B. thuringiensis strains. Finally, SDS-PAGE analysis was optimized to detect protein profiles of crystal proteins obtained from B. thuringiensis isolates. It was found that, 136 of 359 isolates showed B. thuringiensis-like colony morphology and subterminal endospore position. One hundred isolates were screened by PCR and 18 of them were found to contain cry genes (5 cry 1, 3 cry3, and 10 cry 9). However, the cry 2 gene was not detected from any isolates. 16S rRNA based PCR-RFLPfor 18 isolates gave the same restriction pattern as positive controls, indicating that all 18 isolates were B. thuringiensis. SDS-PAGE studies for Cry 9 proteins of the isolates exhibited different protein profile from positive control of B thuringiensis strain

Razvoj metoda za genetičke manipulacije bakterija iz reda Bacillales

Strains from order Bacillales have numerous industrial applications but due to the complexity and non-efficient transformation protocols have not reached their full implementation potential. This Master Thesis was focused on the transformation of selected strains from order Bacillales: Bacillus thuringiensis 407 cry-, Paenibacillus sp., Bacillus simplex, Lysinibacillus fusiformis M5, and Bacillus velezensis FZB42. In order to perform and follow their transformation, two vectors labelled through introduction of a green fluorescent protein gene were constructed by using Gibson assembly. Within transformation protocols special attention was paid to electroporation, suitable for the transformation of B. thuringiensis 407 cry-, and conjugation, optimal for the rest of the tested strains. Further, stability of the two constructed plasmids and two other, previously prepared plasmids, used in the transformation of B. thuringiensis 407 cry-, was tested by using fluorescence and resistance to antibiotics (kanamycin and chloramphenicol). In addition, plasmid gene expression was followed during growth of B. thurin-giensis 407 cry- by stereomicroscopy, root colonization assay, and confocal laser scanning microscopy. Based on obtained results it can be concluded that successfully transformed B. thuringiensis 407 cry- is good candidate for plant-growth-promoting bacterium.Vrste iz roda Bacillales imaju široku primjenu u industrijskoj proizvodnji, ali zbog kom-pleksnosti i neučinkovitosti protokola za njihovu transformaciju još uvijek nisu dosegli svoj puni in-dustrijski potencijal. Ovaj je diplomski rad bio usmjeren na transformaciju odabranih vrsta iz reda Ba-cillales: Bacillus thuringiensis 407 cry-, Paenibacillus sp., Bacillus simplex, Lysinibacillus fusiformis M5 i Bacillus velezensis FZB42. Primjenom Gibsonove metode konstruirana su dva vektora, u koje je kao marker uveden gen za zeleni fluorescirajući protein. Ova su dva vektora korištena za optimiranje protokola za transformaciju pobrojenih vrsta, u okviru kojeg je posebna pozornost posvećena postupku elektroporacije, koji se pokazao učinkovitim za transformaciju B. thuringiensis 407 cry-, i konjugacije, koji je bio optimalan za preostale odabrane vrste iz ovog reda. Nadalje, stabilnost dvaju konstruiranih plazmida kao i dva prethodno pripremljena plazmida, koji su se koristili za transformaciju B. thurin-giensis 407 cry-, testirana je uz primjenu metode za određivanje fluorescencije i rezistencije na antibi-otike (kanamicin i kloramfenikol). Dodatno, praćena je ekspresija gena plazmida tijekom rasta B. thu-ringiensis 407 cry- metodama stereomikroskopije, kolonizacije korijena biljke i konfokalnom la-serskom skenirajućom mikroskopijom. Na temelju dobivenih rezultata može se zaključiti da je uspješno transformirani B. thuringiensis 407 cry- dobar kandidat za grupu bakterija koje pospješuju rast biljaka

Advanced analysis of a cryptochrome mutation's effects on the robustness and phase of molecular cycles in isolated peripheral tissues of Drosophila

BACKGROUND: Previously, we reported effects of the cry(b) mutation on circadian rhythms in period and timeless gene expression within isolated peripheral Drosophila tissues. We relied on luciferase activity driven by the respective regulatory genomic elements to provide real-time reporting of cycling gene expression. Subsequently, we developed a tool kit for the analysis of behavioral and molecular cycles. Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs. RESULTS: Isolated antennal pairs, heads, bodies, wings and forelegs were evaluated under light-dark cycles. In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing. Moreover, among those specimens with detectable rhythmicity, mutant rhythms are significantly weaker than cry(+) controls. In addition, cry(b) alters the phase of period gene expression in these tissues. Furthermore, peak phase of luciferase-reported period and timeless expression within cry(+) samples is indistinguishable in some tissues, yet significantly different in others. We also analyze rhythms produced by antennal pairs in constant conditions. CONCLUSIONS: These analyses further show that circadian clock mechanisms in Drosophila may vary in a tissue-specific manner, including how the cry gene regulates circadian gene expression

Resistência do milho (Zea mays L.) transgênico (Bt) a lagarta-do-cartucho, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae).

No Brasil, a utilização de milho transgênico (Bt) pode reduzir perdas causadas por vários lepidópteros-pragas, equivalentes a aproximadamente 500 milhões de dólares anuais. O objetivo deste trabalho foi avaliar os híbridos de milho Bt disponíveis no mercado americano para resistência à lagarta-do-cartucho do milho (LCM), Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae). Nove híbridos de milho Bt expressando as toxinas Cry 1F, Cry 1A(b), Cry 1 A(c) e Cry 9C, além de um híbrido (MP 704 X 707) expressando resistência natural para a LCM, foram comparados num experimento, em campo, em blocos ao acaso, com parcela subdividida e quatro repetições. Nas parcelas, foram comparados os dez híbridos e, nas subparcelas, as versões Bt versus não-Bt, ou híbrido com resistência natural versus susceptível. A infestação artificial foi realizada 33 dias após a semeadura e as avaliações basearam-se no número de larvas sobreviventes, aos 10 e 15 dias após a infestação, peso das larvas sobreviventes e notas de danos nas plantas. Para todas as variáveis estudadas, os resultados demonstraram diferenças significativas (P=0,05) entre os híbridos avaliados. Também as testemunhas não-Bts diferiram significativamente dos respectivos híbridos Bts, exceto para aqueles expressando a toxina Cry 9C. O híbrido 2722 IMI, expressando a toxina Cry 1F, foi o mais resistente (imune) e os híbridos expressando a toxina Cry 1A(b) e a resistência natural foram moderadamente resistentes. Em geral, os híbridos transgênicos resistentes produziram cerca de 32% a mais de grãos que as testemunhas suscetíveis

Kloning Gen Cry Dari Bacillus Thuringiensis Var. Kurstaki = Cloning Of The Cry Gene From Bacillus Thuringiensis Var. Kurstaki

Bacillus thuringiensis is an entomopathogenic organism. The pathogenic effect is caused by crystalline protein, 5-endotoxin, which is encoded by the cry gene. The aim of this study was to clone the cry gene from B. thuringiensis var. kurstaki with a genomic library approach. Hindi-11 digested B.thuringiensis total DNA was ligated to Hind!!! digested of pUC19, and then used to transform Escherichia coli DH5a. Selection of transformans carrying recombinant was done by a-complementation and a recombinant clone containing the cry gene was further screened by non-radioactive hybridization method using a probe synthesized from the conserved region of the published cry genes. The result suggested that two recombinant clones with the insert size of 3.4 kb and 2.0 kb, respectively, carrying the cry gene. Key words: cloning, cry gene, Bacillus thuringiensi

Behavioral Dissection of the Drosophila Circadian Multioscillator System that Regulates Locomotor Rhythms

The fruit fly, Drosophila melanogaster, shows a bimodal circadian activity rhythm with peaks around light-on and before light-off. This rhythm is driven by seven groups of so-called clock neurons in the brain. To dissect the multioscillatory nature of the Drosophila clock system, the process of reentrainment to a reversed light cycle was examined by using wild-type flies and cry(b) mutant flies that carry a strong loss-of-function mutation in cryptochrome (cry) gene. The wild-type flies showed that the morning peak dissociated into two components, while a substantial fraction of cry(b) flies exhibited dissociation of the evening peak into two components that shifted in different directions. When the temperature cycle was given in constant darkness in such a manner that the thermophase corresponded to the previous night phase, the morning peak also split into two components in wild-type flies. These results suggest that both morning and evening peaks are driven by two separate oscillators that have different entrainability to light and temperature cycles. Examination of the process of reentrainment to a reversed LD in mutant flies that lack some of the four known circadian photoreceptors (compound eyes, ocelli, CRYPTOCHROME [CRY], and Hofbauer-Buchner [H-B] eyelets) revealed that these four photoreceptors play different roles in photic entrainment of the four putative oscillators

Caracterización de cepas de Bacillus thuringiensis berliner y actividad biológica hacia Spodoptera frugiperda (J. E. Smith) (Lepidoptera: noctuidae) y Anticarsia gemmatalis hübner (Lepidoptera: noctuidae).

RESUMEN: Se realizó la caracterización de nueve cepas cubanas de Bacillus thuringiensis según la morfología del cristal, la determinación del patrón de proteínas Cry y la actividad biológica frente a los insectos lepidópteros Spodoptera frugiperda y Anticarsia gemmatalis. Se observó la típica morfología bipiramidal en todas las cepas, y además la presencia de inclusiones cúbicas. El patrón de proteínas Cry obtenido correspondió con el de la cepa estándar internacional de B. thuringiensis var. kurstaki cepa HD1, en el que se observan dos bandas bien definidas correspondientes a la proteína Cry 1 (130 kDa) y Cry 2 (70 kDa). En la evaluación de la actividad biológica las cepas LBT 4 y LBT 7 causaron el 100% de mortalidad frente a S. frugiperda, mientras que las LBT 4, LBT 7, LBT 13 y LBT 47 provocaron el 100% de mortalidad para A. gemmatalis. abstract: This study describes the characterization of nine Cuban Bacillus thuringiensis strains based on crystal morphology, SDS polyacrylamide electrophoresis (PAGE) and insecticidal activity against Spodoptera frugiperda and Anticarsia gemmatalis. Ultrastructural analysis of parasporal bodies of the nine strains showed the typical bipyramidal crystal and cubic inclusion partially embedded in the middle of the bipyramidal crystal. The PAGE analysis showed two bands of 130 kDa and 70 kDa belongs to Cry 1 and Cry 2 protein present to HD1 standard strains B. thuringiensis var. kurstaki. The strains LBT 4 and LBT 7 analyzed in this report showed potential as biological insecticide against S. frugiperda and LBT 4, LBT 7, LBT13 and LBT47 strains showed 100% of mortality to Anticarsia gemmatalis

LARVICIDAL ACTIVITY OF BACILLUS THURINGIENSIS ISOLATED FROM Bt COTTON RHIZOSPHERE SOIL AGAINST ANOPHELES MOSQUITO LARVAE (CULICIDAE)

Objective: The objective of the study was to isolate and identify Bacillus thuringiensis (Bt) from Bt cotton rhizosphere soil and analyze its larvicidal activity against Anopheles mosquito larvae.Methods: Soil samples were collected from Bt cotton field, and B. thuringiensis was isolated and characterized by biochemical and microscopical characterization. Crystal (Cry) proteins were extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and its larvicidal activity was checked by mortality analysis. Effective Bt isolates were identified by 16S rRNA sequencing.Results: A total of 24 isolates were characterized as Bacillus sp. by biochemical characterization. Further, parasporal inclusion and Cry proteins were extracted, and it was quantified by Bradford assay. The precipitated Cry proteins were analyzed by SDS-PAGE and the results have indicates Cry I protein at 100kDa, Cry3 protein at 44kDa, and cytolytic protein (Cyt) at 29kDa in most of the isolates. Larvicidal activity was checked by the 4th instar Anopheles mosquito larvae. Among the tested isolates, RBL10 and RBL20 have shown the highest percentage of larvicidal activity at 96 and 83%, respectively. Further, the two isolates RBL10 and RBL20 were identified as Bt by 16S rRNA sequencing.Conclusion: B. thuringiensis producing Cry and Cyt proteins posses potential larvicidal activity against Anopheles mosquito larvae which has biological and economic importance in mosquito control

Detection of cryIII gene in Local Isolate of Bacillus thuringiensis Using Polymerase Chain Reaction (PCR)

Bacillus thuringiensis is a biological biopesticide that was used for defense against pests. In B. thuringiensis there is a Cry protein that is only toxic to certain insects. This Cry protein is encoded by cry gene. There are many types of cry genes that have been identified, one of which is cryIII gene that is toxic to insects from the Coleoptera group as pests in sweet potato (Ipomoea batatas). The aim of this study was to amplify the cryIII gene from local isolate of B. thuringiensis. The method that can be used  for cryIII gene amplification is Polymerase Chain Reaction (PCR). The primer pair is one of the important factors that determine the success of PCR. From a number of designed primers, the primer pair selected to be used in this study is Cry3B forward 5’-AAAGTGCGGCTATTCGACCA-3’ and Cry3B reverse 5’-CACTTCATCCTGTGACGCCT-3’. This primer pair successfully amplified cryIII gene and showed a DNA band with molecular size approximately 914 base pairs. Gradient PCR needs to be done for optimizing specific amplification of cryIII gene.   Keywords: PCR, primer, sweet potat

A Group Of Phage-like-particles In Three Subspecies Of Bacillus Thuringiensis And Bacillus Medusa

Phage-med-1, a phage-like-particle (approx. 22 nm in diameter) specifically found in stage II sporulating cells of B. medusa and initially reported as RNA containing was studied further. Similar particles, designated as (SLASHCIRC)isr-1, (SLASHCIRC)kyu-1 and (SLASHCIRC)10-2-1, were subsequently found in Bacillus thuringiensis subsp. israelensis (serotype 14), B. thuringiensis subsp. kyushuensis (serotype 11) and B. thuringiensis isolate 73-E-10-2 (serotype 10). These strains share the unique character of producing solely ovoid or round parasporal crystalline inclusion (cry) toxic to mosquito larvae.;An improved purification enabled the phage-like-particles (PLP) close to homogenous to be isolated. Electron microscopic studies showed that all PLP were assembled at early stages of sporulation, while immunoprecipitation showed that at least in B. medusa and subsp. israelensis, the PLP proteins are synthesized at the times the PLP were assembled.;Acrystalliferous (cry(\u27-)) variants of subsp. israelensis isolated by a 42(DEGREES)C curing method were found to produce, upon sporulation, a satellite inclusion (sat) which was subsequently found in the wild type. Cry(\u27-)sat(\u27+) variants were (SLASHCIRC)isr-1 producing while cry(\u27-)sat(\u27-) variants were not. The latter strains also lacked a 68MDa plasmid of the wild type. A cry(\u27+)sat(\u27-) strain was (SLASHCIRC)isr-1 producing. Examination of transformed strains of a cry(\u27-)sat(\u27-) and plasmidless variant revealed that the sat(\u27+) and (SLASHCIRC)isr-1 producing characters could have been co-transformed in a recipient strain which had acquired the 68 MDa plasmid; likewise, the cry(\u27+) and (SLASHCIRC)isr-1 producing characters could have been co-transformed in a strain which had acquired the 75 MDa plasmid known to be associated with the synthesis of the crystal.;B. thuringiensis isolate 73-E-10-2 produced inclusions of varying size. The size distribution of the inclusions was influenced by the composition of the medium and the growth temperature. Inclusions prepared from cultures with higher number of small inclusions-producing cells have relatively less of a 25.5 K proteins and two smaller proteins. These inclusion preparations were also less toxic than those preparations with more of these three proteins. Variants producing solely small non-toxic inclusions lacking the above three proteins and a 140 K protein were devoid of (SLASHCIRC)10-2-1.;The results of this study indicated that the genes responsible for the syntheses of (SLASHCIRC)isr-1 and (SLASHCIRC)10-2-1 could be located on plasmids which also carried the genes determining the syntheses of components of the inclusions. (Abstract shortened with permission of author.