Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats / Abundancia, distribución y expresión de genes de proteínas cristalinas nematicidas en cepas de Bacillus thuringiensis de diversos hábitats

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.(AU)

Strategical isolation of efficient chicken feather-degrading bacterial strains from tea plantation soil sample

Chicken feather waste is generally insufficiently utilized despite its high content of protein, constituting an environmental issue. Biodegradation of the waste with enabling microbes provides an advantageous option among the available solutions. In this study, an efficient whole feather-degrading strain was strategically isolated from a soil sample taken from a local tea plantation that has little or nothing to do with feathers. The strain was identified as Bacillus thuringiensis (designated as FDB-10) according to the cloned complete 16S rRNA sequence. The FDB-10 could efficiently degrade briefly heat-treated whole feather (102 °C, 5 min; up to 90% of a maximum concentration of 30 g/L) in a salt medium supplemented with 0.1 g/L yeast extract within 24 h (37°C, 150 rpm). Addition of carbon sources (glycerol, glucose, starch, Tween 20, Tween 80, 1.25 g/L as glycerol) to the fermentation medium could improve the degradation. However, significant inhibition could be observed when the added carbon source reached the amount usually adopted in the investigation of carbon source preference (1%). Nitrogen source (NH4Cl, (NH4)2SO4, peptone) adversely influenced the performance of the strain. When the molar concentrations of NH4+ were equal for the two salt, the inhibitory effect on degradation of whole feathers was similar. Entirely different from other reported feather-degrading strains showing a preference to melanin-free feather substrates, the strain isolated in this study could degrade melanin-containing feather equally efficiently, and higher protease activity could be detected in the digest mix. As a plus, the strain could degrade feathers in rice wash produced in daily cooking, indicating its potential use in the simultaneous treatment of rice cooker wastewater produced by a rice processing plant. All these results imply that the FDB-10 is a strain with great potential in the biodegradation of feather waste

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Optimización y Validación de una PCR en Tiempo Real para la Rápida Identificación de Bacillus thuringiensis, Simulador de Bacillus anthracis / Optimization and validation of a real-time PCR for rapid identification of Bacillus thuringiensis, surrogate of Bacillus anthracis

INTRODUCCIÓN: Bacillus anthracis es uno de los agentes de guerra biológica más empleados en el mundo. Sin embargo, en los laboratorios de defensa biológica es conveniente utilizar en muchas ocasiones otras bacterias parecidas a Bacillus anthracis pero menos peligrosas o apatógenas. Uno de los principales simuladores de Bacillus anthracis es Bacillus thuringiensis, debido a su elevada homología con B. anthracis y a su nula patogenicidad para el ser humano. OBJETIVOS: El objetivo del presente estudio es desarrollar y validar una PCR en tiempo real para la rápida identificación del ADN de Bacillus thuringiensis, agente empleado muy a menudo en los simulacros para entrenamiento de las Unidades Operativas de toma de muestras NBQ de las FAS. Material y MÉTODOS: La identificación del simulador Bacillus thuringiensis se ha realizado mediante la amplificación y detección con una sonda de hidrólisis de un fragmento de 69 pares de bases del gen cry1A, el cual es específico de esta bacteria. Tras optimizar las condiciones de amplificación probando tres temperaturas diferentes de hibridación/extensión, se procedió a realizar la validación del método desarrollado. RESULTADOS: La nueva PCR en tiempo real desarrollada presentó una eficiencia del 93%, así como una elevada linealidad (coeficiente de regresión R2 0,9993). El límite de detección al 95% de probabilidad fue de 13 equivalentes de genoma completo por reacción. Tanto la inclusividad como la exclusividad del método fueron del 100%. CONCLUSIONES: El método molecular desarrollado por el Laboratorio de Biología Molecular del INTA permite una rápida identificación del ADN de Bacillus thuringiensis, simulador de Bacillus anthracis, con unas elevadas sensibilidad y especificidad analíticas

INTRODUCTION: Bacillus anthracis is the most employed biological warfare agent in the world. However, in biological defense laboratories, many times it is convenient to use other bacteria similar to Bacillus anthracis but less dangerous or non-pathogenic. One of the main surrogates of Bacillus anthracis is Bacillus thuringiensis, due to its high homology with B. anthracis and its null pathogenicity for humans. OBJECTIVE: The objective of the present study is to develope and validate a real-time PCR for the rapid identification of Bacillus thuringiensis DNA, biological agent very often employed in the training of the Operative Units of CBRN sampling of the Armed Forces. METHODS: The identification of Bacillus thuringiensis has been performed by the amplification and detection with a hydrolysis probe of a 69 base pairs fragment of the cry1A gene, which is specific for this bacterium. After optimizing the amplification conditions by testing three different hybridization / extension temperatures, the validation of the new developed method was carried out. RESULTS: The new developed real-time PCR showed an efficiency of 93%, as well as a high linearity (regression coefficient R20.9993). The limit of detection at 95% probability was 13 genome equivalents per reaction. Both the inclusiveness and the exclusivity of the method were 100%. CONCLUSIONS: The molecular method developed at the Molecular Biology Laboratory of INTA allows the rapid identification of Bacillus thuringiensis DNA, surrogate of Bacillus anthracis, with a high analytical sensitivity and specificity

New combinations of cry genes from Bacillus thuringiensis strainsisolated from northwestern Mexico

Twenty eight Bacillus thuringiensis strains isolated from the Tijuana-Ensenada region of northwestern Mexico were analyzed to determine the distribution of cry and cyt genes. Crystal production by the strains was examined by scanning electron microscopy, which showed the predominance of cubic crystals. Alkaline-dissolved and trypsin activated crystals were also analyzed by SDS-PAGE, yielding bands of 40-200 kDa. The cry1 and cry2 genes were molecularly characterized using general and newly designed specific primers in addition to other oligonucleotides (cry3, cry4, cry8, cry9, cry11, Nem, cry25,cry29 and cyt), resulting in the identification of novel gene combinations. The use of specific primers for cry1A, cry1B, cry1C,cry1D, cry1E, cry1F and cry2Aa, cry2Ab, cry2Ac, cry2Ad showed differences in the distribution of cry1 (36 %), cry2 (71 %),and cyt (40 %) in strains from Tijuana-Ensenada compared to other previously studied regions. Bioassays were conducted on Manduca sexta larvae to analyze the Cry insecticidal capacity of the isolated strains. The hemolytic activity of the Cyt toxin from the same strains was assessed in human erythrocytes (AU)

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Evaluación de la toxicidad/patogenicidad de una formulación de Bacillus thuringiensis var israelensis (Bactivec) / Toxicity/pathogenicity evaluation of a Bacillus sphaericus 2362 formulation (Griselesf)

Existen varios agentes microbianos, incluyendo hongos, protozoos, virus y bacterias, que han sido utilizados como mosquiticidas. No obstante, entre esos agentes, el Bacillus thuringiensis es el más potente y ha sido el más ampliamente utilizado. El Bactivec es un biolarvicida producido en Cuba, que contiene Bacillus thuringiensis variedad israelensis como agente activo. Con el objetivo de evaluar su toxicidad/patogenicidad, se administró el Bactivec en una dosis única de 5 x 108 unidades formadoras de colonias por vía oral a ratas, y por vía dérmica a dos grupos de conejos albinos, uno recibiendo una dosis de Bactivec de l x 109 unidades formadoras de colonia por animal, y el otro una dosis de 9.2 x 108 de unidades formadoras de colonia de Bacillus thuringiensis variedad israelensis por animal. En ambos ensayos las observaciones clínicas fueron diarias, y se evaluó el comportamiento del peso corporal. Además, en el ensayo por vía oral se estimó el aclaramiento mediante recolección de las heces fecales, y se evaluó la infectividad mediante toma de muestras de fluidos y órganos. Al final de los ensayos se realizó la necropsia ä todos los animales. No ocurrieron mortalidades, ni evidencias de patogenicidad o toxicidad relacionada con el tratamiento en ninguno de los ensayos. En el ensayo de toxicidad/patogenicidad aguda oral se obtuvo que el microorganismo fue eliminado rápidamente sin provocar infección significativa. Se concluyó que el Bactivec no es patogénico por las vías oral y dérmica a las dosis evaluadas. (AU)