Sample records for bacillus thuringiensis spores
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Plomp, M; Leighton, T; Wheeler, K
2005-02-18
We have utilized atomic force microscopy (AFM) to visualize the native surface topology and ultrastructure of Bacillus thuringiensis and Bacillus cereus spores in water and in air. AFM was able to resolve the nanostructure of the exosporium and three distinctive classes of appendages. Removal of the exosporium exposed either a hexagonal honeycomb layer (B. thuringiensis) or a rodlet outer spore coat layer (B. cereus). Removal of the rodlet structure from B. cereus spores revealed an underlying honeycomb layer similar to that observed with B. thuringiensis spores. The periodicity of the rodlet structure on the outer spore coat of B. cereusmore » was {approx}8 nm, and the length of the rodlets was limited to the cross-patched domain structure of this layer to {approx}200 nm. The lattice constant of the honeycomb structures was {approx}9 nm for both B. cereus and B. thuringiensis spores. Both honeycomb structures were composed of multiple, disoriented domains with distinct boundaries. Our results demonstrate that variations in storage and preparation procedures result in architectural changes in individual spore surfaces, which establish AFM as a useful tool for evaluation of preparation and processing ''fingerprints'' of bacterial spores. These results establish that high-resolution AFM has the capacity to reveal species-specific assembly and nanometer scale structure of spore surfaces. These species-specific spore surface structural variations are correlated with sequence divergences in a spore core structural protein SspE.« less
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Delvecchio, Vito G; Connolly, Joseph P; Alefantis, Timothy G; Walz, Alexander; Quan, Marian A; Patra, Guy; Ashton, John M; Whittington, Jessica T; Chafin, Ryan D; Liang, Xudong; Grewal, Paul; Khan, Akbar S; Mujer, Cesar V
2006-09-01
Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.
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2014-01-01
Response surface methodology using a face-centered cube design was used to describe and predict spore inactivation of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure of six spore-contaminated materials to hot, humid air. For each strain/material pair, an attempt was made to fit a first or second order model. All three independent predictor variables (temperature, relative humidity, and time) were significant in the models except that time was not significant for B. thuringiensis Al Hakam on nylon. Modeling was unsuccessful for wiring insulation and wet spores because there was complete spore inactivation in the majority of the experimental space. In cases where a predictive equation could be fit, response surface plots with time set to four days were generated. The survival of highly purified Bacillus spores can be predicted for most materials tested when given the settings for temperature, relative humidity, and time. These predictions were cross-checked with spore inactivation measurements. PMID:24949256
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Milutinović, Barbara; Höfling, Christina; Futo, Momir; Scharsack, Jörn P.
2015-01-01
Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen. PMID:26386058
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Code of Federal Regulations, 2010 CFR
2010-07-01
... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1011 Viable spores of the... characteristics of the parent strain or contamination by other microorganisms. (3) Each lot of spore preparation... production is a Bacillus thuringiensis strain which does not produce β-exotoxin under standard manufacturing...
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Hariram, Upasana; Labbé, Ronald
2015-03-01
Recent incidents of foodborne illness associated with spices as the vehicle of transmission prompted this examination of U.S. retail spices with regard to Bacillus cereus. This study focused on the levels of aerobic-mesophilic spore-forming bacteria and B cereus spores associated with 247 retail spices purchased from five states in the United States. Samples contained a wide range of aerobic-mesophilic bacterial spore counts (< 200 to 8.3 × 10(7) CFU/g), with 19.1% of samples at levels above 10(5) CFU/g. For examples, paprika, allspice, peppercorns, and mixed spices had high levels of aerobic spores (> 10(7) CFU/g). Using a novel chromogenic agar, B. cereus and B. thuringiensis spores were isolated from 77 (31%) and 11 (4%) samples, respectively. Levels of B. cereus were <3 to 1,600 MPN/g. Eighty-eight percent of B. cereus isolates and 91% of B. thuringiensis isolates possessed at least one type of enterotoxin gene: HBL (hemolysin BL) or nonhemolytic enterotoxin (NHE). None of the 88 isolates obtained in this study possessed the emetic toxin gene (ces). Using commercially available immunological toxin detection kits, the toxigenicity of the isolates was confirmed. The NHE enterotoxin was expressed in 98% of B. cereus and 91% of B. thuringiensis isolates that possessed the responsible gene. HBL enterotoxin was detected in 87% of B. cereus and 100% of B. thuringiensis PCR-positive isolates. Fifty-two percent of B. cereus and 54% of B. thuringiensis isolates produced both enterotoxins. Ninety-seven percent of B. cereus isolates grew at 12°C, although only two isolates grew well at 9°C. The ability of these spice isolates to form spores, produce diarrheal toxins, and grow at moderately abusive temperatures makes retail spices an important potential vehicle for foodborne illness caused by B. cereus strains, in particular those that produce diarrheal toxins.
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Milutinović, Barbara; Höfling, Christina; Futo, Momir; Scharsack, Jörn P; Kurtz, Joachim
2015-12-01
Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Buhr, T L; Young, A A; Bensman, M; Minter, Z A; Kennihan, N L; Johnson, C A; Bohmke, M D; Borgers-Klonkowski, E; Osborn, E B; Avila, S D; Theys, A M G; Jackson, P J
2016-04-01
To develop test methods and evaluate survival of Bacillus thuringiensis kurstaki cry(-) HD-1 and B. thuringiensis Al Hakam spores after exposure to hot, humid air inside of a C-130 aircraft. Bacillus thuringiensis spores were either pre-inoculated on 1 × 2 or 2 × 2 cm substrates or aerosolized inside the cargo hold of a C-130 and allowed to dry. Dirty, complex surfaces (10 × 10 cm) swabbed after spore dispersal showed a deposition of 8-10 log10 m(-2) through the entire cargo hold. After hot, humid air decontamination at 75-80°C, 70-90% relative humidity for 7 days, 87 of 98 test swabs covering 0·98 m(2) , showed complete spore inactivation. There was a total of 1·67 log10 live CFU detected in 11 of the test swabs. Spore inactivation in the 98 test swabs was measured at 7·06 log10 m(-2) . Laboratory test methods for hot, humid air decontamination were scaled for a large-scale aircraft field test. The C-130 field test demonstrated that hot, humid air can be successfully used to decontaminate an aircraft. Transition of a new technology from research and development to acquisition at a Technology Readiness Level 7 is unprecedented. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
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Code of Federal Regulations, 2013 CFR
2013-07-01
... authentic strain of Bacillus thuringiensis Berliner conforming to the morphological and biochemical... million spores into each of five laboratory test mice weighing 17 grams to 23 grams. Such test shall show no evidence of infection or injury in the test animals when observed for 7 days following injection...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... authentic strain of Bacillus thuringiensis Berliner conforming to the morphological and biochemical... million spores into each of five laboratory test mice weighing 17 grams to 23 grams. Such test shall show no evidence of infection or injury in the test animals when observed for 7 days following injection...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... authentic strain of Bacillus thuringiensis Berliner conforming to the morphological and biochemical... million spores into each of five laboratory test mice weighing 17 grams to 23 grams. Such test shall show no evidence of infection or injury in the test animals when observed for 7 days following injection...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... authentic strain of Bacillus thuringiensis Berliner conforming to the morphological and biochemical... million spores into each of five laboratory test mice weighing 17 grams to 23 grams. Such test shall show no evidence of infection or injury in the test animals when observed for 7 days following injection...
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Beuchat, Larry R; Pettigrew, Charles A; Tremblay, Mario E; Roselle, Brian J; Scouten, Alan J
2004-08-01
Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.
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Omotade, T O; Bernhards, R C; Klimko, C P; Matthews, M E; Hill, A J; Hunter, M S; Webster, W M; Bozue, J A; Welkos, S L; Cote, C K
2014-12-01
Decontamination and remediation of a site contaminated by the accidental or intentional release of fully virulent Bacillus anthracis spores are difficult, costly and potentially damaging to the environment. Development of novel decontamination strategies that have minimal environmental impacts remains a high priority. Although ungerminated spores are amongst the most resilient organisms known, once exposed to germinants, the germinating spores, in some cases, become susceptible to antimicrobial environments. We evaluated the concept that once germinated, B. anthracis spores would be less hazardous and significantly easier to remediate than ungerminated dormant spores. Through in vitro germination and sensitivity assays, we demonstrated that upon germination, B. anthracis Ames spores and Bacillus thuringiensis Al Hakam spores (serving as a surrogate for B. anthracis) become susceptible to environmental stressors. The majority of these germinated B. anthracis and B. thuringiensis spores were nonviable after exposure to a defined minimal germination-inducing solution for prolonged periods of time. Additionally, we examined the impact of potential secondary disinfectant strategies including bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UV-B and UV-C radiation, employed after a 60-min germination-induction step. Each secondary disinfectant employs a unique mechanism of killing; as a result, germination-induction strategies are better suited for some secondary disinfectants than others. These results provide evidence that the deployment of an optimal combination strategy of germination-induction/secondary disinfection may be a promising aspect of wide-area decontamination following a B. anthracis contamination event. By inducing spores to germinate, our data confirm that the resulting cells exhibit sensitivities that can be leveraged when paired with certain decontamination measures. This increased susceptibility could be exploited to devise more
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Buhr, T L; Wells, C M; Young, A A; Minter, Z A; Johnson, C A; Payne, A N; McPherson, D C
2013-08-01
To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
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Buhr, T L; Young, A A; Barnette, H K; Minter, Z A; Kennihan, N L; Johnson, C A; Bohmke, M D; DePaola, M; Cora-Laó, M; Page, M A
2015-11-01
To develop test methods and evaluate survival of Bacillus anthracis ∆Sterne or Bacillus thuringiensis Al Hakam on materials contaminated with dirty spore preparations after exposure to hot, humid air using response surface modelling. Spores (>7 log10 ) were mixed with humic acid + spent sporulation medium (organic debris) or kaolin (dirt debris). Spore samples were then dried on five different test materials (wiring insulation, aircraft performance coating, anti-skid, polypropylene, and nylon). Inoculated materials were tested with 19 test combinations of temperature (55, 65, 75°C), relative humidity (70, 80, 90%) and time (1, 2, 3 days). The slowest spore inactivation kinetics was on nylon webbing and/or after addition of organic debris. Hot, humid air effectively decontaminates materials contaminated with dirty Bacillus spore preparations; debris and material interactions create complex decontamination kinetic patterns; and B. thuringiensis Al Hakam is a realistic surrogate for B. anthracis. Response surface models of hot, humid air decontamination were developed which may be used to select decontamination parameters for contamination scenarios including aircraft. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.
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Pathogenicity of Bacillus thuringiensis variety kurstaki to Ixodes scapularis (Acari: Ixodidae)
Zhioua, Elyes; Heyer, Klaus; Browning, M.; Ginsberg, Howard S.; LeBrun, Roger A.
1999-01-01
Pathogenicity of the entomopathogenic bacterium Bacillus thuringiensis var. kurstaki de Barjac & Lemille was tested against the black-legged tick, Ixodes scapularis Say. Engorged larvae dipped in a solution of 108 spores per ml showed 96% mortality, 3 wk post-infection. The LC50 value for engorged larvae (concentration required to kill 50% of ticks) was 107 spores/ml. Bacillus thuringiensis shows considerable potential as a microbial control agent for the management of Ixodes scapularis.
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Myasnik, M; Manasherob, R; Ben-Dov, E; Zaritsky, A; Margalith, Y; Barak, Z
2001-08-01
Susceptibility of Bacillus thuringiensis spores and toxins to the UV-B range (280--330 nm) of the solar spectrum reaching Earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the mosquito larvicidal B. thuringiensis subsp. israelensis were significantly more resistant to UV-B than spores of the lepidopteran-active subsp. kurstaki. Spores of subsp. israelensis were as resistant to UV-B as spores of B. subtilis and more resistant than spores of the closely related B. cereus and another mosquito larvicidal species B. sphaericus. Sensitivity of B. thuringiensis subsp. israelensis spores to UV-B radiation depended upon their culture age; 24-h cultures, approaching maximal larvicidal activity, were still sensitive. Maximal resistance to UV-B was achieved only at 48 h.
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Tayabali, A F; Seligy, V L
2000-01-01
Most contemporary bioinsecticides are derived from scaled-up cultures of Bacillus thuringiensis subspecies israelensis (Bti) and kurstaki (Btk), whose particulate fractions contain mostly B. thuringiensis spores (> 10(12)/L) and proteinaceous aggregates, including crystal-like parasporal inclusion bodies (PIB). Based on concerns over relatedness to B. cereus-group pathogens, we conducted extensive testing of B. thuringiensis (BT) products and their subfractions using seven human cell types. The Bti/Btk products generated nonspecific cytotoxicities involving loss in bioreduction, cell rounding, blebbing and detachment, degradation of immunodetectable proteins, and cytolysis. Their threshold dose (Dt approximately equal.5 times 10(-14)% BT product/target cell) equated to a single spore and a target cell half-life (tLD(50)) of approximately 16 hr. At Dts > 10(4), the tLD(50) rapidly shifted to < 4 hr; with antibiotic present, no component, including PIB-related [delta]-endotoxins, was cytolytic up to an equivalent of approximately 10(9 )Dt. The cytolytic agent(s) within the Bti/Btk-vegetative cell exoprotein (VCP) pool is an early spore outgrowth product identical to that of B. cereus and acting possibly by arresting protein synthesis. No cytolytic effects were seen with VCP from B. subtilis and Escherichia coli. These data, including recent epidemiologic work indicate that spore-containing BT products have an inherent capacity to lyse human cells in free and interactive forms and may also act as immune sensitizers. To critically impact at the whole body level, the exposure outcome would have to be an uncontrolled infection arising from intake of Btk/Bti spores. For humans, such a condition would be rare, arising possibly in equally rare exposure scenarios involving large doses of spores and individuals with weak or impaired microbe-clearance capacities and/or immune response systems. PMID:11049810
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Buhr, T L; Young, A A; Minter, Z A; Wells, C M; McPherson, D C; Hooban, C L; Johnson, C A; Prokop, E J; Crigler, J R
2012-11-01
To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics. Hot, humid air is a potential alternative to conventional chemical decontamination. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
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DelVecchio, Vito G.; Connolly, Joseph P.; Alefantis, Timothy G.; Walz, Alexander; Quan, Marian A.; Patra, Guy; Ashton, John M.; Whittington, Jessica T.; Chafin, Ryan D.; Liang, Xudong; Grewal, Paul; Khan, Akbar S.; Mujer, Cesar V.
2006-01-01
Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Δ-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development. PMID:16957262
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Nano-Mechanical Properties of Heat Inactivated Bacillus anthracis and Bacillus thuringiensis Spores
2008-03-01
Scanner Laser Mirror Cantilever Sample Probe Tip 16 cereus strain 569, and Bacillus globigii var. niger . Zolock determined that there wer...been used to measure the surface elasticities of a variety of microbial organisms including Pseudomonas putida, Bacillus subtilis, Aspergillus ...66:307-311 (2005). Zhao, Liming, David Schaefer, and Mark R. Marten. “Assessment of Elasticity and Topography of Aspergillus nidulans Spores via
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Molecular characterization of Bacillus thuringiensis isolated from diverse habitats of India.
Patel, Ketan D; Chudasama, Chaitanyasinh J; Ingle, Sanjay S
2012-08-01
Bacillus thuringiensis (Bt) strains were isolated from 94 samples from different geographical regions. Novel types of crystalline inclusion bodies were observed from some of the isolates. Crystalline inclusions of bipyramidal, spherical and cuboidal morphology were found produced by most of the isolates. Isolate GS12 showed crystal on one side of spore while isolate GM108 formed crystals on both termini of spore. Isolate GN31 produced large sized bipyramidal crystals. SDS-PAGE analysis of the spore crystal suspension showed major protein bands in the range of 29 and 140 kDa. Two new serovars of Bt viz. GS4 and GN24 having H3abce and H3ab serotype respectively were isolated. Toxicity comparable to the reference strain Bacillus thuringiensis subs. kurstaki (Btk) HD-1 was observed for the isolates GM20, GM17 and MP3 against larvae of Helicoverpa armigera. Some of the isolates harboring cry genes like cry1Ac and cry2 did not show any toxicity towards H. armigera while most of the isolates were harboring cry1, cry1Ac and cry2 gene. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Faille, C; Bénézech, T; Blel, W; Ronse, A; Ronse, G; Clarisse, M; Slomianny, C
2013-04-01
This study was designed to evaluate the respective roles of mechanical and chemical effects on the removal of Bacillus spores during cleaning-in-place. This analysis was performed on 12 strains belonging to the Bacillus cereus group (B. cereus, Bacillus anthracis, Bacillus thuringiensis) or to less related Bacillus species (Bacillus pumilus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus subtilis). Adherent spores were subjected to rinsing-in-place (mechanical action) and cleaning-in-place (mechanical and chemical actions) procedures, the latter involving NaOH 0.5% at 60°C. Results revealed that mechanical action alone only removed between 53 and 89% of the attached spores at a shear stress of 500 Pa. This resistance to shear was not related to spore surface properties. Conversely, in the presence of NaOH at a shear stress of 4 Pa, spores were readily detached, with between 80 and 99% of the adherent spores detached during CIP and the chemical action greatly depended on the strain. This finding suggests that chemical action plays the major role during CIP, whose efficacy is significantly governed by the spore surface chemistry. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Andrews, R. E.; Iandolo, J. J.; Campbell, B. S.; Davidson, L. I.; Bulla, L. A.
1980-01-01
Rocket immunoelectrophoresis was used to quantitate the soluble parasporal crystal of Bacillus thuringiensis subsp. kurstaki. The method described is rapid, reliable, specific, and extremely accurate, and it can be used to measure crystal toxin in commercial microbial insecticides that contain a mixture of spores, vegetative cells, and carrier materials. Images PMID:16345656
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Guidi, Valeria; Patocchi, Nicola; Lüthy, Peter; Tonolla, Mauro
2011-06-01
Recurrent treatments with Bacillus thuringiensis subsp. israelensis are required to control the floodwater mosquito Aedes vexans that breeds in large numbers in the wetlands of the Bolle di Magadino Reserve in Canton Ticino, Switzerland. Interventions have been carried out since 1988. In the present study, the spatial distribution of resting B. thuringiensis subsp. israelensis spores in the soil was measured. The B. thuringiensis subsp. israelensis concentration was determined in soil samples collected along six transects covering different elevations within the periodically flooded zones. A total of 258 samples were processed and analyzed by quantitative PCR that targeted an identical fragment of 159 bp for the B. thuringiensis subsp. israelensis cry4Aa and cry4Ba genes. B. thuringiensis subsp. israelensis spores were found to persist in soils of the wetland reserve at concentrations of up to 6.8 log per gram of soil. Continuous accumulation due to regular treatments could be excluded, as the decrease in spores amounted to 95.8% (95% confidence interval, 93.9 to 97.7%). The distribution of spores was correlated to the number of B. thuringiensis subsp. israelensis treatments, the elevation of the sampling point, and the duration of the flooding periods. The number of B. thuringiensis subsp. israelensis treatments was the major factor influencing the distribution of spores in the different topographic zones (P < 0.0001). These findings indicated that B. thuringiensis subsp. israelensis spores are rather immobile after their introduction into the environment.
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Guidi, Valeria; Patocchi, Nicola; Lüthy, Peter; Tonolla, Mauro
2011-01-01
Recurrent treatments with Bacillus thuringiensis subsp. israelensis are required to control the floodwater mosquito Aedes vexans that breeds in large numbers in the wetlands of the Bolle di Magadino Reserve in Canton Ticino, Switzerland. Interventions have been carried out since 1988. In the present study, the spatial distribution of resting B. thuringiensis subsp. israelensis spores in the soil was measured. The B. thuringiensis subsp. israelensis concentration was determined in soil samples collected along six transects covering different elevations within the periodically flooded zones. A total of 258 samples were processed and analyzed by quantitative PCR that targeted an identical fragment of 159 bp for the B. thuringiensis subsp. israelensis cry4Aa and cry4Ba genes. B. thuringiensis subsp. israelensis spores were found to persist in soils of the wetland reserve at concentrations of up to 6.8 log per gram of soil. Continuous accumulation due to regular treatments could be excluded, as the decrease in spores amounted to 95.8% (95% confidence interval, 93.9 to 97.7%). The distribution of spores was correlated to the number of B. thuringiensis subsp. israelensis treatments, the elevation of the sampling point, and the duration of the flooding periods. The number of B. thuringiensis subsp. israelensis treatments was the major factor influencing the distribution of spores in the different topographic zones (P < 0.0001). These findings indicated that B. thuringiensis subsp. israelensis spores are rather immobile after their introduction into the environment. PMID:21498758
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Raymond, Ben; Elliot, Simon L; Ellis, Richard J
2008-07-01
The Bacillus cereus group comprises a range of micro-organisms with diverse habits, including gut commensals, opportunistic pathogens and soil saprophytes. Using quantitative microbiological methods we tested whether Bacillus thuringiensis (Bt) could reproduce in cadavers of Plutella xylostella killed by Bt, or in the gut of live insects, or be transmitted vertically from females to their offspring. We also tested whether diverse Bt strains could grow in high nutrient broth at a pH similar to that in the larval midgut. Low levels of reproduction were found in insect cadavers but there was no evidence of vertical transmission, or of significant reproduction in live insects. Four strains of B. thuringiensis var. kurstaki and one of B. thuringiensis var. tenebrionis were found to be capable of growth at high pH. Greater spore recovery rates in frass were found in hosts that were resistant or tolerant of infection. We concluded that that spores recovered in frass represent, in general, an ungerminated fraction of ingested inoculum and that germination rates are reduced in unsuitable hosts.
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USDA-ARS?s Scientific Manuscript database
Developing a dual agent biopesticide formulation made with blastospores of Beauveria bassiana (Balsamo) Vuillemin strain GHA (Bb) at the ratio of 1:1 mixed with traditional fermentation production of Bacillus thuringiensis kurstaki (Btk) spores and crystals were studied. Twelve potential formulation...
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USDA-ARS?s Scientific Manuscript database
Developing a dual agent biopesticide formulation made with blastospores of Beauveria bassiana (Balsamo) Vuillemin strain GHA (Bb) at ratio of 0.5:0.5 mixed with traditional fermentation production of Bacillus thuringiensis kurstaki (Bt) spores and crystals were studied. Twelve potential formulation ...
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Rodríguez, Ana Priscila García; Martínez, Marcela Gaytán; Barrera-Cortés, Josefina; Ibarra, Jorge E; Bustos, Fernando Martínez
2015-02-01
Bacillus thuringiensis (Bt) is one of the bioinsecticides used worldwide due to its specific toxicity against target pests in their larval stage. Despite this advantage, its use is limited because of their short persistence in field when exposed to ultra violet light and changing environmental conditions. In this work, microencapsulation has been evaluated as a promising method to improve Bt activity. The objective of this study was to develop and characterize native and modified amaranth starch granules and evaluate their potential application as wall materials in the microcapsulation of B thuringiensis serovar kurstaki HD-1 (Bt- HD1), produced by spray drying. Native amaranth starch granules were treated by hydrolyzation, high energy milling (HEM) and were chemically modified by phosphorylation and succinylation. The size of the Bt microcapsules varied from 12.99 to 17.14 μm adequate to protect the spores of Bt from ultraviolet radiation. The aw coefficient of the microcapsules produced by the modified starches after drying was low (0.14-1.88), which prevent microbial growth. Microcapsules prepared with phosphorylated amaranth starch presented the highest bacterial count and active material yield. Different concentrations of the encapsulated Bt formulation in phosphorylated amaranth starch showed a high level of insecticidal activity when tested on M. sexta larvae and has great potential to be developed as a bioinsecticide formulation, also, the level of toxicity is much higher than that found in some of the products commercially available.
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de Souza Freire, Ingrid; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Martins, Erica Soares; Monnerat, Rose Gomes; Grisolia, Cesar Koppe
2014-01-01
The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually. PMID:25268978
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BOOK REVIEW – BACILLUS THURINGIENSIS: A CORNERSTONE OF MODERN AGRICULTURE BACILLUS THURINGIENSIS
Are you interested in the technical issues surrounding the use of Bacillus thuringiensis pesticidal traits as sprays and as plant incorporated protectants (transgenic crops)? Should the dimensions of human health, ecology, entomology, risk assessment, resistance management, and d...
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Aerosol and Surface Deposition Characteristics of Two Surrogates for Bacillus anthracis Spores
Stapleton, Helen L.
2016-01-01
ABSTRACT Spores of an acrystalliferous derivative of Bacillus thuringiensis subsp. kurstaki, termed Btcry−, are morphologically, aerodynamically, and structurally indistinguishable from Bacillus anthracis spores. Btcry− spores were dispersed in a large, open-ended barn together with spores of Bacillus atrophaeus subsp. globigii, a historically used surrogate for Bacillus anthracis. Spore suspensions (2 × 1012 CFU each of B. atrophaeus subsp. globigii and Btcry−) were aerosolized in each of five spray events using a backpack misting device incorporating an air blower; a wind of 4.9 to 7.6 m s−1 was also flowing through the barn in the same direction. Filter air samplers were situated throughout the barn to assess the aerosol density of the spores during each release. Trays filled with a surfactant in aqueous buffer were placed on the floor near the filter samplers to assess spore deposition. Spores were also recovered from arrays of solid surfaces (concrete, aluminum, and plywood) that had been laid on the floor and set up as a wall at the end of the barn. B. atrophaeus subsp. globigii spores were found to remain airborne for significantly longer periods, and to be deposited on horizontal surfaces at lower densities, than Btcry− spores, particularly near the spray source. There was a 6-fold-higher deposition of Btcry− spores than of B. atrophaeus subsp. globigii spores on vertical surfaces relative to the surrounding airborne density. This work is relevant for selecting the best B. anthracis surrogate for the prediction of human exposure, hazard assessment, and hazard management following a malicious release of B. anthracis. IMPORTANCE There is concern that pathogenic bacteria could be maliciously disseminated in the air to cause human infection and disruption of normal life. The threat from spore-forming organisms, such as the causative agent of anthrax, is particularly serious. In order to assess the extent of this risk, it is important to have a
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Wang, Gaoyan; Manns, David C; Guron, Giselle K; Churey, John J; Worobo, Randy W
2014-06-01
Large-scale purification of the highly hydrophobic bacteriocin thurincin H was accomplished via a novel and simple two-step method: ammonia sulfate precipitation and C18 solid-phase extraction. The inhibition spectrum and stability of thurincin H as well as its antagonistic activity against Bacillus cereus F4552 spores were further characterized. In the purification method, secreted proteins contained in the supernatant of a 40 h incubated culture of B. thuringiensis SF361 were precipitated by 68 % ammonia sulfate and purified by reverse-phase chromatography, with a yield of 18.53 mg/l of pure thurincin H. Silver-stained SDS-PAGE, high-performance liquid chromatography, and liquid chromatography-mass spectrometry confirmed the high purity of the prepared sample. Thurincin H exhibited a broad antimicrobial activity against 22 tested bacterial strains among six different genera including Bacillus, Carnobacterium, Geobacillus, Enterococcus, Listeria, and Staphylococcus. There was no detectable activity against any of the selected yeast or fungi. The bacteriocin activity was stable for 30 min at 50 °C and decreased to undetectable levels within 10 min at temperatures above 80 °C. Thurincin H is also stable from pH 2-7 for at least 24 h at room temperature. Thurincin H is germicidal against B. cereus spores in brain heart infusion broth, but not in Tris-NaCl buffer. The efficient purification method enables the large-scale production of pure thurincin H. The broad inhibitory spectrum of this bacteriocin may be of interest as a potential natural biopreservative in the food industry, particularly in post-processed and ready-to-eat food.
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Martins, Liliane Nachtigall; Lara, Ana Paula de Souza Stori de; Ferreira, Márcio Soares; Nunes, Adrise Medeiros; Bernardi, Daniel; Leite, Fábio Pereira Leivas; Garcia, Flávio Roberto Mello
2018-05-28
Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) is considered to be one of the major pest insects in fruit orchards worldwide. Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) strains are widely used as biological control agents and show high biological activity against different insect species. The objective of this study was to evaluate the biological activity of different strains of B. thuringiensis against A. fraterculus larvae and adults. Bioassays were performed using suspensions of bacterial spores/crystals of B. thuringiensis var. israelensis (Bti), kurstaki (Btk), and oswaldocruzi (Bto) strains at three concentrations [2 × 107, 2 × 108, and 2 × 109 colony-forming units per ml (CFU ml-1)]. At a concentration of 2 × 109 CFU ml-1, a significant larval effect (mortality 60%) was observed when compared with the control treatment. Larvae that ingested spore/crystal suspensions of Bti, Btk, or Bto bacterial strains exhibited significant larval and pupal deformations, leading to a significant decrease (~50%) in the completion of the insects' biological cycle (egg to adult). The B. thuringiensis strains (Bti, Btk, or Bto) at a concentration of 2 × 109 CFU ml-1 in combination with one food attractant (BioAnastrepha 3% or CeraTrap 1.5%) in formulations of toxic baits provided high mortality (mortality > 85%) of A. fraterculus adults 7 d after treatment. However, the Btk strain in combination with CeraTrap 1.5% caused mortality of 40%. On the basis of these results, the native bacterial strains Bti, Btk, and Bto were considered to be promising candidates as biological control agents against A. fraterculus.
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Frederiksen, Kristine; Rosenquist, Hanne; Jørgensen, Kirsten; Wilcks, Andrea
2006-01-01
A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic. PMID:16672488
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Phyllis A.W. Martin; Elizabeth A. Mongeon; Michael B. Blackburn; Dawn E. Gundersen-Rindal
2011-01-01
Bacillus thuringiensis Berliner (Bt) has been applied for gypsy moth (Lymantria dispar L.) control in forests in the northeastern U.S. for many years. The subspecies of Bt that is used (urstaki) is not common in U.S. soil. We attempted to recover Bt from...
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Distribution of phenotypes among Bacillus thuringiensis strains.
Martin, Phyllis A W; Gundersen-Rindal, Dawn E; Blackburn, Michael B
2010-06-01
An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20-86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis of esculin (E; 32.3% of isolates), acid production from salicin (A; 37.4% of isolates), acid production from sucrose (S; 34.0% of isolates), production of phospholipase C or lecithinase (L; 79.7% of isolates), and hydrolysis of starch (T; 85.8% of isolates). With the exception of acid production from salicin and hydrolysis of esculin, which were associated, the traits assorted independently. Of the 64 possible combinations of these six phenotypic characteristics, 15 combinations accounted for ca. 80% of all isolates, with the most common phenotype being TL (23.6% of isolates). Surprisingly, while the biochemical traits generally assorted independently, certain phenotypic traits associated with the parasporal crystal were correlated with certain combinations of biochemical traits. Crystals that remained attached to spores (which tended to be non-toxic to insects) were highly correlated with the phenotypes that included both L and S. Among the 15 most abundant phenotypes characterizing B. thuringiensis strains, amorphous crystals were associated with TLE, TL, T, and Ø (the absence of positive tested biochemical traits). Amorphous crystal types displayed a distinct bias toward toxicity to dipteran insects. Although all common phenotypes included B. thuringiensis isolates producing bipyramidal crystals toxic to lepidopteran insects, those with the highest abundance of these toxic crystals displayed phenotypes TLU, TLUA, TLUAE, and TLAE.
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Spicher, G; Peters, J; Borchers, U
1999-02-01
For the spores of Bacillus subtilis and Bacillus stearothermophilus as well as for spore earth (acc. DIN 58,946 Part 4 of August 1982), the dependence of resistance on the superheating of the steam used to kill germs was determined. A material (glass fibre fleece) was used as the germ carrier which does not superheat on contact with steam. The temperature of the saturated steam was 100 degrees C (B. subtilis) and 120 degrees C (B. stearothermophilus and spore earth). The yardstick for the resistance of the spores or bioindicators was the exposure period of the saturated or superheated steam at which 50% of the treated test objects no longer showed any viable test germs. The spores of Bacillus subtilis were far more sensitive to superheating of steam and reacted far more than the spores of Bacillus stearothermophilus and the germs in the spore earth. When superheating by 4 Kelvin the spores of Bacillus subtilis were approximately 2.5 times more resistant than they were to saturated steam. The resistance of Bacillus stearothermophilus and spore earth was only slightly higher up to superheating by 10 Kelvin. The spores of Bacillus subtilis had the highest resistance during superheating by 29 Kelvin; they were 119 times more resistant than they were to saturated steam. The resistance maximum of the spores of Bacillus stearothermophilus was at an superheating by around 22 Kelvin. However, the spores were only 4.1 times more resistant than they were to saturated steam. When using steam to kill germs, we must expect superheated steam. This raises the question whether the spores of Bacillus stearothermophilus, with their weaker reaction to the superheating of steam, are suitable as test germs for sterilisation with steam in all cases.
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Identifying experimental surrogates for Bacillus anthracis spores: a review
2010-01-01
Bacillus anthracis, the causative agent of anthrax, is a proven biological weapon. In order to study this threat, a number of experimental surrogates have been used over the past 70 years. However, not all surrogates are appropriate for B. anthracis, especially when investigating transport, fate and survival. Although B. atrophaeus has been widely used as a B. anthracis surrogate, the two species do not always behave identically in transport and survival models. Therefore, we devised a scheme to identify a more appropriate surrogate for B. anthracis. Our selection criteria included risk of use (pathogenicity), phylogenetic relationship, morphology and comparative survivability when challenged with biocides. Although our knowledge of certain parameters remains incomplete, especially with regards to comparisons of spore longevity under natural conditions, we found that B. thuringiensis provided the best overall fit as a non-pathogenic surrogate for B. anthracis. Thus, we suggest focusing on this surrogate in future experiments of spore fate and transport modelling. PMID:21092338
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Barboza-Corona, José Eleazar; Delgadillo-Ángeles, Jorge Luis; Castañeda-Ramírez, José Cristóbal; Barboza-Pérez, Uriel Eleazar; Casados-Vázquez, Luz Edith; Bideshi, Dennis K; del Rincón-Castro, Ma Cristina
2014-01-24
The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1. Based on mRNA analyses, up to ~9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (~70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli-pEHchiA74∆sp was ~150 mU/mL, and in E. coli-pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1-pEBchiA74∆sp showed LC₅₀s of 67.30 ng/cm² and 41.45 ng/cm², respectively. Alkali-labile ChiA74
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Xu, Shanwei; Harvey, Amanda; Barbieri, Ruth; Reuter, Tim; Stanford, Kim; Amoako, Kingsley K.; Selinger, Leonard B.; McAllister, Tim A.
2016-01-01
Anthrax outbreaks in livestock have social, economic and health implications, altering farmer’s livelihoods, impacting trade and posing a zoonotic risk. Our study investigated the survival of Bacillus thuringiensis and B. anthracis spores sporulated at 15, 20, or 37°C, over 33 days of composting. Spores (∼7.5 log10 CFU g-1) were mixed with manure and composted in laboratory scale composters. After 15 days, the compost was mixed and returned to the composter for a second cycle. Temperatures peaked at 71°C on day 2 and remained ≥55°C for an average of 7 days in the first cycle, but did not exceed 55°C in the second. For B. thuringiensis, spores generated at 15 and 21°C exhibited reduced (P < 0.05) viability of 2.7 and 2.6 log10 CFU g-1 respectively, as compared to a 0.6 log10 CFU g-1 reduction for those generated at 37°C. For B. anthracis, sporulation temperature did not impact spore survival as there was a 2.5, 2.2, and 2.8 log10 CFU g-1 reduction after composting for spores generated at 15, 21, and 37°C, respectively. For both species, spore viability declined more rapidly (P < 0.05) in the first as compared to the second composting cycle. Our findings suggest that the duration of thermophilic exposure (≥55°C) is the main factor influencing survival of B. anthracis spores in compost. As sporulation temperature did not influence survival of B. anthracis, composting may lower the viability of spores associated with carcasses infected with B. anthracis over a range of sporulation temperatures. PMID:27303388
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Current research efforts with Bacillus thuringiensis
Normand R. Dubois
1991-01-01
The bioassay of 260 strains of Bacillus thuringiensis (Bt) and 70 commercial preparations show that regression coefficient estimates may be as critical as LC5O estimates when evaluating them for future consideration.
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Guidi, Valeria; Lehner, Angelika; Lüthy, Peter; Tonolla, Mauro
2013-01-01
Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lsph) are extensively used in mosquito control programs. These biocides are the active ingredients of a commercial larvicide. Quantitative data on the fate of both Bti and Lsph applied together for the control of mosquitoes in urban drainage structures such as catch basins are lacking. We evaluated the dynamics and persistence of Bti and Lsph spores released through their concomitant application in urban catch basins in southern Switzerland. Detection and quantification of spores over time in water and sludge samples from catch basins were carried out using quantitative real-time PCR targeting both cry4A and cry4B toxin genes for Bti and the binA gene for Lsph. After treatment, Bti and Lsph spores attained concentrations of 3.76 (±0.08) and 4.13 (±0.09) log ml−1 in water, then decreased progressively over time, reaching baseline values. For both Bti and Lsph, spore levels in the order of 105 g−1 were observed in the bottom sludge two days after the treatment and remained constant for the whole test period (275 days). Indigenous Lsph strains were isolated from previously untreated catch basins. A selection of those was genotyped using pulsed field gel electrophoresis of SmaI-digested chromosomal DNA, revealing that a subset of isolates were members of the clonal population of strain 2362. No safety issues related to the use of this biopesticide in the environment have been observed during this study, because no significant increase in the number of spores was seen during the long observation period. The isolation of native Lysinibacillus sphaericus strains belonging to the same clonal population as strain 2362 from catch basins never treated with Lsph-based products indicates that the use of a combination of Bti and Lsph for the control of mosquitoes does not introduce non-indigenous microorganisms in this area. PMID:23390547
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Thomas, P; Sekhar, A C; Mujawar, M M
2014-11-01
To examine whether bacterial spores are vulnerable to impaction injury during standard spread-plating or to other modes of physical impaction. Employing heat-challenged spores of Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Lysinibacillus, Paenibacillus and Brevibacillus spp. from day-4 to day-10 nutrient agar (NA) plates in 50% ethanol, plating the spore suspension to the extent of just drying the agar surface on fresh NA (50-60 s; SP-B) was tested in comparison with the spreader-independent approach of spotting-and-tilt-spreading (SATS), or a brief plating (<10 s; SP-A). Spore CFU was significantly reduced with SP-B in different organisms (23-40%) over SATS independent of the spore size. Comparing 4-, 7- and 10-day-old B. pumilus spores, the former two displayed significant CFU reduction in SP-B indicating a spore age-related effect. Continuous plating for 2-5 min showed a reduction in spore CFU in all organisms depending on plating duration. CFU reduction effect with SP-B was less manifest on refrigerated plates where no friction was experienced but acute on prewarmed and surface-dried plates. Spreader movement over agar surface subsequent to the exhaustion of free moisture proved highly detrimental to spores. A simulated plating study by plating the spores over a plastic film till drying showed a significant reduction in spore CFU. DAPI staining and glass bead-vortexing studies confirmed spore disruption through physical impaction. Bacterial spores are vulnerable to injury during spread-plating or with other forms of physical impaction with variable effects on different genotypes independent of the spore size but altered by spore age. Implications during spore CFU estimations employing spread-plating and during spore surveillance, and the recommendation of SATS as an easier and safer alternative for spore CFU enumeration. © 2014 The Society for Applied Microbiology.
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Measurement of Metabolic Activity in Dormant Spores of Bacillus Species
2015-01-14
SECURITY CLASSIFICATION OF: Spores of Bacillus megaterium and Bacillus subtilis were harvested shortly after release from sporangia, incubated under...Measurement of Metabolic Activity in Dormant Spores of Bacillus Species Report Title Spores of Bacillus megaterium and Bacillus subtilis were...ribosomal RNA when newly harvested Bacillus subtilis spores are incubated at physiological temperatures, as well as some evidence for transcription in
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Worldwide Abundance and Distribution of Bacillus thuringiensis Isolates
Martin, Phyllis A. W.; Travers, Russell S.
1989-01-01
We found the insect control agent Bacillus thuringiensis to be a ubiquitous soil microorganism. Using acetate selection to screen soil samples, we isolated B. thuringiensis in 785 of 1,115 soil samples. These samples were obtained in the United States and 29 other countries. A total of 48% of the B. thuringiensis isolates (8,916 isolates) fit the biochemical description of known varieties, while 52% represented undescribed B. thuringiensis types. Over 60% (1,052 isolates) of the isolates tested for toxicity were toxic to insects in the orders Lepidoptera or Diptera. Soil samples were collected from various habitats, including those habitats with different numbers of insects. The current presence of insects did not predict the presence of B. thuringiensis in a particular soil sample. B. thuringiensis was most abundant in samples from Asia. PMID:16348022
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Schesser, J H; Bulla, L A
1979-05-01
Toxicity of Bacillus thuringiensis parasporal crystals to the Indian meal moth, Plodia interpunctella, is described. The numbers of insects killed were in relation to crystal dry weight. Mortality was determined by comparing adult emergence in diets treated with crystals to emergence in untreated diets. There was only a 30% survival at an application of 0.414 microgram/cm2, and the mean 50% lethal concentration value was found to be 0.299 microgram/cm2. The use of emergence data has provided a reliable and reproducible bioassay for comparing relative toxicities of crystals, spores, and other cellular components to this economically important insect.
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Raber, Ellen; Burklund, Alison
2010-01-01
Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (102 and 106 spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for a biocide, although they were found to be as effective for concentrations of 102 spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult. PMID:20709855
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Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis.
Barboza-Corona, J Eleazar; Vázquez-Acosta, Herminia; Bideshi, Dennis K; Salcedo-Hernández, Rubén
2007-02-01
Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.
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Bacteriocins synthesized by Bacillus thuringiensis: generalities and potential applications
Salazar-Marroquín, Elma Laura; Galán-Wong, Luis J.; Moreno-Medina, Víctor Ricardo; Reyes-López, Miguel Ángel; Pereyra-Alférez, Benito
2016-01-01
The members of the Bacillus thuringiensis group, commonly known as Bt, produce a huge number of metabolites, which show biocidal and antagonistic activity. B. thuringiensis is widely known for synthesizing Cry, Vip and Cyt proteins, active against insects and other parasporins with biocidal activity against certain types of cancerous cells. Nevertheless, B. thuringiensis also synthesizes compounds with antimicrobial activity, especially bacteriocins. Some B. thuringiensis bacteriocins resemble lantibiotics and other small linear peptides (class IIa) from the lactic acid bacteria bacteriocins classification system. Although many bacteriocins produced by Bt have been reported, there is no proper classification for them. In this work, we have grouped these based on molecular weight and functionality. Bacteriocins are small peptides synthesized by bacteria, presenting inhibitory activity against Gram-positive and Gram-negative bacteria and to a lesser extent against fungi. These molecules represent a good study model in the search for microbial control alternatives. Lactic acid bacteria produces a huge number of these types of molecules with great potential. Nonetheless, members of the Bacillus, cereus group, especially B. thuringiensis, emerge as an attractive alternative for obtaining bacteriocins showing novel activities. This review describes the potential applications of B. thuringiensis bacteriocins in the control of foodborne pathogens, environment and medical area. PMID:27340340
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Bacteriocins synthesized by Bacillus thuringiensis: generalities and potential applications.
Salazar-Marroquín, Elma Laura; Galán-Wong, Luis J; Moreno-Medina, Víctor Ricardo; Reyes-López, Miguel Ángel; Pereyra-Alférez, Benito
2016-07-01
The members of the Bacillus thuringiensis group, commonly known as Bt, produce a huge number of metabolites, which show biocidal and antagonistic activity. B. thuringiensis is widely known for synthesizing Cry, Vip and Cyt proteins, active against insects and other parasporins with biocidal activity against certain types of cancerous cells. Nevertheless, B. thuringiensis also synthesizes compounds with antimicrobial activity, especially bacteriocins. Some B. thuringiensis bacteriocins resemble lantibiotics and other small linear peptides (class IIa) from the lactic acid bacteria bacteriocins classification system. Although many bacteriocins produced by Bt have been reported, there is no proper classification for them. In this work, we have grouped these based on molecular weight and functionality. Bacteriocins are small peptides synthesized by bacteria, presenting inhibitory activity against Gram-positive and Gram-negative bacteria and to a lesser extent against fungi. These molecules represent a good study model in the search for microbial control alternatives. Lactic acid bacteria produces a huge number of these types of molecules with great potential. Nonetheless, members of the Bacillus, cereus group, especially B. thuringiensis, emerge as an attractive alternative for obtaining bacteriocins showing novel activities. This review describes the potential applications of B. thuringiensis bacteriocins in the control of foodborne pathogens, environment and medical area.
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Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores
Wood, Joseph P.; Meyer, Kathryn M.; Kelly, Thomas J.; Choi, Young W.; Rogers, James V.; Riggs, Karen B.; Willenberg, Zachary J.
2015-01-01
There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log10 reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially. PMID:26372011
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Nerandzic, Michelle M.; Sunkesula, Venkata C. K.; C., Thriveen Sankar; Setlow, Peter; Donskey, Curtis J.
2015-01-01
Background Due to their efficacy and convenience, alcohol-based hand sanitizers have been widely adopted as the primary method of hand hygiene in healthcare settings. However, alcohols lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We hypothesized that sporicidal activity could be induced in alcohols through alteration of physical or chemical conditions that have been shown to degrade or allow penetration of spore coats. Principal Findings Acidification, alkalinization, and heating of ethanol induced rapid sporicidal activity against C. difficile, and to a lesser extent Bacillus thuringiensis and Bacillus subtilis. The sporicidal activity of acidified ethanol was enhanced by increasing ionic strength and mild elevations in temperature. On skin, sporicidal ethanol formulations were as effective as soap and water hand washing in reducing levels of C. difficile spores. Conclusions These findings demonstrate that novel ethanol-based sporicidal hand hygiene formulations can be developed through alteration of physical and chemical conditions. PMID:26177038
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Probst, Alexander; Facius, Rainer; Wirth, Reinhard; Wolf, Marco; Moissl-Eichinger, Christine
2011-01-01
Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probable interference with life detection experiments. Therefore, space agencies measure the cultivable spore load (“bioburden”) of a spacecraft. A recent study has reported an insufficient recovery of Bacillus atrophaeus spores from Vectran fabric, a typical spacecraft airbag material (A. Probst, R. Facius, R. Wirth, and C. Moissl-Eichinger, Appl. Environ. Microbiol. 76:5148-5158, 2010). Here, 10 different sampling methods were compared for B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to 15.4%). The most efficient method, based on the wipe-rinse technique (foam-spatula protocol; 13.2% efficiency), was then compared to the current European Space Agency (ESA) standard wipe assay in sampling four different kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed the standard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additional experiments were performed by sampling Vectran fabric seeded with seven different spore concentrations and five different Bacillus species (B. atrophaeus, B. anthracis Sterne, B. megaterium, B. thuringiensis, and B. safensis). Among these, B. atrophaeus spores were recovered with the highest (13.2%) efficiency and B. anthracis Sterne spores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on test surfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this study provide a step forward in understanding the spore distribution on and recovery from rough surfaces. The results presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research. PMID:21216908
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1F protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.504 Bacillus thuringiensis Cry1F protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in the food...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are exempted...
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Schesser, J H; Bulla, L A
1979-01-01
Toxicity of Bacillus thuringiensis parasporal crystals to the Indian meal moth, Plodia interpunctella, is described. The numbers of insects killed were in relation to crystal dry weight. Mortality was determined by comparing adult emergence in diets treated with crystals to emergence in untreated diets. There was only a 30% survival at an application of 0.414 microgram/cm2, and the mean 50% lethal concentration value was found to be 0.299 microgram/cm2. The use of emergence data has provided a reliable and reproducible bioassay for comparing relative toxicities of crystals, spores, and other cellular components to this economically important insect. Images PMID:485134
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Distribution of phenotypes among Bacillus thuringiensis strains
USDA-ARS?s Scientific Manuscript database
An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20-86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis...
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Proteomic Analysis of Bacillus thuringiensis Strain 4.0718 at Different Growth Phases
Li, Xiaohui; Ding, Xuezhi; Xia, Liqiu; Sun, Yunjun; Yuan, Can; Yin, Jia
2012-01-01
The growth process of Bacillus thuringiensis Bt4.0718 strain was studied using proteomic technologies. The proteins of Bt whole cells at three phases—middle vegetative, early sporulation, and late sporulation—were extracted with lysis buffer, followed with separation by 2-DE and identified by MALDI-TOF/TOF MS. Bioactive factors such as insecticidal crystal proteins (ICPs) including Cry1Ac(3), Cry2Aa, and BTRX28, immune inhibitor (InhA), and InhA precursor were identified. InhA started to express at the middle vegetative phase, suggesting its contribution to the survival of Bt in the host body. At the early sporulation phase, ICPs started their expression. CotJC, OppA, ORF1, and SpoIVA related to the formation of crystals and spores were identified, the expression characteristics of which ensured the stable formation of crystals and spores. This study provides an important foundation for further exploration of the stable expression of ICPs, the smooth formation of crystals, and the construction of recombinant strains. PMID:22649324
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Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins
NASA Astrophysics Data System (ADS)
Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga
2010-04-01
Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.
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De Respinis, S; Demarta, A; Patocchi, N; Lüthy, P; Peduzzi, R; Tonolla, M
2006-11-01
To determine the fate of viable Bacillus thuringiensis var. israelensis (Bti) spores dispersed in the environment, using a universally applicable molecular detection methodology. Soil samples were spread on growth medium, after a temperature selection of the spores. A PCR amplification of the cry4Aa and cry4Ba insecticidal genes was applied on the colonies. Ribotyping was performed subsequently. This combined molecular method proved to be very specific for Bti, which was easily differentiated from the other B. thuringiensis serovars. A site regularly treated with Vectobac-G was chosen within the 'Bolle di Magadino' natural reserve, and monitored throughout 1 year for the detection of Bti spores. The results showed that the numbers were relatively high after insecticidal applications (1.4 x 10(5) CFU g(-1)), and decreased approx. 10-fold after 220 days. A successive treatment induced a new increase. The results show that yearly repeated use of Vectobac-G does not seem to have a major ecological impact on the 'Bolle di Magadino' natural reserve. Bti spores followed a trend leading to their eventual disappearance from the ecosystem, despite the seasonal application of this biological insecticide for more than a decade. The molecular identification of Bti cells through the PCR analysis of the delta-endotoxins genes coupled to ribotyping, is an innovative method, that has enabled the identification of this organism into wetland environments.
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Ghribi, D; Zouari, N; Jaoua, S
2005-01-01
The present work aimed to increase yields of delta-endotoxin production through adaptation of Bacillus thuringiensis cells to heat shock and sodium chloride and to investigate their involvements in bioinsecticides production improvement. Growing B. thuringiensis cells were heat treated after different incubation times to study the response of the adaptative surviving cells in terms of delta-endotoxin synthesis. Similarly, adaptation of B. thuringiensis cells to sodium chloride was investigated. Adaptation to combined stressors was also evaluated. When applied separately in the glucose-based medium, 20-min heat treatment of 6-h-old cultures and addition of 7 g l(-1) NaCl at the beginning of the incubation gave respectively 38 and 27% delta-endotoxin production improvements. Heat shock improved toxin synthesis yields, while NaCl addition improved delta-endotoxin production by increasing the spore titres without significant effect on toxin synthesis yields. Cumulative improvements (66%) were obtained by combination of the two stressors at the conditions previously established for each one. Interestingly, when the similar approach was conducted by using the large scale production medium based on gruel and fish meal, 17, 8 and 29% delta-endotoxin production improvements were respectively, obtained with heat shock, NaCl and combined stressors. Heat treatment of vegetative B. thuringiensis cells and NaCl addition to the culture media improved bioinsecticides production. Heat treatment increased toxin synthesis yields, while addition of NaCl increased biomass production yields. Cumulative improvements of 66 and 29% were obtained in glucose and economic production media, respectively. Overproduction of bioinsecticides by B. thuringiensis could be obtained by the combination of heat treatment of vegetative cells and addition of NaCl to the culture medium. This should contribute to a significant reduction of the cost of B. thuringiensis bioinsecticides production and
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Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin
2016-01-01
Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis.
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Zhu, Lei; Peng, Donghai; Wang, Yueying; Ye, Weixing; Zheng, Jinshui; Zhao, Changming; Han, Dongmei; Geng, Ce; Ruan, Lifang; He, Jin; Yu, Ziniu; Sun, Ming
2015-09-28
Bacillus thuringiensis has been globally used as a microbial pesticide for over 70 years. However, information regarding its various adaptions and virulence factors and their roles in the entomopathogenic process remains limited. In this work, we present the complete genomes of two industrially patented Bacillus thuringiensis strains (HD-1 and YBT-1520). A comparative genomic analysis showed a larger and more complicated genome constitution that included novel insecticidal toxicity-related genes (ITRGs). All of the putative ITRGs were summarized according to the steps of infection. A comparative genomic analysis showed that highly toxic strains contained significantly more ITRGs, thereby providing additional strategies for infection, immune evasion, and cadaver utilization. Furthermore, a comparative transcriptomic analysis suggested that a high expression of these ITRGs was a key factor in efficient entomopathogenicity. We identified an active extra urease synthesis system in the highly toxic strains that may aid B. thuringiensis survival in insects (similar to previous results with well-known pathogens). Taken together, these results explain the efficient entomopathogenicity of B. thuringiensis. It provides novel insights into the strategies used by B. thuringiensis to resist and overcome host immune defenses and helps identify novel toxicity factors.
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Genomic and transcriptomic insights into the efficient entomopathogenicity of Bacillus thuringiensis
Zhu, Lei; Peng, Donghai; Wang, Yueying; Ye, Weixing; Zheng, Jinshui; Zhao, Changming; Han, Dongmei; Geng, Ce; Ruan, Lifang; He, Jin; Yu, Ziniu; Sun, Ming
2015-01-01
Bacillus thuringiensis has been globally used as a microbial pesticide for over 70 years. However, information regarding its various adaptions and virulence factors and their roles in the entomopathogenic process remains limited. In this work, we present the complete genomes of two industrially patented Bacillus thuringiensis strains (HD-1 and YBT-1520). A comparative genomic analysis showed a larger and more complicated genome constitution that included novel insecticidal toxicity-related genes (ITRGs). All of the putative ITRGs were summarized according to the steps of infection. A comparative genomic analysis showed that highly toxic strains contained significantly more ITRGs, thereby providing additional strategies for infection, immune evasion, and cadaver utilization. Furthermore, a comparative transcriptomic analysis suggested that a high expression of these ITRGs was a key factor in efficient entomopathogenicity. We identified an active extra urease synthesis system in the highly toxic strains that may aid B. thuringiensis survival in insects (similar to previous results with well-known pathogens). Taken together, these results explain the efficient entomopathogenicity of B. thuringiensis. It provides novel insights into the strategies used by B. thuringiensis to resist and overcome host immune defenses and helps identify novel toxicity factors. PMID:26411888
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USDA-ARS?s Scientific Manuscript database
Sprayer comparisons and larval morality assays were conducted following SR450 backpack mist blower and Superhawk XP thermal fogger applications of Vectobac® WDG Bacillus thuringiensis israelensis (Bti) de Barjac against Culex quinquefasciatus Say. Bacillus thuringiensis israelensis was applied at m...
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Siegel, J P; Smith, A R; Novak, R J
2001-03-01
We conducted surveys to identify the species of spore-forming bacteria present in natural and artificial containers. Most of our samples came from Illinois. Identification was based on the cellular fatty acid composition of the bacterial cell wall. In addition, we utilized a custom database for commercially produced strains of Bacillus thuringiensis var. israelensis (Bti) and B. sphaericus, to differentiate between larvicidal isolates with commercial or native origin. Native Bti was present at low levels in almost all habitats but was not recovered from bromeliads and metal containers. In temporary woodland pools, 27.9% of the colonies recovered were native Bti. We did not recover larvicidal B. sphaericus in untreated habitats. VectoBac and VectoLex were applied to tires containing water and the tires were sampled 3 months and 9 months after treatment. Isolates of Bti and B. sphaericus with commercial origin were recovered as long as 9 months after application. We noticed numerous cadavers of Aedes triseriatus in several tires 9 months after treatment with VectoBac. We could not determine if this mortality resulted from recycling of Bti in these tires or whether insecticidal crystal proteins from the original treatment were resuspended. Bacillus thuringiensis var. israelensis isolates with commercial ancestry were recovered from untreated tires 9 months after application. Isolates of larvicidal B. sphaericus that differed from the bacteria in VectoLex were also recovered from untreated tires.
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Tripathi, Monika; Kumar, Arvind; Kalia, Vinay; Saxena, A K; Gujar, Govind
2016-07-01
Both, the tobacco caterpillar Spodoptera litura (Fabricius) and the cotton bollworm Helicoverpa armigera (Hibner), are serious polyphagous pests causing considerable loss to crops. Indiscriminate use of chemical pesticides for controlling them has rather resulted in their resistance development. Microbial pesticides, Bacillus thuringiensis in particular, play an important role in pest management. Here, we isolated Bacillus thuringiensis-like bacteria from the soil samples primarily collected from North East region of India along with some states viz., Haryana, Punjab, Maharashtra, West Bengal and Uttarakhand and studied their toxicity against the above two insect pests at 10 gg/g along with standard strain B. thuringiensis subsp. kurstaki HD-I and at 1 pg/g Pseudomonasfluorescens based MVPII expressing CrylAc toxin and AUG-5. Isolates AUG-5 and GTG-7 proved toxic to more than 75% larvae on the 4h as well as 7h day of the treatment of the neonates of H. armigera. The AUG-5 isolate was also effective against S. litura. Ten effective isolates (AUG-5, GTG-4, GTG-7, GTG-9, GTG-42, GTG-64, GTG-70, GTG-3S, GTG-4S and GTG-6S) were characterized using biochemical and 16S rDNA analysis. Nearly, all the isolates tested positive for utilizing monosaccharides. All selected B. thuringiensis isolates showed resistance to ampicillin and co-trimoxazole except AUG-5 to- co-trimoxazole. AUG-5 and GTG-7 were highly toxic to both insects, and possessed cryl, cry1A and cry2 genes. These isolates AUG-5 and GTG-7 also contained high CrylAc (104.8 and 88.32 ng/mg) and Cry2Ab (3792 and 1305.9 ng/mg), respectively in their spore-crystal complex. Both, AUG-5 and GTG-7 isolates, could be considered for further development as bioinsecticides. The present study has established the diversity and richness of B. thuringiensis-like isolates in soils collected from north-eastern region of India.
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Survival strategies of Bacillus spores in food.
Stecchini, Mara Lucia; Del Torre, Manuela; Polese, Pierluigi
2013-11-01
Control of bacterial spores is one of the major problem in the food preservation. Spores of Bacillus genus are commonly present in different environments, including soil and the gut of insects and animals and, as a result, they can be spread to all kind of foods. Due to their high resistance properties, their complete inactivation in food is often impossible without changing the product characteristics. Surviving spores can germinate and grow out to vegetative cells, with the consequent great risk of food spoilage and food poisoning after consumption. Spores have evolved various mechanisms, including phenotypic variability, to protect themselves from a wide range of damage resulting from food preservation treatments. Even if the phenotypic heterogeneity contributes to increase the chances of survival of Bacillus spore to conventional preservation treatments, in some specific instances, an homogeneous response could be the result of a strategy adopted by the spores to increase resistance to those treatments.
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Wang, Kui; Shu, Changlong; Soberón, Mario; Bravo, Alejandra; Zhang, Jie
2018-04-30
The goal of this work was to perform a systematic characterization of Bacillus thuringiensis (Bt) strains from the Bacillus Genetic Stock Center (BGSC) collection using Multi-Locus Sequence Typing (MLST). Different genetic markers of 158 Bacillus thuringiensis (Bt) strains from 73 different serovars stored in the BGSC, that represented 92% of the different Bt serovars of the BGSC were analyzed, the 8% that were not analyzed were not available. In addition, we analyzed 72 Bt strains from 18 serovars available at the pubMLST bcereus database, and Bt strains G03, HBF18 and Bt185, with no H serovars provided by our laboratory. We performed a systematic MLST analysis using seven housekeeping genes (glpF, gmK, ilvD, pta, pur, pycA and tpi) and analyzed correlation of the results of this analysis with strain serovars. The 233 Bt strains analyzed were assigned to 119 STs from which 19 STs were new. Genetic relationships were established by phylogenetic analysis and showed that STs could be grouped in two major Clusters containing 21 sub-groups. We found that a significant number of STs (101 in total) correlated with specific serovars, such as ST13 that corresponded to nine Bt isolates from B. thuringiensis serovar kenyae. However, other serovars showed high genetic variability and correlated with multiple STs; for example, B. thuringiensis serovar morrisoni correlated with 11 different STs. In addition, we found that 16 different STs correlated with multiple serovars (2-4 different serovars); for example, ST12 correlated with B. thuringiensis serovar alesti, dakota, palmanyolensis and sotto/dendrolimus. These data indicated that only partial correspondence between MLST and serotyping can be established. Copyright © 2018 Elsevier Inc. All rights reserved.
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Garcia-Ramon, Diana C; Molina, C Alfonso; Osuna, Antonio; Vílchez, Susana
2016-04-01
In the present work, the local isolate Bacillus pumilus 15.1 has been morphologically and biochemically characterized in order to gain a better understanding of this novel entomopathogenic strain active against Ceratitis capitata. This strain could represent an interesting biothechnological tool for the control of this pest. Here, we report on its nutrient preferences, extracellular enzyme production, motility mechanism, biofilm production, antibiotic suceptibility, natural resistance to chemical and physical insults, and morphology of the vegetative cells and spores. The pathogen was found to be β-hemolytic and susceptible to penicillin, ampicillin, chloramphenicol, gentamicin, kanamycin, rifampicin, tetracycline, and streptomycin. We also report a series of biocide, thermal, and UV treatments that reduce the viability of B. pumilus 15.1 by several orders of magnitude. Heat and chemical treatments kill at least 99.9 % of vegetative cells, but spores were much more resistant. Bleach was the only chemical that was able to completely eliminate B. pumilus 15.1 spores. Compared to the B. subtilis 168 spores, B. pumilus 15.1 spores were between 2.67 and 350 times more resistant to UV radiation while the vegetative cells of B. pumilus 15.1 were almost up to 3 orders of magnitude more resistant than the model strain. We performed electron microscopy for morphological characterization, and we observed geometric structures resembling the parasporal crystal inclusions synthesized by Bacillus thuringiensis. Some of the results obtained here such as the parasporal inclusion bodies produced by B. pumilus 15.1 could potentially represent virulence factors of this novel and potentially interesting strain.
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Omardien, Soraya; Ter Beek, Alexander; Vischer, Norbert; Montijn, Roy; Schuren, Frank; Brul, Stanley
2018-06-14
An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.
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Prevalence and Toxin Characteristics of Bacillus thuringiensis Isolated from Organic Vegetables.
Kim, Jung-Beom; Choi, Ok-Kyung; Kwon, Sun-Mok; Cho, Seung-Hak; Park, Byung-Jae; Jin, Na Young; Yu, Yong Man; Oh, Deog-Hwan
2017-08-28
The prevalence and toxin characteristics of Bacillus thuringiensis isolated from 39 organic vegetables were investigated. B. thuringiensis was detected in 30 out of the 39 organic vegetables (76.9%) with a mean value of 2.60 log CFU/g. Twenty-five out of the 30 B. thuringiensis isolates (83.3%) showed insecticidal toxicity against Spodoptera exigua . The hblCDA, nheABC , and entFM genes were found to be the major toxin genes, but the ces gene was not detected in any of the tested B. thuringiensis isolates. The hemolysin BL enterotoxin was detected in all 30 B. thuringiensis isolates (100%). The non-hemolytic enterotoxin complex was found in 27 out of 30 B. thuringiensis isolates (90.0%). The B. thuringiensis tested in this study had similar toxin gene characteristics to B. cereus , which possessed more than one toxin gene. B. thuringiensis could have the potential risk of foodborne illness based on the toxin genes and toxin-producing ability.
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Bacillus spore-based oral carriers loading curcumin for the therapy of colon cancer.
Yin, Liang; Meng, Zhan; Zhang, Yuxiao; Hu, Kaikai; Chen, Wuya; Han, Kaibin; Wu, Bao-Yan; You, Rong; Li, Chu-Hua; Jin, Ying; Guan, Yan-Qing
2018-02-10
Oral drug delivery has attracted substantial attention due to its advantages over other administration routes. Bacillus spores, as oral probiotic agents, are applied widely. In this paper, a novel Bacillus spore-based oral colon targeted carrier loading curcumin was developed for colon cancer treatment. Curcumin was linked covalently with the outer coat of Bacillus spore and folate, respectively (SPORE-CUR-FA). Bacillus spores are capable of delivering drugs to the colon area through gastric barrier, taking the advantage of its tolerance to the harsh conditions and disintegration of the outer coat of spores after germination in the colon. The drug release in vitro and in vivo of SPORE-CUR-FA was investigated. Results showed that SPORE-CUR-FA had the characteristics of colon-targeted drug release. Pharmacokinetic studies confirmed that Bacillus spore-based carriers could efficiently improve the oral bioavailability of curcumin. In vitro and in vivo anti-tumor studies showed that SPORE-CUR-FA had substantial ability for inhibiting colon cancer cells. These findings suggest that this Bacillus spore-based oral drug delivery system has a great potential for the treatment of colon cancer. Copyright © 2017 Elsevier B.V. All rights reserved.
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Transferrin Impacts Bacillus thuringiensis Biofilm Levels
Brown, Elrica; Taplin, Martha; Garcia, Angel; Williams-Mapp, Baracka
2016-01-01
The present study examined the impact of transferrin on Bacillus thuringiensis biofilms. Three commercial strains, an environmental strain (33679), the type strain (10792), and an isolate from a diseased insect (700872), were cultured in iron restricted minimal medium. All strains produced biofilm when grown in vinyl plates at 30°C. B. thuringiensis 33679 had a biofilm biomass more than twice the concentration exhibited by the other strains. The addition of transferrin resulted in slightly increased growth yields for 2 of the 3 strains tested, including 33679. In contrast, the addition of 50 μg/mL of transferrin resulted in an 80% decrease in biofilm levels for strain 33679. When the growth temperature was increased to 37°C, the addition of 50 μg/mL of transferrin increased culture turbidity for only strain 33679. Biofilm levels were again decreased in strain 33679 at 37°C. Growth of B. thuringiensis cultures in polystyrene resulted in a decrease in overall growth yields at 30°C, with biofilm levels significantly decreased for 33679 in the presence of transferrin. These findings demonstrate that transferrin impacts biofilm formation in select strains of B. thuringiensis. Identification of these differences in biofilm regulation may be beneficial in elucidating potential virulence mechanisms among the differing strains. PMID:28025643
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Characterization of Cyt2Bc Toxin from Bacillus thuringiensis subsp. medellin
Juárez-Pérez, Victor; Guerchicoff, Alejandra; Rubinstein, Clara; Delécluse, Armelle
2002-01-01
We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus. PMID:11872472
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Phage Display of a Biologically Active Bacillus thuringiensis Toxin
Kasman, Laura M.; Lukowiak, Andrew A.; Garczynski, Stephen F.; McNall, Rebecca J.; Youngman, Phil; Adang, Michael J.
1998-01-01
Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning. PMID:9687463
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BOOK REVIEW: BACILLUS THURINGIENSIS: A CORNERSTONE OF MODERN AGRICULTURE
Are you interested in the technical issues surrounding the use of Bacillus thuringiensis pesticidal traits as sprays and as plant incorporated protectants (transgenic crops)? Should the dimensions of human health, ecology, entomology, risk assessment, resistance management, and d...
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Navas, Laura E; Berretta, Marcelo F; Pérez, Melisa P; Amadio, Ariel F; Ortiz, Elio M; Sauka, Diego H; Benintende, Graciela B; Zandomeni, Rubén O
2014-01-01
We found and characterized two cry8 genes from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. These genes, cry8Kb3 and cry8Pa3, are located in a tandem array within a 13,200-bp DNA segment sequenced from a preparation of total DNA. They encode 1,169- and 1,176-amino-acid proteins, respectively. Both genes were cloned with their promoter sequences and the proteins were expressed separately in an acrystalliferous strain of B. thuringiensis leading to the formation of ovoid crystals in the recombinant strains. The toxicity against larvae of Anthonomus grandis Bh. (Coleoptera: Curculionidae) of a spore-crystal suspension from the recombinant strain containing cry8Pa3 was similar to that of the parent strain INTA Fr7-4. © 2014 S. Karger AG, Basel.
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Prabakaran, G; Hoti, S L
2008-08-01
Immobilized techniques have been used widely for the controlled release formulation of mosquitoes. Among the microbial formulations, polymeric matrices play an important role in the controlled release of microbial pesticide at rates sufficiently effective to kill mosquitoes in the field. The advantage of these matrices is that they enhance the stability of both spores and toxin against pH, temperature variations, and UV irradiation. The disadvantage of using calcium alginate beads is that they are unstable upon contact with phosphate of potassium or sodium ions rich in the mosquito habitats. To overcome these problems, attempts were made to encapsulate Bacillus thuringiensis var. israelensis within alginate by using different multivalent counterions, namely, calcium chloride, zinc sulfate, copper sulfate, cobalt chloride, and ferric chloride, and the beads formed were tested for its mosquito larvicidal activity. Among all the beads tested, zinc alginate beads resulted in maximum larvicidal activity of 98% (+/-1.40 SE) against Culex quinquefasciatus IIIrd instar larvae and maximum spore count of 3.36 x 10(5) (+/-5291.50 SE) CFU/ml. Zinc alginate beads maintained their structure for up to 48 h when shaken vigorously on a rotary shaker at 180 rpm in the presence of 10 mM potassium phosphate buffer (pH 6.8 +/- 0.1). In conclusion, our results suggest that the use of zinc sulfate as counterions to encapsulate B. thuringiensis var. israelensis within alginate may be a potent mosquito control program in the habitats where more phosphate ions are present.
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Devidas, Patil Chandrashekhar; Pandit, Borase Hemant; Vitthalrao, Patil Satish
2014-01-01
Production of indigenous isolate Bacillus thuringiensis sv2 (Bt sv2) was checked on conventional and nonconventional carbon and nitrogen sources in shake flasks. The effects on the production of biomass, toxin production, and spore formation capability of mosquito toxic strain were determined. Toxicity differs within the same strain depending on the growth medium. Bt sv2 produced with pigeon pea and soya bean flour were found highly effective with LC50 < 4 ppm against larvae of Aedes aegypti. These results were comparable with bacteria produced from Luria broth as a reference medium. Cost-effective analyses have revealed that production of biopesticide from test media is highly economical. The cost of production of Bt sv2 with soya bean flour was significantly reduced by 23-fold. The use of nonconventional sources has yielded a new knowledge in this area as the process development aspects of biomass production have been neglected as an area of research. These studies are very important from the point of media optimization for economic production of Bacillus thuringiensis based insecticides in mosquito control programmes. PMID:24592157
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Development of an aerosol surface inoculation method for bacillus spores.
Lee, Sang Don; Ryan, Shawn P; Snyder, Emily Gibb
2011-03-01
A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.
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Wang, Pengxia; Zhu, Yiguang; Zhang, Yuyang; Zhang, Chunyi; Xu, Jianyi; Deng, Yun; Peng, Donghai; Ruan, Lifang; Sun, Ming
2016-06-10
Bacillus thuringiensis and Bacillus cereus are two important species in B. cereus group. The intensive study of these strains at the molecular level and construction of genetically modified bacteria requires the development of efficient genetic tools. To insert genes into or delete genes from bacterial chromosomes, marker-less manipulation methods were employed. We present a novel genetic manipulation method for B. thuringiensis and B. cereus strains that does not leave selection markers. Our approach takes advantage of the relaxase Mob02281 encoded by plasmid pBMB0228 from Bacillus thuringiensis. In addition to its mobilization function, this Mob protein can mediate recombination between oriT sites. The Mob02281 mobilization module was associated with a spectinomycin-resistance gene to form a Mob-Spc cassette, which was flanked by the core 24-bp oriT sequences from pBMB0228. A strain in which the wild-type chromosome was replaced with the modified copy containing the Mob-Spc cassette at the target locus was obtained via homologous recombination. Thus, the spectinomycin-resistance gene can be used to screen for Mob-Spc cassette integration mutants. Recombination between the two oriT sequences mediated by Mob02281, encoded by the Mob-Spc cassette, resulted in the excision of the Mob-Spc cassette, producing the desired chromosomal alteration without introducing unwanted selection markers. We used this system to generate an in-frame deletion of a target gene in B. thuringiensis as well as a gene located in an operon of B. cereus. Moreover, we demonstrated that this system can be used to introduce a single gene or an expression cassette of interest in B. thuringiensis. The Mob/oriT recombination system provides an efficient method for unmarked genetic manipulation and for constructing genetically modified bacteria of B. thuringiensis and B. cereus. Our method extends the available genetic tools for B. thuringiensis and B. cereus strains.
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Bacillus odysseyi sp. nov., a round-spore-forming bacillus isolated from the Mars Odyssey spacecraft
NASA Technical Reports Server (NTRS)
La Duc, Myron T.; Satomi, Masataka; Venkateswaran, Kasthuri
2004-01-01
A round-spore-forming Bacillus species that produces an exosporium was isolated from the surface of the Mars Odyssey spacecraft. This novel species has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus and is a Gram-positive, aerobic, rod-shaped, endospore-forming eubacterium. Ultrathin sections of the spores showed the presence of an exosporium, spore coat, cortex and core. 16S rDNA sequence similarities between this strain, Bacillus fusiformis and Bacillus silvestris were approximately 96% and DNA-DNA reassociation values with these two bacilli were 23 and 17%, respectively. Spores of the novel species were resistant to desiccation, H2O2 and UV and gamma radiation. Of all strains tested, the spores of this strain were the most consistently resistant and survived all of the challenges posed, i.e. exposure to conditions of desiccation (100% survival), H2O2 (26% survival), UV radiation (10% survival at 660 J m(-2)) and gamma radiation (0.4% survival). The name proposed for this novel bacterium is Bacillus odysseyi sp. nov.; the type strain is 34hs-1T (=ATCC PTA-4993T=NRRL B-30641T=NBRC 100172T).
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USDA-ARS?s Scientific Manuscript database
Transgenic crops containing pyramid-stacked genes for Bacillus thuringiensis derived toxins for controlling coleopteran and lepidopteran pests are increasingly common. As part of environmental risk assessments, these crops are evaluated for toxicity against non-target organisms, and for their poten...
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Spatio-Temporal Evolution of Sporulation in Bacillus thuringiensis Biofilm.
El-Khoury, Nay; Majed, Racha; Perchat, Stéphane; Kallassy, Mireille; Lereclus, Didier; Gohar, Michel
2016-01-01
Bacillus thuringiensis can produce a floating biofilm which includes two parts: a ring and a pellicle. The ring is a thick structure which sticks to the culture container, while the pellicle extends over the whole liquid surface and joins the ring. We have followed over time, from 16 to 96 h, sporulation in the two biofilm parts. Sporulation was followed in situ in 48-wells polystyrene microtiterplates with a fluorescence binocular stereomicroscope and a spoIID-yfp transcriptional fusion. Sporulation took place much earlier in the ring than in the pellicle. In 20 h-aged biofilms, spoIID was expressed only in the ring, which could be seen as a green fluorescent circle surrounding the non-fluorescent pellicle. However, after 48 h of culture, the pellicle started to express spoIID in specific area corresponding to protrusions, and after 96 h both the ring and the whole pellicle expressed spoIID. Spore counts and microscopy observations of the ring and the pellicle harvested separately confirmed these results and revealed that sporulation occured 24 h-later in the pellicle comparatively to the ring, although both structures contained nearly 100% spores after 96 h of culture. We hypothesize that two mechanisms, due to microenvironments in the biofilm, can explain this difference. First, the ring experiences a decreased concentration of nutrients earlier than the pellicle, because of a lower exchange area with the culture medium. An second, the ring is exposed to partial dryness. Both reasons could speed up sporulation in this biofilm structure. Our results also suggest that spores in the biofilm display a phenotypic heterogeneity. These observations might be of particular significance for the food industry, since the biofilm part sticking to container walls - the ring - is likely to contain spores and will therefore resist both to washing and to cleaning procedures, and will be able to restart a new biofilm when food production has resumed.
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Kinetics of Germination of Bacillus Spores1
Vary, J. C.; Halvorson, H. O.
1965-01-01
Vary, J. C. (University of Wisconsin, Madison), and H. O. Halvorson. Kinetics or germination of Bacillus spores. J. Bacteriol. 89:1340–1347. 1965.—The kinetics of germination of Bacillus cereus strain T spores was accurately described by McCormick. To study the mechanism of germination, it is necessary to correlate the characteristic changes in a population of germinating spores with the behavior of the individual spores in the same population. Two microscopic events are apparent during germination: microlag, the time interval between the addition of l-alanine to heat-activated spores and the beginning of loss in refractility, and microgermination time, the time for the actual change in refractility to occur. The frequency distributions of both events are skewed, and appear to be independent. The effects of l-alanine concentration, heat activation, and temperature of germination on three parameters, microlag, microgermination, and per cent germination, were microscopically studied. The data are discussed in relation to the mechanism of germination, and a correlation between microlag and microgermination times with the constants of McCormick's equation has been suggested. Images PMID:14293008
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An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.
Xu, X-X; Zhang, Y-Q; Freed, S; Yu, J; Gao, Y-F; Wang, S; Ouyang, L-N; Ju, W-Y; Jin, F-L
2016-12-01
Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.
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Germination of Spores of Astrobiologically Relevant Bacillus Species in High-Salinity Environments.
Nagler, Katja; Julius, Christina; Moeller, Ralf
2016-07-01
In times of increasing space exploration and search for extraterrestrial life, new questions and challenges for planetary protection, aiming to avoid forward contamination of different planets or moons with terrestrial life, are emerging. Spore-forming bacteria such as Bacillus species have a high contamination potential due to their spores' extreme resistance, enabling them to withstand space conditions. Spores require liquid water for their conversion into a growing cell (i.e., spore germination and subsequent growth). If present, water on extraterrestrial planets or moons is likely to be closely associated with salts (e.g., in salty oceans or brines), thus constituting high-salinity environments. Spores of Bacillus subtilis can germinate despite very high salt concentrations, although salt stress does exert negative effects on this process. In this study, germination and metabolic reactivation ("outgrowth") of spores of five astrobiologically relevant Bacillus species (B. megaterium, B. pumilus SAFR-032, B. nealsonii, B. mojavensis, and B. vallismortis) in high salinity (≤3.6 M NaCl) were investigated. Spores of different species exhibited different germination and outgrowth capabilities in high salinity, which strongly depended on germination conditions, especially the exact composition of the medium. In this context, a new "universal" germination trigger for Bacillus spores, named KAGE (KCl, L-alanine, D-glucose, ectoine), was identified, which will be very useful for future comparative germination and outgrowth studies on different Bacillus species. Overall, this study yielded interesting new insights on salt stress effects on spore germination and points out the difficulty of predicting the potential of spores to contaminate salty environments on extraterrestrial celestial bodies. Bacillus species-Spores-Germination-High salinity-Salt stress-NaCl-Inhibition. Astrobiology 16, 500-512.
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Research evaluated the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface material using formaldehyde gas. Spores were dried on seven types of indoor surfaces and exposed to 1100 ppm formaldehyde gas for 10 hr. Fo...
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USDA-ARS?s Scientific Manuscript database
Pryeria sinica Moore (Lepidoptera: Zygaenidae), an invasive pest of Euonymus, is susceptible in the second instar to the Bacillus thuringiensis Berliner product Thuricide®, and to several strains isolated from other B. thuringiensis products. Third instars are also susceptible, while susceptibility...
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Menéndez Díaz, Zulema; Rodríguez Rodríguez, Jinnay; Gato Armas, René; Companioni Ibañez, Ariamys; Díaz Pérez, Manuel; Bruzón Aguila, Rosa Yirian
2012-01-01
the integration of chemical and biological methods is one of the strategies for the vector control, due to the existing environmental problems and the concerns of the community as a result of the synthetic organic insecticide actions. The bacterium called Bacillus thuringiensis var. israelensis in liquid formulation has been widely used in the vector control programs in several countries and has shown high efficacy at lab in Cuba. to determine the susceptibility of Aedes aegypti collected in the municipalities of La Habana province to Bacillus thuringiensis var. israelensis. fifteen Aedes aegypti strains, one from each municipality, were used including larvae and pupas collected in 2010 and one reference strain known as Rockefeller. The aqueous formulation of Bacillus thuringiensis var. israelensis (Bactivec, Labiofam, Cuba) was used. The bioassays complied with the World Health Organization guidelines for use of bacterial larvicides in the public health sector. The larval mortality was read after 24 hours and the results were processed by the statistical system SPSS (11.0) through Probit analysis. the evaluated mosquito strains showed high susceptibility to biolarvicide, there were no significant differences in LC50 values of Ae. aegypti strains, neither in the comparison of these values with those of the reference strain. the presented results indicate that the use of Bacillus thuringiensis var. israelensis continues to be a choice for the control of Aedes aegypti larval populations in La Habana province.
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Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores.
Tseng, Shawn; Abramzon, Nina; Jackson, James O; Lin, Wei-Jen
2012-03-01
Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.
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Development of an Aerosol Surface Inoculation Method for Bacillus Spores ▿
Lee, Sang Don; Ryan, Shawn P.; Snyder, Emily Gibb
2011-01-01
A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 107 CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies. PMID:21193670
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Effects of Mn2+ Levels on the Resistance Properties of Bacillus cereus Spores
2013-01-01
In contrast, Bacillus subtilis spores with over a 200-fold range of protoplast Mn levels exhibited no significant differences in resistance to... Bacillus subtilis . J. Bacteriol. 189:8458-8466. Coleman WH, Zhang P, Li YQ, Setlow P (2010). Mechanism of killing of spores of Bacillus cereus and...Gaidamakova EK, Matrosova VY, Daly MJ, Setlow P (2011). Effects of levels of Mn and Fe on Bacillus subtilis spore resistance, and effects of Mn 2
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Wood, J P; Lemieux, P; Betancourt, D; Kariher, P; Gatchalian, N G
2010-07-01
To obtain needed data on the dry thermal resistance of Bacillus anthracis spores and other Bacillus species for waste incinerator applications. Tests were conducted in a pilot-scale incinerator utilizing biological indicators comprised of spores of Geobacillus stearothermophilus, Bacillus atrophaeus and B. anthracis (Sterne) and embedded in building material bundles. Tests were also conducted in a dry heat oven to determine the destruction kinetics for the same species. In the pilot-scale incinerator tests, B. atrophaeus and G. stearothermophilus demonstrated similar thermal sensitivity, but B. anthracis (Sterne) was less thermally resistant than G. stearothermophilus. For the dry heat oven tests conducted at 175°C, the D-values were 0·4, 0·2 and 0·3 min for B. atrophaeus, B. anthracis (Sterne) and G. stearothermophilus, respectively. Bacillus anthracis (Sterne) possesses similar or less dry heat resistance compared to B. atrophaeus and G. stearothermophilus. Previous studies have demonstrated conditions under which bacterial spores may survive in an incinerator environment. The data from this study may assist in the selection of surrogates or indicator micro-organisms to ensure B. anthracis spores embedded in building materials are completely inactivated in an incinerator. © 2009 The Society for Applied Microbiology, Journal of Applied Microbiology. No claim to US Government works.
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Interactions of transgenic Bacillus thuringiensis insecticidal crops with spiders (Araneae)
USDA-ARS?s Scientific Manuscript database
Genetically modified crops expressing insecticidal proteins from Bacillus thuringiensis (Bt) have dramatically increased in acreage since their introduction in the mid-1990’s. Although the insecticidal mechanisms of Bt target specific pests, concerns persist regarding direct and indirect effects on...
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Hydrazine vapor inactivates Bacillus spores
NASA Astrophysics Data System (ADS)
Schubert, Wayne W.; Engler, Diane L.; Beaudet, Robert A.
2016-05-01
NASA policy restricts the total number of bacterial spores that can remain on a spacecraft traveling to any planetary body which might harbor life or have evidence of past life. Hydrazine, N2H4, is commonly used as a propellant on spacecraft. Hydrazine as a liquid is known to inactivate bacterial spores. We have now verified that hydrazine vapor also inactivates bacterial spores. After Bacillus atrophaeus ATCC 9372 spores deposited on stainless steel coupons were exposed to saturated hydrazine vapor in closed containers, the spores were recovered from the coupons, serially diluted, pour plated and the surviving bacterial colonies were counted. The exposure times required to reduce the spore population by a factor of ten, known as the D-value, were 4.70 ± 0.50 h at 25 °C and 2.85 ± 0.13 h at 35 °C. These inactivation rates are short enough to ensure that the bioburden of the surfaces and volumes would be negligible after prolonged exposure to hydrazine vapor. Thus, all the propellant tubing and internal tank surfaces exposed to hydrazine vapor do not contribute to the total spore count.
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Endotrophic Calcium, Strontium, and Barium Spores of Bacillus megaterium and Bacillus cereus1
Foerster, Harold F.; Foster, J. W.
1966-01-01
Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333–1345. 1966.—Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl2, SrCl2, or BaCl2. Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed “coat fraction” from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH. Images PMID:4956334
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Germination of Spores of Astrobiologically Relevant Bacillus Species in High-Salinity Environments
NASA Astrophysics Data System (ADS)
Nagler, Katja; Julius, Christina; Moeller, Ralf
2016-07-01
In times of increasing space exploration and search for extraterrestrial life, new questions and challenges for planetary protection, aiming to avoid forward contamination of different planets or moons with terrestrial life, are emerging. Spore-forming bacteria such as Bacillus species have a high contamination potential due to their spores' extreme resistance, enabling them to withstand space conditions. Spores require liquid water for their conversion into a growing cell (i.e., spore germination and subsequent growth). If present, water on extraterrestrial planets or moons is likely to be closely associated with salts (e.g., in salty oceans or brines), thus constituting high-salinity environments. Spores of Bacillus subtilis can germinate despite very high salt concentrations, although salt stress does exert negative effects on this process. In this study, germination and metabolic reactivation ("outgrowth") of spores of five astrobiologically relevant Bacillus species (B. megaterium, B. pumilus SAFR-032, B. nealsonii, B. mojavensis, and B. vallismortis) in high salinity (≤3.6 M NaCl) were investigated. Spores of different species exhibited different germination and outgrowth capabilities in high salinity, which strongly depended on germination conditions, especially the exact composition of the medium. In this context, a new "universal" germination trigger for Bacillus spores, named KAGE (KCl, L-alanine, D-glucose, ectoine), was identified, which will be very useful for future comparative germination and outgrowth studies on different Bacillus species. Overall, this study yielded interesting new insights on salt stress effects on spore germination and points out the difficulty of predicting the potential of spores to contaminate salty environments on extraterrestrial celestial bodies.
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NASA Technical Reports Server (NTRS)
2004-01-01
Bacillus thuringiensis (Bt), a natural bacteria found all over the Earth, has a fairly novel way of getting rid of unwanted insects. Bt forms a protein substance (shown on the right) that is not harmful to humans, birds, fish or other vertebrates. When eaten by insect larvae the protein causes a fatal loss of appetite. For over 25 years agricultural chemical companies have relied heavily upon safe Bt pesticides. New space based research promises to give the insecticide a new dimension in effectiveness and applicability. Researchers from the Consortium for Materials Development in Space along with industrial affiliates such as Abott Labs and Pern State University flew Bt on a Space Shuttle mission in the fall of 1996. Researchers expect that the Shuttle's microgravity environment will reveal new information about the protein that will make it more effective against a wider variety of pests.
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[Potential of Bacillus thuringiensis israelensis Berliner for controlling Aedes aegypti].
Polanczyk, Ricardo Antonio; Garcia, Marcelo de Oliveira; Alves, Sérgio Batista
2003-12-01
The importance of the entomopathogenic bacterium Bacillus thuringiensis israelensis in the control of Aedes aegypti is presented. The use and potential of B. thuringiensis israelensis against the mosquito vector of dengue fever is described. Other aspects such as insect's resistance development against chemicals and advantages and constraints of using microbial control are discussed. Emphasis is given to the importance of the use of this bacterium in Brazil, which could contribute significantly to solving the mosquito problem without affecting the environment, humans and others invertebrate organisms in critical regions.
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Bacillus thuringiensis and Its Pesticidal Crystal Proteins
Schnepf, E.; Crickmore, N.; Van Rie, J.; Lereclus, D.; Baum, J.; Feitelson, J.; Zeigler, D. R.; Dean, D. H.
1998-01-01
During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit. PMID:9729609
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Ni, Hong; Zeng, Siquan; Qin, Xu; Sun, Xiaowen; Zhang, Shan; Zhao, Xiuyun; Yu, Ziniu; Li, Lin
2015-01-01
Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed. PMID:25678849
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Measurements of DNA Damage and Repair in Bacillus anthracis Sterne Spores by UV Radiation
2014-09-18
MEASUREMENTS OF DNA DAMAGE AND REPAIR IN BACILLUS ANTHRACIS STERNE SPORES BY UV RADIATION...AFIT-ENP-T-14-S-01 MEASUREMENTS OF DNA DAMAGE AND REPAIR IN BACILLUS ANTHRACIS STERNE SPORES BY UV RADIATION THESIS Presented to the... DAMAGE AND REPAIR IN BACILLUS ANTHRACIS STERNE SPORES BY UV RADIATION Chelsea C. Marcum, BS Approved
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H. T. Dulmage; C. C. Beegle; N. R. Dubois
1985-01-01
The panel discussed various aspects of Bacillus thuringiensis formulations and fermentations and concluded that the only means at present of standardizing these formulations or discovering more potent strains of the Bacillus is through carefully controlled bioassays.
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González-Ponce, Karen S; Casados-Vázquez, Luz E; Salcedo-Hernández, Rubén; Bideshi, Dennis K; Del Rincón-Castro, María C; Barboza-Corona, José E
2017-05-01
In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74∆sp) was fused in frame with the sequence coding for the C-terminal crystallization domain and transcription terminator of cry1Ac. The chimeric gene was expressed under the strong pcytA-p/STAB-SD promoter system in an acrystalliferous Cry - B strain of Bacillus thuringiensis and B. thuringiensis subsp. kurstaki HD73. We showed that the chimeric ChiA74∆sp produced amorphous inclusions in both Cry - B and HD73. In addition to the amorphous inclusions putatively composed of the chimera, bipyramidal Cry1Ac crystals, smaller than the wild-type crystal, were observed in recombinant HD73, and chitinase activity was remarkably higher (75-fold) in this strain when compared with parental HD73. Moreover, we observed that lyophilized samples of a mixture containing Cry1Ac, amorphous inclusions, and spores maintained chitinase activity. Amorphous inclusions could not be separated from Cry1Ac crystals by sucrose gradient centrifugation. Interestingly, the chitinase activity of purified Cry1Ac/amorphous inclusions was 51-fold higher compared to purified Cry1Ac inclusions of parental HD73, indicating that the increased enzymatic activity was due primarily to the presence of the atypical amorphous component. The possibility that the chimera is occluded with the Cry1Ac crystal, thereby contributing to the increased endochitinolytic activity, cannot be excluded. Finally, bioassays against larvae of Spodoptera frugiperda with spore/crystals of HD73 or spore-crystal ChiA74∆sp chimeric inclusions of recombinant HD73 strain showed LC 50 s of 396.86 and 290.25 ng/cm 2 , respectively. Our study suggests a possible practical application of the chimera in formulations of B. thuringiensis-based lepidopteran larvicides.
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Liu, M; Cai, Q X; Liu, H Z; Zhang, B H; Yan, J P; Yuan, Z M
2002-01-01
To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7.0, and so did 17 strains at pH 10.0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml(-1) in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2.35-fold. This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.
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Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity
Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Caballero, Primitivo
2014-01-01
Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities. PMID:25514092
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Gillis, Annika; Mahillon, Jacques
2014-01-01
Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767
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An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals.
Rubio-Infante, Néstor; Moreno-Fierros, Leticia
2016-05-01
Crystal proteins (Cry) produced during the growth and sporulation phases of Bacillus thuringiensis (Bt) bacterium are known as delta endotoxins. These toxins are being used worldwide as bioinsecticides to control pests in agriculture, and some Cry toxins are used against mosquitoes to control vector transmission. This review summarizes the relevant information currently available regarding the biosafety and biological effects that Bt and its insecticidal Cry proteins elicit in mammals. This work was performed because of concerns regarding the possible health impact of Cry toxins on vertebrates, particularly because Bt toxins might be associated with immune-activating or allergic responses. The controversial data published to date are discussed in this review considering earlier toxicological studies of B. thuringiensis, spores, toxins and Bt crops. We discussed the experimental studies performed in humans, mice, rats and sheep as well as in diverse mammalian cell lines. Although the term 'toxic' is not appropriate for defining the effects these toxins have on mammals, they cannot be considered innocuous, as they have some physiological effects that may become pathological; thus, trials that are more comprehensive are necessary to determine their effects on mammals because knowledge in this field remains limited. Copyright © 2015 John Wiley & Sons, Ltd.
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry9C protein... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for residues...
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Translocation and insecticidal activity of Bacillus thuringiensis living inside of plants
Monnerat, Rose Gomes; Soares, Carlos Marcelo; Capdeville, Guy; Jones, Gareth; Martins, Érica Soares; Praça, Lilian; Cordeiro, Bruno Arrivabene; Braz, Shélida Vasconcelos; Dos Santos, Roseane Cavalcante; Berry, Colin
2009-01-01
Summary The major biological pesticide for the control of insect infestations of crops, Bacillus thuringiensis was found to be present naturally within cotton plants from fields that had never been treated with commercial formulations of this bacterium. The ability of B. thuringiensis to colonize plants as an endophyte was further established by the introduction of a strain marked by production of green fluorescent protein (GFP). After inoculation of this preparation close to the roots of cotton and cabbage seedlings, GFP‐marked bacteria could be re‐isolated from all parts of the plant, having entered the roots and migrated through the xylem. Leaves taken from the treated plants were able to cause toxicity when fed to the Lepidoptera Spodoptera frugiperda (cotton) and Plutella xylostella (cabbage). These results open up new horizons for understanding the natural ecology and evolution of B. thuringiensis and use of B. thuringiensis in insect control. PMID:21255282
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Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance. 174.510 Section 174.510 Protection of... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.510 Bacillus thuringiensis...
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Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores
Edmonds, Jason; Lindquist, H. D. Alan; Sabol, Jonathan; Martinez, Kenneth; Shadomy, Sean; Cymet, Tyler; Emanuel, Peter
2016-01-01
The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening. PMID:27123934
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1F protein... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1F protein... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1F protein... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1F protein... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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Vincent D' amico; John D. Podgwaite; Sara Duke; Sara Duke
2004-01-01
Bacillus thuringiensis Berliner var. tenebrionis and B. thuringiensis toxins were assayed against larval and adult Asian longhorned beetles, Anoplophora glabripennis (A. glabripennis). Preliminary in vitro assays showed some toxins to be active on whole midgut preparations in voltage clamp assays and in assays on brush border membrane vesicles formed from midgut...
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USDA-ARS?s Scientific Manuscript database
Bacillus thuringiensis is an entomopathogenic bacterium that can kill a variety of pest insects, but seldom causes epizootics because it replicates poorly in insects. By attempting to repeatedly pass lepidopteran-active B. thuringiensis strains through gypsy moth larvae, we found that only those str...
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Brar, Satinder K; Verma, M; Tyagi, R D; Valéro, J R; Surampalli, R Y
2009-10-01
This study investigated the production of biopesticides, protease and chitinase activity by Bacillus thuringiensis grown in raw wastewater sludge at high solids concentration (30 g/L). The rheology of wastewater sludge was modified with addition of Tween-80 (0.2% v/v). This addition resulted in 1.6 and 1.3-fold increase in cell and spore count, respectively. The maximum specific growth rate (micro(max)) augmented from 0.17 to 0.22 h(-1) and entomotoxicity (Tx) increased by 29.7%. Meanwhile, volumetric mass transfer coefficient (k(L)a) showed marked variations during fermentation, and oxygen uptake rate (OUR) increased 2-fold. The proteolytic activity increased while chitinase decreased for Tween amended wastewater sludge, but the entomotoxicity increased. The specific entomotoxicity followed power law when plotted against spore concentration and the relation between Tx and protease activity was linear. The viscosity varied and volume percent of particles increased in Tween-80 amended wastewater sludge and particle size (D(50)) decreased at the end of fermentation. Thus, there was an increase in entomotoxicity at higher suspended solids (30 g/L) as Tween addition improved rheology (viscosity, particle size, surface tension); enhanced maximum growth rate and OUR.
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Baril, Eugénie; Coroller, Louis; Couvert, Olivier; Leguérinel, Ivan; Postollec, Florence; Boulais, Christophe; Carlin, Frédéric; Mafart, Pierre
2012-05-01
Although sporulation environmental factors are known to impact on Bacillus spore heat resistance, they are not integrated into predictive models used to calculate the efficiency of heating processes. This work reports the influence of temperature and pH encountered during sporulation on heat resistance of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 spores. A decrease in heat resistance (δ) was observed for spores produced either at low temperature, at high temperature or at acidic pH. Sporulation temperature and pH maximizing the spore heat resistance were identified. Heat sensitivity (z) was not modified whatever the sporulation environmental factors were. A resistance secondary model inspired by the Rosso model was proposed. Sporulation temperatures and pHs minimizing or maximizing the spore heat resistance (T(min(R)), T(opt(R)), T(max(R)), pH(min(R)) and pH(opt(R))) were estimated. The goodness of the model fit was assessed for both studied strains and literature data. The estimation of the sporulation temperature and pH maximizing the spore heat resistance is of great interest to produce spores assessing the spore inactivation in the heating processes applied by the food industry. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as plant...
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Leah S. Bauer; Stuart H. Pankratz
1992-01-01
Sequential observations of the ultrastructural effects of Bacillus thuringiensis var. san diego were made on midgut epithelial cells of the cottonwood leaf beetle, Chrysomela scripta F. Larvae imbibed a droplet of B. thuringiensis var. san diego containing endotoxin and live...
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Aims: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Methods and Results: B. anthracis, B. subtilis, and G. Stearothermophilus spores were dried on seven...
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Rogers, J V; Choi, Y W; Richter, W R; Rudnicki, D C; Joseph, D W; Sabourin, C L K; Taylor, M L; Chang, J C S
2007-10-01
To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.
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Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin of...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin of...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin of...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin of...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin of...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry3Bb1 protein... Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated...
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14C Analysis of Protein Extracts from Bacillus Spores
Cappucio, Jenny A.; Sarachine Falso, Miranda J.; Kashgarian, Michaele; Buchholz, Bruce A.
2014-01-01
Investigators of bioagent incidents or interdicted materials need validated, independent analytical methods that will allow them to distinguish between recently made bioagent samples versus material drawn from the archives of a historical program. Heterotrophic bacteria convert the carbon in their food sources, growth substrate or culture media, into the biomolecules they need. The F14C (fraction modern radiocarbon) of a variety of media, Bacillus spores, and separated proteins from Bacillus spores was measured by accelerator mass spectrometry (AMS). AMS precisely measures F14C values of biological materials and has been used to date the synthesis of biomaterials over the bomb pulse era (1955 to present). The F14C of Bacillus spores reflects the radiocarbon content of the media in which they were grown. In a survey of commercial media we found that the F14C value indicated that carbon sources for the media were alive within about a year of the date of manufacture and generally of terrestrial origin. Hence, bacteria and their products can be dated using their 14C signature. Bacillus spore samples were generated onsite with defined media and carbon free purification and also obtained from archived material. Using mechanical lysis and a variety of washes with carbon free acids and bases, contaminant carbon was removed from soluble proteins to enable accurate 14C bomb-pulse dating. Since media is contemporary, 14C bomb-pulse dating of isolated soluble proteins can be used to distinguish between historical archives of bioagents and those produced from recent media. PMID:24814329
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14C Analysis of protein extracts from Bacillus spores.
Cappuccio, Jenny A; Falso, Miranda J Sarachine; Kashgarian, Michaele; Buchholz, Bruce A
2014-07-01
Investigators of bioagent incidents or interdicted materials need validated, independent analytical methods that will allow them to distinguish between recently made bioagent samples versus material drawn from the archives of a historical program. Heterotrophic bacteria convert the carbon in their food sources, growth substrate or culture media, into the biomolecules they need. The F(14)C (fraction modern radiocarbon) of a variety of media, Bacillus spores, and separated proteins from Bacillus spores was measured by accelerator mass spectrometry (AMS). AMS precisely measures F(14)C values of biological materials and has been used to date the synthesis of biomaterials over the bomb pulse era (1955 to present). The F(14)C of Bacillus spores reflects the radiocarbon content of the media in which they were grown. In a survey of commercial media we found that the F(14)C value indicated that carbon sources for the media were alive within about a year of the date of manufacture and generally of terrestrial origin. Hence, bacteria and their products can be dated using their (14)C signature. Bacillus spore samples were generated onsite with defined media and carbon free purification and also obtained from archived material. Using mechanical lysis and a variety of washes with carbon free acids and bases, contaminant carbon was removed from soluble proteins to enable accurate (14)C bomb-pulse dating. Since media is contemporary, (14)C bomb-pulse dating of isolated soluble proteins can be used to distinguish between historical archives of bioagents and those produced from recent media. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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Rogers, J V; Sabourin, C L K; Choi, Y W; Richter, W R; Rudnicki, D C; Riggs, K B; Taylor, M L; Chang, J
2005-01-01
To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Bacillus anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to > or =1000 ppm hydrogen peroxide gas for 20 min. Hydrogen peroxide exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials except G. stearothermophilus on industrial carpet. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with both surrogates. The effectiveness of gaseous hydrogen peroxide on the growth of biological indicators and spore strips was evaluated in parallel as a qualitative assessment of decontamination. At 1 and 7 days postexposure, decontaminated biological indicators and spore strips exhibited no growth, while the nondecontaminated samples displayed growth. Significant differences in decontamination efficacy of hydrogen peroxide gas on porous and nonporous surfaces were observed when comparing the mean log reduction in B. anthracis spores with B. subtilis and G. stearothermophilus spores. These results provide comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using hydrogen peroxide gas.
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NASA Astrophysics Data System (ADS)
AlOtaibi, Saad Aied
2012-12-01
In this study , two strains of Bacillus belonging to two serotypes and four of their transconjugants were screened with respect to their toxicity against lepidopterous cotton pest. . Bacterial transconjugants isolated from conjugation between both strains were evaluated for their transconjugant efficiency caused mortality in Spodoptera littoralis larvae . Two groups of bioinsecticides ; crystals , crystals and spores have been isolated from Bacillusstrains and their transconjugants . Insecticidal crystal protein ( ICP ) was specific for lepidopteran insects because of the toxin sufficient both for insect specificity and toxicity . The toxicities of these two groups against larvae of Spodoptera littoralis was expressed as transconjugant efficiency , which related to the mean number of larvae died expressed as mortality percentage . The results showed transconjugant efficiency in reducing the mean number of Spodoptera littoralis larvae feeding on leaves of Ricinus communis sprayed with bioinsecticides of Bt transconjugants. Most values of positive transconjugant efficiency related to increasing mortality percentage are due to toxicological effects appeared in response to the treatments with crystals + endospores than that of crystals alone .This indicated that crystals + endospores was more effective for increasing mortality percentage than that resulted by crystals . Higher positive transconjugant efficiency in relation to the mid parents and better parent was appeared at 168 h of treatment . The results indicated that recombinant Bacillus thuringiensis are important control agents for lepidopteran pests , as well as , susceptibility decreased with larval development . The results also suggested a potential for the deployment of these recominant entomopathogens in the management of Spodoptera. littoralis larvae .
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Architecture and assembly of the Bacillus subtilis spore coat.
Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter; Malkin, Alexander J
2014-01-01
Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of "nanodot" particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization
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Architecture and Assembly of the Bacillus subtilis Spore Coat
Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter; Malkin, Alexander J.
2014-01-01
Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of “nanodot” particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization
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Resistance: a threat to the insecticidal crystal proteins of Bacillus thuringiensis
Leah S. Bauer
1995-01-01
Insecticidal crystal proteins (also known as d-endotoxins) synthesized by the bacterium Bacillus thuringiensis Berliner (Bt) are the active ingredient of various environmentally friendly insecticides that are 1) highly compatible with natural enemies and other nontarget organisms due to narrow host specificity, 2) harmless to vertebrates, 3) biodegradable in the...
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Bacillus thuringiensis toxins trigger receptor shedding from gypsy moth midgut cells
Algimantas P. Valaitis
2007-01-01
The mechanism of action of the Cry1 insecticidal proteins produced by Bacillus thuringiensis (Bt) begins with the processing of these proteins in the larval gut. After proteolytic activation, the Bt toxins bind to specific midgut receptors and insert into the membrane of the gut epithelial cells, causing insect death.
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Recent progress in Bacillus subtilis spore-surface display: concept, progress, and future.
Wang, He; Wang, Yunxiang; Yang, Ruijin
2017-02-01
With the increased knowledge on spore structure and advances in biotechnology engineering, the newly developed spore-surface display system confers several inherent advantages over other microbial cell-surface display systems including enhanced stability and high safety. Bacillus subtilis is the most commonly used Bacillus species for spore-surface display. The expression of heterologous antigen or protein on the surface of B. subtilis spores has now been practiced for over a decade with noteworthy success. As an update and supplement to other previous reviews, we comprehensively summarize recent studies in the B. subtilis spore-surface display technique. We focus on its benefits as well as the critical factors affecting its display efficiency and offer suggestions for the future success of this field.
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Scanning Surface Potential Microscopy of Spore Adhesion on Surfaces
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lee, Ida; Chung, Eunhyea; Kweon, Hyojin
2012-01-01
The adhesion of spores of Bacillus anthracis - the cause of anthrax and a likely biological threat - to solid surfaces is an important consideration in cleanup after an accidental or deliberate release. However, because of safety concerns, directly studying B. anthracis spores with advanced instrumentation is problematic. As a first step, we are examining the electrostatic potential of Bacillus thuringiensis (Bt), which is a closely related species that is often used as a simulant to study B. anthracis. Scanning surface potential microscopy (SSPM), also known as Kelvin probe force microscopy (KPFM), was used to investigate the influence of relativemore » humidity (RH) on the surface electrostatic potential of Bt that had adhered to silica, mica, or gold substrates. AFM/SSPM side-by-side images were obtained separately in air, at various values of RH, after an aqueous droplet with spores was applied on each surface and allowed to dry before measurements. In the SSPM images, a negative potential on the surface of the spores was observed compared with that of the substrates. The surface potential decreased as the humidity increased. Spores were unable to adhere to a surface with an extremely negative potential, such as mica.« less
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Inactivation of Bacillus spores inoculated in milk by Ultra High Pressure Homogenization.
Amador Espejo, Genaro Gustavo; Hernández-Herrero, M M; Juan, B; Trujillo, A J
2014-12-01
Ultra High-Pressure Homogenization treatments at 300 MPa with inlet temperatures (Ti) of 55, 65, 75 and 85 °C were applied to commercial Ultra High Temperature treated whole milk inoculated with Bacillus cereus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus coagulans, Geobacillus stearothermophilus and Bacillus subtilis spores in order to evaluate the inactivation level achieved. Ultra High-Pressure Homogenization conditions at 300 MPa with Ti = 75 and 85 °C were capable of a spore inactivation of ∼5 log CFU/mL. Furthermore, under these processing conditions, commercial sterility (evaluated as the complete inactivation of the inoculated spores) was obtained in milk, with the exception of G. stearothermophilus and B. subtilis treated at 300 MPa with Ti = 75 °C. The results showed that G. stearothermophilus and B. subtilis have higher resistance to the Ultra High-Pressure Homogenization treatments applied than the other microorganisms inoculated and that a treatment performed at 300 MPa with Ti = 85 °C was necessary to completely inactivate these microorganisms at the spore level inoculated (∼1 × 10(6) CFU/mL). Besides, a change in the resistance of B. licheniformis, B. sporothermodurans, G. stearothermophilus and B. subtilis spores was observed as the inactivation obtained increased remarkably in treatments performed with Ti between 65 and 75 °C. This study provides important evidence of the suitability of UHPH technology for the inactivation of spores in high numbers, leading to the possibility of obtaining commercially sterile milk. Copyright © 2014 Elsevier Ltd. All rights reserved.
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PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis.
Ceron, J; Covarrubias, L; Quintero, R; Ortiz, A; Ortiz, M; Aranda, E; Lina, L; Bravo, A
1994-01-01
A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology. Thirteen highly homologous primers specific to regions within genes encoding seven different subgroups of B. thuringiensis CryI proteins were described. Differentiation among these strains was determined on the basis of the electrophoretic patterns of PCR products. B. thuringiensis strains, isolated from soil samples, were analyzed by PCR technology. Small amounts of bacterial lysates were assayed in two reaction mixtures containing six to eight primers. This method can be applied to rapidly detect the subgroups of CryI proteins that correspond with toxicity to various lepidopteran insects. Images PMID:8117089
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Effects of High Pressure on Bacillus licheniformis Spore Germination and Inactivation
Borch-Pedersen, Kristina; Mellegård, Hilde; Reineke, Kai; Boysen, Preben; Sevenich, Robert; Lindbäck, Toril
2017-01-01
ABSTRACT Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at <300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at >300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process. IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions
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Sudha, S N; Jayakumar, R; Sekar, V
1999-03-01
The abilities of Bacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere were compared; it was found that B. polymyxa colonizes the crop better. This study also showed that B. polymyxa inoculation to rice plants increased the shoot and the root growth of the crop. Efforts were made to introduce the cry1Ac gene of B. thuringiensis subsp. kurstaki into B. polymyxa so that the application of such transgenic B. polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticide and as a biofertilizer. Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain BP113, indicated efficient expression of this gene in the heterologous host. Bioassays with the first instar larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations from BP113 were toxic.
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Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...
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Bacillus thuringiensis Conjugation in Simulated Microgravity
NASA Astrophysics Data System (ADS)
Beuls, Elise; van Houdt, Rob; Leys, Natalie; Dijkstra, Camelia; Larkin, Oliver; Mahillon, Jacques
2009-10-01
Spaceflight experiments have suggested a possible effect of microgravity on the plasmid transfer among strains of the Gram-positive Bacillus thuringiensis, as opposed to no effect recorded for Gram-negative conjugation. To investigate these potential effects in a more affordable experimental setup, three ground-based microgravity simulators were tested: the Rotating Wall Vessel (RWV), the Random Positioning Machine (RPM), and a superconducting magnet. The bacterial conjugative system consisted in biparental matings between two B. thuringiensis strains, where the transfer frequencies of the conjugative plasmid pAW63 and its ability to mobilize the nonconjugative plasmid pUB110 were assessed. Specifically, potential plasmid transfers in a 0-g position (simulated microgravity) were compared to those obtained under 1-g (normal gravity) condition in each device. Statistical analyses revealed no significant difference in the conjugative and mobilizable transfer frequencies between the three different simulated microgravitational conditions and our standard laboratory condition. These important ground-based observations emphasize the fact that, though no stimulation of plasmid transfer was observed, no inhibition was observed either. In the case of Gram-positive bacteria, this ability to exchange plasmids in weightlessness, as occurs under Earth's conditions, should be seen as particularly relevant in the scope of spread of antibiotic resistances and bacterial virulence.
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Bacillus thuringiensis conjugation in simulated microgravity.
Beuls, Elise; Van Houdt, Rob; Leys, Natalie; Dijkstra, Camelia; Larkin, Oliver; Mahillon, Jacques
2009-10-01
Spaceflight experiments have suggested a possible effect of microgravity on the plasmid transfer among strains of the Gram-positive Bacillus thuringiensis, as opposed to no effect recorded for Gram-negative conjugation. To investigate these potential effects in a more affordable experimental setup, three ground-based microgravity simulators were tested: the Rotating Wall Vessel (RWV), the Random Positioning Machine (RPM), and a superconducting magnet. The bacterial conjugative system consisted in biparental matings between two B. thuringiensis strains, where the transfer frequencies of the conjugative plasmid pAW63 and its ability to mobilize the nonconjugative plasmid pUB110 were assessed. Specifically, potential plasmid transfers in a 0 g position (simulated microgravity) were compared to those obtained under 1 g (normal gravity) condition in each device. Statistical analyses revealed no significant difference in the conjugative and mobilizable transfer frequencies between the three different simulated microgravitational conditions and our standard laboratory condition. These important ground-based observations emphasize the fact that, though no stimulation of plasmid transfer was observed, no inhibition was observed either. In the case of Gram-positive bacteria, this ability to exchange plasmids in weightlessness, as occurs under Earth's conditions, should be seen as particularly relevant in the scope of spread of antibiotic resistances and bacterial virulence.
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Laboratory Assessment of the Effects of Bacillus thuringiensis on Native Lepidoptera
John W. Peacock; Dale F. Schweitzer; Jane L. Carter; Normand R. Dubois
1998-01-01
The effect of 2 formulations of Bacillus thuringiensis subsp. kurstaki (Foray 48B and Dipel 8AF) was evaluated on 42 species of native Lepidoptera in laboratory bioassays using instars that are present in the field at the time of gypsy moth suppression applications. Mortality was significant for 27 of the 42 species evaluated...
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A method for in Vivo radiolabeling Bacillus thuringiensis native δ-endotoxin crystals
C. Noah Koller; Leah S. Bauer; Robert M. Hollingworth
1995-01-01
The entomocidal CryIIIA δ-endotoxin protein of Bacillus thuringiensis var. tenebrionis is distinctive in chemistry and host range. In contrast to other δ-endotoxins, the CryIIIA parasporal crystals are toxic within the acidic midgut environment of several coleopteran species, particularly those in the family...
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Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism
Caccia, Silvia; Di Lelio, Ilaria; La Storia, Antonietta; Marinelli, Adriana; Varricchio, Paola; Franzetti, Eleonora; Banyuls, Núria; Tettamanti, Gianluca; Casartelli, Morena; Giordana, Barbara; Ferré, Juan; Gigliotti, Silvia; Pennacchio, Francesco
2016-01-01
Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host. PMID:27506800
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Ennouri, Karim; Ayed, Rayda Ben; Hassen, Hanen Ben; Mazzarello, Maura; Ottaviani, Ennio
2015-12-01
Bacillus thuringiensis (Bt) is a Gram-positive bacterium. The entomopathogenic activity of Bt is related to the existence of the crystal consisting of protoxins, also called delta-endotoxins. In order to optimize and explain the production of delta-endotoxins of Bacillus thuringiensis kurstaki, we studied seven medium components: soybean meal, starch, KH₂PO₄, K₂HPO₄, FeSO₄, MnSO₄, and MgSO₄and their relationships with the concentration of delta-endotoxins using an experimental design (Plackett-Burman design) and Bayesian networks modelling. The effects of the ingredients of the culture medium on delta-endotoxins production were estimated. The developed model showed that different medium components are important for the Bacillus thuringiensis fermentation. The most important factors influenced the production of delta-endotoxins are FeSO₄, K2HPO₄, starch and soybean meal. Indeed, it was found that soybean meal, K₂HPO₄, KH₂PO₄and starch also showed positive effect on the delta-endotoxins production. However, FeSO4 and MnSO4 expressed opposite effect. The developed model, based on Bayesian techniques, can automatically learn emerging models in data to serve in the prediction of delta-endotoxins concentrations. The constructed model in the present study implies that experimental design (Plackett-Burman design) joined with Bayesian networks method could be used for identification of effect variables on delta-endotoxins variation.
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IMPACT OF BT ( BACILLUS THURINGIENSIS ) CROPS ON BAT ACTIVITY IN SOUTH TEXAS AGROECOSYSTEMS
The widespread adoption of transgenic insecticidal crops raises concerns that nontarget species may be harmed and food webs disrupted. The goal of this research is to determine how transgenic Bt (Bacillus thuringiensis) crops impact the activity of Brazilian freetailed bats (Tada...
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Toxicity of "Bacillus thuringiensis var. Kurstaki" to the Painted Lady Butterfly, Vanessa cardui.
ERIC Educational Resources Information Center
Stalter, Richard; Nadal, Gerard; Kincaid, Dwight
2000-01-01
Reports the effects of Bacillus thuringiensis var. Kurstaki (BT), which is highly toxic, to a non-target lepidopteran, the Painted Lady butterfly. Indicates that BT kills some Painted Lady butterfly larvae at the lowest dilution tested after 48 hours. (ASK)
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Recent field experiences with Bacillus thuringiensis in Canada and research needs
Oswald N. Morris
1985-01-01
The CANUSA working group on the use of B.t. against the spruce budworm has prepared a document entitled "Guidelines for the operational use of Bacillus thuringiensis (B.t.) against the spruce budworm" following six years of extensive cooperative field trials in Canada and the U.S.A. (Morris et al 1984). The document summarized below (Table...
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Dong, Yi-Hu; Zhang, Xi-Fen; Xu, Jin-Ling; Zhang, Lian-Hui
2004-01-01
It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages. Here we report that Bacillus thuringiensis suppressed the quorum-sensing-dependent virulence of plant pathogen Erwinia carotovora through a new form of microbial antagonism, signal interference. E. carotovora produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains possess AHL-lactonase, which is a potent AHL-degrading enzyme. B. thuringiensis did not seem to interfere with the normal growth of E. carotovora; rather, it abolished the accumulation of AHL signal when they were cocultured. In planta, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptom development of potato soft rot caused by the pathogen. The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase. While all the seven AHL-lactonase-producing B. thuringiensis strains provided significant protection against E. carotovora infection, Bacillus fusiformis and Escherichia coli strains that do not process AHL-degradation enzyme showed little effect in biocontrol. Mutation of aiiA, the gene encoding AHL-lactonase in B. thuringiensis, resulted in a substantial decrease in biocontrol efficacy. These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections. PMID:14766576
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Evaluation of Surface Sampling for Bacillus Spores Using ...
Report The primary objectives of this project were to evaluate the Aggressive Air Sampling (AAS) method compared to currently used surface sampling methods and to determine if AAS is a viable option for sampling Bacillus anthracis spores.
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USDA-ARS?s Scientific Manuscript database
Strains from a collection of 3,639 diverse Bacillus thuringiensis isolates were classified based on phenotypic profiles resulting from six biochemical tests, including production of amylase (T), lecithinase (L), urease (U), acid from sucrose (S) and salicin (A), and the hydrolysis of esculin (E). St...
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Algimantas P. Valaitis
2008-01-01
The insecticidal Cry proteins produced by Bacillus thuringiensis strains are pore-forming toxins (PFTs) that bind to the midgut brush border membrane and cause extensive damage to the midgut epithelial cells of susceptible insect larvae. Force-feeding B. thuringiensis PFTs to Lymantria dispar larvae elicited...
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Manganese(II)-oxidizing Bacillus spores in Guaymas Basin hydrothermal sediments and plumes.
Dick, Gregory J; Lee, Yifan E; Tebo, Bradley M
2006-05-01
Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA and mnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor.
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Decontamination of Bacillus spores adhered to iron and ...
Journal Article This study examines the effectiveness of decontaminating Bacillus globigii spores attached to corroded iron and cement-mortar coupons with free chlorine at two pH levels, monochloramine, chlorine dioxide, ozone, peracetic acid (PAA) and acidified nitrite, followed by flushing.
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Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis
2012-11-01
REPORT Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis 14. ABSTRACT 16. SECURITY CLASSIFICATION OF... Bacillus subtilis spores with a gerP mutation triggered spore germination via nutrient germinant receptors (GRs) slowly, although this defect was...gerP, Bacillus subtilis , dipicolinic acid Xuan Y. Butzin, Anthony J. Troiano, William H. Coleman, Keren K. Griffiths, Christopher J. Doona, Florence E
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Inactivation of Bacillus anthracis Spores in Soil Matrices with ...
Report This report documents the results of a laboratory study designed to better understand the effectiveness of chlorine dioxide (ClO2) gas to decontaminate soil materials contaminated with Bacillus anthracis spores.
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Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Deng, Jiao-Yu; Cui, Zong-Qiang; Yang, Rui-Fu; Wang, Xu-Ying; Wei, Hong-Ping; Zhang, Xian-En
2013-04-15
There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30 min. This system has a linear range of 4×10³-10⁶ CFU ml⁻¹ and reproducible detection limits of 200 spores mg⁻¹ milk powder and 130 spores mg⁻¹ soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions. Copyright © 2012 Elsevier B.V. All rights reserved.
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Singh, A; Boora, K S; Chaudhary, K
2007-03-01
The persistence and insecticidal activity of native strains of Bacillus thuringiensis was evaluated in formulations containing different additives such as arrow-root powder, carboxy methyl cellulose (CMC), gum acacia, non-food grade (NFG) starch, and soluble starch. Persistence of B. thuringiensis varied with different additives used in the formulations. Among the different additives used, NFG starch provided maximum protection to B. thuringiensis and native strain 42 showed maximum persistence (83%) which was higher than that obtained in commercial formulation. In commercial formulation, the persistence of B. thuringiensis was 47% only after 3 d of spray. The feeding trials conducted on second instar larvae of H. armigera using leaves sprayed with NFG starch formulation revealed 70% larval mortality while commercial formulation showed 50% mortality during the same period.
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Code of Federal Regulations, 2011 CFR
2011-07-01
... in cotton; temporary exemption from the requirement of a tolerance. 174.530 Section 174.530... thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food commodities of cotton, cotton; cotton, undelinted...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... in cotton; temporary exemption from the requirement of a tolerance. 174.530 Section 174.530... thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food commodities of cotton, cotton; cotton, undelinted...
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Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins
Lucena, Wagner A.; Pelegrini, Patrícia B.; Martins-de-Sa, Diogo; Fonseca, Fernando C. A.; Gomes, Jose E.; de Macedo, Leonardo L. P.; da Silva, Maria Cristina M.; Oliveira, Raquel S.; Grossi-de-Sa, Maria F.
2014-01-01
Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity. PMID:25123558
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Decontamination of fluid milk containing Bacillus spores using commercial household products.
Black, D G; Taylor, T M; Kerr, H J; Padhi, S; Montville, T J; Davidson, P M
2008-03-01
Although commercial sanitizers can inactivate bacterial spores in food processing environments, relatively little data exist as to the decontamination of products and surfaces by consumers using commercial household products. Should a large scale bioterrorism incident occur in which consumer food products were contaminated with a pathogenic sporeformer such as Bacillus anthracis, there may be a need to decontaminate these products before disposal as liquid or solid waste. Studies were conducted to test the efficacy of commercial household products for inactivating spores of Bacillus cereus (used as a surrogate for B. anthracis) in vitro and in fluid milk. Validation of the resistance of the B. cereus spores was confirmed with B. anthracis spores. Fifteen commercial products, designed as either disinfectants or sanitizers or as potential sanitizers, were purchased from retail markets. Products selected had one of the following active compounds: NaOCl, HCl, H2O2, acetic acid, quaternary ammonium compounds, ammonium hydroxide, citric acid, isopropanol, NaOH, or pine oil. Compounds were diluted in water (in vitro) or in 2% fat fluid milk, and spores were exposed for up to 6 h. Products containing hypochlorite were most effective against B. cereus spores. Products containing HCl or H2O2 also reduced significant numbers of spores but at a slower rate. The resistance of spores of surrogate B. cereus strains to chlorine-containing compounds was similar to that of B. anthracis spores. Therefore, several household products on the market may be used to decontaminate fluid milk or similar food products contaminated by spores of B. anthracis.
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Raguse, Marina; Fiebrandt, Marcel; Stapelmann, Katharina; Madela, Kazimierz; Laue, Michael; Lackmann, Jan-Wilm; Thwaite, Joanne E.; Setlow, Peter; Awakowicz, Peter
2016-01-01
Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 × 107 spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques. PMID:26801572
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Peng, Donghai; Chen, Shouwen; Ruan, Lifang; Li, Lin; Yu, Ziniu; Sun, Ming
2007-07-01
The aim of this study was to evaluate the toxicology safety of a genetically modified (GM) Bacillus thuringiensis with vegetative insecticidal protein (VIP) gene. Acute and subacute toxicity studies by using its powder preparation were conducted in Wistar rats. The result of the acute study showed the no-observable-adverse-effect level (NOAEL) of this GM B. thuringiensis powder preparation was greater than 5000 mg/kg body weight (BW). In the subacute study, the data analysis of body weight gain, food and water consumptions, clinical observations, haematology, serum biochemistry, organ weight ratios and histopathological findings did not show significant differences between control and treated groups. These results proved the NOAEL of this GM B. thuringiensis powder preparation in subacute test was greater than 5000 mg/kg BW. Since both the acute and subacute oral toxicity were not detected at the highest dose recommended by OECD guidelines, this GM B. thuringiensis could be generally regarded as safe for use in bio-pesticide industry.
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Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.
Munoz, L; Sadaie, Y; Doi, R H
1978-10-10
The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.
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Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry
Waller, David F.; Hew, Brian E.; Holdaway, Charlie; Jen, Michael; Peckham, Gabriel D.
2016-01-01
Portable detection and quantitation methods for Bacillus anthracis (anthrax) spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL), while also providing a good specificity for Bacillus anthracis spores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min) result in short sample-to-signal times (less than an hour). With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field. PMID:27999382
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USDA-ARS?s Scientific Manuscript database
Studies were undertaken to further characterize the previously identified synergistic activity of Bacillus thuringiensis- and Beauveria bassiana-based biopesticides against Colorado potato beetle (CPB). A flowable concentrate of B. thuringiensis morrisoni strain tenebrionis (Bt) (Novodor® FC) and a ...
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Daniel J. Krofcheck; Algimantas P. Valaitis
2010-01-01
After ingestion by susceptible insect larvae, Bacillus thuringiensis (Bt) insecticidal proteins bind to the brush border membranes of gut epithelial cells and disrupt the integrity of the plasma membrane by forming...
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Adhikari, Atin; Yermakov, Michael; Indugula, Reshmi; Reponen, Tiina; Driks, Adam; Grinshpun, Sergey A
2016-05-01
Destruction of bioweapon facilities due to explosion or fire could aerosolize highly pathogenic microorganisms. The post-event air quality assessment is conducted through air sampling. A bioaerosol sample (often collected on a filter for further culture-based analysis) also contains combustion products, which may influence the microbial culturability and, thus, impact the outcome. We have examined the interaction between spores deposited on collection filters using two simulants of Bacillus anthracis [B. thuringiensis (Bt) and B. atrophaeus (referred to as BG)] and incoming combustion products of Al as well as Mg and B·Ti (common ingredient of metalized explosives). Spores extracted from Teflon, polycarbonate, mixed cellulose ester (MCE), and gelatin filters (most common filter media for bioaerosol sampling), which were exposed to combustion products during a short-term sampling, were analyzed by cultivation. Surprisingly, we observed that aluminum combustion products enhanced the culturability of Bt (but not BG) spores on Teflon filters increasing the culturable count by more than an order of magnitude. Testing polycarbonate and MCE filter materials also revealed a moderate increase of culturability although gelatin did not. No effect was observed with either of the two species interacting on either filter media with products originated by combustion of Mg and B·Ti. Sample contamination, spore agglomeration, effect of a filter material on the spore survival, changes in the spore wall ultrastructure and germination, as well as other factors were explored to interpret the findings. The study raises a question about the reliability of certain filter materials for collecting airborne bio-threat agents in combustion environments. Copyright © 2016 Elsevier Inc. All rights reserved.
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Leah S. Bauer
1990-01-01
A standardized laboratory bioassay was used to quantify the lethal and sub-lethal responses of larval and adult cottonwood leaf beetles, Chrysomela scripta F., to Bacillus thuringiensis var. san diego, formulated as M-One standard powder (Mycogen Corporation, San Diego). The median lethal concentration (LC
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Thermal Inactivation of Aerosolized Bacillus subtilis var. niger Spores
Mullican, Charles L.; Buchanan, Lee M.; Hoffman, Robert K.
1971-01-01
A hot-air sterilizer capable of exposing airborne microorganisms to elevated temperatures with an almost instantaneous heating time was developed and evaluated. With this apparatus, aerosolized Bacillus subtilis var. niger spores were killed in about 0.02 sec when exposed to temperatures above 260 C. This is about 500 times faster than killing times reported by others. Extrapolation and comparison of data on the time and temperature required to klll B. subtilis var. niger spores on surfaces show that approximately the same killing time is required as is necessary for spores in air, if corrections are made for the heating time of the surface. PMID:5002138
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Comparison of hand hygiene procedures for removing Bacillus cereus spores.
Sasahara, Teppei; Hayashi, Shunji; Hosoda, Kouichi; Morisawa, Yuji; Hirai, Yoshikazu
2014-01-01
Bacillus cereus is a spore-forming bacterium. B. cereus occasionally causes nosocomial infections, in which hand contamination with the spores plays an important role. Therefore, hand hygiene is the most important practice for controlling nosocomial B. cereus infections. This study aimed to determine the appropriate hand hygiene procedure for removing B. cereus spores. Thirty volunteers' hands were experimentally contaminated with B. cereus spores, after which they performed 6 different hand hygiene procedures. We compared the efficacy of the procedures in removing the spores from hands. The alcohol-based hand-rubbing procedures scarcely removed them. The soap washing procedures reduced the number of spores by more than 2 log10. Extending the washing time increased the spore-removing efficacy of the washing procedures. There was no significant difference in efficacy between the use of plain soap and antiseptic soap. Handwashing with soap is appropriate for removing B. cereus spores from hands. Alcohol-based hand-rubbing is not effective.
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Ndao, Adama; Sellamuthu, Balasubramanian; Gnepe, Jean R; Tyagi, Rajeshwar D; Valero, Jose R
2017-09-02
Pilot-scale Bacillus thuringiensis based biopesticide production (2000 L bioreactor) was conducted using starch industry wastewater (SIW) as a raw material using optimized operational parameters obtained in 15 L and 150 L fermenters. In pilot scale fermentation process the oxygen transfer rate is a major limiting factor for high product yield. Thus, the volumetric mass transfer coefficient (K L a) remains a tool to determine the oxygen transfer capacity [oxygen utilization rate (OUR) and oxygen transfer rate (OTR)] to obtain better bacterial growth rate and entomotoxicity in new bioreactor process optimization and scale-up. This study results demonstrated that the oxygen transfer rate in 2000 L bioreactor was better than 15 L and 150 L fermenters. The better oxygen transfer in 2000 L bioreactor augmented the bacterial growth [total cell (TC) and viable spore count (SC)] and delta-endotoxin yield. Prepared a stable biopesticide formulation for field use and its entomotoxicity was also evaluated. This study result corroborates the feasibility of industrial scale operation of biopesticide production using starch industry wastewater as raw material.
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2008-03-01
slips was first coated with a detergent wash. Commercially available Ivory soap shavings were diluted with sterile Millipore® water in a...environments. This removed controllable variability between the Bacillus species and increased the confidence in continued use of such surrogacy
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Antifungal activities of Bacillus thuringiensis isolates on barley and cucumber powdery mildews.
Choi, Gyung Ja; Kim, Jin-Cheol; Jang, Kyoung Soo; Lee, Dong-Hyun
2007-12-01
Fourteen Bacillus thuringiensis isolates having both insecticidal activity and in vitro antifungal activity were selected and tested for in vivo antifungal activity against tomato late blight, wheat leaf rust, tomato gray mold, and barley powdery mildew in growth chambers. All the isolates represented more than 70% disease control efficacy against at least one of four plant diseases. Specifically, 12 isolates exhibited strong control activity against barley powdery mildew. Under glasshouse conditions, four (50-02, 52-08, 52-16, and 52- 18) of the isolates also displayed potent control efficacy against cucumber powdery mildew. To our knowledge, this is the first report of B. thuringiensis isolates that have disease control efficacy against powdery mildew of barley and cucumber as well as insecticidal activity.
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The Bacillus thuringiensis cyt Genes for Hemolytic Endotoxins Constitute a Gene Family
Guerchicoff, Alejandra; Delécluse, Armelle; Rubinstein, Clara P.
2001-01-01
In the same way that cry genes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, the cyt genes for hemolytic toxins seem to compose another set of highly related genes in Bacillus thuringiensis. Although the occurrence of Cyt hemolytic factors in B. thuringiensis has been typically associated with mosquitocidal strains, we have recently shown that cyt genes are also present in strains with different pathotypes; this is the case for the morrisoni subspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such as B. thuringiensis subsp. israelensis and subsp. morrisoni PG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50 B. thuringiensis strains revealed that cyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of many B. thuringiensis strains. PMID:11229896
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Dielectric Study of the Physical State of Electrolytes and Water Within Bacillus cereus Spores
Carstensen, Edwin L.; Marquis, Robert E.; Gerhardt, Philipp
1971-01-01
Dielectric measurements revealed that dormant spores of Bacillus cereus have extremely low conductivities at high frequencies (50 MHz) and so must contain remarkably low concentrations of mobile ions both within the core and in the surrounding integuments. Activation, germination, and outgrowth were all accompanied by increases in conductivity of the cells and their suspending medium, and this result indicated that intracellular electrolytes had become ionized and leaked from the spores. High-frequency dielectric constants of spores were consistent with normal states for cell water. These values increased during successive stages of development from dormant spore to vegetative bacillus, and they could be directly related to increases in cell water content. In all, the results refuted a model of the dormant spore involving freely mobile, ionized electrolytes and supported a model involving electrostatically bound electrolytes. PMID:4998245
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Vip3C, a Novel Class of Vegetative Insecticidal Proteins from Bacillus thuringiensis
Palma, Leopoldo; Hernández-Rodríguez, Carmen Sara; Maeztu, Mireya; Hernández-Martínez, Patricia; Ruiz de Escudero, Iñigo; Escriche, Baltasar; Muñoz, Delia; Van Rie, Jeroen; Ferré, Juan
2012-01-01
Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm2. PMID:22865065
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Ben Khedher, Saoussen; Jaoua, Samir; Zouari, Nabil
2013-01-01
In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L(-1) starch, 30 g L(-1) soya bean and 9 g L(-1) sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.
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Ben Khedher, Saoussen; Jaoua, Samir; Zouari, Nabil
2013-01-01
In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L−1 starch, 30 g L−1 soya bean and 9 g L−1 sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view. PMID:24516462
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Öztopuz, Özlem; Pekin, Gülseren; Park, Ro Dong; Eltem, Rengin
2018-05-03
Bacillus is an antagonistic bacteria that shows high effectiveness against different phytopathogenic fungi and produces various lytic enzymes, such as chitosanase, chitinase, protease, and gluconase. The aim of this study is to determine Bacillus spp. for lytic enzyme production and to evaluate the antifungal effects of the selected strains for biocontrol of mycotoxigenic and phytopathogenic fungi. A total of 92 endospore-forming bacterial isolates from the 24 fig orchard soil samples were screened for chitosanase production, and six best chitosanolytic isolates were selected to determine chitinase, protease, and N-acetyl-β-hexosaminidase activity and molecularly identified. The antagonistic activities of six Bacillus strains against Aspergillus niger EGE-K-213, Aspergillus foetidus EGE-K-211, Aspergillus ochraceus EGE-K-217, and Fusarium solani KCTC 6328 were evaluated. Fungal spore germination inhibition and biomass inhibition activities were also measured against A. niger EGE-K-213. The results demonstrated that Bacillus mojavensis EGE-B-5.2i and Bacillus thuringiensis EGE-B-14.1i were more efficient antifungal agents against A. niger EGE-K-213. B. mojavensis EGE-B-5.2i has shown maximum inhibition of the biomass (30.4%), and B. thuringiensis EGE-B-14.1i has shown maximum inhibition of spore germination (33.1%) at 12 h. This is the first study reporting the potential of antagonist Bacillus strains as biocontrol agents against mycotoxigenic fungi of fig orchads.
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Code of Federal Regulations, 2013 CFR
2013-07-01
...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... RESIDUES IN FOOD Exemptions From Tolerances § 180.1154 CryIA(c) and CryIC derived delta-endotoxins of... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus...
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Code of Federal Regulations, 2012 CFR
2012-07-01
...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... RESIDUES IN FOOD Exemptions From Tolerances § 180.1154 CryIA(c) and CryIC derived delta-endotoxins of... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus...
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Code of Federal Regulations, 2014 CFR
2014-07-01
...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... RESIDUES IN FOOD Exemptions From Tolerances § 180.1154 CryIA(c) and CryIC derived delta-endotoxins of... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus...
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Code of Federal Regulations, 2010 CFR
2010-07-01
...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... RESIDUES IN FOOD Exemptions From Tolerances § 180.1154 CryIA(c) and CryIC derived delta-endotoxins of... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus...
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Code of Federal Regulations, 2011 CFR
2011-07-01
...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... RESIDUES IN FOOD Exemptions From Tolerances § 180.1154 CryIA(c) and CryIC derived delta-endotoxins of... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus...
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USDA-ARS?s Scientific Manuscript database
Nanotechnology has become one of the most promising new approaches for pest control in recent years. In this research, biocompatible silver nanoparticles (Btk-AgNPs) were synthesised by using the entomopathogenic bacterium, Bacillus thuringiensis kurstaki (Btk) as a low-cost and eco-friendly product...
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76 FR 63298 - Pesticide Products; Registration Applications
Federal Register 2010, 2011, 2012, 2013, 2014
2011-10-12
... thuringiensis subsp. kurstaki strain VBTS 2546 fermentation solids, spores, and insecticidal toxins at 67... ingredient: Bacillus thuringiensis subsp. kurstaki strain VBTS 2546 fermentation solids, spores, and...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of a...
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van Bokhorst-van de Veen, Hermien; Xie, Houyu; Esveld, Erik; Abee, Tjakko; Mastwijk, Hennie; Nierop Groot, Masja
2015-02-01
Bacterial spores are resistant to severe conditions and form a challenge to eradicate from food or food packaging material. Cold atmospheric plasma (CAP) treatment is receiving more attention as potential sterilization method at relatively mild conditions but the exact mechanism of inactivation is still not fully understood. In this study, the biocidal effect by nitrogen CAP was determined for chemical (hypochlorite and hydrogen peroxide), physical (UV) and heat-resistant spores. The three different sporeformers used are Bacillus cereus a food-borne pathogen, and Bacillus atrophaeus and Geobacillus stearothermophilus that are used as biological indicators for validation of chemical sterilization and thermal processes, respectively. The different spores showed variation in their degree of inactivation by applied heat, hypochlorite, hydrogen peroxide, and UV treatments, whereas similar inactivation results were obtained with the different spores treated with nitrogen CAP. G. stearothermophilus spores displayed high resistance to heat, hypochlorite, hydrogen peroxide, while for UV treatment B. atrophaeus spores are most tolerant. Scanning electron microscopy analysis revealed distinct morphological changes for nitrogen CAP-treated B. cereus spores including etching effects and the appearance of rough spore surfaces, whereas morphology of spores treated with heat or disinfectants showed no such changes. Moreover, microscopy analysis revealed CAP-exposed B. cereus spores to turn phase grey conceivably because of water influx indicating damage of the spores, a phenomenon that was not observed for non-treated spores. In addition, data are supplied that exclude UV radiation as determinant of antimicrobial activity of nitrogen CAP. Overall, this study shows that nitrogen CAP treatment has a biocidal effect on selected Bacillus and Geobacillus spores associated with alterations in spore surface morphology and loss of spore integrity. Copyright © 2014 Elsevier Ltd. All
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A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars
Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor
2017-01-01
Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2–1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus. PMID:28298443
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Bacillus spores as building blocks for stimuli-responsive materials and nanogenerators
NASA Astrophysics Data System (ADS)
Sahin, Ozgur; Chen, Xi
2014-03-01
Materials that mechanically respond to external chemical stimuli have applications in a wide range of fields. Inspired by biological systems, stimuli-responsive materials that can oscillate, transport fluid, mimic homeostasis, and undergo complex changes in shape have been previously demonstrated. However, the effectiveness of synthetic stimuli-responsive materials in generating work is limited when compared to mechanical actuators. During studies of bacterial sporulation, we have found that the mechanical response of Bacillus spores to water gradients exhibits an energy density of more than 10 MJ/m3, which is two orders of magnitude higher than synthetic water-responsive materials. We also identified mutations that can approximately double the energy density of the spores, and found that spores can self-assemble into dense, submicron-thick monolayers on substrates such as silicon microcantilevers and elastomer sheets, creating self-assembled actuators that can remotely generate electrical power from an evaporating body of water. The energy conversion mechanism of Bacillus spores may facilitate synthetic stimuli-responsive materials with significantly higher energy densities. We acknowledge support from the U.S. Dept. of Energy Early Career Research Program, the Wyss Institute for Biologically Inspired Engineering, and the Rowland Institute at Harvard.
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Almeida, Jamie L.; Wang, Lili; Morrow, Jayne B.; Cole, Kenneth D.
2006-01-01
Bacillus anthracis spores have been used as biological weapons and the possibility of their further use requires surveillance systems that can accurately and reliably detect their presence in the environment. These systems must collect samples from a variety of matrices, process the samples, and detect the spores. The processing of the sample may include removal of inhibitors, concentration of the target, and extraction of the target in a form suitable for detection. Suitable reference materials will allow the testing of each of these steps to determine the sensitivity and specificity of the detection systems. The development of uniform and well-characterized reference materials will allow the comparison of different devices and technologies as well as assure the continued performance of detection systems. This paper discusses the special requirements of reference materials for Bacillus anthracis spores that could be used for testing detection systems. The detection of Bacillus anthracis spores is based on recognition of specific characteristics (markers) on either the spore surface or in the nucleic acids (DNA). We have reviewed the specific markers and their relevance to characterization of reference materials. We have also included the approach for the characterization of candidate reference materials that we are developing at the NIST laboratories. Additional applications of spore reference materials would include testing sporicidal treatments, techniques for sampling the environment, and remediation of spore-contaminated environments. PMID:27274929
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Survival of Bacillus anthracis spores in fruit juices and wine.
Leishman, Oriana N; Johnson, Miranda J; Labuza, Theodore P; Diez-Gonzalez, Francisco
2010-09-01
Foods have been identified as a potential target for bioterrorism due to their essential nature and global distribution. Foods produced in bulk have the potential to have large batches of product intentionally contaminated, which could affect hundreds or thousands of individuals. Bacillus anthracis spores are one potential bioterrorism agent that may survive pasteurization and remain viable throughout the shelf life of fruit juices and cause disease if consumed. This project examined B. anthracis spore survival in orange, apple, and grape juices, as well as wine. Samples of beverages were inoculated with spores of two nonpathogenic B. anthracis strains at approximately 10(6) CFU/ml, and the spore count was determined periodically during storage for 30 days at 4°C. After this time, the counts of survival spores never declined more than 1 log CFU/ml in any of the beverage types. These results indicate that spores can survive, with little to no loss in viability, for at least a month in fruit juices and wine.
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USDA-ARS?s Scientific Manuscript database
Plodia interpunctella (Hübner) is an important stored grain insect pest worldwide, and the first lepidopteran with reported resistance to Bacillus thuringiensis (Bt) toxins. Since gut bacteria may affect Bt insecticidal activity, we determined whether P. interpunctella lacking gut enterobacteria had...
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Xu Zhou, K; Wisnivesky, F; Wilson, D I; Christie, G
2017-07-01
The influence of variable culture conditions on the size and wet density of spores of Bacillus cereus and Bacillus megaterium were examined in this work. Culture temperature and initial pH was shown to have a significant impact on the size of both species, with increasingly alkaline culture media and elevated culture temperatures resulting in spores that were, on average, up to 25% reduced in volume. Increasing concentrations of inorganic salts in sporulation media exerted differing effects on each species; whereas a fivefold increase in the concentration of all salts resulted in only minor differences to the dimensions of B. cereus spores, B. megaterium spores became more elongated, displaying an average increase in volume of almost 30%. Similarly, as the spore elongated to yield aspect ratios larger than 1·4, their shape changed from typical prolate spheroids to cylinders with hemispherical ends. In contrast with previous studies, culture conditions employed in this study exerted no discernible impact on the wet density of B. cereus or B. megaterium spores. Bacterial spores of the genera Bacillus and Clostridium represent nature's most durable cells in terms of their extreme resistance to a variety of deleterious environments. As a result, they are of concern in the food processing, healthcare and other sectors, and are of increasing biotechnological interest. Improved understanding of variance in spore size, morphology and density may aid the development of certain spore-associated applications (e.g. spore surface display) while contributing to active areas of research such as spore adhesion and resistance to heat. © 2017 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.
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Fighting Ebola with novel spore decontamination technologies for the military.
Doona, Christopher J; Feeherry, Florence E; Kustin, Kenneth; Olinger, Gene G; Setlow, Peter; Malkin, Alexander J; Leighton, Terrance
2015-01-01
subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2.
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Fighting Ebola with novel spore decontamination technologies for the military
Doona, Christopher J.; Feeherry, Florence E.; Kustin, Kenneth; Olinger, Gene G.; Setlow, Peter; Malkin, Alexander J.; Leighton, Terrance
2015-01-01
subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2. PMID:26322021
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Plomp, M; Malkin, A J
2008-06-02
Atomic force microscopy provides a unique capability to image high-resolution architecture and structural dynamics of pathogens (e.g. viruses, bacteria and bacterial spores) at near molecular resolution in native conditions. Further development of atomic force microscopy in order to enable the correlation of pathogen protein surface structures with specific gene products is essential to understand the mechanisms of the pathogen life cycle. We have applied an AFM-based immunolabeling technique for the proteomic mapping of macromolecular structures through the visualization of the binding of antibodies, conjugated with nanogold particles, to specific epitopes on Bacillus spore surfaces. This information is generated while simultaneouslymore » acquiring the surface morphology of the pathogen. The immunospecificity of this labeling method was established through the utilization of specific polyclonal and monoclonal antibodies that target spore coat and exosporium epitopes of Bacillus atrophaeus and Bacillus anthracis spores.« less
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USDA-ARS?s Scientific Manuscript database
The spotted asparagus beetle, Crioceris quatuordecimpunctata (Coleoptera: Chrysomelidae), is one of the most devastating pests of asparagus in China and elsewhere. In this study, we investigated the interaction of Bacillus thuringiensis (Bt) Cry3Aa toxin and the entomopathogenic fungus Beauveria bas...
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A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars.
Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor
2017-05-05
Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3- C -methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM: C -methyltransferase, and NADPH-dependent CDP-3- C -methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3- C -methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3- C -methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3- C -methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C -methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3- C -methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Zhang, Pengfei; Liang, Jintao; Yi, Xuan; Setlow, Peter
2014-01-01
Short exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ∼7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (Trelease) greater than the time of germinant removal (Tb) increased, while the time Tlag − Tb, where Tlag represents the time when rapid DPA release began, was decreased but rapid DPA release times (ΔTrelease = Trelease − Tlag) were increased; Factors affecting average Trelease values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants. PMID:24769693
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Curran, Harold R.; Pallansch, Michael J.
1963-01-01
Curran, Harold R. (U.S. Department of Agriculture, Washington, D.C.), and Michael J. Pallansch. Incipient germination in heavy suspensions of spores of Bacillus stearothermophilus at subminimal growth temperatures. J. Bacteriol. 86:911–918. 1963.—By use of spore (plate) counts and permeability to stain, labilization was followed periodically in heavy suspensions of washed Bacillus stearothermophilus 1518 spores incubated at different temperatures. Although vegetative proliferation did not occur below 38 C, incipient germination was rapid down to 20 C and much slower and incomplete at 14 C. Dilution of the suspension materially reduced the degree and rate of labilization. The degree of washing and use of deionized water had no appreciable influence upon early development of the spores. The results are discussed from the point of view of the possible origin and nature of the germination stimulant. Images PMID:14080801
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Tip-enhanced Raman scattering of bacillus subtilis spores
NASA Astrophysics Data System (ADS)
Rusciano, G.; Zito, G.; Pesce, G.; Sasso, A.; Isticato, R.; Ricca, E.
2015-07-01
Understanding of the complex interactions of molecules at biological interfaces is a fundamental issue in biochemistry, biotechnology as well as biomedicine. A plethora of biological processes are ruled by the molecular texture of cellular membrane: cellular communications, drug transportations and cellular recognition are just a few examples of such chemically-mediated processes. Tip-Enhanced Raman Scattering (TERS) is a novel, Raman-based technique which is ideally suited for this purpose. TERS relies on the combination of scanning probe microscopy and Raman spectroscopy. The basic idea is the use of a metalled tip as a sort of optical nano-antenna, which gives place to SERS effect close to the tip end. Herein, we present the application of TERS to analyze the surface of Bacillus subtilis spores. The choice of this biological systems is related to the fact that a number of reasons support the use of spores as a mucosal delivery system. The remarkable and well-documented resistance of spores to various environmental and toxic effects make them clear potentials as a novel, surface-display system. Our experimental outcomes demonstrate that TERS is able to provide a nano-scale chemical imaging of spore surface. Moreover, we demonstrate that TERS allows differentiation between wilde-type spore and genetically modified strains. These results hold promise for the characterization and optimization of spore surface for drug-delivery applications.
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Laboratory and field studies on the effects of Bacillus thuringiensis on non-target lepidoptera
John Peacock; Stephen Talley; Taylor Williams; Richard. Reardon
1992-01-01
Bacillus thuringiensis (B.t.) is one of the insecticides considered effective for suppression of gypsy moth infestations, and it is considered to one of the most selective in terms of its effects on other insects. Although B.t. is touted to be "environmentally safe", there is a paucity of field data to support this claim, particularly as...
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Dynamic predictive model for growth of Bacillus cereus from spores in cooked beans
USDA-ARS?s Scientific Manuscript database
Kinetic growth data of Bacillus cereus from spores in cooked beans at several isothermal conditions (between 10 to 49C) were collected. Samples were inoculated with approximately 2 log CFU/g of heat-shocked (80C/10 min) spores and stored at isothermal temperatures. B. cereus populations were deter...
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Schelkle, Bettina; Choi, Young; Baillie, Leslie W; Richter, William; Buyuk, Fatih; Celik, Elif; Wendling, Morgan; Sahin, Mitat; Gallagher, Theresa
2017-01-01
Remediation of Bacillus anthracis -contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms.
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Green, M; Heumann, M; Sokolow, R; Foster, L R; Bryant, R; Skeels, M
1990-01-01
Bacillus thuringiensis var. kurstaki (B.t.-k) is a microbial pesticide which has been widely used for over 30 years. Its safety for a human population living in sprayed areas has never been tested. Surveillance for human infections caused by B.t.-k among Lane County, Oregon residents was conducted during two seasons of aerial B.t.-k spraying for gypsy moth control. Bacillus isolates from cultures obtained for routine clinical purposes were tested for presence of Bacillus thuringiensis (B.t.). Detailed clinical information was obtained for all B.t.-positive patients. About 80,000 people lived in the first year's spray area, and 40,000 in the second year's area. A total of 55 B.t.-positive cultures were identified. The cultures had been taken from 18 different body sites or fluids. Fifty-two (95 percent) of the B.t. isolates were assessed to be probable contaminants and not the cause of clinical illness. For three patients, B.t. could neither be ruled in nor out as a pathogen. Each of these three B.t.-positive patients had preexisting medical problems. The level of risk for B.t.-k and other existing or future microbial pesticides in immunocompromised hosts deserves further study. PMID:2356910
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Code of Federal Regulations, 2010 CFR
2010-07-01
... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of a...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn...
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Effect of Hyperbaric Carbon Dioxide on Spores and Vegetative Cells of Bacillus stearothermophilus
1994-05-01
BACILLUS STEAROTHERMOPHILUS DTIC ELECTE JUN131994 D By Chester T. Roskey* Anthony Sikes *Framingham State College Framingham, MA 01701 94-18004...Spores and Vegetative Cells of Bacillus Stearothermophilus 6. AUTHOR(S) Dr. Chester T. Roskey* & Dr. Anthony Sikes 5 FUNDING NUMBERS PR: TB040...SUBJECT TERMS BACILLUS STEAROTHERMOPHILUS THERM0PHILIC BACTERIA THERM0PHILIC SPOILAGE 15. NUMBER OF PAGES 39 16 PRICE CODE 17. SECURITY
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2013-01-01
Background Bacillus anthracis is a pathogen that causes life-threatening disease--anthrax. B. anthracis spores are highly resistant to extreme temperatures and harsh chemicals. Inactivation of B. anthracis spores is important to ensure the environmental safety and public health. The 2001 bioterrorism attack involving anthrax spores has brought acute public attention and triggered extensive research on inactivation of B. anthracis spores. Single-walled carbon nanotubes (SWCNTs) as a class of emerging nanomaterial have been reported as a strong antimicrobial agent. In addition, continuous near infrared (NIR) radiation on SWCNTs induces excessive local heating which can enhance SWCNTs’ antimicrobial effect. In this study, we investigated the effects of SWCNTs coupled with NIR treatment on Bacillus anthracis spores. Results and discussion The results showed that the treatment of 10 μg/mL SWCNTs coupled with 20 min NIR significantly improved the antimicrobial effect by doubling the percentage of viable spore number reduction compared with SWCNTs alone treatment (88% vs. 42%). At the same time, SWCNTs-NIR treatment activated the germination of surviving spores and their dipicolinic acid (DPA) release during germination. The results suggested the dual effect of SWCNTs-NIR treatment on B. anthracis spores: enhanced the sporicidal effect and stimulated the germination of surviving spores. Molecular level examination showed that SWCNTs-NIR increased the expression levels (>2-fold) in 3 out of 6 germination related genes tested in this study, which was correlated to the activated germination and DPA release. SWCNTs-NIR treatment either induced or inhibited the expression of 3 regulatory genes detected in this study. When the NIR treatment time was 5 or 25 min, there were 3 out of 7 virulence related genes that showed significant decrease on expression levels (>2 fold decrease). Conclusions The results of this study demonstrated the dual effect of SWCNTs-NIR treatment on
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Bressuire-Isoard, Christelle; Broussolle, Véronique; Carlin, Frédéric
2018-05-17
Bacterial spores are resistant to physical and chemical insults, which make them a major concern for public health and for industry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Spore resistance and dormancy are important properties for applications in medicine, veterinary health, food safety, crop protection, and other domains. The resistance of bacterial spores results from a protective multilayered structure and from the unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking of dormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims to illustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in which spores form. We look at the physiological and molecular mechanisms underpinning how sporulation media and environment deeply affect spore yield, spore properties like resistance to wet heat and physical and chemical agents, germination, and further growth. For example, spore core water content decreases as sporulation temperature increases, and resistance to wet heat increases. Controlling the fate of Bacillus spores is pivotal to controlling bacterial risks and process efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulation conditions influence spore properties.
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2006-01-01
the sporangium) contributes the com- plex layers of the spore coats that encase the spore DNA. The mother cell dies and begins to fall apart at the end...spores. Bacillus spores contain a number of coat layers and some species posses an additional outermost layer called the exosporium. BA, B. cereus, and B...exosporium is the outermost layer of the BA spores, it likely contains important protein and carbohydrate markers that are recognized by antibodies
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Felker, Daniel L.; Burggraf, Larry W.
2014-01-01
Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating. PMID:24375142
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Szabo, Jeffrey G; Muhammad, Nur; Heckman, Lee; Rice, Eugene W; Hall, John
2012-04-01
Germination was evaluated as an enhancement to decontamination methods for removing Bacillus spores from drinking water infrastructure. Germinating spores before chlorinating cement mortar or flushing corroded iron was more effective than chlorinating or flushing alone.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Han, C S; Xie, G; Challacombe, J F
The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B. cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including B anthracis. Comparative analysis of these two genomesmore » with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Han, Cliff S.; Xie, Gary; Challacombe, Jean F.
The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B.cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including Banthracis. Comparative analysis of these two genomes with othermore » members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.« less
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Bernhards, Casey B.; Chen, Yan; Toutkoushian, Hannah
2014-01-01
Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn2+ or Ca2+ ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation. PMID:25384476
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Muhammad, Nur; Heckman, Lee; Rice, Eugene W.; Hall, John
2012-01-01
Germination was evaluated as an enhancement to decontamination methods for removing Bacillus spores from drinking water infrastructure. Germinating spores before chlorinating cement mortar or flushing corroded iron was more effective than chlorinating or flushing alone. PMID:22267659
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Phyllis A.W. Martin; Robert R. Jr. Farrar; Michael B. Blackburn
2011-01-01
We tested 50 lepidopteran-toxic Bacillus thuringiensis Berliner (Bt) strains with diverse phenotypes for the ability to survive repeated passages through larvae of the gypsy moth, Lymantria dispar (L.), without intervening growth on artificial media. These experiments have revealed a remarkable correlation...
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Detection of Bacillus spores using PCR and FTA filters.
Lampel, Keith A; Dyer, Deanne; Kornegay, Leroy; Orlandi, Palmer A
2004-05-01
Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.
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Morphological and Chemical Studies of the Spores and Parasporal Bodies of Bacillus laterosporus
Fitz-James, Philip C.; Young, I. Elizabeth
1958-01-01
Spores of Bacillus laterosporus were studied to determine the chemical and morphological nature of their basophilic canoe-shaped parasporal bodies. An unusually high phosphorus content of these spores compared to other Bacillus species appeared to be associated with the parasporal body. Preparations of these "canoes" still attached to the spore coats were indeed high in phosphorus, but also in nitrogen. They were free of lipide-soluble and nucleic acid phosphorus and stained for protein. Some 50 per cent of the total nitrogen, but only 6 to 10 per cent of the total P were liberated by extraction with alkali-thioglycollate (pH 11.5) or alkali alone (pH 12.2–12.5). Proteinaceous material was recovered from these alkaline extracts and electron microscopy indicated that there had been a marked loss of "canoe" substance. Extraction with acid, removed some 80 per cent of the phosphorus associated with the "canoes" as orthophosphate. Chromatographic analyses for amino acids indicated some 14 ninhydrin-positive spots in the canoe-coat preparations whereas the whole spores contained at least 16. PMID:13587561
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Schelkle, Bettina; Choi, Young; Baillie, Leslie W.; Richter, William; Buyuk, Fatih; Celik, Elif; Wendling, Morgan; Sahin, Mitat; Gallagher, Theresa
2018-01-01
Remediation of Bacillus anthracis-contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms. PMID:29379472
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Kida, N; Mochizuki, Y; Taguchi, F
2004-01-01
To develop a sporicidal reagent which shows potent activity against bacterial spores not only at ambient temperatures but also at low temperatures. Suspension tests on spores of Bacillus and Geobacillus were conducted with the reagent based on a previously reported agent (N. Kida, Y. Mochizuki and F. Taguchi, Microbiology and Immunology 2003; 47: 279-283). The modified reagent (tentatively designated as the KMT reagent) was composed of 50 mmol l(-1) EDTA-2Na, 50 mmol l(-1) ferric chloride hexahydrate (FeCl(3).6H(2)O), 50 mmol l(-1) potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3. The KMT reagent showed significant sporicidal activity against three species of Bacillus and Geobacillus spores over a wide range of temperature. The KMT reagent had many practical advantages, i.e. activity was much less affected by organic substances than was sodium hypochlorite, it did not generate any harmful gas and it was stable for a long period at ambient temperatures. The mechanism(s) of sporicidal activity of the KMT reagent was considered to be based on active iodine species penetrating the spores with enhanced permeability of the spore cortex by a synergistic effect of acid, ethanol and generated active oxygen. The data suggest that the KMT reagent shows potent sporicidal activity over a wide range temperatures and possesses many advantages for practical applications. The results indicate development of a highly applicable sporicidal reagent against Bacillus and Geobacillus spores.
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Monnerat, R; Martins, E; Praça, L; Dumas, V; Berry, C
2012-02-01
A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC50 values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain.
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Belfiore, C J; Vadlamudi, R K; Osman, Y A; Bulla, L A
1994-04-15
Biopesticides based on the bacterium Bacillus thuringiensis have attracted wide attention as safe alternatives to chemical insecticides. In this paper, we report, for the first time, the identification of a single binding protein from a coleopteran insect, Tenebrio molitor, that is specific for the cryIII toxin of B. thuringiensis. The protein appeared as a single band of 144 kDa on radioligand and immunoblots of total proteins extracted from brush border membrane vesicles of the midgut of T. molitor. Radiolabelled cryIIIA toxin bound to the protein with a Kd value of 17.5 nM and could be specifically blocked by unlabelled toxin but not by toxins from other subspecies of B. thuringiensis. This study lays the groundwork to clone the cryIIIA toxin binding protein and to determine the molecular mechanism(s) of toxin action.
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Code of Federal Regulations, 2010 CFR
2010-07-01
... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of...
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USDA-ARS?s Scientific Manuscript database
Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem...
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Jara, S; Maduell, P; Orduz, S
2006-07-01
To evaluate the distribution of Bacillus thuringiensis strains from maize and bean phylloplane and their respective soils. B. thuringiensis was isolated from the phylloplane and soil of maize and bean from three municipalities in Antioquia, Colombia. Ninety six samples of phylloplane and 24 of soil were analyzed. A total of 214 isolates were obtained from 96 phylloplane samples while 59 isolates were recovered from 24 soil samples. Sixty five per cent and 12% of the phylloplane and soil isolates, respectively, showed activity against Spodoptera frugiperda. These isolates contained delta-endotoxin proteins of 57 and 130 kDa. The most toxic isolates against S. frugiperda had the genotype cry1Aa, cry1Ac, cry1B, and cry1D. In contrast, 27% of the phylloplane isolates and 88% of the soil isolates were active against Culex quinquefasciatus and had protein profiles similar to B. thuringiensis serovar. medellin and B. thuringiensis serovar. israelensis. The most active isolates contain cry4 and cry11 genes. The predominant population of B. thuringiensis on the phylloplane harbored the cry1 gene and was active against S. frugiperda, whereas in soil, isolates harboring cry11 gene and active against C. quinquefasciatus were the majority. The predominance of specific B. thuringiensis populations, both on the leaves and in the soil, suggests the presence of selection in B. thuringiensis populations on the studied environment.
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Hosseini-Abari, Afrouzossadat; Kim, Byung-Gee; Lee, Sang-Hyuk; Emtiazi, Giti; Kim, Wooil; Kim, June-Hyung
2016-12-01
Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO 4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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USDA-ARS?s Scientific Manuscript database
Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are cultivated extensively worldwide. However, their benefits are being eroded by increasingly rapid evolution of resistance in pests. In some previously analyzed strains of three major lepidopteran pests, resistance t...
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A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation
Cote, Christopher K.; Buhr, Tony; Bernhards, Casey B.; Bohmke, Matthew D.; Calm, Alena M.; Esteban-Trexler, Josephine S.; Hunter, Melissa; Katoski, Sarah E.; Kennihan, Neil; Klimko, Christopher P.; Miller, Jeremy A.; Minter, Zachary A.; Pfarr, Jerry W.; Prugh, Amber M.; Quirk, Avery V.; Rivers, Bryan A.; Shea, April A.; Shoe, Jennifer L.; Sickler, Todd M.; Young, Alice A.; Fetterer, David P.; Welkos, Susan L.; McPherson, Derrell; Fountain, Augustus W.
2018-01-01
ABSTRACT In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10−6. Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques. IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication
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A purification and some properties of an insecticidal exotoxin from Bacillus thuringiensis Berliner
Bond, R. P. M.; Boyce, C. B. C.; French, S. J.
1969-01-01
An insecticidal exotoxin from Bacillus thuringiensis var. thuringiensis (Berliner) has been purified. The efficiency of each stage of the purification has been ascertained and the yield of toxic material estimated by means of a quantitative bioassay. It is shown that the exotoxin is an adenine derivative substituted at position 9 and having a molecular weight of approximately 825. It can be dephosphorylated enzymically or chemically under conditions that define the exotoxin as a phosphomonoester. This results in loss of toxicity, both to insects and to mice. Spectroscopic and kinetic data are presented which suggest that a β-ribofuranosyl moiety may be attached to the adenine. Glucose and allomucic acid have been positively identified as hydrolysis fragments from the exotoxin. These results are discussed and compared with the results of others on similar (or possibly identical) compounds. PMID:5820635
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Enzymatic Manganese(II) Oxidation by Metabolically Dormant Spores of Diverse Bacillus Species
Francis, Chris A.; Tebo, Bradley M.
2002-01-01
Bacterial spores are renowned for their longevity, ubiquity, and resistance to environmental insults, but virtually nothing is known regarding whether these metabolically dormant structures impact their surrounding chemical environments. In the present study, a number of spore-forming bacteria that produce dormant spores which enzymatically oxidize soluble Mn(II) to insoluble Mn(IV) oxides were isolated from coastal marine sediments. The highly charged and reactive surfaces of biogenic metal oxides dramatically influence the oxidation and sorption of both trace metals and organics in the environment. Prior to this study, the only known Mn(II)-oxidizing sporeformer was the marine Bacillus sp. strain SG-1, an extensively studied bacterium in which Mn(II) oxidation is believed to be catalyzed by a multicopper oxidase, MnxG. Phylogenetic analysis based on 16S rRNA and mnxG sequences obtained from 15 different Mn(II)-oxidizing sporeformers (including SG-1) revealed extensive diversity within the genus Bacillus, with organisms falling into several distinct clusters and lineages. In addition, active Mn(II)-oxidizing proteins of various sizes, as observed in sodium dodecyl sulfate-polyacrylamide electrophoresis gels, were recovered from the outer layers of purified dormant spores of the isolates. These are the first active Mn(II)-oxidizing enzymes identified in spores or gram-positive bacteria. Although extremely resistant to denaturation, the activities of these enzymes were inhibited by azide and o-phenanthroline, consistent with the involvement of multicopper oxidases. Overall, these studies suggest that the commonly held view that bacterial spores are merely inactive structures in the environment should be revised. PMID:11823231
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USDA-ARS?s Scientific Manuscript database
Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against some coleopteran pests, but improvements are needed in both efficacy and “time to kill” for Cry toxins to become valuable tools for use in integrated pest management. To gain insight into Bt intoxication of Coleoptera, we perfo...
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USDA-ARS?s Scientific Manuscript database
The behavior of target pests has been recognized as an important factor to appropriately define resistance management plans for Bacillus thuringiensis (Bt) crops. However, most data available do not include the possible impact resistance alleles may have on the behavior of pest larvae or adults. To ...
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Malaikozhundan, Balasubramanian; Vinodhini, Jayaraj
2018-01-01
In the present study, we reported the biological control of stored product insect pest, Callosobruchus maculatus using the entomopathogenic bacteria, Bacillus thuringiensis. A significant delay in the larval, pupal and total development period of C. maculatus was observed after treatment with B. thuringiensis at 4 × 10 8 cells/mL. Furthermore, B. thuringiensis are highly effective in the control of C. maculatus and produced 100% mortality at 4 × 10 8 cells/mL. The LC 50 value was estimated to be 3 × 10 7 cells/mL. In addition, a significant decrease in the activity of mid-gut α-amylase, cysteine protease, α & β-glucosidases, lipase, glutathione S-transferase (GST) and lactate dehydrogenase (LDH) was observed after treatment with B. thuringiensis at 4 × 10 8 cells/mL. This study concludes that B. thuringiensis are more effective against C. maculatus and could be used as a potential biological control agent in the management of stored product insect pests in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Detection of Bacillus spores within 15 minutes by surface-enhanced Raman spectroscopy
NASA Astrophysics Data System (ADS)
Shende, Chetan; Inscore, Frank; Huang, Hermes; Farquharson, Stuart; Sengupta, Atanu
2012-06-01
Since the distribution of Bacillus anthracis causing spores through the US Postal System, there has been a persistent fear that biological warfare agents (BWAs) will be used by terrorists against our military abroad and our civilians at home. Despite the substantial effort to develop BWA analyzers, they remain either too slow, produce high falsealarm rates, lack sensitivity, or cannot be fielded. Consequently there remains a need for a portable analyzer that can overcome these limitations as expressed at the 2011 Biological Weapons Convention. To meet this need we have been developing a sample system that selectively binds BWAs and produce surface-enhanced Raman (SER) spectra using portable Raman spectrometers. Here we describe the use of a short peptide ligand functionalized on silver nanoparticles to selectively capture Bacillus cereus spores (a surrogate of B. anthracis) and their subsequent detection by SER spectroscopy. This technique was used to specifically detect B. cereus spores over closely related species like B. subtilis belonging to the same genus within 15 minutes. Sensitivity of the method was demonstrated by detecting 104 B. cereus spores/mL of water. The technology, once developed should prove invaluable for rapid monitoring of BWAs, which will immensely help first responders and emergency personnel in implementing appropriate counter measures.
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Controlling gastrointestinal nematodes in cattle by Bacillus species.
Pinto, Natália Berne; de Castro, Leonardo Mortagua; de Almeida Capella, Gabriela; Motta, Tairan Ourique; de Souza Stori de Lara, Ana Paula; de Moura, Micaele Quintana; Berne, Maria Elisabeth Aires; Leite, Fábio Pereira Leivas
2017-10-15
In this study, we tested the in vitro and in vivo larvicidal activity of Bacillus species against gastrointestinal nematodes in cattle, and their viability in the presence of anthelmintics. For in vitro tests, cattle feces naturally infected with trichostrongylides were incubated with spore suspensions of Bacillus circulans (Bcir), B. thuringiensis var. osvaldocruzi (Bto), B. thuringiensis var. israelensis (Bti) or B. thuringiensis var. kurstaki (Btk). Subsequently, residual larvae were counted and identified. All of the Bacillus species showed 60% or more larvicidal effects. Bcir and Bti were selected to be incubated with anthelmintics (moxidectin, nitroxynil and levamisole), and after 24, 72, and 144h, their viability was evaluated. Bti showed highest drug resistance, maintaining a concentration of 1×10 7 CFU/mL. Based on this result, Bti was selected for in vivo tests on calves naturally infected with gastrointestinal nematodes. The calves were dived into four groups: Group 1, Bti suspension of ∼1×10 9 CFU orally administered; Group 2, Bti suspension of ∼1×10 9 CFU orally administered with levamisole (subcutaneously, 150mg); Group 3, only levamisole (subcutaneously, 150mg), and Group 4 untreated. Then 24 and 48h after treatment, larvae numbers were counted. We observed a reduction of 84%, 100%, and 100% after 48h of treatment, respectively, for Groups 1, 2 and 3 treatments in comparison with the untreated. The tested Bacillus species showed larvicidal activity against bovine trichostrongylides, and its association with anthelmintics. It may serve as a promising integrated alternative for control of gastrointestinal nematodes in cattle. Copyright © 2017 Elsevier B.V. All rights reserved.
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Swider, Catherine; Maguire, Kelly; Rickenbach, Michael; Montgomery, Madeline; Ducote, Matthew J; Marhefka, Craig A
2012-07-01
Following the September 11, 2001 terrorist attacks, letters containing Bacillus anthracis were distributed through the United States postal system killing five people. A complex forensic investigation commenced to identify the perpetrator of these mailings. A novel liquid chromatography/mass spectrometry protocol for the qualitative detection of trace levels of meglumine and diatrizoate in dried spore preparations of B. anthracis was developed. Meglumine and diatrizoate are components of radiographic imaging products that have been used to purify bacterial spores. Two separate chromatographic assays using multiple mass spectrometric analyses were developed for the detection of meglumine and diatrizoate. The assays achieved limits of detection for meglumine and diatrizoate of 1.00 and 10.0 ng/mL, respectively. Bacillus cereus T strain spores were effectively used as a surrogate for B. anthracis spores during method development and validation. This protocol was successfully applied to limited evidentiary B. anthracis spore material, providing probative information to the investigators. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.
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John S. Strazanac; George E. Seidel; Vicki Kondo; Cynthia J. Fritzler; Linda Butler
2007-01-01
Current measures for gypsy moth (Lymantria dispar L.) control emphasize the use of pheromones, growth regulators, and biopesticides. One of the biopesticides, Bacillus thuringiensis kurstaki (Btk), will continue to be necessary for immediate control of gypsy moth and other forest lepidopteran outbreaks. Although...
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Louis Dorais
1985-01-01
I want, during this presentation, to give you a spray program coordinator point of view on Bt and try to explain why things are always different in Quebec. Not always better but always different, even in the application of Bacillus thuringiensis where 4 engine aircrafts were used to control the spruce budworm, Choristoneura funiferana...
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C. N. Koller; L. S. Bauer; R. M. Hollingworth
1992-01-01
Native crystals of Bacillus thuringiensis var. san diego, a coleopteran-specific δ-endotoxin, were metabolically labeled with [35S]methionine. Specific activity was 82,000 CPM/μg (2.44 Ci/mmol). Using a universal buffer formulated with the same ionic strength at every pH, we determined that...
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Characterization of Cry toxins from autochthonous Bacillus thuringiensis isolates from Mexico.
Camacho-Millán, Raquel; Aguilar-Medina, Elsa Maribel; Quezada, Héctor; Medina-Contreras, Óscar; Patiño-López, Genaro; Cárdenas-Cota, Héctor Manuel; Ramos-Payán, Rosalío
Chemical pesticides, widely used in agriculture and vector-borne disease control, have shown toxic effects on the environment and the people in contact with them. Bacillus thuringiensis is a widely used bacterium for alternative and safer control of insect pests. Its toxins are specific for insects but innocuous for mammals and may be used as powerful adjuvants when applied with vaccines. The objective of this work was to characterize some autochthonous B. thuringiensis strains, which could be used for the control of a local pest (Diatraea considerata Heinrich) that affects sugar cane crops in Sinaloa, Mexico. Also, to evaluate these strains as a source of Cry toxins, which may be used in the future as adjuvants for some vaccines. Eight strains from field-collected dead insects were isolated. These were microbiologically identified as B. thuringiensis and confirmed by amplification and sequencing of 16S rDNA. Bioassays were performed to evaluate their pathogenicity against D. considerata, and Cry toxins were identified by proteomic analyses. An increased mortality among larvae infected with strain Bt-D was observed, and its toxin was identified as Cry1Ac. The observed data showed that the selected strain was pathogenic to D. considerata and seemed to produce Cry1Ac protein, which has been reported as an adjuvant in different types of immunization. Copyright © 2017 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.
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Oomes, S J C M; van Zuijlen, A C M; Hehenkamp, J O; Witsenboer, H; van der Vossen, J M B M; Brul, S
2007-11-30
Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated.
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NASA Astrophysics Data System (ADS)
Pujiastuti, Y.; Astuti, D. T.; Afriyani, S. R.; Suparman, S.; Irsan, C.; Sembiring, E. R.; Nugraha, S.; Mulawarman; Damiri, N.
2018-01-01
The objective of the study was to characterize the molecular weight of protein in order to be utilized as biological agent of S. litura and their cell or spores production. To investigate the molecular weight of protein was done by SDS-Page electrophoresis. Growth media used for producing B. thuringiensis were T3, LB broth and agricultural waste. The results showed that the molecular weight of protein ranged from 37 to 140 kDa. In DLM and DLKK23 isolates were found ranging from 37 to 40 kDa and from 110 to 130 kDa, respectively. KJ3R5 and KJ3P1 isolates were obtained having three protein bands ranging from 43 to 45, 73 to 80 and 110 to 130 kDa and 45-50, 75-80 and 130-140 kDa, respectively. It was predicted that isolates B. thuringiensis were belonging to Cry IA, Cry IIA, Cry IVC and Cry15c. These crystal proteins were toxic to S. litura. There was no protein bands found in the two last isolates (KJ3R3 and KJ3J4). Production of spore after sporulation in agricultural waste media ranged from 0.5 to 106 - 2.67 x 107 spores/ml showing medium level of toxicity to S. litura.
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Cruz, Andre Freire; Ishii, Takaaki
2012-01-01
Summary The aim of this research was to isolate and characterize bacteria from spores of arbuscular mycorrhizal fungi (AMF). We designated these bacteria ‘probable endobacteria’ (PE). Three bacterial strains were isolated from approximately 500 spores of Gigaspora margarita (Becker and Hall) using a hypodermic needle (diameter, 200 μm). The bacteria were identified by morphological methods and on the basis of ribosomal gene sequences as Bacillus sp. (KTCIGM01), Bacillus thuringiensis (KTCIGM02), and Paenibacillus rhizospherae (KTCIGM03). We evaluated the effect of these probable endobacteria on antagonistic activity to the soil-borne plant pathogens (SBPPs) Fusarium oxysporum f. sp. lactucae MAFF 744088, Rosellinia necatrix, Rhizoctonia solani MAFF 237426, and Pythium ultimum NBRC 100123. We also tested whether these probable endobacteria affected phosphorus solubilization, ethylene production, nitrogenase activity (NA), and stimulation of AMF hyphal growth. In addition, fresh samples of spores and hyphae were photographed using an in situ scanning electron microscope (SEM) (Quanta 250FEG; FEI Co., Japan). Bacterial aggregates (BAs), structures similar to biofilms, could be detected on the surface of hyphae and spores. We demonstrate that using extraction with an ultrathin needle, it is possible to isolate AMF-associated bacterial species that are likely derived from inside the fungal spores. PMID:23213368
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Barboza-Corona, José Eleazar; de la Fuente-Salcido, Norma; Alva-Murillo, Nayeli; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E
2009-07-02
Antimicrobial therapy is a useful tool to control bovine mastitis caused by Staphylococcus aureus, as consequence an increase in staphylococci resistant cases has been registered. Alternative strategies are desirable and bacteriocins represent attractive control agents to prevent bovine mastitis. The aim of this work was to evaluate the activity of five bacteriocins synthesized by Bacillus thuringiensis against S. aureus isolates associated to bovine mastitis. Fifty S. aureus isolates were recovered from milk composite samples of 26 Holstein lactating cows from one herd during September 2007 to February 2008 in México and susceptibility of those isolates to 12 antibiotics and 5 bacteriocins from B. thuringiensis was evaluated. S. aureus isolates were mainly resistant to penicillin (92%), dicloxacillin (86%), ampicillin (74%) and erythromycin (74%); whereas susceptibility to gentamicin, trimethoprim and tetracycline was detected at, respectively, 92%, 88%, and 72%. All S. aureus isolates showed susceptibility to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287 followed by kenyacin 404, entomocin 420 and tolworthcin 524. Our results showed that S. aureus isolates had differences in the antimicrobial resistance patterns and were susceptible to bacteriocins produced by B. thuringiensis, which could be useful as an alternative method to control bovine mastitis.
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Code of Federal Regulations, 2010 CFR
2010-07-01
... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a...
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Pathway and kinetics of cyhalothrin biodegradation by Bacillus thuringiensis strain ZS-19
Chen, Shaohua; Deng, Yinyue; Chang, Changqing; Lee, Jasmine; Cheng, Yingying; Cui, Zining; Zhou, Jianuan; He, Fei; Hu, Meiying; Zhang, Lian-Hui
2015-01-01
Cyhalothrin is a common environmental pollutant which poses increased risks to non-target organisms including human beings. This study reported for the first time a newly isolated strain, Bacillus thuringiensis ZS-19 completely degraded cyhalothrin in minimal medium within 72 h. The bacterium transformed cyhalothrin by cleavage of both the ester linkage and diaryl bond to yield six intermediate products. Moreover, a novel degradation pathway of cyhalothrin in strain ZS-19 was proposed on the basis of the identified metabolites. In addition to degradation of cyhalothrin, this strain was found to be capable of degrading 3-phenoxybenzoic acid, a common metabolite of pyrethroids. Furthermore, strain ZS-19 participated in efficient degradation of a wide range of pyrethroids including cyhalothrin, fenpropathrinn, deltamethrin, beta-cypermethrin, cyfluthrin and bifenthrin. Taken together, our results provide insights into the mechanism of cyhalothrin degradation and also highlight the promising potentials of B.thuringiensis ZS-19 in bioremediation of pyrethroid-contaminated environment. This is the first report of (i) degradation of cyhalothrin and other pyrethroids by B.thuringiensis, (ii) identification of 3-phenoxyphenyl acetonitrile and N-(2-isoproxy-phenyl)-4-phenoxy-benzamide as the metabolites in the degradation pathway of pyrethroids, and (iii) a pathway of degradation of cyhalothrin by cleavage of both the ester linkage and diaryl bond in a microorganism. PMID:25740758
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Perry, K. Allison; O’Connell, Heather A.; Rose, Laura J.; Noble-Wang, Judith A.; Arduino, Matthew J.
2016-01-01
The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis. Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at −15°C, 5°C, 21°C, or 35°C for 0–7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T0) was determined for each variable. No differences were observed in recovery between swabs held at −15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 102, p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at −15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores. PMID:27213119
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Perry, K Allison; O'Connell, Heather A; Rose, Laura J; Noble-Wang, Judith A; Arduino, Matthew J
The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis . Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T 0 ) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 10 2 , p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.
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Faille, Christine; Jullien, Celine; Fontaine, Francoise; Bellon-Fontaine, Marie-Noelle; Slomianny, Christian; Benezech, Thierry
2002-08-01
The ability of bacterial spores and vegetative cells to adhere to inert surfaces was investigated by means of the number of adherent spores (Bacillus cereus and Bacillus subtilis spores) and Escherichia coli cells and their resistance to cleaning or rinsing procedures (adhesion strength). Six materials (glass, stainless steel, polyethylene high density (PEHD), polyamide-6, polyvinyl chloride, and Teflon) were tested. Slight differences in the number of adherent spores (less than 1 log unit) were observed between materials, but a higher number of adherent E. coli cells was found on the hydrophobic materials PEHD and Teflon. Conversely, the resistance of both B. cereus and B. subtilis spores to a cleaning procedure was significantly affected by the material. Hydrophobic materials were harder to clean. The topography parameter derived from the Abbott-Firestone curve, RVK, and, to a lesser extent, the widely used roughness parameters RA (average roughness) and Rz (maximal roughness), were related to the number of adherent cells. Lastly, the soiling level as well as the adhesion strength were shown to depend largely on the microorganism. The number of adhering B. cereus hydrophobic spores and their resistance to a cleaning procedure were found to be 10 times greater than those of the B. subtilis hydrophilic spores. Escherichia coli was loosely bound to all the materials tested, even after 24 h biofilm formation.
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Berendsen, Erwin M.; Koning, Rosella A.; Boekhorst, Jos; de Jong, Anne; Kuipers, Oscar P.; Wells-Bennik, Marjon H. J.
2016-01-01
Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant) rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores) grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain. PMID:27994575
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Commercial formulations of Bacillus thuringiensis for control of Indian meal moth.
Schesser, J H
1976-01-01
Doses of four commercial formulations and one experimental formulation of Bacillus thuringiensis Berliner were mixed with the diet used to rear colonies of the Indian meal moth Plodia interpunctella (Hübner). Indian meal moth eggs were introduced to the treated diet, and the resultant adult emergence was tabulated. The experimental formulations ranked as follows in efficacy in controlling the Indian meal moth: Dipel (50% lethal concentration [LC50], 25 mg/kg) greater than Bactospeine WP (LC50, 100 mg/kg) greater than Thuricide (LC50, 150 mg/kg) greater than IMC 90007 (LC30, 180 mg/kg) greater than Bactospeine Flowable (LC50, 440 mg/kg). PMID:984828
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Commercial formulations of Bacillus thuringiensis for control of Indian meal moth.
Schesser, J H
1976-10-01
Doses of four commercial formulations and one experimental formulation of Bacillus thuringiensis Berliner were mixed with the diet used to rear colonies of the Indian meal moth Plodia interpunctella (Hübner). Indian meal moth eggs were introduced to the treated diet, and the resultant adult emergence was tabulated. The experimental formulations ranked as follows in efficacy in controlling the Indian meal moth: Dipel (50% lethal concentration [LC50], 25 mg/kg) greater than Bactospeine WP (LC50, 100 mg/kg) greater than Thuricide (LC50, 150 mg/kg) greater than IMC 90007 (LC30, 180 mg/kg) greater than Bactospeine Flowable (LC50, 440 mg/kg).
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Electron Microscope Study of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis
Bechtel, Donald B.; Bulla, Lee A.
1976-01-01
A comprehensive ultrastructural analysis of sporulation and parasporal crystal development is described for Bacillus thuringiensis. The insecticidal crystal of B. thuringiensis is initiated at the start of engulfment and is nearly complete by the time the exosporium forms. The crystal and a heretofore unobserved ovoid inclusion develop without any clear association with the forespore septum, exosporium, or mesosomes. These observations contradict previous hypotheses that the crystal is synthesized on the forespore membrane, exosporium, or mesosomes. Formation of forespore septa involves densely staining, double-membrane-bound, vesicular mesosomes that have a bridged appearance. Forespore engulfment is subpolar and also involves mesosomes. Upon completion of engulfment the following cytoplasmic changes occur: decrease in electron density of the incipient forespore membrane; loss of bridged appearance of incipient forespore membrane; change in stainability of incipient forespore, forespore, and mother cell cytoplasms; and alteration in staining quality of plasma membrane. These changes are involved in the conversion of the incipient forespore into a forespore and reflect “commitment” to sporulation. Images PMID:182671
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Thermal inactivation kinetics of Bacillus coagulans spores in tomato juice.
Peng, Jing; Mah, Jae-Hyung; Somavat, Romel; Mohamed, Hussein; Sastry, Sudhir; Tang, Juming
2012-07-01
The thermal characteristics of the spores and vegetative cells of three strains of Bacillus coagulans (ATCC 8038, ATCC 7050, and 185A) in tomato juice were evaluated. B. coagulans ATCC 8038 was chosen as the target microorganism for thermal processing of tomato products due to its spores having the highest thermal resistance among the three strains. The thermal inactivation kinetics of B. coagulans ATCC 8038 spores in tomato juice between 95 and 115°C were determined independently in two different laboratories using two different heating setups. The results obtained from both laboratories were in general agreement, with z-values (z-value is defined as the change in temperature required for a 10-fold reduction of the D-value, which is defined as the time required at a certain temperature for a 1-log reduction of the target microorganisms) of 8.3 and 8.7°C, respectively. The z-value of B. coagulans 185A spores in tomato juice (pH 4.3) was found to be 10.2°C. The influence of environmental factors, including cold storage time, pH, and preconditioning, upon the thermal resistance of these bacterial spores is discussed. The results obtained showed that a storage temperature of 4°C was appropriate for maintaining the viability and thermal resistance of B. coagulans ATCC 8038 spores. Acidifying the pH of tomato juice decreased the thermal resistance of these spores. A 1-h exposure at room temperature was considered optimal for preconditioning B. coagulans ATCC 8038 spores in tomato juice.
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Silvestri, Erin E.; Perkins, Sarah; Lordo, Robert; Kovacik, William; Nichols, Tonya L.; Bowling, Charlena Yoder; Griffin, Dale W.; Schaefer, Frank W.
2015-01-01
Bacillus species spores have the potential to remain viable in the soil for many years. Lasting environmental contamination following a release is a possibility, and planning for site characterization and remediation activities should consider both indoor-to-outdoor spore transport and outdoor soil as potential exposure pathways.
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Crystal proteins derived from Bacillus thuringiensis (Bt) have been widely used as a method of insect pest management for several decades. In recent years, a transgenic corn expressing the Cry3Bb1 toxin has been successfully used for protection against corn rootworm larvae (Genus...
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John D. Kegg
1985-01-01
Operational programs to suppress gypsy moth populations in residential and recreational areas first began in New Jersey in May of 1980. Bacillus thuringiensis was used on approximately 17,000 acres applied at the dosage rate of 8 B.I.U.'s in one gallon of water per acre. Two treatments approximately one week apart were applied.
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Kreutzweiser, D P; Gringorten, J L; Thomas, D R; Butcher, J T
1996-04-01
Epilithic microbial communities were colonized on leaf disks and exposed to commercial preparations of Bacillus thuringiensis var. kurstaki (Btk) in aquatic microcosms. Responses in terms of microbial respiration, bacterial cell density, protozoan density, and microbial decomposition activity were measured. Test concentrations for treatments with Dipel 64AF and Dipel 8AF in microcosms were the expected environmental concentration (EEC) of 20 IU/ml, 100x the EEC, and 1000x the EEC. Bacterial cell density in the biofilm of leaf disks was significantly increased at concentrations as low as the EEC. There were no concomitant alterations in protozoan density. Microbial respiration was significantly increased, and decomposition activity was significantly decreased, but only at the artificially high concentration of 1000x the EEC. This effect was attributed to the spore-crystal component rather than formulation ingredients. Microbial decomposition of leaf material was also determined in outdoor stream channels treated at concentrations ranging from the EEC to 100x the EEC. Although there tended to be reduced decomposition activity in treated channels, there were no significant differences in mass loss of leaf material between treated and control channels. Various regression, classification, and ordination procedures were applied to the experimental data, and none indicated significant treatment effects. These results from laboratory and controlled field experiments indicate that contamination of watercourses with Btk is unlikely to result in significant adverse effects on microbial community function in terms of detrital decomposition.
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David R. Coyle; Joel D. McMillin; Steven C. Krause; Elwood R. Hart
2000-01-01
Laboratory and field experiments were conducted to determine the efficacy of two Bacillus thuringiensis Berliner formulations, Novodor and Raven, for controlling cottonwood leaf beetle, Chrysomela scripta F. (Coleoptera: Chrysomelidae). In laboratory bioassays, larvae or adults were added to petri dishes containing ...
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NASA Astrophysics Data System (ADS)
Raguse, Marina; Fiebrandt, Marcel; Denis, Benjamin; Stapelmann, Katharina; Eichenberger, Patrick; Driks, Adam; Eaton, Peter; Awakowicz, Peter; Moeller, Ralf
2016-07-01
Low-pressure plasmas have been evaluated for their potential in biomedical and defense purposes. The sterilizing effect of plasma can be attributed to several active agents, including (V)UV radiation, charged particles, radical species, neutral and excited atoms and molecules, and the electric field. Spores of Bacillus subtilis were used as a bioindicator and a genetic model system to study the sporicidal effects of low-pressure plasma decontamination. Wild-type spores, spores lacking the major protective coat layers (inner, outer, and crust), pigmentation-deficient spores or spore impaired in encasement (a late step in coat assembly) were systematically tested for their resistance to low-pressure argon, hydrogen, and oxygen plasmas with and without admixtures. We demonstrate that low-pressure plasma discharges of argon and oxygen discharges cause significant physical damage to spore surface structures as visualized by atomic force microscopy. Spore resistance to low-pressure plasma was primarily dependent on the presence of the inner, and outer spore coat layers as well as spore encasement, with minor or less importance of the crust and spore pigmentation, whereas spore inactivation itself was strongly influenced by the gas composition and operational settings.
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Carvacrol suppresses high pressure high temperature inactivation of Bacillus cereus spores.
Luu-Thi, Hue; Corthouts, Jorinde; Passaris, Ioannis; Grauwet, Tara; Aertsen, Abram; Hendrickx, Marc; Michiels, Chris W
2015-03-16
The inactivation of bacterial spores generally proceeds faster and at lower temperatures when heat treatments are conducted under high pressure, and high pressure high temperature (HPHT) processing is, therefore, receiving an increased interest from food processors. However, the mechanisms of spore inactivation by HPHT treatment are poorly understood, particularly at moderately elevated temperature. In the current work, we studied inactivation of the spores of Bacillus cereus F4430/73 by HPHT treatment for 5 min at 600MPa in the temperature range of 50-100°C, using temperature increments of 5°C. Additionally, we investigated the effect of the natural antimicrobial carvacrol on spore germination and inactivation under these conditions. Spore inactivation by HPHT was less than about 1 log unit at 50 to 70°C, but gradually increased at higher temperatures up to about 5 log units at 100°C. DPA release and loss of spore refractility in the spore population were higher at moderate (≤65°C) than at high (≥70°C) treatment temperatures, and we propose that moderate conditions induced the normal physiological pathway of spore germination resulting in fully hydrated spores, while at higher temperatures this pathway was suppressed and replaced by another mechanism of pressure-induced dipicolinic acid (DPA) release that results only in partial spore rehydration, probably because spore cortex hydrolysis is inhibited. Carvacrol strongly suppressed DPA release and spore rehydration during HPHT treatment at ≤65°C and also partly inhibited DPA release at ≥65°C. Concomitantly, HPHT spore inactivation was reduced by carvacrol at 65-90°C but unaffected at 95-100°C. Copyright © 2014 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Deng, X. T.; Shi, J. J.; Shama, G.; Kong, M. G.
2005-10-01
Current inactivation studies of Bacillus subtilis spores using atmospheric-pressure glow discharges (APGD) do not consider two important factors, namely microbial loading at the surface of a substrate and sporulation temperature. Yet these are known to affect significantly microbial resistance to heat and hydrogen peroxide. This letter investigates effects of microbial loading and sporulation temperature on spore resistance to APGD. It is shown that microbial loading can lead to a stacking structure as a protective shield against APGD treatment and that high sporulation temperature increases spore resistance by altering core water content and cross-linked muramic acid content of B. subtilis spores.
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Dry-heat Resistance of Bacillus Subtilis Var. Niger Spores on Mated Surfaces
NASA Technical Reports Server (NTRS)
Simko, G. J.; Devlin, J. D.; Wardle, M. D.
1971-01-01
Bacillus subtilis var. niger spores were placed on the surfaces of test coupons manufactured from typical spacecraft materials including stainless steel, magnesium, titanium, and aluminum. These coupons were then juxtaposed at the inoculated surfaces and subjected to test pressures of 0, 1000, 5000, and 10,000 psi. Tests were conducted in ambient, nitrogen, and helium atmospheres. While under the test pressure condition, the spores were exposed to 125 C for intervals of 5, 10, 20, 50, or 80 min. Survivor data were subjected to a linear regression analysis that calculated decimal reduction times.
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Cryogenic Irradiation of Bacillus Atrophaeus spores to understand microbial survival on Icy Bodies
NASA Astrophysics Data System (ADS)
Yerby, C. J.; Noell, A. C.; Hodyss, R. P.; Johnson, P. V.; Ponce, A.
2017-12-01
Bacterial Spores are useful indicator organisms for studying the survival of microbes and degradation of biomolecules on the surface of planetary icy bodies. To predict the limits of life's proliferation in space, specifically on icy bodies, it is essential to understand the ability of microbes to withstand photon and particle irradiation at cryogenic temperatures. Bacillus Atrophaeus spores were transferred onto stainless steel coupons by varied processes and subsequently frozen at Europan temperatures (16oK—273oK) in a vacuum at 8.7x10-8 Torr. An argon lamp bombarded the spore-containing coupons with a solar-like radiation spectra for a variety of times, and spores were removed from the coupons and enumerated in culture. To date, (n=43) coupons have been analyzed for spore kill-rates with regards to ice temperature and radiation exposure time. Results will be presented on the effect of cryogenic temperatures in improving radiation resistance of bacterial spores. This works also details methodology improvements by comparing different spore deposition and recovery methods before and after cryogenic irradiation.
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Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination
2009-12-01
exosporium maturation and assembly and suggest a novel role for the exosporium in germination. During starvation, bacteria of the genus Bacillus...Bacillus subtilis, the outermost struc- ture is a protective layer called the coat, which guards the spore against reactive small molecules, degradative ...analysis. Generation of anti-ExsK antibodies. Recombinant ExsK was generated and purified using the pET expression system (Novagen) according to the
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W. A. Smirnoff
1985-01-01
Field tests were conducted in 1980, 1981, and 1982 with a new Bacillus thuringiensis formula called Futura. Tests were conducted with a Grumman AgCat aircraft on 40 ha forest blocks and with a DC-4G aircraft on larger forested areas. Futura which is able to disperse the required 20 x 109 I.U./ha for spruce budworm control in 2....
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Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Obuchowski, Michał; Nidzworski, Dawid
2016-01-01
Effective vaccination against influenza virus infection is a serious problem mainly due to antigenic variability of the virus. Among many of investigated antigens, the extracellular domain of the M2 protein (M2e) features high homology in all strains of influenza A viruses and antibodies against M2e and is protective in animal models; this makes it a potential candidate for generation of a universal influenza vaccine. However, due to the low immunogenicity of the M2e, formulation of a vaccine based on this antigen requires some modification to induce effective immune responses. In this work we evaluated the possible use of Bacillus subtilis spores as a carrier of the Influenza A M2e antigen in mucosal vaccination. A tandem repeat of 4 consensus sequences coding for human—avian—swine—human M2e (M2eH-A-S-H) peptide was fused to spore coat proteins and stably exposed on the spore surface, as demonstrated by the immunostaining of intact, recombinant spores. Oral immunization of mice with recombinant endospores carrying M2eH-A-S-H elicited specific antibody production without the addition of adjuvants. Bacillus subtilis endospores can serve as influenza antigen carriers. Recombinant spores constructed in this work showed low immunogenicity although were able to induce antibody production. The System of influenza antigen administration presented in this work is attractive mainly due to the omitting time-consuming and cost-intensive immunogen production and purification. Therefore modification should be made to increase the immunogenicity of the presented system. PMID:27902762
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Zimmermann, Morgana; Longhi, Daniel A; Schaffner, Donald W; Aragão, Gláucia M F
2014-05-01
The knowledge and understanding of Bacillus coagulans inactivation during a thermal treatment in tomato pulp, as well as the influence of temperature variation during thermal processes are essential for design, calculation, and optimization of the process. The aims of this work were to predict B. coagulans spores inactivation in tomato pulp under varying time-temperature profiles with Gompertz-inspired inactivation model and to validate the model's predictions by comparing the predicted values with experimental data. B. coagulans spores in pH 4.3 tomato pulp at 4 °Brix were sealed in capillary glass tubes and heated in thermostatically controlled circulating oil baths. Seven different nonisothermal profiles in the range from 95 to 105 °C were studied. Predicted inactivation kinetics showed similar behavior to experimentally observed inactivation curves when the samples were exposed to temperatures in the upper range of this study (99 to 105 °C). Profiles that resulted in less accurate predictions were those where the range of temperatures analyzed were comparatively lower (inactivation profiles starting at 95 °C). The link between fail prediction and both lower starting temperature and magnitude of the temperature shift suggests some chemical or biological mechanism at work. Statistical analysis showed that overall model predictions were acceptable, with bias factors from 0.781 to 1.012, and accuracy factors from 1.049 to 1.351, and confirm that the models used were adequate to estimate B. coagulans spores inactivation under fluctuating temperature conditions in the range from 95 to 105 °C. How can we estimate Bacillus coagulans inactivation during sudden temperature shifts in heat processing? This article provides a validated model that can be used to predict B. coagulans under changing temperature conditions. B. coagulans is a spore-forming bacillus that spoils acidified food products. The mathematical model developed here can be used to predict the spoilage
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Catherine Papp Herms; Deborah G. McCullough; Leah S. Bauer; Robert A. Haack; Norman R. Dubois
1997-01-01
We investigated the phenological and physiological susceptibility of the endangered Karner blue butterfly (Lycaeides melissa samuelis) to Bacillus thuringiensis var. kurstaki (Bt), a product widely used for gypsy moth (Lymantria dispar) suppression in Michigan and other infested states. We...
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Reitz, Günther; Li, Zuofeng; Klein, Stuart; Nicholson, Wayne L.
2012-01-01
Abstract The space environment contains high-energy charged particles (e.g., protons, neutrons, electrons, α-particles, heavy ions) emitted by the Sun and galactic sources or trapped in the radiation belts. Protons constitute the majority (87%) of high-energy charged particles. Spores of Bacillus species are one of the model systems used for astro- and radiobiological studies. In this study, spores of different Bacillus subtilis strains were used to study the effects of high energetic proton irradiation on spore survival. Spores of the wild-type B. subtilis strain [mutants deficient in the homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways and mutants deficient in various spore structural components such as dipicolinic acid (DPA), α/β-type small, acid-soluble spore protein (SASP) formation, spore coats, pigmentation, or spore core water content] were irradiated as air-dried multilayers on spacecraft-qualified aluminum coupons with 218 MeV protons [with a linear energy transfer (LET) of 0.4 keV/μm] to various final doses up to 2500 Gy. Spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to proton radiation than wild-type spores, indicating that both HR and NHEJ DNA repair pathways are needed for spore survival. Spores lacking DPA, α/β-type SASP, or with increased core water content were also significantly more sensitive to proton radiation, whereas the resistance of spores lacking pigmentation or spore coats was essentially identical to that of the wild-type spores. Our results indicate that α/β-type SASP, core water content, and DPA play an important role in spore resistance to high-energy proton irradiation, suggesting their essential function as radioprotectants of the spore interior. Key Words: Bacillus—Spores—DNA repair—Protection—High-energy proton radiation. Astrobiology 12, 1069–1077. PMID:23088412
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Code of Federal Regulations, 2014 CFR
2014-07-01
... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed; cotton...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed; cotton...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed; cotton...
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Algimantas P. Valaitis
2011-01-01
The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the...
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Germination Requirements of Bacillus macerans Spores
Sacks, L. E.; Thompson, P. A.
1971-01-01
2-Phenylacetamide is an effective germinant for spores of five strains of Bacillus macerans, particularly in the presence of fructose. Benzyl penicillin, the phenyl acetamide derivative of penicillin, and phenylacetic acid are also good germinants. l-Asparagine is an excellent germinant for four strains. α-Amino-butyric acid is moderately effective. Pyridoxine, pyridoxal, adenine, and 2,6-diaminopurine are potent germinants for NCA strain 7X1 only. d-Glucose is a powerful germinant for strain B-70 only. d-Fructose and d-ribose strongly potentiate germination induced by other germinants (except l-asparagine) but have only weak activity by themselves. Niacinamide and nicotinamide-adenine dinucleotide, inactive by themselves, are active in the presence of fructose or ribose. Effects of pH, ion concentration, and temperature are described. PMID:4251279
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USDA-ARS?s Scientific Manuscript database
The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the coleopteran-specific Cry3Aa toxin from Bacillus thuringiensis (Bt). Larvae digest protein initially with cysteine peptidases in the anterior midgut and further with serine peptidases in middle and poste...
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Agoston, Réka; Soni, Kamlesh A; McElhany, Katherine; Cepeda, Martha L; Zuckerman, Udi; Tzipori, Saul; Mohácsi-Farkas, Csilla; Pillai, Suresh D
2009-03-01
Deliberate or accidental contamination of foods such as milk, soft drinks, and drinking water with infectious agents or toxins is a major concern to health authorities. There is a critical need to develop technologies that can rapidly and efficiently separate and concentrate biothreat agents from food matrices. A key limitation of current centrifugation and filtration technologies is that they are batch processes with extensive hands-on involvement and processing times. The objective of our studies was to evaluate the continuous flow centrifugation (CFC) technique for the rapid separation and concentration of bacterial spores from large volumes of milk. We determined the effectiveness of the CFC technology for concentrating approximately 10(3) bacterial spores in 3.7 liters (1 gal) of whole milk and skim milk, using Bacillus subtilis, Bacillus atrophaeus, and Clostridium sporogenes spores as surrogates for biothreat agents. The spores in the concentrated samples were enumerated by using standard plating techniques. Three independent experiments were performed at 10,000 rpm and 0.7 liters/min flow rate. The mean B. subtilis spore recoveries were 71.3 and 56.5% in skim and whole milk, respectively, and those for B. atrophaeus were 55 and 59.3% in skim and whole milk, respectively. In contrast, mean C. sporogenes spore recoveries were 88.2 and 78.6% in skim and whole milk, respectively. The successful use of CFC to concentrate these bacterial spores from 3.7 liters of milk in 10 min shows promise for rapidly concentrating other spores from large volumes of milk.
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Anilkumar, Konasale J.; Rodrigo-Simón, Ana; Ferré, Juan; Pusztai-Carey, Marianne; Sivasupramaniam, Sakuntala; Moar, William J.
2008-01-01
Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments. PMID:18024681
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VIABILITY OF BACILLUS SUBTILIS SPORES IN ROCKET PROPELLANTS.
GODDING, R M; LYNCH, V H
1965-01-01
The sporicidal activity of components used in liquid and solid rocket propellants was tested by use of spores of Bacillus subtilis dried on powdered glass. Liquid propellant ingredients tested were N(2)O(4), monomethylhydrazine and 1,1-dimethylhydrazine. N(2)O(4) was immediately sporicidal; the hydrazines were effective within several days. Solid propellants consisted of ammonium perchlorate in combination with epoxy resin (EPON 828), tris-1-(2-methyl) aziridinyl phosphine oxide, bis-1-(2-methyl) aziridinyl phenylphosphine oxide, and three modified polybutadiene polymers. There was no indication of appreciable sporicidal activity of these components.
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Viability of Bacillus subtilis Spores in Rocket Propellants
Godding, Rogene M.; Lynch, Victoria H.
1965-01-01
The sporicidal activity of components used in liquid and solid rocket propellants was tested by use of spores of Bacillus subtilis dried on powdered glass. Liquid propellant ingredients tested were N2O4, monomethylhydrazine and 1,1-dimethylhydrazine. N2O4 was immediately sporicidal; the hydrazines were effective within several days. Solid propellants consisted of ammonium perchlorate in combination with epoxy resin (EPON 828), tris-1-(2-methyl) aziridinyl phosphine oxide, bis-1-(2-methyl) aziridinyl phenylphosphine oxide, and three modified polybutadiene polymers. There was no indication of appreciable sporicidal activity of these components. PMID:14264838
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Code of Federal Regulations, 2013 CFR
2013-07-01
... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a plant...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a plant...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a plant...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a plant...
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Peña, Guadalupe; Aguilar Jiménez, Fortino Agustín; Hallal-Calleros, Claudia; Morales-Montor, Jorge; Hernández-Velázquez, Víctor Manuel; Flores-Pérez, Fernando Iván
2013-01-01
Bacillus thuringiensis is a gram-positive soil-dwelling bacterium that is commonly used as a biological pesticide. This bacterium may also be used for biological control of helminth parasites in domestic animals. In this study, we evaluated the possible ovicidal and cestocidal effects of a total protein extract of B. thuringiensis native strains on the zoonotic cestode parasite of dogs, Dipylidium caninum (D. caninum). Dose and time response curves were determined by coincubating B. thuringiensis proteins at concentration ranging from 100 to 1000 μg/mL along with 4000 egg capsules of D. caninum. Egg viability was evaluated using the trypan blue exclusion test. The lethal concentration of toxins on eggs was 600 μg/ml, and the best incubation time to produce this effect was 3 h. In the adult stage, the motility and the thickness of the tegument were used as indicators of damage. The motility was inhibited by 100% after 8 hours of culture compared to the control group, while the thickness of the cestode was reduced by 34%. Conclusively, proteins of the strain GP526 of B. thuringiensis directly act upon D. caninum showing ovicidal and cestocidal effects. Thus, B. thuringiensis is proposed as a potential biological control agent against this zoonosis. PMID:23484087
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Peña, Guadalupe; Aguilar Jiménez, Fortino Agustín; Hallal-Calleros, Claudia; Morales-Montor, Jorge; Hernández-Velázquez, Víctor Manuel; Flores-Pérez, Fernando Iván
2013-01-01
Bacillus thuringiensis is a gram-positive soil-dwelling bacterium that is commonly used as a biological pesticide. This bacterium may also be used for biological control of helminth parasites in domestic animals. In this study, we evaluated the possible ovicidal and cestocidal effects of a total protein extract of B. thuringiensis native strains on the zoonotic cestode parasite of dogs, Dipylidium caninum (D. caninum). Dose and time response curves were determined by coincubating B. thuringiensis proteins at concentration ranging from 100 to 1000 μ g/mL along with 4000 egg capsules of D. caninum. Egg viability was evaluated using the trypan blue exclusion test. The lethal concentration of toxins on eggs was 600 μ g/ml, and the best incubation time to produce this effect was 3 h. In the adult stage, the motility and the thickness of the tegument were used as indicators of damage. The motility was inhibited by 100% after 8 hours of culture compared to the control group, while the thickness of the cestode was reduced by 34%. Conclusively, proteins of the strain GP526 of B. thuringiensis directly act upon D. caninum showing ovicidal and cestocidal effects. Thus, B. thuringiensis is proposed as a potential biological control agent against this zoonosis.
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Wan, Juan; Sun, Xiaowen; Liu, Cheng; Tang, Mengjun; Li, Lin; Ni, Hong
2017-06-01
A triplicate volcanic rock matrix-Bacillus thuringiensis-laccase WlacD (VRMs-Bt-WlacD) dye decolorization system was developed. WlacD was displayed on the B. thuringiensis MB174 cell surface to prepare a whole-cell laccase biocatalyst by using two repeat N-terminal domains of autolysin Mbg (Mbgn) 2 as the anchoring motif. Immunofluorescence microscopic assays confirmed that the fusion protein (Mbgn) 2 -WlacD was anchored on the surface of the recombinant B. thuringiensis MB174. After optimization by a single factor test, L 9 (3 4 )-orthogonal test, Plackett-Burman test, steepest ascent method, and Box-Behnken response surface methodology, the whole-cell specific laccase activity of B. thuringiensis MB174 was improved to 555.2 U L -1 , which was 2.25 times than that of the primary culture condition. Optimized B. thuringiensis MB174 cells were further adsorbed by VRMs to prepare VRMs-Bt-WlacD, an immobilized whole-cell laccase biocatalyst. Decolorization capacity of as-prepared VRMs-Bt-WlacD toward an initial concentration of 500 mg L -1 of an textile dye reactive blue 19 (RB19) aqueous solution reached 72.36% at a solid-to-liquid ratio of 10 g-100 mL. Repeated decolorization-activation operations showed the high decolorization capacity of VRMs-Bt-WlacD and have the potential for large-scale or continuous operations.
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Thermal Inactivation of Bacillus anthracis Spores Using Rapid Resistive Heating
2016-03-24
thermal inactivation research. However, the research conducted to support this thesis utilizes the B.a. Sterne strain which is used in livestock vaccines...methodology conducted for this research including hard surface recovery, thermal inactivation of Bacillus anthracis spores, and the rapid resistive heating...to 500°C range but again, many of the thermal inactivation studies were conducted in the 350 to 2000°C range. Sample plots will be discussed in
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Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action
de Souza, Renata Damásio; Batista, Milene Tavares; Luiz, Wilson Barros; Cavalcante, Rafael Ciro Marques; Amorim, Jaime Henrique; Bizerra, Raíza Sales Pereira; Martins, Eduardo Gimenes; de Souza Ferreira, Luís Carlos
2014-01-01
Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains. PMID:24475289
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An Integrated Civilian Medical Response to Mass Casualty Incidents
2005-05-01
Industrial Hygiene Association Journal. March-April (2003). [5] Ishizaki, K., Shinriki N., and Matsuyama H. Inactivation of bacillus spores by gaseous ozone... Bacillus subtilis spores under controlled humidity, gas concentration, and temperature at atmospheric pressure. Based on the findings of above study...34Decontamination of bacillus thuringiensis spores on selected surfaces by chlorine dioxide gas", Journal of Environmental Health 66: 16-20 6. Jeng, D. K., and
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USDA-ARS?s Scientific Manuscript database
Transgenic plants that expressed Bacillus thuringiensis (Bt) crystalline (Cry) protein toxins can suffer feeding damage from a small number of lepidopteran insect species under field conditions, which has heightened concerns about the durability of pest control tactics. Genomics research has provid...
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Ballardo, Cindy; Barrena, Raquel; Artola, Adriana; Sánchez, Antoni
2017-12-01
In the framework of a circular economy, organic solid wastes are considered to be resources useful for obtaining value-added products. Among other potential uses, biodegradable wastes from agricultural, industrial, and domestic sources are being studied to obtain biopesticides through solid-state fermentation (SSF), mainly at the laboratory scale. The suitability of biowaste (source-selected organic fraction of municipal solid waste) for use as a substrate for Bacillus thuringiensis (Bt) growth under non-sterile conditions in a 10 L SSF reactor was determined in this study. An operational strategy for setting up a semi-continuous process yielding a stabilised organic compost-like material enriched with Bt suitable for use as a soil amendment was developed. Concentrations of 1.7·10 7 -2.2·10 7 and 1.3·10 7 -2.1·10 7 CFU g -1 DM for Bt viable cells and spores, respectively, were obtained in the final material. As the results confirmed, Bt-enriched compost-like material with potential biopesticide properties can be produced from non-sterile biowaste. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Xu Zhou, Ke; Li, Nan; Christie, Graham
2017-01-01
Abstract The adhesion of spores of 3 Bacillus species with distinctive morphologies to stainless steel and borosilicate glass was studied using the fluid dynamic gauging technique. Marked differences were observed between different species of spores, and also between spores of the same species prepared under different sporulation conditions. Spores of the food‐borne pathogen B. cereus were demonstrated to be capable of withstanding shear stresses greater than 1500 Pa when adhered to stainless steel, in contrast to spores of Bacillus subtilis and Bacillus megaterium, which detached in response to lower shear stress. An extended DLVO model was shown to be capable of predicting the relative differences in spore adhesion between spores of different species and different culture conditions, but did not predict absolute values of force of adhesion well. Applying the model to germinating spores showed a significant reduction in adhesion force shortly after triggering germination, indicating a potential strategy to achieve enhanced removal of spores from surfaces in response to shear stress, such as during cleaning‐in‐place procedures. Practical Application Spore‐forming bacteria are a concern to the food industry because they have the potential to cause food‐borne illness and product spoilage, while being strongly adhesive to processing surfaces and resistant to cleaning‐in‐place procedures. This work is of significance to the food processors and manufacturers because it offers insight to the properties of spore adhesion and identifies a potential strategy to facilitate the removal of spores during cleaning procedures. PMID:29125641
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Tn5401, a new class II transposable element from Bacillus thuringiensis.
Baum, J A
1994-01-01
A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulation-deficient variant of Bacillus thuringiensis subsp. morrisoni EG2158 following its insertion into a recombinant plasmid. Sequence analysis of the insert revealed a 4,837-bp transposon with two large open reading frames, in the same orientation, encoding proteins of 36 kDa (306 residues) and 116 kDa (1,005 residues) and 53-bp terminal inverted repeats. The deduced amino acid sequence for the 36-kDa protein shows 24% sequence identity with the TnpI recombinase of the B. thuringiensis transposon Tn4430, a member of the phage integrase family of site-specific recombinases. The deduced amino acid sequence for the 116-kDa protein shows 42% sequence identity with the transposase of Tn3 but only 28% identity with the TnpA transposase of Tn4430. Two small open reading frames of unknown function, designated orf1 (85 residues) and orf2 (74 residues), were also identified. Southern blot analysis indicated that Tn5401, in contrast to Tn4430, is not commonly found among different subspecies of B. thuringiensis and is not typically associated with known insecticidal crystal protein genes. Transposition was studied with B. thuringiensis by using plasmid pEG922, a temperature-sensitive shuttle vector containing Tn5401. Tn5401 transposed to both chromosomal and plasmid target sites but displayed an apparent preference for plasmid sites. Transposition was replicative and resulted in the generation of a 5-bp duplication at the target site. Transcriptional start sites within Tn5401 were mapped by primer extension analysis. Two promoters, designated PL and PR, direct the transcription of orf1-orf2 and tnpI-tnpA, respectively, and are negatively regulated by TnpI. Sequence comparison of the promoter regions of Tn5401 and Tn4430 suggests that the conserved sequence element ATGTCCRCTAAY mediates TnpI binding and cointegrate resolution. The same element is contained within the 53-bp terminal
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NanoSIMS analysis of Bacillus spores for forensics
DOE Office of Scientific and Technical Information (OSTI.GOV)
Weber, P K; Davisson, M L; Velsko, S P
2010-02-23
The threat associated with the potential use of radiological, nuclear, chemical and biological materials in terrorist acts has resulted in new fields of forensic science requiring the application of state-of-the-science analytical techniques. Since the anthrax letter attacks in the United States in the fall of 2001, there has been increased interest in physical and chemical characterization of bacterial spores. While molecular methods are powerful tools for identifying genetic differences, other methods may be able to differentiate genetically identical samples based on physical and chemical properties, as well as provide complimentary information, such as methods of production and approximate date ofmore » production. Microanalysis has the potential to contribute significantly to microbial forensics. Bacillus spores are highly structured, consisting of a core, cortex, coat, and in some species, an exosporium. This structure provides a template for constraining elemental abundance differences at the nanometer scale. The primary controls on the distribution of major elements in spores are likely structural and physiological. For example, P and Ca are known to be abundant in the spore core because that is where P-rich nucleic acids and Cadipicolinic acid are located, respectively. Trace elements are known to bind to the spore coat but the controls on these elements are less well understood. Elemental distributions and abundances may be directly related to spore production, purification and stabilization methodologies, which are of particular interest for forensic investigation. To this end, we are developing a high-resolution secondary ion mass spectrometry method using a Cameca NanoSIMS 50 to study the distribution and abundance of trace elements in bacterial spores. In this presentation we will review and compare methods for preparing and analyzing samples, as well as review results on the distribution and abundance of elements in bacterial spores. We use Nano
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March, Jordon K; Pratt, Michael D; Lowe, Chinn-Woan; Cohen, Marissa N; Satterfield, Benjamin A; Schaalje, Bruce; O'Neill, Kim L; Robison, Richard A
2015-01-01
This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat
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Callahan, Courtney; Fox, Karen; Fox, Alvin
2009-01-01
The Bacillus cereus group includes Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. The small acid-soluble spore protein (SASP) β has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP β of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP α is conserved in sequence among these two species. Neither SASP α nor β for B. thuringiensis, B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP α and β demonstrated consistent MW among B. cereus, B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP α and β were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP α (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP β (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP α-β type, was described here for the first time. PMID:19616612
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The intentional dissemination of Bacillus anthracis (anthrax) spores at multiple locations in the United States in the Fall of 2001 resulted not only in several deaths and illnesses (including psychological effects), but likely changed lifestyles and attitudes, and increased the ...
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USDA-ARS?s Scientific Manuscript database
Studies were made of the extract of Sargassum wightii combined with Bacillus thuringiensis var. israelensis (Bti) for control of the malaria vector Anopheles sundaicus. Treatment of mosquito larvae with 0.001% S. wightii extract indicated median lethal concentrations (LC50) of 88, 73, 134, 156, and...
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Hutchison, Janine R; Erikson, Rebecca L; Sheen, Allison M; Ozanich, Richard M; Kelly, Ryan T
2015-09-21
Bacillus anthracis is the causative agent of anthrax and can be contracted by humans and herbivorous mammals by inhalation, ingestion, or cutaneous exposure to bacterial spores. Due to its stability and disease potential, B. anthracis is a recognized biothreat agent and robust detection and viability methods are needed to identify spores from unknown samples. Here we report the use of smartphone-based microscopy (SPM) in combination with a simple microfluidic incubation device (MID) to detect 50 to 5000 B. anthracis Sterne spores in 3 to 5 hours. This technique relies on optical monitoring of the conversion of the ∼1 μm spores to the filamentous vegetative cells that range from tens to hundreds of micrometers in length. This distinguishing filament formation is unique to B. anthracis as compared to other members of the Bacillus cereus group. A unique feature of this approach is that the sample integrity is maintained, and the vegetative biomass can be removed from the chip for secondary molecular analysis such as PCR. Compared with existing chip-based and rapid viability PCR methods, this new approach reduces assay time by almost half, and is highly sensitive, specific, and cost effective.
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Inactivation of Bacillus subtilis spores by high pressure CO2 with high temperature.
Rao, Lei; Xu, Zhenzhen; Wang, Yongtao; Zhao, Feng; Hu, Xiaosong; Liao, Xiaojun
2015-07-16
The objective of this study was to investigate the inactivation of the Bacillus subtilis spores by high pressure CO2 combined with high temperature (HPCD+HT) and to analyze the clumping effect of the spores on their HPCD+HT resistance. The spores of B. subtilis were subjected to heat at 0.1 MPa and HPCD at 6.5-25 MPa, and 82 °C, 86 °C, and 91 °C for 0-120 min. The spores were effectively inactivated by HPCD+HT, but a protective effect on the spores was also found, which was closely correlated to the pressure, temperature and time. The spores treated by HPCD+HT at 6.5 and 10 MPa exhibited a two-stage inactivation curve of shoulder and log-linear regions whereas the spores at 15-25 MPa exhibited a three-stage inactivation curve of shoulder, log-linear and tailing regions, and these curves were well fitted to the Geeraerd model. Approximately 90% of pyridine-2,6-dicarboxylic acid (DPA) was released after HPCD+HT and the 90% DPA release time depend on the pressure and temperature. Moreover, the spore clumping in suspensions was examined by dynamic light scattering. The particle size of the spore suspensions increased with the increase of pressure, temperature and time, indicating the spore clumping. 0.1% Tween 80 as a surfactant inhibited the spore clumping and increased the inactivation ratio of the spores by HPCD+HT. These results indicated that the spore clumping enhanced the spores' resistance to HPCD+HT and induced a protective effect. Copyright © 2015 Elsevier B.V. All rights reserved.
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USDA-ARS?s Scientific Manuscript database
BACKGROUND: Biopesticides containing Cry insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are effective against many lepidopteran pests, but there is a lack of Bt-based pesticides to efficiently control important coleopteran pests. Based on the reported increase of Bt toxin olig...
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Synthesis of acid-soluble spore proteins by Bacillus subtilis.
Leventhal, J M; Chambliss, G H
1982-12-01
The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.
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Moazamian, Elham; Bahador, Nima; Azarpira, Negar; Rasouli, Manoochehr
2018-04-23
Bacillus thuringiensis is one of the most important microorganisms used against cancer cell lines in latest studies all over the world. This study aims to perform the isolation, molecular identification, and to identify novel B. thuringiensis strains that specifically targeting human cancer cell-killing activities in Iran. A total of 88 B. thuringiensis isolates were recovered from Iran. Upon the treatment of the non-hemolytic crystal proteins by proteinase K, five isolates belonging to three biotypes, thuringiensis, kurstaki and sotto of B. thuringiensis are found to have different cytotoxicity toward HCT-116 and CCRF-CEM cell lines. Digested inclusions of the isolates consisted of one major poly peptide of 34-kDa, as estimated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The structure, molecular identification, and functionality of five isolates inclusion proteins have shown to be closely like to parasporin-2 but their size of activated protein is not similar to this parasporin. It is unclear that discovered damaging proteins are parasporin-2. This 34-kD protein exhibited varying degrees of cytocidal activity toward human colon and blood cancer cells and caused cell swelling and the formation of blebs in the surface of the cells or alteration in cytoskeleton. The soil in the humid and temperate climates of Iran is a good reservoir for parasporin producing B. thuringiensis. The isolated B. thuringiensis strains exhibit specific and different cytocidal activities against human colon and blood cancer cells. Parasporin is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis and these toxins appeared to attack an identical target on human cancer cells.
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Role of YpeB in Cortex Hydrolysis during Germination of Bacillus anthracis Spores
Bernhards, Casey B.
2014-01-01
The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ΔsleB and ΔypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup. PMID:25022853
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Carroll, Alicia Monroe; Plomp, Marco; Malkin, Alexander J.; Setlow, Peter
2008-01-01
The Bacillus subtilis spore coat is a multilayer, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a “rind” that looks like coat material remains. As observed with a phase-contrast microscope, the rinds are spherical or hemispherical structures that appear to be devoid of internal contents. Atomic force microscopy and chemical analyses showed that (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers, (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion, and (iii) the rinds and intact spores do not contain significant levels of silicon. PMID:18689521
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Wu, Songqing; Lan, Yanjiao; Huang, Dongmei; Peng, Yan; Huang, Zhipeng; Xu, Lei; Gelbic, Ivan; Carballar-Lejarazu, Rebeca; Guan, Xiong; Zhang, Lingling; Zou, Shuangquan
2014-02-01
The aim of this study was to explore a cost-effective method for the mass production of Bacillus thuringiensis (Bt) by solid-state fermentation. As a locally available agroindustrial byproduct, spent mushroom substrate (SMS) was used as raw material for Bt cultivation, and four combinations of SMS-based media were designed. Fermentation conditions were optimized on the best medium and the optimal conditions were determined as follows: temperature 32 degrees C, initial pH value 6, moisture content 50%, the ratio of sieved material to initial material 1:3, and inoculum volume 0.5 ml. Large scale production of B. thuringiensis subsp. israelensis (Bti) LLP29 was conducted on the optimal medium at optimal conditions. High toxicity (1,487 international toxic units/milligram) and long larvicidal persistence of the product were observed in the study, which illustrated that SMS-based solid-state fermentation medium was efficient and economical for large scale industrial production of Bt-based biopesticides. The cost of production of 1 kg of Bt was approximately US$0.075.
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Spicher, G; Peters, J
1997-02-01
Biological indicators used to test sterilisation procedures for their efficacy consist of a so-called germ carrier to which the microorganisms used as test organisms adhere. In previous papers we demonstrated that carriers made of filter paper on contact with saturated steam show superheating while carriers made of glass fibre fleece as well as wetted filter paper do not. Using spores of Bacillus subtilis and Bacillus stearothermophilus as test organisms we have now investigated whether and to what extent carrier superheating affects the characteristic values (t50%) of these biological indicators. The indicators were exposed to saturated steam at 100 degrees C (B. subtilis) or 120 degrees C (B. stearothermophilus) under three different exposure conditions: 1. dry (i.e. conditioned to 45% relative humidity before introduction into the sterilising chamber), freely accessible; 2. dry with a substratum and a cover of filter card-board; 3. wet (moistened with twice distilled water before introduction into the sterilising chamber), freely accessible. For previously selected exposure periods, the incidence of indicators with surviving test organisms was determined. The reaction pattern of bioindicators with spores of B. stearothermophilus was different from that of bioindicators with spores of B. subtilis. For B. subtilis, the incidence of bioindicators exhibiting surviving test organisms depended on the nature of the carries as well as on the exposure conditions. On filter paper carriers, t50% increased in the order "wet, freely accessible", "dry, freely accessible", "dry, between filter card-board". On dry and wetted glass fibre fleece, resistance was approximately the same; when the indicators were sandwiched between layers of filter card-board, t50% increased. For B. stearothermophilus, t50% was largely dependent on the carrier material alone. The values obtained for filter paper were invariably much lower than those for glass fibre fleece. As the results show, using
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NASA Technical Reports Server (NTRS)
Venkateswaran, Kasthuri; Kempf, Michael; Chen, Fei; Satomi, Masataka; Nicholson, Wayne; Kern, Roger
2003-01-01
One of the spore-formers isolated from a spacecraft-assembly facility, belonging to the genus Bacillus, is described on the basis of phenotypic characterization, 16S rDNA sequence analysis and DNA-DNA hybridization studies. It is a Gram-positive, facultatively anaerobic, rod-shaped eubacterium that produces endospores. The spores of this novel bacterial species exhibited resistance to UV, gamma-radiation, H2O2 and desiccation. The 18S rDNA sequence analysis revealed a clear affiliation between this strain and members of the low G+C Firmicutes. High 16S rDNA sequence similarity values were found with members of the genus Bacillus and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain FO-92T and Bacillus benzoevorans DSM 5391T was very high. However, molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus, but DNA-DNA hybridization data support the proposal of FO-92T as Bacillus nealsonii sp. nov. (type strain is FO-92T =ATCC BAAM-519T =DSM 15077T).
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USDA-ARS?s Scientific Manuscript database
Cotton and maize delivering insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non-target organisms, especially natural enemies that suppress pest populations. In th...
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USDA-ARS?s Scientific Manuscript database
Bacillus thuringiensis Vip3 proteins are synthesized and secreted during the vegetative growth phase. They are activated by gut proteases, recognize and bind to midgut receptors, form pores and lyse cells. We tested the susceptibility to Vip3Aa and Vip3Ca of Cry1A-, Cry2A-, Dipel- and Vip3-resistant...
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Inactivation of Bacillus spores by the supercritical carbon dioxide micro-bubble method.
Ishikawa, H; Shimoda, M; Tamaya, K; Yonekura, A; Kawano, T; Osajima, Y
1997-06-01
Bacillus spores were effectively inactivated by the supercritical (SC) CO2 micro-bubble method. The micro-bubble SC CO2 treatment of B. cereus, B. subtilis, B. megaterium, B. polymyxa, and B. coagulans at 40 degrees C and 30 MPa for 30 min produced greater reduction (about 3 log cycles of reduction) than a similar treatment without a filter. The SC CO2 treatment of B. polymyxa, B. cereus, and B. subtilis spores at 45 degrees C, 50 degrees C, respectively, and 30 MPa for 60 min resulted in a 6-log cycle reduction of survival. The SC CO2 treatment under the foregoing conditions should offer higher efficiency than that of heat treatment at 100 degrees C for 60 min. In addition, the SC CO2 treatment (30 MPa, 60 degrees C, 30 min) of B. polymyxa and B. cereus spores also produced a 6-log cycle reduction.
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Pinto, Laura M.N.; Dörr, Natália C.; Ribeiro, Ana Paula A.; de Salles, Silvia M.; de Oliveira, Jaime V.; Menezes, Valmir G.; Fiuza, Lidia M.
2012-01-01
The screening of Bacillus thuringiensis (Bt) Cry proteins with high potential to control insect pests has been the goal of numerous research groups. In this study, we evaluated six monogenic Bt strains (Bt dendrolimus HD-37, Bt kurstaki HD-1, Bt kurstaki HD-73, Bt thuringiensis 4412, Bt kurstaki NRD-12 and Bt entomocidus 60.5, which codify the cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1C, cry2A genes respectively) as potential insecticides for the most important insect pests of irrigated rice: Spodoptera frugiperda, Diatraea saccharalis, Oryzophagus oryzae, Oebalus poecilus and Tibraca limbativentris. We also analyzed their compatibility with chemical insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil), which are extensively used in rice crops. The bioassay results showed that Bt thuringiensis 4412 and Bt entomocidus 60.5 were the most toxic for the lepidopterans, with a 93% and 82% mortality rate for S. frugiperda and D. saccharalis, respectively. For O. oryzae, the Bt kurstaki NRD-12 (64%) and Bt dendrolimus HD-37 (62%) strains were the most toxic. The Bt dendrolimus HD-37 strain also caused high mortality (82%) to O. poecilus, however the strains assessed to T. limbativentris caused a maximum rate of 5%. The assays for the Bt strains interaction with insecticides revealed the compatibility of the six strains with the four insecticides tested. The results from this study showed the high potential of cry1Aa and cry1Ba genes for genetic engineering of rice plants or the strains to biopesticide formulations. PMID:24031872
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Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes.
Mattossovich, Rosanna; Iacono, Roberta; Cangiano, Giuseppina; Cobucci-Ponzano, Beatrice; Isticato, Rachele; Moracci, Marco; Ricca, Ezio
2017-11-28
The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-D-xylans to remove successive D-xylose residues from the non-reducing termini. We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes
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NASA Technical Reports Server (NTRS)
Kempf, Michael J.; Chen, Fei; Kern, Roger; Venkateswaran, Kasthuri
2005-01-01
While the microbial diversity of a spacecraft assembly facility at the Jet Propulsion Laboratory (Pasadena, CA) was being monitored, H2O2-resistant bacterial strains were repeatedly isolated from various surface locations. H2O2 is a possible sterilant for spacecraft hardware because it is a low-temperature process and compatible with various modern-day spacecraft materials, electronics, and components. Both conventional biochemical testing and molecular analyses identified these strains as Bacillus pumilus. This Bacillus species was found in both unclassified (entrance floors, anteroom, and air-lock) and classified (floors, cabinet tops, and air) locations. Both vegetative cells and spores of several B. pumilus isolates were exposed to 5% liquid H2O2 for 60 min. Spores of each strain exhibited higher resistance than their respective vegetative cells to liquid H2O2. Results indicate that the H2O2 resistance observed in both vegetative cells and spores is strain-specific, as certain B. pumilus strains were two to three times more resistant than a standard Bacillus subtilis dosimetry strain. An example of this trend was observed when the type strain of B. pumilus, ATCC 7061, proved sensitive, whereas several environmental strains exhibited varying degrees of resistance, to H2O2. Repeated isolation of H2O2-resistant strains of B. pumilus in a clean-room is a concern because their persistence might potentially compromise life-detection missions, which have very strict cleanliness and sterility requirements for spacecraft hardware.
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Zhang, Pengfei; Kong, Lingbo; Setlow, Peter; Li, Yong-qing
2010-01-01
Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELS) were used to investigate dynamic processes during high-temperature treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis in water. Major conclusions from these studies included the following. (i) After spores of all three species were added to water at 80 to 90°C, the level of the 1:1 complex of Ca2+ and dipicolinic acid (CaDPA; ∼25% of the dry weight of the spore core) in individual spores remained relatively constant during a highly variable lag time (Tlag), and then CaDPA was released within 1 to 2 min. (ii) The Tlag values prior to rapid CaDPA release and thus the times for wet-heat killing of individual spores of all three species were very heterogeneous. (iii) The heterogeneity in kinetics of wet-heat killing of individual spores was not due to differences in the microscopic physical environments during heat treatment. (iv) During the wet-heat treatment of spores of all three species, spore protein denaturation largely but not completely accompanied rapid CaDPA release, as some changes in protein structure preceded rapid CaDPA release. (v) Changes in the ELS from individual spores of all three species were strongly correlated with the release of CaDPA. The ELS intensities of B. cereus and B. megaterium spores decreased gradually and reached minima at T1 when ∼80% of spore CaDPA was released, then increased rapidly until T2 when full CaDPA release was complete, and then remained nearly constant. The ELS intensity of B. subtilis spores showed similar features, although the intensity changed minimally, if at all, prior to T1. (vi) Carotenoids in B. megaterium spores' inner membranes exhibited two changes during heat treatment. First, the carotenoid's two Raman bands at 1,155 and 1,516 cm−1 decreased rapidly to a low value and to zero, respectively, well before Tlag, and then the residual 1,155-cm−1 band disappeared, in parallel
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Oie, Shigeharu; Obayashi, Akiko; Yamasaki, Hirofumi; Furukawa, Hiroyuki; Kenri, Tsuyoshi; Takahashi, Motohide; Kawamoto, Keiko; Makino, Sou-ichi
2011-01-01
To evaluate disinfection methods for environments contaminated with bioterrorism-associated microorganism (Bacillus anthracis), we performed the following experiments. First, the sporicidal effects of sodium hypochlorite on spores of five bacterial species were evaluated. Bacillus atrophaeus was the most resistant to hypochlorite, followed in order by B. anthracis, Clostridium botulinum and Clostridium tetani, and Clostridium difficile. Subsequently, using B. atrophaeus spores that were the most resistant to hypochlorite, the sporicidal effects of hypochlorite at lower pH by adding vinegar were evaluated. Hypochlorite containing vinegar had far more marked sporicidal effects than hypochlorite alone. Cleaning with 0.5% (5000 ppm) hypochlorite containing vinegar inactivated B. atrophaeus spores attached to vinyl chloride and plywood plates within 15 s, while that not containing vinegar did not inactivate spores attached to cement or plywood plates even after 1 h. Therefore, the surfaces of cement or plywood plates were covered with gauze soaked in 0.5% hypochlorite containing vinegar, and the sporicidal effects were evaluated. B. atrophaeus spores attached to plywood plates were not inactivated even after 6 h, but those attached to cement plates were inactivated within 5 min. On the other hand, covering the surfaces of plywood plates with gauze soaked in 0.3% peracetic acid and gauze soaked in 2% glutaral inactivated B. atrophaeus spores within 5 min and 6 h, respectively. These results suggest that hypochlorite containing vinegar is effective for disinfecting vinyl chloride, tile, and cement plates contaminated with B. anthracis, and peracetic acid is effective for disinfecting plywood plates contaminated with such microorganism.
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Abstract
The insecticidal toxin encoded by the cry1Ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a...
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USDA-ARS?s Scientific Manuscript database
Field evolved resistance of insect populations to Bacillus thuringiensis (Bt) crystalline (Cry) toxins expressed by crop plants has resulted in reduced control of insect feeding damage to field crops, and threatens the sustainability of Bt transgenic technologies. A single quantitative trait locus ...
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New detection targets for amyloid-reactive probes: spectroscopic recognition of bacterial spores
NASA Astrophysics Data System (ADS)
Jones, Guilford, II; Landsman, Pavel
2005-05-01
We report characteristic changes in fluorescence of amyloid-binding dyes Thioflavin T (TfT), pinacyanol (PIN) and related dyes, caused by their interaction with suspended Bacillus spore cultures (B. subtilis, B thuringiensis). The gain in TfT emission in the presence of spores allowed their immediate detection in aqueous suspensions, with a sensitivity limit of < 105 spores per ml. The spectroscopic signatures are consistent with a large number of binding sites for the two dyes on spore coats. The possible structural relationship of these dye binding loci with characteristic motifs (β-stacks) of amyloid deposits and other misfolded protein formations suggests new designs for probing biocontamination and also for clinical studies of non-microbial human pathogens (e.g., amyloid-related protein aggregates in prion-related transmissible encephalopathies or in Alzheimer's disease). Also reported is a special screening technique that was designed and used herein for calibration of new detection probes and assays for spore detection. It employed spectroscopic interactions between the candidate amyloid stains and poly(vinylpyrrolidone)-coated colloid silica (Percoll) nanoparticles that also display remarkable parallelism with the corresponding dye-amyloid and dye-spore reactivities. Percoll may thus find new applications as a convenient non-biological structural model mimicking the putative probe-targeted motifs in both classes of bioanalytes. These findings are important in the design of new probes and assays for important human pathogens (i.e. bacterial spores and amyloidogenic protein aggregates).
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Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718.
Rang, Jie; He, Hao; Wang, Ting; Ding, Xuezhi; Zuo, Mingxing; Quan, Meifang; Sun, Yunjun; Yu, Ziquan; Hu, Shengbiao; Xia, Liqiu
2015-01-01
Bacillus thuringiensis is a widely used biopesticide that produced various insecticidal active substances during its life cycle. Separation and purification of numerous insecticide active substances have been difficult because of the relatively short half-life of such substances. On the other hand, substances can be synthetized at different times during development, so samples at different stages have to be studied, further complicating the analysis. A dual genomic and proteomic approach would enhance our ability to identify such substances, and particularily using mass spectrometry-based proteomic methods. The comparative analysis for genomic and proteomic data have showed that not all of the products deduced from the annotated genome could be identified among the proteomic data. For instance, genome annotation results showed that 39 coding sequences in the whole genome were related to insect pathogenicity, including five cry genes. However, Cry2Ab, Cry1Ia, Cytotoxin K, Bacteriocin, Exoenzyme C3 and Alveolysin could not be detected in the proteomic data obtained. The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry. This analysis revealed Spo0A~P, SigF, SigE(+), SigK(+) and SigG(+), all known to play an important role in the process of spore formation regulatory network, also were displayed in the proteomic data. Through the comparison of the two data sets, it was possible to infer that some genes were silenced or were expressed at very low levels. For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons. With this comparative study a relatively complete database can be constructed and used to transform hereditary material, thereby prompting the high expression of toxic proteins. A theoretical basis is provided for constructing highly virulent engineered bacteria and for
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Cetin, Huseyin; Oz, Emre; Yanikoglu, Atila; Cilek, James E
2015-06-01
The residual effectiveness of VectoMax® WSP (a water-soluble pouch formulation containing a combination of Bacillus thuringiensis subsp. israelensis strain AM65-52 and B. sphaericus strain ABTS 1743) when applied to septic tanks against 3rd- and 4th-stage larvae of Culex pipiens L. was evaluated in this study. This formulation was evaluated at operational application rates of 1 pouch (10 g) and 2 pouches (20 g) per septic tank. Both application rates resulted in >96% control of larvae for 24 days. Operationally, VectoMax WSP has proven to be a useful tool for the nonchemical control of Culex species in septic tank environments.
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Simple, Inexpensive, and Rapid Way to Produce Bacillus subtilis Spores for the Guthrie Bioassay
Franklin, Martha L.; Clark, William A.
1981-01-01
Esculin agar has been found to be a simple, inexpensive, rapid, and reliable means to promote production of spores of inhibitor-sensitive clones of Bacillus subtilis strains ATCC 6051 and 6633 for use in the Guthrie bioassay screening tests for genetic metabolic disorders. Images PMID:6790564
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March, Jordon K; Pratt, Michael D; Lowe, Chinn-Woan; Cohen, Marissa N; Satterfield, Benjamin A; Schaalje, Bruce; O'Neill, Kim L; Robison, Richard A
2015-10-01
This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat
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Evolution of Bacillus thuringiensis Cry toxins insecticidal activity.
Bravo, Alejandra; Gómez, Isabel; Porta, Helena; García-Gómez, Blanca Ines; Rodriguez-Almazan, Claudia; Pardo, Liliana; Soberón, Mario
2013-01-01
Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.
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Bacillus thuringiensis: A story of a successful bioinsecticide
Bravo, Alejandra; Likitvivatanavong, Supaporn; Gill, Sarjeet S.; Soberón, Mario
2013-01-01
Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture. PMID:21376122
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SporeWeb: an interactive journey through the complete sporulation cycle of Bacillus subtilis.
Eijlander, Robyn T; de Jong, Anne; Krawczyk, Antonina O; Holsappel, Siger; Kuipers, Oscar P
2014-01-01
Bacterial spores are a continuous problem for both food-based and health-related industries. Decades of scientific research dedicated towards understanding molecular and gene regulatory aspects of sporulation, spore germination and spore properties have resulted in a wealth of data and information. To facilitate obtaining a complete overview as well as new insights concerning this complex and tightly regulated process, we have developed a database-driven knowledge platform called SporeWeb (http://sporeweb.molgenrug.nl) that focuses on gene regulatory networks during sporulation in the Gram-positive bacterium Bacillus subtilis. Dynamic features allow the user to navigate through all stages of sporulation with review-like descriptions, schematic overviews on transcriptional regulation and detailed information on all regulators and the genes under their control. The Web site supports data acquisition on sporulation genes and their expression, regulon network interactions and direct links to other knowledge platforms or relevant literature. The information found on SporeWeb (including figures and tables) can and will be updated as new information becomes available in the literature. In this way, SporeWeb offers a novel, convenient and timely reference, an information source and a data acquisition tool that will aid in the general understanding of the dynamics of the complete sporulation cycle.
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Bloomfield, S F; Arthur, M
1992-02-01
Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortex material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.
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Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor.
Hao, Rongzhang; Wang, Dianbing; Zhang, Xian'en; Zuo, Guomin; Wei, Hongping; Yang, Ruifu; Zhang, Zhiping; Cheng, Zhenxing; Guo, Yongchao; Cui, Zongqiang; Zhou, Yafeng
2009-01-01
Since the anthrax spore bioterrorism attacks in America in 2001, the early detection of Bacillus anthracis spores and vegetative cells has gained significant interest. At present, many polyclonal antibody-based quartz crystal microbalance (QCM) sensors have been developed to detect B. anthracis simulates. To achieve a simultaneous rapid detection of B. anthracis spores and vegetative cells, this paper presents a biosensor that utilizes an anti-B. anthracis monoclonal antibody designated to 8G3 (mAb 8G3, IgG) functionalized QCM sensor. Having compared four kinds of antibody immobilizations on Au surface, an optimized mAb 8G3 was immobilized onto the Au electrode with protein A on a mixed self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) and 6-mercaptohexan-1-ol (6-MHO) as adhesive layer. The detection of B. anthracis was investigated under three conditions: dip-and-dry, static addition and flow through procedure. The results indicated that the sensor yielded a distinct response to B. anthracis spores or vegetative cells but had no significant response to Bacillus thuringiensis species. The functionalized sensor recognized B. anthracis spores and vegetative cells specifically from its homophylic ones, and the limit of detection (LOD) reached 10(3)CFU or spores/ml of B. anthracis in less than 30 min. Cyclic voltammogram (CV) and scanning electronic microscopy (SEM) were performed to characterize the surface of the sensor in variable steps during the modification and after the detection. The mAb functionalized QCM biosensor will be helpful in the fabrication of a similar biosensor that may be available in anti-bioterrorism in the future.
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Lin, Yi; Cai, Fu-Ying; Zhang, Guang-Ya
2007-01-01
A quantitative structure-property relationship (QSPR) model in terms of amino acid composition and the activity of Bacillus thuringiensis insecticidal crystal proteins was established. Support vector machine (SVM) is a novel general machine-learning tool based on the structural risk minimization principle that exhibits good generalization when fault samples are few; it is especially suitable for classification, forecasting, and estimation in cases where small amounts of samples are involved such as fault diagnosis; however, some parameters of SVM are selected based on the experience of the operator, which has led to decreased efficiency of SVM in practical application. The uniform design (UD) method was applied to optimize the running parameters of SVM. It was found that the average accuracy rate approached 73% when the penalty factor was 0.01, the epsilon 0.2, the gamma 0.05, and the range 0.5. The results indicated that UD might be used an effective method to optimize the parameters of SVM and SVM and could be used as an alternative powerful modeling tool for QSPR studies of the activity of Bacillus thuringiensis (Bt) insecticidal crystal proteins. Therefore, a novel method for predicting the insecticidal activity of Bt insecticidal crystal proteins was proposed by the authors of this study.
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The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis.
Wang, Katherine H; Isidro, Anabela L; Domingues, Lia; Eskandarian, Haig A; McKenney, Peter T; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O; Eichenberger, Patrick
2009-11-01
Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.
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The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis
Wang, Katherine H.; Isidro, Anabela L.; Domingues, Lia; Eskandarian, Haig A.; McKenney, Peter T.; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N.; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O.; Eichenberger, Patrick
2009-01-01
SUMMARY Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to GFP and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homolog of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement. PMID:19775244
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Cangiano, Giuseppina; Sirec, Teja; Panarella, Cristina; Isticato, Rachele; Baccigalupi, Loredana; De Felice, Maurilio
2014-01-01
The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform. PMID:25239894
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Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.
Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H
1978-09-01
Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Letant, S E; Kane, S R; Murphy, G A
2008-05-30
This note presents a comparison of Most-Probable-Number Rapid Viability (MPN-RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs generated by the Centers for Disease Control and Prevention (CDC) for a multi-center validation study aimed at testing environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were in statistical agreement with the CDC conventional culture method for all three levels of spores tested (10{sup 4}, 10{sup 2}, and 10 spores) even in the presence ofmore » dirt. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols.« less
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Zhang, Pengfei; Kong, Lingbo; Wang, Guiwen; Setlow, Peter; Li, Yong-qing
2011-01-01
Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca2+ with dipicolinic acid (CaDPA) was released rapidly at a highly variable time Tlag, the levels of spore nucleic acids remained nearly unchanged, and the Tlag times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ∼50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before Tlag and reached maximum at a time slightly later than Trelease. However, the fluorescence intensities of wet-heat-inactivated spores were ∼15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment. PMID:21602365
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Draft Genome Sequence of the Spore-Forming Probiotic Strain Bacillus coagulans Unique IS-2
Upadrasta, Aditya; Pitta, Swetha
2016-01-01
Bacillus coagulans Unique IS-2 is a potential spore-forming probiotic that is commercially available on the market. The draft genome sequence presented here provides deep insight into the beneficial features of this strain for its safe use as a probiotic for various human and animal health applications. PMID:27103709
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Hibi, Makoto; Kawashima, Takashi; Kodera, Tomohiro; Smirnov, Sergey V.; Sokolov, Pavel M.; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun
2011-01-01
We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. PMID:21821743
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Code of Federal Regulations, 2014 CFR
2014-07-01
... protein in corn; exemption from the requirement of a tolerance. 174.532 Section 174.532 Protection of...Cry3.1Ab protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn, field; corn...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... protein in corn; exemption from the requirement of a tolerance. 174.532 Section 174.532 Protection of...Cry3.1Ab protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn, field; corn...
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Multigeneration Cross Contamination of Mail with Bacillus Species Spores by Tumbling ▿
Edmonds, Jason; Clark, Paul; Williams, Leslie; Lindquist, H. D. Alan; Martinez, Kenneth; Gardner, Warren; Shadomy, Sean; Hornsby-Myers, Jennifer
2010-01-01
In 2001, envelopes loaded with Bacillus anthracis spores were mailed to Senators Daschle and Leahy as well as to the New York Post and NBC News buildings. Additional letters may have been mailed to other news agencies because there was confirmed anthrax infection of employees at these locations. These events heightened the awareness of the lack of understanding of the mechanism(s) by which objects contaminated with a biological agent might spread disease. This understanding is crucial for the estimation of the potential for exposure to ensure the appropriate response in the event of future attacks. In this study, equipment to simulate interactions between envelopes and procedures to analyze the spread of spores from a “payload” envelope (i.e., loaded internally with a powdered spore preparation) onto neighboring envelopes were developed. Another process to determine whether an aerosol could be generated by opening contaminated envelopes was developed. Subsequent generations of contaminated envelopes originating from a single payload envelope showed a consistent two-log decrease in the number of spores transferred from one generation to the next. Opening a tertiary contaminated envelope resulted in an aerosol containing 103 B. anthracis spores. We developed a procedure for sampling contaminated letters by a nondestructive method aimed at providing information useful for consequence management while preserving the integrity of objects contaminated during the incident and preserving evidence for law enforcement agencies. PMID:20511424
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NASA Technical Reports Server (NTRS)
La Duc, Myron T.; Satomi, Masataka; Agata, Norio; Venkateswaran, Kasthuri
2004-01-01
Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B. cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis. As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species. Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard". We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B. cereus group," including lab strains and environmental isolates. Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors. The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B. cereus group.
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Dybwad, Marius; van der Laaken, Anton L; Blatny, Janet Martha; Paauw, Armand
2013-09-01
Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 10(6) spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.
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van der Laaken, Anton L.; Blatny, Janet Martha; Paauw, Armand
2013-01-01
Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 106 spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min. PMID:23811517
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Tang, Zeli; Shang, Mei; Chen, Tingjin; Ren, Pengli; Sun, Hengchang; Qu, Hongling; Lin, Zhipeng; Zhou, Lina; Yu, Jinyun; Jiang, Hongye; Zhou, Xinyi; Li, Xuerong; Huang, Yan; Xu, Jin; Yu, Xinbing
2016-12-19
Clonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes. We constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome. The B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores. Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic
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USDA-ARS?s Scientific Manuscript database
HTST pasteurization of milk is generally ineffective against spore-forming bacteria such as Bacillus anthracis (BA) but is lethal to its vegetative cells. Crossflow microfiltration (MF), using ceramic membranes with a pore diameter of 1.4 um, has been shown to physically remove somatic cells, vegeta...
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Setlow, B; Korza, G; Blatt, K M S; Fey, J P; Setlow, P
2016-01-01
Determine how supercritical CO2 (scCO2 ) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO2 -PAA, and if spores inactivated by scCO2 -PAA are truly dead. Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO2 -PAA in liquid or dry at 35°C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2 -PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85°C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO2 -PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO2 -PAA sensitive. These findings suggest that scCO2 -PAA inactivates spores by damaging spores' inner membrane. The spore coat provided scCO2 -PAA resistance for both wet and dry spores. DNA protective proteins provided scCO2 -PAA resistance only for dry spores. These results provide information on mechanisms of spore inactivation of and resistance to scCO2 -PAA, an agent with increasing use in sterilization applications. © 2015 The Society for Applied Microbiology.
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NASA Technical Reports Server (NTRS)
Nicholson, Wayne L.; Schuerger, Andrew C.
2005-01-01
Bacterial endospores in the genus Bacillus are considered good models for studying interplanetary transfer of microbes by natural or human processes. Although spore survival during transfer itself has been the subject of considerable study, the fate of spores in extraterrestrial environments has received less attention. In this report we subjected spores of a strain of Bacillus subtilis, containing luciferase resulting from expression of an sspB-luxAB gene fusion, to simulated martian atmospheric pressure (7-18 mbar) and composition (100% CO(2)) for up to 19 days in a Mars simulation chamber. We report here that survival was similar between spores exposed to Earth conditions and spores exposed up to 19 days to simulated martian conditions. However, germination-induced bioluminescence was lower in spores exposed to simulated martian atmosphere, which suggests sublethal impairment of some endogenous spore germination processes.
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Berendsen, Erwin M; Zwietering, Marcel H; Kuipers, Oscar P; Wells-Bennik, Marjon H J
2015-02-01
The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of time-temperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups (p < 0.001) within the B. subtilis group could be identified. One group of strains had spores with an average D120 °C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D120 °C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z-values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Comparison of Fe(VI) (FeO4(2-)) and ozone in inactivating Bacillus subtilis spores.
Makky, Essam A; Park, Gui-Su; Choi, Ik-Won; Cho, Sung-Il; Kim, Hyunook
2011-05-01
The protozoan parasites such as Cryptosporidiumparvum and Giardialamblia have been recognized as a frequent cause of recent waterborne disease outbreaks because of their strong resistance against chlorine disinfection. In this study, ozone and Fe(VI) (i.e., FeO(4)(2-)) were compared in terms of inactivation efficiency for Bacillus subtilis spores which are commonly utilized as an indicator of protozoan pathogens. Both oxidants highly depended on water pH and temperature in the spore inactivation. Since redox potential of Fe(VI) is almost the same as that of ozone, spore inactivation efficiency of Fe(VI) was expected to be similar with that of ozone. However, it was found that ozone was definitely superior over Fe(VI): at pH 7 and 20°C, ozone with the product of concentration×contact time (C¯T) of 10mgL(-1)min inactivate the spores more than 99.9% within 10min, while Fe(VI) with C¯T of 30mgL(-1) min could inactivate 90% spores. The large difference between ozone and Fe(VI) in spore inactivation was attributed mainly to Fe(III) produced from Fe(VI) decomposition at the spore coat layer which might coagulate spores and make it difficult for free Fe(VI) to attack live spores. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.
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Brown, Gary S; Betty, Rita G; Brockmann, John E; Lucero, Daniel A; Souza, Caroline A; Walsh, Kathryn S; Boucher, Raymond M; Tezak, Matthew S; Wilson, Mollye C
2007-07-01
Vacuum filter socks were evaluated for recovery efficiency of powdered Bacillus atrophaeus spores from two non-porous surfaces, stainless steel and painted wallboard and two porous surfaces, carpet and bare concrete. Two surface coupons were positioned side-by-side and seeded with aerosolized Bacillus atrophaeus spores. One of the surfaces, a stainless steel reference coupon, was sized to fit into a sample vial for direct spore removal, while the other surface, a sample surface coupon, was sized for a vacuum collection application. Deposited spore material was directly removed from the reference coupon surface and cultured for enumeration of colony forming units (CFU), while deposited spore material was collected from the sample coupon using the vacuum filter sock method, extracted by sonication and cultured for enumeration. Recovery efficiency, which is a measure of overall transfer effectiveness from the surface to culture, was calculated as the number of CFU enumerated from the filter sock sample per unit area relative to the number of CFU enumerated from the co-located reference coupon per unit area. The observed mean filter sock recovery efficiency from stainless steel was 0.29 (SD = 0.14, n = 36), from painted wallboard was 0.25 (SD = 0.15, n = 36), from carpet was 0.28 (SD = 0.13, n = 40) and from bare concrete was 0.19 (SD = 0.14, n = 44). Vacuum filter sock recovery quantitative limits of detection were estimated at 105 CFU m(-2) from stainless steel and carpet, 120 CFU m(-2) from painted wallboard and 160 CFU m(-2) from bare concrete. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling for biological agents such as Bacillus anthracis.
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Setlow, Barbara; Korza, George; Blatt, Kelly M.S.; Fey, Julien P.; Setlow, Peter
2015-01-01
Aims Determine how supercritical CO2 (scCO2) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO2-PAA, and if spores inactivated by scCO2-PAA are truly dead. Methods and Results Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO2-PAA in liquid or dry at 35°C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2-PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85°C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO2-PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO2-PAA sensitive. Conclusions These findings suggest that scCO2-PAA inactivates spores by damaging spores’ inner membrane. The spore coat provided scCO2-PAA resistance for both wet and dry spores. DNA protective proteins provided scCO2-PAA resistance only for dry spores. Significance and Impact of Study These results provide information on mechanisms of spore inactivation of and resistance to scCO2-PAA, an agent with increasing use in sterilization applications. PMID:26535794
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Dashora, Kavya; Roy, Somnath; Nagpal, Akanksha; Roy, Sudipta Mukhopadhyay; Flood, Julie; Prasad, Anjali Km; Khetarpal, Ravinder; Neave, Suzanne; Muraleedharan, N
2017-03-01
Bacillus thuringiensis (Bt) is a soil bacterium that forms spores containing crystals comprising one or more Cry or Cyt proteins having potential and specific insecticidal activity. Different strains of Bt produce different types of toxins, affecting a narrow taxonomic group of insects. Therefore, it is used in non-chemical pest management, including inherent pest resistance through GM crops. The specificity of action of Bt toxins reduces the concern of adverse effects on non-target species, a concern which remains with chemical insecticides as well. To make use of Bt more sustainable, new strains expressing novel toxins are actively being sought globally. Since Bt is successfully used against many pests including the lepidopteran pests in different crop groups, the insecticidal activity against Samia cynthia (Drury) (Eri silkworm) and Antheraea assamensis Helfer (Muga silkworm) becomes a concern in the state of Assam in India which is a predominantly tea- and silk-producing zone. Though Bt can be used as an effective non-chemical approach for pest management for tea pests in the same geographical region, yet, it may potentially affect the silk industry which depends on silkworm. There is a need to identify the potentially lethal impact (through evaluating their mortality potential) of local Bt strains on key silkworm species in North Eastern India. This will allow the use of existing Bt for which the silkworms have natural resistance. Through this review, the authors aim to highlight recent progress in the use of Bt and its insecticidal toxins in tea pest control and the potential sensitivity for tea- and silk-producing zone of Assam in India.
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Pérez, Melisa P; Sauka, Diego H; Onco, María I; Berretta, Marcelo F; Benintende, Graciela B
Preliminary bioassays with whole cultures (WC) of 124 Bacillus thuringiensis strains were performed with neonate larvae of Anthonomus grandis, a major cotton pest in Argentina and other regions of the Americas. Three exotic and four native strains were selected for causing more than 50% mortality. All of them were β-exotoxin producers. The native strains shared similar morphology of parasporal crystals, similar protein pattern and identical insecticidal gene profiles. These features resembled Lepidoptera-toxic strains. Furthermore, these strains showed a Rep-PCR pattern identical to lepidoptericidal strain HD-1, suggesting that these strains may belong to serovar kurstaki. However, some differences were observed in the plasmid profiles and in the production of β-exotoxin. To determine the culture fractions where the insecticidal metabolites were present, bioassays including resuspended spore-crystal pellets, filtered supernatants (FS) were compared with those of WC. Both fractions tested showed some level of insecticidal activity. The results may suggest that the main toxic factors can be found in FS and could be directly correlated with the presence of β-exotoxin. Based on the bioassays with FS and autoclaved FS, the participation of thermolabile virulence factors such as Cry1I in toxicity is neither discarded. In the selected strains, β-exotoxin would be the major associated virulence factor; therefore, their use in biological control of A. grandis should be restricted. Nevertheless, these strains could be the source of genes (e.g., cry1Ia) to produce transgenic cotton plants resistant to this pest. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.
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Algimantas P. Valaitis; John D. Podgwaite
2011-01-01
Many strains of Bacillus thuringiensis (Bt) produce insecticidal proteins, also referred to as Cry toxins, in crystal inclusions during sporulation. When ingested by insects, the Cry toxins bind to receptors on the brush border midgut epithelial cells and create pores in the epithelial gut membranes resulting in the death of...
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Olga Loseva; Mohamed Ibrahim; Mehmet Candas; C. Noah Koller; Leah S. Bauer; Lee A. Jr. Bulla
2002-01-01
Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance...
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USDA-ARS?s Scientific Manuscript database
Background: Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt) toxin and for discovering new targets for novel toxins for use in pest management. This study analyzed the ES...
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Influence of food matrix on outgrowth heterogeneity of heat damaged Bacillus cereus spores.
Warda, Alicja K; den Besten, Heidy M W; Sha, Na; Abee, Tjakko; Nierop Groot, Masja N
2015-05-18
Spoilage of heat treated foods can be caused by the presence of surviving spore-formers. It is virtually impossible to prevent contamination at the primary production level as spores are ubiquitous present in the environment and can contaminate raw products. As a result spore inactivation treatments are widely used by food producing industries to reduce the microbial spore loads. However consumers prefer mildly processed products that have less impact on its quality and this trend steers industry towards milder preservation treatments. Such treatments may result in damaged instead of inactivated spores, and these spores may germinate, repair, and grow out, possibly leading to quality and safety issues. The ability to repair and grow out is influenced by the properties of the food matrix. In the current communication we studied the outgrowth from heat damaged Bacillus cereus ATCC 14579 spores on Anopore membrane, which allowed following outgrowth heterogeneity of individual spores on broccoli and rice-based media as well as standard and mildly acidified (pH 5.5) meat-based BHI. Rice, broccoli and BHI pH 5.5 media resulted in delayed outgrowth from untreated spores, and increased heterogeneity compared to BHI pH 7.4, with the most pronounced effect in rice media. Exposure to wet heat for 1 min at 95 °C caused 2 log inactivation and approximately 95% of the spores in the surviving fraction were damaged resulting in substantial delay in outgrowth based on the time required to reach a maximum microcolony size of 256 cells. The delay was most pronounced for heat-treated spores on broccoli medium followed by spores on rice media (both untreated and treated). Interestingly, the increase in outgrowth heterogeneity of heat treated spores on BHI pH 7.4 was more pronounced than on rice, broccoli and BHI pH 5.5 conceivably reflecting that conditions in BHI pH 7.4 better support spore damage repair. This study compares the effects of three main factors, namely heat treatment, p
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den Besten, Heidy M W; Berendsen, Erwin M; Wells-Bennik, Marjon H J; Straatsma, Han; Zwietering, Marcel H
2017-07-17
Realistic prediction of microbial inactivation in food requires quantitative information on variability introduced by the microorganisms. Bacillus subtilis forms heat resistant spores and in this study the impact of strain variability on spore heat resistance was quantified using 20 strains. In addition, experimental variability was quantified by using technical replicates per heat treatment experiment, and reproduction variability was quantified by using two biologically independent spore crops for each strain that were heat treated on different days. The fourth-decimal reduction times and z-values were estimated by a one-step and two-step model fitting procedure. Grouping of the 20 B. subtilis strains into two statistically distinguishable groups could be confirmed based on their spore heat resistance. The reproduction variability was higher than experimental variability, but both variabilities were much lower than strain variability. The model fitting approach did not significantly affect the quantification of variability. Remarkably, when strain variability in spore heat resistance was quantified using only the strains producing low-level heat resistant spores, then this strain variability was comparable with the previously reported strain variability in heat resistance of vegetative cells of Listeria monocytogenes, although in a totally other temperature range. Strains that produced spores with high-level heat resistance showed similar temperature range for growth as strains that produced low-level heat resistance. Strain variability affected heat resistance of spores most, and therefore integration of this variability factor in modelling of spore heat resistance will make predictions more realistic. Copyright © 2017. Published by Elsevier B.V.
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Silvestri, Erin E.; Griffin, Dale W.
2017-01-01
This protocol describes the processing steps for 45 g and 9 g soil samples potentially contaminated with Bacillus anthracis spores. The protocol is designed to separate and concentrate the spores from bulk soil down to a pellet that can be used for further analysis. Soil extraction solution and mechanical shaking are used to disrupt soil particle aggregates and to aid in the separation of spores from soil particles. Soil samples are washed twice with soil extraction solution to maximize recovery. Differential centrifugation is used to separate spores from the majority of the soil material. The 45 g protocol has been demonstrated by two laboratories using both loamy and sandy soil types. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol would be robust enough to use at multiple laboratories while achieving comparable recoveries. The 45 g protocol has demonstrated a matrix limit of detection at 14 spores/gram of soil for loamy and sandy soils.
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Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis
Espinasse, Sylvain; Gohar, Michel; Chaufaux, Josette; Buisson, Christophe; Perchat, Stéphane; Sanchis, Vincent
2002-01-01
Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 μg of β-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 μg/ml. However, these amounts of β-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of β-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of β-exotoxin I. PMID:12200263
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Yu, Ziquan; Xiong, Jing; Zhou, Qiaoni; Luo, Haiyan; Hu, Shengbiao; Xia, Liqiu; Sun, Ming; Li, Lin; Yu, Ziniu
2015-02-01
Cry6A toxin from Bacillus thuringiensis is a representative nematicidal crystal protein with a variety of nematicidal properties to free-living nematode Caenorhabditis elegans. Cry6A shares very low homology and different structure with Cry5B, another representative nematicidal crystal protein, and probably acts in a distinct pathway. All these strongly indicate that Cry6A toxin is likely a potent candidate for nematicide. The present study dealt with global investigation to determine the detrimental impacts of Cry6Aa2 toxin on Meloidogyne hapla, a root-knot nematode, and evaluated its biocontrol efficacy in pot experiment. Obtained results indicated that Cry6Aa2 toxin exhibits obvious toxicity to second-stage juvenile of M. hapla, and significantly inhibits egg hatch, motility, and penetration to host plant. Pot experiment suggested that soil drenching with spore-crystal mixture of Cry6Aa2 can clearly lighten the disease of root-knot nematode, including reduction of galling index and egg masses on host plant root, decreasing final population of nematode in soil. Moreover, application of Cry6Aa2 can obviously promote plant growth. These results demonstrated that Cry6Aa2 toxin is a promising nematicidal agent, and possesses great potential in plant-parasitic nematode management and construction of transgenic crop with constant resistance to nematode. Copyright © 2015 Elsevier Inc. All rights reserved.
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Vesley, Donald; Langholz, Ann C.; Rohlfing, Stephen R.; Foltz, William E.
1992-01-01
A biological indicator based on fluorimetric detection within 60 min of a Bacillus stearothermophilus spore-bound enzyme, α-d-glucosidase, has been developed. Results indicate that the enzyme survived slightly longer than spores observed after 24 h of incubation. The new system shows promise for evaluating flash sterilization cycles within 60 min compared with conventional 24-h systems. PMID:16348654
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NASA Astrophysics Data System (ADS)
Lutfiana, Nihayatul; Gama, Zulfaidah Penata
2017-11-01
Dengue is a mosquito-borne viral disease that is transmitted by the female Aedes species. The number of dengue fever cases has increased in many geographic regions including Indonesia and one of them occurred in Bareng Tenes, Malang City, East Java Province. The objective of this research was to identify the potency of B. thuringeinsis isolates from Bareng Tenes, Malang, as the biological agent to control third instar Ae. aegypti larvae and to identify the potential B. thuringiensis isolates based on 16S rDNA sequence. B. thuringiensis was isolated from water and soil from 12 sites in the Bareng Tenes area. Bacterial isolation was performed using B. thuringiensis selective media. Several isolates had similar phenotypic characters with B. thuringiensis used to toxicity test against third instar Ae. aegypti larvae. The LC50-96h value was determined using probit regression. The most effective isolate was identified based on the 16S rDNA sequence, then aligned to the reference isolate using the BLAST program. A phylogeny tree was constructed using the Maximum Likelihood method. This study showed that among 22 isolates of B. thuringiensis, only BA02b, BS04a, and BA03a isolates have similar phenotypic characters with B. thuringiensis. Based on the toxicity test of B. thuringiensis against the third instar of Ae. aegypti larvae, it was indicated that BA02b and BA03a isolates were the potential agents to control Ae. aegypti larvae. BA02b isolate was the most effective B. thuringiensis (LC50-96h = 2,75 x 107 cell/mL). Based on 16S rDNA sequence, BA02b was identified as Bacillus thuringiensis var. Israelensis BGSC4Q2 (99 % similarities).
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Influence of Spore Moisture Content on the Dry-Heat Resistance of Bacillus subtilis var. niger
Angelotti, Robert; Maryanski, James H.; Butler, Thomas F.; Peeler, James T.; Campbell, Jeptha E.
1968-01-01
The dry-heat resistance of Bacillus subtilis var. niger spores located in or on various materials was determined as D and z values in the range of 105 through 160 C. The systems tested included spores located on steel and paper strips, spores located between stainless-steel washers mated together under 150 inch-lb and 12 inch-lb of torque, and spores encapsulated in methylmethacrylate and epoxy plastics. D values for a given temperature varied with the test system. High D values were observed for the systems in which spores were encapsulated or under heavy torque, whereas lower D values were observed for the steel and paper strip systems and the lightly torqued system. Similar z values were obtained for the plastic and steel strip systems (zD = 21 C), but an unusually low z for spores on paper (zD = 12.9 C) and an unusually high z for spores on steel washers mated at 150 inch-lb of torque (zD = 32 C) were observed. The effect of spore moisture content on the D value of spores encapsulated in water-impermeable plastic was determined, and maximal resistance was observed for spores with a water activity (aw) of 0.2 to 0.4. Significantly decreased D values were observed for spores with moisture contents below aw 0.2 or above aw 0.4. The data indicate that the important factors to be considered when measuring the dry heat resistance of spores are (i) the initial moisture content of the spore, (ii) the rate of spore desiccation during heating, (iii) the water retention capacity of the material in or on which spores are located, and (iv) the relative humidity of the system at the test temperature. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 PMID:4968962
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Influence of spore moisture content on the dry-heat resistance of Bacillus subtilis var. niger.
Angelotti, R; Maryanski, J H; Butler, T F; Peeler, J T; Campbell, J E
1968-05-01
The dry-heat resistance of Bacillus subtilis var. niger spores located in or on various materials was determined as D and z values in the range of 105 through 160 C. The systems tested included spores located on steel and paper strips, spores located between stainless-steel washers mated together under 150 inch-lb and 12 inch-lb of torque, and spores encapsulated in methylmethacrylate and epoxy plastics. D values for a given temperature varied with the test system. High D values were observed for the systems in which spores were encapsulated or under heavy torque, whereas lower D values were observed for the steel and paper strip systems and the lightly torqued system. Similar z values were obtained for the plastic and steel strip systems (z(D) = 21 C), but an unusually low z for spores on paper (z(D) = 12.9 C) and an unusually high z for spores on steel washers mated at 150 inch-lb of torque (z(D) = 32 C) were observed. The effect of spore moisture content on the D value of spores encapsulated in water-impermeable plastic was determined, and maximal resistance was observed for spores with a water activity (a(w)) of 0.2 to 0.4. Significantly decreased D values were observed for spores with moisture contents below a(w) 0.2 or above a(w) 0.4. The data indicate that the important factors to be considered when measuring the dry heat resistance of spores are (i) the initial moisture content of the spore, (ii) the rate of spore desiccation during heating, (iii) the water retention capacity of the material in or on which spores are located, and (iv) the relative humidity of the system at the test temperature.
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Rajan, S; Ahn, J; Balasubramaniam, V M; Yousef, A E
2006-04-01
Bacillus amyloliquefaciens is a potential surrogate for Clostridium botulinum in validation studies involving bacterial spore inactivation by pressure-assisted thermal processing. Spores of B. amyloliquefaciens Fad 82 were inoculated into egg patty mince (approximately 1.4 x 10(8) spores per g), and the product was treated with combinations of pressure (0.1 to 700 MPa) and heat (95 to 121 degrees C) in a custom-made high-pressure kinetic tester. The values for the inactivation kinetic parameter (D), temperature coefficient (zT), and pressure coefficient (zP) were determined with a linear model. Inactivation parameters from the nonlinear Weibull model also were estimated. An increase in process pressure decreased the D-value at 95, 105, and 110 degrees C; however, at 121 degrees C the contribution of pressure to spore lethality was less pronounced. The zP-value increased from 170 MPa at 95 degrees C to 332 MPa at 121 degrees C, suggesting that B. amyloliquefaciens spores became less sensitive to pressure changes at higher temperatures. Similarly, the zT-value increased from 8.2 degrees C at 0.1 MPa to 26.8 degrees C at 700 MPa, indicating that at elevated pressures, the spores were less sensitive to changes in temperature. The nonlinear Weibull model parameter b increased with increasing pressure or temperature and was inversely related to the D-value. Pressure-assisted thermal processing is a potential alternative to thermal processing for producing shelf-stable egg products.
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Shull, James J.; Ernst, Robert R.
1962-01-01
The thermal death curve of dried spores of Bacillus stearothermophilus in saturated steam was characterized by three phases: (i) a sharp initial rise in viable count; (ii) a low rate of death which gradually increased; and (iii) logarithmic death at maximal rate. The first phase was a reflection of inadequate heat activation of the spore population. The second and third phases represented the characteristic thermal death curve of the spores in saturated steam. A jacketed steam sterilizer, equipped with a system for initial evacuation of the chamber, was examined for superheat during normal operation. Measurements of spore inactivation and temperature revealed superheat in surface layers of fabrics being processed in steam at 121 C. The high temperature of the fabric surfaces was attributed to absorption of excess heat energy from superheated steam. The superheated steam was produced at the beginning of the normal sterilizing cycle by transfer of heat from the steam-heated jacket to saturated steam entering the vessel. PMID:13988774
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Levy, Caroline; Bornard, Isabelle; Carlin, Frédéric
2011-02-01
Microbial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10(7) spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3×10(4) spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3×10(5) spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed. Copyright © 2010 Elsevier B.V. All rights reserved.
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Effect of Soybean Casein Digest Agar Lot on Number of Bacillus stearothermophilus Spores Recovered †
Pflug, I. J.; Smith, Geraldine M.; Christensen, Ronald
1981-01-01
In recent years it has become increasingly apparent that Bacillus stearothermophilus spores are affected by various environmental factors that influence the performance of the spores as biological indicators. One environmental factor is the recovery medium. The effect of different lots of commercial soybean casein digest agar on the number of colony-forming units per plate was examined in two series of experiments: (i) several lots of medium from two manufacturers were compared in single experiments, and (ii) paired media experiments with four lots of medium were carried out and yielded three-point survivor curves. The results demonstrate that commercial soybean casein digest agar is variable on a lot-to-lot basis. The variation was lowest when recovering unheated or minimally heated spores and increased greatly with the severity of heating. PMID:16345822
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How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis
Poncet, Sandrine; Lazar, Noureddine; Li de la Sierra-Gallay, Inès; Gohar, Michel; Lereclus, Didier; Nessler, Sylvie
2016-01-01
Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection. PMID:27483473
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How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis.
Perchat, Stéphane; Talagas, Antoine; Poncet, Sandrine; Lazar, Noureddine; Li de la Sierra-Gallay, Inès; Gohar, Michel; Lereclus, Didier; Nessler, Sylvie
2016-08-01
Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection.
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Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production.
Posada-Uribe, Luisa F; Romero-Tabarez, Magally; Villegas-Escobar, Valeska
2015-10-01
Bacillus subtilis spores have important biotechnological applications; however, achieving both, high spore cell densities and sporulation efficiencies in fermentation, is poorly reported. In this study, medium components and culture conditions were optimized with different statistical methods to increase spore production of the plant growth promoting rhizobacteria B. subtilis EA-CB0575. Key medium components were determined with Plackett-Burman (PB) design, and the optimum concentration levels of two components (glucose, MgSO4·7H2O) were optimized with a full factorial and central composite design, achieving 1.37 × 10(9) CFU/mL of spore cell density and 93.5 % of sporulation efficiency in shake flask. The optimized medium was used to determine the effect of culture conditions on spore production at bioreactor level, finding that maintaining pH control did not affect significantly spore production, while the interaction of agitation and aeration rates had a significant effect on spore cell density. The overall optimization generated a 17.2-fold increase in spore cell density (8.78 × 10(9) CFU/mL) and 1.9-fold increase in sporulation efficiency (94.2 %) compared to that of PB design. These results indicate the potential of B. subtilis EA-CB0575 to produce both, high spore cell densities and sporulation efficiencies, with very low nutrient requirements and short incubation period which can represent savings of process production.
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Effects of solar ultraviolet radiations on Bacillus subtilis spores and T-7 bacteriophage
NASA Technical Reports Server (NTRS)
Spizizen, J.; Isherwood, J. E.; Taylor, G. R.
1975-01-01
Spores of Bacillus subtilis HA 101 and the DNA polymerase I-defective mutant HA 101 (59)F were exposed to selected wavelengths of solar ultraviolet light and space vacuum during the return of Apollo 16. In addition, coliphage T-7 suspensions were exposed to solar ultraviolet radiation as part of the Microbial Response to Space Environment Experiment. Optical filters were employed to provide different energy levels at wavelengths 254 nm and 280 nm. Dose-response curves for lethal and mutagenic effects were compared with ground-based data. A close parallel was observed between the results of solar radiation and ground tests with spores of the two strains. However, significantly greater inactivation of T-7 bacteriophage was observed after exposure to solar ultraviolet radiation.
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Is the Insect World Overcoming the Efficacy of Bacillus thuringiensis?
Peralta, Cecilia; Palma, Leopoldo
2017-01-18
The use of chemical pesticides revolutionized agriculture with the introduction of DDT (Dichlorodiphenyltrichloroethane) as the first modern chemical insecticide. However, the effectiveness of DDT and other synthetic pesticides, together with their low cost and ease of use, have led to the generation of undesirable side effects, such as pollution of water and food sources, harm to non-target organisms and the generation of insect resistance. The alternative comes from biological control agents, which have taken an expanding share in the pesticide market over the last decades mainly promoted by the necessity to move towards more sustainable agriculture. Among such biological control agents, the bacterium Bacillus thuringiensis (Bt) and its insecticidal toxins have been the most studied and commercially used biological control agents over the last 40 years. However, some insect pests have acquired field-evolved resistance to the most commonly used Bt-based pesticides, threatening their efficacy, which necessitates the immediate search for novel strains and toxins exhibiting different modes of action and specificities in order to perpetuate the insecticidal potential of this bacterium.
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Dry Heat Inactivation of Bacillus subtilis var. niger Spores as a Function of Relative Humidity
Brannen, J. P.; Garst, D. M.
1972-01-01
Dry heat sterilization of Bacillus subtilis var. niger spores at 105 C is enhanced in the relative humidity range 0.03 to 0.2%. D-values of 115 and 125 C are predicted by a kinetic model with parameters set from 105 C data. These predictions are compared to observations. Images PMID:4625341
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Effect of calcium in assay medium on D value of Bacillus stearothermophilus ATCC 7953 spores.
Sasaki, K; Shintani, H; Itoh, J; Kamogawa, T; Kajihara, Y
2000-12-01
The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of D value.
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Marcon, Joelma; Taketani, Rodrigo Gouvêa; Dini-Andreote, Francisco; Mazzero, Giulia Inocêncio; Soares, Fabio Lino; Melo, Itamar Soares; Azevedo, João Lúcio; Andreote, Fernando Dini
2014-01-30
Here, we report the draft genome sequence and the automatic annotation of Bacillus thuringiensis strain BrMgv02-JM63. This genome comprises a set of genes involved in the metabolism of chitin and N-acetylglucosamine utilization, thus suggesting the possible role of this strain in the cycling of organic matter in mangrove soils.
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Xiao, Yutao; Dai, Qing; Hu, Ruqin; Pacheco, Sabino; Yang, Yongbo; Liang, Gemei; Soberón, Mario
2017-01-01
Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm (Helicoverpa armigera), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism. PMID:28082675
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Evaluation of a Stochastic Inactivation Model for Heat-Activated Spores of Bacillus spp. ▿
Corradini, Maria G.; Normand, Mark D.; Eisenberg, Murray; Peleg, Micha
2010-01-01
Heat activates the dormant spores of certain Bacillus spp., which is reflected in the “activation shoulder” in their survival curves. At the same time, heat also inactivates the already active and just activated spores, as well as those still dormant. A stochastic model based on progressively changing probabilities of activation and inactivation can describe this phenomenon. The model is presented in a fully probabilistic discrete form for individual and small groups of spores and as a semicontinuous deterministic model for large spore populations. The same underlying algorithm applies to both isothermal and dynamic heat treatments. Its construction does not require the assumption of the activation and inactivation kinetics or knowledge of their biophysical and biochemical mechanisms. A simplified version of the semicontinuous model was used to simulate survival curves with the activation shoulder that are reminiscent of experimental curves reported in the literature. The model is not intended to replace current models to predict dynamic inactivation but only to offer a conceptual alternative to their interpretation. Nevertheless, by linking the survival curve's shape to probabilities of events at the individual spore level, the model explains, and can be used to simulate, the irregular activation and survival patterns of individual and small groups of spores, which might be involved in food poisoning and spoilage. PMID:20453137
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NASA Astrophysics Data System (ADS)
Astuti, DT; Pujiastuti, Y.; Suparman, SHK; Damiri, N.; Nugraha, S.; Sembiring, ER; Mulawarman
2018-01-01
Bacillus thuringiensis is a gram-positive bacterium that produces crystal proteins toxic (ᴕ-endotoxin) specific to the target insect, but is not toxic to humans and non-target organisms. This study aims to explore the origin of the soil bacterium B. thuringiensis sub-district Sekayu, Banyuasin, South Sumatra and toxicity to larvae of lepidoptera. Fifty soil samples were taken from Musi Banyuasin District, namely 15 from Kayuare strip 2, 20 from Kayuare and 15 from Lumpatan. Isolation, characterization, identification and screening test were conducted in the laboratorium of Pest and Disease, Agricultural Faculty, Sriwijaya University. Isolat codes were given based on the area origin of the samples. Results of the study showed that from 50 isolates of bacteria that had been isolated, there were 15 bacterial isolates, characterized by morphology and physiology the same as B. thuringiensis, which has round colonies, white, wrinkled edges, slippery, elevation arise, aerobic and gram-positive. Of the 15 codes that contain positive isolates of B. thuringiensis, we have obtained several isolates of the following codes: KJ2D5, KJ2N1, KJ2N4, KJ2B3, KJ3R1, KJ3R2, KJ3R3, KJ3R5, KJ3J3, KJ3J4, KJ3P1, DLM5, DLKK12, and DLKK23. Results of screening tests on insects of the Lepidoptera Order showed that there were six isolates that had toxic to Plutella xylostella and Spodoptera litura insects, ie bacterial isolate codes DLM5, KJ3R3, KJ3R5, KJ3J4, KJ3P1, and DLKK23.
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Albert, H; Davies, D J; Woodson, L P; Soper, C J
1998-11-01
The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7.2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.
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Nerandzic, Michelle M; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J
2016-01-01
Background. Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5-2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods. We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results. Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200-2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions. Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin.
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Nerandzic, Michelle M.; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J.
2016-01-01
Background. Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5–2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods. We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results. Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200–2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions. Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin. PMID:26885539
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Effect of Calcium in Assay Medium on D Value of Bacillus stearothermophilus ATCC 7953 Spores
Sasaki, Koichi; Shintani, Hideharu; Itoh, Junpei; Kamogawa, Takuji; Kajihara, Yousei
2000-01-01
The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of D value. PMID:11097939
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USDA-ARS?s Scientific Manuscript database
Transgenic soybeans producing the Cry1Ac insecticidal protein from the bacterium Bacillus thuringiensis (or “Bt”) are currently used to control larvae of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) and the soybean looper [Chrysodeixis includens (Walker)]. The main threat to the sustain...
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Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters
Calfee, M. Worth; Rose, Laura J.; Tufts, Jenia; Morse, Stephen; Clayton, Matt; Touati, Abderrahmane; Griffin-Gatchalian, Nicole; Slone, Christina; McSweeney, Neal
2016-01-01
The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37 mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50% of their total holding capacity. The results were analyzed by one-way ANOVA across material types, presence or absence of dust, and sampling device. The extraction method gave higher relative recoveries than the two vacuum methods evaluated (p ≤ 0.001). On average, recoveries obtained by the vacuum methods were about 30% of those achieved by the extraction method. Relative recoveries between the two vacuum methods were not significantly different (p > 0.05). Although extraction methods yielded higher recoveries than vacuum methods, either HVAC filter sampling approach may provide a rapid and inexpensive mechanism for understanding the extent of contamination following a wide-area biological release incident. PMID:24184312
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de la Fuente-Salcido, Norma M; Casados-Vázquez, Luz Edith; Barboza-Corona, J Eleazar
2013-08-01
Various strains of Bacillus thuringiensis are among the most successful entomopathogenic bacteria used commercially as biopesticides owing to their ability to synthesize insecticidal crystal (Cry) and cytolytic (Cyt) protein toxins during sporulation, and vegetative insecticidal (VIPs) proteins during the vegetative phase of growth. Whereas much is known about the molecular biology of Cry, Cyt, and VIPs, comparatively little is known about other proteins and metabolites synthesized by B. thuringiensis that could also have applied value. Here, we review recent reports on bacteriocins synthesized by this bacterium as they relate to antibacterial activity, molecular genetics, biophysical and biochemical properties, and methods used to separate and purify these antimicrobial peptides. We highlight the potential of bacteriocins for use as food preservatives, antibiotics, plant protection, and plant growth promoters. We suggest that B. thuringiensis could be used not only in biological control of insects but also in other agronomical and industrial areas of public interest.
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Plácido, Alexandra; Coelho, Andreia; Abreu Nascimento, Lucas; Gomes Vasconcelos, Andreanne; Fátima Barroso, Maria; Ramos-Jesus, Joilson; Costa, Vladimir; das Chagas Alves Lima, Francisco; Delerue-Matos, Cristina; Martins Ramos, Ricardo; Marani, Mariela M; Roberto de Souza de Almeida Leite, José
2017-07-01
Transgenic maize produced by the insertion of the Cry transgene into its genome became the second most cultivated crop worldwide. Cry gene from Bacillus thuringiensis kurstaki expresses protein derivatives of crystalline endotoxins which confer insect resistance onto the maize crop. Mandatory labeling of processed food containing or made by genetically modified organisms is in force in many countries, so, it is very urgent to develop fast and practical methods for GMO identification, for example, biosensors. In the absence of an available empirical structure of Cry1A(b)16 protein, a theoretical model was effectively generated, in this work, by homology modeling and molecular dynamics simulations based on two available homologous protein structures. Molecular dynamics simulations were carried out to refine the selected model, and an analysis of its global structure was performed. The refined models of Cry1A(b)16 showed a standard fold and structural characteristics similar to those seen in Bacillus thuringiensis Cry1A(a) insecticidal toxin and Bacillus thuringiensis serovar kurstaki Cry1A(c) toxin. After in silico analysis of Cry1A(b)16, two immunoreactive candidate peptides were selected and specific polyclonal antibodies were produced resulting in antibody-peptide interaction. Biosensing devices are expected to be developed for detection of the Cry1A(b) protein as a marker of transgenic maize in food. Proteins 2017; 85:1248-1257. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
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Cerf, O
1977-01-01
Spores of Bacillus licheniformis 109-2A0 lost their refractility and absorbancy at 640 nm in the presence of metabolizable molecules (L-alanine). The same occurred with spores treated with 4.4 mol/1 hydrogen peroxide, pH 2.0, at 65 degrees C, even after 5 min of treatment. In addition, these transformations could be promoted after 2 min of treatment by inorganic ions (KI). This possibility occurs following a kinetics of activation. Thermodynamic parameters showed this activation to be combined with a molecular re-organization. Loss of refractility or absorbancy, induced by L-ala or KI, was inhibited by inhibitors of membrane functions or of L-alanine dehydrogenase, enzyme of which a noticeable activity was demonstrated in treated spores. Only 10% of spore calcium leaked during the treatment. Therefore loss of refractility or absorbancy caused by molecules metabolizable or not seemed to correspond to a physiological germination. The first even of the metabolic, as well as or the ionic germination could well be a modification of the spore membrane proton-motive force.
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Involvement of Superoxide Dismutase in Spore Coat Assembly in Bacillus subtilis
Henriques, Adriano O.; Melsen, Lawrence R.; Moran, Charles P.
1998-01-01
Endospores of Bacillus subtilis are enclosed in a proteinaceous coat which can be differentiated into a thick, striated outer layer and a thinner, lamellar inner layer. We found that the N-terminal sequence of a 25-kDa protein present in a preparation of spore coat proteins matched that of the Mn-dependent superoxide dismutase (SOD) encoded by the sodA locus. sodA is transcribed throughout the growth and sporulation of a wild-type strain and is responsible for the SOD activity detected in total cell extracts prepared from B. subtilis. Disruption of the sodA locus produced a mutant that lacked any detectable SOD activity during vegetative growth and sporulation. The sodA mutant was not impaired in the ability to form heat- or lysozyme-resistant spores. However, examination of the coat layers of sodA mutant spores revealed increased extractability of the tyrosine-rich outer coat protein CotG. We showed that this condition was not accompanied by augmented transcription of the cotG gene in sporulating cells of the sodA mutant. We conclude that SodA is required for the assembly of CotG into the insoluble matrix of the spore and suggest that CotG is covalently cross-linked into the insoluble matrix by an oxidative reaction dependent on SodA. Ultrastructural analysis revealed that the inner coat formed by a sodA mutant was incomplete. Moreover, the outer coat lacked the characteristic striated appearance of wild-type spores, a pattern that was accentuated in a cotG mutant. These observations suggest that the SodA-dependent formation of the insoluble matrix containing CotG is largely responsible for the striated appearance of this coat layer. PMID:9573176
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2012-01-01
Background Two entomopathogenic fungi, Isaria fumosorosea and Metarhizium anisopliae, and one bacterium, Bacillus thuringiensis, were tested for their ability to cause mortality of Formosan subterranean termites (FST), Coptotermes formosanus (Shiraki), after liquid exposure, and for their lack of propensity to repel FST. Results The fungus Isaria fumosorosea at 108 spores/ml caused 72.5% mortality on day 7, significantly higher than the control and 106 spores/ml treatment. On day 14, the 106 and 108 concentrations caused 38.8% and 92.5% mortality, respectively, significantly higher than the control. On day 21, 82.5% and 100% of the termites were killed by the 106 and 108 treatments, respectively. I. fumosorosea did not repel termites at 106 nor 108 spores/g in sand, soil or sawdust. The fungus Metarhizium anisopliae at 108 spores/ml caused 57.5% mortality on day 7, 77.5% mortality on day 14 and 100% mortality on day 21. Conclusions On all three days the rate of mortality was significantly higher than that of the control and 106 spores/ml treatment with I. fumosorosea. Neither I. fumosorosea nor M. anisopliae caused repellency of FST in sand, soil or sawdust. The bacterium Bacillus thuringiensis did not cause significant mortality on days 7, 14 or 21. When termites were exposed to cells of B. thuringiensis in sawdust and when termites were exposed to a mixture of spores and cells in sand, a significantly higher number remained in the control tubes. Repellency was not seen with B. thuringiensis spores alone, nor with the above treatments in the other substrates. PMID:23241169
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Fajardo-Cavazos, Patricia; Link, Lindsey; Melosh, H Jay; Nicholson, Wayne L
2005-12-01
An important but untested aspect of the lithopanspermia hypothesis is that microbes situated on or within meteorites could survive hypervelocity entry from space through Earth's atmosphere. The use of high-altitude sounding rockets to test this notion was explored. Granite samples permeated with spores of Bacillus subtilis strain WN511 were attached to the exterior telemetry module of a sounding rocket and launched from White Sands Missile Range, New Mexico into space, reaching maximum atmospheric entry velocity of 1.2 km/s. Maximum recorded temperature during the flight was measured at 145 degrees C. The surfaces of the post-flight granite samples were swabbed and tested for recovery and survival of WN511 spores, using genetic markers and the unique DNA fingerprint of WN511 as recovery criteria. Spore survivors were isolated at high frequency, ranging from 1.2% to 4.4% compared with ground controls, from all surfaces except the forward-facing surface. Sporulation-defective mutants were noted among the spaceflight survivors at high frequency (4%). These experiments constitute the first report of spore survival to hypervelocity atmospheric transit, and indicate that sounding rocket flights can be used to model the high-speed atmospheric entry of bacteria-laden artificial meteorites.
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NASA Astrophysics Data System (ADS)
Fajardo-Cavazos, Patricia; Link, Lindsey; Melosh, H. Jay; Nicholson, Wayne L.
2005-12-01
An important but untested aspect of the lithopanspermia hypothesis is that microbes situated on or within meteorites could survive hypervelocity entry from space through Earth's atmosphere. The use of high-altitude sounding rockets to test this notion was explored. Granite samples permeated with spores of Bacillus subtilis strain WN511 were attached to the exterior telemetry module of a sounding rocket and launched from White Sands Missile Range, New Mexico into space, reaching maximum atmospheric entry velocity of 1.2 km/s. Maximum recorded temperature during the flight was measured at 145°C. The surfaces of the post-flight granite samples were swabbed and tested for recovery and survival of WN511 spores, using genetic markers and the unique DNA fingerprint of WN511 as recovery criteria. Spore survivors were isolated at high frequency, ranging from 1.2% to 4.4% compared with ground controls, from all surfaces except the forward-facing surface. Sporulation-defective mutants were noted among the spaceflight survivors at high frequency (4%). These experiments constitute the first report of spore survival to hypervelocity atmospheric transit, and indicate that sounding rocket flights can be used to model the high-speed atmospheric entry of bacteria-laden artificial meteorites.
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Cetin, Huseyin; Dechant, Peter; Yanikoglu, Atila
2007-06-01
Efficacy of tank mixtures of commercial Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) water-dispersable granule (WDG) formulations was evaluated in septic tanks, against Culex pipiens L. (Diptera: Culicidae) larvae. VectoLex WDG (Bs) + VectoBac WDG (Bti) were evaluated at various ratios from 488 g/ha VectoLex + 250 g/ha VectoBac up to 3,000 g/ha of each. All applications and ratios delivering VectoLex WDG at a rate equal to or greater than 988 g/ha provided more than 90% control for 28 days after treatment. The lowest dose provided this level of control for at least 7 days after treatment, with greater than 80% control after 2 wk. These results suggest that a retreatment interval of 2 wk is recommended with the lowest dose and retreatment intervals of 4 or more wk are recommended with the doses equal to or higher than 988 g/ha VectoLex + 250 g/ha VectoBac.
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Armada, E; Probanza, A; Roldán, A; Azcón, R
2016-03-15
This study evaluates the responses of Lavandula dentata under drought conditions to the inoculation with single autochthonous arbuscular mycorrhizal (AM) fungus (five fungal strains) or with their mixture and the effects of these inocula with a native Bacillus thuringiensis (endophytic bacteria). These microorganisms were drought tolerant and in general, increased plant growth and nutrition. Particularly, the AM fungal mixture and B. thuringiensis maximized plant biomass and compensated drought stress as values of antioxidant activities [superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase APX)] shown. The AMF-bacteria interactions highly reduced the plant oxidative damage of lipids [malondialdehyde (MDA)] and increased the mycorrhizal development (mainly arbuscular formation representative of symbiotic functionality). These microbial interactions explain the highest potential of dually inoculated plants to tolerate drought stress. B. thuringiensis "in vitro" under osmotic stress does not reduce its PGPB (plant growth promoting bacteria) abilities as indole acetic acid (IAA) and ACC deaminase production and phosphate solubilization indicating its capacity to improve plant growth under stress conditions. Each one of the autochthonous fungal strains maintained their particular interaction with B. thuringiensis reflecting the diversity, intrinsic abilities and inherent compatibility of these microorganisms. In general, autochthonous AM fungal species and particularly their mixture with B. thuringiensis demonstrated their potential for protecting plants against drought and helping plants to thrive in semiarid ecosystems. Copyright © 2015 Elsevier GmbH. All rights reserved.
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Structural Insights into Bacillus thuringiensis Cry, Cyt and Parasporin Toxins
Xu, Chengchen; Wang, Bi-Cheng; Yu, Ziniu; Sun, Ming
2014-01-01
Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively. PMID:25229189
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Tavassoli, Setareh; Hinc, Krzysztof; Iwanicki, Adam; Obuchowski, Michal; Ahmadian, Gholamreza
2013-03-01
The production of highly efficient, recyclable and cost-effective enzymes is one of the most important goals in industrial biotechnology. Bacterial spores are highly resistant to harsh environmental conditions, easy to produce and are suitable for manipulation of genetic materials. These features make them a very efficient tool for biotechnology. Here, we show the use bacterial spores for presentation of functional enzyme. Spore coat display was used to produce a biocatalyst, which expresses β-galactiosidase (LacA). This enzyme is commonly used to produce lactose-free milk for lactose intolerant individuals. The lacA gene from Bacillus subtilis strain 168 was expressed on the surface of B. subtilis RH101(ΔcotC) spores using CotC as protein carrier. Presence of LacA protein is verified by western blotting. Results of β-galactiosidase assay show that the expressed enzyme retained its activity in condition of freezing and drying, as well as after recovery from the reaction's mixture.
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Leski, Tomasz A.; Caswell, Clayton C.; Pawlowski, Marcin; Klinke, David J.; Bujnicki, Janusz M.; Hart, Sean J.; Lukomski, Slawomir
2009-01-01
The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are present in B. anthracis strains. Examination of bclABCDE sequences identified polymorphisms in bclB alleles of the B. cereus group organisms. These sequence polymorphisms allowed specific detection of B. anthracis strains by PCR using both genomic DNA and purified Bacillus spores in reactions. By exploiting the length variation of the bcl alleles it was demonstrated that the combined bclABCDE PCR products generate markedly different fingerprints for the B. anthracis Ames and Sterne strains. Moreover, we predict that bclABCDE length polymorphism creates unique signatures for B. anthracis strains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept for B. anthracis detection and fingerprinting, which can be used alone or in combination with previously established typing platforms. PMID:19767469
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Ahmed, Ashraf M; Hussein, Hamdy I; El-Kersh, Talat A; Al-Sheikh, Yazeed A; Ayaad, Tahany H; El-Sadawy, Hanan A; Al-Mekhlafi, Fahd A; Ibrahim, Mohamed S; Al-Tamimi, Jameel; Nasr, Fahd A
2017-06-01
The incidence of mosquito-borne diseases and the resistance of mosquitoes to conventional pesticides have recently caused a panic to the authorities in the endemic countries. This study was conducted to identify native larvicidal biopesticides against Culex pipiens for utilization in the battle against mosquito-borne diseases. Larvicidal activities of new indigenous Bacillus thuringiensis isolates and crude toxin complexes (TCs) of two nematode bacterial-symbionts, Photorhabdus luminescens akhurstii (HRM1) and Ph. luminescens akhurstii (HS1) that tested against Cx. pipiens . B. thuringiensis isolates were recovered from different environmental samples in Saudi Arabia, and the entomopathogenic nematodes, Heterorhabditis indica (HRM1) and He. sp (HS1) were isolated from Egypt. Larvicidal activities (LC 50 and LC 95 ) of the potentially active B. thuringiensis strains or TCs were then evaluated at 24 and 48h post-treatment. Three B. thuringiensis isolates were almost as active as the reference B. thuringiensis israelensis (Bti-H14), and seven isolates were 1.6-5.4 times more toxic than Bti-H14. On the other hand, the TCs of the bacterial symbionts, HRM1 and HS1, showed promising larvicidal activities. HS1 showed LC 50 of 2.54 folds that of HRM1 at 24h post-treatment. Moreover, histopathological examinations of the HS1-treated larvae showed deformations in midgut epithelial cells at 24h post-treatment. Synergistic activity and molecular characterization of these potentially active biocontrol agents are currently being investigated. These results may lead to the identification of eco-friend mosquito larvicidal product(s) that could contribute to the battle against mosquito-borne diseases.
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Ahmed, Ashraf M; Hussein, Hamdy I; El-Kersh, Talat A; Al-Sheikh, Yazeed A; Ayaad, Tahany H; El-Sadawy, Hanan A; Al-Mekhlafi, Fahd A; Ibrahim, Mohamed S; Al-Tamimi, Jameel; Nasr, Fahd A
2017-01-01
Background: The incidence of mosquito-borne diseases and the resistance of mosquitoes to conventional pesticides have recently caused a panic to the authorities in the endemic countries. This study was conducted to identify native larvicidal biopesticides against Culex pipiens for utilization in the battle against mosquito-borne diseases. Methods: Larvicidal activities of new indigenous Bacillus thuringiensis isolates and crude toxin complexes (TCs) of two nematode bacterial-symbionts, Photorhabdus luminescens akhurstii (HRM1) and Ph. luminescens akhurstii (HS1) that tested against Cx. pipiens. B. thuringiensis isolates were recovered from different environmental samples in Saudi Arabia, and the entomopathogenic nematodes, Heterorhabditis indica (HRM1) and He. sp (HS1) were isolated from Egypt. Larvicidal activities (LC50 and LC95) of the potentially active B. thuringiensis strains or TCs were then evaluated at 24 and 48h post-treatment. Results: Three B. thuringiensis isolates were almost as active as the reference B. thuringiensis israelensis (Bti-H14), and seven isolates were 1.6–5.4 times more toxic than Bti-H14. On the other hand, the TCs of the bacterial symbionts, HRM1 and HS1, showed promising larvicidal activities. HS1 showed LC50 of 2.54 folds that of HRM1 at 24h post-treatment. Moreover, histopathological examinations of the HS1-treated larvae showed deformations in midgut epithelial cells at 24h post-treatment. Conclusion: Synergistic activity and molecular characterization of these potentially active biocontrol agents are currently being investigated. These results may lead to the identification of eco-friend mosquito larvicidal product(s) that could contribute to the battle against mosquito-borne diseases. PMID:29062851
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A Novel Tenebrio molitor Cadherin Is a Functional Receptor for Bacillus thuringiensis Cry3Aa Toxin*
Fabrick, Jeff; Oppert, Cris; Lorenzen, Marcé D.; Morris, Kaley; Oppert, Brenda; Jurat-Fuentes, Juan Luis
2009-01-01
Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera. PMID:19416969
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Babolmorad, Ghazal; Emtiazi, Giti; Emamzadeh, Rahman
2014-05-01
S-layer is a self-assemble regularly crystalline surface that covers major cell wall component of many bacteria and archaea and exhibits a high metal-binding capacity. We have studied the effect of the calcium ions and type of solid support (glass or mica) on the structure of the S-layers from Bacillus coagulans HN-68 and Bacillus thuringiensis MH14 upon simple methods based on light microscopy and AFM. Furthermore, the Fourier transform infrared spectroscopy (FTIR) study is indicated that the calcium-S-layer interaction occurred mainly through the carboxylate groups of the side chains of aspartic acid (Asp) and glutamic acid (Glu) and nitrogen atoms of Lys, Asn, and histidine (His) amino acids and N-H groups of the peptide backbone. Studied FTIR revealed that inner faces of S-layer are mainly negative, and outer faces of S-layer are mainly positive. Probably, calcium ions with positive charges bound to the carboxyl groups of Glu and Asp. Accordingly, calcium ions are anchored in the space between the inner faces of S-layer with negative charge and the surface of mica with negative charge. This leads to regular arrangement of the S-layer subunits.
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Arteaga, M E; Mancebo, A; Molier, T; Gómez, D; González, C; Bada, A M; González, B; Rojas, N M; Rodríguez, G
2014-02-01
Bacillus thuringiensis (Bt) is the best known and most widely used of all pesticidal microbes. The aim of this study was to assess the toxicity of a new formulation of Bacillus thuringiensis var israelensis SH-14 in rats through acute dermal toxicity, dermal and eye irritation experiments. The acute dermal toxicity and dermal and eye irritation studies were performed using rabbits according to the United States Environmental Protection Agency guidelines 885.3100, 870.2500 and 870.2500, respectively. The skin sensitization study was carried out in accordance to the EPA OPPTS 870.2600 using guinea pigs. There was no mortality and no evidence of treatment-related toxicity in acute dermal toxicity test. No dermal responses, including erythema/eschar or edema, were found in rabbits treated with the new formulation of Bti SH-14. Minimum response was observed after eye application of test substance. No skin sensitization reactions were observed after the challenge with the new formulation of Bti SH-14 in the Bti SH-14-treated guinea pigs. In summary, the present study demonstrated that the new formulation of Bti SH-14 is not acutely toxic via dermal route, has low eye irritation and would not cause dermal irritation or hypersensitivity to tested animals. Copyright © 2013 Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hutchison, Janine R.; Erikson, Rebecca L.; Sheen, Allison M.
Rapid, cost-effective bacterial detection systems are needed to respond to potential biothreat events. Here we report the use of smartphone-based microscopy in combination with a simple microfluidic incubation device to detect 5000 Bacillus anthracis spores in 3 hours. This field-deployable approach is compatible with real-time PCR for secondary confirmation.
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Hyun-woo Park; Baoxue Ge; Leah S. Bauer; Brian A. Federici
1998-01-01
The insecticidal activity of Bacillus thuringiensis strains toxic to coleopterous insects is due to Cry3 proteins assembled into small rectangular crystals. Toxin synthesis in these strains is dependent primarily upon a promoter that is active in the stationary phase and a STAB-SD sequence that stabilizes the cry3 transcript-ribosome complex. Here we show that...
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USDA-ARS?s Scientific Manuscript database
The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South America. In 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were established usin...
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Plaut, Roger D; Staab, Andrea B; Munson, Mark A; Gebhardt, Joan S; Klimko, Christopher P; Quirk, Avery V; Cote, Christopher K; Buhr, Tony L; Rossmaier, Rebecca D; Bernhards, Robert C; Love, Courtney E; Berk, Kimberly L; Abshire, Teresa G; Rozak, David A; Beck, Linda C; Stibitz, Scott; Goodwin, Bruce G; Smith, Michael A; Sozhamannan, Shanmuga
2018-04-01
The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.
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Johnson, D E; Brookhart, G L; Kramer, K J; Barnett, B D; McGaughey, W H
1990-03-01
Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.
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USDA-ARS?s Scientific Manuscript database
A technique is described where an atmospheric pressure-thermal desorption (AP-TD) device and electrospray ionization (ESI)-mass spectrometry are coupled and used for the rapid analysis of Bacillus spores in complex matrices. The resulting AP-TD/ESI-MS technique combines the generation of volatile co...
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Peng, Donghai; Zhou, Chenfei; Chen, Shouwen; Ruan, Lifang; Yu, Ziniu; Sun, Ming
2008-01-01
The aim of the present study is to evaluate the toxicology safety to mammals of a genetically modified (GM) Bacillus thuringiensis with an additional N-acyl homoserine lactones gene (aiiA), which possesses insecticidal activity together with restraint of bacterial pathogenicity and is intended for use as a multifunctional biopesticide. Safety assessments included an acute oral toxicity test and 28-d animal feeding study in Wistar rats, primary eye and dermal irritation in Zealand White rabbits, and delayed contact hypersensitivity in guinea pigs. Tests were conducted using spray-dried powder preparation. This GM product showed toxicity neither in oral acute toxicity test nor in 28-d animal feeding test at a dose of 5,000 mg/kg body weight. During the animal feeding test, there were no significant differences in growth, food and water consumption, hematology, blood biochemical indices, organ weights, and histopathology finding between rats in controls and tested groups. Tested animals in primary eye and dermal irritation and delayed contact hypersensitivity test were also devoid of any toxicity compared to controls. All the above results demonstrated that the GM based multifunctional B. thuringiensis has low toxicity and low eye and dermal irritation and would not cause hypersensitivity to laboratory mammals and therefore could be regarded as safe for use as a pesticide.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kabilan, S.; Suffield, S. R.; Recknagle, K. P.
Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived respectively from computed tomography (CT) and µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation–exhalation breathingmore » conditions using average species-specific minute volumes. Two different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the nasal sinus compared to the human at the same air concentration of anthrax spores. In contrast, higher spore deposition was predicted in the lower conducting airways of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology for deposition.« less
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Mason, J M; Setlow, P
1987-01-01
Spores of Bacillus subtilis strains which carry deletion mutations in one gene (sspA) or two genes (sspA and sspB) which code for major alpha/beta-type small, acid-soluble spore proteins (SASP) are known to be much more sensitive to heat and UV radiation than wild-type spores. This heat- and UV-sensitive phenotype was cured completely or in part by introduction into these mutant strains of one or more copies of the sspA or sspB genes themselves; multiple copies of the B. subtilis sspD gene, which codes for a minor alpha/beta-type SASP; or multiple copies of the SASP-C gene, which codes for a major alpha/beta-type SASP of Bacillus megaterium. These findings suggest that alpha/beta-type SASP play interchangeable roles in the heat and UV radiation resistance of bacterial spores. Images PMID:3112127
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Killing of Bacillus Megaterium Spores by X-rays at the Phosphorus K-edge
NASA Technical Reports Server (NTRS)
Richmond, Robert C.; Frigo, Sean P.; Ehret, Charles F.; Rose, M. Franklin (Technical Monitor)
2001-01-01
This study continues a progression of experiments on the radiation-induced killing of bacterial spores that began at the Argonne National Laboratory in 1957. A series of aliquots of Bacillus megaterium spores were prepared onto polycarbonate filters and irradiated with photons of 2159 eV compared to 2140 eV energy on the 2-IDB beamline at the Advanced Photon Source. Flux density was approximately 10(exp 18) photons/sec/sq mm. The phosphorous K-edge absorption spectrum in these spores was determined to peak at 2159 eV, wheras 2140 eV was determined to be outside that absorption spectrum. Spores on filters were irradiated at ambient conditions, and were either immediately plated for colony formation after irradiation, or were held for postirradiation exposure to oxygen prior to plating. Slopes of survival curves from the four conditions of irradiation, i.e., two photon energies each comparing immediate plating vs postirradiation holding, were used for quantitative determination of differences in rates of spore killing over a range of radiation doses. It was found that spores irradiated at the phosphorus K-edge were killed 20% more efficiently than when irradiated with 2140 eV photons, and this was true for both immediate plating and postirradiation holding in air. Postirradiation holding in air increased killing efficiency by about 12% for both photon energies compared to plating immediately after irradiation. The increase of killing efficiency with postirradiation holding is less than expected from earlier experiments using relatively low-flux X-rays, and raises the possibility of dose-mitigation by radical-radical recombination in the case of high-flux X-rays from the synchrotron.
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Killing of Bacillus Megaterium Spores by X-Rays at the Phosphorus K-Edge
NASA Technical Reports Server (NTRS)
Richmond, Robert C.; Frigo, Sean P.; Ehret, Charles F.; Rose, M. Franklin (Technical Monitor)
2001-01-01
This study continues a progression of experiments on the radiation-induced killing of bacterial spores that began at the Argonne National Laboratory in 1957. A series of aliquots of Bacillus megaterium spores were prepared onto polycarbonate filters and irradiated with photons of 2159 eV compared to 2140 eV energy on the 2-IDB beamline at the Advanced Photon Source. Flux density was approximately 10 photons/sec/mm . The phosphorous K-edge absorption spectrum in these spores was determined to peak at 2159 eV, wheras 2140 eV was determined to be outside that absorption spectrum. Spores on filters were irradiated at ambient conditions, and were either immediately plated for colony formation after irradiation, or were held for postirradiation exposure to oxygen prior to plating. Slopes of survival curves from the four conditions of irradiation, i.e., two photon energies each comparing immediate plating vs postirradiation holding, were used for quantitative determination of differences in rates of spore killing over a range of radiation doses. It was found that spores irradiated at the phosphorus K-edge were killed 20% more efficiently than when irradiated with 2140eV photons, and this was true for both immediate plating and postirradiation holding in air. Postirradiation holding in air increased killing efficiency by about 12% for both photon energies compared to plating immediately after irradiation. The increase of killing efficiency with postirradiation holding is less than expected from earlier experiments using relatively low-flux X-rays, and raises the possibility of dose-mitigation by radical-radical recombination in the case of high-flux X-rays from the synchrotron.
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[Ecological safety of mosquitocidal biocides based on Bacillus thuringiensis israelensis].
Rydzanicz, Katarzyna; Lonc, Elzbieta
2010-01-01
Bacillus thuringiensis israelensis (Bti) has been developed into many products for the biological control of dipteran larvae, including mosquitoes (Culicidae), black flies (Simuliidae), and midges (Chironomidae) in various parts of the World. Bti appears to pose significantly less of a risk than other chemical pesticides used for mosquito control and eradication programs. Bioproducts based on Bti are highly selective with short environmental persistence, and thus they have very little potential to cause damage to populations of non-target organisms. So far, no example of an unexpected pathogenic organism being developed in the field as well as no examples of resistance to Bti both laboratory and field populations of mosquitoes have been documented. There are some indications that large declines in insect biomass can occur after long-term use of Bti in freshwater wetlands. However, no evidence for permanent damage to ecosystem function has been found. Organisms that utilized insects for food, adapted to the declines and either switched to other food sources or migrate (birds) outside of the treated zones to acquire insects. Even though over 40 tons of Bti have been applied in West Africa alone, no indications of human health or non-target effects have been reported.
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Das, Ranjit; Li, Guiying; Mai, Bixian; An, Taicheng
2018-06-04
Laccase has been applied extensively as a biocatalyst to remove different organic pollutants. This study characterized a spore-laccase from the bisphenol A (BPA)-degrading strain Bacillus sp. GZB. The spore-laccase was encoded with 513 amino acids, containing spore coat protein A (CotA). It showed optimal activity at 70 °C and pH = 7.2 in presence of 2, 6-dimethoxyphenol. At 60 °C, optimal activity was also seen at pH = 3.0 and pH = 6.8 with 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine, respectively. The spore-laccase was stable at high temperature, at acidic to alkaline pH values, and in the presence of different organic solvents. Spore-laccase activity was increased by introducing Cu 2+ , Mg 2+ , and Na + , but was strongly inhibited by Fe 2+ , Ag + , l-cysteine, dithiothreitol, and NaN 3 . The cotA gene was cloned and expressed in E. coli BL21 (DE3); the purified protein was estimated as having a molecular weight of ~63 kDa. Different synthetic dyes and BPA were effectively decolorized or degraded both by the spore laccase and recombinant laccase. When BPA oxidation was catalyzed using laccase, there was an initial formation of phenoxy radicals and further oxidation or CC bond cleavage of the radicals produced different organic acids. Detailed reaction pathways were developed based on nine identified intermediates. The acute toxicity decreased gradually during BPA degradation by laccase. This study is the first report about a genus of Bacillus that can produce a highly active and stable laccase to degrade BPA. Copyright © 2018 Elsevier B.V. All rights reserved.
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Raguse, Marina; Reitz, Günther; Okayasu, Ryuichi; Li, Zuofeng; Klein, Stuart; Setlow, Peter; Nicholson, Wayne L.
2014-01-01
The roles of various core components, including α/β/γ-type small acid-soluble spore proteins (SASP), dipicolinic acid (DPA), core water content, and DNA repair by apurinic/apyrimidinic (AP) endonucleases or nonhomologous end joining (NHEJ), in Bacillus subtilis spore resistance to different types of ionizing radiation including X rays, protons, and high-energy charged iron ions have been studied. Spores deficient in DNA repair by NHEJ or AP endonucleases, the oxidative stress response, or protection by major α/β-type SASP, DPA, and decreased core water content were significantly more sensitive to ionizing radiation than wild-type spores, with highest sensitivity to high-energy-charged iron ions. DNA repair via NHEJ and AP endonucleases appears to be the most important mechanism for spore resistance to ionizing radiation, whereas oxygen radical detoxification via the MrgA-mediated oxidative stress response or KatX catalase activity plays only a very minor role. Synergistic radioprotective effects of α/β-type but not γ-type SASP were also identified, indicating that α/β-type SASP's binding to spore DNA is important in preventing DNA damage due to reactive oxygen species generated by ionizing radiation. PMID:24123749
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Bacillus thuringiensis: a successful insecticide with new environmental features and tidings.
Jouzani, Gholamreza Salehi; Valijanian, Elena; Sharafi, Reza
2017-04-01
Bacillus thuringiensis (Bt) is known as the most successful microbial insecticide against different orders of insect pests in agriculture and medicine. Moreover, Bt toxin genes also have been efficiently used to enhance resistance to insect pests in genetically modified crops. In light of the scientific advantages of new molecular biology technologies, recently, some other new potentials of Bt have been explored. These new environmental features include the toxicity against nematodes, mites, and ticks, antagonistic effects against plant and animal pathogenic bacteria and fungi, plant growth-promoting activities (PGPR), bioremediation of different heavy metals and other pollutants, biosynthesis of metal nanoparticles, production of polyhydroxyalkanoate biopolymer, and anticancer activities (due to parasporins). This review comprehensively describes recent advances in the Bt whole-genome studies, the last updated known Bt toxins and their functions, and application of cry genes in plant genetic engineering. Moreover, the review thoroughly describes the new features of Bt which make it a suitable cell factory that might be used for production of different novel valuable bioproducts.
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Code of Federal Regulations, 2012 CFR
2012-07-01
... popilliae conforming to the morphological and biochemical characteristics of Bacillus popilliae as described... million spores into each of five laboratory test mice weighing 17 grams to 23 grams. Such test shall show no evidence of infection of injury in the test animals when observed for 7 days following injection...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... popilliae conforming to the morphological and biochemical characteristics of Bacillus popilliae as described... million spores into each of five laboratory test mice weighing 17 grams to 23 grams. Such test shall show no evidence of infection of injury in the test animals when observed for 7 days following injection...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... popilliae conforming to the morphological and biochemical characteristics of Bacillus popilliae as described... million spores into each of five laboratory test mice weighing 17 grams to 23 grams. Such test shall show no evidence of infection of injury in the test animals when observed for 7 days following injection...
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Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner
2001-01-01
BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...
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Genotoxic action of sunlight upon Bacillus subtilis spores: monitoring studies at Tokyo, Japan.
Munakata, N
1989-12-01
Samples of Bacillus subtilis spores dried on membrane filter were exposed to natural sunlight from solar-noon time at Tokyo. The survival and mutation induction of wild-type (UVR) and repair-deficient (UVS) spores were determined on 66 occasions since 1979. Two of the values were considered to be useful in monitoring solar UV intensity; the inverse of the time (in minutes) of exposure to kill 63% of the UVS spores ("sporocidal index") and the induced mutation frequency at 60 minutes of exposure of the UVR spores ("mutagenic index"). Both values were varied greatly due to time of a year, weather and other conditions. Estimates of year-round changes under clear skies were obtained by connecting the maximum values attained in these years. In these curves, there are more than 7-fold differences in the genotoxicity between winter and summer months, with major increases observed in early spring and decreases through autumn. Using a series of UV cut-off filters, the wavelengths most effective for the sporocidal actions were estimated to be in the range of 308-325 nm, shorter wavelengths being effective when the genotoxicity was higher. Sunburn meter of Robertson-Berger type seems to respond to slightly longer wavelength components of the solar spectrum. However, a reasonable correlation was obtained between the reading of the meter and the sporocidal index.
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Zhou, Bin; Wirsching, Peter; Janda, Kim D
2002-04-16
A naive, human single-chain Fv (scFv) phage-display library was used in bio-panning against live, native spores of Bacillus subtilis IFO 3336 suspended in solution. A direct in vitro panning and enzyme-linked immunosorbent assay-based selection afforded a panel of nine scFv-phage clones of which two, 5B and 7E, were chosen for further study. These two clones differed in their relative specificity and affinity for spores of B. subtilis IFO 3336 vs. a panel of spores from 11 other Bacillus species/strains. A variety of enzyme-linked immunosorbent assay protocols indicated these scFv-phage clones recognized different spore epitopes. Notably, some spore epitopes markedly changed between the free and microtiter-plate immobilized state as revealed by antibody-phage binding. An additional library selection procedure also was examined by constructing a Fab chain-shuffled sublibrary from the nine positive clones and by using a subtractive panning strategy to remove crossreactivity with B. licheniformis 5A24. The Fab-phage clone 52 was improved compared with 5B and was comparable to 7E in binding B. subtilis IFO 3336 vs. B. licheniformis 5A24, yet showed a distinctive crossreactivity pattern with other spores. We also developed a method to directly detect individual spores by using fluorescently labeled antibody-phage. Finally, a variety of "powders" that might be used in deploying spores of B. anthracis were examined for antibody-phage binding. The strategies described provide a foundation to discover human antibodies specific for native spores of B. anthracis that can be developed as diagnostic and therapeutic reagents.
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Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters.
Calfee, M Worth; Rose, Laura J; Tufts, Jenia; Morse, Stephen; Clayton, Matt; Touati, Abderrahmane; Griffin-Gatchalian, Nicole; Slone, Christina; McSweeney, Neal
2014-01-01
The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50% of their total holding capacity. The results were analyzed by one-way ANOVA across material types, presence or absence of dust, and sampling device. The extraction method gave higher relative recoveries than the two vacuum methods evaluated (p≤0.001). On average, recoveries obtained by the vacuum methods were about 30% of those achieved by the extraction method. Relative recoveries between the two vacuum methods were not significantly different (p>0.05). Although extraction methods yielded higher recoveries than vacuum methods, either HVAC filter sampling approach may provide a rapid and inexpensive mechanism for understanding the extent of contamination following a wide-area biological release incident. Published by Elsevier B.V.
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USDA-ARS?s Scientific Manuscript database
Crystalline (Cry) toxins from Bacillus thuringiensis (Bt) control insect feeding damage on crop plants via foliar applications or by expression within transgenic plants, but continued Bt use is threatened by the buildup of insect resistance traits. Aminopeptidase N (apn) gene family members encode m...
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The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid.
Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie
2016-01-01
Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 10(4) spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites.
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The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid
Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie
2016-01-01
Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites. PMID:26858699
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NASA Astrophysics Data System (ADS)
Nuding, Danielle L.; Gough, Raina V.; Venkateswaran, Kasthuri J.; Spry, James A.; Tolbert, Margaret A.
2017-10-01
Observed features such as recurring slope lineae suggest that liquid water may exist on the surface and near-subsurface of Mars today. The presence of this liquid water, likely in the form of a brine, has important implications for the present-day water cycle, habitability, and planetary protection policies. It is possible that this water is formed, at least partially, by deliquescence of salts, a process during which hygroscopic salts absorb water vapor from the atmosphere and form a saturated liquid brine. We performed laboratory experiments to examine the ability of Bacillus subtilis (B-168) spores, alone or mixed with calcium perchlorate salt (Ca(ClO4)2), to form liquid water via deliquescence under Mars-relevant conditions. Spore survival after exposure to these conditions was examined. An environmental chamber was used to expose the samples to temperature and relative humidity (RH) values similar to those found on Mars, and Raman microscopy was used to identify the phases of water and salt that were present and to confirm the presence of spores. We found that B-168 spores did not condense any detectable water vapor on their own during the diurnal cycle, even at 100% RH. However, when spores were mixed with perchlorate salt, the entire sample deliquesced at low RH values, immersing the spores in a brine solution during the majority of the simulated martian temperature and humidity cycle. After exposure to the simulated diurnal cycles and, in some cases, perchlorate brine, the impact of each environmental scenario on spore survival was estimated by standard plate assay. We found that, if there are deliquescent salts in contact with spores, there is a mechanism for the spores to acquire liquid water starting with only atmospheric water vapor as the H2O source. Also, neither crystalline nor liquid Ca(ClO4)2 is sporicidal despite the low water activity.
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Strauß, Jakob Friedrich; Crain, Philip; Schulenburg, Hinrich; Telschow, Arndt
2016-08-01
Most mathematical models on the evolution of virulence are based on epidemiological models that assume parasite transmission follows the mass action principle. In experimental evolution, however, mass action is often violated due to controlled infection protocols. This "theory-experiment mismatch" raises the question whether there is a need for new mathematical models to accommodate the particular characteristics of experimental evolution. Here, we explore the experimental evolution model system of Bacillus thuringiensis as a parasite and Caenorhabditis elegans as a host. Recent experimental studies with strict control of parasite transmission revealed that one-sided adaptation of B. thuringiensis with non-evolving hosts selects for intermediate or no virulence, sometimes coupled with parasite extinction. In contrast, host-parasite coevolution selects for high virulence and for hosts with strong resistance against B. thuringiensis. In order to explain the empirical results, we propose a new mathematical model that mimics the basic experimental set-up. The key assumptions are: (i) controlled parasite transmission (no mass action), (ii) discrete host generations, and (iii) context-dependent cost of toxin production. Our model analysis revealed the same basic trends as found in the experiments. Especially, we could show that resistant hosts select for highly virulent bacterial strains. Moreover, we found (i) that the evolved level of virulence is independent of the initial level of virulence, and (ii) that the average amount of bacteria ingested significantly affects the evolution of virulence with fewer bacteria ingested selecting for highly virulent strains. These predictions can be tested in future experiments. This study highlights the usefulness of custom-designed mathematical models in the analysis and interpretation of empirical results from experimental evolution. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kabilan, Senthil; Suffield, Sarah R.; Recknagle, Kurtis P.
Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditionsmore » using average species-specific minute volumes. The highest exposure concentration was modeled in the rabbit based upon prior acute inhalation studies. For comparison, human simulation was also conducted at the same concentration. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways compared to the human at the same air concentration of anthrax spores. As a result, higher particle deposition was predicted in the conducting airways and deep lung of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology.« less
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Wood, Joseph P; Calfee, Michael Worth; Clayton, Matthew; Griffin-Gatchalian, Nicole; Touati, Abderrahmane; Egler, Kim
2013-04-15
The purpose of this study was to evaluate the sporicidal (inactivation of bacterial spores) effectiveness and operation of a fogging device utilizing peracetic acid/hydrogen peroxide (PAA). Experiments were conducted in a pilot-scale 24 m(3) stainless steel chamber using either biological indicators (BIs) or bacterial spores deposited onto surfaces via aerosolization. Wipe sampling was used to recover aerosol-deposited spores from chamber surfaces and coupon materials before and after fogging to assess decontamination efficacy. Temperature, relative humidity, and hydrogen peroxide vapor levels were measured during testing to characterize the fog environment. The fog completely inactivated all BIs in a test using a 60 mL solution of PAA (22% hydrogen peroxide/4.5% peracetic acid). In tests using aerosol-deposited bacterial spores, the majority of the post-fogging spore levels per sample were less than 1 log colony forming units, with a number of samples having no detectable spores. In terms of decontamination efficacy, a 4.78 log reduction of viable spores was achieved on wood and stainless steel. Fogging of PAA solutions shows potential as a relatively easy to use decontamination technology in the event of contamination with Bacillus anthracis or other spore-forming infectious disease agents, although additional research is needed to enhance sporicidal efficacy. Published by Elsevier B.V.
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Ehrhardt, Christopher J; Chu, Vivian; Brown, TeeCie; Simmons, Terrie L; Swan, Brandon K; Bannan, Jason; Robertson, James M
2010-03-01
The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 omega9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation.
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Ehrhardt, Christopher J.; Chu, Vivian; Brown, TeeCie; Simmons, Terrie L.; Swan, Brandon K.; Bannan, Jason; Robertson, James M.
2010-01-01
The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 ω9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation. PMID:20097814
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Setlow, B; Cabrera-Martinez, R-M; Setlow, P
2004-01-01
To determine the mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (beta-MUG) by germinating and outgrowing spores of Bacillus species. Spores of B. atrophaeus (formerly B. subtilis var. niger, Fritze and Pukall 2001) are used as biological indicators of the efficacy of ethylene oxide sterilization by measurement of beta-MUG hydrolysis during spore germination and outgrowth. It was previously shown that beta-MUG is hydrolysed to 4-methylumbelliferone (MU) during the germination and outgrowth of B. atrophaeus spores (Chandrapati and Woodson 2003), and this was also the case with spores of B. subtilis 168. Germination of spores of either B. atrophaeus or B. subtilis with chloramphenicol reduced beta-MUG hydrolysis by almost 99%, indicating that proteins needed for rapid beta-MUG hydrolysis are synthesized during spore outgrowth. However, the residual beta-MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for beta-MUG uptake and hydrolysis. With B. subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, beta-MUG hydrolysis during spore germination and outgrowth was decreased >99.9%. This indicated that beta-MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of beta-MUG, beta-MUG-6-phosphate (beta-MUG-P). This was further demonstrated by the lack of detectable glucosidase activity on beta-MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on beta-MUG-P was readily detected. Dormant B. subtilis 168 spores had low levels of at least four phosphoglucosidases active on beta-MUG-P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP). These enzymes were also detected in spores germinating and outgrowing with beta-MUG, but levels of BglH were the highest, as this enzyme's synthesis was induced ca 100-fold
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Du, Cuiying; Cao, Shiyun; Shi, Xiangyu; Nie, Xiangtao; Zheng, Jinshui; Deng, Yun; Ruan, Lifang; Peng, Donghai; Sun, Ming
2017-02-24
trans -Aconitic acid (TAA) is an isomer of cis -aconitic acid (CAA), an intermediate of the tricarboxylic acid cycle that is synthesized by aconitase. Although TAA production has been detected in bacteria and plants for many years and is known to be a potent inhibitor of aconitase, its biosynthetic origins and the physiological relevance of its activity have remained unclear. We have serendipitously uncovered key information relevant to both of these questions. Specifically, in a search for novel nematicidal factors from Bacillus thuringiensis , a significant nematode pathogen harboring many protein virulence factors, we discovered a high yielding component that showed activity against the plant-parasitic nematode Meloidogyne incognita and surprisingly identified it as TAA. Comparison with CAA, which displayed a much weaker nematicidal effect, suggested that TAA is specifically synthesized by B. thuringiensis as a virulence factor. Analysis of mutants deficient in plasmids that were anticipated to encode virulence factors allowed us to isolate a TAA biosynthesis-related ( tbr ) operon consisting of two genes, tbrA and tbrB We expressed the corresponding proteins, TbrA and TbrB, and characterized them as an aconitate isomerase and TAA transporter, respectively. Bioinformatics analysis of the TAA biosynthetic gene cluster revealed the association of the TAA genes with transposable elements relevant for horizontal gene transfer as well as a distribution across B. cereus bacteria and other B. thuringiensis strains, suggesting a general role for TAA in the interactions of B. cereus group bacteria with nematode hosts in the soil environment. This study reveals new bioactivity for TAA and the TAA biosynthetic pathway, improving our understanding of virulence factors employed by B. thuringiensis pathogenesis and providing potential implications for nematode management applications. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Oves, Mohammad; Khan, Mohammad Saghir; Zaidi, Almas
2012-01-01
The study was navigated to examine the metal biosorbing ability of bacterial strain OSM29 recovered from rhizosphere of cauliflower grown in soil irrigated consistently with industrial effluents. The metal tolerant bacterial strain OSM29 was identified as Bacillus thuringiensis following 16S rRNA gene sequence analysis. In the presence of the varying concentrations (25–150 mgl−1) of heavy metals, such as cadmium, chromium, copper, lead and nickel, the B. thuringiensis strain OSM29 showed an obvious metal removing potential. The effect of certain physico-chemical factors such as pH, initial metal concentration, and contact time on biosorption was also assessed. The optimum pH for nickel and chromium removal was 7, while for cadmium, copper and lead, it was 6. The optimal contact time was 30 min. for each metal at 32 ± 2 °C by strain OSM29. The biosorption capacity of the strain OSM29 for the metallic ions was highest for Ni (94%) which was followed by Cu (91.8%), while the lowest sorption by bacterial biomass was recorded for Cd (87%) at 25 mgl−1 initial metal ion concentration. The regression coefficients obtained for heavy metals from the Freundlich and Langmuir models were significant. The surface chemical functional groups of B. thuringiensis biomass identified by Fourier transform infrared (FTIR) were amino, carboxyl, hydroxyl, and carbonyl groups, which may be involved in the biosorption of heavy metals. The biosorption ability of B. thuringiensis OSM29 varied with metals and was pH and metal concentration dependent. The biosorption of each metal was fairly rapid which could be an advantage for large scale treatment of contaminated sites. PMID:24115905
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Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Wang, Xu-Ying; Fleming, Joy; Bi, Li-Jun; Yang, Rui-Fu; Zhang, Xian-En
2015-05-15
Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5 min of chromatography and a total time of 20 min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6×10(4) spores/g milk powder, 2×10(5) spores/g starch and 5×10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
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Vergis, James M.; Cote, Christopher K.; Bozue, Joel; Alem, Farhang; Ventura, Christy L.; Welkos, Susan L.
2013-01-01
Bacillus anthracis spores are the infectious form of the organism for humans and animals. However, the approved human vaccine in the United States is derived from a vegetative culture filtrate of a toxigenic, nonencapsulated B. anthracis strain that primarily contains protective antigen (PA). Immunization of mice with purified spore proteins and formalin-inactivated spores (FIS) from a nonencapsulated, nontoxigenic B. anthracis strain confers protection against B. anthracis challenge when PA is also administered. To investigate the capacity of the spore particle to act as a vaccine without PA, we immunized mice subcutaneously with FIS from nontoxigenic, nonencapsulated B. cereus strain G9241 pBCXO1−/pBC210− (dcG9241), dcG9241 ΔbclA, or 569-UM20 or with exosporium isolated from dcG9241. FIS vaccination provided significant protection of mice from intraperitoneal or intranasal challenge with spores of the virulent B. anthracis Ames or Ames ΔbclA strain. Immunization with dcG9241 ΔbclA FIS, which are devoid of the immunodominant spore protein BclA, provided greater protection from challenge with either Ames strain than did immunization with FIS from BclA-producing strains. In addition, we used prechallenge immune antisera to probe a panel of recombinant B. anthracis Sterne spore proteins to identify novel immunogenic vaccine candidates. The antisera were variably reactive with BclA and with 10 other proteins, four of which were previously tested as vaccine candidates. Overall our data show that immunization with FIS from nontoxigenic, nonencapsulated B. cereus strains provides moderate to high levels of protection of mice from B. anthracis Ames challenge and that neither PA nor BclA is required for this protection. PMID:23114705
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Dash, Hirak R; Mangwani, Neelam; Das, Surajit
2014-02-01
Bacillus thuringiensis PW-05 was isolated from the Odisha coast and was found to resist 50 ppm of Hg as HgCl2 as well as higher concentrations of CdCl2, ZnSO4, PbNO3 and Na2HAsO4. Resistance towards several antibiotics, viz amoxycillin, ampicillin, methicillin, azithromycin and cephradine (CV) was also observed. The mer operon possessed by most of the mercury-resistant bacteria was also found in this isolate. Atomic absorption spectroscopy revealed that the isolate can volatilize >90 % of inorganic mercury. It showed biofilm formation in the presence of 50 ppm HgCl2 and can produce exopolysaccharide under same conditions. The isolate was found to volatilize mercury efficiently under a wide range of environmental parameters, i.e. pH (7 to 8), temperature (25 °C to 40 °C) and salinity (5 to 25 ppt). merA gene expression has been confirmed by real-time reverse transcriptase PCR study. Fourier transform infrared study revealed that -SH and -COOH groups play a major role in the process of adaptation to Hg. Hence, this isolate B. thuringiensis PW-05 shows an interesting potential for bioremediation of mercury.
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Aghaee, Mohammad-Amir; Godfrey, Larry D
2015-02-01
Rice water weevil (Lissorhoptrus oryzophilus Kushel) is the most damaging insect pest of rice in the United States. Larval feeding on the roots stunt growth and reduce yield. Current pest management against the weevil in California relies heavily on pyrethroids that can be damaging to aquatic food webs. Examination of an environmentally friendly alternative biopesticide based on Bacillus thuringiensis spp. galleriae chemistry against rice water weevil larvae showed moderate levels of activity in pilot studies. We further examined the performance of different formulations of Bt.galleriae against the leading insecticide used in California rice, λ-cyhalothrin. The granular formulation performed as well as the λ-cyhalothrin in use in California in some of our greenhouse and field studies. This is the first reported use of B. thuringiensis spp. galleriae against rice water weevil. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.