Rapid HPLC assay for the .beta.-exotoxin of Bacillus thuringiensis

Campbell, David P.; Dieball, Donald E.; Brackett, John M.

doi:10.1021/jf00073a033

Cited by 23 publications

(15 citation statements)

References 4 publications

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“…Interestingly, another peak was present in HD1-1 which was missing from dBl:1. The presumptive exotoxin peak in dBl:1 was unchanged by autoclaving at pH 6.5, but disappeared upon autoclaving at pH 3, conditions expected to destroy exotoxin (5).…”

Section: Resultsmentioning

confidence: 92%

“…840 system (Waters Associates, Inc., Milford, Mass.). The basic procedure of Campbell et al (5) was followed, except that the separation was performed on a wide-pore C18 reverse-phase column (no. 218TP54; VYDAC, Hesperia, Calif.) instead of the Waters ,u-Bondapak C18 column.…”

Section: Methodsmentioning

confidence: 99%

“…Campbell et al developed a method which they report to be both fast and specific for P-exotoxin, even in complex fermentation broths (5). We used a modification of their method for this study.…”

mentioning

confidence: 99%

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Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography

et al. 1990

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An improved high-performance liquid chromatography separation was developed to detect and quantify beta-exotoxin production in Bacillus thuringiensis culture supernatants. Exotoxin production was assigned to a plasmid in five strains, from three subspecies (B. thuringiensis subsp. thuringiensis serotype 1, B. thuringiensis subsp. tolworthi serotype 9, and B. thuringiensis subsp. darmstadiensis serotype 10). A new exotoxin, called type II beta-exotoxin in this report, was discovered in B. thuringiensis subsp. morrisoni serotype 8ab, purified, and partially characterized. This material is more specific than type I beta-exotoxin and is very active against the Colorado potato beetle, Leptinotarsa decemlineata.

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Section: Resultsmentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

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Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography

et al. 1990

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“…These materials were compared with a partially purified technical powder of thuringiensin (80.7% purity, as determined by HPLC) (Campbell et al, 1987). A liquid preparation containing 30mg mL -1 (3.0%) active thuringiensin.…”

Section: Exotoxin Preparationsmentioning

confidence: 99%

The effects of exotoxin (Thuringiensin) from Bacillus thuringiensis on Meloidogyne incognita and Caenorhabditis elegans

Premachandran

Rehberger

1992

Plant Soil

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A partially purified preparation as well as two formulations of exotoxin from Bacillus thuringiens& (thuringiensin) were evaluated for nematicidal activity. The methods used in our evaluations included direct contact nematicidal assays, hatching tests, infection tests in seed pouches using the cucumber/ root-knot nematode (Meloidogyne incognita) system, and greenhouse test using the root-knot nematode. While contact nematicidal activity was not observed against juveniles of M. incognita, 100% mortality occurred when the free-living nematode, Caenorhabdit& elegans, was used as the test organism. Nematode infection evaluations in the seed pouch assay showed reduced root galling at relatively high concentrations (>10 mg kg i). Greenhouse assays indicated significant reduction in the soil population. However, the degree of control in relation to the amount of material applied, as measured by the gall numbers, larvae from soil/roots, and plant growth parameters, was not considered adequate. Data on the plant response in relation to treatment with different formulations of the toxin are presented.

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“…␤-Exotoxin I displays some toxicity to mammalian cells (1,22) and has been banned from public use based on World Health Organization advice (31). However, unless a bioassay or high-performance liquid chromatography (HPLC) analysis (6,13,14) is performed, it is impossible to predict whether a strain produces ␤-exotoxin I. Previous studies have shown that ␤-exotoxin I production is often linked to the presence of plasmids bearing cry genes (cry plasmids); several experiments have shown that the ability to secrete ␤-exotoxin I and the ability to produce crystals were transferred together to Bacillus cereus and B. thuringiensis recipient strains by conjugation (21,24).…”

mentioning

confidence: 99%

Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis

Espinasse

Gohar

Chaufaux

et al. 2002

Appl Environ Microbiol

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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 g of ␤-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 g/ml. However, these amounts of ␤-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of ␤-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 ␤-exotoxin I.Bacillus thuringiensis is a sporogenic soil bacterium which forms characteristic crystalline inclusions composed of insecticidal crystal (Cry) proteins that are highly specific for the larvae of several insect pests (27). Because of this, B. thuringiensis has been widely used in biological pest control (19, 22a). Many isolates also produce an assortment of various other virulence factors that are secreted into the culture medium (9). These factors include the vegetative insecticidal proteins Vip1, Vip2, and Vip3, which do not display any sequence homology with each other or with any known protein (10,29,30), the Cry1I toxin (17), and ␤-exotoxin I (2, 5, 15, 23), a nonproteinaceous toxin. Unlike the Vip and Cry toxins, ␤-exotoxin I is not specific and thus may have detrimental effects on nontarget organisms (16,22); it is particularly active against dipteran species, but it is also active against coleopteran, lepidopteran, and some nematode species (12). The mechanism of action of ␤-exotoxin I is not fully understood. However, this toxin is an adenine nucleotide analogue (11) that has been found to interfere with RNA polymerase (1, 28). Thus, it has been proposed that this molecule inhibits the synthesis of RNA by competing with ATP for binding sites, thereby affecting insect molting and pupation and causing teratological effects at sublethal doses (4, 16). ␤-Exotoxin I displays some toxicity to mammalian cells (1, 22) and has been banned from public use based on World Health Organization advice (31). However, unless a bioassay or high-performance liquid chromatography (HPLC) analysis (6,13,14) is performed, it is impossible to predict whether a strain produces ␤-exotoxin I. Previous studies have shown that ␤-exotoxin I production is often linked to the presence of plasmids bearing cry genes (cry plasmids); several experiments have shown that the ability to secrete ␤-exotoxin I and the ability to produce crystals were transferred together to Bacillus cereus and B. thuringiensis recipient strains by conjugation (21, 24). Conversely, strains that had lost the capacity to synthesize crystal toxins following loss of cry plasmids also were unable to secrete ␤-exotoxin I, although the parental str...

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Rapid HPLC assay for the .beta.-exotoxin of Bacillus thuringiensis

Cited by 23 publications

References 4 publications

Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography

Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography

The effects of exotoxin (Thuringiensin) from Bacillus thuringiensis on Meloidogyne incognita and Caenorhabditis elegans

Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis

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