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Applied and Environmental Microbiology, September 2002, p. 4182-4186, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4182-4186.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Sylvain Espinasse,1 Michel Gohar,1,2 Josette Chaufaux,1 Christophe Buisson,1 Stéphane Perchat,1 and Vincent Sanchis1,3*

Unité de Recherches de Lutte Biologique, INRA La Minière, 78285 Guyancourt,1 Unité de Biochimie Microbienne, Institut Pasteur, 75724 Paris Cedex 15, France,3 Aventis Crop Science, B9000 Ghent, Belgium2

Received 14 February 2002/ Accepted 29 May 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 ß-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 ß-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of ß-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 ß-exotoxin I.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 ß-exotoxin I (2, 5, 15, 23), a nonproteinaceous toxin. Unlike the Vip and Cry toxins, ß-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 ß-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). ß-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 ß-exotoxin I. Previous studies have shown that ß-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 ß-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 ß-exotoxin I, although the parental strains had the capacity to produce high levels of this compound. ß-Exotoxin I production has been linked to the presence of plasmids of various sizes expressing Cry proteins with apparent molecular masses of 150, 140, 67, 23, and 21 kDa (18, 21). However, no direct relationship between the presence of specific cry plasmids and ß-exotoxin I production has been established, and it is not known whether Cry toxins are associated with ß-exotoxin I other than in a strain-specific fashion.

In this study we assessed the abilities of 640 natural B. thuringiensis strains, isolated from different places around the world, to produce ß-exotoxin I by examining their crystal toxin profiles in order to determine whether the ability to synthesize ß-exotoxin I is associated with the presence of a particular cry gene.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, electrophoretic Cry profiles, and supernatant preparations for toxicity assays.
The Institut National de la Recherche Scientifique (INRA) collection contains 1,260 natural B. thuringiensis strains, including isolates from 101 countries, which were isolated from various sources (soil, insects, dust, plants, animal waste, etc.). These strains were characterized as B. thuringiensis primarily on the basis of the ability to produce crystals during sporogenesis; they are conserved as spores in 12% (vol/vol) glycerol suspensions at -20°C. Spore crystal preparations from 640 strains randomly selected from this collection were individually grown in liquid HCT medium (20) at 30°C for 48 h. They were then analyzed directly by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Spores and crystals were centrifuged, washed in distilled water, and subjected to Bradford protein quantification. They were then treated with 1% SDS and electrophoresed on 10.5% polyacrylamide gels. The sizes of the major protein bands obtained for each strain were recorded (8). The culture supernatants were also tested against the insect Anthonomus grandis. Supernatants were harvested from cultures at the midsporulation stage, grown in 100 ml of liquid Luria-Bertani nutrient broth at 30°C, and inoculated by using single colonies previously reisolated on agar plates. Supernatants were filtered through 0.2-µm-pore-size Nalgene filter units (Nalgene) and stored at -20°C until the bioassay was performed.

Insect bioassay.
We used a free ingestion technique to assess the toxicity to A. grandis of bacterial culture supernatant preparations. Each supernatant was incorporated into an artificial diet at a final concentration of 8%. The diet consisted of a mixture of soya flour, cotton flour, wheat germ oil, cholesterol, sugar, vitamins, and agar. It was poured into a sterile 24-well plate. One egg of A. grandis was placed on the food in each well. At 25°C, control larvae developed into adults when they were given untreated food for 3 weeks. A. grandis was highly sensitive to ß-exotoxin I, as the concentration of toxin required to kill 50% of A. grandis larvae was 6 µg/ml, as calculated by PROBIT analysis (26) with 95% confidence intervals (2 to 10 µg/ml). In bioassays, culture supernatants were considered toxic when the level of mortality at 3 weeks was more than 80%, whereas the level of mortality in the controls was less than 10%.

PCR amplification of cry1B, vip2, vip3, and cry1I.
The oligonucleotides used in this study are listed in Table 1. The specific primers used to detect the cry1I, vip2, and vip3 genes were designed from the cry1I, vip2, and vip3 gene sequences present in the databases by using Primer Select from the DNAStar software (DNAStar Inc.). The primers used for specific detection of cry1B were the previously described primers CJ8 and CJ9 (7). PCR amplifications were performed by using the general procedure described here to search for vip2, vip3, cry1I, and cry1B genes in wild-type B. thuringiensis cells. DNA templates were obtained from cells grown overnight on agar plates and resuspended in 100 µl of distilled H2O. Cell membranes were disrupted by freezing at -70°C and immersion in a 98°C water bath (heat shock). The DNA solution (2 µl) was mixed with 0.5 µl of each primer (0.1 µM), 0.5 U of Taq polymerase, water (50 µl), MgCl2, and buffer according to the manufacturer's instructions (Gibco-BRL). The PCR conditions were as follows: denaturation for 5 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 50°C, and 1.5 min at 72°C and then a final elongation step consisting of 10 min at 72°C.


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  		TABLE 1. Oligonucleotides used in this study

 
Only fragments of the expected size after migration on a 1% agarose gel were considered to result from specific amplification. PCR results were considered to be negative if no amplicon was obtained after two attempts with independent DNA preparations.

Detection and quantification of ß-exotoxin I.
ß-Exotoxin I was extracted from the culture supernatant by solvent extraction and was quantified by HPLC, as previously described (13). Briefly, for solvent extraction, acetone was added to the exotoxin in 0.2 ml of culture supernatant to a final concentration of 90%, and the mixture was centrifuged. The pellet was solubilized in 0.2 ml of double-distilled H2O. Acetonitrile was added to a final concentration of 40%, and the mixture was then centrifuged. The pellet was discarded, and the acetonitrile concentration in the supernatant was increased to 90%. The precipitate was collected by centrifugation, and the pellet was solubilized in 100 µl of 50 mM potassium phosphate buffer (pH 2.5). For HPLC, we injected 20 µl of the sample into a Lichrospher (Merck) C18 end-capped column (4 by 250 mm). A 5 to 15% methanol gradient in 50 mM potassium phosphate buffer (pH 2.5) was applied for 10 min. The flow rate was 1 ml/min, and UV absorption was monitored at 260 nm. ß-Exotoxin I eluted at 5.5 min. The detection limit of this method for ß-exotoxin I was 2 µg/ml. A standard sample of ß-exotoxin I (70% pure) was kindly provided by I. Thiery from Laboratoire des bactéries entomopathogènes (Institut Pasteur, Paris, France).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of Cry toxins in natural strains producing ß-exotoxin I.
We analyzed the supernatant toxicities and crystal contents of 640 strains randomly selected from our collection of 1,260 strains (8). We assessed the toxicity of culture supernatants of each of the strains individually against A. grandis. As ß-exotoxin I was toxic at a concentration of 5 µg/ml or more, we used this value as a threshold to discriminate potential Exo+ strains from Exo- strains. ß-Exotoxin I was then characterized and precisely quantified by HPLC in all possible Exo+ strains. We found 58 strains whose culture supernatants were toxic to A. grandis, and each of these strains secreted more than 10 µg of ß-exotoxin I per ml (generally more than 50 µg/ml) into the culture supernatant, accounting for the activity against this species. We also determined precisely the amounts of ß-exotoxin I present in the culture supernatants of 118 of the nontoxic isolates using the same quantification method. We found that 55 of these nontoxic strains produced between 2 and 5 µg of ß-exotoxin I per ml and that 63 produced less than 2 µg/ml. These amounts of ß-exotoxin I were below the threshold for detectable toxicity against A. grandis in our test conditions.

Analysis of the crystal protein profiles of the 640 strains revealed many different electrophoretic patterns (Fig. 1). Most of the patterns were consistent with those reported previously for the nontoxic Cry15 proteins (40 and 45 kDa), the Cry2 proteins (66 kDa) active against Lepidoptera and Diptera, the Cry3A protein (70 and 73 kDa) active against Coleoptera, and the Cry1 proteins (130- to 140-kDa polypeptides) active against Lepidoptera (27). However, many isolates contained crystals with unusual protein profiles. We created the following categories based on the major protein bands observed: 40 and 45 kDa, 66 kDa, 70 and 73 kDa, 128, 130, or 132 kDa, 140 kDa, and unusual profiles. These categories roughly reflected the Cry diversity of the B. thuringiensis strains (Table 2). We investigated whether there was a correlation between ß-exotoxin I production and a particular protein profile by comparing the crystal protein profiles of the 58 Exo+ strains with those obtained for the whole sample. Chi-square analysis showed that there was a highly significant difference between the Exo+ strains and the whole strain collection and indicated that there was a strong link (P < 0.0001) between ß-exotoxin I production and the presence of a 140-kDa polypeptide in the crystals of a given strain. Conversely, there was a strong negative correlation between the presence of the 40- and 45-kDa, 66-kDa, and 128-, 130-, or 132-kDa Cry proteins and the presence of ß-exotoxin I (P < 0.0001, P < 0.0021, and P < 0.0068, respectively). Finally, 13% of the strains that produced unusual protein profiles produced ß-exotoxin I, but the difference was not significant (P = 0.1719).



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  		FIG. 1. SDS-10.5% PAGE of B. thuringiensis crystal proteins representative of the main profiles listed in Table 2. (A) Lane 1, strain LM63 (70 and 73 kDa); lane 2, strain LM651B (66 and 130 kDa); lane 3, strain LM381 (40 and 45 kDa); lane 4, strain LM380 (132 kDa); lane 5, LM 379 (66, 130, and 132 kDa); lane 6, strain LM378B (140 kDa). (B) Examples of unusual profiles. Lane 7, strain LM86 (98 kDa); lane 8, strain LM85 (34, 40, 45, and 80 kDa); lane 9, strain LM72 (32, 80, 130, and 140 kDa); lane 10, strain LM567 (28, 68, 125, and 135 kDa); lane 11, strain LM54 (36 and 90 kDa); lane 12, strain LM347 (85 and 135 kDa). Lanes M contained molecular mass markers, whose positions are indicated on the left.

 

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  		TABLE 2. Crystal protein profiles of B. thuringiensis strains based on ß-exotoxin I productiona

 
Genotypic characterization of strains that produced 140-kDa protein bands.
We further investigated the 39 Exo+ and 2 Exo- strains that produced crystals containing 140-kDa Cry toxins in order to better characterize them genetically. The only known Cry toxin that produces a protoxin band at about 140 kDa on SDS-PAGE gels is Cry1B (3, 27). On another hand, in a previous study S. Espinasse, J. Chaufaux, C. Buisson, S. Perchat, M. Gohar, D. Bourguet, and V. Sanchis, submitted for publication), we showed that there was a significant association between the presence of vip2 genes and ß-exotoxin I production in B. thuringiensis populations. We also found a negative correlation between ß-exotoxin I production and the presence of vip3 or cry1I genes encoding two other secreted insecticidal toxins. We therefore carried out PCR with primers specific for cry1B, vip2, vip3, and cry1I genes in these strains (Table 3). The cry1B gene was detected in all 39 strains that produced more than 10 µg of ß-exotoxin I per ml. All of these strains also generated amplicons for vip2 genes during PCR, indicating that there is a strong association between ß-exotoxin I production and the presence of vip2 and cry1B in a given strain. The two strains that produced only small amounts of ß-exotoxin I but whose crystals contained a 140-kDa polypeptide did not contain these genes. The vip3 and cry1I genes, although present in more than 50% of natural B. thuringiensis isolates, were underrepresented among the Exo+ strains harboring cry1B.


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  		TABLE 3. Genotypic characterization of the strains producing 140-kDa crystal proteinsa

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the genetic determinants of ß-exotoxin I have not yet been characterized, ß-exotoxin I production has been associated with the presence of plasmids harboring cry genes (18, 21). Ozawa and Iwahana (24), by transferring a single 62-MDa plasmid from a B. thuringiensis subsp. darmstadiensis strain by conjugation, even managed to demonstrate production of both ß-exotoxin I and crystals in a strain of B. cereus, indicating that at least some of the ß-exotoxin I genetic elements and cry genes were present on a single plasmid.

The toxic compound produced by the 58 strains whose supernatants were toxic to A. grandis was clearly identified as ß-exotoxin I by using HPLC. In bioassays with A. grandis, in which the larvae ingested crude supernatants, the typical inhibition of growth and molting was observed, followed by death, as described in several studies on ß-exotoxin I (2). Quantification by HPLC indicated that in all cases the concentration of ß-exotoxin I in the supernatants was more than 10 µg/ml. These results indicated that the toxicity of these strains was due to ß-exotoxin I, eliminating possible confusion with any other insecticidal metabolite, such as ß-exotoxin II (21). In addition, we found that 46% of a sample of 118 B. thuringiensis strains whose supernatants were not toxic to A. grandis also produced small but detectable amounts of ß-exotoxin I (between 2 and 5 µg/ml). We did not check for lower levels of ß-exotoxin I production, but a larger number of ß-exotoxin I-producing B. thuringiensis strains would probably be identified if the detection threshold for this compound was lowered. Given the large number of natural isolates that produce a constant low level of ß-exotoxin I and given that in our culture conditions 9% of B. thuringiensis strains have the capacity to produce high levels of this compound, understanding the environmental factors or mutations that enhance ß-exotoxin I production seems to be important.

We found a highly significant association between the presence of the cry1B gene and ß-exotoxin I production. Such an association was previously reported by Perani et al. (25), but these authors did not find a statistical relationship between the ability to produce this metabolite and the presence of genes of the cry1B subfamily. In our study, only two strains that produced 140-kDa crystal protein bands did not give an amplicon with cry1B-specific primers (7), which suggests that these strains produce a unique 140-kDa polypeptide that is different from Cry1B. Remarkably, the 39 strains that produced large amounts of ß-exotoxin I also gave amplicons with vip2 primers, suggesting that cry1B, vip2 (29), and the genetic determinants of ß-exotoxin I are located on the same plasmid. In contrast, the vast majority of B. thuringiensis strains that produce 40- and 45-kDa, 66-kDa, and 128-, 130-, or 132-kDa crystal proteins, potentially corresponding to various known Cry toxins active against Lepidoptera, did not produce ß-exotoxin I or produced only small amounts. Thus, certain Cry toxins are clearly only rarely associated with the determinants necessary to promote production of high levels of ß-exotoxin I, suggesting that the genes encoding these proteins are present on different plasmids. We identified no strains that produced ß-exotoxin I along with 70- and 73-kDa crystal proteins. However, we studied only a limited number of strains producing this profile, too few to establish a significant correlation for this category of strains.

In summary, this study shows that, in B. thuringiensis, production of high levels of ß-exotoxin I is linked to the presence of cry plasmids preferentially harboring the cry1B crystal protein gene. We also found a strong association between some determinants that enhance production of ß-exotoxin I and the cry1B and vip2 genes, which suggests that these genes are generally located on the same plasmid. Further work should aim to characterize the various plasmid-borne and chromosomal genes involved in the regulation and/or biosynthesis of ß-exotoxin I.


   ACKNOWLEDGMENTS
 
We thank Didier Lereclus, in whose laboratory this work was conducted. We also thank Alex Edelman & Associates for editing the English manuscript. We thank Sophie Fleurdépine for technical assistance with PCR experiments. We are indebted to Jeroen Van Rie for providing the primers used for PCR.

This work was supported by the Institut Pasteur and INRA. Sylvain Espinasse was supported by a grant from Aventis Crop Science, Ghent, Belgium.


   FOOTNOTES
 
* Corresponding author. Mailing address: Unité de Biochimie Microbienne, Institut Pasteur, 25/28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33 1 45 68 88 12. Fax: 33 1 45 68 89 38. E-mail: vsanchis{at}pasteur.fr. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Beebee, T., A. Korner, and R. P. Bond. 1972. Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus thuringiensis. The direct observation of nucleoplasmic ribonucleic acid polymerase activity in intact nuclei. Biochem. J. 127:619-634.[Medline]
  2. Bond, R. P., and C. B. C. Boyce. 1971. The thermostable exotoxin of Bacillus thuringiensis, p. 275-301. In H. D. Burges (ed.), Microbial control of insects and mites. Academic Press, London, United Kingdom.
  3. Brizzard, B. L., and H. R. Whiteley. 1988. Nucleotide sequence of an additional crystal protein gene cloned from Bacillus thuringiensis subsp. thuringiensis. Nucleic Acids Res. 16:2723-2724.[Free Full Text]
  4. Burgerjon, A., G. Biache, and P. Cals. 1969. Teratology of the Colorado potato beetle, Leptinotarsa decemlineata, as provoked by larval administration of the thermostable toxin of Bacillus thuringiensis. J. Invertebr. Pathol. 14:274-278.[CrossRef]
  5. Burgerjon, A., and H. De Barjac. 1967. Another serotype (4:4a, 4c) of Bacillus thuringiensis which produces thermostable exotoxin. J. Invertebr. Pathol. 9:574-577.[CrossRef]
  6. Campbell, D., D. Dieball, and J. Brackett. 1987. Rapid HPLC assay for the beta-exotoxin of Bacillus thuringiensis. J. Agric. Food. Chem. 35:156-158.[CrossRef]
  7. Ceron, J., L. Covarrubias, R. Quintero, A. Ortiz, M. Ortiz, E. Aranda, L. Lina, and A. Bravo. 1994. PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis. Appl. Environ. Microbiol. 60:353-356.[Abstract/Free Full Text]
  8. Chaufaux, J., M. Marchal, N. Gilois, I. Jehanno, and C. Buisson. 1997. Research on natural strains of Bacillus thuringiensis in different biotopes throughout the world. Can. J. Microbiol. 43:337-343.
  9. De Maagd, R. A., A. Bravo, and N. Crickmore. 2001. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet. 17:193-199.[CrossRef][Medline]
  10. Estruch, J. J., G. W. Warren, M. A. Mullins, G. J. Nye, J. A. Craig, and M. G. Koziel. 1996. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc. Natl. Acad. Sci. USA 93:5389-5394.[Abstract/Free Full Text]
  11. Farkas, J., K. Sebesta, K. Horska, Z. Samek, J. Dollijs, and F. Storm. 1969. The structure of exotoxin of Bacillus thuringiensis var. gelechiae. Collect. Czech. Chem. Commun. 34:1118-1120.
  12. Gevrey, J., and J. Euzéby. 1966. Pouvoir inhibiteur de l'exotoxine de Bacillus thuringiensis berliner var. thuringiensis à l'encontre des formes pré-imaginales des strongles digestifs—etude in vitro. Bull. Soc. Sci. Vet. Med. Comp. Lyon 68:111-119.
  13. Gohar, M., and S. Perchat. 2001. Sample preparation for ß-exotoxin determination in Bacillus thuringiensis cultures by reversed-phase high-performance liquid chromatography. Anal. Biochem. 298:112-117.[CrossRef][Medline]
  14. Hernandez, C. S., J. Ferre, and I. Larget-Thiery. 2001. Update on the detection of beta-exotoxin in Bacillus thuringiensis strains by HPLC analysis. J. Appl. Microbiol. 90:643-647.[CrossRef][Medline]
  15. Horska, K., J. Vankova, and K. Sebesta. 1975. Determination of exotoxin in Bacillus thuringiensis cells. Z. Naturforsch. 30:120-123.
  16. Ignoffo, C. M., and B. G. Gregory. 1972. Effects of B. thuringiensis ß-exotoxin on larval maturation, adult longevity, fecundity, and egg viability in several species of lepidoptera. Environ. Entomol. 1:269-272.
  17. Kostichka, K., G. W. Warren, M. Mullins, A. D. Mullins, N. V. Palekar, J. A. Craig, M. G. Koziel, and J. J. Estruch. 1996. Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase. J. Bacteriol. 178:2141-2144. (Erratum, 178:3990.)[Abstract/Free Full Text]
  18. Kuzina, L. V., A. L. Andreeva, E. M. Aslanian, A. P. Dobritsa, E. B. Danilova, and E. A. Bocharov. 1993. Study of the nature of determinants for synthesis of delta-endotoxin and ß-exotoxin in Bacillus thuringiensis subsp. thuringiensis strains—bitoxybacillin producers. Mol. Gen. Mikrobiol. Virusol. 1993:20-24.
  19. Lambert, B., and M. Peferoen. 1992. Insecticidal promise of Bacillus thuringiensis. Facts and mysteries about a successful biopesticide. Bioscience 42:112-122.[CrossRef]
  20. Lecadet, M.-M., M. O. Blondel, and J. Ribier. 1980. Generalized transduction in Bacillus thuringiensis var. Berliner 1715, using bacteriophage CP54-Ber. J. Gen. Microbiol. 121:203-212.[Medline]
  21. Levinson, B. L., K. J. Kasyan, S. S. Chiu, T. C. Currier, and J. M. J. Gonzalez. 1990. Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography. J. Bacteriol. 172:3172-3179.[Abstract/Free Full Text]
  22. Mackedonski, V. V., N. Nikolaev, K. Sebesta, and A. A. Hadjiolov. 1972. Inhibition of ribonucleic acid biosynthesis in mice liver by the exotoxin of Bacillus thuringiensis. Biochim. Biophys. Acta 272:56-66.[Medline]
  23. Navon, A. 2000. Bacillus thuringiensis application in agriculture, p. 355-369. In J. F. Charles, A. Delecluse, and C. Nielsen-Leroux (ed.), Entomopathogenic bacteria: from laboratory to field application. Kluwer Academic Publishers, Dordrecht, The Netherlands.
  24. Ohba, M., A. Tantichodok, and K. Aizawa. 1980. Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria. J. Invertebr. Pathol. 38:26-32.
  25. Ozawa, K., and H. Iwahana. 1986. Involvement of a transmissible plasmid in heat-stable exotoxin and delta-endotoxin production in Bacillus thuringiensis subspecies darmstadiensis. Curr. Microbiol. 13:337-340.[CrossRef]
  26. Perani, M., A. H. Bishop, and A. Vaid. 1998. Prevalence of beta-exotoxin, diarrhoeal toxin and specific delta-endotoxin in natural isolates of Bacillus thuringiensis. FEMS Microbiol. Lett. 160:55-60.[Medline]
  27. Raymond, M., G. Prato, and D. Ratsira. 1993. PROBIT analysis of mortality assays displaying quantal response. Praxème SARL (3. 3), Saint Georges d'Orques, France.
  28. Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775-806.[Abstract/Free Full Text]
  29. Sebesta, K., and K. Horska. 1970. Mechanism of inhibition of DNA-dependent RNA polymerase by exotoxin of Bacillus thuringiensis. Biochim. Biophys. Acta 209:357-376.[Medline]
  30. Warren, G. W. 1997. Vegetative insecticidal proteins: novel proteins for control of corn pests, p. 109-121. In N. B. Carozzi and M. Koziel. (ed.), Advances in insect control: the role of transgenic plants. Taylors & Francis Ltd., London, United Kingdom.
  31. Warren, G. W., M. G. Koziel, and M. A. Mullins. June 1998. Auxiliary proteins for enhancing the insecticidal activity of pesticidal proteins. U.S. patent 5770696.
  32. World Health Organization. 1999. Guidelines specification for bacterial larvicides for public health use. Publication W. H. O./CDS/CPC/WHOPES/99.2. Report of the W. H. O. Informal Consultation. World Health Organization, Geneva, Switzerland.

Applied and Environmental Microbiology, September 2002, p. 4182-4186, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4182-4186.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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