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Correlation between serovars of Bacillus thuringiensis and type I β-exotoxin production
Journal of
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 82 (2003) 57–62 www.elsevier.com/locate/yjipa
Correlation between serovars of Bacillus thuringiensis and type I b-exotoxin production Carmen Sara Hern andez,a Clara Martınez,b,1 Manuel Porcar,b,2 Primitivo Caballero,b and Juan Ferrea,* a
Departament de Gen etica, Facultad de Ciencias Biologicas, Universitat de Val encia, 46100 Burjassot (Valencia), Spain b Departamento de Producci on Agraria, Universidad P ublica de Navarra, Pamplona, Spain Received 7 November 2002; accepted 2 December 2002
Abstract b-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce b-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and b-exotoxin production. A specific HPLC assay for type I b-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of b-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed b-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as ‘‘common,’’ ‘‘seldom,’’ or ‘‘rare’’ b-exotoxin producers. The serovar-dependent b-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovardependent regulation of plasmid-encoded genes. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: b-Exotoxin; Bacillus thuringiensis; Flagellar (H) antigen; Serovar; Serotype; HPLC; Bacterial toxins
1. Introduction b-Exotoxin, the thermostable exotoxin produced by Bacillus thuringiensis, was one of the first broad spectrum vegetative factors described in this bacterium. Like other extra-cellular insecticidal toxins, b-exotoxin is expressed during vegetative growth. This compound is an ATP analog, and this similarity makes b-exotoxin an inhibitor of DNA-dependent RNA polymerases (Farkas et al., 1976; Sebesta and Horsk a, 1970; Sebesta *
Corresponding author. Fax: +34-96-398-3029. E-mail address: [email protected] (J. Ferre). 1 Present address: Equipe de Morphogenese Cellulaire et Progression Tumorale. Institut Curie, UMR 144 CNRA 12, rue Lhomond, 75005 Paris, France. 2 Present address: Interactions Moleculaires Flavivirus-H^ otes, Departement de Virologie, Institut Pasteur, 25 Rue du Doctor Roux, 75724 Paris, France.
and Sternbach, 1970). Because of toxicity to vertebrates, most commercial preparations of B. thuringiensis are prepared from isolates that lack the ability to produce b-exotoxin (McClintock et al., 1995). Several laboratories use high-performance liquid chromatography (HPLC) methods to detect and quantify b-exotoxin in fermentation and formulation samples as an alternative to the house fly bioassay, which is more tedious and time consuming, and suffers from several limitations, including inaccurate potency estimations of impure toxin and poor reproducibility (Johnson and Peterson, 1983). Two types of b-exotoxin have been found by HPLC (Levinson et al., 1990). Type I b-exotoxin is composed of adenosine, glucose, and allaric acid (Farkas et al., 1969). The pathway leading to this toxin is expected to be controlled, in part, by chromosomal loci involved in other cellular metabolite pathways, such as nucleic acid
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C. Sara Hernandez et al. / Journal of Invertebrate Pathology 82 (2003) 57–62
metabolism. In addition, a gene required for type I b-exotoxin synthesis or secretion has been reported to be plasmid encoded. This plasmid may also carry one or more d-endotoxin genes (Espinasse et al., 2002a,b; Levinson et al., 1990). Very few studies have dealt with type II b-exotoxin and its structure remains unknown. Nowadays, the most widely accepted classification system of B. thuringiensis strains is based on the flagellar (H) antigens (De Barjac, 1981; De Barjac and Bonnefoi, 1962; Lecadet et al., 1999). b-Exotoxin production was initially thought to be limited to certain flagellar serovars, and a utilization of this observation as one of the criteria for the classification of B. thuringiensis had even been attempted (Heimpel, 1967; Krieg, 1968). However, in a study to determine the production of b-exotoxin in isolates from silkworm litters of sericultural farms and soils in Japan, Ohba et al. (1981) concluded that ‘‘the heat-stable exotoxin production is a strain-specific property rather than a serovar (subspecies)-specific property.’’ This has also been confirmed in other studies (Hern andez et al., 2001), though preliminary results in our laboratories, along with results reported by other authors, seem to indicate that there is some kind of relationship between the type of serovar to which a strain belongs and the capacity of b-exotoxin production. To carry out a systematic study on the relationship between B. thuringiensis serovars (based on the H antigen) and type I b-exotoxin production, we have applied HPLC analysis to 100 strains belonging to four serovars of B. thuringiensis: aizawai, morrisoni, kurstaki, and thuringiensis. The first two serovars had already been analyzed in a previous study using an alternative assay (the fly bioassay) (Ohba et al., 1981). Serovar kurstaki was chosen because it is the serovar of the strains used in many commercial B. thuringiensis products, and serovar thuringiensis because several strains of this serovar have been reported to produce type I b-exotoxin (Hern andez et al., 2001; Levinson et al., 1990).
2. Materials and methods 2.1. Determination of the H antigen and strain selection Serological identification of strains belonging to two B. thuringiensis collections (from the laboratory of Biochemical Genetics and Biotechnology of the Universitat de Valencia, and the LEAPI from Universidad P ublica de Navarra) was carried out with the agglutination method described by Laurent et al. (1996) using the collection of H antisera provided by the Institut Pasteur (Paris, France). Among the strains belonging to serovars thuringiensis (H1), kurstaki (H3a, 3b, 3c),
aizawai (H7), and morrisoni (H8a, 8b), 25 strains from each were randomly selected. 2.2. Bacterial culture and sample preparation Cultures from the different B. thuringiensis strains were grown from a single cell colony in 5 ml of CCY medium (Stewart et al., 1981) for 48 h at 30 °C. After centrifugation at 1800g, 1 ml of the supernatant was transferred to a new tube and centrifuged at 9000g for 15 min. The resulting supernatant was autoclaved at 120 °C for 30 min and filtered through a 45 lm filter unit (MILLEX-HV, Millipore). 2.3. HPLC analysis Type I b-exotoxin determination was based on the method of Campbell et al. (1987). The autoclaved supernatants were subjected to HPLC analysis on a Waters l-Bondapak C18 column (30 cm 3:9 mm i.d., Millipore) as described by Hernandez et al. (2001). The mobile phase was 50 mM KH2 PO4 (pH 3.0) in Milli Q quality water (Millipore, Bedford, MA), at a flow rate of 2 ml/ min at 25 °C. Supernatants were used straight, with an injection volume of 20 ll and peak detection at 260 nm. B. thuringiensis strains were considered as type I b-exotoxin producers when they showed a major peak with the retention time of the standard and a minor peak corresponding to its dephosphorilated form. No other apparent differences were detected among chromatograms of supernatant samples (Fig. 1). 2.4. Source of type I b-exotoxin standard and peak identification The standard was kindly supplied by Isabelle Thiery (Institut Pasteur, France) and was obtained from strain HD2 ‘‘BERLINER,’’ the type-strain of B. thuringiensis serovar thuringiensis. Type I b-exotoxin concentration in the standard was estimated spectrophotometrically at 260 nm (Levinson et al., 1990). The final concentration of the b-exotoxin solution injected into HPLC was 25 lg/ml. Upon injection of type I b-exotoxin standard, the retention time of the major peak (observed at 260 nm) was around 3 min. A minor peak with a retention time of approximately 4.6 min was also observed (Fig. 1). The identity of these peaks was assessed by incubating the type I b-exotoxin standard sample with alkaline phosphatase and then analyzing the product by HPLC (Campbell et al., 1987; Hernandez et al., 2001; Sebesta et al., 1969). After this treatment, the main peak diminished and the peak with the longer retention time increased, indicating that the peak with retention time around 3 min corresponded to type I
C. Sara Hernandez et al. / Journal of Invertebrate Pathology 82 (2003) 57–62
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Fig. 1. Detection of type I b-exotoxin by HPLC. (A) b-Exotoxin standard; (B) example with a nonproducing-exotoxin strain belonging to serovar aizawai; (C) example with a b-exotoxin producing strain belonging to serovar thuringiensis. The b-exotoxin peak appears at retention time of 3.0 min, and the dephosphorilated form of b-exotoxin is observed at approximately 4.6 min.
b-exotoxin and the latter peak was its dephosphorilated form.
3. Results The frequency of type I b-exotoxin production in strains within a given serovar is shown in Table 1. Production of type I b-exotoxin was found in a high proportion of the 25 strains tested from serovar thuringiensis (H1) (84%), whereas very few type I b-exotoxin producing strains were found from the 25 strains belonging to serovar kurstaki (H3a, 3b, 3c) (8%). None of the 25 strains each from serovars aizawai (H7) and morrisoni (H8a, 8b) showed type I b-exotoxin production. Type I b-exotoxin levels in those strains producing this metabolite ranged from 5.6 to 29 lg/ml (Fig. 2), with
a mean value of 10:8 4:5 lg=ml. Most strains produced levels around the mean value, except strain MA10.30 (serovar thuringiensis) which showed approximately three times more production of b-exotoxin than the mean value.
4. Discussion Studies relating b-exotoxin production and the serovar type of B. thuringiensis strains are very limited and most of them included very few strains. In the present work, we carried out a systematic study to shed light on the possible relationship between type I b-exotoxin production and the type of serovar to which a determined strain belongs. Among the 100 strains analyzed (from serovars thuringiensis, kurstaki, aizawai, and morrisoni) most of the strains belonging to the serovar
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Table 1 Frequency of b-exotoxin production among isolates of B. thuringiensis belonging to different serovars in several studies H antigen
Serovar
b-Exotoxin production in B. thuringiensisa Type strainb
Isolates
1 3a, 3c 3a, 3b, 3c 4a, 4b 4a, 4c 5a, 5b 5a, 5c 6 7 8a, 8b 9 10a, 10b 12
thuringiensis alesti kurstaki sotto kenyae galleriae canadensis entomocidus aizawai morrisoni tolworthi darmstadiensis thompsoni
Ohba et al. (1981)
Levinson et al. (1990)
Hernandez et al. (2001)
This study
Total
%c
1/1 0/147 1/1 0/105 4/24 0/3 0/1 0/1 0/75 0/47
1/1
3/6
21/25
1/3
0/2 0/1 1/1
2/25
26/33 0/147 4/31 0/106 5/25 0/3 0/2 0/1 0/100 0/75 2/2 31/35 0/1
79 0 13 0 20
29/32
0/1
0/1 1/1 2/2
0/2 1/1 0/1 0/1
0/25 0/25
0 0 89
+ ) ) ) ) ) ) ) ) )d + + )
a
No. of b-exotoxin producing strains/No. of strains tested. Presence (+) or absence ()) of type I b-exotoxin by HPLC in type strains from the International Enthomopathogenic Bacillus Collection, Institut Pasteur, Paris (Hernandez et al., 2001). c Percentage has only been determined for those serovars represented by at least 25 strains. d Absence of type I b-exotoxin by HPLC. This type strain induced insect mortality in bioassays (Hernandez et al., 2001; Levinson et al., 1990; Ohba et al., 1981). b
Fig. 2. Levels of type I b-exotoxin in supernatants from B. thuringiensis strains producing this metabolite.
thuringiensis and just a few belonging to serovar kurstaki produced type I b-exotoxin. Type I b-exotoxin levels in producing strains were similar to those previously described (Hern andez et al., 2001) and they differed from values reported by other authors using different culture media (Espinasse et al., 2002a; Levinson et al., 1990). Combining our results with those from previous studies (Table 1), B. thuringiensis serovars can be classified in three categories according to the frequency of b-exotoxin producing strains: ‘‘common,’’ ‘‘seldom,’’ and ‘‘rare’’ b-exotoxin producers. If only results involving at least 25 strains from a given serovar are considered,
thuringiensis and darmstadiensis would be classified as ‘‘common’’ producers, kurstaki and kenyae as ‘‘seldom’’ producers, and alesti, sotto, aizawai, and morrisoni as ‘‘rare’’ producers. The classification of B. thuringiensis strains into serovars was developed on the basis of flagellar antigens (De Barjac and Bonnefoi, 1962; De Barjac and Franchon, 1990). Nevertheless, the biochemical and genetic basis of the flagellar antigens system in B. thuringiensis remains totally unknown. This fact makes especially difficult to find a direct relationship between serovardetermining genes and other genes in B. thuringiensis.
C. Sara Hernandez et al. / Journal of Invertebrate Pathology 82 (2003) 57–62
Flagellin genes (which are candidate for serovar determination) are chromosomal (Ankarloo et al., 1996; Chen and Helmann, 1994; L€ ovgren et al., 1998), whereas the involvement of a large plasmid in b-exotoxin production has been shown by curing and conjugation-like transformation experiments (Levinson et al., 1990; Ozawa and Iwahana, 1986). Besides the occurrence of one or several plasmid genes controlling b-exotoxin production or secretion, it is likely that type I b-exotoxin synthesis shares many steps of its pathway with that of some other essential metabolites (as, for example, ATP), and a plasmid-encoded function is involved in the final structure of this exotoxin or in its secretion. Recently, it has been proposed that the genetic determinants responsible for b-exotoxin production found in Crydependent plasmids are regulatory elements (Espinasse et al., 2002b). The association of type I b-exotoxin production to certain serovars somehow implies a relationship between the plasmid content and the serovar. This relationship was also found by other authors, which stated that ‘‘when strains of the same subspecies are compared, similarities in plasmid content are obvious’’ (Carlton and Gonz alez, 1985). Even though mating can occur between cells from different serovars (Gonz alez et al., 1982), Baum and Gonz alez (1992) proposed a limited plasmid exchange among serovars to explain the apparent ‘‘serovar-dependent’’ distribution of B. thuringiensis plasmid profiles. Another observation that relates plasmid-encoded properties to a given serovar is that most highly active mosquitocidal strains from serovar israelensis have the dipteran specific toxin genes located in a 137 kb plasmid (originally described as a 72 MDa plasmid) (Ben-Dov et al., 1996, 1999; Gonz alez and Carlton, 1984; Ward and Ellar, 1983). The acquisition of a plasmid encoding a gene (or genes) that enabled the cell to produce type I b-exotoxin must have been an adaptive event during the evolution of B. thuringiensis. The situation observed currently may be a result of plasmid compatibility with the rest of the chromosomal genes and other bacterial plasmids. Alternatively, evolutionary divergence of B. thuringiensis serovars might have been a later event than the acquisition of plasmid encoding b-exotoxin production. A different explanation for the observed serovar-dependent b-exotoxin production is that this relationship may reflect differences, among serovars, in the expression level of one or more of the genes responsible for bexotoxin synthesis. A serovar-dependent regulation of the plasmid-encoded d-endotoxin genes has been recently reported (Cheng et al., 1999) and a similar regulation system could also control the expression of bexotoxin synthesis genes in those strains carrying the appropriate plasmid. Due to its wide spectrum of activity, in many countries b-exotoxin is prohibited by law in commercial
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products using spores and crystals as the active components (Meadows, 1993). Although type I b-exotoxin production is a property of any B. thuringiensis strain, a strong correlation between its production/lack-of-production is found with the type of serovar. This observation is a useful tool once the H antigen has been determined, since a particular strain belonging to serovars thuringiensis or darmstadiensis will have a much higher chance to produce this metabolite than if it belongs to other serovars. However, the final test for the presence or absence of this metabolite will require direct b-exotoxin determination by any analytical means.
Acknowledgments This research was supported by the Spanish Comisi on Interministerial de Ciencia y Tecnologıa (CICYT), and by the contract with INDUSTRIAS AFRASA, S. A., under the Plan Tecnol ogico de la Comunidad Valenciana (IMPIVA).
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De Barjac, H., Franchon, E., 1990. Classification of Bacillus thuringiensis strains. Entomophaga 35, 233–240. Espinasse, S., Gohar, M., Chaufaux, J., Buisson, C., Perchat, S., Sanchis, V., 2002a. Correspondence of high levels of b-exotoxin I and the presence of cry1B in Bacillus thuringiensis. Appl. Environ. Microbiol. 68, 4182–4186. Espinasse, S., Gohar, M., Lereclus, D., Sanchis, V., 2002b. An ABC transporter from Bacillus thuringiensis is essential for b-exotoxin I production. J. Bacteriol. 184, 5848–5854. Farkas, J., Sebesta, K., Horska, K., Samek, Z., Dolejs, L., Sorm, F., 1969. The structure of exotoxin of Bacillus thuringiensis var. gelechiae. Collect. Czech. Chem. Commun. 34, 1118–1120. Farkas, J., Sebesta, K., Horska, K., Samek, Z., Dolejs, L., Sorm, F., 1976. Structure of thuringiensin, the thermostable exotoxin from Bacillus thuringiensis. Collect. Czech. Chem. Commun. 42, 909–929. Gonz alez Jr., J.M., Carlton, B.C., 1984. A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis. Plasmid 11, 28–38. Gonz alez Jr., J.M., Brown, B.J., Carlton, B.C., 1982. Transfer of Bacillus thuringiensis plasmids coding for d-endotoxin among strains of Bacillus thuringiensis and B. cereus. Proc. Natl. Acad. Sci. USA 79, 6951–6955. Heimpel, A.M., 1967. A critical review of Bacillus thuringiensis var. thuringiensis Berliner and other crystalliferous bacteria. Annu. Rev. Entomol. 12, 287–322. Hern andez, C.S., Ferre, J., Larget-Thiery, I., 2001. Update on the detection of b-exotoxin in Bacillus thuringiensis strains by HPLC analysis. J. Appl. Microbiol. 90, 643–647. Johnson, D.E., Peterson, R.E., 1983. Limitations of HPLC for the detection of b-exotoxin in culture filtrates of Bacillus thuringiensis. Eur. J. Appl. Microbiol. Biotechnol. 17, 231–234. Krieg, A., 1968. A taxonomic study of Bacillus thuringiensis Berliner. J. Invertebr. Pathol. 12, 366–378. Laurent, P., Ripoteau, H., Dumanoir, V.C., Franchon, E., Lecadet, M.-M., 1996. A micromethod for serotyping Bacillus thuringiensis. Lett. Appl. Microbiol. 22, 259–261. Lecadet, M.-M., Franchon, E., Dumanoir, V.C., Ripoteau, H., Hamon, S., Laurent, P., Thiery, I., 1999. Updating the H-antigen classification of Bacillus thuringiensis. J. Appl. Microbiol. 86, 660–672.
Levinson, B.L., Kasyan, K.J., Chiu, S.S., Currier, T.C., Gonzalez Jr., J.M., 1990. Identification of b-exotoxin production, plasmids encoding b-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography. J. Bacteriol. 172, 3172–3179. L€ ovgren, A., Carlson, C.R., Eskils, K., Kolstø, A.B., 1998. Localization of putative virulence genes on a physical map of the Bacillus thuringiensis subsp. gelechiae chromosome. Curr. Microbiol. 37, 245–250. McClintock, J., Schaffer, C.R., Sjoblad, R.D., 1995. A comparative review of the mammalian toxicity of Bacillus thuringiensis-based pesticides. Pestic. Sci. 45, 95–105. Meadows, M.P., 1993. Bacillus thuringiensis in the Environment: Ecology and risk assessment. In: Entwistle, P.F., Cory, J.S., Bailey, M.J., Higgs, S. (Eds.), Bacillus thuringiensis, an Environmental Biopesticide: Theory and Practice. Wiley & Sons Ltd., West Sussex, England, pp. 193–220. Ohba, M., Tantichodok, A., Aizawa, K., 1981. Production of heatstable exotoxin by Bacillus thuringiensis and related bacteria. J. Invertebr. Pathol. 38, 26–32. Ozawa, K., Iwahana, H., 1986. Involvement of a transmissible plasmid in heat-stable exotoxin and d-endotoxin production in Bacillus thuringiensis subsp. darmstadiensis. Curr. Microbiol. 13, 337–340. Sebesta, K., Horska, K., 1970. Mechanism of inhibition of DNAdependent RNA polymerase by exotoxin of Bacillus thuringiensis. Biochem. Biophys. Acta 209, 357–376. Sebesta, K., Sternbach, H., 1970. The specificity of inhibition of DNAdependent RNA polymerase by Bacillus thuringiensis exotoxin. FEBS Lett. 8, 223–235. Sebesta, K., Horska, K., Vankova, J., 1969. Inhibition of the novo RNA synthesis by the insecticidal exotoxin of Bacillus thuringiensis var. gelechiae. Collect. Czech. Chem. Commun. 34, 891–900. Stewart, G.S.A.B., Jonhstone, K., Hagelbert, E., Ellar, D.J., 1981. Commitment of bacterial spores to germinate. Biochem. J. 198, 101–106. Ward, E.S., Ellar, D.J., 1983. Assignment of the d-endotoxin gene of Bacillus thuringiensis var. israelensis to a specific plasmid by curing analyses. FEBS Lett. 158, 49–59.
INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 82 (2003) 57–62 www.elsevier.com/locate/yjipa
Correlation between serovars of Bacillus thuringiensis and type I b-exotoxin production Carmen Sara Hern andez,a Clara Martınez,b,1 Manuel Porcar,b,2 Primitivo Caballero,b and Juan Ferrea,* a
Departament de Gen etica, Facultad de Ciencias Biologicas, Universitat de Val encia, 46100 Burjassot (Valencia), Spain b Departamento de Producci on Agraria, Universidad P ublica de Navarra, Pamplona, Spain Received 7 November 2002; accepted 2 December 2002
Abstract b-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce b-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and b-exotoxin production. A specific HPLC assay for type I b-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of b-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed b-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as ‘‘common,’’ ‘‘seldom,’’ or ‘‘rare’’ b-exotoxin producers. The serovar-dependent b-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovardependent regulation of plasmid-encoded genes. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: b-Exotoxin; Bacillus thuringiensis; Flagellar (H) antigen; Serovar; Serotype; HPLC; Bacterial toxins
1. Introduction b-Exotoxin, the thermostable exotoxin produced by Bacillus thuringiensis, was one of the first broad spectrum vegetative factors described in this bacterium. Like other extra-cellular insecticidal toxins, b-exotoxin is expressed during vegetative growth. This compound is an ATP analog, and this similarity makes b-exotoxin an inhibitor of DNA-dependent RNA polymerases (Farkas et al., 1976; Sebesta and Horsk a, 1970; Sebesta *
Corresponding author. Fax: +34-96-398-3029. E-mail address: [email protected] (J. Ferre). 1 Present address: Equipe de Morphogenese Cellulaire et Progression Tumorale. Institut Curie, UMR 144 CNRA 12, rue Lhomond, 75005 Paris, France. 2 Present address: Interactions Moleculaires Flavivirus-H^ otes, Departement de Virologie, Institut Pasteur, 25 Rue du Doctor Roux, 75724 Paris, France.
and Sternbach, 1970). Because of toxicity to vertebrates, most commercial preparations of B. thuringiensis are prepared from isolates that lack the ability to produce b-exotoxin (McClintock et al., 1995). Several laboratories use high-performance liquid chromatography (HPLC) methods to detect and quantify b-exotoxin in fermentation and formulation samples as an alternative to the house fly bioassay, which is more tedious and time consuming, and suffers from several limitations, including inaccurate potency estimations of impure toxin and poor reproducibility (Johnson and Peterson, 1983). Two types of b-exotoxin have been found by HPLC (Levinson et al., 1990). Type I b-exotoxin is composed of adenosine, glucose, and allaric acid (Farkas et al., 1969). The pathway leading to this toxin is expected to be controlled, in part, by chromosomal loci involved in other cellular metabolite pathways, such as nucleic acid
0022-2011/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0022-2011(02)00199-4
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C. Sara Hernandez et al. / Journal of Invertebrate Pathology 82 (2003) 57–62
metabolism. In addition, a gene required for type I b-exotoxin synthesis or secretion has been reported to be plasmid encoded. This plasmid may also carry one or more d-endotoxin genes (Espinasse et al., 2002a,b; Levinson et al., 1990). Very few studies have dealt with type II b-exotoxin and its structure remains unknown. Nowadays, the most widely accepted classification system of B. thuringiensis strains is based on the flagellar (H) antigens (De Barjac, 1981; De Barjac and Bonnefoi, 1962; Lecadet et al., 1999). b-Exotoxin production was initially thought to be limited to certain flagellar serovars, and a utilization of this observation as one of the criteria for the classification of B. thuringiensis had even been attempted (Heimpel, 1967; Krieg, 1968). However, in a study to determine the production of b-exotoxin in isolates from silkworm litters of sericultural farms and soils in Japan, Ohba et al. (1981) concluded that ‘‘the heat-stable exotoxin production is a strain-specific property rather than a serovar (subspecies)-specific property.’’ This has also been confirmed in other studies (Hern andez et al., 2001), though preliminary results in our laboratories, along with results reported by other authors, seem to indicate that there is some kind of relationship between the type of serovar to which a strain belongs and the capacity of b-exotoxin production. To carry out a systematic study on the relationship between B. thuringiensis serovars (based on the H antigen) and type I b-exotoxin production, we have applied HPLC analysis to 100 strains belonging to four serovars of B. thuringiensis: aizawai, morrisoni, kurstaki, and thuringiensis. The first two serovars had already been analyzed in a previous study using an alternative assay (the fly bioassay) (Ohba et al., 1981). Serovar kurstaki was chosen because it is the serovar of the strains used in many commercial B. thuringiensis products, and serovar thuringiensis because several strains of this serovar have been reported to produce type I b-exotoxin (Hern andez et al., 2001; Levinson et al., 1990).
2. Materials and methods 2.1. Determination of the H antigen and strain selection Serological identification of strains belonging to two B. thuringiensis collections (from the laboratory of Biochemical Genetics and Biotechnology of the Universitat de Valencia, and the LEAPI from Universidad P ublica de Navarra) was carried out with the agglutination method described by Laurent et al. (1996) using the collection of H antisera provided by the Institut Pasteur (Paris, France). Among the strains belonging to serovars thuringiensis (H1), kurstaki (H3a, 3b, 3c),
aizawai (H7), and morrisoni (H8a, 8b), 25 strains from each were randomly selected. 2.2. Bacterial culture and sample preparation Cultures from the different B. thuringiensis strains were grown from a single cell colony in 5 ml of CCY medium (Stewart et al., 1981) for 48 h at 30 °C. After centrifugation at 1800g, 1 ml of the supernatant was transferred to a new tube and centrifuged at 9000g for 15 min. The resulting supernatant was autoclaved at 120 °C for 30 min and filtered through a 45 lm filter unit (MILLEX-HV, Millipore). 2.3. HPLC analysis Type I b-exotoxin determination was based on the method of Campbell et al. (1987). The autoclaved supernatants were subjected to HPLC analysis on a Waters l-Bondapak C18 column (30 cm 3:9 mm i.d., Millipore) as described by Hernandez et al. (2001). The mobile phase was 50 mM KH2 PO4 (pH 3.0) in Milli Q quality water (Millipore, Bedford, MA), at a flow rate of 2 ml/ min at 25 °C. Supernatants were used straight, with an injection volume of 20 ll and peak detection at 260 nm. B. thuringiensis strains were considered as type I b-exotoxin producers when they showed a major peak with the retention time of the standard and a minor peak corresponding to its dephosphorilated form. No other apparent differences were detected among chromatograms of supernatant samples (Fig. 1). 2.4. Source of type I b-exotoxin standard and peak identification The standard was kindly supplied by Isabelle Thiery (Institut Pasteur, France) and was obtained from strain HD2 ‘‘BERLINER,’’ the type-strain of B. thuringiensis serovar thuringiensis. Type I b-exotoxin concentration in the standard was estimated spectrophotometrically at 260 nm (Levinson et al., 1990). The final concentration of the b-exotoxin solution injected into HPLC was 25 lg/ml. Upon injection of type I b-exotoxin standard, the retention time of the major peak (observed at 260 nm) was around 3 min. A minor peak with a retention time of approximately 4.6 min was also observed (Fig. 1). The identity of these peaks was assessed by incubating the type I b-exotoxin standard sample with alkaline phosphatase and then analyzing the product by HPLC (Campbell et al., 1987; Hernandez et al., 2001; Sebesta et al., 1969). After this treatment, the main peak diminished and the peak with the longer retention time increased, indicating that the peak with retention time around 3 min corresponded to type I
C. Sara Hernandez et al. / Journal of Invertebrate Pathology 82 (2003) 57–62
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Fig. 1. Detection of type I b-exotoxin by HPLC. (A) b-Exotoxin standard; (B) example with a nonproducing-exotoxin strain belonging to serovar aizawai; (C) example with a b-exotoxin producing strain belonging to serovar thuringiensis. The b-exotoxin peak appears at retention time of 3.0 min, and the dephosphorilated form of b-exotoxin is observed at approximately 4.6 min.
b-exotoxin and the latter peak was its dephosphorilated form.
3. Results The frequency of type I b-exotoxin production in strains within a given serovar is shown in Table 1. Production of type I b-exotoxin was found in a high proportion of the 25 strains tested from serovar thuringiensis (H1) (84%), whereas very few type I b-exotoxin producing strains were found from the 25 strains belonging to serovar kurstaki (H3a, 3b, 3c) (8%). None of the 25 strains each from serovars aizawai (H7) and morrisoni (H8a, 8b) showed type I b-exotoxin production. Type I b-exotoxin levels in those strains producing this metabolite ranged from 5.6 to 29 lg/ml (Fig. 2), with
a mean value of 10:8 4:5 lg=ml. Most strains produced levels around the mean value, except strain MA10.30 (serovar thuringiensis) which showed approximately three times more production of b-exotoxin than the mean value.
4. Discussion Studies relating b-exotoxin production and the serovar type of B. thuringiensis strains are very limited and most of them included very few strains. In the present work, we carried out a systematic study to shed light on the possible relationship between type I b-exotoxin production and the type of serovar to which a determined strain belongs. Among the 100 strains analyzed (from serovars thuringiensis, kurstaki, aizawai, and morrisoni) most of the strains belonging to the serovar
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Table 1 Frequency of b-exotoxin production among isolates of B. thuringiensis belonging to different serovars in several studies H antigen
Serovar
b-Exotoxin production in B. thuringiensisa Type strainb
Isolates
1 3a, 3c 3a, 3b, 3c 4a, 4b 4a, 4c 5a, 5b 5a, 5c 6 7 8a, 8b 9 10a, 10b 12
thuringiensis alesti kurstaki sotto kenyae galleriae canadensis entomocidus aizawai morrisoni tolworthi darmstadiensis thompsoni
Ohba et al. (1981)
Levinson et al. (1990)
Hernandez et al. (2001)
This study
Total
%c
1/1 0/147 1/1 0/105 4/24 0/3 0/1 0/1 0/75 0/47
1/1
3/6
21/25
1/3
0/2 0/1 1/1
2/25
26/33 0/147 4/31 0/106 5/25 0/3 0/2 0/1 0/100 0/75 2/2 31/35 0/1
79 0 13 0 20
29/32
0/1
0/1 1/1 2/2
0/2 1/1 0/1 0/1
0/25 0/25
0 0 89
+ ) ) ) ) ) ) ) ) )d + + )
a
No. of b-exotoxin producing strains/No. of strains tested. Presence (+) or absence ()) of type I b-exotoxin by HPLC in type strains from the International Enthomopathogenic Bacillus Collection, Institut Pasteur, Paris (Hernandez et al., 2001). c Percentage has only been determined for those serovars represented by at least 25 strains. d Absence of type I b-exotoxin by HPLC. This type strain induced insect mortality in bioassays (Hernandez et al., 2001; Levinson et al., 1990; Ohba et al., 1981). b
Fig. 2. Levels of type I b-exotoxin in supernatants from B. thuringiensis strains producing this metabolite.
thuringiensis and just a few belonging to serovar kurstaki produced type I b-exotoxin. Type I b-exotoxin levels in producing strains were similar to those previously described (Hern andez et al., 2001) and they differed from values reported by other authors using different culture media (Espinasse et al., 2002a; Levinson et al., 1990). Combining our results with those from previous studies (Table 1), B. thuringiensis serovars can be classified in three categories according to the frequency of b-exotoxin producing strains: ‘‘common,’’ ‘‘seldom,’’ and ‘‘rare’’ b-exotoxin producers. If only results involving at least 25 strains from a given serovar are considered,
thuringiensis and darmstadiensis would be classified as ‘‘common’’ producers, kurstaki and kenyae as ‘‘seldom’’ producers, and alesti, sotto, aizawai, and morrisoni as ‘‘rare’’ producers. The classification of B. thuringiensis strains into serovars was developed on the basis of flagellar antigens (De Barjac and Bonnefoi, 1962; De Barjac and Franchon, 1990). Nevertheless, the biochemical and genetic basis of the flagellar antigens system in B. thuringiensis remains totally unknown. This fact makes especially difficult to find a direct relationship between serovardetermining genes and other genes in B. thuringiensis.
C. Sara Hernandez et al. / Journal of Invertebrate Pathology 82 (2003) 57–62
Flagellin genes (which are candidate for serovar determination) are chromosomal (Ankarloo et al., 1996; Chen and Helmann, 1994; L€ ovgren et al., 1998), whereas the involvement of a large plasmid in b-exotoxin production has been shown by curing and conjugation-like transformation experiments (Levinson et al., 1990; Ozawa and Iwahana, 1986). Besides the occurrence of one or several plasmid genes controlling b-exotoxin production or secretion, it is likely that type I b-exotoxin synthesis shares many steps of its pathway with that of some other essential metabolites (as, for example, ATP), and a plasmid-encoded function is involved in the final structure of this exotoxin or in its secretion. Recently, it has been proposed that the genetic determinants responsible for b-exotoxin production found in Crydependent plasmids are regulatory elements (Espinasse et al., 2002b). The association of type I b-exotoxin production to certain serovars somehow implies a relationship between the plasmid content and the serovar. This relationship was also found by other authors, which stated that ‘‘when strains of the same subspecies are compared, similarities in plasmid content are obvious’’ (Carlton and Gonz alez, 1985). Even though mating can occur between cells from different serovars (Gonz alez et al., 1982), Baum and Gonz alez (1992) proposed a limited plasmid exchange among serovars to explain the apparent ‘‘serovar-dependent’’ distribution of B. thuringiensis plasmid profiles. Another observation that relates plasmid-encoded properties to a given serovar is that most highly active mosquitocidal strains from serovar israelensis have the dipteran specific toxin genes located in a 137 kb plasmid (originally described as a 72 MDa plasmid) (Ben-Dov et al., 1996, 1999; Gonz alez and Carlton, 1984; Ward and Ellar, 1983). The acquisition of a plasmid encoding a gene (or genes) that enabled the cell to produce type I b-exotoxin must have been an adaptive event during the evolution of B. thuringiensis. The situation observed currently may be a result of plasmid compatibility with the rest of the chromosomal genes and other bacterial plasmids. Alternatively, evolutionary divergence of B. thuringiensis serovars might have been a later event than the acquisition of plasmid encoding b-exotoxin production. A different explanation for the observed serovar-dependent b-exotoxin production is that this relationship may reflect differences, among serovars, in the expression level of one or more of the genes responsible for bexotoxin synthesis. A serovar-dependent regulation of the plasmid-encoded d-endotoxin genes has been recently reported (Cheng et al., 1999) and a similar regulation system could also control the expression of bexotoxin synthesis genes in those strains carrying the appropriate plasmid. Due to its wide spectrum of activity, in many countries b-exotoxin is prohibited by law in commercial
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products using spores and crystals as the active components (Meadows, 1993). Although type I b-exotoxin production is a property of any B. thuringiensis strain, a strong correlation between its production/lack-of-production is found with the type of serovar. This observation is a useful tool once the H antigen has been determined, since a particular strain belonging to serovars thuringiensis or darmstadiensis will have a much higher chance to produce this metabolite than if it belongs to other serovars. However, the final test for the presence or absence of this metabolite will require direct b-exotoxin determination by any analytical means.
Acknowledgments This research was supported by the Spanish Comisi on Interministerial de Ciencia y Tecnologıa (CICYT), and by the contract with INDUSTRIAS AFRASA, S. A., under the Plan Tecnol ogico de la Comunidad Valenciana (IMPIVA).
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