APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2002, p. 1228–1231 0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.3.1228–1231.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 68, No. 3
Characterization of Cyt2Bc Toxin from Bacillus thuringiensis subsp. medellin Victor Juárez-Pérez,1 Alejandra Guerchicoff,2 Clara Rubinstein,2 and Armelle Delécluse1* Laboratoire des Bactéries et Champignons Entomopathogènes, Institut Pasteur, 75724 Paris Cedex 15, France,1 and Facultad de Ciencias Exactas y Naturales, Departamento de Quı´mica Biológica, Ciudad Universitaria, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina2 Received 29 August 2001/Accepted 2 January 2002
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. Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus are currently used to combat the mosquitoes responsible for several tropical diseases including dengue, yellow fever, and malaria. Several screening programs aiming to isolate new strains that could replace or be used in rotation with these two bacteria have been set up. One of the strains isolated, Bacillus thuringiensis subsp. medellin 163-131, is almost as toxic as B. thuringiensis subsp. israelensis but produces different polypeptides (13). Crystals from B. thuringiensis subsp. medellin are composed of several proteins, including Cry11Bb, Cry29A, Cry30A, and Cyt1Ab, which have been fully characterized elsewhere (6). B. thuringiensis mosquitocidal toxins belong to two structurally different groups: the Cry family, with specific activity, and the Cyt family, the polypeptides of which are cytolytic and hemolytic. Based on amino acid identity, two cytolytic classes of Cyt toxins have been identified, Cyt1 and Cyt2, and several cytolytic toxins from each class (Cyt1Aa and Cyt1Ab, Cyt1Ba, Cyt2Aa, and Cyt2Ba and Cyt2Bb) have been characterized from various mosquitocidal B. thuringiensis strains (for a review see reference 6). The Cyt toxins are cytolytic to a wide variety of insect and mammalian cells, including erythrocytes (16). Maximal toxicity requires proteolytic processing by insect midgut proteases of both the amino and carboxyl termini of the proteins (1). The cytolytic toxins are also mosquitocidal, with Cyt1Aa and Cyt2Aa displaying the highest activity (11). The level of activity of Cyt toxins depends on the mosquito larvae tested, Aedes, Anopheles, or Culex species (6). Cyt1Aa generally has lower larvicidal activity than do the mosquitocidal Cry toxins, at least against Aedes and Culex species (4, 5). However, the Cyt1Aa
toxins greatly contribute to the overall toxicity of the native crystals through synergistic interactions with Cry polypeptides (5, 19). In addition, Cyt toxins have been implicated previously as major factors in the lack of resistance to B. thuringiensis subsp. israelensis in laboratory-selected Culex quinquefasciatus populations (8). Thus, Cyt proteins may be useful for combating insecticide resistance and for increasing the activity of microbial insecticides. We report here the cloning and expression of a gene encoding a new variant of the Cyt2B family, Cyt2Bc, from B. thuringiensis subsp. medellin. The hemolytic and mosquitocidal activities of Cyt2Bc were determined and compared to those of Cyt1Aa and Cyt2Ba, the characteristic toxins of the two families. MATERIALS AND METHODS Strains and plasmids. B. thuringiensis subsp. medellin strain 163-131 (serotype H30) and Bacillus thuringiensis subsp. thuringiensis strain SPL407 were obtained from the IEBC collection held by the Laboratoire des Bactéries et Champignons Entomopathogènes (Institut Pasteur, Paris, France). B. thuringiensis strain 4Q7(pWF45) (19) was used as the source of Cyt1Aa protein (kindly provided by Brian Federici). Escherichia coli TGI [K-12; ⌬(lac-proAB) supE thi hsdD5/F⬘ (traD36 proA⫹ proB⫹ lacIq lacZ⌬M15)] was used for the cloning of the toxin gene. For initial cloning of the toxin gene, we used the plasmid pBluescript SK(⫹) (Stratagene). The shuttle vector pHT315 (2) was used for expression in B. thuringiensis. Plasmid pCYT2Ba was obtained after subcloning the 1.5-kb SacIEcoRI fragment from plasmid pRX80 (7) into pHT315 digested with SacI and EcoRI. B. thuringiensis SPL407 cells were transformed as previously described (12). B. thuringiensis transformants were selected and grown on Luria-Bertani medium supplemented with 10 g of erythromycin/ml. Cloning and sequencing of the toxin gene. Total DNA from B. thuringiensis subsp. medellin strain 163-131 was extracted as previously described (14) and digested, and the resulting fragments, subjected to electrophoresis, were transferred to an N⫹ nylon membrane (Amersham). The membrane was probed with a PCR fragment obtained from total B. thuringiensis subsp. medellin DNA using primers specific for cyt2 genes, as previously published (10). An EcoRI fragment of approximately 6 kb was found to hybridize with this probe. The PCR probe was then used to screen a library made by inserting 5- to 7-kb
* Corresponding author. Mailing address: Laboratoire des Bactéries et Champignons Entomopathogènes, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: (33) 1 40 61 31 80. Fax: (33) 1 40 61 30 44. E-mail:
[email protected]. 1228
B. THURINGIENSIS SUBSP. MEDELLIN Cyt2Bc
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FIG. 1. Restriction map of the recombinant plasmids pCYT2BC1 and pCYT2BC2 containing the cyt2Bc gene. The position and direction of transcription of the identified genes are indicated. The vectors pBluescript SK(⫹) and pHT315 are represented by hatched and solid bars, respectively. The asterisks indicate that sites have been lost. Abbreviations: E, EcoRI; H, HindIII; N, NsiI.
fragments of EcoRI-digested total DNA into the pBluescript SK(⫹) plasmid. A positive clone, TGI(pCYT2Bc1), was isolated and used for further studies. The recombinant contains a 6.6-kb fragment (Fig. 1). The entire cloned fragment was sequenced by primer walking on an automatic sequencer (model 310; Applied Biosystems) using the dRhodamine terminator cycle sequencing ready reaction kit (Applied Biosystems). A 1.8-kb NsiI fragment containing the cyt2Bc gene was subcloned into the shuttle vector pHT315 digested with PstI and treated with calf intestine phosphatase. The resulting plasmid was designated pCYT2Bc2 (Fig. 1). Protein analysis. Inclusions were purified from sporulated B. thuringiensis strains by ultracentrifugation on discontinuous sucrose gradients, as previously described (13). Protein concentrations were determined by the Bradford assay (Bio-Rad). Purified crystals were assayed following solubilization in 0.05 N NaOH for 1 h at 37°C and centrifugation at 10,000 ⫻ g to recover the supernatant. Purified crystals were loaded onto sodium dodecyl sulfate (SDS)–15% polyacrylamide gels and subjected to electrophoresis. Separated proteins were stained with Coomassie brilliant blue or transferred to nitrocellulose membranes. Membranes were probed with rabbit antisera directed against Cyt1Aa or Cyt2Ba, diluted 1 in 2,000. Peroxidase-conjugated secondary antibodies were used for detection with the ECL Western blotting system kit (Amersham). Mosquitocidal and hemolytic activity assays. The mosquitoes used were from colonies of Aedes aegypti strain Bora-Bora, Anopheles stephensi strain ST15, Culex pipiens strain Montpellier, and C. quinquefasciatus strain GeoR resistant to the B. sphaericus binary toxin (18) reared in the laboratory as previously described (13). Purified inclusions were diluted in 150 ml of deionized water in plastic cups and tested in duplicate against 25 fourth-instar larvae of Aedes and Culex and thirdinstar larvae of Anopheles. Each bioassay was repeated at least five times. Larval mortality was recorded after 48 h, and 50% lethal concentrations were determined by Probit analysis. The hemolytic activity of purified crystals was determined on sheep red blood cells, as previously described (13). The toxins used for hemolytic assays were prepared as follows. Crystals were solubilized by incubation in 50 mM Na2CO3 (pH 10.5)–10 mM dithiothreitol for 1 h at 37°C. The mixture was centrifuged at 10,000 ⫻ g for 10 min, and the supernatant was incubated at 37°C in 10% (wt/wt) proteinase K (Eurobio) or trypsin (Serva). Aliquots were removed after 1, 2, and 16 h of incubation. Proteinase K activity was blocked by adding 1 mM Pefabloc; trypsin activity was blocked by adding immobilized trypsin inhibitor (Pierce), as recommended by the manufacturer. Nucleotide sequence accession number. The nucleotide sequence data for the 6.6-kb fragment from pCYT2Bc1 are available from the EMBL-GenBank nucleotide sequence databases under accession no. AJ251979.
RESULTS Sequence analysis. The gene encoding the Cyt2B-like protein from B. thuringiensis subsp. medellin was cloned into
pBluescript SK(⫹) as described in Materials and Methods. The isolated recombinant plasmid had a 6.6-kb EcoRI insert (Fig. 1). The nucleotide sequence of this insert was determined in both directions, and six open reading frames (ORFs) were identified (Fig. 1). An ORF of 780 bp encoded the Cyt2B-like protein, which consists of 260 amino acids with a predicted molecular mass of 29,697 Da. We identified, downstream from this ORF and transcribed in the same orientation, the sequences encoding P21 and Cyt1Ab1 previously described by Thiéry et al. (15). Three ORFs with sequences homologous to the insertion sequence IS240 were found in the vicinity of these cyt genes. The first and last sequences encode polypeptides of 204 and 187 amino acids, respectively, and correspond to partial IS240 elements, although both have inverted repeats of 14 nucleotides. The element located between these partial elements is 861 bp long. It encodes a polypeptide of 235 amino acids and corresponds to a whole IS240 element with inverted repeat sequences of 13 bp. The Cyt2B-like toxin from B. thuringiensis subsp. medellin was found to be 91.5% identical to Cyt2Ba and 85% identical to Cyt2Bb. The Cyt2B-like toxin from B. thuringiensis subsp. medellin fits into the Cyt dendrogram at a level suggesting that it represents a new tertiary rank. It was therefore designated Cyt2Bc. Expression of cyt2Bc in a crystal-negative strain of B. thuringiensis. The cyt2Bc gene was located on a 1.8-kb NsiI fragment (Fig. 1), which was subcloned into the shuttle vector pHT315 to facilitate expression in the SPL407 Cry⫺ strain of B. thuringiensis (see Materials and Methods). Inclusions from strain SPL407(pCYT2Bc2) were purified and further analyzed by SDS-polyacrylamide gel electrophoresis, along with inclusions purified from strains 4Q7(pWF45) and SPL407(pCYT2Ba) containing proteins Cyt1Aa and Cyt2Ba from B. thuringiensis subsp. israelensis, respectively. All three polypeptides migrated to similar positions, corresponding to a molecular mass of 28 kDa (Fig. 2A). Immunological cross-reactions among Cyt2Bc, Cyt2Ba, and Cyt1Aa were studied by Western blotting (Fig. 2B). Cyt2Bc was not recognized by an antiserum directed against Cyt1Aa, and neither was Cyt2Ba. Cyt2Bc re-
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JUÁREZ-PÉREZ ET AL.
APPL. ENVIRON. MICROBIOL. TABLE 1. Hemolytic activities of solubilized and activated Cyt2Bc toxina Activity of toxin:
Treatmentb
Cyt2Bc
Cyt2Ba
c
Solubili- ⬎390 zation
31.2 (25.9–37.5) 0.147 (0.138–0.157)
Proteinase K 1h ⬎200c 2h ⬎200c 14 h ⬎200c Trypsin 1h 2h 14 h
Cyt1Aa
⬎250c ⬎250c ⬎250c
9.7 (8.5–11.5) 8.5 (7.6–9.6) 4.5 (4.2–4.9)
0.058 (0.053–0.063) 0.064 (0.059–0.069) 0.054 (0.051–0.058)
3.0 (2.7–3.3) 1.4 (1.3–1.6) 0.9 (0.8–0.9)
0.023 (0.021–0.024) 0.034 (0.032–0.036) 0.026 (0.025–0.027)
a Doses giving 50% hemolysis are expressed in micrograms of protein. Values are the means of at least three experiments. Numbers in parentheses are 95% confidence limits, as determined by Probit analysis. b Proteins were solubilized and activated as described in Materials and Methods. c At this concentration, no hemolytic activity was detected.
FIG. 2. Protein analysis of Cyt2Bc-containing inclusions. (A) Purified inclusions corresponding to 10 g of protein were subjected to electrophoresis in 15% polyacrylamide gels containing SDS and stained with Coomassie brilliant blue. Lane 1, Cyt2Bc; lane 2, Cyt2Ba; lane 3, Cyt1Aa. (B) Purified inclusions were subjected to electrophoresis (as described above) and then transferred onto a nitrocellulose filter. Incubation with antiserum raised against either Cyt2Ba (a) or Cyt1Aa (b) and detection of immunoreactive polypeptides were performed as described in Materials and Methods. Lane 1, Cyt2Bc (1 g); lane 2, Cyt2Ba (0.1 g); lane 3, Cyt1Aa (5 g); lane 4, Cyt2Bc (5 g); lane 5, Cyt2Ba (5 g); lane 6, Cyt1Aa (0.05 g). Molecular mass is indicated in kilodaltons at the left of panel A and in the center of panel B.
acted with the antiserum directed against Cyt2Ba, but the signal was only about 1/10 as strong as that obtained for Cyt2Ba. Cyt1Aa was not recognized by this antiserum. Biological activity of the Cyt2Bc protein. Purified inclusions containing Cyt2Bc were assayed for hemolytic activity against sheep red blood cells (see Materials and Methods). Their activity was compared with those of Cyt1Aa and Cyt2Ba (Table 1). Cyt2Bc was not hemolytic if it was only solubilized, in contrast to Cyt1Aa and Cyt2Ba. None of the Cyt2B toxins was hemolytic after activation with proteinase K, even after a 16-h incubation. Only trypsin treatment led to Cyt2Bc activity. A similar effect was observed for Cyt2Ba. Whatever the time of exposure, Cyt2Bc was always less active than were Cyt2Ba (by a factor of 3 to 5) and Cyt1Aa (by a factor of 150 to 400). Purified inclusions containing Cyt2Bc were also assayed
against mosquito larvae (as described in Materials and Methods) and compared with Cyt1Aa and Cyt2Ba (Table 2). Cyt2Bc was active against all species tested, particularly on C. quinquefasciatus resistant to the binary toxin of B. sphaericus. Against this species, it was as active as Cyt2Ba. In contrast, against susceptible C. pipiens, A. aegypti, and A. stephensi, Cyt2Bc was only about half as active as Cyt2Ba. Cyt2Bc was less toxic than Cyt1Aa (by a factor of 4 to 12), whatever the mosquito species. DISCUSSION We report the cloning and characterization of a gene encoding a hemolytic and mosquitocidal protein from the highly mosquitocidal strain B. thuringiensis subsp. medellin. This 29,697-Da protein is the third element of the Cyt2 family, designated Cyt2Bc. The cyt2Bc gene is located 2 kb upstream from the previously described p21 and cyt1Ab genes (15) and is transcribed in the same orientation. The location of this cluster is different from that in B. thuringiensis subsp. israelensis, in which cyt1Aa and cyt2Ba are transcribed in opposite orientations and are separated from each other by about 24 kb (3). Two IS240-like insertion sequences flank the cyt2Bc gene, in a transposon-like TABLE 2. Mosquitocidal activities of Cyt2Bc toxina Mosquito species
Aedes aegypti Culex pipiens Culex quinquefasciatus GeoR Anopheles stephensi a
Activity of toxin: Cyt2Bc
Cyt2Ba
Cyt1Aa
7.0 (5.4–9.0) 7.3 (5.8–9.3) 1.8 (1.4–2.3)
3.6 (2.7–4.9) 5.0 (3.7–6.9) 1.8 (1.4–2.4)
1.0 (0.8–1.3) 0.6 (0.4–0.8) 0.4 (0.3–0.5)
11.0 (8.4–14.5)
5.5 (4.3–7.1)
2.7 (2.1–3.5)
Values correspond to the 50% lethal concentration at 48 h (see Materials and Methods) and are expressed in micrograms of protein. Means are from at least five independent experiments. Numbers in parentheses are 95% confidence limits, as determined by Probit analysis.
B. THURINGIENSIS SUBSP. MEDELLIN Cyt2Bc
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structure. The cyt2Bc-p21-cyt1Ab toxin cluster is also contained within such a structure. Guerchicoff et al. (9) recently demonstrated that cyt2 genes are widely distributed among B. thuringiensis strains and found that all but 1 of 18 strains positive for cyt2 were also positive for IS240. Further work is required to elucidate the role of this insertion sequence in the dispersion of cyt2 genes. Although the two toxins are highly similar in sequence, antibodies directed against Cyt2Ba did not fully recognize Cyt2Bc, indicating that the sequence differences probably concern important epitopes. None of the Cyt2 proteins were recognized by the antiserum directed against Cyt1Aa, but the level of sequence identity between Cyt1 and Cyt2 families is probably too low. Immunodetection is, therefore, not a powerful tool for analyzing the Cyt content of a strain. Reverse transcription-PCR experiments would probably give more accurate results, although this strategy would provide little information about the level of production of the corresponding protein. Differences in hemolytic or mosquitocidal activities were observed between Cyt2 proteins, despite their high level of similarity. Cyt2Ba was active upon solubilization, but only very weakly. In contrast, solubilization did not result in Cyt2Bc activity. Only trypsin treatment revealed the activity of this toxin. None of the Cyt2B toxins was active after proteinase K processing. Al-yahyaee and Ellar (1) demonstrated that trypsin processing at the N terminus of Cyt1Aa occurred after residue Arg-25. This residue is present in both Cyt2B toxins. They also found that the potential cleavage site for proteinase K was located just after residue Arg-30, which is absent from both Cyt2B proteins. This may account for the lack of hemolytic activity of proteinase K-treated Cyt2B proteins. Cyt2Bc is the least active toxin against susceptible mosquitoes but has activity similar to that of Cyt2Ba on resistant larvae. It has already been reported elsewhere that a few amino acids are responsible for a considerable change in toxicity of Cyt1Aa (17). Mutagenesis analysis could be used to identify the amino acids responsible for the lower toxicity of Cyt2Bc. Such experiments should be performed to construct new Cyt2B variants with greater toxicity. ACKNOWLEDGMENTS We thank Sylviane Hamon for help with mosquito bioassays and Vidalia Patricio for mosquito rearing. We also thank Daniel Zeigler for the phylogenetic protein comparison.
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