Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rang, C. Articles by Frutos, R. Search for Related Content PubMed PubMed Citation Articles by Rang, C. Articles by Frutos, R. Agricola Articles by Rang, C. Articles by Frutos, R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2005, p. 6276-6281, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6276-6281.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Vip3-Related Protein from Bacillus thuringiensis

Cécile Rang,^1, Patricia Gil,¹ Nathalie Neisner,¹ Jeroen Van Rie,¹ and Roger Frutos^2*

Bayer BioScience N.V., Technologiepark 38, 9052 Gent, Belgium,¹ CIRAD, TA 30/D, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France²

Received 11 February 2005/ Accepted 18 May 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
A novel vip3-related gene was identified in Bacillus thuringiensis. This novel gene is 2,406 bp long and codes for a 91-kDa protein (801 amino acids). This novel protein exhibits between 61 and 62% similarity with Vip3A proteins and is designated Vip3Ba1. Vip3Ba1 has several specific features. Differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but are more frequent in the C-terminal part from amino acid 456 onward. The regions containing the two proteolytic processing sites, which are highly conserved among the Vip3A toxins, are markedly different in Vip3Ba1. The pattern DCCEE (Asp Cys Cys Glu Glu) is repeated four times between position 463 and 483 in Vip3Ba1, generating the sequence 463-DCCEEDCCEEDCCEEDCCEE-483. This sequence, which is rich in negatively charged amino acids, also contains 73% of the cysteines present in Vip3Ba1. This repeated sequence is not present in Vip3A proteins. The Vip3Ba1protein was produced in Escherichia coli and tested against Ostrinia nubilalis and Plutella xylostella, and it generated significant growth delays but had no larvicidal effect, indicating that its host range might be different than that of Vip3A proteins.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacillus thuringiensis is the most extensively used biopesticide worldwide. In addition, this bacterium is currently the sole source of toxin genes for the development of insect-resistant transgenic plants (4, 8, 26). In large part, the insecticidal activity of B. thuringiensis is associated with its ability to synthesize a crystalline parasporal inclusion body containing highly specific insecticidal proteins, referred to as Cry and Cyt proteins (21). The mode of action of the Cry proteins involves solubilization of the crystal, processing of the protoxins by intestinal proteases, and recognition of a binding site on the midgut brush border membrane surface, followed by pore formation and cell lysis, leading ultimately to insect death (16, 21, 25).

Other kinds of insecticidal proteins have been described in B. thuringiensis and Bacillus cereus, among which are the vegetative insecticidal proteins or Vip proteins (1, 6, 7, 22, 27, 28). The Vip proteins are secreted during vegetative growth and do not exhibit any similarity with the Cry or Cyt toxins. Currently, all Vip-related sequences that have been described fall into three different families, Vip1, Vip2, and Vip3 (3). A classification of these proteins into three classes, seven subclasses, and further subdivisions was recently proposed by the Bacillus thuringiensis nomenclature committee (3). The Vip1 and Vip2 proteins are the two components of a binary toxin that exhibits toxicity to coleopterans. Vip1Aa1 and Vip2Aa1 are very active against corn rootworms, particularly Diabrotica virgifera virgifera and Diabrotica longicornis (10, 27). Vip3 proteins have a different host range, which includes several major lepidopteran pests (1, 6, 7, 22, 28). Like Cry toxins, Vip3A proteins must be activated by proteases prior to recognition at the surface of the midgut epithelium of specific 80-kDa and 100-kDa membrane proteins different from those recognized by Cry toxins (15). Apoptosis was initially suggested as a mode of action, but it was recently shown that like Cry toxins, activated Vip3A toxins are pore-forming proteins capable of making stable ion channels in the membrane (15). The possibility of different modes of action of Vip proteins against different pests has also been suggested (22).

Owing to the difference in the mode of action compared with Cry toxins, Vip proteins are good candidates for resistance management strategies involving stacking or rotation of proteins with different insecticidal mechanisms. Stable expression of vip genes in plants has been achieved, and transgenic maize lines expressing a plant-optimized vip3Aa1 gene and exhibiting toxicity to several major lepidopteran pests have been developed (6, 9). A transgenic cotton variety (Cot102) expressing a vip3A gene has also been created (5).

The search for novel vip sequences might prove to be useful for increasing the target range of future insecticidal transgenic plants while allowing resistance management by associating vip genes with the functionally different cry genes. Along with this approach, we describe here cloning, sequencing, and expression of a novel vip gene encoding a protein having very specific features, and we suggest the designation vip3Ba1 for this gene.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and plasmids.
B. thuringiensis strain ucr8 was isolated from soil samples and grown in usual medium [2% Bacto peptone, 50 mM K₂HPO₄, 0.5 mM MgSO₄, 0.01 mM MnSO₄, 0.05 mM ZnSO₄, 0.05 mM Fe₂(SO₄)₃, 1 mM CaCl₂, pH 7.4] supplemented with 1% glucose as previously described (11). Escherichia coli DH5 and BL21-Lambda DE3 were used as host strains for transformation. pGEM-T Easy (Promega) and pCR2.1TOPO were used for cloning PCR products, and pBluescript KS(+) and pBC KS(+) (Stratagene) were used as general cloning vectors. The pET11a expression vector was used for expression in E. coli.

Molecular techniques.
Standard recombinant DNA techniques were used, as described by Sambrook et al. (20). For PCR screening, total DNA was extracted as previously described (12), and each PCR was performed with a 50-µl (final volume) mixture containing 500 ng of total DNA, 2 µl of a 100 mM deoxynucleoside triphosphate (dNTP) mixture, 2 µl of a 100 mM MgCl₂ solution, 0.5 U of Taq DNA polymerase, 20 pmol of the Vip+ sense primer (5'-GCACAAGAGCCTTACCAAGT-3'), and 20 pmol of the the Vip– antisense primer (5'-TTAGCTTAGCCTCATAT-3'). The primers were designed to amplify conserved regions in vip3A genes in order to amplify all vip3A genes. Further characterization was done by differential physical mapping of cloned PCR products. The PCR conditions were as follows: denaturation for 4 min at 95°C, 30 cycles of denaturation for 1 min at 94°C, annealing for 45 s at 45°C, and extension for 90 s at 72°C, and a final extension at 72°C for 10 min. PCR amplification with the Vip– and Vip+ primers yielded a 1,113-bp PCR product. Two DNA libraries were prepared from total DNA digested by XbaI and HincII. Digestion was performed as recommended by the supplier, and the digested DNA was fractionated on a 10% to 40% sucrose gradient as previously described (29). Standard procedures were used for agarose gel electrophoresis, Southern blotting, probe labeling (by random priming), hybridization, cloning, and colony transfer (20). DNA sequences were obtained using the standard chain termination technique with an Applied Biosystems 370A nucleotide sequence analyzer from plasmid DNA extracted with the Wizard Plus SV Minipreps DNA purification system.

Cloning of vip3Ba1 into pET11a.
For insertion into the pET11a expression vector, the full-length vip3Ba1 gene was obtained by PCR amplification from the native ucr8 strain with the sense primer Vip3B+ (5'-GCATATGAACATGAATAATACTAA-3') and the antisense primer Vip3B– (5'-TCAGATTTTTGTTAAAATAT-3'). The sequence GCAT was added immediately upstream from the native ATG of the vip3Ba1 gene in order to create the NdeI CATATG site in the 5' end of the Vip3B+ sense primer. The PCR was performed with a 50-µl (final volume) mixture containing 500 ng of total DNA, 20 pmol of each primer, 1.5 µl of a 10 mM dNTP mixture, 1 µl of a 50 mM MgSO₄ solution, and 1.25 U of Taq polymerase Pfx Platinum (Gibco). After initial denaturation at 94°C for 2 min, 25 amplification cycles of denaturation for 15 s at 94°C, annealing for 30 s at 42°C, and elongation for 150 s at 68°C were performed. After completion of the 25 cycles, 1 µl of a 10 mM dNTP mixture and 5 U of Taq DNA polymerase (Invitrogen) were added, and an additional extension step consisting of 10 min at 72°C was performed. The 2.4-kb PCR product was cloned into the pCR2.1TOPO vector using standard procedures. A clone was selected and, after full-length sequencing of the insert to ensure that no mismatch was present in the insert, was designated pTOPO207. An internal NdeI site was present in the vip3Ba1 gene, and therefore the GCA codon at position 1744 coding for alanine 581 was modified by site-directed mutagenesis to the codon GCT, also coding for alanine, to eliminate the NdeI site without changing the amino acid. No mismatch was present, and the construct was designated pTOPO207b. A 2.4-kb NdeI-BamHI fragment from pTOPOb was inserted into the pET11a vector by using standard procedures (20) to obtain plasmid pET209, which contained the full-length vip3Ba1 gene under the control of the T7 promoter. pET209 was mapped with restriction enzymes and transformed into BL21-Lambda DE3 to obtain E. coli BL21(pET209).

Computer analysis of nucleic and protein sequences.
Contigs, a consensus sequence, and similarity matrices were generated using the Vector NTI package (Informax). Alignments of DNA and protein sequences were obtained using Clustal X (24). Trees were constructed from Clustal X data using Treeview (18).

Expression and production of proteins.
Strain E. coli BL21(pETR209) and control strain E. coli BL21(pET11) were grown overnight at 37°C and 200 rpm on LB medium containing 100 µg/ml of ampicillin. A 1/100 dilution of this culture in 20 ml of LB medium containing ampicillin (100 µg/ml) was incubated at 37°C until the optical density at 600 nm was 0.6. Expression was then induced by addition of 1 mM (final concentration) IPTG, and incubation was continued for 2.5 h at three temperatures, 37°C, 30°C, and 25°C, in the same medium with shaking (200 rpm). Samples were centrifuged at 10,000 rpm for 10 min at 4°C. The pellets were resuspended in 0.1 volume of 50 mM Tris-HCl (pH 8), 2 mM EDTA, 100 µg/ml lysozyme and incubated at 30°C for 15 min prior to sonication in ice (two 1-min pulses at 100 W, separated by a 30-s incubation on ice) and centrifugation at 10,000 rpm for 10 min at 4°C. The protein concentration was estimated by using the method of Bradford (2). The supernatant was collected and stored at –80°C.

Analysis of proteins by SDS-PAGE.
Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (13). Ten micrograms of total soluble proteins and pelleted proteins was separated on a 10% polyacrylamide gel and stained with Coomassie brilliant blue R-250. Broad-range molecular weight standards from Biolabs were used as standards. A colony of E. coli BL21(DE3) bearing only the pET11a plasmid was used as a control.

Insect bioassays.
Bioassays were conducted with neonates of the European corn borer (Ostrinia nubilalis) and second-instar larvae of the diamondback moth (Plutella xylostella). Bioassays with O. nubilalis were conducted in Multiwell-48 plates (Corning Costar Corp.) by using the Vanderzant diet, as previously described (14). Twenty-five microliters of culture supernatant was layered on top of the diet, and the excess water was removed by evaporation for 1 h in a laminar flow hood. Bioassays were conducted with soluble proteins at 25°C, and the effects were observed after 7 days. The following two control experiments were performed: (i) diet with no treatment and (ii) diet treated with 25 µl of culture supernatant of a nontransformed strain. Eighteen larvae were used for each experiment, with five repeats. Bioassays with P. xylostella were performed by using the method of Tabashnik et al. (23), as previously described (17). The bioassays were performed five times with 50 larvae each time.

Nucleotide sequence accession number.
The sequence of the gene encoding the Vip3Ba1 protein has been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) under accession number AY823271.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of a putative variant of Vip3A.
Screening of a collection of Bacillus strains with primers Vip+ and Vip– revealed the presence of a 1-kb PCR product for strain ucr8, which was cloned in pGEM-T Easy for sequencing. Seven clones were analyzed by digestion with EcoRI, and two patterns were observed. Six of the clones, 8V1 to 8V6, had two EcoRI sites that generated 200-bp and 900-bp fragments, whereas clone 8VA had only one EcoRI site (data not shown). The sequence of the insert present in clones 8V1 to 8V6 was identical to that of the vip3Aa1 gene. The sequence of the PCR fragment present in clone 8VA, designated fragment Vip3Ba1, was different from that of vip3Aa1 and other vip genes. Alignment of the predicted expression product of fragment Vip3Ba1 with the various Vip3A proteins showed that the sequence of fragment Vip3Ba1 from those of the other Vip3A proteins (data not shown).

Cloning and sequencing of the vip3Ba1 gene.
The full-length vip3Ba1 gene was cloned and reconstructed from two different DNA libraries. An XbaI library generated a clone, pUCR200, carrying a ca. 4.3-kb insert which hybridized with fragment Vip3Ba1 used as a probe. The sequence of clone pUCR200 showed that this clone contained the first 1,374 bases of the vip3Ba1 gene and 2,905 bases of the 5' flanking region. An additional HincII library was constructed, from which clone pUCR201, which hybridized with a 0.5-kb EcoRV fragment from pUCR200, was isolated and sequenced. pUCR201 carried a ca. 3.8-kb insert containing the last 1,239 bases of vip3Ba1 between the HincII site and the stop codon, which included a 220-bp HincII/XbaI fragment common to pUCR200 and pUCR201. In addition, pUCR201 carried 2,592 bp of the 3' flanking region of the vip3Ba1 gene. The full-length vip3Ba1 gene was 2,406 bp long and coded for an 801-amino-acid Vip3Ba1 protein.

Comparison of the Vip3Ba1 toxin with other Vip proteins.
The predicted sequence of the Vip3Ba1 protein was compared to the sequences of other Vip proteins (3), including Vip1Aa1, Vip1Aa2, Vip1Ab1, Vip1Ba1, Vip1Ba2, Vip1Bb1, Vip1Ca1, Vip1Da1, Vip2Aa1, Vip2Aa2, Vip2Ab1, Vip2Ac1, Vip2Ad1, Vip2Ba1, Vip2Ba2, Vip2Bb1, Vip3Aa1, Vip3Aa2, Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vip3Aa9, Vip3Aa10, Vip3Aa11, Vip3Aa12, VipAa13, Vip3Aa14, Vip3Aa15, Vip3Aa16, Vip3Aa17, Vip3Ab1, Vip3Ac1, Vip3Ad1, Vip3Ad2, Vip3Ae1, Vip3Af1, and Vip3Ag1. The vip3Aa8 gene described in the GenBank database (accession number AAK97481) is missing a stop codon and might not be a full-length gene. Therefore, Vip3Aa8 was not considered in the alignment. The dendrogram in Fig. 1 shows that there are three separate clusters associated with high bootstrap values (982 to 1,000) and that these clusters comprise the Vip1, Vip2, and Vip3 proteins. Vip3Ba1 branches separately from all other groups. This separate position of Vip3Ba1 is associated with the maximum bootstrap value, 1,000 (Fig. 1). The Vip2 proteins exhibit 21% to 22% similarity with Vip3Ba1, whereas the Vip1 toxins exhibit levels of similarity ranging from 8% to 22% (see Table S1 in the supplemental material). As expected, the Vip3 proteins are more closely related to Vip3Ba1 and exhibit 61% to 62% similarity with Vip3Ba1 (see Table S2 in the supplemental material). When a novel protein exhibits 45% to 78% similarity with the most closely related Vip protein, the primary rank (i.e., the Arabic first number) is conserved, but the secondary rank (uppercase letter) is changed to the next letter in alphabetical order. The proposed designation of this novel protein is therefore Vip3Ba1. This designation was validated by the B. thuringiensis nomenclature committee (3).

View larger version (12K): [in this window] [in a new window]

FIG. 1. Dendrogram showing the relationship of Vip3Ba1 to other Vip proteins. Bootstrap values associated which the clustering of Vip1 toxins, Vip2 toxins, Vip3A toxins, and Vip3Ba1 are indicated at the nodes.

To determine the extent of the sequence differences, Vip3Ba1 was aligned with Vip3Aa1, Vip3Ab1, Vip3Ab1, Vip3Ac1, Vip3Ad1, Vip3Ae1, Vip3Af1, and Vip3Ag1, which represented all subclasses of the Vip3 proteins (Fig. 2.). The differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but the rate is higher between amino acid 456 and the C terminus of the protein (Fig. 2). The N terminus of the putative signal sequence of Vip3Ba1 is, with the exception of the first two amino acids, identical to that of all Vip3 toxins except Vip3Aa1. The Vip3Ba1 sequence differs from the highly conserved putative signal sequence of Vip3Aa proteins by replacement of the sequence MNKNNTKLST by the sequence MNNTKLNA (Fig. 2). Although different, this sequence has the high asparagine content that characterizes the N-terminal end of signal sequences in Bacillus (6, 19). The last 23 amino acids of the signal sequence are identical in all Vip3 sequences (Fig. 2). The complete putative signal sequence of Vip3Ba1 is therefore MNNTKLNARALPSFIDYFNGIYGFATGIKDI.

View larger version (108K): [in this window] [in a new window]

FIG. 2. Alignment of the Vip3Ba1 sequence with the sequence of a representative of each subclass of Vip3A toxins. Asterisks above the sequences indicate divergent amino acids. Extra amino acids in insertion/deletion regions are indicated by boldface type. The gray boxes indicate regions described in the text. The gray box labeled S indicates the signal peptide sequence. The gray boxes labeled P indicate the proteolytic processing sites characterized in Vip3Aa1 (6, 7). The gray box labeled I indicates the beginning of the 66-kDa insecticidal fragment characterized in Vip3Aa1 (6, 7).

Vip3Aa proteins are processed at two lysine-rich sites (6, 7). The first processing site is located in Vip3Aa1 at lysine 198 in the sequence 192-SSKVKK-199 (Fig. 2). This sequence in the other Vip3A proteins is either 192-TLKVKK-199 or 192-TLKVKE-199. This region is significantly different in Vip3Ba1, in which it is replaced by the sequence 192-NPKINQ-199 (Fig. 2). The 66-kDa insecticidal moiety of Vip3Aa starts at glycine 200 and contains the sequence 199-GSPADIL-207 (6). The corresponding region in the other Vip3A proteins is 199-SSPADIL-207 (Fig. 2). This region is not conserved in Vip3Ba1, in which the sequence 198-NFTEDVI-206 is found instead (Fig. 2). This sequence, which is highly conserved among Vip3A toxins, is not present in Vip3Ba1. The second processing site in Vip3A is located at amino acid 455 and results in the release of a 33-kDa fragment ranging from residue 200 to residue 455 (6). In Vip3A proteins the sequence of this second processing site is either DLNKKKVESS or DLNKTKVESS (Fig. 2). Vip3Ba1 also differs from the Vip3A proteins at this level, although the difference is less, and contains the sequence DLNKTILESW, in which only the first lysine is present (Fig. 2).

Another major difference between Vip3Ba1 and the Vip3A toxins is the pattern DCCEE (Asp Cys Cys Glu Glu), which is repeated four times between position 463 and position 483 to generate the sequence 463-DCCEEDCCEEDCCEEDCCEE-483 (Fig. 2). These repeats are located immediately downstream from the region corresponding to the second processing site of Vip3A toxins (Fig. 2). The two main characteristics of this repeated sequence, which is absent in all Vip1, Vip2, and Vip3A proteins, are high contents of negatively charged amino acids (D and E) and cysteines. Of the 11 cysteines found in Vip3Ba1, 8 (73%) are present in the repeated sequence. The region located downstream from this repeated sequence, a region known to be involved in resistance to proteases in Vip3Aa1 (6, 7), is also significantly different in Vip3Ba1 and the Vip3A proteins.

Expression and production of recombinant Vip3Ba1.
Owing to the potential presence of a vip3Aa1 gene in strain ucr8, expression of Vip3Ba1 was analyzed in the recombinant strain E. coli BL21(pET209) rather than in the native B. thuringiensis strain. After induction, strain E. coli BL21(pET209) produced a major protein of the expected size, ca. 91 kDa (Fig. 3).

View larger version (118K): [in this window] [in a new window]

FIG. 3. SDS-PAGE analysis of the production of Vip3Ba1 by a recombinant E. coli strain. Lanes 1, 2, 3, 5, and 6 contained bacterial cells bearing the vip3Ba1 gene. Lane 1, whole cells; lane 2, whole cells, 3 h of induction; lane 3, whole cells, 3 h of induction; lane 4, control bacterial strain bearing the expression pET11a vector; lane 5, supernatant after sonication, 24 h of induction; lane 6, pellet after sonication, 24 h of induction; lane MW, molecular mass marker (200, 116, 97, 66, and 45 kDa). The arrowhead indicates the position of the ca. 91-kDa vip3Ba1 expression product.

Assessment of the insecticidal activity of Vip3Ba1.
The soluble protein extract from the recombinant E. coli BL21(pET209) strain had an effect on both O. nubilalis and P. xylostella. No mortality was recorded, but larval growth was impaired in both species. O. nubilalis larvae exposed to the soluble protein extract from E. coli BL21(pET209) remained at the first-instar stage for 7 days. The total weight of all the larvae was 0.0055 g after 7 days. During the same time neonate larvae exposed to the soluble protein extract from the control E. coli BL21(pET11) strain reached the third-instar stage, and the total weight was 0.1124 g. Similarly, second-instar larvae of P. xylostella exposed to the soluble protein extract from strain E. coli BL21(pET209) did not grow and remained at the second-instar stage, whereas larvae exposed to the soluble protein extract from strain E. coli BL21(pET11) reached the third-instar stage after 4 days.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here we report the presence in B. thuringiensis of a novel type of vip3-related gene encoding a protein that is significantly different from the previously known Vip3 proteins. All Vip proteins that have been described in the literature or in databases fall into three families, Vip1, Vip2, and Vip3 (3). The novel protein described here exhibits at most 62% amino acid sequence similarity with the Vip3A proteins. Based on recent recommendations for the nomenclature of Vip proteins (3), this protein is designated Vip3Ba1.

Vip3Ba1 has several specific sequence features. First, it does not contain the two highly conserved, lysine-rich processing sites (6, 7) which are present in Vip3A proteins. Another specific feature is the presence of the four repeats of the DCCEE sequence, which is not present in any other Vip protein. Sixty percent of the 20 amino acids of this repeated sequence are either aspartic acid (D) or glutamic acid (E), making the sequence a highly negatively charged region. This repeat sequence contains 8 the 11 cysteine residues (73%) in the protein. It is likely that some of these cysteines are involved in disulfide bridges and may play a role in the stability of Vip3Ba1. The C-terminal end of Vip3Ba1 is highly divergent from that of the Vip3A toxins. This region is conserved among Vip3 proteins and has been reported to be crucial for resistance to proteases (6, 7). It is not known whether the C-terminal end of Vip3Ba1 has a similar role.

Here we describe the discovery and partial characterization of a novel Vip protein, the Vip3Ba1 protein. Further studies are needed to determine its host range and assess whether the differences in the amino acid sequence translate into toxicity to insects different than the toxicity exhibited by Vip3A proteins. Unlike Vip3A, which is insecticidal to P. xylostella (1, 22), Vip3Ba1 resulted in only a growth delay. Deletion analyses of both the N terminus and the C terminus might provide more detailed information about the insecticidal moiety of the protein, as they have for Vip3A proteins (22). Since Vip3Ba1 is closely related to Vip3A proteins, it most likely acts as a single toxin. However, due to the failure to demonstrate lethal activity, the involvement of a partner protein cannot be formally excluded. Further investigation is therefore needed to unravel the mode of action of Vip3Ba1 and to understand the role of the specific features observed.

 
This work was jointly supported by Cirad and Bayer BioScience N.V.

 
* Corresponding author. Mailing address: Cirad, TA 30/D, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France. Phone: (33) 4 67 59 39 62. Fax: (33) 4 67 59 39 60. E-mail: roger.frutos{at}cirad.fr.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: UR 1199, Laboratoire de Protéomique, INRA, 2 Place Viala, 34060 Montpellier Cedex 1, France.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Bhalla, R., M. Dalal, S. K. Panguluri, B. Jagadish, A. D. Mandaokar, A. K. Singh, and P. A. Kumar. 2005. Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis FEMS Microbiol. Lett. 243:467-472.[CrossRef][Medline]
2
2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
3
3. Crickmore, N., D. R. Zeigler, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, A. Bravo, and D. H. Dean. 2005. Bacillus thuringiensis toxin nomenclature. [Online.] http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/.
4
4. de Maagd, R. A., D. Bosch, and W. Stiekema. 1999. Bacillus thuringiensis toxin-mediated insect resistance in plants. Trends Plant Sci. 4:9-13.[CrossRef][Medline]
5
5. Environmental Protection Agency. 2003. Bacillus thuringiensis Vip3A insect control protein as expressed in event COT102: notice of filing a pesticide petition to establish an exemption from the requirement of a tolerance for a certain pesticide chemical in or on food. Fed. Regist. 68:66422-66425.
6
6. Estruch, J. J., M. G. Koziel, C. G. Yu, N. M. Desai, G. J. Nye, and G. W. Warren. 1998. Plant pest control. Patent WO 9844137.
7
7. 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]
8
8. Frutos, R., C. Rang, and M. Royer. 1999. Managing insect resistance to plants producing Bacillus thuringiensis toxins. Crit. Rev. Biotechnol. 19:227-276.[CrossRef]
9
9. Gay, P. B. 1997. Method of protecting crop plants against insect pests. Patent WO9726339.
10
10. Han, S., J. A. Craig, C. D. Putnam, N. B. Carozzi, and J. A. Tainer. 1999. Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex. Nat. Struct. Biol. 6:932-936.[CrossRef][Medline]
11
11. Itoua-Apoyolo, C., L. Drif, J. M. Vassal, H. De Barjac, J. P. Bossy, F. Leclant, and R. Frutos. 1995. Isolation of multiple subspecies of Bacillus thuringiensis from a population of the European sunflower moth, Homoeosoma nebulella. Appl. Environ. Microbiol. 61:4343-4347.[Abstract]
12
12. Juarez-Perez, V. M., M. D. Ferrandis, and R. Frutos. 1997. PCR-based approach for detection of novel Bacillus thuringiensis cry genes. Appl. Environ. Microbiol. 63:2997-3002.[Abstract]
13
13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
14
14. Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. Van Audenhove, J. Van Rie, A. Van Vliet, and M. Peferoen. 1996. A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae. Appl. Environ. Microbiol. 62:80-86.[Abstract]
15
15. Lee, M. K., F. S. Walters, H. Hart, N. Palekar, and J. S. Chen. 2003. The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab -endotoxin. Appl. Environ. Microbiol. 69:4648-4657.[Abstract/Free Full Text]
16
16. Masson, L., B. E. Tabashnik, Y. B. Liu, R. Brousseau, and J. L. Schwartz. 1999. Helix 4 of the Bacillus thuringiensis Cry1Aa toxin lines the lumen of the ion channel. J. Biol. Chem. 274:31996-32000.[Abstract/Free Full Text]
17
17. Monnerat, R., L. Masson, R. Brousseau, M. Puzstai-Carey, D. Bordat, and R. Frutos. 1999. Differential activity and activation of Bacillus thuringiensis insecticidal proteins in diamondback moth, Plutella xylostella. Curr. Microbiol. 39:159-162.[CrossRef][Medline]
18
18. Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358.[Free Full Text]
19
19. Pugsley, A. P. 1993. The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57:50-108.[Abstract/Free Full Text]
20
20. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
21
21. 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]
22
22. Selvapandiyan, A., N. Arora, R. Rajagopal, S. K. Jalali, T. Venkatesan, S. P. Singh, and R. Bhatnagar. 2001. Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis. Appl. Environ Microbiol. 67:5855-5858.[Abstract/Free Full Text]
23
23. Tabashnik, B. E., N. L. Cushing, N. Finson, and M. W. Johnson. 1990. Field-develoment of resistance to Bacillus thuringiensis in diamond-back moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 83:1671-1676.
24
24. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882.
25
25. Vachon, V., G. Préfontaine, F. Coux, C. Rang, L. Marceau, L. Masson, R. Brousseau, R. Frutos, J. L. Schwartz, and R. Laprade. 2002. Role of helix three in pore formation by the Bacillus thuringiensis insecticidal toxin Cry1Aa. Biochemistry 41:6178-6184.[CrossRef][Medline]
26
26. Van Rie, J. 2000. Bacillus thuringiensis and its use in transgenic insect control technologies. Int. J. Med. Microbiol. 290:463-469.[Medline]
27
27. Warren, G. W. 1997. Vegetative insecticidal proteins: novel proteins for control of corn pests, p. 109-121. In N. Carozzi and M. Koziel (ed.), Advances in insect control: the role of transgenic plants. Taylor and Francis, London, United Kingdom.
28
28. Yu, C. G., M. A. Mullins, G. W. Warren, M. G. Koziel, and J. J. Estruch. 1997. The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects. Appl. Environ Microbiol. 63:532-536.[Abstract]
29
29. Yuan, Z., C. Rang, R. C. Maroun, V. Juarez-Perez, R. Frutos, N. Pasteur, C. Vendrely, J. F. Charles, and C. Nielsen-Leroux. 2001. Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin. Eur. J. Biochem. 268:2751-2760.[Medline]

---

Applied and Environmental Microbiology, October 2005, p. 6276-6281, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6276-6281.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Fang, J., Xu, X., Wang, P., Zhao, J.-Z., Shelton, A. M., Cheng, J., Feng, M.-G., Shen, Z. (2007). Characterization of Chimeric Bacillus thuringiensis Vip3 Toxins. Appl. Environ. Microbiol. 73: 956-961 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rang, C. Articles by Frutos, R. Search for Related Content PubMed PubMed Citation Articles by Rang, C. Articles by Frutos, R. Agricola Articles by Rang, C. Articles by Frutos, R.