Characterization of Chimeric Bacillus thuringiensis Vip3 Toxins

Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.     

Isolation,cloning, and overexpression of vip3Aa gene isolated from a local Bacillus thuringiensis

Vegetative insecticidal protein (Vip) is a newly discovered family of toxin protein isolated from Bacillus thuringiensis (Bt). An 88.5-kDa Vip3Aa protein was secreted by a local strain of the bacterium during the vegetative growth phase. The full length of the coding region ‘2.3 kbp’ of the vip3Aa gene was isolated from plasmid DNA, cloned in pGEM-T vector and finally cloned in pQE-30 expression vector. Nucleotide sequence revealed 98% homology with that of the previously isolated genes. Expression of the vip3Aa in Escherichia coli was carried out and the expressed protein was detected in the concentrated supernatant, not in the pellet. This indicated that vip3Aa is secreted into the culture medium. Expressed protein was purified, blotted, and assayed against the cotton leaf worm Spodoptera littoralis. The LC₅₀ was found to be 142.4 µ/mL while the LC₅₀ was 90 ppm for the wild strain. These results suggest the use of either the isolated Bt strains or the expressed vip3Aa in an integrated pest management program against lepidopteran insect pests.     

<Emphasis Type="Italic">Prays oleae</Emphasis> Midgut Putative Receptor of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Vegetative Insecticidal Protein Vip3LB Differs from that of Cry1Ac Toxin

Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins.     

Heterologous Expression of Bacillus thuringiensis Vegetative Insecticidal Protein-Encoding Gene vip3LB in Photorhabdus temperata Strain K122 and Oral Toxicity against the Lepidoptera Ephestia kuehniella and Spodoptera littoralis

Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB is a Bacillus thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we focused for the first time on the heterologous expression of vip3LB gene in Photorhabdus temperata strain K122. Firstly, Western blot analyses of whole cultures of recombinant Photorhabdus temperata showed that Vip3LB was produced and appeared lightly proteolysed. Cellular fractionation and proteinase K proteolysis showed that in vitro-cultured recombinant Photorhabdus temperata K122 accumulated Vip3LB in the cell and appeared not to secrete this protein. Oral toxicity of whole cultures of recombinant Photorhabdus temperata K122 strains was assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Unlike the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth. These results demonstrate that the heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata could be considered as an excellent tool for improving Photorhabdus insecticidal activities.     

Characterization of Bacillus thuringiensis isolates from soil and small mammals that harbour vip3A gene homologues

The insecticidal and psychrotropic potential of 132 new isolates of Bacillus thuringiensis from northeastern Poland (74 from animals and 58 from soil) were determined by screening these for vip and cry genes encoding, respectively, vegetative insecticidal proteins (Vip) and Cry proteins, and cspA that encoded the CspA cold shock protein that confers psychrotropy in Bacillus species. The vip3A gene, encoding Vip3A toxic to lepidopterans, was found in ~5% of the isolates from animals and ~17% the isolates from soil, whereas coleopteran-specific vip1 and vip2 genes were present in 8% of the isolates from soil. Nucleotide sequences of vip3A-specific amplicons were highly conserved, with only a few containing minor differences from vip3A. Despite the high level of vip3A conservation, isolates harbouring the gene demonstrated a high level of heterogeneity based on whole-cell genomic DNA RFLP analysis with pulsed-field gel electrophoresis (PFGE) and plasmid profiling. Eight isolates positive for vip3A contained cry1 and six also harboured the cry2 gene, which encodes an endotoxin toxic to lepidopteran insects. However, none of these isolates contained cry genes coding for proteins toxic to coleopteran or dipteran insects. Due to the known potential for synergistic interactions between Vip and Cry proteins, the isolates positive for vip3A and cry genes may be used in resistance management strategies directed against lepidopteran larvae. Finally, all of the B. thuringiensis vip3A-positive isolates harboured the cspA gene, but only two were confirmed to be psychrotrophs.     

苏云金芽孢杆菌vip3A基因的检测及保守性分析

Vip3A蛋白是苏云金芽孢杆菌(Bacillus thuringiensis,Bt)在营养期分泌的一类新型杀虫蛋白。用PCR方法从114个Bl菌株和41个Bl标准菌株中筛选到39株即约25％的菌株含有vip3A基因。利用所制备的Vip3A蛋白的多克隆抗体对以上含有vip3A基因的Bt菌株进行Western印迹分析,发现多数PCR反应为阳性的菌株都产生89kD大小的蛋白,其中有4株没有Vip3A蛋白的表达。从以上菌株中挑选2个对夜蛾科害虫具有较高和较低毒力的菌株,即S101和6ll,并分别进行vip3A基因的克隆和测序,再与GenBank上所登录的其它6个全长vip3A基因和2个已报道的但未登录GenBank的vip3A基因进行核苷酸和氨基酸序列比较,结果表明,vip3A是一个极其保守的基因。将以上所克隆的2个却3A基因即vip3A—S101和vip3A-611分别插入表达载体pQE30构建了表达质粒pOTP-S101和pOTP-6ll,转化到大肠杆菌M15,经lmmol／L IPTG诱导后均表达89kD大小的Vip3A蛋白。蛋白可溶性试验表明,Vip3A-S101和Vip3A-611分别有48％和35％的蛋白是可溶的。将Vip3A-S101和Vip3A-6ll蛋白和已报道的Vip3A—S184蛋白对初孵斜纹夜蛾(Spodoptera litura)幼虫进行生物测定,结果表明,3个Vip3A蛋白对斜纹夜蛾幼虫毒力没有显著性差异,这说明了Vip3A个别氨基酸的变化对蛋白的杀虫活性没有影响。     

Novel Vip3-Related Protein from Bacillus thuringiensis

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.     

Molecular characterization of a novel vegetative insecticidal protein from Bacillus thuringiensis effective against sap-sucking insect pest

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on LC(50) values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an ~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.     

Increase of the Bacillus thuringiensis Secreted Toxicity Against Lepidopteron Larvae by Homologous Expression of the vip3LB Gene During Sporulation Stage

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against several agricultural insect pests. The fact that the expression of vip3 genes occurs only during the vegetative growth phase of Bacillus thuringiensis is a limiting factor in term of production level. Therefore, extending the synthesis of the Vip proteins to the sporulation phase is a good alternative to reach high levels of toxin synthesis. In this study, we have demonstrated that the maximal production of the secreted Vip3LB (also called Vip3Aa16) during the growth of the wild-type strain B. thuringiensis BUPM 95 is reached at the end of the vegetative growth phase, and that the protein remains relatively stable in the culture supernatant during the late sporulation stages. The vip3LB gene was cloned and expressed under the control of the sporulation dependant promoters BtI and BtII in B. thuringiensis BUPM 106 (Vip3(-)) and BUPM 95 (Vip3(+)) strains. The examination of the culture supernatants during the sporulation phase evidenced the synthesis of Vip3LB and its toxicity against the second-instars larvae of the Lepidopteron insect Spodoptera littoralis for the recombinant BUPM 106. Moreover, there was an increase of the Vip3LB synthesis level and an enhancement of the oral toxicity for the recombinant BUPM 95 resulting from the expression of the vip3LB gene during both the vegetative and sporulation phases and the relative stability of the Vip3LB protein.     

Effects of Mycobacterium bovis on monocyte-derived macrophages from bovine tuberculosis infection and healthy cattle

A PCR-restriction fragment length polymorphism (PCR-RFLP) method for identifying vegetative insecticidal protein (vip) 1-type genes from Bacillus cereus was developed by designing specific primers based on the conserved regions of the genes to amplify vip1-type gene fragments. PCR products were digested with endonuclease AciI, and four known vip1-type genes were identified. Vip1Ac and vip1Aa-type genes appeared in 17 of 26 B. cereus strains. A novel vip1-type gene, vip1Ac1, was identified from B. cereus strain HL12. The vip1Ac1 and vip2Ae3 genes were co-expressed in Escherichia coli strain BL21 by vector pCOLADuet-1. The binary toxin showed activity only against Aphis gossypii (Homoptera), but not for Coleptera (Tenebrio molitor, Holotrichia oblita), Lepidoptera (Spodoptera exigua, Helicoverpa armigera, and Chilo suppressalis), Diptera (Culex quinquefasciatus). The LC(50) of this binary toxin for A. gossypii is 87.5 (34.2-145.3) ng mL(-1) . This is probably only the second report that Vip1 and Vip2 binary toxin shows toxicity against homopteran pests. The PCR-RFLP method developed could be very useful for identifying novel Vip1-Vip2-type binary toxins, and the novel binary toxins, Vip1Ac1 and Vip2Ae3, identified in this study may have applications in biological control of insects, thus avoiding potential problems of resistance.     

`Cloning of <Emphasis Type="Italic">vip1/vip2</Emphasis> genes and expression of Vip1Ca/Vip2Ac proteins in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Forty-one Bacillus thuringiensis (Bt) standard reference strains and 118 Bt local isolates were screened for vip1/vip2 genes by PCR amplification, with only three strains (HD201, HD109 and HD12) producing the desired bands. Southern blot showed that vip1/vip2 genes were located on a 10 Kb EcoRV fragment of their total DNAs. Furthermore, the vip1Ca/vip2Ac genes were cloned from a partial genomic library of HD201. Sequence homologous analysis revealed that vip2Ac gene was highly conserved and encoded a protein possibly having ADP-ribosyltransferase activity, and that vip1Ca gene was of low homology, especially at its 3-terminus. Western blot showed that Vip1Ca and Vip2Ac proteins could be detected from middle logarithmic phase to the stationary phase in Bt HD201. However, bioassays of HD201 supernatants exhibited no activity against Culex quinquefasciatus, Spodoptera exigua, S. litura, Helicoverpa amigera and Tenebrio molitor larvae. Whether Vip1Ca and Vip2Ac proteins have any toxicity to other susceptible targets still needs to be investigated.     

Expression of vip1/vip2 genes in Escherichia coli and Bacillus thuringiensis and the analysis of their signal peptides

AIMS: To determine the expression time courses and high expression level of Vip2A(c) and Vip1A(c) in Bacillus thuringiensis, and survey their insecticidal toxicity and insecticidal spectrum. METHODS AND RESULTS: A kind of new vegetative insecticidal toxin genes encoded by a single operon from B. thuringiensis had been cloned and sequenced. The individual genes, 5-terminus truncated genes and the operon were respectively expressed in Escherichia coli. Only N-terminus deleted Vip2A(c) and Vip1A(c) proteins could be purified by Ni-NTA agarose, while others were processed and their N-terminal signal peptides were cleaved. The individual genes and the operon were also expressed in B. thuringiensis. Both proteins were mostly secreted into the cell supernatants. The expression level of Vip1A(c) was influenced because of the interruption of vip2A(c) gene on the operon. Bioassays showed that neither separate protein nor both performed any toxicity against tested lepidopteran and coleopteran insects. CONCLUSIONS: Vip2A(c) and Vip1A(c) have similar secretion mechanism in E. coli and B. thuringiensis. Vip1A(c) remained its high expression level only when being expressed with vip2A(c) gene as an operon in B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of vip2A(c) and vip1A(c) genes in E. coli and B. thuringiensis were investigated. This would help to make clear the secretion mechanism of VIP proteins and study the function of ADP-ribosyltransferase Vip2.     

Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera)

We developed a protocol for obtaining high yields (10-15 mg per 1100 ml of culture supernatant) of highly purified (up to 95%) Vip3Aa protein from HD-1 cultures. The protocol is based on acetone precipitation of supernatant protein, followed by HPLC fractionation (DEAE-5PW column) and several concentration steps. Our protocol resulted in higher yields and purity of Vip3Aa than a previously published method [Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a 353 novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of 354 activities against lepidopteran insects. Proc. Nat. Acad. Sci. USA 93, 5389-5394.]. This was achieved by using acetone rather than ammonium sulfate for precipitation of proteins from culture supernatants, and a shallow rather than a steep NaCl gradient for elution of the toxin, and by conducting all the purification steps at low temperature to prevent toxin degradation. In bioassays of the purified protein, Choristoneura fumiferana and Lymantria dispar larvae were less susceptible than Spodopteraexigua (10- and ∼100-fold, respectively). A B. thuringiensis var. kurstaki strain HD-1 from which the vip3Aa gene had been deleted (EG12414) showed reduced toxicity to S. exigua relative to the unmodified parental strain (EG2001), but not to L. dispar or C. fumiferana. We interpret these results as indicating that the Vip3Aa toxin does not contribute measurably to pathogenicity of HD-1 in these species.     

Multilocus sequence typing for phylogenetic view and <Emphasis Type="Italic">vip</Emphasis> gene diversity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains of the Assam soil of North East India

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes’ crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.     

Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis

Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.     

Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis

The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.     

Screening of vip genes from a Spanish Bacillus thuringiensis collection and characterization of two Vip3 proteins highly toxic to five lepidopteran crop pests

A Spanish Bacillus thuringiensis strain collection was screened for the presence of vip genes. One hundred strains from a Canary Island collection were screened for vip1 and vip2 genes and 7% contained potentially novel vip1 and vip2-like genes, as indicated by the low degree of similarity with previously known vip1 and vip2 genes. Four hundred strains from a collection originating from the Spanish mainland were screened for vip3 genes and 14.5% of them contained potentially novel vip3-like genes. Reconstruction of the full-length vip sequences could only be achieved for two vip3 gene variants encoding 789 and 787 amino acid proteins that were designated as Vip3Aa45 and Vip3Ag4, respectively. These proteins showed 82% pairwise identity between them and differed from Vip3Aa1 in the putative signal peptide, two specific proteolytic processing sites and the 66-kDa insecticidal fragment. The purified proteins were tested against nine lepidopteran pest species and displayed toxicity, expressed as mean lethal concentration, for five of them. The two toxins were highly toxic for Lobesia botrana (∼1–2 μg/ml) and Spodoptera littoralis (∼20 ng/cm²), moderately toxic for Spodoptera exigua (∼100–300 ng/cm²), and varied greatly in their toxicity for Mamestra brassicae or Chrysodeixis chalcites, with high toxicity for Vip3Aa45 in M. brassicae (∼40 ng/cm²) and for Vip3Ag4 in C. chalcites (∼45 ng/cm²).     

苏云金杆菌辅助蛋白P20对营养期杀虫蛋白Vip3A表达的影响

为检测苏云金杆菌辅助蛋白P20对Vip3A表达和杀虫活性的影响,将p20基因与vip3A基因相连构建了重组质粒pHVP20,然后电激转化至Bt中进行了共表达,以仅携带vip3A基因的质粒pHPT3作为对照质粒。Westernblot结果显示,当vip3A基因和p20基因在Bt无晶体缺陷株CryB中共表达时,Vip3A蛋白的最大表达量约是其在CryB(pHPT3)菌株中单独表达的1.5倍。生物测定结果表明,CryB(pHVP20)和CryB(pHPT3)菌株对初孵斜纹夜蛾幼虫的LC50值分别为48.79μg/mL和78.00μg/mL,这说明P20蛋白可以促进vip3A基因在Bt中的表达,但对提高Vip3A蛋白的杀虫毒力没有显著性帮助。     

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli

AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli. METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184. Both were transformed and expressed in E. coli. About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space. In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm. Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S. litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus. CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E. coli and might be required for toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E. coli was investigated. These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B. thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein.     

Ser-Substituted Mutations of Cys Residues in Bacillus thuringiensis Vip3Aa7 Exert a Negative Effect on its Insecticidal Activity

Vegetative insecticidal proteins (VIPs), which were produced by Bacillus thuringiensis during its vegetative growth stage, display a broad insecticidal spectrum to Lepidoptera larvae. Sequence alignment of the Vip3A-type indicates that three cysteine residues were conserved in Vip3A-type proteins. To determine whether these conserved cysteine residues contributed to the insecticidal activity, the three residues were respectively substituted with serine in the Vip3Aa7 protein by site-directed mutagenesis. Bioassays using the third instar larvae of Plutella xylostella showed that the toxicity of C401S and C507S mutants were completely abolished. To find out the inactivity reason of mutants, three mutants and the wild-type Vip3Aa7 were treated with trypsin. The results indicated that the C507S mutant was rapidly cleaved and resulted in decrease of the 62?kDa toxic core fragment. These results indicated that the replacement of the Cys⁵⁰⁷ with a Ser⁵⁰⁷ caused decrease in C507S resistance against trypsin degradation. It is suggesting a possible association between insecticidal activity and trypsin sensitivity of Vip3A proteins. This study serves a guideline for the study of Vip3A protein structure and active mechanism.