Jiguo Gao

Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer : Holotrichia parallela

A new Bacillus thuringiensis strain, Bt185, was isolated from HeBei soil samples in China. Observations after transmission electron microscopy found that the strain produced spherical parasporal inclusions similar to that of the B. thuringiensis subsp. japonensis Buibui strain, which showed toxicity to both Anomala corpulenta and Popillia japonica. The plasmid profile seen on an agarose gel revealed that Bt185 contained six large bands of 191 kb, 161 kb, 104 kb, 84 kb, 56 kb, and 37 kb. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis revealed one major band with an estimated molecular mass of 130 kDa. Polymerase chain reaction–restriction fragment length polymorphism results showed that a novel cry8-type gene sequence was found in the Bt185 strain. When we screened for this novel gene sequence, an additional novel cry8-type gene was isolated, having a partial sequence of 2340 bp and encoding a protein of 780 amino acids. Bioassay results showed that Bt185 had no toxicity against several Coleopteran and Lepidopteran pests. However, Bt185 exhibited toxicity against larvae of the Asian cockchafer, Holotrichia parallela. This is the first report of the occurrence of a Bacillus strain that has insecticidal activity against Holotrichia parallela larvae.

Isolation and characterization of atrazine-degrading strain Shewanella sp. YJY4 from cornfield soil.

Atrazine has been used worldwide for over 50 years as a chemical herbicide. A strain of bacteria, YJY4 which utilizes atrazine as its sole nitrogen source for growth was isolated from agricultural black soil in northeastern China. 16S rDNA sequencing identified YJY4 as a Shewanella sp. PCR analysis and sequencing confirmed that YJY4 contained atrazine‐degrading atzA, atzB and atzC genes. These genes revealed high similarity with those in Pseudomonas sp. ADP and Arthrobacter sp. TC1. The strain YJY4 was observed to degrade atrazine (100 mg l−1) to cyanuric acid completely after 36 h. To the best of our knowledge, YJY4 was the first reported Shewanella sp. to grow in pure culture with atrazine serving as a sole source of nitrogen. Therefore, YJY4 may help with atrazine biodegradation and may become an abundant resource of atrazine degradation strains.

Effects of Site-Mutations Within the 22 kDa No-Core Fragment of the Vip3Aa11 Insecticidal Toxin of Bacillus thuringiensis

Bacillus thuringiensis vegetative insecticidal proteins (VIPs) are not homologous to other known Cry proteins, and they act against lepidopteran larvae via a unique process. All reported studies on the mode of action of Vip3 proteins have been performed on the Vip3A family, mostly on the Vip3Aa subfamily. Vip3Aa proteins are activated by midgut proteases, and they cross the peritrophic membrane and bind specific proteins in apical membrane epithelial midgut cells, which results in pore formation and, eventually, death to the insects. Some studies of trypsin-activated protein (core fragment) and the full-length protein show differences in mortality on the same insect species. The N-terminus of Vip3A proteins is responsible for the translocation of the protein across the cell membrane. To determine whether the N-terminus of Vip3Aa11 proteins contribute to insecticidal activity, we exchanged Vip3Aa11 residues with Vip3Aa39 no-core fragment residues using site-directed mutagenesis. Bioassays showed that the toxicity of S9N, S193T, and S194L mutants displayed approximately one- and twofold increases in toxicity against Helicoverpa armigera. Mutant protein R115H demonstrated a threefold decrease in toxicity. This work serves as a guideline for the study of the Vip3Aa11 no-core fragment protein insecticidal mechanism.

An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1-Type Genes

ABSTRACT The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.

Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests

ABSTRACT The insecticidal activities and specificities of the Vip3Aa proteins derived from different Bt strains are very different, although the similarities between these proteins are higher than 95%. In this study, we hypothesised that the differences in Vip3Aa11 and Vip3Aa39 C-terminal amino acids determine their differences in insecticidal activity against three Lepidoptera insects. To find the amino acid residues associated with insecticidal activity, nine different amino acid residues of Vip3Aa11 were substituted with the corresponding amino acid residues from Vip3Aa39 by site-directed mutagenesis. The toxicity of each protein was estimated by bioassays, and the results demonstrated that the mutant Y784N lost its insecticidal activity against three insects (Agrotis ipsilon, Helicoverpa armigera, and Spodoptera exigua). The insecticidal activity of S543N, I544L, and S686R against S. exigua increased 5-fold, 2.65-fold, and 8.98-fold, while the toxicity to H. armigera and A. ipsilon slightly decreased compared with that of the Vip3Aa11 protein. These findings indicate that the amino acid residues Ser543, Ile544, Thr685, Ser686, Arg704, Ile780, and Tyr784 may be insecticidal activity-related residues. Additionally, the trypsin activation of the four mutants indicated that all proteins can form a 62-kDa core fragment, except Y784N. A possible association between the insecticidal activity and trypsin sensitivity of Vip3A proteins is suggested.

Cry78Aa, a novel Bacillus thuringiensis insecticidal protein with activity against Laodelphax striatellus and Nilaparvata lugens

Transgenic plants expressing insecticidal proteins originating from Bacillus thuringiensis (Bt) have successfully been used to control lepidopteran and coleopteran pests with chewing mouthparts. However, only a handful of Bt proteins have been identified that have bioactivity against sap sucking pests (Hemiptera), including aphids, whiteflies, plant bugs and planthoppers. A novel Bt insecticidal protein with significant toxicity against a hemipteran insect pest is described here. The gene encoding the 359 amino acid, 40.7 kDa protein was cloned from strain C9F1. After expression and purification of the toxin, its median lethal concentration (LC50) values against Laodelphax striatellus and Nilaparvata lugens were determined as 6.89 μg/mL and 15.78 μg/mL respectively. Analysis of the toxin sequence revealed the presence of both Toxin_10 and Ricin_B_Lectin domains.

Sip1Ab gene from a native Bacillus thuringiensis strain QZL38 and its insecticidal activity against Colaphellus bowringi Baly

ABSTRACT In this study, we collected 540 soil samples from northeast China and isolated the wild-type strain of Bacillus thuringiensis (Bt) by identifying and cloning 9 Bt strains that expressed the secreted insecticidal protein (Sip) gene. We selected the strain QZL38 for further study. The sip gene was identified from the Bt strain QZL38 using polymerase chain reaction (PCR). We sequenced a 1095-base pair fragment of DNA that encodes 364 amino acid residues of a 41.18 kDa pro-toxin and compared it with the registered Sip1Ab protein amino acid residue sequence. The sequence was submitted to GenBank with the accession no. KP231523, and the gene was named sip1Ab. The Sip1Ab protein expressed in Escherichia coli showed insecticidal activity against Colaphellus bowringi Baly, with an LC50 of 1.051 μg mL−1. To identify the active fragment of the Sip1Ab toxin, four pairs of primers with different truncation positions were designed, and the recombinant proteins were expressed in E. coli. The truncated Sip protein expressed in E. coli showed insecticidal activity against C. bowringi Baly. The insecticidal activity of the recombinant proteins against C. bowringi Baly from the Sip1Ab signal peptide after removal of 30 amino acid residues showed an LC50 of 1.078 μg mL−1. Sip proteins may play an important role in the prevention and control of the C. bowringi Baly.

In Silico Structure-Based Identification and Validation of Key Residues of Vip3Aa Involving in Lepidopteran Brush Border Receptor Binding

The vegetative insecticidal proteins (VIPs) of Bacillus thuringiensis (Bt) have a broad-spectrum insecticidal activity against Lepidopteran pests and no cross-resistance with the insecticidal crystal protein Cry protein. So there are great potentials for the control of agricultural pests and the resolution of resistance problems. The structural information of Vip3Aa protein and the predicted key amino acid sites on the C-terminal domain of Vip3Aa were analyzed with the methods of bioinformatics such as homology modeling and molecular docking. Site-directed mutagenesis was used to replace these amino acids with alanine, and there was difference in the activities of the mutant protein and Vip3Aa protein. Y619A had improved insecticidal activity against Helicoverpa armigera, but the toxicity of W552A and E627A to Helicoverpa armigera was significantly reduced. The mutants of W552A and E627A had reduced insecticidal activity against Spodoptera exigua. This study demonstrated that the C-terminal domain played an important role in the function of Vip3Aa protein toxin, and the deletion of the side chain of key residues had a significant effect on the activity of the insecticidal protein. This study provides the theoretical basis for revealing the relationship between the structure and function of Vip3Aa protein.

Identification and cloning of two insecticidal protein genes from Bacillus thuringiensis strain S185

Bacillus thuringiensis (Bt) is the most widely applied type of microbial pesticide due to its high specificity and environmental safety. The activity of Bt is largely attributed to the insecticidal crystal protein encoded by the cry genes. Different insecticidal crystal proteins of Bt have different bioactivity against distinct agricultural insect pests, and combination of these proteins not only increases insecticidal activity, but also overcomes and delays development of resistance. A Bt strain, S185, was isolated from a soil sample collected in Songfeng Shan district, Heilongjiang Province, China. Bt S185 has highly specific insecticidal activity against Coleoptera, and was determined to contain cry8 -type genes by peptide mess fingerprint (PMF) analysis. Application of polymerase chain reaction-restricted fragment length polymorphisms (PCR-RFLP) analysis further determined the genotype due to the high homology of cry8Ea1 and cry8Fa1 genes. Through the full-length primers design, two insecticidal crystal protein genes cry8Ca and cry8Ea were obtained. Using prokaryotic cloning vectors, the recombinant plasmids pEB-cry8Ca and pEB-cry8Ea were transferred into expression host strain Escherichia coli Rosetta, thus the two genes were successfully expressed in heterologous bacteria. Keywords: Bacillus thuringiensis , peptide mess fingerprint, identification, clone, insecticidal crystal protein. African Journal of Biotechnology , Vol 13(25) 2484-2490