• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.517-522
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• 1998

The cry1 gene content of a collection of Bacillus thuringiensis strains, which include new isolates from Malaysia and Indonesia, was determined by a multiplex PCR using a set of eight oligonucleotide forward primers specific to cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1Ca, cry1Da, cry1Ea, and cry1Fa genes, and two reverse primers, one specific to cry1Ab and the other common to the remaining cry1 genes. Two-thirds of the 59 strains screened were cry1 positive and contained one to four different genes. The cry gene profiles correlated well with toxicities of the strains to lepidopteran insects. The method can be used for rapid screening of a large number of new isolates as the total DNA extracted by boiling cells from single colonies can be used directly in the PCR. However, it is not suitable for follow-up monitoring of specific commercial strains after application in the field as the PCR product profiles of these strains could not be differentiated from those of new isolates.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.710-715
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• 2005

A novel recombinant baculovirus, Bactrus, was constructed by the insertion of the Bacillus thuringiensis cry1Ac gene between two polyhedrin genes of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of the polyhedrin gene promoter. Polyhedra produced by Bactrus in insect cells were incorporated with 130 kDa of polyhedrin-Cry1Ac-polyhedrin fusion protein, and 30 kDa of intact polyhedrin, resulting from a homologous recombination between two polyhedrin genes, was also expressed. The insecticidal activity of Bactrus against Spodoptera exigua larvae was similar to that of AcNPV, but it showed significantly higher toxicity towards Plutella xylostella larvae in comparison with that of AcNPV. The expression level of fusion protein and the insecticidal activity of recombinant polyhedra produced by the Bactrus against P. xylostella larvae were decreased after serial passages. In conclusion, the Bactrus had improved insecticidal activity and returned to wild-type AcNPV after several passages.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.729-735
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• 2012

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.152-161
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• 2015

CodY is a highly conserved protein in low G+C gram-positive bacteria that regulates genes involved in sporulation and stationary-phase adaptation. Bacillus thuringiensis is a grampositive bacterium that forms spores and parasporal crystals during the stationary phase. To our knowledge, the regulatory mechanism of CodY in B. thuringiensis is unknown. To study the function of CodY protein in B. thuringiensis, BMB171codY^- was constructed in a BMB171 strain. A shuttle vector containing the ORF of cry1Ac10 was transformed into BMB171 and BMB171codY^-, named BMB171cry1Ac and BMB171codY^-cry1Ac, respectively. Some morphological and physiological changes of codY mutant BMB171codY^-cry1Ac were observed. A comparative proteomic analysis was conducted for both BMB171codY^-cry1Ac and BMB171cry1Ac through two-dimensional gel electrophoresis and MALDI-TOF-MS/MS analysis. The results showed that the proteins regulated by CodY are involved in microbial metabolism, including branched-chain amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, and energy metabolism. Furthermore, we found CodY to be involved in sporulation, biosynthesis of poly-β-hydroxybutyrate, growth, genetic competence, and translation. According to the analysis of differentially expressed proteins, and physiological characterization of the codY mutant, we performed bacterial one-hybrid and electrophoretic mobility shift assay experiments and confirmed the direct regulation of genes by CodY, specifically those involved in metabolism of branched-chain amino acids, ribosomal recycling factor FRR, and the late competence protein ComER. Our data establish the foundation for in-depth study of the regulation of CodY in B. thuringiensis, and also offer a potential biocatalyst for functions of CodY in other bacteria.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.154-158
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• 2005

Twenty-three Bacillus thuringiensis strains isolated from Korea were screened to detect the cry-type genes using PCR with 21 specific oligonucleotide primers. Eight strains contained distinct multiple crystal genes; cry1Aa2, cry1Ab1, cry1Ac1 and cry2Aa1. These results indicate that the strains coincided with the B. thuringiensis subsp. kurstaki strain. The other 15 strains were not recognised to the 21 specific primers.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.15-20
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• 2007

To clone novel cry1-type genes from the Bacillus thuringiensis K1 isolate, about 2.4-kb-long PCR fragments were amplified with two primer sets of ATG1-F/N400-R and 1BeATG1-F/N400-R. Using PCR-RFLP, three novel cry1-type genes, cry1-1, cry1-7, and cry1-44, were obtained from B. thuringiensis K1 and the complete coding sequences of these novel genes were analyzed. The Cry1-1, Cry1-7, and Cry1-44 proteins showed maximum similarities of about 78.0%, 99.7%, and 91.0% with the Cry1Ha1, Cry1Be1, and Cry1Ac2 proteins, respectively. These novel cry1-type genes were expressed using a baculovirus expression vector system and their insecticidal activities were investigated. Whereas all three novel genes were toxic to Plutella xylostella larvae, only Cry1-1 showed insecticidal activity against Spodoptera exigua larvae.

• The Korean Journal of Pesticide Science
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• v.14 no.4
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• pp.446-455
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• 2010

The characteristics of parasporal inclusion body from Bacillus thuringiensis subsp. kurstaki KB099 isolate which is high bioactive to the tobacco cutworm, Spodoptera litura, were examined. Parasporal inclusion of B. thuringiensis subsp. kurstaki KB099 isolate showed only 1 band at 130 kDa compared with B. thuringiensis subsp. kurstaki HD-l isolate producing 2 protein bands at 130 kDa and 60 kDa from by SDS-PAGE analysis without any enzyme treatment. Also, we confirmed that gut extract of sensitive S. litura KB099 isolate had digested only 60 kDa ${\delta}$-endotoxin protein. When the digestive enzyme of sensitive insect responsible for parasporal inclusion from KB099 and HD-l isolate was treated to each of them, protein band 60 KDa of KB099 was maintained up to 12 hours but all bands of HD-l were disappeared within 6 hours. In KB099 isolate, 6 genes (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D and Cry1I) were identified by PCR analysis. Also, $Cry^-$ mutant of KB099 isolate was investigated by phase- contrast microscope, SDS-PAGE and PCR.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.317-324
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• 2009

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is one of the most commonly used in the development of genetically modified (GM) crops. In this research, we analyzed Bt rice showing lepidopteran pest-resistance. The Bt gene is a synthetic Cry1Ac composed of optimal codons for plants, and the Bt protein is targeted to the chloroplast by a transit peptide. Three Cry1Ac rice events (C103-3, C127-1, and C7-1) were analyzed for molecular characterization. C103-3 contains two copies of T-DNA where the left border (LB) region is truncated. Both C7-1 and C127-1 have a single copy of T-DNA, but a part of the vector backbone DNA is inserted into the genome of C127-1; thus, only C7-1 had intact T-DNA. Progenies of C7-1 crossed with the original cultivar, Nakdong, and double-haploid lines from anther culture of lines crossed with the elite cultivar, Dongjin, were analyzed for T-DNA flanking genomic DNA and genotyping. Results showed that an intact T-DNA region without the vector backbone was inserted into the genome and was stably inherited through generations. The C7-1 homozygous event could be used as breeding material to develop GM rice with pest resistance.

• International Journal of Industrial Entomology and Biomaterials
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• v.6 no.1
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• pp.81-85
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• 2003

To verify importance of the intervening sequence between the ribosome binding site (RBS) and the initiation codon for expression of Bacillus thuringiensis $\delta$-endotoxin, the pProMu, containing SphI and NcoIsites between RBS and the initiation codon of the cry1Ac gene, and the deletion derivatives of pProMu were constructed and transformed into the B. thuringiensis subsp. kurstaki $Cry^{-B}$ strain. The pProMu-ΔSphIhad identical six bases of intervening sequence to pProAc though the arrangement of sequence was different. Other mutants containing pProMu had 1 or 10 or 14 bases between RBS and the initiation codon. Among deletion mutants, only ProMu-ΔSphI/CB only produced 130 kDa typical bipyramidal crystals like those seen for ProAc/CB. However, ProMu/CB, $ProMu-{\Delta}NcoI$, and ProMu-ΔSphI＋NcoIdid not produce Cry1Ac crystals. In conclusion, the results suggest that 6-base intervening sequence was important for expression of cry1-type class gene. Furthermore, spacing effect of the intervening sequences may play an important role in expression of individual crystal proteins in B. thuringiensis without doubt.

• Research in Plant Disease
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• v.15 no.3
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• pp.202-208
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• 2009

The genetically modified leaffolder-resistant (Cry1Ac1) rice plant was evaluated for the changes of resistance by comparing the occurrence of major diseases with a japonica type Korean rice variety, Nakdong which was the mother plant of the transgenic rice event, in greenhouse and field conditions. There was no difference in the occurrence of sheath blight and Helminthosporium blight between the two varieties in the fields. We couldn't find any difference of resistance for fungal blast and bacterial leaf blight by artificial inoculation in greenhouse. There was also no difference in the susceptibility to sheath blight in artificial inoculation tests confirming the results in the fields. The possibility of gene transfer of Bar and Cry1Ac1 from the genetically modified rice plant to naturally infected pathogens such as Fusarium moniliforme and Pyricularia oryzae in the field conditions was tested by PCR. And the possible transfer of those genes by continuous inoculation of Xanthomonas oryzae pv. oryzae and Rhizoctonia solani was also tested. However, we couldn't find any possibility of transfer of the genes in natural and artificial conditions.