Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58)is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine.The Fourier-transform infrared (FT-IR)spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma).A bioassay shows that the LC50 of a Bacillus thuringiensis (Bt)formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml,which is similar to that of the Bt formulation without UV treatment,however,it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58.The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation.This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation.     

Expression of mel gene improves the UV resistance of Bacillus thuringiensis

Aims:  To improve ultraviolet (UV) resistance of Bacillus thuringiensis for increasing the duration of the Bt product applied in the field, a genetically engineered strain Bt TD841 that produced both melanin and Cry1A protein was constructed, and its UV resistance was evaluated in the laboratory.
Methods and Results:  Melanin quantitative analysis revealed that the recombinant strain Bt TD841 could synthesize 0.15 mg melanin ml⁻¹ sporulated culture. Atomic force microscopy confirmed the production of diamond crystal and SDS-PAGE results showed the expression of the 130 kDa Cry1A protein. Bioassay results demonstrated that the LC₅₀ value of Bt TD841 was 3.69 μl ml⁻¹ against Helicoverpa armigera and the UV resistance of this recombinant was enhanced 9.7-fold compared to its parental strain Bt HC42 after 4-h UV irradiation.
Conclusion:  Expression of the mel gene can significantly increase UV resistance of B. thuringiensis.
Significance and Impact of the Study:  This is the first report on genetically engineered Bt strain with co-expression of melanin and the insecticidal crystal proteins gene, and the results may offer a practical solution for improving the photoprotection of Bt products in field application.     

Melanin pigment formation and increased UV resistance in Bacillus thuringiensis following high temperature induction

The pigment melanin is well known to protect against the damaging effects of UV radiation. In this study, we show that thirty-five of thirty-seven tested Bacillus thuringiensis strains possess the potential to produce melanin in the presence of L-tyrosin at elevated temperature (42 degrees C). These findings offer a method of protecting insecticidal toxins produced by B. thuringiensis from UV degredation and may therefore have important applications in the field of crop protection. Toxicity assays on Heliothis armigera suggested that the insecticidal activity of B. thuringiensis that produced melanin was significantly higher after UV irradiation than when melanin was not produced.     

Investigation of the Cry4B-Prohibitin Interaction in Aedes aegypti Cells

Bacillus thuringiensis (Bt) produces insecticidal toxins active against insects. Cry4B, one of the major insecticidal toxins produced by Bt subsp. israelensis, is highly toxic to mosquitoes in the genus Aedes: the major vectors of dengue, yellow fever, and chikungunya. Previous work has shown that Cry4B binds to several mid-gut membrane proteins in Aedes aegypti larvae including prohibitin, a protein recently identified as a receptor that also mediates entry of dengue virus into Aedes cells. This study confirms the interaction between Cry4B and prohibitin by co-immunoprecipitation analysis and demonstrates colocalization of prohibitin and Cry4B by confocal microscopy. While activated Cry4B toxin showed high larvicidal activity, it was not cytotoxic to two Aedes cell lines, allowing determination of its effect on dengue virus infectivity in the absence of Cry4B-induced cell lysis. Pre-exposure of Aedes cells to Cry4B resulted in a significant reduction in the number of infected cells compared to untreated cells.     

Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli.

Escherichia coli JM103 cells harboring expression plasmid pTB1 or pKC6 synthesized the 130- and 135-kilodalton insecticidal proteins, respectively, of Bacillus thuringiensis subsp. aizawai IPL7, and both products accumulated as cytoplasmic inclusion bodies. Amorphous inclusions which contained contaminating proteins, together with the corresponding insecticidal proteins, were formed in cultures at 37 degrees C, but bipyramidal crystals practically free of contaminants were observed at 30 degrees C. Although 9.8% of the amino acids were substituted between these two proteins, both protein crystals had the same shape as those of the parental B. thuringiensis strain, which produced both proteins.     

Restraining Erwinia virulence by expression of N-acyl homoserine lactonase gene pro3A-aiiA in Bacillus thuringiensis subsp leesis

To widen the biological control function of a genetically modified Bacillus thuringiensis subsp leesis strain BMB-005, an acyl homoserine lactonase (AHL lactonase) gene aiiA transcribed by the promoter of insecticidal crystal protein coding gene cry3A, was transformed into strain BMB-005. The amount of AHL lactonase protein produced by transformant BMB821A was 2.4-fold more than that produced by BMB-005. AHL-degradation assay showed that transformant BMB821A could degrade more AHLs molecules than the original strain BMB-005. The result of Erwinia carotovora pathogenicity test showed that the parental strain BMB-005 had no restraint of Erwinia infection, but the transformants exhibited strong restraint of E. carotovora infection on potato slices and cactus stems. Insecticidal bioassay against lepidopteran Spodoptera exigua showed that both strain BMB-005 and transformant BMB821A were toxic to S. exigua. The toxicity of transformant BMB821A (LC(50) was 3.8) was a little attenuated comparing with the toxicity of the original strain BMB-005 (LC(50) was 2.9). The B. thuringiensis strain BMB-005 has high toxicity against Helicoverpa armigera, Plutella xylostella, and S. exigua. This work provided new strategy for developing genetically engineered multi-functional B. thuringiensis strain that possesses insecticidal activity together with restraint of bacterial pathogenicity.     

Cloning and partial characterization of zwittermicin A resistance gene cluster from Bacillus thuringiensis subsp. kurstaki strain HD1

AIM: The study seeks to shed light on the aminopolyol, broad-spectrum antibiotic zwittermicin A gene cluster of Bacillus thuringiensis subsp. kurstaki HD1 and to identify any new uncharacterized genes with an eventual goal to establish a better understanding of the resistance gene cluster. METHODS AND RESULTS: We screened 51 serovars of B. thuringiensis by PCR and identified 12 zmaR-positive strains. The zmaR-positive B. thuringiensis subsp. kurstaki HD1 strain displayed inhibition zones against indicator fungal strain Phytophthora meadii and bacterial strain Erwinia herbicola as well as against Rhizopus sp., Xanthomonas campestris and B. thuringiensis subsp. finitimus. The zmaR gene cluster of strain HD1 was partially cloned using a lambda library and was extensively characterized based on the information available from a study performed on a similar group of genes in Bacillus cereus. CONCLUSIONS: Three of the five genes in the zwittermicin gene cluster, including the zmaR gene, had counterparts in B. cereus, and the other two were new members of the B. thuringiensis zmaR gene cluster. SIGNIFICANCE AND IMPACT OF THE STUDY: The two new genes were extensively analysed and the data is presented. Understanding antifungal activity of B. thuringiensis may help us to design suitable Cry toxin delivery agents with antifungal activity as well as enhanced insecticidal activity.     

Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58) is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine. The Fourier-transform infrared (FT-IR) spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma). A bioassay shows that the LC₅₀ of a Bacillus thuringiensis (Bt) formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml, which is similar to that of the Bt formulation without UV treatment, however, it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58. The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation. This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation. Translated from Microbiology, 2006, 33(1): 42–45 [译自: 微生物学通报]     

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。     

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7 in B. subtilis MI113 and B. stearothermophilus SIC1 was examined. Production of the protein (130 kilodaltons [KDa]) was analyzed by its reaction with antibody against the insecticidal proteins of the parental B. thuringiensis. When the original gene containing its own promoter was subcloned in B. subtilis, only a small amount of the protein was produced. Therefore, both the promoter for the B. stearothermophilus alpha-amylase gene and the insecticidal protein gene were inserted in a repA (low-copy-number) plasmid to yield the recombinant plasmid pTBT-Pamy. B. subtilis MI113 carrying pTBT-Pamy produced more of the 130-kDa protein (about 10(4) molecules per cell) at 37 degrees C. In contrast, B. stearothermophilus SIC1 carrying pTBT-Pamy produced a small amount of 130-kDa protein (10(2) to 10(3) molecules per cell) at 55 degrees C.     

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile.

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7 in B. subtilis MI113 and B. stearothermophilus SIC1 was examined. Production of the protein (130 kilodaltons [KDa]) was analyzed by its reaction with antibody against the insecticidal proteins of the parental B. thuringiensis. When the original gene containing its own promoter was subcloned in B. subtilis, only a small amount of the protein was produced. Therefore, both the promoter for the B. stearothermophilus alpha-amylase gene and the insecticidal protein gene were inserted in a repA (low-copy-number) plasmid to yield the recombinant plasmid pTBT-Pamy. B. subtilis MI113 carrying pTBT-Pamy produced more of the 130-kDa protein (about 10(4) molecules per cell) at 37 degrees C. In contrast, B. stearothermophilus SIC1 carrying pTBT-Pamy produced a small amount of 130-kDa protein (10(2) to 10(3) molecules per cell) at 55 degrees C.     

Bacillus thuringiensis: a biotechnology model

This paper is on the different biotechnological approaches that have been used to improve Bacillus thuringiensis (Bt) for the control of agricultural insect pests and have contributed to the successful use of this biological control agent; it describes how a better knowledge of the high diversity of Bt strains and toxins genes together with the development of efficient host-vector systems has made it possible to overcome a number of the problems associated with Bt based insect control measures. First we present an overview of the biology of Bt and of the mode of action of its insecticidal toxins. We then describe some of the progress that has been made in furthering our knowledge of the genetics of Bt and of its insecticidal toxin genes and in the understanding of their regulation. The paper then deals with the use of recombinant DNA technology to develop new Bt strains for more effective pest control or to introduce the genes encoding partial-endotoxins directly into plants to produce insect-resistant trangenic plants. Several examples describing how biotechnology has been used to increase the production of insecticidal proteins in Bt or their persistence in the field by protecting them against UV degradation are presented and discussed. Finally, based on our knowledge of the mechanism of transposition of the Bt transposon Tn4430, we describe the construction of a new generation of recombinant strains of Bt, from which antibiotic resistance genes and other non-Bt DNA sequences were selectively eliminated, using a new generation of site-specific recombination vectors. In the future, continuing improvement of first generation products and research into new sources of resistance is essential to ensure the long-term control of insect pests. Chimeric toxins could also be produced so as to increase toxin activity or direct resistance towards a particular type of insect. The search for new insecticidal toxins, in Bt or other microorganisms, may also provide new weapons for the fight against insect damage.     

The chromosome map of Bacillus thuringiensis subsp. canadensis HD224 is highly similar to that of the Bacillus cereus type strain ATCC 14579

Abstract A physical map of the Bacillus thuringiensis subsp. canadensis HD224 chromosome based on Asc I, Not I, and Sfi I restriction sites has been established. The chromosome map of 4.3 Mb was similar to a revised map of the chromosome of the B. cereus type strain ATCC 14579, except that the B. thuringiensis subsp. canadensis HD224 chromosome lacked a Not I site and had two additional Asc I sites. The positions of 27 probes were identical in the common macromap. A probe for the insecticidal toxin gene, cryIA , hybridized only to the B. thuringiensis subsp. canadensis HD224 chromosome. The Bss HII ribotype patterns were almost identical confirming the similarity between the two strains.     

Formation of Crystalline delta-Endotoxin or Poly-beta-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of delta-endotoxin on M medium, which contained 330 mug of potassium per ml, but not on ST and ST-a media, each of which contained only 11 mug of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-beta-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH(2)PO(4) (3.7 mM) led to the formation of crystalline delta-endotoxin. The replacement of KH(2)PO(4) with equimolar amounts of KCl, KNO(3), and potassium acetate or an equivalent amount of K(2)SO(4) had a similar effect, whereas the addition of an equimolar amount of NaH(2)PO(4) or NH(4)H(2)PO(4) did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced delta-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-beta-hydroxybutyric acid granules on the media containing 相似文献   

Evolutionary trade-offs of insect resistance to Bacillus thuringiensis crops: fitness cost affecting paternity

Evolution of resistance to insecticides provides a useful model for examining fitness trade-offs associated with adaptation to stress. Here, we examined male reproductive costs in pink bollworm (Pectinophora gossypiella) resistant to an insecticidal protein of Bacillus thuringiensis (Bt) produced by transgenic cotton, using contrasts between two pairs of related susceptible and resistant strains. Without competition for access to females, no costs affecting reproductive success of resistant males were observed. Resistant and susceptible males had similar mating frequency and fertility. Additionally, fecundity of females mated to resistant and susceptible males was comparable. In competition for matings with virgin females, resistant and susceptible males had comparable success in one strain, whereas susceptible males tended to mate more often than resistant males in the other. However, irrespective of strain origin, resistant males that mated first sired significantly less offspring than susceptible males that mated first. The reduced first-male paternity in resistant males may involve reduced sperm precedence caused by mutations in a cadherin gene linked with resistance to Bt cotton.     

Novel cloning vectors for Bacillus thuringiensis.

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.     

Novel cloning vectors for Bacillus thuringiensis

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.     

Complete genome sequence of Bacillus thuringiensis subsp. chinensis strain CT-43

Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.