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.     

Isolation,purification and physicochemical characterization of water‐soluble Bacillus thuringiensis melanin

Melanins are widely used in medicine, pharmacology, cosmetics and other fields. Although several technologies for the purification of water‐insoluble dioxyphenylalanine (DOPA) melanins have been described, a source of water‐soluble melanin is highly desirable. Here we describe an effective procedure for the isolation and purification of water‐soluble melanin using the culture medium of Bacillus thuringiensis subsp. galleriae strain K1. Water‐soluble melanin from this organism has an isoelectric point (pI = 3.0–3.2) and was purified optimally by adsorbtion using the IA‐1r resin and elution as a concentrated solution. The purified melanin obtained exhibited a similar infra‐red absorbtion spectrum to synthetic melanin and contained quinolic and phenolic structures and an amino acid content of around 20% after acid hydrolysis. The molecular weight of the purified melanin determined by SDS‐PAGE was 4 kDa and the electromagnetic spin resonance spectrum of the purified microbial melanin was a slightly asymmetric singlet without hyperfine structure with about 7 Gauss width of the line between points of the maximum incline and g = 2.006. The concentration of paramagnetic centers in melanin is 0.21 × 10¹⁸ spin/g. The results obtained provide a rapid, simple and inexpensive method for the large scale purification of water soluble melanin that may have widespread applications.     

苏云金杆菌NU-2原生质体复合诱变的研究

通过对苏云金杆菌NU-2的原生质体进行紫外线和LiCl的复合诱变,筛选到1株发酵周期可以从44 h缩短到40.3 h,晶体蛋白含量提高10.03%的突变株NU-2-16。实验所确定的NU-2原生质体形成和再生的最佳条件为将菌体培养10 h后再经活化5 h,溶菌酶终浓度为1.5 mg/mL,pH 7.0,42℃水浴中处理60 min。     

Bacillus thuringiensis and neem seed oil (Azadirachta indica) effects on the potato tuber moth Phthorimaea operculella zeller in the field and stores

A comparative evaluation for the efficacy of Bacillus thuringiensis and neem seed oil on Phthorimaea operculella has been carried out in the field and store. These two preparations were almost equally effective on the potato tuber moth infestation. The percentage of infestation was reduced through successive application of either preparations in the field up to harvest. No synergism was observed upon using combination of the two preparations. In the store, neem seed oil (500 ppm) was highly protective and was as effective as sevin. A combination of both neem and B.t. (Delfin) significantly protects the tubers. This suggests the possible use of either neem seed oil or B.t. in combating the insect pest in the field or during storage.     

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.     

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.     

Characterisation of lepidopteran-active Bacillus thuringiensis isolates recovered from infected larvae

Abstract

As a part of an ongoing nationwide programme focused on finding novel strains of Bacillus thuringiensis (Bt) that are toxic to some of the major pests that impact economically important crops, we initiated a search for Bt isolates native to Syria. We succeeded in assembling a collection of 40 Bt isolates recovered from infected larvae of Galleria mellonella, Helicoverpa armigera and Ephestia kuehniella. Light microscopy showed that all isolates produce bipyramidal and cuboidal crystal proteins. The 50% lethal concentration of the spore-crystal mixture of the 40 isolates against E. kuehniella larvae varied from 3 to more than 200 µg g^?1. A comparison of the LC₅₀ values of the tested isolates with the reference strain Bt kurstaki HD-1 (20.55 µg g^?1), showed that some of these isolates have a similar or up to six times higher toxicity potential. PCR screening revealed that all obtained isolates contain cry1 and cry2 genes, whereas only four contain cry9. Moreover, the proteins of 130 and 65/70 Kda encoded by these genes were detected in the SDS-PAGE of the purified parasporal bodies. Flagellar serotyping classified 30 as serovar kurstaki, six isolates serovar aizawai, one isolate cross-reacted with more than one H3 antisera and three were not typeable. Assays of toxicity of the aizawai isolates against third instar of G. mellonella showed that four, which contain cry9, have almost similar toxicity to the commercial strain Bt aizawai B401. Therefore, these isolates could be adopted for future applications to control G. mellonella. Moreover, this study contributes to our knowledge of Bt diversity in Syria where to date very few collections have been described.     

苏云金杆菌酪氨酸酶基因转化子黑色素的研究

苏云金杆菌(Bacillusthuringiensis,Bt)是使用广泛的杀虫细菌。将构建的含有嗜麦芽假单胞菌酪氨酸酶基因的重组质粒导入苏云金杆菌体内,使后者获得了稳定产生黑色素的能力。所产黑色素是无定形物质,在大多数有机和无机溶剂中溶解度低或不溶解,在碱性溶液中有较好的溶解性,在中性及弱酸性溶液中溶解度低,pH<4时沉淀;可用过氧化氢或次氯酸钠氧化,也能被银离子或硫化氢还原;含有天然物质中少见的自由基,并能清除由紫外线诱导产生的自由基;能吸收所有波长的光线,在紫外波长下吸收值最大,能有效地保护紫外辐射对蛋白质及DNA等生物大分子物质的损害,具有很强的抗辐射作用。使转化子抗辐射能力显著增强,杀虫活性稳定保持;能抵抗病毒的侵袭,生长旺盛;对畜禽安全。     

Occurrence of three different binding sites for Bacillus thuringiensis δ-endotoxins in the midgut brush border membrane of the potato tuber moth,phthorimaea operculella (zeller)

The potato tuber moth is susceptible to at least three insecticidal crystal proteins (ICPs) from Bacillus thuringiensis: CrylA(b), CrylB, and CrylC. To design useful combinations of toxin genes either in transgenic plants or in new genetically modified B. thuringiensis strains, it is necessary to determine the binding characteristics of the different ICPs so as not to combine a pair sharing the same binding site. This has been accomplished using two different techniques: ¹²⁵I-labeling of the ICPs with further measurement of the radioactivity bound to brush border membrane vesicles, and microscopic visualization of the bound ICPs by enzyme-linked reagents such as antibodies or streptavidin using biotinylated ICPs. Our results show that CrylA(b), CrylB, and CrylC bind to different sites in the brush border membrane of midgut epithelial cells. Also, the affinity of the binding sites for the ICPs and their concentration in brush border membrane vesicles has been determined in a laboratory strain and a storage collected population. No significant differences were found between these two strains. © 1994 Wiley-Liss, Inc.     

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

Endogenous protease-activated 66-kDa toxin from Bacillus thuringiensis subsp. kurstaki active against Spodoptera littoralis

The anti-lepidopteran toxin from sporulated Bacillus thuringiensis subsp. kurstaki cells, generated by the proteolytic action of endogenous protease(s) on the protoxin, was purified and studied to identify the effect of such proteolysis on the biochemical nature of the toxin. The active toxin was purified employing anion-exchange chromatography to absolute homogeneity, as indicated by SDS-PAGE and Western blotting. Antisera to the purified toxin (66 kDa) crossreacted with the protoxin (132 kDa) confirming its origin from protoxin. The purified toxin with a pI of 7.95 was derived from the N-terminal region of the protoxin (pI 7.6). Circular dichroism data revealed that the toxin has significant secondary structure and it undergoes pH dependent conformational change. Unlike the toxin generated by exogenous proteases such as trypsin, etc., the endogenous protease(s) activated toxin is highly lethal to a tolerant insect variety of the lepidopteran order, Spodoptera littoralis.     

苏云金杆菌生物农药生产和应用现状

苏云金杆菌（Bacillus thuringiensis,Bt）生物农药在全国乃至全世界都是被公认为最安全、最有效、工业化程度最高、最廉价因而也是应用范围最广、使用量最大的微生物杀虫剂,而中外专家和政府在设施害虫IPM计划和无公害农产品生产的推广杀虫剂首选就是Bt。因此,它在生物防治的作用和地位不可忽略。我国生产推广应用的Bt厂家一直对国内外Bt产品的应用状况十分关注。为此,本文就多年来国内Bt生产菌株及其防治对象,生产厂家及其生产能力、市场范围以及应用水平,存在问题以及发展前景等方面做综述。     

苏云金芽胞杆菌HD-73菌株sigL基因突变体的特性分析

[目的]通过分析苏云金芽胞杆菌sigL基因突变体的特征,进一步明确sigL基因在苏云金芽胞杆菌中的功能.[方法]测定了苏云金芽胞杆菌HD-73菌株,sigL基因缺失突变菌株和互补菌株在不同营养成分的培养基中的生长曲线以及在不同氮源条件下的生长情况.分别将调控aco操纵子(编码3-羟基丁酮脱氢酶系统)的转录调节基因acoR和调控bkd操纵子(编码催化支链脂肪酸合成的酶系统)的转录调节基因bkdR的启动子与lacZ基因融合,并转入出发菌株和sigL突变体中,测定β-半乳糖苷酶的活性.[结果]sigL突变体不能利用精氨酸、脯氨酸、缬氨酸、异亮氨酸、谷氨酰胺、苯丙氨酸、蛋氨酸、色氨酸为唯一的氮源;β-半乳糖苷酶的活性分析表明:在sigL突变体中acoR基因和bkdR基因的启动子活性降低.序列比对分析表明:Bt中的AcoR和BkdR的蛋白结构域与依赖于σL的转录调节因子的保守序列相似.[结论]苏云金芽胞杆菌中sigL基因的缺失可能阻碍了某些重要碳、氮源参与的代谢途径.在Bt中AcoR和BkdR是依赖于σL的转录调节因子.     

Characterisation of endophytic Bacillus thuringiensis strains isolated from wheat plants as biocontrol agents against wheat flag smut

To explore effective and ecofriendly means of controlling wheat flag smut (WFS) using biocontrol agents, three endophytic strains (58-2-1, 37-1 and YC-1) of Bacillus sp. were isolated from winter wheat plants in China and identified as Bacillus thuringiensis based on their 16S rDNA sequences as well as phenotypic characteristics. Four morphological (leaf length, root length, dry weight and tiller numbers) and one physiological [root vigour (RV)] parameters of wheat plants treated by strains 58-2-1 and 37-1 were significantly enhanced compared to the control. The soluble sugar contents in the roots of wheat samples treated by the two strains were significantly lower than the control. The resistance of wheat varieties to WFS was investigated by inoculation tests. Of the 12 wheat varieties tested, 6 (Yunhan-618, Bainongaikang-58, Kaimai-20, Zhengmai-9023, 04-zhong-36 and Yanzhan-4110) were identified as WFS-highly resistant (HR), 3 (Pumai-9, Jinboshi-1 and Yunong-202) WFS-moderately resistant (MR), 1 (Yubao-1) WFS-susceptible (S) and 2 (Yumai-012 and Yunong-416) WFS-highly susceptible (HS) varieties. The Urocystis tritici-induced yield loss on the S/HS varieties was significantly higher than that on the HR/MR ones. The strains 58-2-1 and 37-1 had control efficacies of 6.7–100% (av.54.8%) and 33.3–100% (av. 66.5%) on 9 and 7 out of 12 varieties, respectively. The strains 58-2-1 and 37-1 had enhanced yields of 10.2–54.9% (av. 32.9%) and 2.8–43.4% (av. 24.8%) on 10 and 8 out of 12 varieties, respectively. This is the first report on endophytic B. thuringiensis strains isolated from wheat plants with the abilities to suppress WFS and to enhance yields on multiple wheat varieties.     

A novel strain of Bacillus thuringiensis (NCIMB 40152) active against coleopteran insects

Abstract: A novel Bacillus thuringiensis strain, NCIMB 40152, was isolated from dead Tenebrio molitor L. larvae. The isolate is endowed with specific insecticide activity against the Colorado potato beetle ( Leptinotarsa decemlineata (Say), Coleoptera, Crysomelidae), but has no effect against lepidopteran or dipteran insects.
During the sporulation process it produces diamond-shaped crystals and small inclusions. Rare flat, square crystals are alsò produced. The strain can be differentiated from the other reported B. thuringiensis strains active against Coleoptera on the basis of physiological and biochemical tests, flagellar serotype and toxin electrophoretic patterns.
NCIMB 40152 toxin is easily extractable from spore/crystal preparations in neutral distilled water without detergents or reducing agents.     

A pangenomic study of Bacillus thuringiensis

Bacillus thuringiensis (B.thuringiensis) is a soil-dwelling Gram-positive bacterium and its plasmid-encoded toxins (Cry) are commonly used as biological alternatives to pesticides.In a pangenomic study,we sequenced seven B.thuringiensis isolates in both high coverage and base quality using the next-generation sequencing platform.The B.thuringiensis pangenome was extrapolated to have 4196 core genes and an asymptotic value of 558 unique genes when a new genome is added.Compared to the pangenomes of its closely related species of the same genus,B.thuringiensis pangenome shows an open characteristic,similar to B.cereus but not to B.anthracis; the latter has a closed pangenome.We also found extensive divergence among the seven B.thuringiensis genome assemblies,which harbor ample repeats and single nucleotide polymorphisms (SNPs).The identities among orthologous genes are greater than 84.5％ and the hotspots for the genome variations were discovered in genomic regions of 2.3-2.8 Mb and 5.0-5.6 Mb.We concluded that high-coverage sequence assemblies from multiple strains,before all the gaps are closed,are very useful for pangenomic studies.     

苏云金芽孢杆菌的一个新亚种(Btsubsp.sinensis)

198 9年自云南昆明市石林的红棕壤中分离到数株苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎ ｓｉｓ,Ｂｔ)菌株[1] ,对其中的一株ＹＫ30 0 4进行了生物学特性、杀虫特性研究及分类鉴定。1　材料与方法1.1　供鉴定的Ｂｔ菌株由云南昆明市石林的红棕壤中分离的苏云金芽孢杆菌ＹＫ30 0 4菌株。1.2　标准Ｂｔ菌株血清型Ｈ1 Ｈ4 1、Ｈ4 4 Ｈ55及Ｈ57 Ｈ69标准Ｂｔ菌株由法国巴斯德研究院ＤｒＬｅｃａｄｅｔ提供 ,其余为本实验室保存。1.3　生物测定用昆虫小菜蛾 (Ｐｌｕｔｅｌｌａｘｙｌｏｓｔｅｌｌａ) 3龄幼虫 ;斜纹夜盗蛾 (Ｐｒ…     

Characterisation of silver nanoparticles synthesised by Bacillus thuringiensis as a nanobiopesticide for insect pest control

Nanotechnology has become one of the most promising new approaches for pest control in recent years. In this research, biocompatible silver nanoparticles (Btk-AgNPs) were synthesised by using the entomopathogenic bacterium, Bacillus thuringiensis kurstaki (Btk) as a low-cost and eco-friendly production system. The AgNP samples exhibited a brownish-yellow colour that is characteristic for silver nanoparticles synthesis. Btk-synthesised AgNPs were produced using both the supernatant and pellet of Bt culture at various concentrations and AgNP particles were characterised by UV-Vis spectrophotometer and Dynamic Light Scattering (DLS). The variation of hydrodynamic diameter (D_h) and UV-Vis spectra of silver particles produced by various concentration of culture showed that production of AgNPs was maximised when using 20% for either supernatant or pellet treatments of Bt of culture and the size of particles was around 85?nm for both. The insecticidal efficacy of Btk-synthesised AgNPs against larvae of the cabbage looper, Trichoplusia ni (Hübner) and black cutworm, Agrotis ipsilon (Hufnagel) was tested. Results demonstrated that the treatments of either Btk-synthesised AgNP(s) made with Bt supernatant or Btk-synthesised AgNP(p) using Bt pellet were found to be significantly more virulent toward larvae of T. ni than to A. ipsilon.