Surface display of recombinant proteins on Bacillus thuringiensis spores

The insecticidal protoxin from Bacillus thuringiensis has been shown to be a major component of the spore coat. We have developed a novel surface display system using B. thuringiensis spores in which the N-terminal portion of the protoxin is replaced with a heterologous protein. The expression vector with a sporulation-specific promoter was successfully used to display green fluorescent protein and a single-chain antibody (scFv) gene that encodes anti-4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (anti-phOx) antibody. The spores that carry the anti-phOx antibody can bind to phOx specifically.     

Analysis of non-active engineered Bacillus thuringiensis crystal proteins

Abstract Crystal proteins of Bacillus thuringiensis are known for their insecticidal specificity. This specificity is, to a large extent, determined by the interaction of the proteins with high-affinity binding sites on the epithelial membrane of the midgut of sensitive insects. In particular, domain II of the three domains of the toxic moiety has been implicated in specificity. To determine which sequences of the protein are involved in binding, loops of domain II which terminate in the molecular apex of CryIA(b) were replaced by the corresponding regions of CryIE, a protein with different binding characteristics and insect specificity. In contrast to expression of the wild-type genes, expression of the mutant alleles in Escherichia coli resulted in the formation of biologically inactive, insoluble aggregates. Although these aggregates could be solubilized in vitro using urea, in contrast to the wild-type CryIA(b), the mutant proteins did not correctly refold as is shown by their increased protease sensitivity and lack of biological activity. The results indicate that engineering CryI proteins, based on the CryIIIA structure, is likely to prove difficult, particularly since the conformation of CryIIIA and CryI proteins might differ in domain II.     

Production of alginate beads by emulsification/internal gelation. II. Physicochemistry

Alginate microspheres were produced by emulsification/internal gelation of alginate sol dispersed within vegetable oil. Gelification was initiated within the alginate sol by a reduction in pH (7.5 to 6.5), releasing calcium from an insoluble complex. Smooth, spherical beads with the narrowest size dispersion were obtained when using low-guluronic-acid and low-viscosity alginate and a carbonate complex as the calcium vector. A more finely dispersed form of the complexed calcium within the alginate sol promotes a more homogeneous gelification. Microsphere mean diameters ranging from 50 m to 1000 m were obtained with standard deviations ranging from 35% to 45% of the mean.     

Production of alginate beads by emulsification/internal gelation. I. Methodology

Summary Small diameter alginate beads (microspheres) were formed via internal gelation of alginate solution emulsified within vegetable oil. Gelation was initiated by addition of an oil-soluble acid thereby reducing the pH of the alginate solution and releasing soluble Ca²⁺ from the citrate complex. Smooth, spherical, micron-sized beads were formed. The mean diameter ranged from 200 to 1000 m, controlled by the reactor impeller design and rotational speed. The technique has potential for large-scale and continuous applications in immobilization.Correspondence to: R. J. Neufeld     

杀蚊苏云金芽孢杆菌及其晶体蛋白研究进展

自从发现苏云金芽孢杆菌Bacillusthuringiensis(Bt)具有杀蚊活性以来,目前已发现多种Bt亚种或血清型对蚊虫具有杀虫活性,同时也发现了一些新的杀蚊晶体蛋白。在对杀蚊晶体蛋白的分子结构进行研究的基础上,对其的作用机理有了一定的了解。近年来利用DNA重组技术显著提高杀蚊晶体蛋白的合成和将不同菌种的杀蚊晶体蛋白进行联合表达,为有效控制蚊虫危害展示广阔前景。     

Correlation between alkaline activation of Bacillus thuringiensis var. kurstaki spores and crystal production

The spores of crystal-forming (Cry⁺) and non-crystal-forming (Cry^-) strains of Bacillus thuringiensis var. kurstaki and Bacillus cereus were tested for the ability to be activated by 0.1 m K₂CO₃ (pH 10). Only the spores of crystal-forming strains could be activated, and this phenotype was independent of whether crystals were present with the spores in the activation solution. The spores of a B. thuringiensis var. kurstaki strain that is temperature sensitive for protoxin accumulation could be activated by the alkaline solution when produced at the permissive temperature, whereas spores produced at the nonpermissive temperature were not activated. The results indicate that protoxin in the spore coat is responsible for the alkaline-activation phenotype and may serve an ecological function for the organism.     

苏云金芽孢杆菌杀虫晶体蛋白超量表达的机制

杀虫晶体蛋白是苏云金芽孢杆菌主要杀虫成分,进一步提高杀虫晶体蛋白的表达量是苏云金芽杆菌高效工程菌构建的主要途径。本文讨论了ｃｒｙ基因启动子活性、ｍＲＮＡ稳定性、不同ｃｒｙ基因间的协同表达发及伴了孢晶体的形成等几个方面在转录水平或转录后水平上对杀虫晶体蛋白表达的影响。     

On the formation of crystal proteins during sporulation in Bacillus thuringiensis var. thuringiensis

The sporulation potential of Bacillus subtilis as a function of position in the cell cycle was determined by transferring cells from growth medium to sporulation medium at various times during growth. Growth was induced by incubating heat-activated spores in rich medium or by diluting stationary phase vegetative cultures with fresh growth medium. The results supported earlier observations that sporulation potential is cell cycle dependent. The rise in sporulation potential was studied by exposing cultures to the inhibitors of cell wall and protein synthesis, vancomycin and chloramphenicol. The delay in the appearance of the peak of sporulation potential caused by these inhibitors compared with the reported lack of effect of nalidixic acid, indicates that the appearance of sporulation potential requires synthesis of a macromolecular component other than deoxyribonucleic acid. The effect of nalidixic acid in preventing the decline of the sporulation potential was compared with the effect of high temperature on a mutant temperature sensitive for the initiation of DNA replication. It was found that prevention of chromosome completion with nalidixic acid maintained a high sporulation potential, whereas prevention of chromosome re-initiation in the temperature sensitive mutant did not affect the decline in sporulation potential as the cells enter stationary phase.Abbreviations NAL Nalidixic acid - HPUra 6-(p-hydroxyphenylazo)-uracil - VAN Vancomycin - CAM Chloramphenicol - BHI Brain heart infusion broth - c.f.u. Colony forming units     

Inactivation of Bacillus thuringiensis spores by ultraviolet and visible light.

The inactivation of Bacillus thuringiensis spores and spores treated with two protectants, one proteinaceous and the other a commercial product, Shade, at wavelengths of the near-ultraviolet and visible spectra and at 254 nm is described. Determination of the inactivating wavelengths may be used to establish an efficient sunlight protective system for B. thuringiensis when used as a microbial insecticide.     

Biochemistry and genetics of insect resistance to Bacillus thuringiensis insecticidal crystal proteins

Abstract Current knowledge of biochemical mechanisms of insect resistance to Bacillus thuringiensis is reviewed. Available information on resistance inheritance and on patterns of cross-resistance is included. Modification of the binding sites for B. thuringiensis insecticidal crystal proteins has been found in different populations of three insect species. This resistance mechanism seems to be inherited as a single recessive or partially recessive major gene, and the resistance levels reached are high. Altered proteolytic processing of B. thuringiensis crystal proteins has been suggested to be involved in one case of resistance. From the available data it seems that binding site modification is the most significant resistance mechanism under field conditions.     

Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.     

Swelling behaviour of alginate–chitosan microcapsules prepared by external gelation or internal gelation technology

Swelling behaviour is one of the important properties for microcapsules made by hydrogels, which always affects the diffusion and release of drugs when the microcapsules are applied in drug delivery systems. In this paper, alginate–chitosan microcapsules were prepared by different technologies called external or internal gelation process respectively. With the volume swelling degree (S_w) as an index, the effect of properties of chitosan on the swelling behaviour of both microcapsules was investigated. It was demonstrated that the microcapsules with low molecular weight and high concentration of chitosan gave rise to low S_w. Considering the need of maintaining drug activity and drug loading, neutral pH and short gelation time were favorable. It was also noticed that S_w of internal gelation microcapsules was lower than that of external gelation microcapsules, which was interpreted by the structure analysis of internal or external gelation Ca–alginate beads with the aid of confocal laser scanning microscope.     

苏云金芽孢杆菌Bt9875晶体蛋白诱导HL-60细胞凋亡

[目的]研究苏云金芽孢杆菌(Bacillus thuringiensis,Bt)Bt9875菌株晶体蛋白对人急性髓细胞性白血病细胞HL-60的影响.[方法]采用MTT比色、荧光显微观察、DNA凝胶电泳、流式细胞术等方法来检测不同浓度的Bt9875晶体蛋白处理后HL-60细胞的凋亡特征.[结果]Bt9875晶体蛋白对HL-60细胞的生长具有明显的抑制作用,且随着蛋白质浓度的增加对HL-60细胞生长抑制愈加明显,而对正常人外周血单个核细胞(PBMC)无作用;荧光显微镜下观察发现经该蛋白作用后HL-60细胞核的形态呈现凋亡特征;流式细胞术分析表明,HL-60细胞经100 μg/mL晶体蛋白作用后,凋亡率达到52%;琼脂糖凝胶电泳显示细胞DNA呈梯状降解.[结论]初步证明了Bt9875晶体蛋白在体外能够明显抑制HL-60细胞的增长,并诱导其凋亡,这为苏云金芽抱杆菌晶体蛋白的应用开创了新的思路.