苏云金芽孢杆菌转座因子研究进展

近年来的研究发现苏云金芽孢杆菌转座因子和许多毒力因子可能是紧密联系的。由于转座因子的特殊性质,使它们在现代农业生物技术中有着广泛的应用前景,科学家对苏云金芽孢杆菌转座因子的研究也在不断深入。本文主要针对苏云金芽孢杆菌转座因子的研究进展进行综述,并对发展前景进行展望。     

Advances in plant biotechnology and its adoption in developing countries

Developing countries are already benefiting and should continue to benefit significantly from advances in plant biotechnology. Insect-protected cotton containing a natural insecticide protein from Bacillus thuringiensis (Bt cotton) is providing millions of farmers with increased yields, reduced insecticide costs and fewer health risks. Many other useful plant biotechnology products that can benefit poor farmers and consumers are in the research and development pipelines of institutions in developing countries, and should soon reach farmers' fields.     

The biotechnology of Bacillus thuringiensis

One of the challenges in the application of biotechnology to pest control is the identification of agents found in nature which can be used effectively. Biotechnology offers the potential of developing pesticides based on such agents which will provide environmentally sound and economically feasible insect control alternatives. Such an agent, the insect pathogen Bacillus thuringiensis, is the subject of intense investigations in several laboratories. Insecticides which use the entomocidal properties of B. thuringiensis are currently produced and sold worldwide; new products are currently in the development stage. Herein, the biology and genetics of B. thuringiensis and the problems associated with current products are critically reviewed with respect to biotechnology. Moreover, the economic and regulatory implications of technologically advanced products are evaluated.     

Cover Caption

To answer the question of whether phylogenetic relatedness can act as a proxy for insect response to Bacillus thuringiensis, the susceptibility of eleven colonies of lepidoptera representing different families, genera, species and colonies was compared. While species within genera had similar responses, genera within families did not. Two laboratory colonies of one species differed, suggesting a colony divergence. We conclude that caution is needed in using surrogate species to assess Bacillus thuringiensis effects on non‐target insects. (see pages 803‐812). Imaged by E. P. J. Burgess, Plant & Food Research, New Zealand     

台湾的微生物防治

化学防治自1940年代后虽然为农业和医界害虫防治带来许多的经济效益。相对的。因其对非标的生物(有益生物,野生生物．家畜及农场操作人员)有毒害作用．及其残留量(residue)可在空气中．土壤,水中发现．又可经作物带给消费。也使我们得考虑其安全性．     

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.     

Persistence of Bacillus thuringiensis subsp. kurstaki in Urban Environments following Spraying

Bacillus thuringiensis subsp. kurstaki is applied extensively in North America to control the gypsy moth, Lymantria dispar. Since B. thuringiensis subsp. kurstaki shares many physical and biological properties with Bacillus anthracis, it is a reasonable surrogate for biodefense studies. A key question in biodefense is how long a biothreat agent will persist in the environment. There is some information in the literature on the persistence of Bacillus anthracis in laboratories and historical testing areas and for Bacillus thuringiensis in agricultural settings, but there is no information on the persistence of Bacillus spp. in the type of environment that would be encountered in a city or on a military installation. Since it is not feasible to release B. anthracis in a developed area, the controlled release of B. thuringiensis subsp. kurstaki for pest control was used to gain insight into the potential persistence of Bacillus spp. in outdoor urban environments. Persistence was evaluated in two locations: Fairfax County, VA, and Seattle, WA. Environmental samples were collected from multiple matrices and evaluated for the presence of viable B. thuringiensis subsp. kurstaki at times ranging from less than 1 day to 4 years after spraying. Real-time PCR and culture were used for analysis. B. thuringiensis subsp. kurstaki was found to persist in urban environments for at least 4 years. It was most frequently detected in soils and less frequently detected in wipes, grass, foliage, and water. The collective results indicate that certain species of Bacillus may persist for years following their dispersal in urban environments.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light.

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Correlation between insecticidal and antibiotic activities of Bacillus thuringiensis parasporal crystals

The work was concerned with studying the insecticide and antibiotic activities of solutions containing parasporal crystals of several Bacillus thuringiensis subspecies. The correlation coefficients of these values were determined. A direct correlation was established between the insecticide and antibiotic activities of solutions of parasporal crystals from several B. thuringiensis subspecies. Therefore, the quality of bacterial insecticides can be controlled by assaying the antibiotic activity of polypeptide crystals in biotechnology.     

苏云金芽胞杆菌转座因子的转座机理及其与杀虫晶体蛋白基因的关系

各种苏云金芽胞杆菌在杀虫毒力和杀虫谱上有很大差异。研究表明,这种特异性的杀虫毒力与存在于苏云金芽胞杆菌内的转座因子有密切关系,不同类型的转座因子其转座方式各异,总的来说可分为３种,即同源重组、转座重组和特异位点重组。这种转座过程的发生往往伴随着苏云金芽胞杆菌杀虫晶体蛋白的变异,这在基因工程菌的构建和杀虫多样性的研究上有着重要意义。     

The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.     

Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system.

The cry genes of Bacillus thuringiensis encode a diverse group of crystal-forming proteins that exhibit insecticidal activity, particularly against the larvae of lepidopteran, coleopteran, and dipteran insects. The efficacy of B. thuringiensis-based biopesticides may be improved through the genetic manipulation of these genes. A gene transfer system has been developed for the introduction and maintenance of cloned insecticidal cry genes on small plasmids in B. thuringiensis. This vector system combines a B. thuringiensis plasmid replicon and an indigenous site-specific recombination system that allows for the selective removal of ancillary or foreign DNA from the recombinant bacterium after introduction of the Cry-encoding plasmid. The site-specific recombination system is useful for engineering strains with unique combinations of cry genes, resulting in new active ingredients with improved insecticidal properties.     

Transformation of Bacillus thuringiensis by electroporation

Abstract A simple and reliable method of transforming Bacillus thuringiensis is described. This protocol, based on high-voltage electro-transformation (electroporation) in the presence of polyethylene glycol, allows introduction of plasmid DNA in most of the Bacillus thuringiensis strains tested. Efficiencies vary between 10² and 10⁵ transformants per μg DNA, depending on the strain or the replicon used.     

The construction of Bacillus thuringiensis strains expressing novel entomocidal delta-endotoxin combinations.

Using our recently reported method of electroporation to transform Bacillus thuringiensis [Bone & Ellar (1989) FEMS Microbiol. Lett. 58, 171-178], cloned B. thuringiensis entomocidal delta-endotoxin genes have been introduced into several native B. thuringiensis strains. In many cases the resulting transformants expressed both their native toxins and the cloned toxin, producing strains with broader toxicity spectra. The introduction of the var. tenebrionis toxin gene into B. thuringiensis var. israelensis resulted in a strain with activity against Pieris brassicae (cabbage white butterfly), an activity which neither parent strain possesses. We discuss further the possibility of synergism and also the problems associated with introducing cloned DNA by this method.     

Identification and characterization of strains of Bacillus thuringiensis by genomic fingerprinting using biotinylated phage M13 DNA]

Genomic DNA of the entomopathogenic bacterium Bacillus thuringiensis was analyzed by the genomic fingerprinting technique. The biotin-labeled single-stranded DNA of the phage M13 was used as a marker of hypervariable sequences. A procedure for analyzing the differentiation among various Bacillus thuringiensis strains was developed. Characteristic patterns of fingerprints were obtained for several strains, the main representatives of subspecies that are most frequently used in the manufacture of bacterial insecticides, such as subsp. thuringiensis, subsp. kurstaki, and subsp. galleriae. Because no essential differences were revealed in band patterns upon comparing fingerprints of crystal-producing bacterial strains with those of acrystallic mutants, it was assumed that the loss of crystal-producing ability in the insect pathogen Bacillus thuringiensis is not connected with significant rearrangement of its genome.     

Microbial control and biotechnology research on Bacillus thuringiensis in China

The current status of production and application of biopesticides for pest control in China is briefly reviewed, with a focus on research advances in microbial control with Bacillus thuringiensis (Bt). These have led to improvements in Bt production, exploitation of Bt gene resources, and development of engineered Bt insecticides and transgenic Bt crops that have expanded host ranges and increased efficacy against target pests. Both conventional and biotechnology approaches need to be employed to achieve further progress in discovery, production technology, formulation processing, development of quality standards and recommended use patterns.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Tubular bioreactors: case study of bioreactor performance for industrial production and scientific research

Application of bioreactors is dominated by industrial production with the consequence that bioreactors also for scientific purposes are mainly used following an empiric pragmatic approach. For the sake of a breakthrough in biotechnology in general, and especially for advanced process development, a more systematic approach is emphasized here. This methodology in bioreactor performance studies is explained and the meaning clarified in a case study of a new type of tubular bioreactor. The central role of so-called "model bioreactors" in bench-scale applications is illustrated as a powerful contribution to the optimal design of bioreactors in technical scale. Pilot plant data in case of a tubular reactor for the production of ethanol with Zymomonas and biopesticides with Bacillus thuringiensis are presented.