RUI: Temporal Expression and Targeting of Parasporal Inclusion Proteins of Bacillus Thuringiensis Subspecies Finitimus

苏云金芽孢杆菌（BT）是一种革兰氏阳性孢子菌，可产生多种生物农药蛋白，对主要作物病虫害媒介具有活性（毒性）。根据生物体的不同，这些杀虫蛋白在指数期晚期、静止期或孢子期积聚在细胞质中，形成结晶包涵体。通常，包涵蛋白在营养细胞或孢子细胞内形成，在孢子形成的自溶阶段结束时分泌或释放。只有少数BT菌株和某些其他相关的芽孢杆菌物种例外。在有限BT亚种的情况下，形成了两个结晶包裹体。组成这两种包涵体的主要蛋白质大小相似，但具有独特的抗原决定因子，形成于孢子形成的不同时间阶段，并在细胞内相对于外孢子的不同位置积累。一种是较小的内含物，在孢子外形成并独立于孢子释放到环境中。第二个主要包涵体和孢子本身一起被内孢子的外孢子所包围。包涵体和孢子一起被释放，包涵体在自溶后仍附着在孢子上。在数千种已知的细菌菌株中，这种副孢子包涵体的外孢子封闭是罕见的。外孢子内靶向副孢子包涵蛋白的机制尚不清楚，这是本研究项目的主题。本项目的目的是利用有限亚种的拟孢子包涵蛋白来确定包涵蛋白的内在特性和包涵蛋白在外孢子封闭时表达的环境的相对贡献。克隆技术将用于分离外孢子封闭蛋白和游离副孢子包涵蛋白基因。对基因进行测序，鉴定和比较两个基因的调控区域。利用分子技术交换两个基因上的调控区域，然后监测包涵位点和积累位点，以衡量合成时间对包涵蛋白外孢子封闭的相对影响。克隆的基因将通过电穿孔引入不同的细胞环境（孢子+，包涵体-变体），并监测包涵体相对于外孢子的积累位置，以确定细胞环境对副孢子包涵蛋白靶向的相对贡献。编码序列的区域将被交换，位点定向诱变将被用来改变编码序列，以确定靶向是否具有序列特异性。基于副孢子内含物的生物农药是商业化生产的，用于控制经济上和医学上重要的害虫。该生物农药或以干粉形式分布，或其内含蛋白的毒性成分在转基因植物中表达。生物农药的有效性受到环境可用性的限制，并且持续使用已选择了抗虫菌株。增加可获得性和减少抗虫菌株的选择将提高天然农药的有效性。特别是外孢子封闭的副孢子包裹体比自由包裹体更不容易受到环境退化的影响。外孢囊赋予孢子包涵体疏水性，并保护包裹体不被降解。因此，暴露孢子延长了包裹体-孢子复合物在环境中的可用性，使复合物集中在水环境的空气-水界面，并产生自然的“时间释放”效应。因此，了解副孢子包涵体的外孢子封闭机制对于改善天然农药向环境的传递和控制其表达具有重要的经济和农业意义。

Bacillus thuringiensis (BT) is a Gram positive sporulating bacterium that produces a variety of biopesticidal proteins that are active against (toxic to) major crop pests and disease vectors. Depending on the organism, these pesticidal proteins accumulate in the cytoplasm of cells during late exponential phase, stationary phase, or sporulation, to form crystalline inclusions. Typically, inclusion proteins are formed inside the vegetative or sporulating cell and either secreted or liberated at the end of the autolysis stage of sporulation. Exceptions to this rule have been noted in only a few strains of BT and in certain other related Bacillus species. In the case of the BT subspecies finitimus, two crystalline inclusions are formed. The major proteins comprising each of these two inclusions are similar in size but have unique antigenic determinants, are formed at different temporal stages of sporulation, and accumulate at different sites in the cell with respect to the exosporium. One, a minor inclusion, forms outside the exosporium and is released into the environment independently of the spore. The second, major, inclusion and the spore itself are together surrounded by the endospore's exosporium. The inclusion and the spore are released together, and the inclusion remains attached to the spore after autolysis. Of the thousands of known strains of the bacterium, such exosporium enclosure of the parasporal inclusions is a rare event. The mechanism for targeting parasporal inclusion proteins within an exosporium is not known, and is the subject of this research project.The objective of this project is to use the parasporal inclusion proteins of subspecies finitimus to determine the relative contribution of the intrinsic properties of inclusion proteins and the environment in which the protein is expressed upon exosporium enclosure. Cloning techniques will be used to isolate the genes for exosporium enclosed and free parasporal inclusion proteins. The genes will be sequenced and the regulatory regions of the two genes will be identified and compared. Molecular techniques will be used to exchange the regulatory regions on the two genes and then monitor the site of inclusion and accumulation as a measure of the relative effects of time of synthesis on exosporium enclosure of inclusion protein. The cloned genes will be introduced into alternative cell environments (sporulation+, inclusion- variants) by electroporation and the site of inclusion accumulation with respect to the exosporium will be monitored to determine the relative contribution of the cell environment on targeting of parasporal inclusion proteins. Regions of the coding sequences will be exchanged and site directed mutagenesis will be used to alter the coding sequences to determine if targeting is sequence specific.Biopesticides based on parasporal inclusions are commercially produced to control economically and medically important pests. The biopesticide is either distributed as a dried powder or the toxic component of the inclusion protein is expressed in transgenic plants. The effectiveness of the biopesticides is limited by environmental availability and continued use has selected pest resistant strains. Increased availability and decreased selection of pest resistant strains will improve the effectiveness of the natural pesticide. In particular, exosporium enclosed parasporal inclusions are less susceptible to environmental degradation than free inclusions. The exosporium bestows hydrophobicity on the spore-inclusion complex, and protects the enclosed inclusion from degradation. The exposporium thus prolongs the availability of the inclusion-spore complex in the environment, causes the complex to concentrate at the air-water interface of an aqueous environment, and creates a natural "time-release" effect. Understanding the mechanism of exosporium enclosure of parasporal inclusions thus has economic and agricultural significance as a means of improving the delivery of the natural pesticide to the environment and controlling its expression.