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形成细菌，其产生多种生物杀虫蛋白，这些生物杀虫蛋白对主要作物害虫和疾病媒介具有活性（毒性）。 取决于生物体，这些杀虫蛋白在指数期晚期、稳定期或孢子形成期间积累在细胞的细胞质中，以形成结晶内含物。 通常，包涵体蛋白在营养细胞或孢子形成细胞内形成，并且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属物种注意到了这一规律。 在BT亚种finitimus的情况下，形成了两个结晶夹杂物。 包含这两种内含物的主要蛋白质大小相似，但具有独特的抗原决定簇，在孢子形成的不同时间阶段形成，并积累在细胞中相对于孢子外壁的不同位点。 一种是小内含物，形成于孢子外壁外，并独立于孢子释放到环境中。 第二，主要的，内含物和孢子本身一起被内生孢子的孢子外壁包围。 内含物和孢子一起释放，并且内含物在自溶后仍然附着在孢子上。 在数千种已知的细菌菌株中，这种伴孢内含物的外孢壁封闭是罕见的事件。 外孢壁内的伴孢包涵体蛋白的靶向机制尚不清楚，这是本研究项目的主题，本项目的目的是使用亚种finitimus的伴孢包涵体蛋白来确定包涵体蛋白的内在性质和蛋白质在外孢壁封闭后表达的环境的相对贡献。 克隆技术将用于分离外孢壁封闭和游离伴孢包含蛋白的基因。 将对基因进行测序，并鉴定和比较两种基因的调控区。 分子技术将用于交换两个基因上的调控区，然后监测包含和积累的位点，作为合成时间对外孢壁包裹包含蛋白的相对影响的量度。 将通过电穿孔将克隆的基因引入替代细胞环境（孢子形成+，包涵体-变体）中，并监测相对于孢子外壁的包涵体积累位点，以确定细胞环境对靶向伴孢包涵体蛋白的相对贡献。 编码序列的区域将被交换，并且定点诱变将被用于改变编码序列，以确定靶向是否是序列特异性的。 生物农药或者作为干粉分布，或者在转基因植物中表达包含蛋白的毒性组分。 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抗害虫菌株。 增加可用性和减少对害虫抗性菌株的选择将提高天然杀虫剂的有效性。特别是，外孢壁封闭的伴孢内含物比自由内含物更不易受环境降解的影响。 外孢壁赋予孢子-内含物复合物疏水性，并保护所包围的内含物免于降解。 因此，该芽孢杆菌降低了内含物-芽孢复合物在环境中的可用性，导致该复合物在水性环境的空气-水界面处浓缩，并产生天然的"时间释放"效应。因此，了解伴孢内含物的外孢壁封闭机制具有经济和农业意义，作为改善天然农药向环境的递送和控制其表达的手段。

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.