Genetic and Immunological Analysis of Gas Vesicles in Halobacterium Halobium

盐杆菌气体囊泡的遗传和免疫学分析

• 批准号: 9221144
• 负责人: Elizabeth Stuart
• 金额: $ 30.99万
• 依托单位: University of Massachusetts Amherst
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9221144&HistoricalAwards=false
• 关键词: Genetic; Immunological; Analysis; Gas; Vesicles

9221144 DasSarma Gas vesicles are buoyant intracellular organelles found in many aquatic bacteria including halobacteria, cyanobacteria, and methanogens. The vesicles are filled with ambient gas and surrounded by a membrane composed only of protein (no lipids). Gas vesicles come in a variety of shapes but generally contain a cylindrical mid-section with conical ends. Although their exact function is controversial, their buoyancy induces cells to float and thereby increases the availability of light for photosynthesis and oxygen for respiration. Biosynthesis of gas vesicles proceeds bidirectionally starting from the apicies of the cones with incorporation of additional subunits in the central region. The vesicle membrane is impermeable to water and accumulation of gases in the vesicle interior is thought to occur only as a result of passive diffusion of dissolved gases in the medium. Our analysis of high-frequency gas vesicle-deficient mutants of the halophilic archaebacterium, Halobacterium halobium, resulted in the identification of a cluster of thirteen genes, gvpMLKJIHGREDACN, involved in gas vesicle synthesis. We are studying the function of these genes in gas vesicle synthesis using a combinaton of molecular genetic methodology that we have recently developed for H. halobium and immunocytochemical localization techniques. We will utilize an E. coli-H.halobium shuttle plasmid containing the gvp gene cluster and specifying gas vesicle synthesis in H. halobium for genetic analysis by linker scanning and cassette insertion mutagenesis. The effects of mutations on gas vesicle synthesis will be analyzed by electron microscopy of vesicles and vesicle fragments. Antibody probes will be used for localization of gvp products by Western blotting analysis and electron microscopic analysis after immunogold staining vesicles, vesicle fragments and ultrathin frozen sections of cells. These studies should provide definitive information on the function of gvp gene products and a detailed picture of gas vesicle structure and synthesis. %%% These studies should lay the groundwork for genetically engineering bacteria to float, a process with significant potential for applications in biotechnology. ***

小行星9221144 气体囊泡是在许多水生细菌中发现的具有浮力的细胞内细胞器，包括嗜盐菌、蓝藻和产甲烷菌。 囊泡充满环境气体，并被仅由蛋白质（无脂质）组成的膜包围。 气体囊泡有各种形状，但通常包含具有圆锥形末端的圆柱形中间部分。 虽然它们的确切功能是有争议的，但它们的浮力诱导细胞漂浮，从而增加光合作用的光和呼吸的氧气的可用性。 生物合成的气泡进行双向开始的视锥细胞的附加亚基在中央区域的掺入。 囊泡膜对水是不可渗透的，并且囊泡内部的气体积聚被认为仅由于介质中溶解的气体的被动扩散而发生。 我们的高频气泡缺陷突变体的嗜盐古细菌，Halobacterium halobium的分析，导致在鉴定的一组13个基因，gvpMLKJIHGREDACN，参与气泡的合成。 我们正在研究这些基因在气泡合成中的功能，使用我们最近为H.嗜盐菌和免疫细胞化学定位技术。 我们将使用一个E。coli-H.halobium穿梭质粒含有gvp基因簇，并指定在H. 用于通过接头扫描和盒式插入诱变进行遗传分析的盐生菌。 突变对气泡合成的影响将通过电子显微镜的囊泡和囊泡碎片进行分析。 抗体探针将用于在免疫金染色囊泡、囊泡片段和细胞的冰冻切片后通过Western印迹分析和电子显微镜分析定位gvp产物。 这些研究将提供gvp基因产物功能的确定性信息，以及气泡结构和合成的详细图像。 这些研究应该为基因工程细菌漂浮奠定基础，这是一个在生物技术中具有巨大应用潜力的过程。 ***