Genetic and Immunological Analysis of Gas Vesicles in Halobacterium Halobium

[221144] DasSarma气体囊泡是在许多水生细菌中发现的浮力胞内细胞器，包括盐细菌、蓝藻细菌和产甲烷菌。囊泡充满环境气体，并被仅由蛋白质（无脂质）组成的膜包围。气体囊泡有多种形状，但通常包含一个圆柱形的中间部分和圆锥形的末端。尽管它们的确切功能存在争议，但它们的浮力诱导细胞漂浮，从而增加光合作用所需的光和呼吸所需的氧气。气体囊泡的生物合成是双向进行的，从球果的尖端开始，在中心区域加入额外的亚基。囊泡膜不透水，气体在囊泡内部的积聚被认为是溶解气体在介质中被动扩散的结果。通过对嗜盐古细菌（Halobacterium halobium）高频气体囊泡缺陷突变体的分析，我们鉴定出了一个由13个基因组成的基因簇gvpMLKJIHGREDACN，这些基因与气体囊泡合成有关。我们正在研究这些基因在气体囊泡合成中的功能，使用我们最近为盐芽孢杆菌开发的分子遗传学方法和免疫细胞化学定位技术的组合。我们将使用大肠杆菌。含gvp基因簇的盐生菌穿梭质粒，并在盐生菌中指定气囊合成，用于连接子扫描和盒式插入诱变的遗传分析。突变对气体囊泡合成的影响将通过囊泡和囊泡片段的电镜分析。免疫金染色细胞囊泡、囊泡片段和超薄冷冻切片后，利用抗体探针进行免疫印迹分析和电镜分析，定位gvp产品。这些研究将提供关于gvp基因产物功能的明确信息，以及气体囊泡结构和合成的详细图像。这些研究应该为基因工程细菌的漂浮奠定基础，这一过程在生物技术中具有重大的应用潜力。＊＊＊

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. ***