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. ***
