Giant unilamellar vesicles (GUVs) are vesicles>1 μm in diameter that provide an environment in which the effect of a confined reaction volume on intravesicular reactions can be investigated. By synthesizing EmrE, a multidrug transporter from Escherichia coli, as a model membrane protein using a reconstituted in vitro transcription-translation system inside GUVs, we investigated the effect of a confined volume on the synthesis and membrane integration of EmrE. Flow cytometry was used to analyze multiple properties of the vesicles and to quantify EmrE synthesis inside GUVs composed of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. We found that EmrE was synthesized and integrated into the GUV membrane in its active form. We also found that the ratio of membrane-integrated EmrE to total synthesized EmrE increased with decreasing vesicle volume; this finding is explained by the effect of an increased surface-area-to-volume ratio in smaller vesicles. In vitro membrane synthesis inside GUVs is a useful approach to study quantitatively the properties of membrane proteins and their interaction with the membrane under cell-mimicking environments.

The surfactants tested in this study lysed Bacilus subtilis 168 cells at the logarithmic growth phase. Results obtained with inhibitors and a mutant that had defective autolytic enzymes suggested that cell lysis resulted from the deregulation of autolysin activity. The addition of surfactants at sublytic concentrations produced twisted cells, filamented cells, or both. Autolysins extracted with 5 M LiCl from the cell wall fraction and lysozyme added to cells that were treated with surfactants restored the apparently normal cell rod morphology, suggesting that surfactants interfere with the role of autolysins in normal construction of the cell envelope. The rates of cellular autolysis and autolysin activity remaining in growing cells after exposure to a surfactant at a sublytic concentration decreased, although the rate of turnover of cell wall peptidoglycan was the same as that of control cells. Surfactants were suggested to interact with the regulatory system of autolysins and, thus, to affect the activities of autolysins in B. subls cells and to cause either morphological changes or cell autolysis, depending on the concentration of surfactants.

Ishida2, Akinori Ikeuchi2, lzumi Kurnagaii. Mit$uo Umetsui (i7bhoku univ. tech., 2Tbyota Central RdiD tab) Cellulose is naturally occuning plant cell wall constituent and most abundant po]ysaccharides on earth. An economically viable process for converting this biomass to fermentable sugar is a prerequisite for the development ofcellulosic biomass-based bio-refineries for the production of food, feed, chemieals and fuels. Cel]ulDse can be converted to fermentable sugar by microbial cellulose degrading enzyrnes, so ca]]ed ce]lulase. Ce]]u]ases are general]y modular proteins with an independent catalytic domain (CD) and a caTbohydrate-binding module (CBM) and in some bacteria, CDs with different functions are clustered onagiantscaffoldproteincontainingCBM,viacoheisin-dockerininteractionto eencient degradation, so called cellulosome. Recently, we proposcd a new design of artificial cellulosome to enhance ce]]ulase activity with streptavidin or streptavidin modified CdSe quantum dot as a scaffold via biotin-avidin interaction.Activity of the cluster enzyme with two kind of CBD indicated that depend on combination of scafTbld and CBD,