|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1999 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1998 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1997 : ¥1,200,000 (Direct Cost : ¥1,200,000)
We investigated drug extrusion system and ion extrusion systems in Vibrio parahaemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis from structural and functional points of view. Regarding drug extrusion systems, we found several new drug efflux systems, NorM from V. parahaemolyticus, MexXY from P. aeruginosa and EbrAB from B. subtilis. Drug (ethidium) efflux via the NorM was stimulated by Na+. Imposition of inwardly directed Na+ gradient in energy starved cells that were preloaded with ethidium elicited efflux of ethidium. Imposition of inwardly directed ethidium gradient in energy starved cells that were preloaded with Na+ elicited efflux of Na+. These results clearly indicate that drug is extruded from cells in exchange for Na+, namely the NorM is a drug/Na+ antiporter. This is the first example of drug/Na+ antiporter in biological world. Judging from sequence similarity found in databases, it seems that there are some drug/Na+ antiporters in some other bacteria. The MexXY was found to be three-components type multidrug efflux pump. This system is involved in resistance of P. aeruginosa against erythromycin, aminoglycosides and β-lactams. Regarding another efflux systems, ion efflux systems, we found several Na+ efflux systems and analyzed them, Mnh from S. aureus, NhaB from V. parahaemolyticus and NhaG from B. subtilis. These systems extrude Na+ in exchange for H+. The Mnh system is a novel type Na+/H+ antiporter, multisubunit type antiporter. We also analyzed ion efflux from E. coli cells using patch clamp method. This method enabled direct measurement of ion flux in bacterial cells.