|Budget Amount *help
¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1998 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1997 : ¥2,000,000 (Direct Cost : ¥2,000,000)
(i) Enterococcus hirae ntp operon encodes both a vacuolar ATPase, which transports Na+ as well as Li+, and KtrII K+ transport system. A plasmid pHE-CAT1, in which the chloramphenicol acetyltransferase gene (CAT) was placed downstream the ntp promoter, was introduced into a mutant totally defective in Na+ extrusion. The CAT activity of this transformant was increased preferentially by elevating the concentration of Na+, but not Li+, in medium. This selectivity means that the vacuolar ATPase operates for establishing the Na+ concentration gradient useful for various Na+-coupled energy transducing systems not for extruding toxic lithium ion.
(ii) Effect of medium pH on expression of the ntp operon was also examined by the CAT activity and western blotting. The CAT activity was significantly increased by elevating medium pH highly alkaline, correlating well with the increase in the amounts of the ATPase subunits observed by western blotting. E.hirae has another Na+ extrusion system : Na+/H+ antiporter, but this system operates for sodium extrusion at low pH because the proton motive force is negligible at high pH.Therefore, high pH-responsive expression of this operon is physiologically important for this organism.
(iii) Since the vacuolar Li+-translocating ATPase is not induced by Li+, the growth of E.hirae was inhibited by more than 50 mM LiCl in media. When cell were adapted to high Na+ medium where V-ATPase was highly induced, cells grew well in the presence of 50 mM LiCl. We expect that Na+specific regulatory mutant for expression of the ntp operon was included among Li+-tolerant mutant.