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Molecular genetics on expression of the ntp operon encoding vacuolar Na^+-ATPase

编码液泡Na+-ATP酶的ntp操纵子表达的分子遗传学

基本信息

批准号：
09680614
负责人：
KAKINUMA Yoshimi
金额：
$ 1.86万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680614/
关键词：
Vacuolar ATPase Sodium ion pH Gene expression Promoter ATPase

项目摘要

(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.

(i)人肠球菌的操纵子编码空泡atp酶（运输Na+和Li+）和KtrII K+运输系统。将氯霉素乙酰转移酶基因（CAT）置于ntp启动子下游的质粒ph - cat1导入Na+挤出完全缺陷的突变体。培养基中Na+浓度的升高优先提高了CAT活性，而Li+浓度的升高对CAT活性没有影响。这种选择性意味着液泡atp酶的作用是建立对各种Na+偶联能量转导系统有用的Na+浓度梯度，而不是挤压有毒的锂离子。（ii）通过CAT活性和western blotting检测培养基pH对ntp操纵子表达的影响。高碱性培养基pH值显著提高了CAT活性，这与western blotting观察到的atp酶亚基数量的增加有很好的相关性。E.hirae具有另一种Na+挤压系统：Na+/H+反转运体，但该系统在低pH下进行钠挤压，因为质子动力在高pH下可以忽略不计。因此，该操纵子的高pH响应性表达对该生物具有重要的生理意义。（iii）由于液泡Li+-易位atp酶不受Li+的诱导，培养液中超过50 mM的LiCl对E.hirae的生长有抑制作用。当细胞适应于高Na+培养基时，v - atp酶被高度诱导，细胞在50 mM LiCl存在下生长良好。我们期望在Li+耐受突变体中包括Na+特异性调控ntp操纵子表达的突变体。