Rektorschek M, Buhmann A, Weeks D, Schwan D, Bensch K W, Eskandari S, Scott D, Sachs G, Melchers K
Department of Molecular Biology, Byk Gulden Pharmaceuticals Konstanz, Germany.
Mol Microbiol. 2000 Apr;36(1):141-52. doi: 10.1046/j.1365-2958.2000.01835.x.
ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3',4', 5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB-kanR-EFGH operon all showed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited access of urea to internal urease at neutral pH. Measurement of 14C-urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.
ureI编码幽门螺杆菌的一种内膜蛋白。研究了细菌内膜和UreI在这种胃部微生物的酸保护及胞质脲酶活性调节中的作用。当该生物体在酸性pH值下暴露于质子载体（3,3',4',5-四氯水杨酰苯胺；TCS）时脲酶受到不可逆抑制，这表明内膜可保护脲酶免受酸的影响。通过用无启动子的卡那霉素抗性标记基因（kanR）替换脲酶基因簇的ureI基因，构建了几种幽门螺杆菌菌株的同基因ureI敲除突变体。携带修饰后的ureAB-kanR-EFGH操纵子的突变体在体外中性pH值下均表现出野生型水平的脲酶活性。这些突变体能够抵抗pH值>4.0的培养基，但不能抵抗pH值<4.0的培养基。野生型细菌在pH值<4.0时表现出高水平的脲酶活性，而这种能力在ureI突变体中并未保留，导致代谢受到抑制和细胞死亡。用源自质粒的幽门螺杆菌ureI进行的基因互补实验恢复了野生型特性。在用0.01%去污剂（聚氧乙烯-8-月桂基醚；C12E8）处理的结构完整但通透性增加的细菌中发现脲酶活性的激活，这表明在中性pH值下尿素进入内部脲酶受到膜的限制。对注射了ureI cRNA的非洲爪蟾卵母细胞中14C-尿素摄取的测量表明，只有注射的卵母细胞中的摄取表现出酸激活。因此，UreI介导的尿素摄取加速介导了在酸性条件下细胞内脲酶活性的增加。这种尿素通透性的增加对于幽门螺杆菌在pH值<4.0的环境中存活至关重要。不依赖ureI的脲酶活性可能足以维持细菌在pH值>4.0时的活力。
