A major Li+ extrusion system NhaB of Pseudomonas aeruginosa:: Comparison with the major Na+ extrusion system NhaP
A major Li+ extrusion system NhaB of Pseudomonas aeruginosa:: Comparison with the major Na+ extrusion system NhaP
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DOI：
10.1111/j.1348-0421.2004.tb03520.x
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发表时间：
2004-01-01
影响因子：
2.6
通讯作者：
Tsuchiya, T
中科院分区：
文献类型：
--
作者：
Kuroda, T;Fujita, N;Tsuchiya, T
从铜绿假单胞菌的染色体 DNA 中克隆了编码 Li+ 挤出系统的基因，并在大肠杆菌细胞中表达。该基因使大肠杆菌 KNabc 细胞能够生长，但由于缺乏主要的 Na+(Li+)/H+ 反向转运蛋白，这些细胞在 10 mM LiCl 或 0.1 M NaCl 存在的情况下无法生长。我们在大肠杆菌 KNabc 细胞制备的膜囊泡中检测到了 Li+/H+ 和 Na+/H+ 反向转运活性，该细胞含有携带克隆基因的质粒。该逆向转运蛋白的活性依赖于 pH 值，最佳 pH 活性在 pH 7.5 至 8.5 之间。这些特性表明该反向转运蛋白与我们之前报道的来自铜绿假单胞菌的 Na+/H+ 反向转运蛋白 NhaP 不同，NhaP 对 Na+ 具有相当的特异性，但不能有效地挤出 Li+。该基因被测序并鉴定出开放阅读框（ORF）。从ORF推导的氨基酸序列显示出与大肠杆菌和副溶血弧菌的NhaB Na+/H+逆向转运蛋白的同源性（约60%同一性和90%相似性）。因此，我们将反向转运蛋白指定为铜绿假单胞菌的 NhaB。携带来自铜绿假单胞菌的 nhaB 基因的大肠杆菌 KNabc 能够在 10 至 50 mM LiCl 存在下生长，但携带 nhaP 的 KNabc 在这些条件下无法生长。来自铜绿假单胞菌的 NhaB 的反向转运活性是在大肠杆菌中产生的，并且对 Li+ 和 Na+ 的表观 Km 值分别为 2.0 mM 和 1.3 mM。阿米洛利抑制反向转运活性，Li+ 和 Na+ 的 Ki 值分别为 0.03 mM 和 0.04 mM。
A gene encoding a Li+ extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells. The gene enabled growth of E. coli KNabc cells, which were unable to grow in the presence of 10 mM LiCl or 0.1 M NaCl because of the lack of major Na+(Li+)/H+ antiporters. We detected Li+/H+ and Na+/H+ antiport activities in membrane vesicles prepared from E. coli KNabc cells that harbored a plasmid carrying the cloned gene. Activity of this antiporter was pH-dependent with an optimal pH activity between pH 7.5 and 8.5. These properties indicate that this antiporter is different from NhaP, an Na+/H+ antiporter from P aeruginosa that we reported previously, and that is rather specific to Na+ but it cannot extrude Li+ effectively. The gene was sequenced and an open reading frame (ORF) was identified. The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na+/H+ antiporters of E. coli and Vibrio parahaemolyticus. Thus, we designated the antiporter as NhaB of P aeruginosa. E. coli KNabc carrying the nhaB gene from P aeruginosa was able to grow in the presence of 10 to 50 mM LiCl, although KNabc carrying nhaP was unable to grow in these conditions. The antiport activity of NhaB from P aeruginosa was produced in E. coli and showed apparent Km values for Li+ and Na+ of 2.0 mM and 1.3 mM, respectively. The antiport activity was inhibited by amiloride with a Ki value for Li+ and Na+ of 0.03 mM and 0.04 mM, respectively.