A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore
A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore
复制标题
DOI：
10.1046/j.1365-2958.1997.2951656.x
复制
发表时间：
1997-03-01
影响因子：
3.6
通讯作者：
Krulwich, TA
中科院分区：
文献类型：
--
作者：
Cheng, JB;Guffanti, AA;Krulwich, TA
在pH升高条件下，通过Na+抑制生长而分离得到的枯草芽孢杆菌（Bacillus subtilis）转座突变体JC901缺乏能量依赖性Na+挤压。通过碱性转移后细胞质pH的调节来评估，突变体JC901对Na+依赖的pH稳态的能力相对于野生型菌株没有受到影响。该转位位点位于基因natB的3'末端附近，该基因预计编码膜蛋白natB。NatB在其c端具有六个假定的跨膜区域，并且显示出与真核Na+/H+交换器区域的适度序列相似性。序列和Northern blot分析表明，natB与上游基因natA形成一个操纵子。预测的natA产物是atp结合蛋白家族的成员，是atp结合盒（ABC）或交通atp酶类型的运输系统的组成部分。受natAB启动子控制的lacZ基因的表达表明，乙醇和原载体羰基氰化物对氯苯基腙（CCCP）诱导了该操纵子的表达，Na+和K+的诱导作用较弱，而胆碱和高浓度蔗糖则没有诱导作用。克隆到重组质粒（pJY1）中的natAB基因的恢复，补充了突变体JC901在pH升高时的Na+敏感表型，并显著提高了突变体在pH 7下对乙醇和CCCP生长抑制的抗性；然而，没有将乙醇从表达natAB的细胞中排除，因此乙醇抗性不是由natAB依赖的乙醇外排引起的。pJY1突变体的转化显著增强了Na+外排能力，CCCP进一步刺激了Na+外排能力。在没有CCCP的情况下，K+刺激natab介导的Na+外排。通过Rb-86（+）摄取监测，同时发生了依赖natab的K+摄取；这种摄取被CCCP抑制，因此是继发于最初的电致Na+外排。一株枯草芽孢杆菌突变株（BsAJ96）的大部分natA和全部natB被特异霉素耐药基因盒取代，其表型特性与JC901相同。在厌氧条件下，利用一株编码f1f0 - atp酶（BD99-A）的atp基因缺失的枯草芽孢杆菌，葡萄糖以砷酸盐敏感的方式激活Na+排斥；在atp和natAB基因均缺失的菌株（BsAJ96-A）中不存在这种排除能力。我们得出结论，NatAB是一种可诱导的ABC转运系统，它催化atp依赖的电致Na+挤出，而不需要机械耦合的质子或K+摄取。这是一种新的Na+挤压模式，假设在排除细胞毒性Na+和K+摄取的二次刺激中起诱导作用，特别是当膜作为离子渗透屏障的功能被诸如醇或解偶联剂等药物损害时。
A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na+ at elevated pH, was deficient in energy-dependent Na+ extrusion. The capacity of the mutant JC901 for Na+-dependent pH homeostasis was unaffected relative to the wild-type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3'-terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane-spanning regions at its C-terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP-binding proteins that are components of transport systems of the ATP-binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natAB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p-chlorophenylhydrazone (CCCP), and, more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na+-sensitive phenotype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol-resistance does not result from NatAB-dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+ efflux, which was further stimulated by CCCP. In the absence of CCCP, NatAB-mediated Na+ efflux was stimulated by K+. Concomitant NatAB-dependent K+ uptake occurred, as monitored by Rb-86(+) uptake; this uptake was inhibited by CCCP and is thus secondary to the primary, electrogenic Na+ efflux. A B. subtilis mutant strain (BsAJ96) in which most of natA and all of natB was replaced by a spectinomycin-resistance-gene cassette exhibited phenotypic properties identical to JC901. Under anaerobic conditions, using a strain of B. subtilis deleted in atp genes encoding the F1F0-ATPase (BD99-A), glucose energized Na+ exclusion in an arsenate-sensitive manner; this exclusion capacity was absent in a strain deleted both in atp and natAB genes (BsAJ96-A). We conclude that NatAB is an inducible, ABC transport system that catalyses ATP-dependent electrogenic Na+ extrusion without mechanistically coupled proton or K+ uptake. This is a novel mode of Na+ extrusion that is hypothesized to play an inducible role in exclusion of cytotoxic Na+ and in the secondary stimulation of K+ uptake, especially when the function of the membrane as an ion-permeability barrier is compromised by agents such as alcohols or uncouplers.