Sardesai A A, Gowrishankar J
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
J Bacteriol. 2001 Jan;183(1):86-93. doi: 10.1128/JB.183.1.86-93.2001.
Transcription of the K(+) transport operon kdp in Escherichia coli is induced during K(+)-limited growth by the action of a dual-component phosphorelay regulatory system comprised of a sensor kinase (integral membrane protein), KdpD, and a DNA-binding response regulator (cytoplasmic protein), KdpE. In this study, we screened for new dke (named dke for decreased kdp expression) mutations (in loci other than kdpDE) that led to substantially decreased kdp expression. One dke mutation was shown to be in hns, encoding the nucleoid protein H-NS. Another dke mutation was mapped to trxB (encoding thioredoxin reductase), and an equivalent reduction in kdp expression was demonstrated also for trxA mutants that are deficient in thioredoxin 1. Exogenously provided dithiothreitol rescued the kdp expression defect in trxB but not trxA mutants. Neither trxB nor trxA affected gene regulation mediated by another dual-component system tested, EnvZ-OmpR. Mutations in genes dsbC and dsbD did not affect kdp expression, suggesting that the trx effects on kdp are not mediated by alterations in protein disulfide bond status in the periplasm. Reduced kdp expression was observed even in a trxB strain that harbored a variant KdpD polypeptide bearing no Cys residues. A trxB hns double mutant was even more severely affected for kdp expression than either single mutant. The dke mutations themselves had no effect on strength of the signal controlling kdp expression, and constitutive mutations in kdpDE were epistatic to hns and trxB. These results indicate that perturbations in cytoplasmic thiol oxidation status and in levels of the H-NS protein exert additive effects, direct or indirect, at a step(s) upstream of KdpD in the signal transduction pathway, which significantly influence the magnitude of KdpD kinase activity obtained for a given strength of the inducing signal for kdp transcription.
在大肠杆菌中，K⁺转运操纵子kdp的转录在K⁺限制生长期间，通过由传感器激酶（整合膜蛋白）KdpD和DNA结合反应调节因子（细胞质蛋白）KdpE组成的双组分磷酸化信号转导调节系统的作用而被诱导。在本研究中，我们筛选了导致kdp表达大幅降低的新dke（因kdp表达降低而命名为dke）突变（位于kdpDE以外的基因座）。一个dke突变被证明位于hns，其编码类核蛋白H-NS。另一个dke突变被定位到trxB（编码硫氧还蛋白还原酶），并且对于缺乏硫氧还蛋白1的trxA突变体，也证明了kdp表达有同等程度的降低。外源提供的二硫苏糖醇挽救了trxB突变体中的kdp表达缺陷，但不能挽救trxA突变体中的缺陷。trxB和trxA均不影响由另一个测试的双组分系统EnvZ-OmpR介导的基因调控。dsbC和dsbD基因中的突变不影响kdp表达，这表明trx对kdp的影响不是由周质中蛋白质二硫键状态的改变介导的。即使在携带不含半胱氨酸残基的变体KdpD多肽的trxB菌株中，也观察到kdp表达降低。trxB hns双突变体的kdp表达受到的影响比任何一个单突变体都更严重。dke突变本身对控制kdp表达的信号强度没有影响，并且kdpDE中的组成型突变对hns和trxB是上位性的。这些结果表明，细胞质硫醇氧化状态和H-NS蛋白水平的扰动在信号转导途径中KdpD上游的一个或多个步骤上直接或间接发挥累加效应，这显著影响了对于给定强度的kdp转录诱导信号所获得的KdpD激酶活性的大小。
