Kalman L V, Gunsalus R P
Department of Microbiology, University of California, Los Angeles.
J Bacteriol. 1989 Jul;171(7):3810-6. doi: 10.1128/jb.171.7.3810-3816.1989.
Fumarate reductase catalyzes the final step of anaerobic electron transport in Escherichia coli when fumarate is used as a terminal electron acceptor. Transcription of the fumarate reductase operon (frdABCD) was repressed when cells were grown in the presence of either of the preferred terminal electron acceptors, oxygen or nitrate, and was stimulated modestly by fumarate. We have previously identified a locus called frdR which pleiotropically affects nitrate repression of fumarate reductase, trimethylamine N-oxide reductase, and alcohol dehydrogenase gene expression and nitrate induction of nitrate reductase expression (L. V. Kalman and R. P. Gunsalus, J. Bacteriol. 170:623-629, 1988). Transformation of various frdR mutants with plasmids identified two complementation groups, indicating that the frdR locus is composed of two genes. One class of mutants was not completely restored to wild-type frdA-lacZ expression or nitrate reductase induction when complemented with multicopy narX+ plasmids, whereas low-copy narX+ plasmid-containing strains were. A second class of frdR mutants was identified and shown to correspond to a previously described gene, narL (frdR2). Complementation of these strains with multicopy narL+ plasmids resulted in superrepression of frdA-lacZ expression and moderate elevation of nitrate reductase expression. Multicopy plasmids containing both narL+ and narX+ or only narL+ were able to complement narL mutants, whereas narX+ plasmids complemented narX mutants only when present in a copy number approximately equal to that of narL. Both narL and narX mutants retained normal oxygen control of frdA-lacZ expression. Both types of mutants are pleiotropic, as evidenced by derepressed levels of the fumarate reductase and trimethylamine N-oxide reductase enzymes and by defective induction of nitrate reductase when cells were grown in the presence of nitrate. These results indicate that both the narL and narX gene products must be present in a defined ratio in the cell. We conclude that these proteins interact to effect normal nitrate control of the anaerobic electron transport-associated operons. From these studies, we propose that narX encodes a nitrate sensor protein while narL encodes a DNA-binding regulatory protein which together function in a manner analogous to other two-component regulatory systems.
当延胡索酸用作末端电子受体时，延胡索酸还原酶催化大肠杆菌厌氧电子传递的最后一步。当细胞在氧气或硝酸盐这两种首选末端电子受体存在的情况下生长时，延胡索酸还原酶操纵子（frdABCD）的转录受到抑制，而延胡索酸会适度刺激其转录。我们之前鉴定出一个名为frdR的位点，它对延胡索酸还原酶、三甲胺N-氧化物还原酶和乙醇脱氢酶基因表达的硝酸盐抑制以及硝酸盐还原酶表达的硝酸盐诱导具有多效性影响（L. V. 卡尔曼和R. P. 冈萨卢斯，《细菌学杂志》170:623 - 629，1988年）。用质粒转化各种frdR突变体鉴定出两个互补组，这表明frdR位点由两个基因组成。一类突变体在用多拷贝narX⁺质粒互补时，frdA - lacZ表达或硝酸盐还原酶诱导并未完全恢复到野生型水平，而含有低拷贝narX⁺质粒的菌株则恢复到了野生型水平。鉴定出了第二类frdR突变体，并证明其对应于一个先前描述的基因narL（frdR2）。用多拷贝narL⁺质粒互补这些菌株会导致frdA - lacZ表达的超抑制以及硝酸盐还原酶表达的适度升高。含有narL⁺和narX⁺的多拷贝质粒或仅含有narL⁺的多拷贝质粒能够互补narL突变体，而narX⁺质粒只有在其拷贝数与narL大致相等时才能互补narX突变体。narL和narX突变体对frdA - lacZ表达均保留正常的氧气调控。这两种类型的突变体都是多效性的，当细胞在硝酸盐存在的情况下生长时，延胡索酸还原酶和三甲胺N-氧化物还原酶的酶水平去抑制以及硝酸盐还原酶诱导缺陷就证明了这一点。这些结果表明，narL和narX基因产物在细胞中必须以特定比例存在。我们得出结论，这些蛋白质相互作用以实现对厌氧电子传递相关操纵子的正常硝酸盐调控。从这些研究中，我们提出narX编码一种硝酸盐传感蛋白，而narL编码一种DNA结合调节蛋白，它们共同发挥作用的方式类似于其他双组分调节系统。
