Bauer E, Kaspar T, Fischer H M, Hennecke H
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland.
J Bacteriol. 1998 Aug;180(15):3853-63. doi: 10.1128/JB.180.15.3853-3863.1998.
Many nitrogen fixation-associated genes in the soybean symbiont Bradyrhizobium japonicum are regulated by the transcriptional activator NifA, whose activity is inhibited by aerobiosis. NifA is encoded in the fixR-nifA operon, which is expressed at a low level under aerobic conditions and induced approximately fivefold under low-oxygen tension. This induction depends on a -24/-12-type promoter (fixRp1) that is recognized by the sigma54 RNA polymerase and activated by NifA. Low-level aerobic expression and part of the anaerobic expression originates from a second promoter (fixRp2) that overlaps with fixRp1 and depends on an upstream DNA region (UAS) located around position -68 (H. Barrios, H. M. Fischer, H. Hennecke, and E. Morett, J. Bacteriol. 177:1760-1765, 1995). A protein binding to the UAS was previously postulated to act as an activator. This protein has now been purified, and the corresponding gene (regR) has been cloned. On the basis of the predicted amino acid sequence, RegR belongs to the family of response regulators of two-component regulatory systems. We identified upstream of the regR gene an additional gene (regS) encoding a putative sensor kinase. A regR mutant was constructed in which neither a specific UAS-binding activity nor fixRp2-dependent transcript formation and fixR'-'lacZ expression was detected in aerobically grown cells. Anaerobic fixR'-'lacZ expression was also decreased in regR mutants to about 10% of the level observed in the wild type. Similarly, regR mutants showed only about 2% residual nitrogen fixation activity, but unlike nodules induced by nifA mutants, the morphology of those nodules was normal, displaying no signs of necrosis. While regR mutants grew only slightly slower in free-living, aerobic conditions, they displayed a strong growth defect under anaerobic conditions. The phenotypic properties of regS mutants differed only marginally, if at all, from those of the wild type, suggesting the existence of a compensating sensor activity in these strains. The newly identified RegR protein may be regarded as a master regulator in the NifA-dependent network controlling nif and fix gene expression in B. japonicum.
大豆共生体日本慢生根瘤菌中许多与固氮相关的基因受转录激活因子NifA调控，NifA的活性受好氧作用抑制。NifA由fixR-nifA操纵子编码，该操纵子在有氧条件下低水平表达，在低氧张力下诱导表达约增加五倍。这种诱导依赖于一个-24/-12型启动子（fixRp1），它由σ54 RNA聚合酶识别并被NifA激活。低水平的有氧表达和部分厌氧表达源自第二个启动子（fixRp2），它与fixRp1重叠，依赖于位于约-68位的上游DNA区域（UAS）（H. Barrios、H. M. Fischer、H. Hennecke和E. Morett，《细菌学杂志》177:1760 - 1765，1995年）。先前推测一种与UAS结合的蛋白质作为激活剂起作用。现在该蛋白质已被纯化，并且相应的基因（regR）已被克隆。根据预测的氨基酸序列，RegR属于双组分调节系统的应答调节因子家族。我们在regR基因上游鉴定出另一个编码假定传感激酶的基因（regS）。构建了一个regR突变体，在有氧生长的细胞中未检测到特异性UAS结合活性、fixRp2依赖的转录形成和fixR'-'lacZ表达。regR突变体中厌氧fixR'-'lacZ表达也降至野生型观察水平的约10%。同样，regR突变体仅显示约2%的残余固氮活性，但与nifA突变体诱导的根瘤不同，那些根瘤的形态正常，没有坏死迹象。虽然regR突变体在自由生活的有氧条件下生长仅略慢，但在厌氧条件下表现出强烈的生长缺陷。regS突变体的表型特性与野生型仅略有不同，表明这些菌株中存在补偿性传感活性。新鉴定的RegR蛋白可被视为控制日本慢生根瘤菌中nif和fix基因表达的NifA依赖网络中的主调节因子。
