Expression of the fixR-nifA Operon in Bradyrhizobium japonicum Depends on a New Response Regulator, RegR

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Abstract
Many nitrogen fixation-associated genes in the soybean symbiontBradyrhizobium japonicum are regulated by the transcriptional activator NifA, whose activity is inhibited by aerobiosis. NifA is encoded in the fixR-nifAoperon, 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 ς54 RNA polymerase and activated by NifA. Low-level aerobic expression and part of the anaerobic expression originates from a second promoter (fixRp2) that overlaps withfixRp1 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. AregR 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′-′lacZexpression was also decreased in regR mutants to about 10% of the level observed in the wild type. Similarly, regRmutants 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. WhileregR mutants grew only slightly slower in free-living, aerobic conditions, they displayed a strong growth defect under anaerobic conditions. The phenotypic properties of regSmutants 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 controllingnif and fix gene expression in B. japonicum.