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

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 (fixRp 1) 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 (fixRp 2) that overlaps withfixRp 1 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 fixRp 2-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.

Sinorhizobium medicae genes whose regulation involves the ActS and/or ActR signal transduction proteins

“…To our knowledge this is the first report of a second two-component system, in addition to FixLJ, that modulates expression of the fixK-fixNOQP and nifA genes in Sinorhizobium. Unlike Sinorhizobium, the microaerobic induction of nifA in B. japonicum is independent of FixLJ, but absolutely requires the ActSR homologs, RegSR [11]. In B. japonicum, therefore, an intact RegSR system is essential for symbiotic N 2 fixation [11], while the FixLJ system is also essential through its induction of the FixNOQP system through FixK2 (Fig.…”

“…GUS assays were performed on the indicated strains of S. medicae and S. meliloti carrying the fixN2 gusA reporter plasmid pPFIX1 ActR homologs from Rhodobacter spp. and B. japonicum activate nifA, encoding the nitrogen fixation regulator NifA, in response to microaerobiosis [11,23]. Like fixK, the nifA gene of S. meliloti is also positively regulated by FixJ in microoxic conditions [45].…”

“…RegBA act as a typical two-component system, with the membrane-spanning RegB histidine kinase phosphorylating RegA and allowing the activated regulator to modulate transcription by interacting with specific DNA targets [3][4][5]. RegBA homologs have been discovered in other closely related anoxygenic phototrophs [6][7][8][9], plant-associated and soil bacteria [10][11][12][13][14][15] and more recently in pathogens like Brucella melitensis [16] and Mycobacterim tuberculosis [17].…”

“…RegBA homologs regulate a diverse range of metabolic processes including photosystem formation [9,[18][19][20], CO 2 assimilation [21], nitrogen assimilation and hydrogen uptake [22,23], respiration and electron transport [24,25] and formaldehyde assimilation [26]. In the root nodule bacteria, the RegSR system from Bradyrhizobium japonicum is responsible for microaerobic induction of the nitrogen fixation regulator NifA, and regulation of CO 2 assimilation [11,27]. RegBA homologs from Rhodobacter sphaeroides regulate redoxsensitive processes in response to perturbations in reductant flow through the electron transport chain [20,28,29].…”

Regulators of nonsulfur purple phototrophic bacteria and the interactive control of CO2assimilation, nitrogen fixation, hydrogen metabolism and energy generation

“…Both RegSR and ActSR share similarities with the Reg/Prr system with respect to the pathways they regulate. RegSR positively regulates the expression of the fixR-ifA operon in B. japonicum under both aerobic and anaerobic conditions through the binding of RegR to an UAS within the fixR promoter [119]. A regR mutant showed an impaired ability to grow in the free-living state anaerobically and is unable to fix nitrogen symbiotically [119].…”