Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins.

Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox-responsive transcriptional activator NifA which is encoded in the fixR-nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position -68 in the fixR-nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity-purified RegR and a truncated soluble variant of RegS (RegS(C)), both as N-terminally His(6)-tagged proteins. RegS(C) autophosphorylated when incubated with [gamma-(32)P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegS(C) exhibited phosphatase activity on RegR-phosphate. Chemical stability tests and site-specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N-terminal but not the C-terminal domain of RegR interacts with RegS(C). Band-shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA-binding activity compared with dephosphorylated RegR or the mutant protein RegR-D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two-component regulatory systems.

Emmerich R ，Panglungtshang K ，Strehler P ，Hennecke H ，Fischer HM ... -  《european journal of biochemistry》

Expression of the fixR-nifA operon in Bradyrhizobium japonicum depends on a new response regulator, RegR.

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.

Bauer E ，Kaspar T ，Fischer HM ，Hennecke H ... -  《-》

An imperfect inverted repeat is critical for DNA binding of the response regulator RegR of Bradyrhizobium japonicum.

RegR is the response regulator of the RegSR two-component regulatory system in Bradyrhizobium japonicum. The only target known so far is the fixR-nifA operon, encoding the redox-responsive transcription factor NifA, which activates many genes required for symbiotic nitrogen fixation in soybean nodules. In previous in vivo studies, we identified a 32 bp upstream activating sequence located around position -68, which is essential for RegR-dependent expression of the fixR-nifA operon. Here, we used an in vitro binding-site selection assay (SELEX) to more precisely define the DNA-binding specificity of RegR. The selected sequences comprised an imperfect inverted repeat (GCGGC-N(5)-GTCGC) which is highly similar to an imperfect inverted repeat in the fixR UAS (GCGAC-N(5)-GACGC). In a parallel approach, band-shift experiments were performed with oligonucleotides comprising defined point or deletion mutations in the fixR UAS. This led to the identification of 11 critical nucleotides within a 17 bp minimal RegR binding site centered at position -64 upstream of the fixR-nifA transcription start site. Notably, all 11 critical nucleotides were located either within the half sites of the inverted repeat (four nucleotides in each half site) or in the 5 bp spacer that separates the half sites (three nucleotides). Based on these results, we defined a DNA motif comprising those nucleotides that are critical for RegR binding (RegR box; 5'-GNG(A)(G)C(A)(G)TTNNGNCGC-3'). A comparison of the RegR box with functional binding sites of the RegR-like regulator RegA of Rhodobacter capsulatus revealed considerable similarities. Thus, the RegR box may assist in the identification of new RegR target genes not only in B.japonicum but also in other alpha-proteobacteria possessing RegR-like response regulators.

Emmerich R ，Strehler P ，Hennecke H ，Fischer HM ... -  《-》

Evidence for a functional similarity between the two-component regulatory systems RegSR, ActSR, and RegBA (PrrBA) in alpha-Proteobacteria.

:The symbiotic bacteria Bradyrhizobium japonicum and Sinorhizobium meliloti, and the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodovulum sulfidophilum, Roseobacter denitrificans and Rhodobacter sphaeroides possess homologous two-component regulatory systems, namely RegSR, ActSR, RegBA and PrrBA. The respective response regulators of these bacteria control expression of different regulons that are involved in N2 fixation, CO2 fixation, photosynthesis or acid tolerance. We therefore asked whether the regulators are functionally exchangeable or whether they have disparate functions in the different species, despite the amino acid sequence similarity. In this study, we showed that purified B. japonicum RegR bound in vitro to genuine DNA targets for Rba. capsulatus RegA, and that RegA was phosphorylated in vitro when RegSc (a soluble variant of the sensor kinase RegS) was added to an Escherichia coli extract containing overexpressed RegA. In vivo, RegA and S. meliloti ActR activated transcription of the B. japonicum fixR-nifA operon, normally a target for RegR. The genes for both regulators, regA and actR, were able to complement a B. japonicum regR mutant with respect to the formation of a nitrogen-fixing symbiosis with soybean. Vice versa, RegR activated in Rba. capsulatus the expression of the photosynthesis operon puc, normally a target for RegA. In conclusion, the results show that B. japonicum RegR, Rba. capsulatus RegA, and S. meliloti ActR are functionally similar.

Emmerich R ，Hennecke H ，Fischer HM 《ARCHIVES OF MICROBIOLOGY》

The global response regulator RegR controls expression of denitrification genes in Bradyrhizobium japonicum.

Torres MJ ，Argandoña M ，Vargas C ，Bedmar EJ ，Fischer HM ，Mesa S ，Delgado MJ ... -  《PLoS One》