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Symbiotic [N.sub.2] fixation in Bradyrhizobium japonicum is controlled by a complex transcription factor network. Part of it is a hierarchically arranged cascade in which the two-component regulatory system FixLJ, in response to a moderate decrease in oxygen concentration, activates the fix[K.sub.2] gene. The Fix[K.sub.2] protein then activates not only a number of genes essential for microoxic respiration in symbiosis (fixNOQP and fixGHIS) but also further regulatory genes (rpo[N.sub.1], nnrR, and fix[K.sub.1]). The results of transcriptome analyses described here have led to a comprehensive and expanded definition of the FixJ, Fix[K.sub.2], and Fix[K.sub.1] regulons, which, respectively, consist of 26, 204, and 29 genes specifically regulated in microoxically grown cells. Most of these genes are subject to positive control. Particular attention was addressed to the Fix[K.sub.2]-dependent genes, which included a bioinformatics search for putative Fix[K.sub.2] binding sites on DNA (Fix[K.sub.2] boxes). Using an in vitro transcription assay with RNA polymerase holoenzyme and purified Fix[K.sub.2] as the activator, we validated as direct targets eight new genes. Interestingly, the adjacent but divergently oriented fix[K.sub.1] and cycS genes shared the same Fix[K.sub.2] box for the activation of transcription in both directions. This recognition site may also be a direct target for the Fix[K.sub.1] protein, because activation of the cycS promoter required an intact fix[K.sub.1] gene and either microoxic or anoxic, denitrifying conditions. We present evidence that cycS codes for a c-type cytochrome which is important, but not essential, for nitrate respiration. Two other, unexpected results emerged from this study: (i) specifically Fix[K.sub.1] seemed to exert a negative control on genes that are normally activated by the [N.sub.2] fixation-specific transcription factor NifA, and (ii) a larger number of genes are expressed in a Fix[K.sub.2]-dependent manner in endosymbiotic bacteroids than in culture-grown cells, pointing to a possible symbiosis-specific control.

RegSR-like proteins, members of the family of two-component regulatory systems, are present in a large number of proteobacteria in which they globally control gene expression mostly in a redox-responsive manner. The controlled target genes feature an enormous functional diversity. In Bradyrhizobium japonicum, the facultative root nodule symbiont of soybean, RegSR activate the transcription of the nitrogen fixation regulatory gene nifA, thus forming a RegSR-NifA cascade which is part of a complex regulatory network for gene regulation in response to changing oxygen concentrations. Whole-genome transcription profiling was performed here in order to assess the full regulatory scope of RegSR. The comparative analysis of wild-type and [DELTA]regR cells grown under oxic and microoxic conditions revealed that expression of almost 250 genes is dependent on RegR, a result that underscores the important contribution of RegR to oxygen- or redox-regulated gene expression in B. japonicum. Furthermore, transcription profiling of [DELTA]regR bacteroids compared with wild-type bacteroids revealed expression changes for about 1,200 genes in young and mature bacteroids. Incidentally, many of these were found to be induced in symbiosis when wild-type bacteroids were compared with free-living, culture-grown wild-type cells, and they appeared to encode diverse functions possibly related to symbiosis and nitrogen fixation. We demonstrated direct RegR-mediated control at promoter regions of several selected target genes by means of DNA binding experiments and in vitro transcription assays, which revealed six novel direct RegR target promoters.

Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont, possesses a heme uptake system encoded by the gene cluster hmuVUT-hmuR-exbBD-tonB. Transcription of the divergently oriented hmuT and hmuR genes was previously found to be induced by iron limitation and to depend on a 21-bp promoter-upstream iron control element (ICE). Here, we show by deletion analysis that the full-length ICE is needed for this type of positive control. Additional genes associated with ICE-like motifs were identified in the B. japonicum genome, of which bll6680 and blr7895 code for bacterioferritin and rubrerythrin homologs, respectively. Transcription start site mapping revealed that their ICEs directly overlap with either the -10 promoter region or the transcription initiation site, suggesting an involvement of the ICE in negative control of both genes. Consistent with this inference was the observed down-regulation of both genes under iron limitation, which in the case of bll6680 was shown to require an intact ICE motif. Using a yeast one-hybrid system, we demonstrated in vivo interaction of the iron response regulator (Irr) with all three ICEs. Moreover, specific in vitro binding of purified Irr protein to the ICE motifs of bll6680 and blr7895 was shown in electrophoretic mobility shift experiments. A genome-wide survey for iron-regulated genes with a custom-made Affymetrix gene chip revealed 17 genes to be induced and 68 to be repressed under iron-replete conditions. Remarkably, ICE-like motifs are associated with a large subset of those B. japonicum genes. We propose the ICE as an important cis-acting element in B. japonicum which represents the DNA-binding site for the Irr protein and, depending on its location within promoter regions, is involved in positive or negative control of the associated iron-regulated genes.

In Bradyrhizobium japonicum, the [N.sub.2]-fixing root nodule endosymbiont of soybean, a group of genes required for microaerobic, anaerobic, or symbiotic growth is controlled by Fix[K.sub.2], a key regulator that is part of the FixLJ-Fix[K.sub.2] cascade. Fix[K.sub.2] belongs to the family of cyclic AMP receptor protein/fumarate and nitrate reductase (CRP/FNR) transcription factors that recognize a palindromic DNA motif (CRP/FNR box) associated with the regulated promoters. Here, we report on a biochemical analysis of Fix[K.sub.2] and its transcription activation activity in vitro. Fix[K.sub.2] was expressed in Escherichia coil and purified as a soluble N-terminally histidine-tagged protein. Gel filtration experiments revealed that increasing the protein concentration shifts the monomer-dimer equilibrium toward the dimer. Purified Fix[K.sub.2] productively interacted with the B. japonicum [[alpha].sup.80]-RNA polymerase holoenzyme, but not with E. coli [[alpha].sup.70]-RNA polymerase holoenzyme, to activate transcription from the B. japonicum fixNOQP, fixGHIS, and hem[N.sub.2] promoters in vitro. Furthermore, Fix[K.sub.2] activated transcription from the E. coli FF(-41.5) model promoter, again only in concert with B. japonicum RNA polymerase. All of these promoters are so-called class II CRP/FNR-type promoters. We showed by specific mutagenesis that the Fix[K.sub.2] box at nucleotide position -40.5 in the hem[N.sub.2] promoter, but not that at -78.5, is crucial for activation both in vivo and in vitro, which argues against recognition of a potential class III promoter. Given the lack of any evidence for the presence of a cofactor in purified Fix[K.sub.2], we surmise that Fix[K.sub.2] alone is sufficient to activate in vitro transcription to at least a basal level. This contrasts with all well-studied CRP/FNR-type proteins, which do require coregulators.

Two oxygen-responsive regulatory systems controlling numerous symbiotic genes in Bradyrhizobium japonicum were assayed in free-living cultures for their capacity to activate target genes under different oxygen conditions. NifA- and FixLJ-controlled target genes showed disparate relative expression patterns. Induction of NifA-dependent genes was observed only at oxygen concentrations below 2% in the gas phase, whereas that of FixLJ-controlled targets progressively increased when the oxygen concentration was lowered from 21 to 5, 2, or 0.5%. We propose that this reflects a response to a gradient of increasing oxygen deprivation as bacteria invade their host during root nodule development.

Research demonstrates hemN-like gene (hemN) 1 and 2 in Bradyrhizobium japonicum involved in the heme biosynthesis in bacteria. Mutants of only hemN1 gene are able to grow anaerobically under nitrate-respiring conditions and fix nitrogen in symbiotic association with soybean.

Results reveal that the nitrogen fixation regulatory protein NifA is an activator of three other genes, termed nrg for NifA-regulated gene, besides being an activator of nitrogen fixation genes in Bradyrhizobium japonicum as shown by competitive DNA-RNA hybridization.

Research has been conducted on the bacteria regulate chaperone gene expression by CIRCE-HrcA system. The use of the complementation approach in Bradyrhizobium japonicum gene cloning is described.

Regulation of the synthesis of the key enzyme of biological nitrogen fixation that is the dinitrogenase complex substantially varies in terms of different environmental signals and the method by which they are transduced to the level of gene expression. Experiments done to purify the protein binding to the fixR-nifA upstream activation sites in the soybean Bradyrhizobium japonicum have resulted in the identification of a two-component regulatory system called regSR, which is a new element in the nifA regulatory cascade of the soybean.

The heat shock response in Bradyrhizobium japonicum is mediated by the numerous regulatory factors. The amino acid sequence of the RpoH genes of the microbe is similar to proteobacteria proteins that belong to the alpha subgroup. The rpoH gene is clustered with five additional heat shock genes that is responsible for allowing the heat shock induction of groESL which has a highly conserved inverted-repeat structure. A conserved sequence was also detected between the transcriptional and first translational start sites of the rpoH gene.

Two positively controlled overlapping promoters, recognized by different RNA polymerase holoenzymes, Esigma(super 54) and Esigma(super 92), transcribes the fixRnifA operon which encodes the Bradyrhizobium japonicum NifA protein, the central regulator for nitrogen fixation gene expression. One promoter is autoactivated by NifA while the other is regulated by the oxygen.

Nitrogen fixation in three rhizobial bacteriae, Rhizobium meliloti, Azorhizobium caulinodans and Bradyrhizobium japonicum is controlled by complicated genetic regulatory networks. Low-oxygen conditions in the environment stimulate translocation of nitrogen fixation genes in the bacteria through five regulatory proteins which transmit the signals. The regulatory activity of proteins is modulated by dephosphorylation and phosphorylation. The proteins indirectly involve the genes of bacterial species in regulating nitrogen fixation process.