Symbiotic N 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 fixK 2 gene. The FixK 2 protein then activates not only a number of genes essential for microoxic respiration in symbiosis (fixNOQP and fixGHIS) but also further regulatory genes (rpoN 1 , nnrR, and fixK 1 ). The results of transcriptome analyses described here have led to a comprehensive and expanded definition of the FixJ, FixK 2 , and FixK 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 FixK 2 -dependent genes, which included a bioinformatics search for putative FixK 2 binding sites on DNA (FixK 2 boxes). Using an in vitro transcription assay with RNA polymerase holoenzyme and purified FixK 2 as the activator, we validated as direct targets eight new genes. Interestingly, the adjacent but divergently oriented fixK 1 and cycS genes shared the same FixK 2 box for the activation of transcription in both directions. This recognition site may also be a direct target for the FixK 1 protein, because activation of the cycS promoter required an intact fixK 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 FixK 1 seemed to exert a negative control on genes that are normally activated by the N 2 fixation-specific transcription factor NifA, and (ii) a larger number of genes are expressed in a FixK 2 -dependent manner in endosymbiotic bacteroids than in culture-grown cells, pointing to a possible symbiosisspecific control.
Under a shortage of oxygen, bacterial growth can be faced mainly by two ATP-generating mechanisms: (i) by synthesis of specific high-affinity terminal oxidases that allow bacteria to use traces of oxygen or (ii) by utilizing other substrates as final electron acceptors such as nitrate, which can be reduced to dinitrogen gas through denitrification or to ammonium. This bacterial respiratory shift from oxic to microoxic and anoxic conditions requires a regulatory strategy which ensures that cells can sense and respond to changes in oxygen tension and to the availability of other electron acceptors. Bacteria can sense oxygen by direct interaction of this molecule with a membrane protein receptor (e.g., FixL) or by interaction with a cytoplasmic transcriptional factor (e.g., Fnr). A third type of oxygen perception is based on sensing changes in redox state of molecules within the cell. Redox-responsive regulatory systems (e.g., ArcBA, RegBA/PrrBA, RoxSR, RegSR, ActSR, ResDE, and Rex) integrate the response to multiple signals (e.g., ubiquinone, menaquinone, redox active cysteine, electron transport to terminal oxidases, and NAD/NADH) and activate or repress target genes to coordinate the adaptation of bacterial respiration from oxic to anoxic conditions. Here, we provide a compilation of the current knowledge about proteins and regulatory networks involved in the redox control of the respiratory adaptation of different bacterial species to microxic and anoxic environments.
In Bradyrhizobium japonicum, a gene named nnrR was identified which encodes a protein with high similarity to FNR/CRP-type transcriptional regulators. Mutant strains carrying an nnrR null mutation were unable to grow anaerobically in the presence of nitrate or nitrite, and they lacked both nitrate and nitrite reductase activities. Anaerobic activation of an nnrR-lacZ fusion required FixLJ and FixK 2 . In turn, N oxide-mediated induction of nir and nor genes encoding nitrite and nitric oxide reductase, respectively, depended on NnrR. Thus, NnrR expands the FixLJ-FixK 2 regulatory cascade by an additional control level which integrates the N oxide signal required for maximal induction of the denitrification genes.
In Bradyrhizobium japonicum, the N 2 -fixing root nodule endosymbiont of soybean, a group of genes required for microaerobic, anaerobic, or symbiotic growth is controlled by FixK 2 , a key regulator that is part of the FixLJ-FixK 2 cascade. FixK 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 FixK 2 and its transcription activation activity in vitro. FixK 2 was expressed in Escherichia coli 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 FixK 2 productively interacted with the B. japonicum 80 -RNA polymerase holoenzyme, but not with E. coli 70 -RNA polymerase holoenzyme, to activate transcription from the B. japonicum fixNOQP, fixGHIS, and hemN 2 promoters in vitro. Furthermore, FixK 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 FixK 2 box at nucleotide position ؊40.5 in the hemN 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 FixK 2 , we surmise that FixK 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.
The nosRZDFYLX gene cluster for the respiratory nitrous oxide reductase from Bradyrhizobium japonicum strain USDA110 has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ, the structural gene, nosD, nosF, nosY, nosL, and nosX were detected. The deduced amino acid sequence exhibited a high degree of similarity to other nitrous oxide reductases from various sources. The NosZ protein included a signal peptide for protein export. Mutant strains carrying either a nosZ or a nosR mutation accumulated nitrous oxide when cultured microaerobically in the presence of nitrate. Maximal expression of a P nosZ-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Microaerobic activation of the fusion required FixLJ and FixK(2).
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