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Bradyrhizobium japonicum NnrR, a denitrification regulator, expands the FixLJ-FixK2 regulatory cascade.

ABSTRACT: 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.

SUBMITTER: Mesa S

PROVIDER: S-EPMC161565 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Bradyrhizobium japonicum NnrR, a denitrification regulator, expands the FixLJ-FixK2 regulatory cascade.

Mesa Socorro S Bedmar Eulogio J EJ Chanfon Astrid A Hennecke Hauke H Fischer Hans-Martin HM

Journal of bacteriology 20030701 13

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 re ...[more]

PMID: 12813094

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Project description: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 symbiosis-specific control.

Project description:Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544), or cy2 (bll2388), genes involved in nitric oxide detoxification (blr2806-09), copper homeostasis (copCAB), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK-like gene (bll3466), and bll4130 which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC. P1 matched with the previously mapped 5M-bM-^@M-^Yend of norC mRNA which we demonstrate in this work to be under FixK2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c-staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS. A total of eight Affymetrix GeneChips are included in this study. Per strain (wild-type B. japonicum 110spc4, delta regR mutant 2426) four biological replicates were processed and analyzed. Strains were grown under anoxic conditions in Bergersen minimal medium supplemented with succinate as carbon source and KNO3 as terminal electron acceptor. For comparison, a microarray expression dataset generated with B. japonicum wild-type (Hauser et al., Mol. Genet. Genomics 278:255-271, 2007) and delta regR mutant cells (Lindemann et al., J. Bacteriol. 189:8928-8943, 2007) was used.

Dataset Information

Bradyrhizobium japonicum NnrR, a denitrification regulator, expands the FixLJ-FixK2 regulatory cascade.

Publications

Bradyrhizobium japonicum NnrR, a denitrification regulator, expands the FixLJ-FixK2 regulatory cascade.

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