Socorro Mesa

Bacterial Adaptation of Respiration from Oxic to Microoxic and Anoxic Conditions: Redox 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.

Comprehensive Assessment of the Regulons Controlled by the FixLJ-FixK2-FixK1 Cascade in Bradyrhizobium japonicum

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.

Molecular characterization of nosRZDFYLX genes coding for denitrifying nitrous oxide reductase of Bradyrhizobium japonicum

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 PnosZ-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 FixK2.

Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).

Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum.

The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (P(norC)-lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O(2)) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The P(norC)-lacZ fusion was not expressed in the B. japonicum fixK(2) mutant strain 9043, but complementation of the mutant with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45.5 bp downstream of the centre of a putative binding site for the transcription factor FixK(2).

The Global Response Regulator RegR Controls Expression of Denitrification Genes in Bradyrhizobium japonicum

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 cy 2 (bll2388), and genes involved in nitric oxide detoxification (blr2806-09) and 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 5′end 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.

Transcription activation in vitro by the Bradyrhizobium japonicum regulatory protein FixK(2)

In Bradyrhizobium japonicum, the N2-fixing root nodule endosymbiont of soybean, a group of genes required for microaerobic, anaerobic, or symbiotic growth is controlled by FixK2, a key regulator that is part of the FixLJ-FixK2 cascade. FixK2 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 FixK2 and its transcription activation activity in vitro. FixK2 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 FixK2 productively interacted with the B. japonicum sigma80-RNA polymerase holoenzyme, but not with E. coli sigma70-RNA polymerase holoenzyme, to activate transcription from the B. japonicum fixNOQP, fixGHIS, and hemN2 promoters in vitro. Furthermore, FixK2 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 FixK2 box at nucleotide position -40.5 in the hemN2 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 FixK2, we surmise that FixK2 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.

Posttranslational control of transcription factor FixK2, a key regulator for the Bradyrhizobium japonicum–soybean symbiosis

Rhizobial FixK-like proteins play essential roles in activating genes for endosymbiotic life in legume root nodules, such as genes for micro-oxic respiration. In the facultative soybean symbiont, Bradyrhizobium japonicum, the FixK2 protein is the key player in a complex regulatory network. The fixK2 gene itself is activated by the 2-component regulatory system FixLJ in response to a moderate decrease of the oxygen tension, and the FixK2 protein distributes and amplifies this response to the level of approximately 200 target genes. Unlike other members of the cAMP receptor protein family, to which FixK2 belongs, the FixK2 protein does not appear to be modulated by small effector molecules. Here, we show that a critical, single cysteine residue (C183) near the DNA-binding domain of FixK2 confers sensitivity to oxidizing agents and reactive oxygen species. Oxidation-dependent inactivation occurs not only in vitro, as shown with cell-free transcription assays, but also in vivo, as shown by microarray-assisted transcriptome analysis of the FixK2 regulon. The oxidation mechanism may involve a reversible dimerization by intermolecular disulfide-bridge formation and a direct, irreversible oxidation at the cysteine thiol, depending on the oxidizing agent. Mutational exchange of C183 to alanine renders FixK2 resistant to oxidation, yet allows full activity, shown again both in vitro and in vivo. We hypothesize that posttranslational modification by reactive oxygen species is a means to counterbalance the cellular pool of active FixK2, which would otherwise fill unrestrictedly through FixLJ-dependent synthesis.

Emerging complexity in the denitrification regulatory network of Bradyrhizobium japonicum

Bradyrhizobium japonicum is a Gram-negative soil bacterium symbiotically associated with soya bean plants, which is also able to denitrify under free-living and symbiotic conditions. In B. japonicum, the napEDABC, nirK, norCBQD and nosRZDYFLX genes which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide respectively are required for denitrification. Similar to many other denitrifiers, expression of denitrification genes in B. japonicum requires both oxygen limitation and the presence of nitrate or a derived nitrogen oxide. In B. japonicum, a sophisticated regulatory network consisting of two linked regulatory cascades co-ordinates the expression of genes required for microaerobic respiration (the FixLJ/FixK2 cascade) and for nitrogen fixation (the RegSR/NifA cascade). The involvement of the FixLJ/FixK2 regulatory cascade in the microaerobic induction of the denitrification genes is well established. In addition, the FNR (fumarase and nitrate reduction regulator)/CRP(cAMP receptor protein)-type regulator NnrR expands the FixLJ/FixK2 regulatory cascade by an additional control level. A role for NifA is suggested in this process by recent experiments which have shown that it is required for full expression of denitrification genes in B. japonicum. The present review summarizes the current understanding of the regulatory network of denitrification in B. japonicum.

NifA is required for maximal expression of denitrification genes in Bradyrhizobium japonicum.

In Bradyrhizobium japonicum the napEDABC, nirK, norCBQD and nosRZDYFLX genes, which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide, respectively, are required for denitrification. Microaerobic induction of these genes depends on fixLJ and fixK2, whose products form the FixLJ-FixK2 regulatory cascade. In B. japonicum, a second oxygen-responsive regulatory cascade mediated by the nitrogen fixation regulatory protein, NifA, has been described. In this study, we show that disruption of nifA caused a growth defect in B. japonicum cells, when grown under denitrifying conditions, and decreased activity of periplasmic nitrate and nitrite reductase enzymes was also observed. Furthermore, expression of napE-lacZ, nirK-lacZ or norC-lacZ transcriptional fusions, as well as levels of nirK transcripts were significantly reduced in the nifA mutant after incubation under nitrate-respiring conditions. Haem c staining analyses revealed that NifA is required for full synthesis of the NapC and NorC proteins, which are required for denitrification. A B. japonicum rpoN1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor sigma54, was able to grow anaerobically with nitrate as terminal electron acceptor and showed wild-type levels of nitrate and nitrite reductase activities. We propose that the nitrogen fixation regulatory protein, NifA, is involved in the maximal expression of the denitrification genes in B. japonicum. This influence is independent of sigma54.