Pleiotropic Effects of PhaR Regulator in Bradyrhizobium diazoefficiens Microaerobic Metabolism.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

Regulation of the Emissions of the Greenhouse Gas Nitrous Oxide by the Soybean Endosymbiont Bradyrhizobium diazoefficiens.

The greenhouse gas nitrous oxide (N2O) has strong potential to drive climate change. Soils are a major source of N2O, with microbial nitrification and denitrification being the primary processes involved in such emissions. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model microorganism to study denitrification, a process that depends on a set of reductases, encoded by the napEDABC, nirK, norCBQD, and nosRZDYFLX genes, which sequentially reduce nitrate (NO3-) to nitrite (NO2-), nitric oxide (NO), N2O, and dinitrogen (N2). In this bacterium, the regulatory network and environmental cues governing the expression of denitrification genes rely on the FixK2 and NnrR transcriptional regulators. To understand the role of FixK2 and NnrR proteins in N2O turnover, we monitored real-time kinetics of NO3-, NO2-, NO, N2O, N2, and oxygen (O2) in a fixK2 and nnrR mutant using a robotized incubation system. We confirmed that FixK2 and NnrR are regulatory determinants essential for NO3- respiration and N2O reduction. Furthermore, we demonstrated that N2O reduction by B. diazoefficiens is independent of canonical inducers of denitrification, such as the nitrogen oxide NO3-, and it is negatively affected by acidic and alkaline conditions. These findings advance the understanding of how specific environmental conditions and two single regulators modulate N2O turnover in B. diazoefficiens.

Fine-Tuning Modulation of Oxidation-Mediated Posttranslational Control of Bradyrhizobium diazoefficiens FixK2 Transcription Factor.

FixK2 is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK2 gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK2 activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK2, we replaced C183 for aspartic acid. Purified C183D FixK2 protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK2, expression of a fixNOQP'-'lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK2 encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK2 gene expression levels and FixK2 protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK2 encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK2 in vivo.

Effect of Copper on Expression of Functional Genes and Proteins Associated with Bradyrhizobium diazoefficiens Denitrification.

Nitrous oxide (N2O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N2O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N2, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N2O emissions from agricultural soils.

Dissection of FixK2 protein-DNA interaction unveils new insights into Bradyrhizobium diazoefficiens lifestyles control.

The FixK2 protein plays a pivotal role in a complex regulatory network, which controls genes for microoxic, denitrifying, and symbiotic nitrogen-fixing lifestyles in Bradyrhizobium diazoefficiens. Among the microoxic-responsive FixK2 -activated genes are the fixNOQP operon, indispensable for respiration in symbiosis, and the nnrR regulatory gene needed for the nitric-oxide dependent induction of the norCBQD genes encoding the denitrifying nitric oxide reductase. FixK2 is a CRP/FNR-type transcription factor, which recognizes a 14 bp-palindrome (FixK2 box) at the regulated promoters through three residues (L195, E196, and R200) within a C-terminal helix-turn-helix motif. Here, we mapped the determinants for discriminatory FixK2 -mediated regulation. While R200 was essential for DNA binding and activity of FixK2 , L195 was involved in protein-DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK2 box at the fixNOQP promoter impaired transcription activation by FixK2 , which was residual when a second mutation affecting the box palindromy was introduced. The substitution of nucleotide 11 within the NnrR box at the norCBQD promoter allowed FixK2 -mediated activation in response to microoxia. Thus, position 11 within the FixK2 /NnrR boxes constitutes a key element that changes FixK2 targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier.

Disparate response to microoxia and nitrogen oxides of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes.

Expression of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes requires low oxygen (O2) tension and nitrate (NO3-), through a regulatory network comprised of two coordinated cascades, FixLJ-FixK2-NnrR and RegSR-NifA. To precisely understand how these signals are integrated in the FixLJ-FixK2-NnrR circuit, we analyzed ß-Galactosidase activities from napE-lacZ, nirK-lacZ and norC-lacZ fusions, and performed analyses of NapC and NorC levels as well as periplasmic nitrate reductase (Nap) activity, in B. japonicum wildtype and fixK2 and nnrR mutant backgrounds. While microoxic conditions (2% O2 at headspace) were sufficient to induce expression of napEDABC and nirK genes and this control depends on FixK2, norCBQD expression requires, in addition to microoxia, nitric oxide gas (NO) and both FixK2 and NnrR transcription factors. Purified FixK2 protein directly interacted and activated transcription in collaboration with B. japonicum RNA polymerase (RNAP) from the napEDABC and nirK promoters, but not from the norCBQD promoter. Further, recombinant NnrR protein bound exclusively to the norCBQD promoter in an O2-sensitive manner. Our work suggest a disparate regulation of B. japonicum denitrifying genes expression with regard to their dependency to microoxia, nitrogen oxides (NOx), and the regulatory proteins FixK2 and NnrR. In this control, expression of napEDABC and nirK genes requires microoxic conditions and directly depends on FixK2, while expression of norCBQD genes relies on NO, being NnrR the candidate which directly interacts with the norCBQD promoter.

Metabolomic Profiling of Bradyrhizobium diazoefficiens-Induced Root Nodules Reveals Both Host Plant-Specific and Developmental Signatures.

Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection-time-of-flight mass spectrometry analysis the metabolome of (i) nodules and roots from four different B. diazoefficiens host plants; (ii) soybean nodules harvested at different time points during nodule development; and (iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by independent transcriptomics data. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.

The structure of Bradyrhizobium japonicum transcription factor FixK2 unveils sites of DNA binding and oxidation.

FixK2 is a regulatory protein that activates a large number of genes for the anoxic and microoxic, endosymbiotic, and nitrogen-fixing life styles of the α-proteobacterium Bradyrhizobium japonicum. FixK2 belongs to the cAMP receptor protein (CRP) superfamily. Although most CRP family members are coregulated by effector molecules, the activity of FixK2 is negatively controlled by oxidation of its single cysteine (Cys-183) located next to the DNA-binding domain and possibly also by proteolysis. Here, we report the three-dimensional x-ray structure of FixK2, a representative of the FixK subgroup of the CRP superfamily. Crystallization succeeded only when (i) an oxidation- and protease-insensitive protein variant (FixK2(C183S)-His6) was used in which Cys-183 was replaced with serine and the C terminus was fused with a hexahistidine tag and (ii) this protein was allowed to form a complex with a 30-mer double-stranded target DNA. The structure of the FixK2-DNA complex was solved at a resolution of 1.77 Å, at which the protein formed a homodimer. The precise protein-DNA contacts were identified, which led to an affirmation of the canonical target sequence, the so-called FixK2 box. The C terminus is surface-exposed, which might explain its sensitivity to specific cleavage and degradation. The oxidation-sensitive Cys-183 is also surface-exposed and in close proximity to DNA. Therefore, we propose a mechanism whereby the oxo acids generated after oxidation of the cysteine thiol cause an electrostatic repulsion, thus preventing specific DNA binding.

Surface Plasmon Resonance as a Tool to Elucidate the Molecular Determinants of Key Transcriptional Regulators Controlling Rhizobial Lifestyles.

Bacteria must be provided with a battery of tools integrated into regulatory networks, in order to respond and, consequently, adapt their physiology to changing environments. Within these networks, transcription factors finely orchestrate the expression of genes in response to a variety of signals, by recognizing specific DNA sequences at their promoter regions. Rhizobia are host-interacting soil bacteria that face severe changes to adapt their physiology from free-living conditions to the nitrogen-fixing endosymbiotic state inside root nodules associated with leguminous plants. One of these cues is the low partial pressure of oxygen within root nodules.Surface plasmon resonance (SPR) constitutes a technique that allows to measure molecular interactions dynamics at real time by detecting changes in the refractive index of a surface. Here, we implemented the SPR methodology to analyze the discriminatory determinants of transcription factors for specific interaction with their target genes. We focused on FixK2, a CRP/FNR-type protein with a central role in the complex oxygen-responsive regulatory network in the soybean endosymbiont Bradyrhizobium diazoefficiens. Our study unveiled relevant residues for protein-DNA interaction as well as allowed us to monitor kinetics and stability protein-DNA complex. We believe that this approach can be employed for the characterization of other relevant transcription factors which can assist to the better understanding of the adaptation of bacteria with agronomic or human interest to their different modes of life.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

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 FixK(2) protein is the key player in a complex regulatory network. The fixK(2) gene itself is activated by the 2-component regulatory system FixLJ in response to a moderate decrease of the oxygen tension, and the FixK(2) 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 FixK(2) belongs, the FixK(2) 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 FixK(2) 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 FixK(2) 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 FixK(2) 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 FixK(2), which would otherwise fill unrestrictedly through FixLJ-dependent synthesis.

Application of biostimulant products and biological control agents in sustainable viticulture: A review.

Current and continuing climate change in the Anthropocene epoch requires sustainable agricultural practices. Additionally, due to changing consumer preferences, organic approaches to cultivation are gaining popularity. The global market for organic grapes, grape products, and wine is growing. Biostimulant and biocontrol products are often applied in organic vineyards and can reduce the synthetic fertilizer, pesticide, and fungicide requirements of a vineyard. Plant growth promotion following application is also observed under a variety of challenging conditions associated with global warming. This paper reviews different groups of biostimulants and their effects on viticulture, including microorganisms, protein hydrolysates, humic acids, pyrogenic materials, and seaweed extracts. Of special interest are biostimulants with utility in protecting plants against the effects of climate change, including drought and heat stress. While many beneficial effects have been reported following the application of these materials, most studies lack a mechanistic explanation, and important parameters are often undefined (e.g., soil characteristics and nutrient availability). We recommend an increased study of the underlying mechanisms of these products to enable the selection of proper biostimulants, application methods, and dosage in viticulture. A detailed understanding of processes dictating beneficial effects in vineyards following application may allow for biostimulants with increased efficacy, uptake, and sustainability.

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.

The nitric oxide response in plant-associated endosymbiotic bacteria.

Nitric oxide (NO) is a gaseous signalling molecule which becomes very toxic due to its ability to react with multiple cellular targets in biological systems. Bacterial cells protect against NO through the expression of enzymes that detoxify this molecule by oxidizing it to nitrate or reducing it to nitrous oxide or ammonia. These enzymes are haemoglobins, c-type nitric oxide reductase, flavorubredoxins and the cytochrome c respiratory nitrite reductase. Expression of the genes encoding these enzymes is controlled by NO-sensitive regulatory proteins. The production of NO in rhizobia-legume symbiosis has been demonstrated recently. In functioning nodules, NO acts as a potent inhibitor of nitrogenase enzymes. These observations have led to the question of how rhizobia overcome the toxicity of NO. Several studies on the NO response have been undertaken in two non-dentrifying rhizobial species, Sinorhizobium meliloti and Rhizobium etli, and in a denitrifying species, Bradyrhizobium japonicum. In the present mini-review, current knowledge of the NO response in those legume-associated endosymbiotic bacteria is summarized.

Bacterial nitric oxide metabolism: Recent insights in rhizobia.

Nitric oxide (NO) is a reactive gaseous molecule that has several functions in biological systems depending on its concentration. At low concentrations, NO acts as a signaling molecule, while at high concentrations, it becomes very toxic due to its ability to react with multiple cellular targets. Soil bacteria, commonly known as rhizobia, have the capacity to establish a N2-fixing symbiosis with legumes inducing the formation of nodules in their roots. Several reports have shown NO production in the nodules where this gas acts either as a signaling molecule which regulates gene expression, or as a potent inhibitor of nitrogenase and other plant and bacteria enzymes. A better understanding of the sinks and sources of NO in rhizobia is essential to protect symbiotic nitrogen fixation from nitrosative stress. In nodules, both the plant and the microsymbiont contribute to the production of NO. From the bacterial perspective, the main source of NO reported in rhizobia is the denitrification pathway that varies significantly depending on the species. In addition to denitrification, nitrate assimilation is emerging as a new source of NO in rhizobia. To control NO accumulation in the nodules, in addition to plant haemoglobins, bacteroids also contribute to NO detoxification through the expression of a NorBC-type nitric oxide reductase as well as rhizobial haemoglobins. In the present review, updated knowledge about the NO metabolism in legume-associated endosymbiotic bacteria is summarized.

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.

Autoregulation of fixK(2) gene expression in Bradyrhizobium japonicum.

Several essential Bradyrhizobium japonicum genes for a symbiotic, nitrogen-fixing root-nodule symbiosis are positively controlled under micro-oxic conditions by the FixLJ-FixK(2) regulatory cascade. Negative control is exerted by reactive oxygen species at the level of the FixK(2) protein. Furthermore, we noticed that fixK (2) gene expression is increased in a fixK (2) mutant, suggesting that FixK(2) in the wild type has a negative effect, directly or indirectly, on its own expression. To possibly understand this effect, the transcription pattern of the fixLJ-bll2758-fixK (2) gene region was examined more closely. While fixK (2) gene transcription is activated by FixJ, the bll2758 gene is transcribed from its own promoter in a FixK(2)-dependent manner, and there is no read-through transcription from bll2758 into fixK (2). The bll2758-encoded protein is predicted to be a stand-alone receiver domain of a response regulator, making it a prime candidate for exerting an inhibitory role on the expression of fixK (2). Transcriptome profiling of a bll2758 knock-out mutant revealed, however, that neither fixK (2) itself nor any of the known FixJ- and FixK(2)-dependent target genes is significantly affected in their expression. This precludes a role of the bll2758 product as a so-called FixT-like protein in the inhibition of FixLJ function, as was proposed for Sinorhizobium meliloti and Caulobacter crescentus. Instead, we rationalize that other transcription factors, whose genes are activated by FixK(2), might be involved in the negative autoregulation of fixK (2) gene expression.

Expanding the Regulon of the Bradyrhizobium diazoefficiens NnrR Transcription Factor: New Insights Into the Denitrification Pathway.

Denitrification in the soybean endosymbiont Bradyrhizobium diazoefficiens is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK2-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK2 and NnrR exert disparate control on expression of core denitrifying systems encoded by napEDABC, nirK, norCBQD, and nosRZDFYLX genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in B. diazoefficiens, we compared the transcriptional profile of an nnrR mutant with that of the wild type, both grown under anoxic denitrifying conditions. This approach revealed more than 170 genes were simultaneously induced in the wild type and under the positive control of NnrR. Among them, we found the cycA gene which codes for the c 550 soluble cytochrome (CycA), previously identified as an intermediate electron donor between the bc 1 complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in cycA neither affected nosZ gene expression nor NosZ protein steady-state levels. Furthermore, cycA, nnrR and its proximal divergently oriented nnrS gene, are direct targets for FixK2 as determined by in vitro transcription activation assays. The dependence of cycA expression on FixK2 and NnrR in anoxic denitrifying conditions was validated at transcriptional level, determined by quantitative reverse transcription PCR, and at the level of protein by performing heme c-staining of soluble cytochromes. Thus, this study expands the regulon of NnrR and demonstrates the role of CycA in the activity of the nitrous oxide reductase, the key enzyme for nitrous oxide mitigation.

An Integrated Systems Approach Unveils New Aspects of Microoxia-Mediated Regulation in Bradyrhizobium diazoefficiens.

The adaptation of rhizobia from the free-living state in soil to the endosymbiotic state comprises several physiological changes in order to cope with the extremely low oxygen availability (microoxia) within nodules. To uncover cellular functions required for bacterial adaptation to microoxia directly at the protein level, we applied a systems biology approach on the key rhizobial model and soybean endosymbiont Bradyrhizobium diazoefficiens USDA 110 (formerly B. japonicum USDA 110). As a first step, the complete genome of B. diazoefficiens 110spc4, the model strain used in most prior functional genomics studies, was sequenced revealing a deletion of a ~202 kb fragment harboring 223 genes and several additional differences, compared to strain USDA 110. Importantly, the deletion strain showed no significantly different phenotype during symbiosis with several host plants, reinforcing the value of previous OMICS studies. We next performed shotgun proteomics and detected 2,900 and 2,826 proteins in oxically and microoxically grown cells, respectively, largely expanding our knowledge about the inventory of rhizobial proteins expressed in microoxia. A set of 62 proteins was significantly induced under microoxic conditions, including the two nitrogenase subunits NifDK, the nitrogenase reductase NifH, and several subunits of the high-affinity terminal cbb 3 oxidase (FixNOQP) required for bacterial respiration inside nodules. Integration with the previously defined microoxia-induced transcriptome uncovered a set of 639 genes or proteins uniquely expressed in microoxia. Finally, besides providing proteogenomic evidence for novelties, we also identified proteins with a regulation similar to that of FixK2: transcript levels of these protein-coding genes were significantly induced, while the corresponding protein abundance remained unchanged or was down-regulated. This suggested that, apart from fixK 2, additional B. diazoefficiens genes might be under microoxia-specific post-transcriptional control. This hypothesis was indeed confirmed for several targets (HemA, HemB, and ClpA) by immunoblot analysis.

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.