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.

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.

Economic Improvement of Artisanal Fishing by Studying the Survival of Discarded Plectorhinchus mediterraneus.

Europe calls for the end to fisheries discards, which means bringing all caught fish (subject to minimum sizes or quotas) to land. This decision is beneficial to the ecosystem, since it forces the selectivity of the fishing gears to improve. However, artisanal fishermen find themselves in a vulnerable situation where their subsistence depends on catches with small profit margins. An exemption to this landing obligation exists, as it is also ruled that those animals whose survival is scientifically guaranteed may be returned to the sea. Here we study the survival of Plectorhinchus mediterraneus captured by hookline and gillnet, as well as their physiological recovery. Survival exceeds 93% in both cases. The physiological assessment of primary (cortisol) and secondary (energy mobilization, acid-base and hydromineral balance, and immune system) stress responses indicates that surviving animals are able to recover after fishing. Thus, we propose the optimal size of capture of this species to achieve greater economic benefit. For this, we rely on the prices according to size in recent years, as well as on the growth curves of the species. In this way, by releasing fish of less than 1 kg, the current benefits could be multiplied between 2.3 and 9.6 times. This pilot study lays the groundwork for regulating artisanal fisheries through scientific data related to survival of discards along with information on the sale prices.

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.

Nuclear Receptors (PPARs, REV-ERBs, RORs) and Clock Gene Rhythms in Goldfish (Carassius auratus) Are Differently Regulated in Hypothalamus and Liver.

The circadian system is formed by a network of oscillators located in central and peripheral tissues that are tightly linked to generate rhythms in vertebrates to adapt the organism to the cyclic environmental changes. The nuclear receptors PPARs, REV-ERBs and RORs are transcription factors controlled by the circadian system that regulate, among others, a large number of genes that control metabolic processes for which they have been proposed as key genes that link metabolism and temporal homeostasis. To date it is unclear whether these nuclear receptors show circadian expression and which zeitgebers are important for their synchronization in fish. Therefore, the objective of this study was to investigate whether the two main zeitgebers (light-dark cycle and feeding time) could affect the synchronization of central (hypothalamus) and peripheral (liver) core clocks and nuclear receptors in goldfish. To this aim, three experimental groups were established: fish under a 12 h light-12 h darkness and fed at Zeitgeber Time 2; fish with the same photoperiod but randomly fed; and fish under constant darkness and fed at Circadian Time 2. After one month, clock genes and nuclear receptors expression in hypothalamus and liver and circulating glucose were studied. Clock genes displayed daily rhythms in both tissues of goldfish if the light-dark cycle was present, with shifted-acrophases of negative and positive elements, as expected for proper functioning clocks. In darkness-maintained fish hypothalamic clock genes were fully arrhythmic while the hepatic ones were still rhythmic. Among studied nuclear receptors, in the hypothalamus only nr1d1 was rhythmic and only when the light-dark cycle was present. In the liver all nuclear receptors were rhythmic when both zeitgebers were present, but only nr1d1 when one of them was removed. Plasma glucose levels showed significant rhythms in fish maintained under random fed regimen or constant darkness, with the highest levels at 1-h postprandially in all groups. Altogether these results support that hypothalamus is mainly a light-entrained-oscillator, while the liver is a food-entrained-oscillator. Moreover, nuclear receptors are revealed as clear outputs of the circadian system acting as key elements in the timekeeping of temporal homeostasis, particularly in the liver.

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.

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.

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.

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.

First evidence on the role of palmitoylethanolamide in energy homeostasis in fish.

The objective of this study was to investigate the role of palmitoylethanolamide (PEA) in the regulation of energy homeostasis in goldfish (Carassius auratus). We examined the effects of acute or chronic intraperitoneal treatment with PEA (20 µg·g-1 body weight) on parameters related to food intake and its regulatory mechanisms, locomotor activity, glucose and lipid metabolism, and the possible involvement of transcription factors and clock genes on metabolic changes in the liver. Acute PEA treatment induced a decrease in food intake at 6 and 8 h post-injection, comparable to that observed in mammals. This PEA anorectic effect in goldfish could be mediated through interactions with leptin and NPY, as PEA increased hepatic expression of leptin aI and reduced hypothalamic expression of npy. The PEA chronic treatment reduced weight gain, growth rate, and locomotor activity. The rise in glycolytic potential together with the increased potential of glucose to be transported into liver suggests an enhanced use of glucose in the liver after PEA treatment. In addition, part of glucose may be exported to be used in other tissues. The activity of fatty acid synthase (FAS) increased after chronic PEA treatment, suggesting an increase in the hepatic lipogenic capacity, in contrast with the mammalian model. Such lipogenic increment could be linked with the PEA-induction of REV-ERBα and BMAL1 found after the chronic treatment. As a whole, the present study shows the actions of PEA in several compartments related to energy homeostasis and feeding behavior, supporting a regulatory role for this N-acylethanolamine in fish.

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.

Diurnal Profiles of N-Acylethanolamines in Goldfish Brain and Gastrointestinal Tract: Possible Role of Feeding.

N-acylethanolamines (NAEs) are a family of endogenous lipid signaling molecules that are involved in regulation of energy homeostasis in vertebrates with a putative role on circadian system. The aim of this work was to study the existence of daily fluctuations in components of NAEs system and their possible dependence on food intake. Specifically, we analyzed the content of oleoylethanolamide (OEA), palmitoylethanolamide (PEA), stearoylethanolamide (SEA), their precursors (NAPEs), as well as the expression of nape-pld (NAEs synthesis enzyme), faah (NAEs degradation enzyme), and pparα (NAEs receptor) in gastrointestinal and brain tissues of goldfish (Carassius auratus) throughout a 24-h cycle. Daily profiles of bmal1a and rev-erbα expression in gastrointestinal tissues were also quantified because these clock genes are also involved in lipid metabolism, are PPAR-targets in mammals, and could be a link between NAEs and circadian system in fish. Gastrointestinal levels of NAEs exhibited daily fluctuations, with a pronounced and rapid postprandial increase, the increment being likely caused by food intake as it is not present in fasted animals. Such periprandial differences were not found in brain, supporting that NAEs mobilization occurs in a tissue-specific manner and suggesting that these three NAEs could be acting as peripheral satiety signals. The abundance of pparα mRNA displayed a daily rhythm in the intestine and the liver, suggesting a possible rhythmicity in the NAEs functionality. The increment of pparα expression during the rest phase can be related with its role stimulating lipid catabolism to obtain energy during the fasting state of the animals. In addition, the clock genes bmal1a and rev-erbα also showed daily rhythms, with a bmal1a increment after feeding, supporting its role as a lipogenic factor. In summary, our data show the existence of all components of NAEs system in fish (OEA, PEA, SEA, precursors, synthesis and degradation enzymes, and the receptor PPARα), supporting the involvement of NAEs as peripheral satiety signals.

Rhizobium etli Produces Nitrous Oxide by Coupling the Assimilatory and Denitrification Pathways.

More than two-thirds of the powerful greenhouse gas nitrous oxide (N2O) emissions from soils can be attributed to microbial denitrification and nitrification processes. Bacterial denitrification reactions are catalyzed by the periplasmic (Nap) or membrane-bound (Nar) nitrate reductases, nitrite reductases (NirK/cd 1Nir), nitric oxide reductases (cNor, qNor/ CuANor), and nitrous oxide reductase (Nos) encoded by nap/nar, nir, nor and nos genes, respectively. Rhizobium etli CFN42, the microsymbiont of common bean, is unable to respire nitrate under anoxic conditions and to perform a complete denitrification pathway. This bacterium lacks the nap, nar and nos genes but contains genes encoding NirK and cNor. In this work, we demonstrated that R. etli is able to grow with nitrate as the sole nitrogen source under aerobic and microoxic conditions. Genetic and functional characterization of a gene located in the R. etli chromosome and annotated as narB demonstrated that growth under aerobic or microoxic conditions with nitrate as nitrogen source as well as nitrate reductase activity requires NarB. In addition to be involved in nitrate assimilation, NarB is also required for NO and N2O production by NirK and cNor, respectively, in cells grown microoxically with nitrate as the only N source. Furthermore, ß-glucuronidase activity from nirK::uidA and norC::uidA fusions, as well as NorC expression and Nir and Nor activities revealed that expression of nor genes under microoxic conditions also depends on nitrate reduction by NarB. Our results suggest that nitrite produced by NarB from assimilatory nitrate reduction is detoxified by NirK and cNor denitrifying enzymes that convert nitrite into NO which in turn is reduced to N2O, respectively.

The Hemoglobin Bjgb From Bradyrhizobium diazoefficiens Controls NO Homeostasis in Soybean Nodules to Protect Symbiotic Nitrogen Fixation.

Legume-rhizobia symbiotic associations have beneficial effects on food security and nutrition, health and climate change. Hypoxia induced by flooding produces nitric oxide (NO) in nodules from soybean plants cultivated in nitrate-containing soils. As NO is a strong inhibitor of nitrogenase expression and activity, this negatively impacts symbiotic nitrogen fixation in soybean and limits crop production. In Bradyrhizobium diazoefficiens, denitrification is the main process involved in NO formation by soybean flooded nodules. In addition to denitrification, nitrate assimilation is another source of NO in free-living B. diazoefficiens cells and a single domain hemoglobin (Bjgb) has been shown to have a role in NO detoxification during nitrate-dependent growth. However, the involvement of Bjgb in protecting nitrogenase against NO in soybean nodules remains unclear. In this work, we have investigated the effect of inoculation of soybean plants with a bjgb mutant on biological nitrogen fixation. By analyzing the proportion of N in shoots derived from N2-fixation using the 15N isotope dilution technique, we found that plants inoculated with the bjgb mutant strain had higher tolerance to flooding than those inoculated with the parental strain. Similarly, reduction of nitrogenase activity and nifH expression by flooding was less pronounced in bjgb than in WT nodules. These beneficial effects are probably due to the reduction of NO accumulation in bjgb flooded nodules compared to the wild-type nodules. This decrease is caused by an induction of expression and activity of the denitrifying NO reductase enzyme in bjgb bacteroids. As bjgb deficiency promotes NO-tolerance, the negative effect of NO on nitrogenase is partially prevented and thus demonstrates that inoculation of soybean plants with the B. diazoefficiens bjgb mutant confers protection of symbiotic nitrogen fixation during flooding.

Time-Lag in Feeding Schedule Acts as a Stressor That Alters Circadian Oscillators in Goldfish.

The circadian system controls temporal homeostasis in all vertebrates. The light-dark (LD) cycle is the most important zeitgeber ("time giver") of circadian system, but feeding time also acts as a potent synchronizer in the functional organization of the teleost circadian system. In mammals is well known that food intake during the rest phase promotes circadian desynchrony which has been associated with metabolic diseases. However, the impact of a misalignment of LD and feeding cycles in the entrainment of fish circadian oscillators is largely unknown. The objective of this work was to investigate how a time-lag feeding alters temporal homeostasis and if this could be considered a stressor. To this aim, goldfish maintained under a 12 h light-12 h darkness were fed at mid-photophase (SF6) or mid-scotophase (SF18). Daily rhythms of locomotor activity, clock genes expression in hypothalamus, liver, and head kidney, and circulating cortisol were studied. Results showed that SF6 fish showed daily rhythms of bmal1a and clock1a in all studied tissues, being in antiphase with rhythms of per1 genes, as expected for proper functioning clocks. The 12 h shift in scheduled feeding induced a short phase advance (4-5-h) of the clock genes daily rhythms in the hypothalamus, while in the liver the shift for clock genes expression rhythms was the same that the feeding time shift (∼12 h). In head kidney, acrophases of per genes underwent a 12-h shift in SF18 animals, but only 6 h shift for clock1a. Plasma cortisol levels showed a significant daily rhythm in animals fed at SF6, but not in SF18 fish fed, which displayed higher cortisol values throughout the 24-h. Altogether, results indicate that hypothalamus, liver, and head kidney oscillate in phase in SF6 fish, but these clocks are desynchronized in SF18 fish, which could explain cortisol alterations. These data reinforce the hypothesis that the misalignment of external cues (daily photocycle and feeding time) alters fish temporal homeostasis and it might be considered a stressor for the animals.

Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes.

Nitric oxide (NO) is a signaling molecule with multiple functions in plants. Given its critical importance and reactivity as a gaseous free radical, we have examined NO production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate. Also, in this context, we critically assess previous and current views of NO production and detection in nodules. EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb2+NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature. Consequently, bacteroids are a major source of NO, denitrification enzymes are required for NO homeostasis, and Lb2+NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb2+NO is artifactually generated in nodule extracts or in intact nodules not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not by those of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrate- and arginine-independent pathways for NO production. Together, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants.

Overexpression of the periplasmic nitrate reductase supports anaerobic growth by Ensifer meliloti.

The alfalfa endosymbiont Ensifer meliloti strain1021 is known to be an incomplete denitrifier due to its inability to grow anoxically using nitrate as respiratory substrate to produce ATP and grow under anoxic conditions. Although this bacterium contains and expresses the complete set of denitrification genes napEFDABC, nirK, norECBQD and nosRZDFYLX encoding the periplasmic nitrate reductase (Nap), Cu-containing nitrite reductase (NirK), c-type nitric oxide (cNor) and nitrous oxide reductase (Nos), respectively, the reasons of its inability to grow under anoxic conditions are still very poorly understood. In the present study, we have constructed an E. meliloti strain overexpressing napEFDABC genes (Nap+) and demonstrated that this strain is able to grow through anaerobic nitrate respiration. Furthermore, Nap+ showed increased NapC levels as well as Nap, Nir and cNor activities and higher capacity to produce NO and N2O compared to wild-type cells. These results suggest that the inability of E. meliloti to grow under anaerobic conditions using nitrate as electron acceptor is attributable to a limitation in the expression of the periplasmic nitrate reductase.

First evidence of nocturnin in fish: two isoforms in goldfish differentially regulated by feeding.

Nocturnin (NOC) is a unique deadenylase with robust rhythmic expression involved in the regulation of metabolic processes in mammals. Currently, the possible presence of NOC in fish is unknown. This report aimed to identify NOC in a fish model, the goldfish ( Carassius auratus), and to study the possible regulation of its expression by feeding. Two partial-length cDNAs of 293 and 223 bp, named nocturnin-a ( noc-a) and nocturnin-b ( noc-b), were identified and found to be highly conserved among vertebrates. Both mRNAs show a similar widespread distribution in central and peripheral tissues, with higher levels detected for noc-a compared with noc-b. The periprandial expression profile revealed that noc-a mRNAs rise sharply after a meal in hypothalamus, intestinal bulb, and liver, whereas almost no changes were observed for noc-b. Food deprivation was found to exert opposite effects on the expression of both NOCs (generally inhibitory for noc-a, and stimulatory for noc-b) in the three mentioned tissues. A single meal after a 48-h food deprivation period reversed (totally or partially) the fasting-induced decreases in noc-a transcripts in all studied tissues and the increases in noc-b expression in the intestinal bulb. Together, this study offers the first report of NOC in fish and shows a high dependence of its expression on feeding and nutritional status. The differential responses to feeding of the two NOCs raise the possibility that they might be underlying different physiological mechanisms (e.g., food intake, lipid mobilization, energy homeostasis) in fish.

FixK2 Is the Main Transcriptional Activator of Bradyrhizobium diazoefficiens nosRZDYFLX Genes in Response to Low Oxygen.

The powerful greenhouse gas, nitrous oxide (N2O) has a strong potential to drive climate change. Soils are the major source of N2O and microbial nitrification and denitrification the main processes involved. The soybean endosymbiont Bradyrhizobium diazoefficiens is considered a model to study rhizobial denitrification, which depends on the napEDABC, nirK, norCBQD, and nosRZDYFLX genes. In this bacterium, the role of the regulatory cascade FixLJ-FixK2-NnrR in the expression of napEDABC, nirK, and norCBQD genes involved in N2O synthesis has been previously unraveled. However, much remains to be discovered regarding the regulation of the respiratory N2O reductase (N2OR), the key enzyme that mitigates N2O emissions. In this work, we have demonstrated that nosRZDYFLX genes constitute an operon which is transcribed from a major promoter located upstream of the nosR gene. Low oxygen was shown to be the main inducer of expression of nosRZDYFLX genes and N2OR activity, FixK2 being the regulatory protein involved in such control. Further, by using an in vitro transcription assay with purified FixK2 protein and B. diazoefficiens RNA polymerase we were able to show that the nosRZDYFLX genes are direct targets of FixK2.