Nitric oxide (NO) has gained interest as a major signaling molecule during plant development and in response to environmental cues. Formation of NO during symbiotic interactions has been reported, but the role and sources of NO in nodules remain unclear. In this work, the involvement of denitrification, performed by the symbiont Bradyrhizobium japonicum, in NO formation in soybean nodules in response to flooding conditions has been investigated by inoculating plants with napA-, nirK-, or norC-deficient mutants. Levels of nitrosylleghemoglobin (LbNO) in flooded nirK and norC nodules were significantly higher than those observed in wild-type nodules. In addition, nirK and norC nodules accumulated more nitrite and NO, respectively, than wild-type nodules. By contrast, levels of LbNO, nitrite, and NO in flooded napA nodules were lower than in wild-type nodules. These results suggest that LbNO formation in soybean nodules in response to flooding conditions is caused by nitrite and NO generated from periplasmic nitrate reductase (Nap) and also containing nitrite reductase (NirK) denitrification enzymes. Flooding caused a decrease of nifH expression and nitrogenase activity in wild-type and norC nodules but not in napA or nirK nodules. Incubation of wild-type and norC nodules with a NO scavenger counteracted the effect of flooding. Under free-living conditions, beta-galactosidase activity from a nifD'-'lacZ fusion decreased in a norC mutant, which also accumulated NO in the medium. These results suggest that NO formed by Cu-containing nitrite reductase in soybean nodules in response to flooding has a negative effect on expression of nitrogenase. We propose that Lb has a major role in detoxifying NO and nitrite produced by bacteroidal denitrification in response to flooding conditions.

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The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B. japonicum Nos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3(-))-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3(-) , nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3(-) addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3(-). Thus, although NasST is known to regulate the NO3(-)-mediated response of NO3(-) assimilation genes in bacteria, our results show that NasST regulates the NO3(-) -mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B. japonicum.

NO (nitric oxide) is a signal molecule involved in diverse physiological processes in cells which can become very toxic under certain conditions determined by its rate of production and diffusion. Several studies have clearly shown the production of NO in early stages of rhizobia-legume symbiosis and in mature nodules. In functioning nodules, it has been demonstrated that NO, which has been reported as a potent inhibitor of nitrogenase activity, can bind Lb (leghaemoglobin) to form LbNOs (nitrosyl-leghaemoglobin complexes). These observations have led to the question of how nodules overcome the toxicity of NO. On the bacterial side, one candidate for NO detoxification in nodules is the respiratory Nor (NO reductase) that catalyses the reduction of NO to nitrous oxide. In addition, rhizobial fHbs (flavohaemoglobins) and single-domain Hbs which dioxygenate NO to form nitrate are candidates to detoxify NO under free-living and symbiotic conditions. On the plant side, sHbs (symbiotic Hbs) (Lb) and nsHbs (non-symbiotic Hbs) have been proposed to play important roles as modulators of NO levels in the rhizobia-legume symbiosis. In the present review, current knowledge of NO detoxification by legume-associated endosymbiotic bacteria is summarized.

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Nitrate respiration by the N(2)-fixing symbiotic bacteria Bradyrhizobium japonicum USDA110 is mediated by a Nap (periplasmic nitrate reductase) encoded by the napEDABC genes. Expression of a transcriptional fusion of the nap promoter region to the reporter gene lacZ, P(napE)-lacZ, was very low in aerobically grown cells of USDA110, but expression was induced approx. 3-fold when the cells were cultured under microaerobic conditions, and 12-fold when nitrate was added to the microaerobic incubation medium. The P(napE)-lacZ fusion was not expressed in the fixL 7403, fixJ 7360 and fixK(2) 9043 mutant strains. Microaerobic induction of the P(napE)-lacZ fusion was retained in the nnrR 8678 mutant, but no increase in beta-galactosidase activity was observed upon nitrate addition. Western-blot and Methyl Viologen-dependent nitrate reductase activity assays showed that synthesis and activity of the catalytic NapA subunit in USDA110 was similar to that in the napC 0906 and nirK GRK308 mutant strains incubated microaerobically with nitrate. These results suggest that nitrate and nitrite, which are not reduced by the napC 0906 and nirK GRK308 mutant cells respectively, induced the synthesis and activity of NapA; conversely, formation of endogenous NO was not required for induction of Nap expression.

When soil oxygen levels decrease, some bradyrhizobia use denitrification as an alternative form of respiration. Bradyrhizobium diazoefficiens (nos+) completely denitrifies nitrate (NO3−) to dinitrogen, whereas B. japonicum (nos−) is unable to reduce nitrous oxide to dinitrogen. We found that anaerobic growth with NO3− as the electron acceptor was significantly lower in B. japonicum than in B. diazoefficiens, and this was not explained by the absence of nos in B. japonicum. Our results indicate that the reason for the limited growth of B. japonicum is weak NO3− reduction due to impaired periplasmic nitrate reductase activity, which may rely on posttranscriptional events.

Rhizobium etli CFN42 is unable to use nitrate for respiration and lacks nitrate reductase activity as well as the nap or nar genes encoding respiratory nitrate reductase. However, genes encoding proteins closely related to denitrification enzymes, the norCBQD gene cluster and a novel nirKnirVnnrRnnrU operon are located on pCFN42f. In this study, we carried out a genetic and functional characterization of the reductases encoded by the R. etli nirK and norCB genes. By gene fusion expression analysis in free-living conditions, we determined that R. etli regulates its response to nitric oxide through NnrR via the microaerobic expression mediated by FixKf. Interestingly, expression of the norC and nirK genes displays a different level of dependence for NnrR. A null mutation in nnrR causes a drastic drop in the expression of norC, while nirK still exhibits significant expression. A thorough analysis of the nirK regulatory region revealed that this gene is under both positive and negative regulation. Functional analysis carried out in this work demonstrated that reduction of nitrite and nitric oxide in R. etli requires the reductase activities encoded by the norCBQD and nirK genes. Levels of nitrosylleghemoglobin complexes in bean plants exposed to nitrate are increased in a norC mutant but decreased in a nirK mutant. The nitrate-induced decline in nitrogenase-specific activity observed in both the wild type and the norC mutant was not detected in the nirK mutant. This data indicate that bacterial nitrite reductase is an important contributor to the formation of NO in bean nodules in response to nitrate.