No abstract

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.

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.

The net effects of soil biota on exotic invaders can be variable, in part, because net effects are produced by many interacting mutualists and antagonists. Here we compared mutualistic and antagonistic biota in soils collected in the native, expanded, and invasive range of the black locust tree, Robinia pseudoacacia. Robinia formed nodules in all soils with a broad phylogenetic range of N-fixing bacteria, and leaf N did not differ among the different sources of soil. This suggests that the global expansion of Robinia was not limited by the lack of appropriate mutualistic N-fixers. Arbuscular mycorrhizal fungi (AMF) from the native range stimulated stronger positive feedbacks than AMF from the expanded or invasive ranges, a biogeographic difference not described previously for invasive plants. Pythium taxa collected from soil in the native range were not more pathogenic than those from other ranges; however, feedbacks produced by the total soil biota were more negative from soils from the native range than from the other ranges, overriding the effects of AMF. This suggests that escape from other pathogens in the soil or the net negative effects of the whole soil community may contribute to superior performance in invaded regions. Our results suggest that important regional evolutionary relationships may occur among plants and soil biota, and that net effects of soil biota may affect invasion, but in ways that are not easily explained by studying isolated components of the soil biota.

Denitrification is an alternative form of respiration in which bacteria sequentially reduce nitrate or nitrite to nitrogen gas by the intermediates nitric oxide and nitrous oxide when oxygen concentrations are limiting. In Bradyrhizobium japonicum, the N(2)-fixing microsymbiont of soya beans, denitrification depends on the napEDABC, nirK, norCBQD, and nosRZDFYLX gene clusters encoding nitrate-, nitrite-, nitric oxide- and nitrous oxide-reductase respectively. Mutational analysis of the B. japonicum nap genes has demonstrated that the periplasmic nitrate reductase is the only enzyme responsible for nitrate respiration in this bacterium. Regulatory studies using transcriptional lacZ fusions to the nirK, norCBQD and nosRZDFYLX promoter region indicated that microaerobic induction of these promoters is dependent on the fixLJ and fixK(2) genes whose products form the FixLJ-FixK(2) regulatory cascade. Besides FixK(2), another protein, nitrite and nitric oxide respiratory regulator, has been shown to be required for N-oxide regulation of the B. japonicum nirK and norCBQD genes. Thus nitrite and nitric oxide respiratory regulator adds to the FixLJ-FixK(2) cascade an additional control level which integrates the N-oxide signal that is critical for maximal induction of the B. japonicum denitrification genes. However, the identity of the signalling molecule and the sensing mechanism remains unknown.

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.

No abstract

The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66?5 bp downstream of the centre of a putative FNR-like binding site.