Exploring the role of the haemoglobin from Bradyrhizobium diazoefficiens in nitric oxide detoxification during free-living and endosymbiotic lifestyles

Abstract

Nitric oxide (NO) is a diatomic gas, formed by an oxygen and nitrogen atom, with an unpaired electron, which turns it into a radical (Bartberger et al., 2002). This molecule may react with different oxygen species to form reactive nitrogen species (RNS) (Earnshaw and Greenwood, 1997). NO is present in all living organisms where it has different roles depending on its concentration. At low concentrations (nmolar levels) NO acts as a signalling molecule, while at higher concentrations (µmolar levels) it has a toxic effect (Toledo and Augusto, 2012). In bacteria, there are many sources of NO, being denitrification and dissimilatory reduction of nitrate to ammonium (DNRA) the main respiratory sources of NO (reviewed by Torres et al., 2016). Due to the toxic effect of NO, bacteria possess several systems and enzymes to remove it. Among these systems, the best studied and most important proteins for NO detoxification are the haemoglobins. In prokaryotes, three types of haemoglobins have been identified: flavohaemoglobins (fHbs), single domain haemoglobins (sdHbs) and truncated haemoglobins (tHbs) (reviewed by Poole, 2005; Stern and Zhu, 2014; Gell, 2018). Bradyrhizobium diazoefficiens is a Gram-negative α-proteobacterium, belonging to the Rhizobiales order that establishes symbiotic associations with soybean plants (Glycine max). This symbiotic association takes places inside the nodules, which are specialized structures in the roots. In the nodules, the differentiated forms of rhizobium, the bacteroids, are responsible for fixing atmospheric N2 by the action of the nitrogenase enzyme. B. diazoeficciens is also able to grow under oxygen limiting conditions using nitrate as the only nitrogen source, to assimilate it and to use it as final electron acceptor through nitrate respiration that constitutes the first step of the denitrification pathway. In addition to fix N2 in symbiosis, B. diazoefficiens is also capable of performing denitrification process inside soybean nodules. In this bacterium, denitrification reactions are catalyzed by the periplasmic nitrate reductase (Nap), nitrite reductase (NirK), nitric oxide reductase (Nor) and nitrous oxide reductase (Nos), enzymes which reduce nitrate to N2 through the formation of NO and nitrous oxide (N2O) as gaseous intermediates. These enzymes are encoded by the napEDABC (Delgado et al., 2003), nirK (Velasco et al., 2001), norCBQD (Mesa et al., 2002) and nosRZDYFLX (Velasco et al., 2004) genes, respectively (reviewed by Bedmar et al., 2005, 2013). Previous studies carried out in the Nitrogen Metabolism Group from Department of Soil Microbiology and Symbiotic Systems (Estación Experimental del Zaidín, CSIC), showed that hypoxia and nitrate induce NO synthesis in soybean nodules, being denitrification in bacteroids the major process involved in its formation. In this context, the presence of NO detoxification mechanisms in the nodules is necessary, since it has been evidenced that NO inhibits nitrogenase activity, as well as expression of nifH gene, responsible for its synthesis. The denitrifying enzyme Nor has been proposed as the principal protein implicated in NO removal in soybean nodules (Sánchez et al., 2010). Besides to denitrify, B. diazoefficiens is able to assimilate nitrate in free living conditions through the expression of a coordinated system for nitrate assimilation and NO detoxification, which is encoded by the narK-bjgb-flp-nasC operon. This operon is responsible for the synthesis of a nitrate/nitrite (NarK) transporter, a haemoglobin (Bjgb), a NAD(P)H dependent flavoprotein (Flp) and an assimilatory nitrate reductase (NasC). Close to those genes, there is another gene cluster that includes genes coding for an assimilatory nitrite reductase (NirA) and a nitrate/nitrite response regulatory system (NasST). Earlier studies have revealed that Bjgb is a single domain haemoglobin involved in NO detoxification in cells grown under free-living conditions (Cabrera et al., 2016). In this Doctoral Thesis, the role in vitro of B. diazoefficiens Bjgb and Flp in NO detoxification has been demonstrated. To achieve this goal, bjgb and flp genes from B. diazoefficiens have been cloned into expression plasmids to subsequently over-express and purify Bjgb and Flp proteins. UV-Vis spectroscopy characterization of purified Bjgb and Flp showed the ability of Flp to reduce Bjgb. Once Bjgb is reduced, the haem group of this protein is capable to bind NO. These results have proved the ability of Bjgb to bind NO in vitro confirming the role of this protein to detoxify NO in vivo. The function of the lysine 52 of Bjgb has also been investigated by carrying out a site directed mutation of this lysine by an alanine (K52A Bjgb). The role in vivo of this K52A Bjgb mutant has been investigated by complementing a B. diazoefficiens bjgb mutant with two plasmids, one that over-expresses the native Bjgb and another that over-expresses the K52A Bjgb mutant protein. The analysis of Nor expression by using a norC-lacZ transcriptional fusion, detection of NorC protein and measuring the capacity of N2O production, the product of Nor, demonstrated that the lysine-52 haem-iron ligand from Bjgb has a critical role on nor expression and consequently on NO homeostasis in vivo. In this Doctoral Thesis, the involvement of Bjgb from B. diazoefficiens in the symbiotic interaction with soybean plants and in NO homeostasis in the nodules has also been investigated. By using the isotopic 15N dilution technique and analyzing expression of nitrogenase, we have demonstrated that soybean plants inoculated with the bjgb mutant strain had greater tolerance to flooding than those plants inoculated with the parental strain. This beneficial effect is probably due to the reduction of NO accumulation in nodules produced by the bjgb mutant in response to flooding, as compared to parental flooded nodules. The decrease in NO accumulation could be resulting from the induction of expression and activity of the nitric oxide reductase enzyme (Nor), which is the main protein involved in NO removal in soybean nodules. Finally, the involvement of nitrate assimilation in the B. diazoeficciens-Glycine max symbiosis has also been investigated in this Doctoral Thesis. To achieve this goal, B. diazoeficciens mutants defective in nasC and nirA genes, encoding the assimilatory nitrate and nitrite reductases, respectively, have been used to inoculate soybean plants. By analyzing nitrogenase activity and leghemoglobin content of the nodules, we found that inoculation with the nirA mutant confers protection of nitrogen fixation to flooding, compared to those plants inoculated with parental strain. However, no differences in nitrogen fixation were observed in plants inoculated with the nasC mutant and subjected to flooding compared to those plants inoculated with the parental strain. These results, together with the analysis of nitrate reductase activity in the bacteroids, suggest that the assimilatory nitrate reductase NasC has not a relevant role in nitrate reduction in bacteroids being the periplasmic nitrate reductase (NapA) the main enzyme involved. Interestingly, expression of narK gene, the first gene of the operon that codes for the enzymes implicated in nitrate assimilation, was significantly induced in nodules from a nifH mutant strain defective in the Fe-protein from nitrogenase complex. We also analyzed NH4+ production capacity by the bacteroids. As expected, nifH bacteroids from plants grown in the absence of nitrate, were unable to produce NH4+ due to the lack of nitrogenase activity. However, when plants were grown with nitrate, bacteroids from the nifH mutant were able to produce NH4+. These results suggest that nitrate assimilation by bacteroids might have a relevant role in those nodules where nitrogen fixation is impaired. The results obtained during this Doctoral Thesis have contributed to increase the knowledge about the function in vivo and in vitro of Bjgb from B. diazoefficiens in NO detoxification, both in free living conditions and in symbiotic association with soybean plants. In addition, the putative role of B. diazoefficiens NasC and NirA assimilatory nitrate and nitrite reductases in soybean nodules has also been established.