A coordinated pathway for nitrate assimilation and nitric oxide detoxification in Bradyrhizobium diazoefficiens

Abstract

Nitrogen (N) is one of the essential compounds, with a great demand by living organisms. Frequently, N is a limiting factor because, even it constitutes 78% of the air composition (N2), only few organisms, the diazotrophs, are able to fix it (Martinez-Espinosa et al., 2011). Mainly, N-assimilation by organisms is provided from different nitrogenous molecules present in the environment, that form part of the nitrogen cycle, being the most abundant molecule, nitrate (NO3-). NO3- assimilation is a ubiquitous process carried out by prokaryotic, fungi, algae and higher plants. This process is a key to mobilizing this compound, whose accumulation causes environmental and public health problems (Guerrero et al., 1981). Biochemically, the assimilative reduction of NO3- always occurs by a nitrate reductase enzyme with an active molybdenum centre, where NO3- is reduced to NO2-. Next, NO2- is reduced to ammonium (NH4+) by a nitrite reductase enzyme with a sirohaem group at its active site. Finally, NH4+ is incorporated into carbon skeletons to be used in cell metabolism (Moreno-Vivián et al., 2011). Microorganisms can also use NO3- as final acceptor of electrons in respiratory chains. Denitrification is the most important, in which NO3- is reduced to N2 through the formation of NO2-, nitric oxide (NO) and nitrous oxide (N2O) as intermediates, being NO2- and mainly NO toxic for the cells (Stern & Zhu, 2014). This Doctoral Thesis is focus in the rizobia species Bradyrhizobium diazoefficiens, an α-proteobacteria from the Rhizobiales order, able to grow with NO3- as sole N-source, both to assimilate it, and to use as the final electron acceptor, through denitrification. B. diazoefficiens is known mainly for its capacity to associate symbiotically with soybean plants (Glycine max). The symbiotic association between the plant and B. diazoefficiens is established in specialized structures of the plant root, called nodules, where the biological N-fixation as well as denitrification occurs (Bedmar et al., 2013). Previous studies carried out by the Nitrogen Metabolism Group of the Department of Soil Microbiology and Symbiotic Systems from Estación Experimental del Zaidín (CSIC-Granada) showed that hypoxia and NO3- induce NO formation inside the nodules (Sanchez et al., 2010). Due to the inhibitory effect of NO on nitrogenase activity, it is expected the presence of mechanisms to eliminate NO within the nodule. Studies by Meakin et al. (2006) suggested the presence in nodules, of others NO detoxification systems in addition to the denitrification enzyme NO reductase. In the B. diazoefficiens genome, we could find the presence of an haemoglobin, Bjgb; encoded by blr2807 (bjgb), which showed high homology with the single domain haemoglobins Vgb and Cgb of Vitreoscilla stercoraria and Campylobacter jejuni, whose role in NO detoxification were already demonstrated (Sánchez et al., 2011). In the same cluster where bjgb is located, there are other genes putative roles in NO3- assimilation; blr2803-05 (nrtABC), that encodes an ABC type nitrate transporter; blr2806 (narK), responsible for a NO3-/NO2- transporter; blr2808 (flp), encoding a flavoprotein; and blr2809 (nasC), implicated in the synthesis of the assimilatory NO3- reductase. On the other hand, at other location in the chromosome, we have identified genes also involved in NO3-/NO2- assimilation; bll4571 (nirA), encoding the assimilatory NO2- reductase; and bll4572-73 (nasST), responsible for a NO3-/NO2- responsive regulator. By using different experimental approaches, in this Doctoral Thesis we have been able to demonstrate the implication of blr2806-09 and bll4571-73 genes in NO3- and NO2- assimilation in B. diazoefficiens. As well as the function of the Bjgb, as a protein involved in detoxification of NO, molecule that is generated by NasC during NO3- assimilation. These findings propose the identification of a new coordinated system for NO3- and NO2- assimilation and NO detoxification described by first time in bacteria. In addition, we have also investigated the regulation of this system as well as identified the regulators involved in its expression. In this Thesis, we have constructed B. diazoefficiens in-frame deletion mutant strains in blr2806-09 and bll4571-73 genes and we have performed the phenotypic characterization of the mutants. In fact, we have demonstrated that NasC and NirA proteins are the catalytic subunits for NO3- and NO2- assimilatory reductases, respectively. Both nasC and nirA mutant strains, were unable to grow with NO3- as the sole N-source. In the case of the nirA mutant, it was not able to grow with NO2- and accumulated it in the presence of NO3- in the medium, which indicates that the NO3-assimilation is blocked at NO2- reduction level. This hypothesis was confirmed by quantification of NO3- and NO2- reductase activity in both mutants, while the nasC mutant lacks NO3- reductase activity, but NO2- reductase remains. The opposite occurs in the nirA mutant strain. We have also shown that the flavoprotein (Flp) is also involved in NO3- assimilation, given the incapacity of the flp mutant to grow with NO3- as the only N-source. We propose that Flp is implicated in the electron transfer to NasC, but not to NirA. In this Thesis, we have also demonstrated the implication of Bjgb in NO detoxification given the growth decrease it observed in anaerobiosis with NO3- compare to the parental strain. Moreover, in the presence of a NO-donor, bjgb strain showed a delay in growth, like a flp mutant, indicating that possibly the electrons needed by Bjgb to remove NO are provided by Flp. In addition, the NO-dependent nor genes expression is induced in a bjgb mutant, probably due to an increase in NO concentration in this strain. The induction of nor in the bjgb mutant was also verified by measuring NO consumption activity and N2O production in this strain, parameters that were higher than those from the wild-type cells. NarK, possibly acts as an additional level of control to remove intracellular NO2-, which might lead to NO production. However, under our experimental conditions, elimination of NO2- from inside the cell by NarK causes a limitation on growth with NO3- or NO2- as sole N-sources. In this work, we have identified that the narK-bjgb-flp-nasC gene cluster is transcribed as a polycistronic unit under the control of a promoter upstream of narK, with a transcription start site identified by 5'-RACE, and that nirA gene is controlled by a promoter located in the intergenic space before it. The narK promoter region presents some regulatory elements such as FNR box, and also NtrC boxes or the formation of hairpins with the ANTAR sequences at the start of mRNA that were found in narK as well as nirA promoter regions. The functionality of the regulatory elements found has been confirmed by using mutant strains for the candidate transcriptional regulators involved. In fact, a mutant strain defective in NtrC, the transcriptional regulator involved in the general regulation of N-compounds, was unable to grow with NO3- as sole N-source, and also showed a growth defect with NO2-, as well as decreased rates of NO3- and NO2- reductases activities and expression of a narK-lacZ and nirA-lacZ transcriptional fusions. Similarly, the NO3-/NO2- response regulator nasT mutant strain was unable to grow with NO3- and NO2-, confirming the implication of the hairpins present in the mRNA of these genes in the premature transcriptional termination in the absence of NasT. Finally, the dependence of the narK and nirA promoters on the sigma factor σ54 of the RNA polymerase has been verified, since a mutant defective in the rpoN1/2 genes encoding this factor σ54, also exhibited a defect in growth, as well as in NO3- and NO2- reductases activities and gene expression. By concluding, in this Thesis, we have performed a detailed characterization, at the physiological, biochemical and regulatory levels, of a new coordinated system for NO3- assimilation and NO detoxification in B. diazoefficiens. As far as we know, this is the first time where a coordinated system like this has been described in bacteria.