Bradyrhizobium japonicum is a Gram-negative soil bacterium symbiotically associated with soya bean plants, which is also able to denitrify under free-living and symbiotic conditions. In B. japonicum, the napEDABC, nirK, norCBQD and nosRZDYFLX genes which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide respectively are required for denitrification. Similar to many other denitrifiers, expression of denitrification genes in B. japonicum requires both oxygen limitation and the presence of nitrate or a derived nitrogen oxide. In B. japonicum, a sophisticated regulatory network consisting of two linked regulatory cascades co-ordinates the expression of genes required for microaerobic respiration (the FixLJ/FixK2 cascade) and for nitrogen fixation (the RegSR/NifA cascade). The involvement of the FixLJ/FixK2 regulatory cascade in the microaerobic induction of the denitrification genes is well established. In addition, the FNR (fumarase and nitrate reduction regulator)/CRP(cAMP receptor protein)-type regulator NnrR expands the FixLJ/FixK2 regulatory cascade by an additional control level. A role for NifA is suggested in this process by recent experiments which have shown that it is required for full expression of denitrification genes in B. japonicum. The present review summarizes the current understanding of the regulatory network of denitrification in B. japonicum.
Rhizobia are Gram-negative nitrogen-fixing bacteria which establish symbiotic associations with legume plants leading to the formation of so-called nodules on legume roots and on the stems of some aquatic legumes. After invasion of the plant cells via a complex signalling pathway between bacteria and plant, rhizobia stop dividing and undergo differentiation into nitrogen-fixing bacteroids. In recent years, it has emerged that many rhizobia species have genes that encode enzymes required for some, or all, of the four reductase reactions for denitrification . Although the ability to denitrify may enhance rhizobia survival and growth capability in anaerobic soils, only Bradyrhizobium japonicum and Azorhizobium caulinodans have been shown to grow under oxygen-limiting conditions, with nitrate, through the denitrification pathway. The soya bean symbiont B. japonicum is the only rhizobium species that has been identified as being able to denitrify under both free-living and symbiotic conditions. In B. japonicum, denitrification is dependent on the napEDABC, nirK, norCBQD and nosRZDYFLX genes that encode Nap (periplasmic nitrate reductase), NirK (copper-containing nitrite reductase), cNor (c-type nitric oxide reductase) and Nos (nitrous oxide reductase) enzymes respectively . In B. japonicum, cytochrome c550, encoded by cycA, is involved in electron transfer to NirK. This is shown by the inability of a cycA mutant to consume nitrite and, consequently, to grow under denitrifying conditions with nitrate or nitrite as the final electron acceptor . It has been proposed that the cycS gene of B. japonicum, a gene which encodes a soluble cytochrome c6 of approx. 9 kDa, has a role in denitrification, although the precise biochemical function of CycS is currently not known . Neither azurin- nor pseudoazurin-like copper proteins have been annotated in the genome sequence of B. japonicum (http://www.kazusa.jp/rhizobase/).
Similar to many other denitrifiers, expression of denitrification genes in B. japonicum requires both oxygen limitation and the presence of nitrate or a derived nitrogen oxide . In B. japonicum, a sophisticated regulatory network, consisting of two linked regulatory cascades, co-ordinates the expression of genes required for microaerobic respiration (the FixLJ/FixK2 cascade) and for nitrogen fixation (the RegSR/NifA cascade). In these two cascades, different oxygen-sensing mechanisms are responsible for a stepwise activation of downstream events . A moderate decrease in the oxygen concentration in the gas phase to 5% is sufficient to activate expression of FixLJ/FixK2-dependent targets . However, in the RegSR/NifA cascade, the low-oxygen-responsive NifA protein activates the transcription of essential symbiotic nitrogen-fixation genes at an oxygen concentration at, or below, 0.5% in the gas phase. In the present paper, the involvement of the FixJL/FixK2/NnrR and the RegSR/NifA cascades in the regulation of the B. japonicum denitrification process is reviewed.
Involvement of the FixJL/FixK2/NnrR regulatory cascade
Microaerobic induction of transcriptional fusions from the nap, nir, nor and nos promoter regions to the lacZ reporter gene depends on the fixLJ and fixK2 genes whose products form the FixLJ/FixK2 regulatory cascade [2,6]. FixLJ is a two-component regulatory system consisting of the haem-based sensor-kinase FixL and the FixJ response regulator. One of the known targets of FixJ in B. japonicum is fixK2, whose product encodes the FNR (fumarase and nitrate reduction regulator)/CRP (cAMP receptor protein)-type transcriptional regulator FixK2. Apart from being activated by FixJ-phosphate, the fixK2 expression is subjected to negative autoregulation [7,8] (Figure 1).
Regulatory network of B. japonicum denitrification
In addition to denitrification genes, other microaerobically induced targets of FixK2 include the operon fixNOQP which encodes the cbb3 terminal oxidase that support microaerobic respiration under free-living and symbiotic conditions, operon fixGHIS, several haem biosynthesis genes (hemA, hemB, hemN1 and hemN2) and some hydrogen oxidation genes (hup genes) . FixK2 also activates rpoN1 and the regulatory genes fixK1 and nnrR (Figure 1). rpoN1 encodes the alternative σ factor (σ54) of the RNA polymerase, which acts in concert with NifA to activate gene expression. Thus RpoN1 represents the link between the two regulatory cascades (Figure 1). The FixK2-activated fixK1 gene encodes an oxygen-sensitive FNR-like protein that is not essential for symbiotic nitrogen fixation or for anaerobic nitrate respiration. However both processes are affected in B. japonicum fixLJ–fixK2 mutants . Finally, FixK2 activates nnrR which encodes the FNR/CRP-type regulator NnrR . Regulatory studies indicate that nitrogen oxide-mediated induction of nap, nir and nor genes depended on NnrR [6,9]. Thus NnrR expands the FixLJ/FixK2 regulatory cascade by an additional control level, which integrates the nitrogen oxide signal required for maximal induction of the denitrification genes (Figure 1). Induction of the norCBQD promoter is completely abolished in the absence of a functional nnrR gene. By contrast, microaerobic induction of the nap or nirK promoters is retained in a nnrR mutant background, implying that the nap or nirK and the norCBQD promoters exhibit slight differences with regard to their dependence on FixK2 [6,9]. In this context, recent results from our group have demonstrated that purified FixK2 activates transcription from nap- or nirK-dependent promoters but not from nor-dependent promoter (E. Bueno and M.J. Delgado, unpublished work). By contrast, isothermal titration calorimetry allowed us to demonstrate that NnrR bound to a specific DNA fragment from the promoter region of the norCBQD genes, but not to those from the napEDABC and nirK genes. This interaction requires anaerobic conditions, but not the presence of a nitrogen oxide (E. Robles and E.J.Bedmar, unpublished work). In support of these observations, a genome-wide transcription profiling of B. japonicum fixJ and fixK2 mutant strains grown under free-living microaerobic conditions has shown that napEDABC, nirK and nnrR, but not norCBQD, are the targets of the fixK2 regulatory gene .
Involvement of the RegSR/NifA regulatory cascade
Activation of the RegSR/NifA cascade is initiated by the RegSR two-component regulatory system which induces expression of the fixR-nifA operon under aerobic and anaerobic conditions . Transcription of fixR-nifA is controlled by two overlapping, but functionally distinct, promoters, fixRp1 and fixRp2, that are dependent on NifA and RegSR respectively [11,12] (Figure 1). RegSR is a member of the family of two-component regulatory redox-responsive proteins present in a large number of α-proteobacteria. These proteins are named RegSR in B. japonicum, RegBA in Rhodobacter capsulatus, Rhodovulum sulfidophilum and Roseobacter denitrificans, PrrBA in Rhodobacter sphaeroides, ActSR in Sinorhizobium meliloti and Agrobacterium tumefaciens, and RoxSR in Pseudomonas aeruginosa . In Rhodobacter species, the RegBA/PrrBA regulon encodes proteins involved in numerous energy-generating and energy-utilizing processes such as photosynthesis, carbon fixation, nitrogen fixation, hydrogen utilization, aerobic and anaerobic respiration, denitrification, electron transport, and aerotaxis . In R. capsulatus, the membrane-bound sensor-kinase protein RegB contains an H-box site of autophosphorylation (His225), a highly conserved quinone-binding site (the heptapeptide consensus sequence GGXXNPF which is conserved in all known RegB homologues) and a conserved redox-active cysteine residue (Cys265, located in a ‘redox box’) capable of regulating the activity of RegB through forming an intermolecular disulfide bond in response to the redox state . In R. capsulatus, the redox control of RegB probably involves both a direct inhibition of kinase activity by oxidized ubiquinone as well as an alteration of the redox state of Cys265 . As for response regulator, RegA is a protein containing conserved domains that are typical in two-component response regulators as a phosphate-accepting aspartate residue, an ‘acid box’ containing two highly conserved aspartate residues and a helix–turn–helix DNA-binding motif . Both phosphorylated and unphosphorylated forms of the cognate response regulator RegA are capable of activating or repressing a variety of genes in the regulon. In B. japonicum, RegS possesses a highly conserved quinone-binding site and a conserved redox-active cysteine which suggests that the redox state of the membrane-localized quinone pool or the redox-active cysteine might be involved in redox-sensing. However the precise nature of the signal that is transduced by the B. japonicum RegSR is unknown. RegSR is required for the aerobic and anaerobic expression of the fixR-fixA operon. Interestingly, a regR mutation reduces expression of fixR-nifA operon and consequently nitrogen fixation activity . In contrast with RegSR, on the basis of indirect evidence, NifA was suggested to sense oxygen or redox conditions directly, probably via a metal cofactor . Under micro-oxic or anoxic conditions, active NifA enhances its own synthesis via induction of the fixRp1 promoter. In addition, it activates expression of the NifA regulon that include the nif and fix genes, which are directly involved in nitrogen fixation, and also genes that are indirectly related to nitrogen fixation or have unknown function in this process [15,16] (Figure 1). The RegSR  and the NifA regulons  of B. japonicum were unravelled by a genome-wide transcriptome analysis, which identified numerous new genes controlled by the RegSR/NifA cascade.
Recent results from our group show that the NifA regulatory protein is required for maximal expression of napEDABC, nirK and norCBQD genes, suggesting a role for RegSR/NifA regulatory cascade in the control of the denitrification process in B. japonicum . In that study, it was shown that disruption of nifA caused a growth defect in B. japonicum cells when grown under denitrifying conditions, as well as decreased activity of Nap and Nir enzymes. Furthermore, expression of napE–lacZ, nirK–lacZ or norC–lacZ transcriptional fusions, as well as levels of nirK transcripts, were significantly reduced in the nifA mutant after incubation under nitrate-respiring conditions. Conversely, a B. japonicum rpoN1/2 mutant, lacking both copies of the gene encoding the alternative σ factor (σ54), was able to grow anaerobically with nitrate as the terminal electron acceptor, and also showed wild-type levels of nitrate and nitrite reductase activities. These results suggest that the influence of NifA on denitrification genes is independent of σ54 .
Haem c staining analyses of membranes from cells grown anaerobically in a complete medium [YEM (yeast extract/mannitol)], supplemented with nitrate, confirmed that FixK2 is absolutely required for expression of NapC and NorC proteins (Figure 2A, lanes 4 and 5) and that maximal expression of these proteins was not reached in the nifA mutant (Figure 2A, lanes 1 and 3). However, in these experiments, no differences in NapC and NorC expression were observed in a B. japonicum regR mutant compared with the wild-type expression levels (Figure 2A, lanes 1 and 2). Supporting this observation, in microarray experiments performed to characterize the B. japonicum RegSR regulon, neither the nap nor the nor genes were identified as RegR-dependent genes after growth under micro-oxic conditions in complete YEM medium . It might be possible that growth conditions producing redox potential variations in the cells are necessary to demonstrate the involvement of RegR on denitrification. To explore further the possibility of redox-dependent regulation of B. japonicum denitrification, we analysed the effect of reduced and oxidized carbon substrates on the expression of NapC and NorC denitrification proteins. Cells were grown anaerobically with nitrate as the terminal electron acceptor in a minimum medium containing a reduced carbon source, such as butyrate, or an oxidized carbon source, such as succinate. Expression levels of NorC in wild-type cells grown on succinate were significantly higher than those observed in cells grown on butyrate (Figure 2B, lanes 1 and 3). Interestingly, when cells were grown under denitrifying conditions with succinate, there was a significant decrease in the expression of NorC in the regR mutant compared with the expression levels detected in the wild-type strain (Figure 2B, lanes 3 and 4). Under these growth conditions, NapC expression was not detected in the presence of either butyrate or succinate as carbon source. Under denitrifying conditions with succinate, expression of FixP, which is a 32 kDa c-type cytochrome associated with the cytochrome cbb3 complex, was significantly lower in membranes from the regR mutant than in those from the wild-type (Figure 2B, lanes 3 and 4). These findings confirm previous results where, by using a fixP’–‘lacZ fusion, B. japonicum cbb3 oxidase was expressed in a redox-dependent manner. Maximal expression was observed when cells were grown under denitrifying conditions in the presence of an oxidized carbon source and required the regulatory protein RegR . The results shown in Figure 2(B) suggest that expression of B. japonicum NorC subunit of the nitric oxide reductase is under redox control and RegR is involved in this control. It is known that assimilation of reduced carbon substrates, which generate more reducing equivalents than assimilation of oxidized carbon sources, increases the reduction state of the UQ (ubiquinone) pool . Under our experimental conditions, the assimilation of oxidized carbon substrates might change the redox status of the UQ pool inducing NorC expression. Alternatively, a change in the electron flow through the transport chain associated with the denitrification pathway might modulate the activity of RegSR and consequently control the expression of NorC. A similar redox-related mechanism has been proposed in R. sphaeroides where the activity of PrrBA depends on the electron transfer through the cytochrome cbb3 oxidase . However, the mechanism involved in the control of B. japonicum denitrification by RegR is, at the moment, unknown.
Haem c staining in B. japonicum membranes
In support of our results, it has been shown that PrrB/PrrA in R. sphaeroides and ActR in A. tumefaciens are involved in denitrification [23,24]. In R. sphaeroides, disruption of prrA or prrB causes a significant decrease in both nirK expression and Nir activity . In A. tumefaciens, it has been shown that purified ActR binds to the nirK promoter, but not to the nor or nnrR promoters . Further investigations are needed in B. japonicum to demonstrate the involvement of RegR on norCBQD gene expression and to establish whether these genes are direct, or indirect, targets of RegR.
Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).
This work was supported by Junta de Andalucía [grant numbers P07-CVI-3177 and RMN-4746], partially funded by Fondo Europeo de Desarrollo Regional, Ciencia y Tecnología para el Desarrollo [grant number 107PICO312] and the Ministerio de Ciencia e Innovación [grant number AGL2010-18607]. Support from the Junta de Andalucía [grant number BIO-275] is also acknowledged. M.J.T. was supported by a fellowship from the Consejo Superior de Investigaciones Cientificas programme I3P.