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 N2-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 fixK2 genes whose products form the FixLJ–FixK2 regulatory cascade. Besides FixK2, 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–FixK2 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.
- Bradyrhizobium japonicum
- denitrification genes
- nitrate respiration
- nitrite and nitric oxide respiratory regulator
- two-component regulatory system
The genera Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium, collectively referred to as rhizobia, are members, among others, of the bacterial order Rhizobiales of the Alphaproteobacteria . They are Gram− soil bacteria with the unique ability to induce the formation of nodules on legume roots and stems of some species and on specific organs. Within the nodules, vegetative bacteria transform into specialized cells, the so-called bacteroids, which synthesize nitrogenase, the enzyme responsible for the reduction of N2 to ammonia (NH4+).
As free-living cells, rhizobia are aerobic microorganisms that utilize O2 as the final electron acceptor of the respiratory chain for the generation of ATP. Whereas 250 μM of dissolved oxygen is measured in the air, the oxygen concentration in soil is constantly fluctuating because of biological, climatological and mechanical factors. Moreover, pO2 within the nodules is extremely low, ranging from 3 to 22 nM. When oxygen becomes limiting, denitrifying bacteria are capable of sequentially reducing nitrate (NO3−) or nitrite (NO2−) to N2, through the intermediates nitric oxide (NO) and nitrous oxide (N2O) in a four-reaction process catalysed by nitrate-, nitrite-, nitric oxide- and nitrous-oxide reductase respectively. Reduction of nitrogen oxides during denitrification is coupled with energy conservation and permits cell growth under anoxic conditions. Reviews covering the physiology, biochemistry and molecular genetics of denitrification have been published elsewhere [2–5].
Denitrification among rhizobia is rare, and only B. japonicum, the microsymbiont of soya beans, has been shown to reduce NO3− simultaneously to NH4+ and N2 when cultured microaerobically with nitrate as the terminal electron acceptor and sole source of nitrogen . In the present study, we report on the structural and regulatory genes involved in the endosymbiotic bacterium B. japonicum USDA110 denitrification pathway.
The napEDABC genes encoding the Nap (periplasmic nitrate reductase)
The first step of denitrification, the two-electron reduction of nitrate to nitrite, is catalysed by nitrate reductase. Two types of enzymes have been found in denitrifying bacteria, the respiratory Nar (membrane-bound nitrate reductase) and the Nap [2,7,8].
In a shotgun cloning experiment, a DNA fragment of B. japonicum was sequenced and found to contain the napEDABC genes  (Figure 1). The deduced primary sequences of NapA, napB and NapC have between 46 and 76% identity with the translated sequences from other denitrifiers. napA encodes the catalytic subunit containing the molybdopterin guanine-dinucleotide cofactor (MGD) and a [4Fe-4S] cluster, napB an electron-transfer subunit, dihaem cytochrome c, and napC a membrane-bound c-type tetrahaem cytochrome respectively. The N-terminal region of NapA contains a twin-arginine motif diagnostic for a large number of metalloproteins that are exported to the periplasm through the Tat (twin arginine translocon) system . 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. DNA sequences showing homology with those published coding for the Nar system (narGHI genes) have not been found in the complete genome sequence of B. japonicum USDA110 (, see also http://www.kazusa.or.jp/rhizobase). Because a napA mutant was incapable of growing under nitrate-respiring conditions, lacked nitrate reductase activity and did not show the NapA component, the B. japonicum Nap system is the primary enzyme respiring nitrate under anoxic conditions. The rhizobia Pseudomonas sp. G-179 (actually R. galegae) , Sinorhizobium meliloti (http://www.kazusa.or.jp/rhizobase) and Azospirillum brasilense  contain a Nap enzyme, and lack a Nar enzyme. Neither of the enzymes have been detected in Mesorhizobium loti strain MAFF 303090 (http://www.kazusa.or.jp/rhizobase) or Rhizobium etli CFN42 (N. Gómez-Hernández, M. Delgado and L. Girard, personal communication).
The nirK gene encoding the Cu-containing Nir (nitrite reductase)
Although denitrification is initiated by respiratory (dissimilatory) nitrate reduction, this reaction is not unique to denitrification since it also occurs in ammonification and assimilatory nitrate reduction. Thus it is considered that the defining reaction in denitrification is the one-electron reduction of nitrite to the first gaseous intermediate, NO, catalysed by respiratory Nir. Two distinct types of Nir are found in denitrifying bacteria: one contains a c-type and a d1-type haem as the redox-active centre, and the other contains Cu as the redoxactive transition metal. In contrast with the complex organization of the genes encoding the cd1-type Nir, e.g. nirSTBMCFDLGH in Pseudomonas stutzeri, a single gene, nirK, is responsible for the synthesis of Cu-Nir in other denitrifiers. The structural and functional characteristics of both enzymes have been reviewed [2,14]. cd1-type and Cu-Nir are never found together in a single species. In B. japonicum, an nirK gene was identified (Figure 1) whose deduced primary sequence has greater than 68% identity with translated sequences of nirK genes from other denitrifiers . NirK is soluble and apparently exported to the periplasm via the Tat translocon, as judged by a twin-arginine motif present in its N-terminal sequence. Implication of B. japonicum nirK in denitrification occurred when nirK mutants were shown to be incapable of growing when cultured under microaerobic conditions in the presence of either nitrate or nitrite. In those mutants, however, Nap was an active enzyme because cells accumulated nitrite during incubation .
Among rhizobia, nirK is present in R. sullae (formerly R. hedysari) , S. meliloti (http://www.kazusa.or.jp/rhizobase), Pseudomonas sp. G-179  and R. etli CFN42 . In contrast, A. brasilense has been shown to contain a cd1-type Nir .
The norCBQD genes encoding the Nor (nitric oxide reductase)
The two-electron reduction of NO to N2O involves formation of an N–N bond, a reaction catalysed by a Nor. Of the two types of Nor identified in denitrifying bacteria, one type, referred to as cNor, receives electrons from cytochrome c or pseudoazurin, and the other type, referred to as qNor, receives electrons from quinol [2,19,20].
In B. japonicum, the nor genes are organized in the norCBQDE gene cluster (Figure 1), in which norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of cNor respectively; norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product is of unknown function . Sequence analysis of the norCBQD genes revealed that norB has sufficient sequence similarity to terminal oxidases to include cNor within the family of haem-copper oxidases ( and references therein). Inspection of the complete genome sequence of B. japonicum shows the existence of an open reading frame, blr3212, whose sequence has more than 60% identity with norE genes from various denitrifiers. The NorE protein also has similarity to CoxIII, the third subunit of the aa3-type cytochrome c oxidases.
Mutational analysis indicated that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. Each norC and norB insertional mutant accumulated NO during incubation under microaerobic conditions, and the norC mutant lacked the c-type cytochrome found in membranes from wild-type cells .
The norCBQD gene cluster has also been found in S. meliloti. Genetic information for the synthesis of cNor is also present in R. etli CFN42 (N. Gómez-Hernández, M. Delgado and L. Girard, personal communication).
The nosRZDFYLX genes coding for Nos (nitrous oxide reductase)
The last step of denitrification involves a triple N–N bond formation, a reaction catalysed by a Nos. The enzyme was first isolated from Ps. stutzeri and the corresponding structural gene was designated nosZ, which is also the first denitrification gene whose nucleotide sequence was determined . Additional genes required for associated regulatory and electron transfer components, and information for metal processing and protein assembly or maturation are encoded by the nosRZDFYL tatE operon . The B. japonicum nos genes (Figure 1) were identified using a major internal portion of the Ps. stutzeri nosZ gene as a probe , and found to be organized in the nosRZDFYLX gene cluster . nosR codes for an integral membrane protein with six transmembrane helices and a C-terminal cytoplasmic domain containing two cysteine clusters similar to the [4Fe-4S] binding motifs of several bacterial ferredoxins ( and references therein). In Ps. stutzeri, NosR is required for the transcription activation of the nos promoter . nosZ encodes the monomers of NosZ, whose deduced primary sequence has between 50 and 77% identity with the translated sequences of the nosZ gene from other denitrifiers. In its N-terminus, a twin-arginine motif indicates that NosZ is exported via the Sec-independent Tat translocon. The NosZ sequence also contains the site signature believed to bind the copper metal ligand. The NosD, -F, -Y and L proteins show significant homology to the bacterial ATP-binding-cassette (ABC) transporter systems, which are typically composed of a cytoplasmic ATP/GTP binding protein (NosF), a transmembrane protein (NosY) and one or two periplasmic components (NosD and NosL). Because NosZ has its own Tat-related periplasmic signal sequence, it is unlikely that the ABC-transporter is used for transport of Nos.
B. japonicum strains carrying either a nosZ or a nosR mutation grew well when cultured microaerobically with nitrate as the final electron acceptor. Nevertheless, Nos was not an active enzyme because cells accumulated nitrous oxide during growth . Apart from B. japonicum and S. meliloti, no other rhizobia have been described to contain a Nos enzyme.
Regulation of denitrification genes
In B. japonicum, maximal expression of denitrification genes requires both the absence of oxygen and the presence of nitrate or a derived N-oxide. Microaerobic induction of transcription from the nir, nor and nos promoter regions depends on the fixLJ and fixK2 genes [15,21,24], whose products form the FixLJ–FixK2 regulatory cascade . FixLJ is a two-component regulatory system consisting of the haem-based sensor kinase FixL and the FixJ response regulator. The only known target of FixJ in B. japonicum is fixK2 whose product encodes the Fnr (fumarate and nitrate reductase)- and Crp (cAMP receptor protein)-type transcriptional regulator FixK2, which was also shown to activate genes involved in microaerobic and anaerobic metabolism . Homologous sequences to the consensus DNA binding sites of the Fnr and FixK proteins (5′-TTGAT-N4-ATCAA/GTCAA-3′ respectively [2,28,29]) are present in the promoter region of each denitrification gene.
Recently, the nnrR gene has been identified whose product, the NnrR (nitrite and nitric oxide respiratory regulator) protein, shares between 47 and 76% identity with other Fnr/FixK-type transcriptional activators . NnrR lacks in its N-terminus the cysteine motif that is characteristic for redox-responsive Fnr-like proteins, and contains in the C-terminus a predicted helix-turn-helix motif likely to be involved in DNA binding. NnrR-like proteins, including B. japonicum NnrR, constitute a consolidated group within the Fnr–Crp regulators with the distinguishing feature that most of them have a histidine susbstitution for the glutamate in the E-SR sequence of the putative DNA recognition helix [30,31]. Mutant strains carrying an nnrR null mutation were unable to grow microaerobically in the presence of nitrate or nitrite, and they lacked both nitrate and Nir activities. Regulatory studies indicated that, in addition to microaerobiosis, nitrate, or an N-oxide derived from it, presumably either NO2− or NO or both, are required for maximal induction of the B. japonicum denitrification genes. nnrR does not control its own expression and was not expressed in fixLJ and fixK2 mutants. A model compatible with these results has been proposed which places NnrR in the FixLJ–FixK2 cascade downstream of the FixK2 (Figure 2) .
Symbiotic expression of the nir, nor and nos denitrification genes has been shown for the first time in soya-bean root nodules following histochemical detection of β-galactosidase in root nodules and further confirmation of activity in isolated bacteroids .
We thank H. Hennecke and H.M. Fischer for their continuous support. Work on denitrification genes in our laboratory is supported by grants BMC2002-04126-C03-02 and FIT-050000-2001-30 from Dirección General de Investigación and PAI/CVI-275 from Junta de Andalucía.
The 10th Nitrogen Cycle Meeting 2004: Focused Meeting held at the University of East Anglia, Norwich, U.K., 2–4 September 2004. Edited by C.S. Butler (Newcastle upon Tyne, U.K.) and D.J. Richardson (Norwich, U.K.). Sponsored by the COST (European Cooperation in the field of Scientific and Technical Research) Office and the ESF (European Science Foundation).
Abbreviations: CRP, cAMP receptor protein; Fnr, fumarate and nitrate reductase; Nap, periplasmic nitrate reductase; Nar, membrane-bound nitrate reductase; Nir, nitrite reductase; NnrR, nitrite and nitric oxide respiratory regulator; Nor, nitric oxide reductase; Nos, nitrous oxide reductase; Tat, twin arginine translocon
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