INTRODUCTION

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Denitrification is an anaerobic respiratory process, found in many genera of bacteria, whereby nitrate is reduced sequentially to nitrite, nitric oxide, nitrous oxide, and ultimately dinitrogen gas by a set of corresponding oxidoreductases (5). The last reduction in the pathway, that of nitrous oxide to nitrogen, is performed by nitrous oxide reductase (NosZ), a soluble dimeric enzyme located in the periplasm. NosZ contains two spectroscopically distinct copper centers, Cu_A, at which the enzyme is thought to receive electrons from small redox proteins and Cu_Z, believed to be the catalytic center (13). Recently, two lines of evidence which suggest that the structural complexity of nitrous oxide reductase is reflected in its biosynthetic pathway have emerged. First, the enzyme contains an unusually long periplasmic targeting sequence containing a so-called twin-arginine motif (3), indicating that the enzyme belongs to a family of metalloproteins in which targeting and metal insertion are coupled by a Sec-independent secretory mechanism (28). Second, the structural gene for nitrous oxide reductase (nosZ) is part of a cluster of genes whose products are involved in the maturation of the enzyme. In the organism Pseudomonas stutzeri, nosZ is followed by the genes nosD, -F, -Y, and -L, and it has been suggested that NosD, -F, and -Y form a multisubunit ABC-type transporter which catalyzes the transport and insertion of copper to the Cu_Z center (36). NosL is predicted to be an outer membrane lipoprotein. The NosL protein from P. stutzeri contains sequence motifs for a disulfide isomerase protein (12), but these are absent in NosL from Sinorhizobium meliloti and mutations of nosL yielded no apparent phenotype in either organism (7, 12). Similar nos gene clusters have been identified in the organisms Bradyrhizobium japonicum (GenBank accession number AJ002531), Achromobacter cycloclastes (20), and Sinorhizobium meliloti (17), and there is evidence from partial sequence data that this operon structure is conserved in Paracoccus denitrificans (16) and Pseudomonas aeruginosa (35). Each of these clusters also contain the nosR gene immediately upstream of nosZ. NosR is predicted to be an integral membrane protein with six transmembrane helices and a C-terminal cytoplasmic domain containing sequence motifs for two [4Fe-4S] clusters (9). Insertion mutagenesis of nosR in P. stutzeri resulted in failure to transcribe the nosZ gene.

In B. japonicum, A. cycloclastes, and S. meliloti, an additional nos gene, nosX, has been identified downstream of nosZDFYL. Tn5 mutagenesis of the S. meliloti nosX gene resulted in a Nos phenotype (7), but the molecular basis for the defect is unknown. NosX is predicted to be a soluble periplasmic protein, but to date there has been no detailed characterization of NosX or of the phenotype exhibited by the nosX mutant. A homologue of nosX designated nirX has been identified in the P. denitrificans nir (nitrite reductase) gene cluster (29). Inactivation of this gene yielded no apparent phenotype, suggesting that another protein, possibly a counterpart of nosX, takes over the role of NirX. The aim of the present study was to test this hypothesis and to determine the function of the NosX and NirX proteins during denitrification.

	MATERIALS AND METHODS

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Bacterial strains and growth media. Escherichia coli TG1 (27) was used routinely for cloning procedures. E. coli HB101(pRK2020) was used as the helper strain in the conjugative transfer of plasmids via triparental mating. E. coli strains were grown aerobically in Luria-Bertani medium. P. denitrificans Pd1222 (10) was grown aerobically in brain heart infusion broth (Oxoid) or anaerobically in minimal succinate medium containing 25 mM succinate and 100 mM KNO₃ (29). Antibiotics were used at different concentrations, as follows: ampicillin, 100 µg ml¹; kanamycin, 25 µg ml¹ (E. coli) or 100 µg ml¹ (P. denitrificans); rifampin, 100 µg ml¹; and streptomycin, 25 µg ml¹ (E. coli) or 100 µg ml¹ (P. denitrificans).

DNA manipulations. Routine cloning procedures were performed according to standard protocols (1). Fusions of chromosomal promoter-lacZ and the nosZ gene were carried out using the suicide plasmid pBK11 as described previously (31). The construction of marked and unmarked mutations in the nirX and nosX genes is described in Results and was performed as described previously (32). Identification and characterization of the P. denitrificans nirX gene has been described previously (29). The nosX gene was identified by subcloning and sequencing of plasmid pFUH21 and cosmid pJLA601 (gifts from S. J. Ferguson, University of Oxford), as described in Results. Generation of subclones in M13mp18 or M13mp19 and automated sequencing of single-stranded DNA were performed as described previously (14).

Protein techniques and enzyme assays. Total soluble cell extracts were prepared by French press and ultracentrifugation as previously described (29). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli (19) using a Bio-Rad Mini Protean II apparatus. Immunoblotting of nitrous oxide reductase was performed as described previously (25), using a rabbit polyclonal antiserum against Paracoccus pantotrophus nitrous oxide reductase for the primary antibody (a gift from B. Berks, University of East Anglia) (4) and goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate (Sigma). The -galactosidase activity of promoter-lacZ fusion strains was measured as described previously (21). Nitrous oxide reductase activity in whole cells using succinate as the electron donor was measured by monitoring the steady-state redox level of cytochrome c as described previously (6). Nitrite was assayed colorimetrically according to a method described elsewhere (22).

Analysis of nitrous oxide. Gas samples (150 µl) were withdrawn from the headspace of denitrifying cultures and injected in a gas chromatograph (type 8500; Perkin-Elmer) equipped with a J&W Scientific column (type GS-Q). Helium was used as carrier gas. Concentrations of nitrous oxide in each sample were calculated from standards of pure nitrous oxide.

EPR and optical spectroscopy. Electron paramagnetic resonance (EPR) spectra were recorded on a Varian 109 EPR spectrometer equipped with a home-built He flow system. Optical spectra were recorded on an Aminco DW2000 spectrophotometer.

Nucleotide sequence accession number. The nucleotide sequence of the nosX gene has been deposited in the GenBank database under accession number AJ010260.

	RESULTS

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Isolation and sequence analysis of the nosX gene. As the nosX gene is located in the downstream part of the nos operon in other organisms, we decided to determine whether P. denitrificans also contained a nosX gene in this region. A 1.3-kbp HindIII-SalI fragment of the plasmid pFUH21 carrying part of the nosZ gene from P. denitrificans was cloned into the suicide vector pRVS3 and transferred to P. denitrificans via conjugation. Strains that had integrated the construct via homologous recombination were isolated and shown to contain two truncated versions of nosZ separated by pRVS3 (not shown). These strains were used to recover the plasmid as a recircularized chromosomal PstI fragment containing the nosZ gene and approximately 4.6 kbp of downstream DNA. A map of the nos locus along with the recombination event are presented in Fig. 1. Sequencing at the 3' end of the fragment revealed a partial open reading frame with substantial identity to both nirX of P. denitrificans and the previously identified nosX genes of other denitrifying bacteria. The P. denitrificans nosX gene sequence was completed using subclones from the cosmid pJLA601. It encoded a protein of 304 amino acids, with a molecular mass of 32.1 kDa. The NosX and NirX protein sequences from P. denitrificans showed 37% identity and 49% similarity.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Maps of the nos gene cluster in P. denitrificans wild-type strain (top) and a strain that had integrated suicide vector pRVS3 with a copy of the nosZ gene via homologous recombination (bottom). The PstI sites used for recovery of the plasmid along with adjacent chromosomal DNA are indicated. The position of a kanamycin resistance cassette, a 1.2-kbp PstI fragment from the plasmid pUC4K (Kmr) in the nosX mutant, is also indicated.

View larger version (69K): [in this window] [in a new window]   FIG. 2.   Multiple alignment of the NosX protein family. The alignment was constructed using the programs ClustalX and GeneDoc (23). The twin-arginine motif is indicated by asterisks. The consensus sequence shown on top includes identical amino acid residues found in all sequences (uppercase) and those found uniquely in the NosX sequences (lowercase). PdnirX, P. denitrificans Pd1222; PdnosX, P. denitrificans Pd1222; SmnosX, S. meliloti; AcnosX, A. cycloclastes; RcnosX, R. capsulatus; BjnosX, B. japonicum. -, amino acid residue is absent.

Construction and analysis of nosX, nirX, and double-mutant strains. To investigate the phenotype of nosX and nirX mutants, mutations were introduced into each gene both separately and in combination. A marked nirX mutant strain, Pd76.21, was already available (29). In the case of nosX, a PstI site was first introduced in the middle of that gene at nucleotide position 469 relative to its start codon using a PCR approach with primers that contained a PstI site at the corresponding position. This site was used for the introduction of a kanamycin cassette as a 1.2-kbp PstI fragment from the plasmid pUC4K. The nosX mutant strain was named Pd101.21. To construct a double nosX nirX mutant, an unmarked deletion was first created in nirX by exchanging the chromosomally integrated kanamycin resistance cassette with a plasmid-borne nirX copy from which a 0.2-kbp NruI-HincII fragment had been removed. The unmarked nirX mutant strain, Pd76.31, was then mutated by the insertion of the kan gene into nosX as described previously, resulting in the nirX nosX::Km^r double mutant, Pd92.36. All mutant strains were analyzed using Southern blotting to confirm that the correct recombinational events had taken place.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Growth curve of P. denitrificans Pd1222 (open triangles), compared with those of the mutant strains Pd76.21 (open squares), Pd101.21 (open circles), and Pd92.36 (closed triangles), during denitrifying growth.

View larger version (10K): [in this window] [in a new window]   FIG. 4.   Nitrous oxide reductase activity in whole cells of Pd1222 (A) compared to that of Pd76.21 (B) Pd101.21 (C), and Pd92.36 (D). Cell suspensions (with a turbidity at 660 nm of 50) of anaerobically grown cultures were incubated with succinate under a stream of nitrogen gas to allow full reduction of c-type cytochromes. Changes in the absorption of light at 552 nm, which is diagnostic for the redox state of c-type cytochromes, were recorded in time using an Aminco spectrophotometer. At the points indicated, aliquots of buffer saturated with nitrous oxide were added to the suspensions.

View larger version (52K): [in this window] [in a new window]   FIG. 5.   Lane 1, prestained protein molecular weight standards, the sizes of which are shown to the left of the figure; lanes 2, 3, 4, and 5, immunodetection of nitrous oxide reductase in total soluble extracts from Pd1222, Pd76.21, Pd101.21, and Pd92.36, respectively.

EPR spectroscopy of nirX nosX double mutant. The EPR spectrum of total soluble extract from wild-type P. denitrificans cells in the oxidized state showed four lines separated by 3.83 mT with a g_z value of 2.17, characteristic for Cu_A of NosZ (18) or cytochrome c oxidase (34) (Fig. 6A). The relative intensity of the lines is consistent with a 1:2:3:4 ratio, as expected for the first four of the seven spectral lines arising from a dinuclear mixed-valence copper center. The g- perpendicular region of the spectrum is obscured by resonances of unknown origin and has been omitted from the figure. The features mentioned above are absent from the spectrum of the soluble fraction obtained from Pd92.36. Therefore, we conclude that the Cu_A center is absent from NosZ in the nirX nosX mutant strain. A similar conclusion can be drawn from the optical spectra (Fig. 6B). The reduced minus oxidized spectrum of the wild-type strain shows a broad (weak) absorbance peak around 800 nm, which is diagnostic for Cu_A. This band is lacking from the spectrum of the nirX nosX mutant.

View larger version (11K): [in this window] [in a new window]   FIG. 6.   (A) EPR spectra of total soluble cell extracts from wild-type (Pd1222) and nirX nosX mutant (Pd92.36) strains, oxidized with solid potassium ferricyanide. CuA is the water-soluble fragment of cytochrome caa3 from Bacillus subtilis, shown here for comparison. The vertical lines are spaced by 3.83 mT, the hyperfine coupling value for both the CuA center of nitrous oxide reductase and CuA in the B. subtilis fragment. Spectra are corrected for differences in protein concentrations. The origin of the peaks around 290 and 295 mT in the mutant is unknown. Experimental conditions: frequency, 9.235 GHz; modulation amplitude, 1.0 mT; microwave power, 2.0 mW; temperature, 39K. (B) Optical reduced minus oxidized spectra of total soluble cellular extracts from the wild-type strain (Pd1222) and the nirX nosX mutant strain (Pd92.36). When isolated, the soluble extracts were found to be completely reduced. For oxidation a small amount of solid potassium ferricyanide was added. The absorbance maximum around 800 nm in the wild-type strain is ascribed to CuA and that at 650 to 700 nm to cytochrome cd1. The spectra were corrected for differences in protein concentrations.

	DISCUSSION

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P. denitrificans is the first organism reported to date that contains two homologues of the NosX protein. Thus far P. denitrificans is also unique in containing a second homologue (named nirI) of the gene nosR within the nir gene cluster (29). Insertion mutagenesis in either of the two genes nirX or nosX results in no observable phenotype. This is in contrast to the observation that Tn5 mutagenesis of nosX from S. meliloti gives a Nos-deficient phenotype and suggests that there may be no second homologue of nosX in the latter organism. Despite its position in the P. denitrificans nir gene cluster, nirX appears not to play an essential role in expression of functional nitrite reductase, nor is nosX essential with respect to nitrous oxide reductase activity. However, mutagenesis of both nirX and nosX eliminates nitrous oxide reductase activity in P. denitrificans, implying that the two proteins are functional homologues and that each can take over the other's role.

Our analyses of the nirX nosX double mutant of P. denitrificans showed that the deficiency in nitrous oxide reduction was the consequence of a failure to activate rather than to express nitrous oxide reductase. The analyses of the promoter-reporter gene fusions as well as the Western analyses have shown that nosZ transcription and translation levels in the double mutant were comparable to those in the wild-type strain. NosZ produced by the double mutant, however, was inactive in nitrous oxide reduction regardless of whether endogenous substrates or ascorbate was used as the electron donor. EPR spectral analyses showed that the signals that are diagnostic for Cu_A were completely absent from the soluble fraction of the double mutant cells grown anaerobically with nitrate, whereas they were present in that of the wild-type cells. The most likely explanation for the observation that NosZ was inactive in the double mutant is therefore that its Cu_A center was not properly assembled. It is then tempting to speculate that the NosX and NirX have their task in the transport of copper to NosZ or in catalyzing its insertion into the metal binding site. However, the absence of the Cu_A center might also be the result of a secondary effect of the mutations rather than the primary cause or it may result from a concomitant failure to assemble the Cu_Z center in NosZ.

An alignment of all known NosX protein sequences revealed several highly conserved blocks, but these did not indicate a possible function of NosX when used to search for similar regions in other protein sequences. However, the N-terminal region of the NosX proteins contains the sequence (S/T/N)-R-R-R-(F/A/L/M)-(I/L), corresponding closely to the twin-arginine motif (S/T)-R-R-X-F-L-K. This conserved motif has been identified in a large number of periplasmic metalloproteins that contain complex cofactors and are exported to the periplasm via the Sec-independent TAT translocon (3). Therefore, we hypothesize that NosX is exported via this pathway in a partially folded state.

One intriguing aspect of NosX is its absence from the nos gene cluster of P. stutzeri. A BLAST search of the emerging sequence from the P. aeruginosa and Neisseria gonorrhoeae genomes, both of which contain functional nos operons, also failed to reveal a NosX-like sequence. It may be that a NosX homologue remains to be discovered in these organisms or that the mechanism of Cu_A insertion differs between bacteria. However, there is evidence that Cu_A insertion into P. stutzeri nitrous oxide reductase is an enzyme-catalyzed event. Expression of the nosZ gene from P. stutzeri in E. coli yielded an apoprotein lacking all copper centers, into which copper could be reconstituted in vitro (33). This form of the enzyme was catalytically inactive and contained only Cu_A, but the Cu_A center was spectroscopically distinct from the native protein. A similar result was achieved by reconstitution of purified nitrous oxide reductase from which the copper centers had been chemically removed (8). In contrast, the characteristic electronic spectrum of Cu_A could be obtained when a translocation-incompetent mutant of nitrous oxide reductase in which the twin-arginine motif was mutated (R20D) was reconstituted with copper, but the insertion process was slow and resulted in a weak retention of copper (11). These data led to the suggestion that the twin-arginine motif of NosZ may confer both translocational competence and delivery to a specific system involved with copper insertion.