INTRODUCTION

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Two major pathways of nitrite reduction to ammonia in members of the family Enterobacteriaceae have been described (reviewed by Cole [6]). The main nitrite reductase activity in Escherichia coli is contributed by the cytoplasmic NADH-nitrite reductase, which detoxifies the nitrite formed as the product of nitrate reduction. The less active pathway in most E. coli strains is the electrogenic, formate-dependent nitrite reductase (Nrf pathway; reviewed in references 6 and 19). Both enzymes in E. coli are induced by anaerobiosis and nitrite and fulfill a dissimilatory rather than an assimilatory role (5).

The nir operon in E. coli encodes the NADH-dependent nitrite reductase and consists of five open reading frames (ORFs): nirB, nirD, nirE, nirC, and cysG (21). Essential and sufficient for NADH-dependent nitrite reduction are NirB and NirD, the two subunits of the enzyme (11), and CysG, which is essential for biosynthesis of the siroheme prosthetic group (12, 27, 32). Expression of the nir operon is totally dependent on fumarate and nitrate reductase regulation (FNR) (28), the global E. coli transcription regulator of anaerobically controlled genes (10). FNR-dependent transcription is modulated by two two-component regulatory systems (NarX-NarL and NarQ-NarP), which appear to coordinate transcriptional responses to nitrate and nitrite (7, 22, 33).

High concentrations of nitrate and nitrite in foodstuffs and drinking water can cause severe health problems. A potential application of microorganisms for denitrification has directed attention to the food-grade organism Staphylococcus carnosus, which has been used for a long time in the production of raw fermented sausages due to its ability to reduce nitrate to nitrite, which is essential for development of the typical red color. The nitrate- and nitrite-reducing system of the organism has been characterized physiologically and may have dissimilatory functions (17). Recently, Pantel et al. (20) identified the narGHJI operon, which encodes the dissimilatory nitrate reductase of S. carnosus. The enzyme shows similarity to the corresponding E. coli enzyme with respect to sequence characteristics, induction factors (anaerobiosis, nitrate, and nitrite), and energy gain. Nitrite is converted to ammonia in S. carnosus (17) only in the absence of oxygen and nitrate and if the initial nitrite concentration does not exceed 10 mM (17).

In this paper, we identify and characterize the S. carnosus nitrite reductase operon, which is comprised of five genes: nirR, sirA, nirB, nirD, and sirB. In accordance with the physiological results, the sequence characteristics of the nitrite reductase NirBD resembles the NADH-dependent nitrite reductase of E. coli. SirA and SirB appear to be necessary for biosynthesis of the siroheme prosthetic group. The protein NirR shows no convincing similarity to proteins with known functions, and we attempted to determine its potential function in the enzymatic process or in regulation. Furthermore, we analyzed the intriguing regulation of the nitrite-reducing system in response to oxygen and nitrite.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results AND Discussion References

Media and culture conditions. The bacterial strains were grown at 37°C in basic medium (BM) (9) or in modified BM (17). Aerobic cultures were incubated on a rotary shaker at 160 rpm. Anaerobic cultures were incubated in screw-cap bottles with slow stirring (100 rpm). The medium was supplemented with Oxyrase (20 ml/liter of medium; Oxyrase Inc., Mansfield, Ohio) to create anoxic conditions.

Bacterial strains, plasmids, and plasmid constructions. S. carnosus TM300 (24) was used as the wild-type strain for transposon mutagenesis and also as a control strain and cloning host. E. coli SURE (Stratagene) served as the cloning host for vectors constructed for sequencing and for pRB473 derivatives. This shuttle vector, a derivative of pRB373 (4), was used to construct complementation vectors. The E. coli vectors pUC18 (29) and pBluescriptII KS(+) (Stratagene) were used for subcloning DNA fragments for sequencing.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Map of the locus comprising genes involved in nitrite reduction in S. carnosus. The DNA fragments isolated from partial gene libraries or by inverse PCR and used for sequencing are shown above the gene map together with the names of the plasmids in which these fragments were cloned. Please note that plasmid pRBnirC comprises only the 1.56-kb HindIII/EcoRI fragment. Below the gene map, the different fragments cloned in pRB473 are depicted together with the names of the respective plasmid constructs that were used for complementation analysis and for homologous recombination. The restriction sites of enzymes indicated in parentheses belong to the vector pRB473. Restriction sites with a subscript "i" are inserted by means of PCR. The location of the transposon Tn917 in the transposon mutant nir1 is marked in the gene map (). The promoter (Pnir) in front of the nir operon was mapped by primer extension analysis (Fig. 3), while the promoters upstream of sirA (Pstat, stationary-phase promoter) and nirC (PnirC) are postulated. Putative transcription terminors () are indicated.

Transposon mutagenesis and screening of mutants affected in nitrite reduction. S. carnosus TM300 harboring pTV1ts, which contains the transposon Tn917 (33), was grown in BM containing chloramphenicol (20 µg/ml) and erythromycin (20 µg/ml) at 30°C to an optical density at 578 nm (OD₅₇₈) of 1. The culture was then diluted 100-fold into BM supplemented with erythromycin (2.5 µg/ml) and cultivated for 16 h at 42°C. After two further cycles of cultivation at 42°C (first cycle, 500-fold dilution, 8 h of cultivation; second cycle, 100-fold dilution, 16 h of cultivation), the cells were spread on BM agar plates containing erythromycin (4 µg/ml) and incubated for 1 day at 37°C. Erythromycin-resistant and chloramphenicol-sensitive (Em^r Cm^s) mutants were screened for their ability to reduce nitrite in microtiter plates containing modified BM supplemented with nitrite (1 mM). To decrease the oxygen tension, the medium was overlaid with paraffin oil. The inoculated microtiter plates were incubated at 37°C with shaking (120 rpm). The presence or absence of nitrite was analyzed after 20 h of incubation.

Nitrite reductase activity. Nitrite reductase activity in intact cells was assayed with glucose as the electron donor, as described by Neubauer and Götz (17). Nitrite reductase activity in cell extracts was monitored as nitrite-specific NADH oxidation as described previously (17). For reconstitution of nitrite reductase activity, cells of strains M12 (pRP-ABDB) and nir1 were grown anaerobically with nitrite, harvested in the mid-exponential phase, washed twice with buffer (50 mM MOPS [morpholinepropanesulfonic acid], 150 mM NaCl, pH 7.0), and then lysed with lysostaphin at 37°C in equal volumes either separately or together. The lysates were incubated for up to 40 min. After subsequent centrifugation, the supernatants comprising mainly the cytosolic fractions were used for the determination of NADH-dependent nitrite reductase activity.

Nitrite reduction after shift from aerobiosis to anaerobiosis. Cells were grown aerobically in modified BM supplemented either without or with nitrate (20 mM) or nitrite (2 mM). At an OD₅₇₈ of 3, chloramphenicol (200 µM) or rifampin (200 µM) was added to one-third of each culture and the aerobic incubation was continued for 20 min. Subsequently, the nine different cell batches were washed twice with modified BM with or without antibiotic to remove nitrate and nitrite. All washing steps were performed at 4°C to minimize protein synthesis or enzyme activation at this stage. The cells were then resuspended to an OD₅₇₈ of 2.8 in modified BM with or without chloramphenicol and switched to anaerobiosis by the addition of Oxyrase, and the tubes were transferred to a 37°C water bath. The experiment was started by addition of nitrite (600 µM without antibiotics, 100 µM with antibiotic). The nitrite concentrations in the different batches were monitored for up to 75 min.

DNA preparation, transformation, and molecular techniques. Chromosomal DNAs from staphylococci were isolated according to the method of Marmur (15). E. coli plasmid DNA was prepared with a Qiafilter plasmid midi kit 100 (Qiagen, Hilden, Germany) according to the protocol supplied by the manufacturer. S. carnosus plasmid DNA was prepared essentially as described for E. coli, with the exception that lysostaphin (Sigma) was added to the lysis buffer (to 12.5 µg/ml). S. carnosus was transformed either by protoplast transformation (9) or by electroporation (2). Other molecular techniques followed established protocols (23).

DNA amplification by PCR, and DNA sequencing and analysis. Vent polymerase (New England BioLabs, Schwalbach, Germany) was used for PCRs. rTrh (recombinant Thermus thermophilus) DNA polymerase XL and an X-large PCR kit were used for inverse PCR according to the protocol supplied by the manufacturer (Perkin-Elmer). When the PCR product was used for sequencing, the sequence was verified by direct sequencing of chromosomal DNA. Double-stranded plasmid and chromosomal DNAs were sequenced by using the dideoxy procedure, a Thermo Sequenase Fluorescent labeled primer cycle sequencing kit (Amersham), and a Li-Cor DNA Sequencer (Lincoln Cooperation). The program Gapped BLAST (1) was used for protein similarity searches.

Isolation of the nir promoter region. A 1.9-kb EcoRI fragment (Fig. 1) from the wild-type genome adjacent to the SnaBI fragment was amplified by inverse PCR with ligated chromosomal EcoRI fragments as the template. The primers PraenirPCR1, 5'-GCCCCTTTGTTATCATCCAGCAC-3', and PraenirPCR4, 5'-GCAAAGGCACGATGGTAAAATAATGCTCGCC-3', bind to either of the two sides of the 0.2-kb overlapping region in opposite directions.

Homologous recombination. For insertional inactivation, major parts of nirR and sirA were replaced by a 1.5-kb ermB fragment (3). For this, ermB from plasmid pEC2 (3), isolated by cleavage with EcoRI and PstI, with flanking regions of 1.3 kb upstream and 2.7 kb downstream of the disrupted genes was cloned in pRB473, yielding pRBC12 (Fig. 1). The 1.3-kb fragment upstream of nirR was amplified by inverse PCR with the primers MutORF1, 5'-GCTCGCCTTCTGCTGAATTCTTACGTATAACTTGCGG-3', and PraenirL2, 5'-GCCCCTTTGTTATCATCCAGCAC-3', and with chromosomal DNA of wild-type S. carnosus as the template after EcoRI digestion and ligation. The introduced EcoRI site and a naturally occurring HindIII site were used for cloning. The downstream region was cloned in two fragments. The 0.8-kb fragment directly downstream was amplified with the primers MutORF2, 5'-GATATAGCAGCTGCAGATGTTGTGATTATCGCGACGG-3', and NirBint, 5'-CACGCAGCATATCTCCTGCTTGACGG-3', and digested with AvaI and PstI. The adjacent downstream fragment was obtained by AvaI cleavage of pR-AB. The four different fragments were ligated into HindIII- and AvaI-digested pRB473, cloned in E. coli SURE, and transformed into wild-type S. carnosus.

mRNA manipulations. Total RNA was isolated as described by Sizemore et al. (26) from S. carnosus cells grown aerobically or anaerobically without or with nitrite (2 mM). IRD 800-labeled oligonucleotides (obtained from MWG-Biotech) complementary to positions +108 to +79 of the nirR gene relative to the transcription start site were used for primer extension reactions according to the method of Wagner et al. (30). For Northern blot analyses 15 µg of total RNA was separated according to size by electrophoresis through a denaturing agarose gel (final formaldehyde concentration, 1.06 M) and then transferred to a positively charged nylon membrane (Boehringer Mannheim). A DIG RNA labeling kit (SP6/T7) (Boehringer Mannheim) was used for preparing an RNA probe. As a probe, an 870-bp EcoRV fragment of the nir operon comprising the entire sirA gene, approximately 300 bp of the 5' end of nirB, and approximately 100 bp of the 3' end of nirR was chosen. Hybridization was carried out overnight at 68°C with 100 ng of RNA probe per ml of DIG Easy Hyb (Boehringer Mannheim). The subsequent washing steps and detection with CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1^3,7]decan}-4-yl)phenyl phosphate] (Boehringer Mannheim) were carried out as recommended by the manufacturers. Digoxigenin-labeled RNA molecular weight marker I (Boehringer Mannheim) was used as a standard.

Analytical determinations. The nitrite concentration was measured colorimetrically by the method of Nicholas and Nason (18) as modified by Showe and DeMoss (25). The protein concentration was determined as described by Lowry et al. (13) in the presence of sodium dodecyl sulfate (8) with bovine serum albumin as the standard.

Nucleotide sequence accession number. The sequence of the genes involved in nitrite reduction has been assigned GenBank accession no. AF029224.

	RESULTS AND Discussion

Top Abstract Introduction Materials and Methods Results AND Discussion References

The nitrite reductase operon. (i) Isolation of nitrite reductase-negative Tn917 insertion mutants of S. carnosus. In order to identify genes involved in nitrite reduction in S. carnosus TM300, transposon mutagenesis was performed and 530 of the obtained mutants were analyzed in a rapid screening for their ability to reduce nitrite during anaerobic growth. Twenty-seven mutants (nir1 to nir27) were unable to reduce nitrite, which was confirmed by anaerobic cultivation with different concentrations of nitrite (0.1, 1, and 10 mM). Nitrite did not decrease during 20 h of incubation. It is unlikely that the nitrite reductase-negative phenotype is due to a general defect in anaerobic metabolism since the nitrate reductase activity of the mutants was unaffected.

(ii) Molecular characterization of the mutants. The transposons in the genomes of mutants were localized by digesting chromosomal DNAs with various restriction endonucleases and probing the fragments with Tn917-specific DNA. The restriction patterns of the mutants were similar, indicating that the transposon hit the same chromosomal DNA region. Being representative of the various mutants, the transposon and flanking DNA of nir1 was cloned in pUC18 on a 4.5-kb SalI/EcoRI fragment from a partial library enriched for 3- to 6-kb fragments; the insert was identified by Southern hybridization. A similar strategy was used to isolate the corresponding intact DNA region from wild-type S. carnosus. After Southern hybridization with a 1.7-kb AvaI/EcoRI fragment flanking Tn917 in nir1, a 5.1-kb SnaBI fragment was cloned into the HincII site of pUC18, yielding pUCnir1-1, which was identified by Southern hybridization and restriction analysis. The SnaBI fragment was sequenced, and four ORFs transcribed in the same direction (Fig. 1) were identified. Potential ribosome-binding-site sequences were found at appropriate distances from the predicted start codons.

(iii) Sequence analysis. In a computer-aided similarity search, the deduced amino acid sequences of the proteins revealed similarity to those of proteins involved in nitrite reduction (Table 1). The second and third ORFs encode proteins similar to NirB and NirD, respectively, the subunits of the dissimilatory NADH-dependent nitrite reductase of E. coli. The presence of NirBD is in agreement with our physiological results (17). The first ORF encodes a protein similar in sequence to the N terminus of E. coli CysG (residues 5 to 153), and the fourth ORF encodes a protein similar in sequence to the C-terminal portion of CysG (residues 216 to 450); the ORFs were termed sirA and sirB, respectively, to indicate that the enzyme activities of the trifunctional CysG might be attributed to two enzymes in S. carnosus. In E. coli, the C terminus of CysG catalyzes the S-adenosylmethionine-dependent methylation of uroporphyrinogen III to dihydrosirohydrochlorin, and the N terminus is important for pyridine-dinucleotide-dependent dehydrogenation of dihydrosirohydrochlorin to sirohydrochlorin and for the subsequent insertion of Fe²⁺ (31). It has been speculated that the E. coli enzyme arose by a gene fusion between a uroporphyrinogen III methylase and the oxidase and chelatase enzyme (31). Thus, the presence of the two separate enzymes in S. carnosus further supports this theory.

(iv) Complementation studies. When nir1 was complemented with the genes sirA, nirB, nirD, and sirB together with a putative promoter region of 209 nucleotides (plasmid pR-ABDB) (Fig. 1), the rate of nitrite reduction during anaerobic growth was only 30% of that of the wild type and nitrite reduction started at the beginning of the stationary phase. Therefore, we speculated that the plasmid lacks the primary promoter region. The delayed nitrite reduction might be due to an additional weak promoter upstream of sirA or nirB that is switched on in the stationary phase. In nir1 complemented with only sirA and nirB (plasmid pR-AB) (Fig. 1) the rate of nitrite reduction was even lower (6%), suggesting that the transposon has a polar effect on nirD and sirB transcription and that no additional internal promoters are present upstream of nirD or sirB.

(v) Identification of the nir promoter region. A potential ORF upstream of sirA that was incomplete on the SnaBI fragment was identified. An EcoRI chromosomal fragment that overlaps the SnaBI fragment was amplified by inverse PCR (see Materials and Methods), cloned, and sequenced. The ORF upstream of sirA was identified as the first gene of the nir operon. The gene product, NirR, displays low similarity to the deduced amino acid sequence of Bacillus subtilis ylnE (Table 1). Interestingly, ylnE is located between two genes, ylnD and ylnF, which are similar to sirB and sirA of S. carnosus, respectively (Table 1). Interestingly, ylnE is located between two genes, ylnD and ylnF, which are similar to sirB and sirA of S. carnosus, respectively (Table 1). Computer analyses predict that NirR is a cytoplasmic protein with an estimated molecular weight of 28,035. A region upstream of nirR contains the putative nir operon promoter, which was confirmed by primer extension analysis (see below, Fig. 3).

(vi) Construction and analysis of a nirR sirA mutant. To analyze the role of nirR and sirA in nitrite reduction, a mutant strain (M12) was constructed by homologous recombination in which the majority of the two genes was replaced by an erythromycin resistance cassette (plasmid pRBC12-B) (Fig. 1). The inserted ermB gene was transcribed divergently from the nitrite reductase genes (Fig. 1). Proper recombination was checked by Southern blot analysis (data not shown). As expected, nitrite reduction in mutant M12 was completely abolished.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Levels of nitrate and nitrite reduction during anaerobic growth of wild-type S. carnosus (filled circles) and of the complementation clones M12 (pR-PBDB), M12 (pR-PABDB), and M12 (pR-PNO) (open triangles, filled squares, and open circles, respectively) in comparison to levels in the nirR sirA mutant M12 (open squares). The cells were cultivated anaerobically in modified BM supplemented with 500 µM nitrite and 400 µM nitrate. During growth, the OD578 (A) and levels of reduction in nitrate (monitored as nitrite increase) and nitrite (monitored as nitrite decrease) (B) were determined as described in Materials and Methods.

(vii) Functional analysis of NirR. The amino acid sequence of NirR revealed no obvious DNA-binding motif and no similarity to any family of proteins in the PROSITE database. However, it should be mentioned that the N-terminal part of NirR (residues 22 to 90) reveals some identity (28%) to parts of the central domain of the transcriptional activator NifA of Bradyrhizobium japonicum (residues 349 to 419). The central domain is involved in interaction with ⁵⁴ (16).

Regulation of nitrite reduction. Aerobically growing cells of S. carnosus are unable to reduce nitrite, and nitrite was unable to induce nitrite reductase activity in the presence of oxygen (17), suggesting that induction of nitrite reductase is strictly coupled to anaerobiosis. In the absence of oxygen, nitrite further increases nitrite reductase activity (17). To analyze this induction process, we investigated the expression of nitrite reductase in more detail.

(i) Transcription initiation at the nir promoter in response to oxygen and nitrite. Primer extension experiments with RNA isolated from cells grown anaerobically with nitrite revealed a predominant start site 17 nucleotides upstream of the predicted start codon of nirR (Fig. 3). Besides this predominant site, also other weaker transcription initiation sites were visible further upstream. For the predominant transcription initiation site, the sequences of the corresponding deduced 10 region (TACAAT) and 35 region (TTCACA) differ from the optimal consensus sequence for ⁷⁰-dependent promoters by one nucleotide each (boldface). The 35 region is located in an inverted repeat centered at 38.5 (TGTGAATXXATTCACA). Whether this palindrome is a binding site for a regulatory protein is not known.

View larger version (71K): [in this window] [in a new window]   FIG. 3.   Primer extension analysis of the regulation of the nir promoter. The figure shows the reverse transcripts obtained with an IRD 800-labeled nir-specific primer (0.5 pmol) and 20 µg of total RNA (1 of 3 µl was loaded on the gel) isolated from mid-exponential-growth-phase S. carnosus wild-type cells grown in modified BM under aerobic (Ae) or anaerobic (An) conditions and in the absence or presence of nitrite (2 mM). Lanes A, C, G, and T contained the respective sequencing reaction mixtures. The arrow indicates the predominant +1 site.

(ii) Northern blot analyses. Coinciding with the results from the primer extension studies, Northern blot analyses revealed small amounts of full-length transcript of the nir operon (approximately 5 kb) in cells grown aerobically with nitrite but no full-length transcript in cells grown aerobically without nitrite (Fig. 4). In cells grown anaerobically, large amounts of full-length transcript were detectable and the amount of full-length transcript seemed to be slightly greater when nitrite was present. The amount of full-length transcript in anaerobically grown cells does not reflect the rate of transcription initiation from the predominant start site. We speculate that this result might be due to transcription initiation from different start sites and/or that another unknown factor(s) favors large amounts of full-length transcript during anaerobic growth.

View larger version (57K): [in this window] [in a new window]   FIG. 4.   Northern blot analyses of the nir operon. Northern hybridization was performed as described in Materials and Methods. Total RNA (15 µg) isolated from cells grown aerobically (Ae) or anaerobically (An) with (+) or without () nitrite were transferred to a positively charged nylon membrane (Boehringer Mannheim). Northern hybridization was carried out with a digoxigenin-labeled RNA probe (comprising the entire sirA gene, approximately 300 bp of the 5' end of nirB, and approximately 100 bp of the 3' end of nirR). For chemiluminescence detection, CSPD (Boehringer Mannheim) was used. The full-length transcript of the nir operon (approximately 5 kb) is indicated by an arrow. As a standard, 90 ng of digoxigenin-labeled RNA molecular weight marker I (Boehringer Mannheim) was used.

(iii) Other oxygen-regulated steps are involved in nir expression and Nir activity. Both primer extension studies and Northern blot analyses confirmed the presence of transcript in cells grown aerobically with nitrite which clearly lack nitrite reductase activity. This suggests that an additional mechanism(s) controls induction of nitrite reductase activity. Several steps are conceivable: (i) reversible or irreversible inhibition of the nitrite-reducing system by oxygen, (ii) competition for electrons with the aerobic electron transfer chain, (iii) oxygen-controlled regulation of translation, and (iv) oxygen-controlled enzyme folding and/or assembly.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Reversible inhibition of nitrite reductase activity by oxygen. Induced cells (grown anaerobically with 2 mM nitrite) were incubated aerobically (for 10 min [filled circles], and for 60 min [filled triangles]) or anaerobically (for 60 min [open triangles]). After this preincubation, the conditions were switched to anaerobiosis and the ability of the cells to reduce nitrite was measured as nitrite decrease. In parallel, nitrite decrease was detected in the induced cells incubated aerobically (filled squares).

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Influence of rifampin on induction of nitrite reductase activity after a shift from aerobiosis to anaerobiosis. After aerobic precultivation without (circles) or with (triangles) nitrite, no antibiotic or 200 µM rifampin was added to equal parts of each culture and the incubation was continued for 15 min. Subsequently, the remaining nitrite was removed by centrifugation and two washing steps. Cells were resuspended in modified BM with or without rifampin and incubated anaerobically at 37°C. After addition of nitrite, samples were taken at different time points and the amount of nitrite was monitored.

The transporter NirC. Upstream of the nir operon, we identified another ORF, which encodes a putative integral transmembrane protein of 276 amino acids (estimated molecular weight, 30,800). From hydrophobicity plots, it was estimated to have six membrane-spanning segments. The protein shows similarity to the putative E. coli nitrite transporter NirC (6) (Table 1). The E. coli nirC gene is located in the nir operon (21). A putative transcription termination structure was identified downstream of S. carnosus nirC (G = 30.2 kcal/mol).

Construction and analysis of a nirC mutant. By homologous recombination of plasmid pRBC-RAB onto the chromosome (Fig. 1), we constructed a mutant strain (MC11) in which nirC was replaced by an erythromycin resistance cassette transcribed in the direction opposite to that of the nitrite reductase genes. Proper recombination was confirmed by Southern blot analysis (data not shown). Nitrite reduction during anaerobic growth and growth at high concentrations of nitrite (100 mM) in this mutant were unaffected. To analyze a function in nitrite uptake, nitrite reduction in the nirC mutant was analyzed with increasing pH. Nitrite uptake at physiological pH might take place by passive diffusion of nitrous acid (14). Since the concentration of available nitrous acid decreases (pK_a, 3.4) as the pH of the medium is raised, the contribution of active transport might be expected only at alkaline pH. However, levels of nitrite reduction at pH 8.6 and 9.6 were similar in the nirC mutant and in the wild type (80% of the activity at pH 7.2). Thus, NirC is not responsible for nitrite uptake at alkaline pH values.

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Model of nir expression and Nir activity. Three different oxygen-regulated steps are postulated and indicated by arrows. Whether the second regulated step is at the level of translation, protein folding, assembly, or insertion of prosthetic groups is not known. The detailed function of NirR for promoter activity is unclear.