Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan 1
Author for correspondence: Akinobu Okabe. Tel: +81 87 891 2129. Fax: +81 87 891 2129. e-mail: microbio{at}kms.ac.jp
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ABSTRACT |
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Keywords: Clostridium perfringens, nitrate reductase, molybdopterin guanine dinucleotide, ferredoxin-like protein, NADH oxidase
Abbreviations: NaR, nitrate reductase; NarA, nitrate reductase of Clostridium perfringens; NiR, nitrite reductase; Mo-MGD, a molybdenummolybdopterin guanine dinucleotide complex
The GenBank accession number for the sequence reported in this paper is AB017192.
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INTRODUCTION |
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Both assimilatory and respiratory NaRs are molybdoenzymes containing a molybdopterin cofactor and an ironsulfur cluster. They differ in that the former is soluble in the cytoplasm, while the latter is membrane bound. They also differ in the regulation of enzyme synthesis: the synthesis of the former is controlled by the availability of fixed nitrogen, e.g. glutamine and ammonium ions (Lin & Stewart, 1998 ), while the synthesis of the latter is independent of their availability. Another type of NaR, which is more similar to assimilatory NaR than to respiratory NaR (Berks et al., 1995
), has been identified in the periplasm, although its physiological function has not yet been established.
Nitrate reduction in strict anaerobes has attracted much less interest and is not well understood mainly because of the lack of suitable bacteria for physiological and genetic studies. Clostridium perfringens, a strict anaerobic pathogen, lives in soil, water, and the intestines of humans and other animals. Some strains of the organism can reduce nitrate to ammonium ions (Hasan & Hall, 1975 ). NaR (Seki-Chiba & Ishimoto, 1977
) and NiR (Sekiguchi et al., 1983
) of C. perfringens, hereinafter designated NarA and NirA, respectively, were purified and their biochemical properties were characterized. NarA is a soluble molybdoenzyme whose molecular mass was estimated to be approximately 80 kDa (Seki-Chiba & Ishimoto, 1977
; Seki et al., 1987
). The enzyme can catalyse NAD(P)H-dependent nitrate reduction in the presence of rubredoxin (Seki et al., 1989
) and a partially purified NAD(P)H-rubredoxin reductase (Seki et al., 1988
). Its location and molecular size are more similar to those of assimilatory NaRs than to those of respiratory NaRs. However, induction of the enzyme activity by nitrate is independent of the presence of ammonium ions in the culture medium, as in the case of respiratory NaRs (Hasan & Hall, 1975
). NirA does not contain siroheme, unlike assimilatory NiRs, or haem or copper, unlike respiratory NiRs. Its molecular mass, 54 kDa, is significantly less than those reported for assimilatory and respiratory NiRs (63 kDa for higher plant NiRs, and 88140 kDa for fungal and bacterial NiRs) (Sekiguchi et al., 1983
). The nitrite reduction by NirA generates hydroxylamine as the main product and possibly nitric oxide, but not ammonium ions (Sekiguchi et al., 1983
). Thus, nitrate reduction in C. perfringens differs in many respects from assimilatory and respiratory nitrate reduction. This unique nitrate-reduction system has been considered not to be linked to nitrate assimilation but to energy production: this is called nitrate fermentation (Takahashi et al., 1963
; Ishimoto et al., 1974
; Hasan & Hall, 1975
).
NarA and NirA were well studied until the late 1980s. Since then, however, no further work has been conducted and much of this system remains to be elucidated. In an attempt to determine the molecular characteristics of NarA and NirA, we have undertaken the cloning and sequencing of their genes (narA and nirA). Nitrate is reduced by bacteria to toxic compounds when it is ingested along with meals (Calmels et al., 1996 ; Vermeer et al. , 1998
). Considering that C. perfringens inhabits the intestines of humans and other animals, and also that it may generate potentially harmful byproducts of nitrate reduction such as hydroxylamine and nitrous oxides (Sekiguchi et al., 1983
), genetic analysis of nitrate reduction in C. perfringens is also of medical importance. Moreover, it would provide insights into the evolutionary relationships between the fermentative and other nitrate-reduction enzymes. In this study we have characterized the nar operon of C. perfringens and the downstream genes involved in molybdopterin synthesis, and compare them with already characterized homologous genes.
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METHODS |
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Media and culture conditions.
C. perfringens PB6K was precultured in cooked-meat medium (Nissui Pharmaceuticals) at 37 °C for 12 h. The preculture was diluted 200-fold with fresh medium, GAM (Nissui Pharmaceuticals) or PYG broth (7·5 g polypeptone, 5 g yeast extract, 2·5 g glucose and 50 µl thioglycolic acid l -1; Seki-Chiba & Ishimoto, 1977 ) and incubated at 37 °C. KNO3 was added to the medium to a final concentration of 0·2% (w/v), when necessary. Transformed E. coli cells were selected on LB plates containing 20 g LB broth base (Gibco) and 15 g agar per litre, supplemented with 100 µg ampicillin ml-1, 0·2 mM IPTG (Wako Pure Chemicals) and 0·004% (w/v) X-Gal (Wako Pure Chemicals).
Assaying of NarA activity.
Cultures were collected by centrifugation at 12000 g for 10 min at 4 °C. The cells were washed three times with 10 mM Tris/HCl (pH 8·0) by centrifugation. The cell suspension was passed twice through a French pressure cell at 10000 p.s.i. (6·9 MPa), followed by centrifugation at 18000 g for 20 min. The supernatant was used as the crude extract. NarA activity was determined using methyl viologen as a substrate, as described by Seki-Chiba & Ishimoto (1977) . Enzyme activity was expressed as nmol nitrite formed min-1 . Protein concentrations were determined using the BCA protein assay reagent (Pierce) with BSA as a standard. All assays were carried out in triplicate.
SDS-PAGE and zymography.
SDS-PAGE on a 7·5% polyacrylamide gel and staining with Coomassie brilliant blue R were performed as described by Laemmli (1970) . To detect NarA activity by zymography, proteins were first separated on a gel under non-denaturing conditions and visualized by means of substrate-dependent oxidation of reduced methyl viologen, as described by Seki-Chiba & Ishimoto (1977)
.
Purification of NarA.
C. perfringens PB6K was grown in 1 l PYG medium containing 0·2% (w/v) KNO3 at 37 °C for 9 h. A crude extract was prepared as described above. Purification was carried out by the method of Seki-Chiba & Ishimoto (1977) with the following modification. Briefly, ammonium sulfate was added to the crude extract to give 40% saturation, followed by centrifugation at 15000 g at 4 °C for 20 min. Ammonium sulfate was added to the supernatant to give 80% saturation and the precipitate was collected by centrifugation. The precipitate was dissolved in 15 ml 10 mM Tris/HCl (pH 8·0) and dialysed against the same buffer. The dialysed sample was applied to a DEAE Sephadex A-25 column (2·2x15 cm; Amersham Pharmacia Biotech). Proteins were eluted with a 400 ml linear gradient of 00·3 M NaCl in Tris/HCl (pH 8·0). Enzyme activity and the absorbance at 280 nm were monitored. The fractions eluted in the active peak were collected and the pooled fraction was dialysed against 10 mM Tris/HCl (pH 8·0). The dialysed sample was applied to an ion-exchange FPLC column (MonoQ, 1 ml bed volume; Amersham Pharmacia Biotech), that had been pre-equilibrated with 20 mM Tris/HCl (pH 8·0). Proteins were eluted with a 20 ml linear gradient of 00·3 M NaCl in the same buffer. The enzyme was eluted as a single peak at 170 mM NaCl. This step was repeated three times and the active fractions were combined.
Determination of the N-terminal amino acid sequence.
The purified NarA was subjected to SDS-PAGE, followed by transfer to a PVDF membrane (Trans-Blot transfer medium; Bio-Rad). Proteins were visualized by staining with Coomassie brilliant blue R and the corresponding protein band was cut out. The excised membrane was wetted with acetonitrile and incubated in 100 mM Tris/HCl (pH 8·3) containing 6 M guanidine hydrochloride and 2 mg DTT ml-1 at 37 °C for 2 h. Alkylation of cysteine residues was performed by incubation at 37 °C for 2 h after the addition of 4-vinylpyridine to a final concentration of 1% (w/v). After washing five times with 50% (v/v) methanol, the membrane was applied to a protein sequencer (model 492; Perkin Elmer) to determine the N-terminal amino acid sequence.
Cloning of a portion of the narA gene.
A pair of degenerate oligonucleotide primers was designed to amplify a portion of the narA gene by PCR. Their sequences, 5'-ACITGYAAYTAYTGYGCIYT-3' and 5'- CKYTTIATYTTICCRTCYTC-3', correspond to the amino acid sequences of Tyr7Leu13 and Glu21Arg 27, respectively, of NarA. C. perfringens chromosomal DNA was prepared as described previously (Matsushita et al., 1994 ) and used as the template. PCR was performed with a thermal cycler (model TC1, Perkin Elmer), and the PCR product was purified from an acrylamide gel and cloned into the pT7Blue T-vector (Novagen), as described elsewhere (Matsushita et al., 1999
). The resulting plasmid, which was designated pNR1, was transformed into E. coli NovaBlue.
Cloning of the nar operon.
The insert DNA in pNR1 was amplified, labelled with digoxigenin-11- dUTP (Roche Diagnostics) by PCR as described previously (Matsushita et al., 1999 ) and used as a DNA probe. The chromosomal DNA was digested with PstI and then subjected to Southern hybridization at 55 °C. DNA fragments around the positive signal were recovered from an agarose gel and ligated into pUC19. E. coli DH5
was transformed with the ligation mixture. The colonies were screened by PCR as described previously (Matsushita et al., 1999
). Of the plasmids recovered from positive clones, a plasmid named pNR4 produced a PCR amplification product of the expected size. This plasmid was confirmed by nucleotide sequencing to contain the insert DNA, of which the deduced amino acid sequence coincided with that determined for the purified NarA. This plasmid contained a 9·5 kb Pst I fragment. A further downstream region was detected on Southern hybridization using chromosomal DNA digested with HincII and a DNA probe prepared by PCR amplification of the region corresponding to nt 92869416 of the PstI fragment. The 6·1 kb hybridizing fragment was cloned into pUC19, as described above. The resulting plasmid was designated pNR7.
DNA manipulations and nucleotide sequencing.
Restriction endonucleases were purchased from Takara Shuzo, Toyobo and New England Biolabs. The DNA ligation kit was a product of Takara Shuzo. All recombinant DNA procedures were carried out as described by Sambrook et al. (1989) . The nucleotide sequence was determined by the dideoxy chain-termination method (Sanger et al., 1977
), using an automated nucleotide sequencer (model ABI PRISM 377, Perkin Elmer). Plasmid template DNA was prepared using the Wizard plus miniprep DNA purification system (Promega). A Thermo Sequenase fluorescent-labelled primer cycle-sequencing kit with 7-deaza dGTP (Amersham Pharmacia Biotech) and M13 dye primers (Perkin Elmer) were used for sequencing. An ABI PRISM dye-terminator cycle- sequencing ready reaction kit with AmpliTaq DNA polymerase FS (Perkin Elmer) and various synthetic primers were also used to determine ambiguous nucleotides and to fill in sequence gaps.
Southern hybridization.
C. perfringens DNA (0·2 µg) was digested with appropriate restriction enzymes, applied to a 0·8% agarose mini-gel and electrophoresed at 100 V for 1 h. Treatment of the gel and transfer of DNA to a nylon membrane (Hybond-N; Amersham Pharmacia Biotech) were performed as described previously (Matsushita et al., 1994 ). After hybridization at 50 °C, the hybridized probes were detected with anti- digoxigenin-alkaline phosphatase Fab fragments (Roche Diagnostics) and a chemiluminescent dye (Lumi-Phos 530; Lumigen).
Northern hybridization and primer-extension analysis.
C. perfringens cells were grown in GAM broth with or without 0·2% KNO3 at 37 °C for 2 h until the culture reached an OD600 of 0·8. Total RNA was prepared by the SDS-phenol method (Aiba et al., 1981 ) and a sample (1 µg) was separated on a denaturing agarose gel (1%). Hybridization was carried out at 50 °C using DNA fragments within the narA (nt 46295605), narB (nt 61156528), narC (nt 68457119), mobB (nt 78308261) and moeA (nt 92869416) genes as DNA probes. RNA molecular weight marker II (Roche Diagnostics) was used as size markers. Primer-extension experiments were performed using the same RNA sample as described above. A 30 nt primer, 5'- GAGCACAATAGTTACAAGTAGATTGTATTC-3', which is complementary to nt +8 to +37 of the narA gene coding region, was 5' end labelled with [
-32P]dATP [4·5 kCi/mmol -1 (166·5 TBq mmol-1); ICN Biochemicals] and hybridized with total RNA from C. perfringens cells. The hybrids were extended with reverse transcriptase (Superscript RT; Life Technologies) and the extension products were electrophoresed on a sequencing gel as described by Ba-Thein et al. (1996)
.
Amino acid sequence similarity search.
A search for similar protein sequences was carried out using the BLAST algorithm (Altschul et al., 1997 ) at the Center for Information Biology (National Institute of Genetics, Mishima, Japan). The default settings in the BLAST program were used without prefiltering low compositional complexity regions.
Expression of the narB and narC genes in E. coli.
The narB gene was expressed in E. coli as follows. The DNA fragment corresponding to NarB aa 1137 was amplified using the two primers 5'-CATATGAAAAGAATAAAAATCAATAGAGAT-3' and 5'-GGATCC TTATTCTTCTACCTCCTCTAAAAGT-3', which contained NdeI and BamHI sites, respectively (underlined). A 423 bp Nde IBamHI fragment containing the coding region of the narB gene was inserted into expression vector pET-11a. The resulting plasmid, named pET-NarB, was transformed into E. coli BL21(DL3), after the nucleotide sequence of the narB gene had been verified. The transformant was grown in LB broth containing 100 µg ampicillin ml-1 at 37 °C. When the cultures reached an OD600 of 0·6, IPTG was added to a final concentration of 1 mM. After incubation for 4 h, cells were harvested by centrifugation at 5000 g for 5 min at 4 °C. The cells were washed once with 10 mM phosphate buffer (pH 7·0) containing 1 mM EDTA, disrupted by two passages through a French pressure cell at 10000 p.s.i. (6·9 MPa) and centrifuged at 13000 g for 20 min. The precipitates containing large amounts of NarB inclusion bodies were washed three times with 10 mM phosphate buffer (pH 7·0) containing 4% Triton X-100 and 1 mM EDTA by centrifugation at 25000 g for 20 min to remove membrane proteins. To remove residual Triton X- 100, the precipitates were washed twice with distilled water. The NarB protein was solubilized with 50 mM Tris/HCl (pH 8·5) containing 8 M urea and 10 mM DTT. The NarB protein recovered in the supernatant fraction was dialysed against the same buffer containing 4 M urea and 10 mM DTT. The dialysed sample was applied to a Sephadex G75 column (Amersham Pharmacia Biotech) and the protein recovered in the void fraction was used as the purified recombinant NarB protein, since this was shown to be homogeneous on SDS-PAGE.
The narC gene was expressed in E. coli as follows. The DNA fragment corresponding to the coding region of the narC gene was amplified using two PCR primers 3'-GGATCC ATGAGATATATTGTTGTGGGGGCA-3' and 5'-CTCGAG TTAATATGTGTACTCTAAATTTTC-3', which contained BamHI and XhoI sites, respectively (underlined). A 1·2 kb BamHIXhoI fragment was inserted into the pGEX- 4T-2 vector (Amersham Pharmacia Biotech) to produce the NarC protein as a glutathione S-transferase fusion protein. After the nucleotide sequence of the fusion gene had been verified, the resulting plasmid, named pGST-NarC, was transformed into E. coli DH5. The production and purification of the fusion protein were performed as described by the manufacturer.
Immunological methods.
Polyclonal antibodies were raised in ddY mice by subcutaneous injection of 40 µg recombinant NarB or NarC protein in Freunds complete adjuvant (Amersham Pharmacia Biotech). Booster injections were given after 3 weeks and bleeding was performed after 4 weeks. Prior to use for Western blots, the antisera were preadsorbed with a large excess of a cell extract prepared from E. coli BL21(DE3) cells harbouring pET-11a overnight at 4 °C. The preadsorbed antisera were used at 1:100 dilution. For Western-blot analysis, 1 ml cultures of C. perfringens were centrifuged in a microfuge. The cell pellets were resuspended in 100 µl SDS-containing sample buffer and boiled for 3 min. Twenty microlitres of a sample was subjected to SDS-PAGE on either a 12·5 or 15% polyacrylamide gel and the proteins were blotted onto a nitrocellulose membrane. The replicates were incubated with mouse antiserum against the NarB or NarC protein and then subjected to immunostaining with goat anti-mouse IgGalkaline phosphatase conjugate (Bio-Rad), and nitro blue tetrazolium (Sigma) and BCIP (5- bromo-4-chloro-3-indolyl phosphate, p-toluidine salt; Wako Pure Chemicals) as chromogenic substrates.
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RESULTS |
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Identification of the nar operon
There are two putative rho-independent transcriptional terminators immediately upstream of the narA gene and downstream of the mobB gene, respectively, suggesting that the narA, narB, narC and mobB genes are transcribed as a polycistronic operon. Although a -10 consensus sequence, TATAAT, is present upstream of the narA gene, there is no sequence indicative of a -35 region. To determine the transcriptional-start site of this operon, primer-extension analysis was performed on cells grown in GAM broth with and without KNO3 . As shown in Fig. 3a , NarA activity increased only slightly in the medium without KNO3. On the contrary, the enzyme activity increased markedly in the presence of KNO3, indicating that the enzyme synthesis is induced by KNO3. RNA was prepared from the two cultures at an OD600 of 0·8. No positive band was detected for RNA from cells grown in the absence of KNO3. On the other hand, RNA from cells grown in the presence of KNO3 gave a positive signal, which clearly indicated the transcriptional-start site was A at position +1 (Fig. 3b
). Northern-blot analysis was also performed on the same RNA samples using the DNA fragments from the narA, narB, narC and mobB genes as DNA probes. All blots had a band corresponding to approximately 5300 nt for the RNA from the cells grown in the presence of KNO3 but not for that in the absence of KNO3 (Fig. 3c
). When Northern-blot analysis was performed using the DNA fragment within the moeA gene, a band was detected at the location corresponding to approximately 4800 nt, being distinct from that of narABCmobB transcripts (data not shown). These results indicate that the four genes are transcribed to give a polycistronic mRNA from the narA promoter.
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The amino acid sequence deduced from the narC gene exhibits homology to those of NADH peroxidase (Poisson P value 1·2e-15) and sulfide dehydrogenase (Poisson P value 0·018). Their alignment revealed that NarC contains FAD- and NADH-binding domains, which were detected in the two enzymes on crystallographic analysis (Fig. 4c). These structural characteristics of NarB and NarC, and the linkage of the three genes suggest that they function to reduce nitrate in a cooperative manner such that an electron from NADH is first accepted by NarC, then transferred to NarB and finally to NarA.
Expression of the narB and narC genes
C. perfringens contains ferredoxin (Seki et al., 1979 ) and rubredoxin (Seki et al., 1989
), both of which may function as an electron carrier for NarA (Seki et al., 1988
, 1989
). Although it is clear that the narB gene is transcribed from the narA promoter upon induction by KNO3, the possibility that NarB is not synthesized cannot be ruled out. To determine whether or not NarB is produced upon induction, cells were grown in the presence and absence of KNO3, collected at an OD600 of 2·0 and subjected to Western-blot analysis (Fig. 5a
). A band with the same mobility as that of the NarB protein was detected for the cells grown in the presence of KNO3 but not for cells grown in its absence. However, other band(s) were also detected for both the induced and uninduced cells. They may be unknown protein(s) which are immunologically related to NarB or associated with the NarB protein. Synthesis of the NarC protein in induced and uninduced cells was also examined by Western-blot analysis (Fig. 5b
). Antiserum was prepared from mice immunized with a GST-NarC fusion protein, because the fusion protein was insensitive to cleavage by thrombin. A band corresponding to a 45 kDa polypeptide, which coincided with the molecular mass predicted from the narC gene, was detectable for the induced cells. A band with the same mobility is weakly present in the uninduced cells. Since low but significant levels of nitrate reductase activity were detectable in the uninduced cultures grown at an OD600 of 2·0 (Fig. 3a
), small amounts of NarB and NarC seem to be produced in the uninduced cells. The discrepancy that the NarC protein but not the NarB protein was detectable for the uninduced cells could be due to the difference in the immunoreactivity of the two antisera used. Thus, it seems very likely that the expression of the narB and narC genes at the protein level is coregulated with that of the narA gene.
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DISCUSSION |
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The organization of the nar operon suggests a functional relationship between NarA, NarB and NarC, i.e. the latter two proteins are linked to NarA. On the other hand, the in vitro reconstitution experiment performed by Seki et al. (1988) strongly suggested that rubredoxin, a 6 kDa ironsulfur protein (Seki et al., 1989
), serves as an intermediary electron carrier for NAD(P)H-linked nitrate reduction, and that ferredoxin reduced by pyruvate dehydrogenase is linked to NarA instead of hydrogenase in the presence of nitrate. Their results, however, do not rule out a possible functional relationship between NarA, NarB and NarC. Considering the results of both our genetic analysis and their in vitro study, we assume that an electron-transfer pathway from NADH to NarA via NarC and NarB could be the main route of nitrate reduction, at least upon induction by KNO 3, although ferredoxin and rubredoxin would also serve as intermediary electron carriers to NarA. To prove this, the NarA activity in the presence of NarB and NarC must be compared with that in their system.
Comparison of NaRs and their electron carriers between the three different nitrate-reduction systems would be informative for understanding their evolutionary relationships. B. subtilis possesses both respiratory and assimilatory NaRs, which are encoded by narG (Cruz Ramos et al., 1995 ; Hoffmann et al., 1995
) and nasC (Ogawa et al. , 1995
), respectively. The deduced amino acid sequence of C. perfringens NarA exhibits higher similarity to NasC than to NarG, indicating that C. perfringens NarA is more closely related to assimilatory NaRs. NasB, NADH dehydrogenase of B. subtilis, possesses three ironsulfur clusters, while Klebsiella oxytoca NADH dehydrogenase, which is homologous to B. subtilis NasB, lacks such a cluster (Fig. 6
). Instead, a region homologous to the clusters resides at the N-terminus of NaR of the latter organism (Fig. 6
). Based on the homology between these ironsulfur clusters and TodB, an electron-transfer subunit in the NADH-dependent toluene degradation system of Pseudomonas putida, the current K. oxytoca NaR and B. subtilis NADH-dependent dehydrogenase are postulated to have resulted from fusion with an intermediate electron-carrier protein containing an ironsulfur cluster (Lin & Stewart, 1998
). Our finding that NarB is the most likely candidate for an intermediate electron carrier for NarA strengthens the fusion hypothesis, which might be extended to the origin of eukaryotic assimilatory NaRs containing all three components corresponding to NarA, NarB and NarC of C. perfringens (Campbell & Kinghorn, 1990
).
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ACKNOWLEDGEMENTS |
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Received 8 April 1999;
revised 13 August 1999;
accepted 27 August 1999.