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• W2119712943 abstract "The periplasmic nitrate reductase (Nap) is wide-spread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His6-tagged NapF protein was overproduced in Escherichiacoli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF– strain of R. sphaeroides. The His6-tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2′-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF– strain phenotype, and the NapF-dependent reactivation of the 2,2′-dipyridyl-treated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA. The periplasmic nitrate reductase (Nap) is wide-spread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His6-tagged NapF protein was overproduced in Escherichiacoli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF– strain of R. sphaeroides. The His6-tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2′-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF– strain phenotype, and the NapF-dependent reactivation of the 2,2′-dipyridyl-treated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA. The periplasmic nitrate reduction (Nap) 1The abbreviations used are: Nap, periplasmic nitrate reductase; AmpR, ampicillin resistance; BSA, bovine serum albumin; DP, 2,2′-dipyridyl; DTT, dithiothreitol; IPTG, isopropyl β-d-thiogalactoside; LB, Luria-Bertani broth; MGD, molybdopterin guanine dinucleotide cofactor; MV, methyl viologen; NifS, A. vinelandii cysteine desulfurase. system has been found in many different bacteria, and several physiological roles have been proposed depending on the organism, such as redox control to dissipate excess of reductant, anaerobic and aerobic denitrification, adaptation to anaerobic growth, and scavenging nitrate in nitrate-limiting environments (1Moreno-Vivián C. Cabello P. Martínez-Luque M. Blasco R. Castillo F. J. Bacteriol. 1999; 181: 6573-6584Crossref PubMed Google Scholar, 2Sears H.J. Sawers G. Reilly A. Berks B.C. Ferguson S.J. Richardson D.J. Microbiology. 2000; 146: 2977-2985Crossref PubMed Scopus (64) Google Scholar, 3Potter L.C. Millington P. Griffiths L. Thomas G.H. Cole J.A. Biochem. J. 1999; 344: 77-84Crossref PubMed Scopus (133) Google Scholar, 4Gavira M. Roldán M.D. Castillo F. Moreno-Vivián C. J. Bacteriol. 2002; 184: 1693-1702Crossref PubMed Scopus (49) Google Scholar, 5Delgado M.J. Bonnard N. Tresierra-Ayala A. Bedmar E.J. Müller P. Microbiology. 2003; 149: 3395-3403Crossref PubMed Scopus (74) Google Scholar). The function of the Rhodobacter sphaeroides DSM158 Nap system, which is encoded by the napKEFDABC transcription unit (6Reyes F. Roldán M.D. Klipp W. Castillo F. Moreno-Vivián C. Mol. Microbiol. 1996; 19: 1307-1318Crossref PubMed Scopus (82) Google Scholar, 7Reyes F. Gavira M. Castillo F. Moreno-Vivián C. Biochem. J. 1998; 331: 897-904Crossref PubMed Scopus (49) Google Scholar), is to regulate the cellular redox state by dissipating excess of reductant in cells growing under phototrophic conditions or with highly reduced carbon sources, such as butyrate or caproate (4Gavira M. Roldán M.D. Castillo F. Moreno-Vivián C. J. Bacteriol. 2002; 184: 1693-1702Crossref PubMed Scopus (49) Google Scholar, 8Roldán M.D. Reyes F. Moreno-Vivián C. Castillo F. Curr. Microbiol. 1994; 29: 241-245Crossref Scopus (52) Google Scholar). The Nap enzyme is a heterodimer composed of NapA, the Mo-bisMGD-containing catalytic subunit that also presents an N-terminal [4Fe-4S] center, and NapB, a diheme cytochrome c. NapC is a membrane-anchored cytochrome c that mediates the physiological electron transfer from the membrane quinol pool to the NapAB periplasmic heterodimer. The Nap system of R. sphaeroides also includes two putative small transmembrane proteins of unknown function (NapK and NapE), a soluble protein (NapD), which could be required for the maturation of the enzyme, and NapF, a putative 16-kDa cysteine-rich protein that has been suggested to bind four [4Fe-4S] centers (6Reyes F. Roldán M.D. Klipp W. Castillo F. Moreno-Vivián C. Mol. Microbiol. 1996; 19: 1307-1318Crossref PubMed Scopus (82) Google Scholar, 7Reyes F. Gavira M. Castillo F. Moreno-Vivián C. Biochem. J. 1998; 331: 897-904Crossref PubMed Scopus (49) Google Scholar). The R. sphaeroides wild-type strain lacks nitrite reductase, thus nitrite production can be used as an indicative of nitrate reductase activity in vivo. The mutational analysis on the R. sphaeroides nap genes reveals that the napABC genes are essential for periplasmic nitrate reduction, because mutants defective in these genes are unable to produce nitrite in vivo. Although the function of the NapF protein in the R. sphaeroides Nap system remains unknown, a mutation of the napF gene indicates that this protein is also required for in vivo nitrate reduction (7Reyes F. Gavira M. Castillo F. Moreno-Vivián C. Biochem. J. 1998; 331: 897-904Crossref PubMed Scopus (49) Google Scholar). In addition, loss of NapF results in a considerable decrease of the Nap activity assayed with methyl viologen (MV) as artificial electron donor (7Reyes F. Gavira M. Castillo F. Moreno-Vivián C. Biochem. J. 1998; 331: 897-904Crossref PubMed Scopus (49) Google Scholar). As reduced viologens seem to transfer electrons directly to the active site of the enzyme (9Jones R.W. Garland P.B. Biochem. J. 1977; 164: 199-211Crossref PubMed Scopus (208) Google Scholar, 10Buc J. Santini C.L. Blasco F. Giordani R. Cárdenas M.L. Chippaux M. Cornish-Bowden A. Giordano G. Eur. J. Biochem. 1995; 234: 766-772Crossref PubMed Scopus (25) Google Scholar), it is unlikely that NapF participates in electron transfer to the periplasmic NapAB complex, even more if NapF is a cytoplasmic protein. Accordingly to this, the R. sphaeroides napC– strain is unable to produce nitrite in vivo but shows the same MV-dependent nitrate reductase activity as the wild-type strain (6Reyes F. Roldán M.D. Klipp W. Castillo F. Moreno-Vivián C. Mol. Microbiol. 1996; 19: 1307-1318Crossref PubMed Scopus (82) Google Scholar, 7Reyes F. Gavira M. Castillo F. Moreno-Vivián C. Biochem. J. 1998; 331: 897-904Crossref PubMed Scopus (49) Google Scholar). However, the subcellular localization of NapF is controversial due to the presence of a conserved twin arginine motif, which could be involved in the periplasmic targeting of the protein, although this motif is not followed by a hydrophobic region (7Reyes F. Gavira M. Castillo F. Moreno-Vivián C. Biochem. J. 1998; 331: 897-904Crossref PubMed Scopus (49) Google Scholar). The Escherichia coli Nap system includes two iron-sulfur proteins, NapG and NapH, in addition to NapF (11Grove J. Tanapongpipat S. Thomas G. Griffihs L. Crooke H. Cole J.A. Mol. Microbiol. 1996; 19: 467-481Crossref PubMed Scopus (145) Google Scholar). Although napFGH genes are not essential for periplasmic nitrate reduction in E. coli (12Potter L.C. Cole J.A. Biochem. J. 1999; 344: 69-76Crossref PubMed Google Scholar), the loss of NapF results in a severe growth defect in a NapG+H+ strain, but not in an napGH deletion mutant (13Brondijk T.C.H. Fiegen D. Richardson D.J. Cole J.A. Mol. Microbiol. 2002; 44: 245-255Crossref PubMed Scopus (80) Google Scholar). It has been proposed that electrons can flow from menaquinol to the NapC subunit in E. coli. A membrane-anchored NapGH complex could act as a proton translocating dehydrogenase transferring electrons from ubiquinol and catalyzing an effective electron transfer to the periplasmic NapAB complex. Nevertheless, no function has been assessed to NapF in the Nap system of E. coli (11Grove J. Tanapongpipat S. Thomas G. Griffihs L. Crooke H. Cole J.A. Mol. Microbiol. 1996; 19: 467-481Crossref PubMed Scopus (145) Google Scholar, 12Potter L.C. Cole J.A. Biochem. J. 1999; 344: 69-76Crossref PubMed Google Scholar, 13Brondijk T.C.H. Fiegen D. Richardson D.J. Cole J.A. Mol. Microbiol. 2002; 44: 245-255Crossref PubMed Scopus (80) Google Scholar). Fe-S clusters act as cofactors in many different proteins such as electron carriers, environmental sensors, substrate transporters, or regulatory proteins participating in control of gene expression (14Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (277) Google Scholar, 15Bauer C.E. Elsen S. Bird T.H. Annu. Rev. Microbiol. 1999; 53: 495-523Crossref PubMed Scopus (201) Google Scholar). Spontaneous in vitro Fe-S cluster assembly can occur, but in vivo Fe-S assembly requires accessory proteins (16Frazzon J. Dean D.R. Curr. Opin. Chem. Biol. 2003; 7: 166-173Crossref PubMed Scopus (188) Google Scholar). Studies of Fe-S cluster assembly in the Azotobacter vinelandii nitrogenase revealed the requirement of two proteins, NifS and NifU (14Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (277) Google Scholar). Homologues of these two proteins are found in almost all organisms, from bacteria to humans. NifS is a pyridoxal phosphate-dependent cysteine desulfurase that mobilizes sulfur from l-cysteine. NifU is the Fe-S cluster scaffold upon which the nascent cluster is constructed to be transfer to an apo-protein (14Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (277) Google Scholar). In E. coli, the isc and suf operons are required for Fe-S biogenesis of different iron-sulfur proteins (17Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 18Urbina H.D. Silberg J.J. Hoff K.G. Vickery L.E. J. Biol. Chem. 2001; 276: 44521-44526Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 19Loiseau L. Ollagnier-de-Choudens S. Nachin L. Fontecave M. Barras F. J. Biol. Chem. 2003; 278: 38352-38359Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 20Outten F.W. Djaman O. Storz G. Mol. Microbiol. 2004; 52: 861-872Crossref PubMed Scopus (341) Google Scholar). The Isc system is the housekeeping Fe-S cluster assembly, whereas the Suf system is important for Fe-S biogenesis under stressful conditions. In this system, SufA plays a role as scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins, SufS is a cysteine desulfurase that mobilizes the sulfur atom from cysteine and provides it to the cluster, and SufE binds to SufS and is responsible for a 50-fold stimulation of the cysteine desulfurase activity of SufS (19Loiseau L. Ollagnier-de-Choudens S. Nachin L. Fontecave M. Barras F. J. Biol. Chem. 2003; 278: 38352-38359Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In this study we report the heterologous expression in E. coli of the R. sphaeroides NapF protein fused to an N-terminal His6 motif. NapF purification allowed the study of its spectroscopic properties and the isolation of anti-NapF antibodies raised in rabbit to assess the subcellular localization of the native protein in R. sphaeroides. Demonstration of the implication of the R. sphaeroides NapF protein in the assembly of the iron-sulfur center of the catalytic subunit (NapA) is also presented. Bacterial Strains and Plasmids—The bacterial strains and plasmids used in this work are listed in Table I. The E. coli strains were cultured in Luria-Bertani (LB) medium or on LB agar plates at 37 °C (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). pBluescript and pSVB25 were used routinely in gene manipulation, and the pKPD60 vector was used for the construction of alkaline phosphatase gene fusions. E. coli cells harboring any of the plasmids used in this work were cultured in the presence of ampicillin at a final concentration of 100 μg ml–1.Table IBacterial strains and plasmids used in this workStrains and plasmidsPhenotype or characteristicsSource or referenceBacterial strainsR. sphaeroides DSM158Wild type, Nap+21Kerber N.L. Cárdenas J. J. Bacteriol. 1982; 150: 1091-1097Crossref PubMed Google ScholarR. sphaeroides DSM158 (pFR86 Km1/2)NapF--defective strains7Reyes F. Gavira M. Castillo F. Moreno-Vivián C. Biochem. J. 1998; 331: 897-904Crossref PubMed Scopus (49) Google ScholarE. coli JM109Host strain for plasmids carrying the pQE derivatives22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarE. coli DH5αLac-, host strain for plasmids carrying the lacZ or phoA genes22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarE. coli XL1-blueHost strain for plasmids carrying the phoA gene22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarPlasmidspBluescript SK(+/-)Cloning vector, AmpR, lacZ, f1ori, T7 promoteraAmpR, ampicillin-resistance marker.StratagenepSVB25Cloning vector, AmpR, Lac+23Arnold W. Pühler A. Gene (Amst.). 1988; 70: 171-179Crossref PubMed Scopus (99) Google ScholarpGEM-TPoly-T cloning vector for PCR fragments, AmpRPromegapKPD60From pKK223-3 with a cycA81'-phoA fusion, AmpR24Stoll R. Page M.D. Sambongi Y. Ferguson S.J. Microbiology. 1996; 142: 2577-2585Crossref PubMed Scopus (14) Google ScholarpQE32Cloning vector carrying the His6 motif, AmpRQiagenpFR242.17-kb PstI-BamHI fragment with the napKEFD genes cloned into pUC18▵ SalI, AmpR6Reyes F. Roldán M.D. Klipp W. Castillo F. Moreno-Vivián C. Mol. Microbiol. 1996; 19: 1307-1318Crossref PubMed Scopus (82) Google ScholarpALTERSite-directed mutagenesis vector, AmpRPromegapALTER-PB2.17-kb PstI/BamHI fragment with the napKEFD genes cloned into pALTER, AmpRThis workpKPD60XFrom pKPD60 with a napF26'-phoA fusionThis workpKPD60▵ SXFrom pKPD60 with a deletion of the cycA sequenceThis workpKPD60B*X*From pKPD60 with a napA41'-phoA fusionThis workpFR242.17-kb PstI-BamHI fragment cloned into pUC18▵ SalI, AmpR6Reyes F. Roldán M.D. Klipp W. Castillo F. Moreno-Vivián C. Mol. Microbiol. 1996; 19: 1307-1318Crossref PubMed Scopus (82) Google ScholarpQE32/napF0.8-kb BamHI fragment with the napFD genes cloned into pQE32This worka AmpR, ampicillin-resistance marker. Open table in a new tab Enzyme Assays, Analytical Methods, and Spectroscopic Analyses— Nitrate reductase was assayed with reduced methyl viologen as artificial electron donor, and the nitrite produced in the reaction was determined colorimetrically (25Moreno-Vivián C. Cárdenas J. Castillo F. FEMS Microbiol. Lett. 1986; 34: 105-109Crossref Scopus (17) Google Scholar). Alkaline phosphatase activity was assayed in subcellular fractions of E. coli with p-nitrophenyl phosphate (24Stoll R. Page M.D. Sambongi Y. Ferguson S.J. Microbiology. 1996; 142: 2577-2585Crossref PubMed Scopus (14) Google Scholar, 26Sambongi Y. Stoll R. Ferguson S.J. Mol. Microbiol. 1996; 19: 1193-1204Crossref PubMed Scopus (54) Google Scholar). Malate dehydrogenase activity was measured in subcellular fractions of R. sphaeroides by following NADH oxidation in a spectrophotometer at 340 nm (27Smith A.F. Bergmeyer H.U. Bergmeyer J. Gral M. Methods of Enzymatic Analysis. 3. Verlagchemie, Weinheim, Germany1983: 166-171Google Scholar). Succinate dehydrogenase was assayed in subcellular fractions of R. sphaeroides by the phenazine methosulfate-dependent reduction of dichlorophenolindophenol (28Carithers R.P. Yoch D.C. Arnon D.I. J. Biol. Chem. 1977; 252: 7461-7467Abstract Full Text PDF PubMed Google Scholar). Labile sulfur determination was performed by a reaction with N-N-dimethyl-p-phenylenediamine and FeCl3 following described methods (29Fogo J.K. Popowsky M. Anal. Chem. 1949; 21: 732-734Crossref Scopus (364) Google Scholar, 30Beinert H. Anal. Biochem. 1983; 131: 373-378Crossref PubMed Scopus (401) Google Scholar) and using a calibration curve with Na2S·9H2O in NaOH (under nitrogen atmosphere) as standard. Labile iron was assayed in samples heated at 80 °C for 10 min, by reaction with sodium methasulfite and bathophenanthroline, as previously described (31Nilsson U.A. Bassen M. Sävman K. Kjellmer I. Free Radic. Res. 2002; 36: 677-684Crossref PubMed Scopus (60) Google Scholar), and using a calibration curve with FeSO4·7H2Oas standard. To determine iron and sulfur labile in the apo-NapF reconstitution assay, low molecular mass iron and sulfide compounds, not bond by the protein, were removed by gel filtration. Protein concentration was estimated by the Lowry procedure using bovine serum albumin (BSA) as standard (32Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The oxidized and reduced UV-visible spectra of NapF were obtained by oxidizing the protein with potassium ferricyanide or using dithionite as reductant, respectively. The spectra were recorded in a DU7500 (Beckman) spectrophotometer. The form B molybdopterin derivative was extracted from the native NapA protein and the dipyridyl-treated NapA samples by a modification of the procedure of Johnson and Rajagopalan (33Johnson J.L. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6856-6860Crossref PubMed Scopus (273) Google Scholar). 1 ml of sample (0.5 mg ml–1 of enzyme) in 10 mm Tris-HCl (pH 7.0) was acidified to pH 2.5 with concentrated HCl. The samples were incubated in a boiling water bath for 20 min and centrifuged at 19,000 × g for 10 min. Fluorescence excitation and emission spectra of the supernatants were recorded using a PerkinElmer Life Sciences LS-5 luminescence spectrometer. Surface-enhanced laser desorption ionization mass spectrometry was used to determine, through time of flight, the His6-tagged NapF molecular mass by using an immobilized metal affinity capture surface. Subcellular Fractionation—Subcellular fractionations of E. coli and R. sphaeroides were carried out as previously described (34McEwan A.G. Jackson J.B. Ferguson S.J. Arch. Microbiol. 1984; 137: 344-349Crossref Scopus (73) Google Scholar). Cells were harvested by centrifugation and washed in 100 ml of 0.05 m Tris-HCl buffer (pH 8.0). A cell pellet was obtained by centrifugation and resuspended in 50 ml of sucrose buffer (75 mm Tris-HCl, pH 8.0; 20 mm EDTA; 100 mm NaCl; 0.5 m sucrose) and 100 μgml–1 lysozyme and then incubated for 30 min at 30 °C. After centrifugation for 30 min at 19,000 × g, the supernatant (periplasmic fraction) was separated from the pellet (spheroplasts), which was resuspended in 75 mm Tris-HCl, pH 8.0, buffer. The spheroplasts were then broken by cavitation (3 pulses of 5 s at 90 watts), and the cell extract was centrifuged at 200,000 × g for 45 min. Then, the supernatant (cytoplasmic fraction) was separated from the pellet (membrane fraction). Several enzymatic activities were measured as markers of purity of each subcellular fraction of R. sphaeroides. Thus, periplasmic nitrate reductase (25Moreno-Vivián C. Cárdenas J. Castillo F. FEMS Microbiol. Lett. 1986; 34: 105-109Crossref Scopus (17) Google Scholar) was found in the periplasmic fraction, malate dehydrogenase activity (27Smith A.F. Bergmeyer H.U. Bergmeyer J. Gral M. Methods of Enzymatic Analysis. 3. Verlagchemie, Weinheim, Germany1983: 166-171Google Scholar) was detected in the cytoplasmic fraction, and succinate dehydrogenase (28Carithers R.P. Yoch D.C. Arnon D.I. J. Biol. Chem. 1977; 252: 7461-7467Abstract Full Text PDF PubMed Google Scholar) was found in the membrane fraction. Western Blots and Heme or Protein Staining—For electrophoretic separation, samples were loaded onto polyacrylamide gels, with 14% (w/v) resolving gels and 5% stacking gels. These gels were used in Western blots, heme analysis, or protein staining with Coomassie Brilliant Blue or silver. Immunoprobing analyses to detect the His6-tagged NapF protein in E. coli were performed by using monoclonal antipolyhistidine clone his-1 from mouse ascites fluid as primary antibody and anti-mouse IgG alkaline phosphatase conjugate from goat as second antibody. Immunoprobing analyses to detect the native NapF protein in R. sphaeroides were carried out with polyclonal anti-NapF antibodies raised in rabbit and anti-rabbit IgG alkaline phosphatase conjugate from goat. Immunoprobing analyses to detect the NapA protein in R. sphaeroides were carried out with polyclonal anti-NapA antibodies and anti-rabbit IgG alkaline phosphatase conjugate from goat. Heme staining gels were carried out with dimetoxybenzidine dihydrochloride (35Francis R.T. Becker R.R. Anal. Biochem. 1984; 136: 509-514Crossref PubMed Scopus (154) Google Scholar). The reaction was developed with 0.5 m sodium citrate (pH 4.4) and 300 μl of 30% H2O2. Silver staining gels were performed in the presence of sodium acetate, sodium thiosulfate, 25% glutaraldehyde, 2.5% silver nitrate, and 37% formaldehyde (36Heukeshoven J. Dernick R. Electrophoresis. 1985; 6: 103-112Crossref Scopus (1242) Google Scholar). DNA Manipulations—DNA manipulations were performed by using standard procedures (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A fusion between the 5′-end of the napF gene and the alkaline phosphatase gene (phoA) from the plasmid pKPD60 (24Stoll R. Page M.D. Sambongi Y. Ferguson S.J. Microbiology. 1996; 142: 2577-2585Crossref PubMed Scopus (14) Google Scholar) has been performed. For this purpose, the 2.17-kb PstI/BamHI fragment that contains the R. sphaeroides napKEFD genes and the 5′-end of napA was ligated into the vector pALTER to generate pALTER-PB. To create in the 26th triplet of the napF gene a recognition site for the restriction enzyme XhoI, an amplification by PCR with the primers 5′-TGTCGGCCTCGAGCGTCCAGGGC-3′ (XhoI site underlined) and 5′-CCACGCACTTTGCCTCGAGATC-3′ was performed. The restriction enzyme XhoI was used to digest the isolated PCR product, generating a 1.2-kb fragment that contains the napKE genes and the 5′-end of the napF gene. This fragment was then ligated into the pKPD60 vector previously digested with XhoI and SalI to generate the pKPD60X construct. In the alkaline phosphatase measurements, the pKPD60 vector was used as a positive control, because this plasmid contains the signal peptide of the periplasmic cytochrome c550 of Paracoccus denitrificans fused to the phoA gene. A negative control was performed by digestion of the pKPD60 vector with XhoI and SalI and further re-ligation to generate the pKPD60ΔX plasmid, thus removing a 0.5-kb fragment, which contains the cytochrome c550 signal peptide. A napA-phoA gene fusion was also constructed by inserting the 2.17-kb PstI/BamHI fragment, which contains the napKEFD genes and the 5′-end region of the napA gene, into the pSVB25 vector to generate pSVB25X60. This construct was digested with BamHI, and the linear fragment was partially filled-in with the Klenow polymerase in the presence of dGTP and dATP. Thus, compatible ends were generated to be cloned into the vector pKPD60, previously digested with XhoI and partially filled-in with the Klenow polymerase in the presence of dCTP and dTTP, to produce the last construct pKPD60B*X* with the desired in-frame napA-phoA gene fusion. The plasmids pKPD60, pKPD60X, pKPD60ΔX, and pKPD60B*X* were sequenced to check that all the constructions were made in the correct reading frame, and were introduced into E. coli strains DH5α and XL1-blue. To generate the His6-tagged NapF recombinant protein, the PCR mutagenesis technique was performed by using as template the plasmid pFR24 carrying the napKEFD genes and the 5′-end of the napA gene, which presents a BamHI restriction site, and the primers: 5′-CTGACAGCCGTGGATCCTATCCCGC-3′ (BamHI site underlined) and 5′-GGAAACAGCTATGACCATG-3′. The PCR product was digested with BamHI and cloned into pQE32, to generate pQE32/napF. This plasmid was sequenced to confirm that the His6 tag was correctly fused to NapF. The napF gene sequence has been deposited in the EMBL, GenBank™, and DDBJ Nucleotide Sequence Databases under the accession number Z46806. Overproduction and Purification of NapF—The E. coli JM109 strain harboring the pQE32/napF plasmid was cultured anaerobically at 37 °C. Anaerobic conditions were achieved by filling completely 500-ml screw-capped bottles with LB culture medium supplemented with ampicillin (100 μg ml–1). When the cultures reached an A600 of 0.4, a heat-shock of 10 min at 42 °C was carried out and 50 μm of IPTG were added, keeping the cultures at 25 °C overnight for the NapF induction. The cytoplasmic fraction of E. coli containing the heterologous NapF protein was loaded onto a nickel-nitrilotriacetic acid-agarose (Qiagen) column, and an imidazole gradient was performed (up to 250 mm imidazole) to elute the recombinant His6-tagged NapF protein. Isolation and Purification of the Anti-NapF Polyclonal Antibodies— Polyclonal antibodies raised against the purified NapF were obtained by following the method previously described by Diez and López-Ruiz (37Diez J. López-Ruiz A. Arch. Biochem. Biophys. 1989; 268: 707-715Crossref PubMed Scopus (13) Google Scholar). A volume of 250 μl containing 135 μg of purified NapF in 50 mm Tris-HCl, pH 8.0, was diluted up to 2 ml in phosphate-buffered saline buffer (135 mm NaCl, 2.6 mm KCl, 1.5 mm KH2PO4, 8 mm Na2HPO4, pH 8.0), mixed with 2 ml of adjuvant complete Freund (Difco Laboratories), and homogenized to obtain an uniform sample. The immunization process was carried out during 59 days. Rabbit-blood samples were collected in the presence of heparin, centrifuged at 4 °C and 19,000 × g for 15 min, and after incubation at 56 °C for 15 min, were frozen at –80 °C until use. Immunoglobulins were partially purified by a 40% ammonium sulfate precipitation. The pellet was washed with 1.75 m ammonium sulfate and resuspended in 17.5 mm phosphate buffer, pH 7.0. Lipoproteins were eliminated by centrifugation, and the supernatant was loaded onto an ionic exchange DEAE-Sephacel column with the phosphate buffer described above. The fractions that contributed at the first peak at 280 nm were collected, representing the IgG fraction. The anti-NapF antibodies were stored at 4 °C until use. In Vitro Reconstitution of the Iron-Sulfur Centers of NapF—The assembly of the iron-sulfur clusters to the apo-NapF protein was performed under anaerobic conditions (nitrogen atmosphere) in a total volume of 1 ml, by adding: 12.5 mm Tris-HCl buffer, pH 7.4; 50 mm KCl; 5 nmol of NapF; 1 mm l-cysteine; 2.5 mm DTT; 0.3 nmol of cysteine desulfurase (NifS) from A. vinelandii; and 2 mm ferrous ammonium sulfate, as previously described (38Khoroshilova N. Beinert H Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2499-2503Crossref PubMed Scopus (173) Google Scholar). The reconstitution was monitored in a Hitachi U-3310 spectrophotometer by recording scans between 350 and 700" @default.
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• W2119712943 date "2004-11-01" @default.
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• W2119712943 title "NapF Is a Cytoplasmic Iron-Sulfur Protein Required for Fe-S Cluster Assembly in the Periplasmic Nitrate Reductase" @default.
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