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Originally published In Press as doi:10.1074/jbc.M708097200 on October 24, 2007

J. Biol. Chem., Vol. 282, Issue 51, 37016-37025, December 21, 2007
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Evidence for nifU and nifS Participation in the Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase*Formula

Dehua Zhao, Leonardo Curatti, and Luis M. Rubio1

From the Department of Plant and Microbial Biology, University of California, Berkeley, California 94720

Received for publication, September 28, 2007 , and in revised form, October 24, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] cluster, the NifDK component carries two complex metalloclusters, the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster. FeMo-co, located at the active site of NifDK, is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom. To investigate whether NifUS are required for FeMo-co biosynthesis and to understand at what level(s) they might participate in this process, we analyzed the effect of nifU and nifS mutations on the formation of active NifB protein and on the accumulation of NifB-co, an isolatable intermediate of the FeMo-co biosynthetic pathway synthesized by the product of the nifB gene. The nifU and nifS genes were required to accumulate NifB-co in a nifN mutant background. This result clearly demonstrates the participation of NifUS in NifB-co synthesis and suggests a specific role of NifUS as the major provider of [Fe-S] clusters that serve as metabolic substrates for the biosynthesis of FeMo-co. Surprisingly, although nifB expression was attenuated in nifUS mutants, the assembly of the [Fe-S] clusters of NifB was compensated by other non-nif machinery for the assembly of [Fe-S] clusters, indicating that NifUS are not essential to synthesize active NifB.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The [Fe-S] clusters carried by the protein components of the molybdenum nitrogenase endow this enzyme with the ability to perform N2 fixation. The heterotetrameric NifDK protein component ({alpha}2β2 dinitrogenase) contains the iron-molybdenum cofactor (FeMo-co)2 within the active site in the {alpha}-subunit (NifD) and has the [8Fe-7S] P-cluster at the interface of the {alpha}- and β-subunits (1). The homodimeric NifH (dinitrogenase reductase) contains a [4Fe-4S] cubane and a site for Mg-ATP binding and hydrolysis (2). These Fe-S clusters of nitrogenase play a critical function in electron transfer and in the reduction of substrates driven by the free energy liberated from Mg-ATP hydrolysis (3). The [4Fe-4S] cluster carried by NifH is relatively ubiquitous in nature, but the P-cluster and FeMo-co are unique and regarded as some of the most complex metalloclusters known in biology. FeMo-co is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom (4-6).

A systematic genetic and biochemical analysis, mostly in Azotobacter vinelandii and Klebsiella pneumoniae, has revealed complex and specialized cellular biosynthetic pathways for the maturation of the nitrogenase component proteins (see Refs. 7-9 for reviews). The products of the nitrogen fixation (nif) genes nifU and nifS are required to achieve full activity of both nitrogenase component proteins. A. vinelandii nifU or nifS deletion mutants exhibited a 15-fold reduction in NifH activity and a 4-fold reduction in NifDK activity (10). Similar to A. vinelandii, nifS mutants of K. pneumoniae exhibited negligible NifH activity and a 25-fold reduction in NifDK activity (11). Because both nitrogenase components are [Fe-S] proteins, it was promptly suggested that NifU and NifS are involved in the formation of [Fe-S] clusters for NifH and NifDK (10). Later on, in vivo and in vitro experiments demonstrated that NifU and NifS are involved in the assembly of the [4Fe-4S] cluster of NifH (12-14).

A series of studies by Johnson and co-workers (reviewed in Ref. 15) showed that NifU and NifS work in concert to synthesize [Fe-S] clusters under nitrogen fixing conditions and that their roles represent a specialization of the roles performed by the homologous proteins IscU and IscS in general [Fe-S] cluster assembly. Each [Fe-S] cluster assembly machinery minimally consists of a sulfur-providing cysteine desulfurase and a molecular scaffold where [2Fe-2S] or [4Fe-4S] clusters are transiently assembled prior transfer to target apo-proteins. NifS is a pyridoxal phosphate-containing enzyme that catalyzes the desulfurization of L-cysteine to provide sulfur for [Fe-S] cluster formation (16, 17), and NifU serves as the molecular scaffold for the NifS-directed assembly of [Fe-S] clusters (18, 19). Indeed, in vitro experiments showed that NifU could transfer a [4Fe-4S] cluster to apoNifH3 and reconstitute an active holo-NifH (14).

The involvement of NifU and NifS in the assembly of the P-cluster and the FeMo-co embedded within the NifDK protein is less clear. Strains lacking both nifU and nifS exhibited a 10-fold decrease in NifDK activity that could not be recovered by addition of FeMo-co (10). Complementation by FeMo-co addition is a characteristic property of apoNifDK containing the P-clusters but lacking FeMo-co. The phenotype of nifUS mutants thus suggests that the absence of NifUS mostly impairs P-cluster synthesis but does not clarify whether nifUS mutants are capable of synthesizing FeMo-co.

Other nif genes, nifB, nifE, nifH, nifN, nifQ, nifV, and nifX, have been shown to be involved in the biosynthesis of FeMo-co (7, 8). The nifB gene encodes a SAM-radical protein required to synthesize NifB-co, an [Fe-S] cluster of unknown structure that serves as a biosynthetic intermediate during the early steps of FeMo-co biosynthesis (20-22). Unlike the wild-type strain, nifN or nifE mutant strains accumulate a measurable amount of NifB-co under nitrogen fixing growing conditions, because the FeMo-co biosynthetic pathway is interrupted at the level of NifB-co processing. NifB-co can be isolated from cytoplasmic membranes of a K. pneumoniae nifN mutant strain by treatment with the detergent Sarkosyl (20). Radiolabeling experiments with 55Fe and 35S isotopes have shown that iron and sulfur from NifB-co are transferred to FeMo-co during cofactor synthesis in vitro (21). However, it is not known whether NifB-co is the only source of iron and sulfur to FeMo-co. NifB-co contains neither molybdenum nor an organic acid, such as homocitrate (20).

A standing hypothesis has been that the simple [2Fe-2S] or [4Fe-4S] clusters assembled by NifU and NifS could serve as metabolic substrates during the early steps of FeMo-co synthesis (i.e. the synthesis of NifB-co) (7-9). However, direct experimental evidence supporting this hypothesis is lacking. The impairment of nifU and nifS mutant strains to synthesize active NifDK protein could be the result of cumulative effect over the activity of some of the [Fe-S] cluster-containing proteins that are involved in FeMo-co synthesis and NifDK maturation, for example NifH, NifB, or NifEN. To address this question, we have investigated here the effect of nifU and nifS mutations on the accumulation of NifB-co and on the activity of the NifB protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
K. pneumoniae Strains and Growth Conditions—K. pneumoniae strains UN (wild type) and UN1217 (nifN4536::mu) have been previously described (23). Strains generated during the course of this study are described in supplemental Table S1. Growth in minimal medium, nif derepression, cell collection, and cell breakage has been described (20). For growth on plates, LC medium (1% Tryptone, 0.5% yeast extract, and 0.5% NaCl) was solidified with separately autoclaved 1.5% agar solution. Kanamycin (50 µg/ml), spectinomycin (100 µg/ml), chloramphenicol (17.5 µg/ml), and ampicillin (25 µg/ml) were added as required. A. vinelandii strain UW45 (nifB mutant) (24) was cultivated in 20-liter carboys and derepressed for nitrogenase expression as described before (25). Escherichia coli DH5{alpha}, BL21, and S17-1 strains were grown in Luria-Bertani medium at 37 °C with shaking (200 rpm). For growth of E. coli on plates, medium solidified with 1.5% agar was used. Antibiotics were used at standard concentrations (26).

Plasmid Constructions and DNA Manipulations—Plasmid constructions, PCR, and transformation of E. coli were carried out by standard methods (26). Plasmids generated during the course of this work are listed in supplemental Table S1. Isolation of genomic DNA from K. pneumoniae strains was carried out using the DNAeasyTM Tissue Kit (Qiagen). Procedures for K. pneumoniae transformation (27), conjugation, and gene replacement (28) have been described.

Generation of K. pneumoniae nifU and nifS Mutant Strains—Mutations in nifU or nifS consisted of in-frame deletions spanning the complete amino acid coding sequences of nifU or nifS without altering any other DNA sequence in the nif gene cluster (Fig. 1). These deletions were constructed on suicide plasmids to force recombination with the genomic DNA of K. pneumoniae UN1217 and to promote exchange of gene alleles. Oligonucleotides were designed to amplify by PCR the nif DNA regions flanking nifU or nifS using K. pneumoniae genomic DNA as template. DNA fragments were amplified using Pfu DNA polymerase and ligated into the corresponding restriction sites of plasmid pEP185.2 to construct pRHB288 ({Delta}nifU), pRHB289 ({Delta}nifS), and pRHB290 ({Delta}nifUS), respectively. In pRHB288 ({Delta}nifU), the 1.9-kb KpnI DNA fragment containing part of nifN and the complete nifX was blunt-end ligated to the 2.5-kb PstI DNA fragment containing the nifSVW genes. In pRHB289 ({Delta}nifS), the 2.73-kb KpnI DNA fragment containing part of nifN and the complete nifX and nifU genes was blunt-end-ligated to the 1.26-kb PstI DNA fragment containing the complete nifVW genes. In pRHB290 ({Delta}nifUS), the 1.9-kb KpnI DNA fragment containing part of nifN and the complete nifX gene was blunt-end ligated to the 1.8-kb NotI DNA fragment containing the complete nifVWZ genes. Plasmid pRHB291 was generated from pRHB290 by replacing the 630-bp DNA fragment between two BamHI restriction sites within nifX and nifV by the 1.2-kb kanamycin resistance cassette from pUC4K. Fidelity of all constructions was confirmed by sequencing both DNA strands.

The nifU and nifS mutations were introduced into the chromosome of strain UN1217 by allelic exchange events. First, UC0 was generated by conjugation of K. pneumoniae UN1217 with E. coli S17-1 (pRHB291) followed by selection of a Kmr Cms phenotype. After isolating genomic DNA from resulting Kmr Cms colonies, incorporation and segregation of mutant allele into the chromosome was checked by PCR. Second, pRHB288 ({Delta}nifU), pRHB289 ({Delta}nifS), or pRHB290 ({Delta}nifUS) were transferred to strain UC0 by conjugation to generate UC1, UC2, and UC3, respectively. Ampr Cmr clones with plasmids integrated into the chromosome by single crossover events were selected and confirmed by PCR analysis. Third, selected Ampr Cmr clones were continuously cultured in liquid LC medium containing 25 µg/ml ampicillin for more than 100 generations to enrich for cells having the second allelic exchange. Cultures were then diluted and plated onto solid LC medium containing 25 µg/ml ampicillin. Kms Cms colonies (which had a second allelic exchange event) were selected, and the deletions in nifU, nifS, or nifU and nifS were confirmed by PCR analysis (Fig. 1).

Genetic Complementation of {Delta}nifU, {Delta}nifS, and {Delta}nifUS Mutant Strains—To perform genetic complementation analysis of {Delta}nifU and {Delta}nifS mutants, strains UC1, UC2, and UC3 were transformed with plasmid pRHB257 according to (27). Plasmid pRHB257 is a derivative of the low copy number plasmid pEXT21 that carries wild-type nifUS genes. Plasmid pRHB257 was generated by cloning a 2275-bp BamHI and HindIII DNA fragment, which covers from the restriction enzyme site at the 3'-end of nifX to the stop codon of nifS, into the BamHI and HindIII sites of pEXT21 (Fig. 1).

Generation and Expression of GST-NifB Fusion Proteins in K. pneumoniae—NifB from K. pneumoniae was expressed as a glutathione S-transferase (GST) fusion protein. The chimera was constructed in the pRHB153 plasmid, a derivative of plasmid pGEX-4T-3 (GE Healthcare) (29). The nifB gene was PCR-amplified from the chromosome of K. pneumoniae UN1217 using oligonucleotides nifB-N1 5'-CCCCATATGACTTCCTGCTCCTCTTTTTCTGG-3' and nifB-C1 5'-GGGCTCGAGTCAGGCGACCCCCTTATGCG-3' as primers. The nifB gene cartridge was then digested with NdeI and XhoI and ligated into the corresponding sites of plasmid pRHB153 to generate plasmid pRHB233.

Two strategies were used to express gst-nifB at different cellular levels. First, to keep gst-nifB expression at wild-type levels, the chromosomal copy of nifB was replaced by a gst-nifB allele so that expression was controlled by the natural nifB promoter. A 1-kb DNA fragment containing the nifA gene, the nifB promoter (PnifB), and a XbaI restriction site at the 5'-end, was PCR-amplified from the chromosome of K. pneumoniae UN1217 using oligonucleotides nifA-m1 (5'-CCCCTCTAGAATCGCCAACGCCATCCACCATAAT-3') and nifA-c1 (5'-GGTCGTACCTTCGTGGTTGGGC-3') as primers. In addition, a 2.1-kb DNA fragment containing the gst-nifB gene and a SacI restriction site at the 3'-end was PCR-amplified from pRHB233 using oligonucleotides RNF17 (5'-ATGTCCCCTATACTAGGTTATTGGAAATTAAG-3') and nifB-c3 (5'-CCGAGCTCTCAGGCGACCCCCTTATGCGGCAA-3') as primers. Both DNA fragments were ligated into the XbaI and SacI sites of the suicide plasmid pDS132, which carries an sacB gene (30), to generate plasmid pRHB292. Plasmid pRHB292 was transferred to strains UN (wild type), UN1217 (nifN::mu), and UC3 ({Delta}nifUS nifN::mu) to generate strains UC4, UC5, and UC8, respectively. After selecting Cmr clones, the integration of pRHB292 into the chromosome was confirmed by PCR analysis. Clones with integrated pRHB292 were cultured in liquid LC medium and subsequently plated onto solid LB medium supplemented with 5% sucrose to select for plasmid excision in sucrose-resistant colonies. Substitution of gst-nifB for nifB was confirmed by PCR analysis of chromosomal DNA isolated from strains UC4, UC5, and UC8. Cells from K. pneumoniae UC5 and UC8 strains were grown, derepressed for nitrogenase, and collected by standard procedures (20).

The second strategy aimed at boosting expression of gst-nifB up to levels that facilitated purification of NifB. To achieve this, the wild-type copy of nifB was removed from the chromosome of UN1217, and the gst-nifB gene was expressed from plasmid pRHB233 so that expression was controlled by an isopropyl 1-thio-β-D-galactopyranoside (IPTG)-inducible Ptac promoter. A 1-kb XbaI-EcoRI DNA fragment containing nifA, and a 1-kb EcoRI-SphI DNA fragment containing nifQ, were amplified from UN1217 genomic DNA by PCR and ligated into the XbaI and SphI sites of pDS132 to generate plasmid pRHB235 ({Delta}nifB). Plasmid pRHB235 was transferred to strains UN (wild type), UN1217 (nifN::mu), and UC3 ({Delta}nifUS nifN::mu) to generate strains UC9, UC10, and UC11, respectively, by a procedure analogous to that described above for gst-nifB replacement. Finally, plasmid pRHB233 was transferred to UC9, UC10, and UC11 mutant strains for GST-NifB expression under different genetic background generating strains UC16, UC17, and UC18, respectively. Cells from K. pneumoniae UC16, UC17, and UC18 strains were subjected to IPTG induction (5 µM IPTG) and nif derepression at the same time.

Generation of a K. pneumoniae nifENX Mutant Strain—A K. pneumoniae {Delta}nifENX mutant strain (UC15) was generated. A nifTY-nifU DNA fragment having a complete deletion of the nifENX operon deleted was first cloned in plasmid pDS132 and then introduced into the chromosome of K. pneumoniae UN by allelic exchange to generate UC15. A 986-bp nifTY DNA fragment carrying XbaI restriction site at the 5'-end and EcoRI restriction site at the 3'-end was amplified by PCR using primers nifT-N2 (5'-CCCTCTAGATGCCCCGCGTCATGCGGCGGCAG-3') and nifY-C2 (5'-CCCGAATTCGAGCGTAACGTGGGGAAGAGCGTCC-3'). A 1053-bp nifU DNA fragment carrying EcoRI restriction site at the 5'-end and an SphI restriction site at the 3'-end were amplified by PCR using primers nifU-p (5'-CCCGAATTCGATCCGGACCCGCGCCGCTAGCC-3') and nifU-C3 (5'-CCCGCATGCTCAGGCCGCCACCACTTCCATATAA-3'). Both DNA fragments were digested by the corresponding restriction enzymes and co-ligated into the XbaI and SphI sites of pDS132 to generate plasmid pRHB294. Transfer of pRHB294 into K. pneumoniae UN, clone selection, and segregation of {Delta}nifENX mutation were performed using plasmid pDS132 as described above. Deletion of nifENX genes from the chromosome of UC15 was confirmed by PCR analysis. Strain UC15 did not exhibit nitrogenase activity in vivo, as expected.

Purification of GST-NifB from K. pneumoniae Cells—GST-NifB proteins were purified from cells of strains UC5, UC8, UC17, and UC18 by affinity chromatography using GSH-Sepharose resin (GE Healthcare). For preparation of GST-NifB, 25 g of collected cell paste was resuspended in 80 ml of 2x buffer A (10 mM sodium phosphate, 1.8 mM potassium phosphate buffer, pH 8.5, 140 mM NaCl, 2.7 mM KCl, 10% glycerol, 5 mM β-mercaptoethanol, 0.02% n-dodecyl-β-D-maltopyranoside, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 5 µg/ml DNaseI, and 1 mM DTH). Cells were disrupted by 10 cycles of sonication (1 min per cycle) using a Fisher Sonic Dismembrator 550 equipped with a 12-mm tip at 25% power output inside an anaerobic glove box. After adjusting pH of lysate to pH 7.4, cell debris was removed by centrifugation at 27,000 x g for 30 min in a Beckman Ti-50.2 ultracentrifuge rotor. The clarified cell-free extract (supernatant) was applied onto a 5-ml GSH-Sepharose column. The column was then washed with three column volumes of buffer A supplemented with 1% Triton X-100 and 10 column volumes of buffer A to remove contaminants. The GST-NifB was eluted from the column applying three column volumes of buffer B (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 0.02% n-dodecyl-β-D-maltopyranoside, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 10 mM reduced glutathione, and 1 mM DTH). Eluted GST-NifB protein was concentrated to 3-5 mg/ml inside an anaerobic glove box using an Amicon cell equipped with YM100 Millipore ultrafiltration membranes. Purification of the GST-NifB fusion protein was accomplished within 6 h at 16 °C. Purified NifB preparations were drop-frozen and stored in liquid nitrogen.

In Vivo and in Vitro Nitrogenase Activities—In vivo nitrogenase activity was determined by ethylene production at 30 °C for 30 min in 1-ml culture samples as previously described (31). NifDK activity in cell-free extracts was obtained after titration with an excess of the complementary component, NifH, as described (32). Specific activity is defined as nanomoles of ethylene formed per min/mg of protein in the extract.

In Vitro FeMo-co-dependent or NifB-co-dependent Apo-NifDK Activation Assays—Protocols for the isolation of FeMo-co (33) and NifB-co (20) have been described previously. Preparation of crude NifB-co extracts was carried out according to previous study (20) with a modified cell breakage procedure. K. pneumoniae cells in a 3-ml suspension were broken inside an anaerobic glove box by sonication for 2 min and 15% power output using a Fisher Sonic Dismembrator 550 equipped with a 3-mm tip. Crude NifB-co extract refers to NifB-co solubilized from lysed cells of K. pneumoniae strains with Sarkosyl detergent (n-lauroyl sarcosine) but not purified through further chromatographic steps.

Assays for NifB-co dependent in vitro activation of apo-NifDK present in extracts of A. vinelandii strain UW45 (nifB) were performed as described before (20) with modifications. 9-ml serum vials sealed with stoppers were repeatedly evacuated and flushed with argon gas and rinsed with 0.3 ml of anaerobic buffer. The complete reactions contained: 100 µl of 25 mM Tris-HCl buffer, pH 7.5, 10 µl of 1 mM Na2MoO4, 20 µl of 5 mM homocitrate, 200 µl of ATP-regenerating mixture (containing 3.6 mM ATP, 6.3 mM MgCl2, 51 mM phosphocreatine, 20 units/ml creatine phosphokinase, and 6.3 mM DTH), 200 µl of UW45 cell-free extracts ({approx}3 mg of protein), and 50 µl of crude NifB-co extract from the K. pneumoniae strain being analyzed. The reactions were incubated at 30 °C for 35 min to allow for the FeMo-co synthesis and insertion reactions. The resulting activation of apoNifDK present in UW45 extract was analyzed by the acetylene reduction assay after adding 0.8 ml of ATP-regenerating mixture and an excess of purified NifH (0.2 mg of protein) (32).

FeMo-co precursor activity present in purified GST-NifB preparations was analyzed in the UW45 crude extract-based assay as described above except that purified GST-NifB substituted for NifB-co in the reaction mixture. FeMo-co insertion assays into apoNifDK were performed as described in (34).

Apo-NifDK Activation Assay with Purified Components—FeMo-co synthesis and apoNifDK4 activation assays with purified components were set in 406-µl reaction mixtures inside 9-ml anaerobic vials. Apo-NifDK reconstitution was dependent on the NifB-co activity associated with purified NifB protein and therefore no extra source of iron, sulfur, or SAM was added to the assay. 200 µl of ATP-regenerating mixture, 100 µl of 25 mM Tris-HCl, pH 7.4, 20 µl of 5 mM homocitrate, and 10 µl of 1 mM Na2MoO4 were mixed, and the mixture was incubated at room temperature for 10 min. Reactions were then initiated by adding the purified protein components, namely precursor-free NifEN, NifH, apoNifDK, and GST-NifB, to final concentrations of 1.1 µM, 14.3 µM, 1.1 µM, and 4.5 µM, respectively. Precursor-free NifEN was purified from A. vinelandii strain UW243 (29), NifH was purified from A. vinelandii strain DJ884, and apoNifDK was purified from A. vinelandii strain DJ1143 (35). Additional 25 mM Tris-HCl, pH 7.4, buffer was added to bring the reaction volume to 406 µl when needed. Reaction mixtures were incubated for 35 min at 30 °C in a rotary shaker. The resulting activation of apoNifDK present in the reaction mixtures was routinely analyzed by the acetylene reduction assay after addition of excess NifH and ATP-regenerating mixture (32).

SDS-PAGE, Anoxic Native Gel Electrophoresis, and Immunoblot Analysis—The procedure for SDS-PAGE has been described (36). Immunoblot analysis was performed as described by Brandner et al. (37). Purified preparations of K. pneumoniae NifB were used to raise anti-NifB antibodies at Capralogics Inc. (Hardwick, MA). For anoxic native gel electrophoresis, proteins were separated on gels with superimposed 7-20% acrylamide and 0-20% sucrose gradients as described (29). Native gels were then stained for proteins with Coomassie R-250 by standard procedures, or stained for iron as described before (38).

UV-visible Spectroscopy—The UV-visible spectra were determined using a Shimadzu UV-1601 spectrophotometer.

Miscellaneous Assays—Protein concentrations were determined by the bicinchoninic acid method using bovine serum albumin as standard (39). Iron content in purified GST-NifB preparations was determined according to a previous study (40).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
K. pneumoniae nifU and nifS Mutants Fail to Accumulate NifB-co—Strain UN1217 carries a mutation in nifN that impairs FeMo-co synthesis and results in the accumulation of NifB-co activity (20). NifB-co activity levels in K. pneumoniae strains that combine mutations in nifN with deletions of nifU, nifS, or both nifU and nifS were determined and are presented in Table 1. NifB-co activity levels in extracts of K. pneumoniae strains were determined by using the NifB-co-dependent FeMo-co synthesis and apoNifDK activation assay (20). This assay is based on the biochemical complementation of a cell-free extract from a {Delta}nifB A. vinelandii strain (UW45) with a sample containing NifB-co activity. In this assay, FeMo-co is synthesized in vitro and inserted into apoNifDK present in the UW45 extract to reconstitute nitrogenase activity. Crude NifB-co preparations from cell-free extracts of strains UC1 ({Delta}nifU nifN::mu), UC2 ({Delta}nifS nifN::mu), and UC3 ({Delta}nifUS nifN::mu) were unable to reconstitute NifDK activity, indicating that they did not accumulate enough NifB-co to support NifB-co-dependent FeMo-co synthesis (Table 1).


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  		TABLE 1
Determination of NifB-co activity in K. pneumoniae {Delta}nifU, {Delta}nifS, and {Delta}nifUS mutant strains Details for the complete NifB-co dependent FeMo-co synthesis and apoNifDK activation assay can be found under "Experimental Procedures." The amount of apo-NifDK from UW45 extracts reconstituted in vitro was determined by measuring acetylene reduction activity of matured NifDK. Activation of apoNifDK present in UW45 extracts with an excess of isolated FeMo-co gives 13 nmol of ethylene formed per min/mg of protein in the extract.

 
Genetic complementation experiments confirmed that the lack of NifB-co activity in extracts of strains UC1, UC2, and UC3 was specifically due to mutations in nifU and nifS genes. The nifU and nifS genes were cloned into the low copy plasmid pEXT21 under the control of the nifU promoter to generate plasmid pRHB257, which in turn was used to transform strains UC1, UC2, and UC3 (Fig. 1). Table 1 shows that NifB-co preparations from extracts of strains UC1, UC2 and UC3, carrying pRHB257, have the ability of support NifB-co-dependent apoNifDK activation to levels similar to those observed in strain UN1217, indicating that the expression of nifU and nifS from pRHB257 restores the intracellular accumulation of NifB-co. These results demonstrate that NifU and NifS were required for the accumulation of NifB-co in a nifN mutant background.

NifB Overexpression Partially Overcomes the Effect of nifUS Deletion on NifB-co Accumulation—Two strategies were used to express NifB at different levels and examine the relationship between NifB protein levels and NifB-co activity (Fig. 2). The first was to substitute gst-nifB for nifB in the chromosome of K. pneumoniae while keeping wild-type expression levels (strains UC4, UC5, and UC8). This strategy allowed examination of GST-NifB and NifB-co levels under normal nif regulatory conditions. The second was to delete nifB from the chromosome and introduce a plasmid carrying a gst-nifB gene under an IPTG-inducible Ptac promoter (strains UC16, UC17, and UC18). This strategy uncouples nifB expression from nif transcriptional regulation. In addition, NifB expression is enhanced allowing purification and analysis of purified GST-NifB from different genetic backgrounds (see below).

Strains UC4 (gst-nifB), UC5 (nifN::mu, gst-nifB), and UC8 ({Delta}nifUS nifN::mu, gst-nifB) were generated from the parental strains UN, UN1217, and UC3, respectively (Fig. 2). Incorporation of the GST tag into NifB did not impair in vivo nitrogenase activity in K. pneumoniae UC4 cells. K. pneumoniae strain UC4, which expresses GST-NifB protein in a wild-type genetic background, exhibited in vivo nitrogenase activity (943 nmol of C2H4 formed/h/A600) very similar to that of the wild-type UN strain (817 nmol of C2H4 formed/h/A600).


Figure 1
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  		FIGURE 1.
Scheme showing the genetic organization of K. pneumoniae strains UN1217, UC0, UC1, UC2, and UC3 in the chromosomal region around nifU and nifS. Plasmid pRHB257, used for genetic complementation analysis, contains the nifU and nifS genes cloned into pEXT21.

 


Figure 2
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  		FIGURE 2.
GST-NifB protein levels in nifN::mu and nifN::mu {Delta}nifUS genetic backgrounds. A, scheme showing the wild-type chromosomal region around nifB and the genetic constructions to express gst-nifB from the PnifB promoter in the chromosome (strains UC4, UC5, and UC8) or from the Ptac promoter present in plasmid pRHB233 (strains UC16, UC17, and UC18). B, GST-NifB levels in nitrogenase-derepressed cells of UC5 (nifN::mu and gst-nifB), UC8 (nifN::mu {Delta}nifUS and gst-nifB), UC17 (nifN::mu {Delta}nifB and Ptac-gst-nifB), and UC18 (nifN::mu {Delta}nifB {Delta}nifUS and Ptac-gst-nifB). Cells from nif-derepressed cultures were collected and boiled in SDS loading buffer. UC17 and UC18 cultures were nif-derepressed and IPTG-induced. To help compare GST-NifB levels between strains, the amount of UC5 and UC8 cells loaded in the gel was 7-fold higher than UC17 and UC18. After SDS-PAGE, proteins in the gel were transferred to nitrocellulose membranes, and GST-NifB was detected by immunoblot developed with antibodies to NifB and quantified by densitometry.

 
Fig. 2B shows that strain UC8 accumulates 10-fold less GST-NifB than strain UC5, suggesting an effect of {Delta}nifUS mutation in either expression or stability of GST-NifB. In addition, UC8 cells were severely affected in the accumulation of NifB-co, the metabolic product of NifB activity. Although UC5 cells exhibited similar NifB-co activity levels to those of the parental strain UN1217, NifB-co activity extracted from UC8 cells was remarkably low and consistently undistinguishable from the background of the UW45 extracts used in that particular assay (Table 2).


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  		TABLE 2
NifB-co activity in K. pneumoniae nifN::mu and {Delta}nifUS nifN::mu strains expressing GST-NifB Crude NifB-co extracts were prepared as described under "Experimental Procedures." Reaction conditions were the same as those in Table 1. Values are the averages of two to seven assays performed separately. Specific activities are expressed as nanomoles of ethylene formed per min/mg of protein in UW45 extract. Activation of apoNifDK present in UW45 extracts with an excess of isolated NifB-co gives 12.9 ± 1.0 nmol ethylene formed per min/mg of protein in the extract.

 
Several differences were apparent in strain UC17 in which GST-NifB expression was driven by the tac promoter instead of the endogenous nifB promoter. First, GST expression (Fig. 2B) and NifB-co activity (Table 2) increased 10- and 2-fold, respectively. The fact that UC17 cells overexpressing NifB contain higher levels of NifB-co suggests that the NifB levels present in a wild-type strain could be a limiting factor for NifB-co synthesis. Second, the {Delta}nifUS mutation did not have a major effect in the in vivo levels of GST-NifB protein (compare GST-NifB levels in UC17 versus UC18 to those in UC5 versus UC8), which suggests that the effect of the {Delta}nifUS mutation on GST-NifB levels observed in UC8 was mainly due to changes in expression rather than in NifB stability. Third, in a {Delta}nifUS genetic background, UC18 cells overexpressing GST-NifB exhibited more NifB-co activity than UC8 cells (Table 2), uncovering a NifUS-independent pathway that can partially substitute for the function of NifUS when NifB is overexpressed.

The Products of nifU and nifS Are Not Required to Synthesize a Functional NifB Protein—The failure of strains UC1, UC2, and UC3 to accumulate NifB-co in a nifN background led to several hypothetical scenarios: (i) it is possible that NifU and NifS directly provide the [Fe-S] cluster substrates for NifB activity to transform into NifB-co, (ii) it is possible that NifU and NifS are involved in the synthesis of a functional NifB protein, which itself is an [Fe-S] protein or, (iii) a combination of both scenarios. To discriminate between these possibilities, we compared the iron content, UV-visible absorption spectrum, and specific activity of NifB protein purified from a nifN mutant strain to those of NifB purified from a mutant strain lacking nifN and nifUS.

GST-NifB proteins were purified from cells of strains UC17 ({Delta}nifB nifN::mu, Ptac-gst-nifB) and UC18 ({Delta}nifB {Delta}nifUS nifN::mu, Ptac-gst-nifB) that had been derepressed for nitrogenase activity as described under "Experimental Procedures" (Fig. 3A). A typical purification procedure yielded 15 mg of GST-NifB protein from 45 g of K. pneumoniae cells. The NifB purification yields from UC17 and UC18 cells were similar, what was consistent with the similar levels of in vivo NifB expression in UC17 and UC18 strains, as determined by immunoblot analysis (Fig. 2B). Hereafter, we refer to NifB preparations as GST-NifB and {Delta}nifUS GST-NifB when purified from UC17 or UC18 cells, respectively. Purified preparations of GST-NifB and {Delta}nifUS GST-NifB contained similar amounts of iron (10.3 ± 1.1 mol of iron/mol GST-NifB monomer compared with 8.7 ± 0.1 mol of iron/mol {Delta}nifUS GST-NifB monomer) and showed similar UV-visible absorption spectra (Fig. 3B), suggesting that nifU and nifS are not essential for the synthesis of the [Fe-S] clusters of NifB. As an SAM-radical enzyme, each NifB monomer is expected to contain at least one permanent [4Fe-4S] cluster coordinated by three cysteine residues and a molecule of SAM. Thus, the iron content of purified GST-NifB preparations would be enough to account for the presence of the permanent [4Fe-4S] cluster and additional [Fe-S] cluster(s).


Figure 3
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  		FIGURE 3.
Purification of GST-NifB proteins from K. pneumoniae cells. A, SDS-PAGE analysis of GST-NifB as purified from strains UC17 and UC18. Lane 1, molecular weight markers; lane 2, GST-NifB (3.2 µg) purified from UC17 (nifN::mu {Delta}nifB and Ptac-gst-nifB); lane 3, {Delta}nifUS GST-NifB (3.2 µg) purified from UC18 (nifN::mu {Delta}nifB {Delta}nifUS and Ptac-gst-nifB). The lower arrow points to endogenous K. pneumoniae GST protein present in GST-NifB preparations. B, UV-visible spectra of GST-NifB and {Delta}nifUS GST-NifB proteins as isolated in 1 mM DTH. Excess DTH was removed by gel-filtration chromatography before recording the spectra. The A400 to A280 ratios were 0.19 for both GST-NifB and {Delta}nifUS GST-NifB.

 


Figure 4
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  		FIGURE 4.
Activation of apoNifDK present in UW45 (nifB) extracts with purified GST-NifB proteins. Purified GST-NifB or {Delta}nifUS GST-NifB proteins were used as FeMo-co precursor sources for FeMo-co synthesis and the activation of apoNifDK present in UW45 extracts. Reaction details are described under "Experimental Procedures." UW45 extracts were titrated for apoNifDK activation with 0.28-4.4 µM GST-NifB (•) or with 0.24-3.8 µM {Delta}nifUS GST-NifB ({blacksquare}), respectively. Activation of apoNifDK present in UW45 extracts with an amount of isolated NifB-co equivalent to 20 µM iron resulted in 12.9 ± 0.5 nmol of ethylene formed per minute/mg of protein in the extract.

 
Purified GST-NifB was active in the in vitro FeMo-co synthesis assay in which GST-NifB substitutes for NifB-co. Titration of an FeMo-co synthesis reaction based on UW45 cell-free extract with purified GST-NifB shows that non-saturating concentrations of {Delta}nifUS GST-NifB provided 63 ± 4% precursor activity compared with the GST-NifB protein (Fig. 4). This result suggests that the load of FeMo-co precursor in purified {Delta}nifUS GST-NifB was less than in purified GST-NifB and is consistent with the slightly lower iron content exhibited by {Delta}nifUS GST-NifB preparations. Both titrations saturated at similar activity levels at which another component of the reaction mixture becomes limiting. The FeMo-co synthesis assay based on UW45 extracts contains 3.2 mM DTH. It has been shown that neither NifS (41) nor NifU (19) are functional in the presence of 2 mM DTH. Thus, it is unlikely that an in vitro activity of NifUS could load GST-NifB with a FeMo-co precursor under the conditions of this assay.

To further rule out in vitro complementation of NifU and NifS present in an UW45 extract and confirm that purified GST-NifB proteins carried FeMo-co precursors, GST-NifB and {Delta}nifUS GST-NifB were assayed for in vitro FeMo-co synthesis in a reaction with purified components (Table 3). Both forms of GST-NifB proteins produced similar apoNifDK activation levels in the assay with purified components. Unlike what has been reported for A. vinelandii-purified NifB protein (22), the GST-NifB protein, as purified from K. pneumoniae cells, did not require addition of iron, sulfur, and SAM for in vitro activity. Thus, GST-NifB appeared to be loaded with FeMo-co precursor activity (presumably NifB-co). In fact, addition of 0.42 mM (NH4)2Fe(SO4)2, 0.42 mM Na2S, and 0.88 mM SAM to these reaction mixtures resulted in a slight decrease in the level of reconstituted NifDK activity (data not shown).


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  		TABLE 3
GST-NifB activities in the in vitro FeMo-co synthesis system with purified components Details for in vitro FeMo-co synthesis and apoNifDK activation with purified components are described under "Experimental Procedures." Reaction mixtures contain 1.1 µM precursor-free NifEN, 14.3 µM NifH, 1.1 µM apoNifDK, and 4.5 µM GST-NifB. The amount of in vitro activated apoNifDK was determined by measuring its acetylene reduction activity after adding excess NifH. Values are the averages of at least two assays performed separately. Specific activities are expressed as nanomoles of ethylene formed per min/mg of apoNifDK.

 
Accumulation of FeMo-co precursor on K. pneumoniae NifB was not caused by the interruption of FeMo-co synthesis pathway in a nifN::mu strain, because GST-NifB purified from a wild-type genetic background (strain UC16) also carried FeMo-co precursor activity (Table 3).

The above results can be summarized as follows: (i) NifU and NifS are not required for the synthesis of a functional SAM-radical NifB protein, (ii) GST-NifB preparations purified from a {Delta}nifUS strain carry substantial amounts of FeMo-co precursor (presumably NifB-co), and (iii) nifU and nifS mutants do not synthesize enough NifB-co as to observe its accumulation in a strain (nifN::mu) unable to process it into FeMo-co. These results strongly support the hypothesis in which NifU and NifS are the main providers of [Fe-S] cluster substrates that are converted into NifB-co by the activity of NifB. They also indicate that, in the absence of NifU and NifS, another cellular [Fe-S] cluster assembly machinery is able to support low levels of NifB-co synthesis.

Role of NifX in NifB-co Accumulation—Although the NifB protein co-purifies with a small fraction of NifB-co activity present in cellular extracts of K. pneumoniae UN1217 (nifN::mu), most of the NifB-co activity pool accumulates elsewhere in the cell, probably in a protein-bound form. The main candidate would be NifX, which is known to be an NifB-co-binding protein (42). The transfer of NifB-co from NifB to NifX was tested in vitro, and the results are shown in Fig. 5. Incubation of purified GST-NifB with NifX followed by separation of both proteins by anoxic native gel electrophoresis results in the transfer of an entity containing iron from NifB to NifX (Fig. 5, lanes 1 and 2). This iron-containing moiety presumably represents NifB-co, because a NifX/NifB-co complex generated in vitro by incubating together purified samples NifX and NifB-co migrates to the same position in the gel (Fig. 5, lane 6). Importantly, NifX has been shown not to bind free iron (29). Purified {Delta}nifUS GST-NifB was also able to transfer iron to NifX (Fig. 5, lanes 3 and 4), consistent with the presence of NifB-co activity in {Delta}nifUS GST-NifB preparations. This result shows that NifB-co can be transferred from NifB to NifX in vitro and suggests this could also be the case in vivo.

To test whether the pool of NifB-co activity accumulated in nif-derepressed UN1217 cells was dependent on the presence of NifX, we analyzed the accumulation of NifB-co activity in a K. pneumoniae {Delta}nifENX mutant strain (UC15). Table 4 shows that strain UC15 exhibited similar levels of NifB-co activity than strain UN1217, indicating that NifX was not essential for NifB-co accumulation in vivo. In addition, the phenotype of UC15 completely rules out the possibility that NifB-co activity could be associated to the remaining NifE polypeptide of UN1217 (nifN::mu). Thus, the majority of the NifB-co pool in strain UC15 and, probably, in UN1217 might be bound to an unidentified carrier other than the NifB, NifX, or NifEN proteins.


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  		TABLE 4
Comparison of NifB-co activity in K. pneumoniae nifN::mu and {Delta}nifENX strains Crude NifB-co extracts were prepared as described under "Experimental Procedures." Reaction conditions were the same as those in Table 1. Values are the averages of two to four assays performed separately. Specific activities are expressed as nanomoles of ethylene formed per min/mg of protein in UW45 extract.

 


Figure 5
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  		FIGURE 5.
Anoxic native gel showing the transfer of iron from GST-NifB to NifX. Samples from purified GST-NifB and NifX preparations were incubated anaerobically on ice for 30 min and then loaded onto an anoxic native gel to separate the proteins. Lane 1, 0.4 nmol of GST-NifB; lane 2, 0.4 nmol of GST-NifB plus 1.7 nmol of NifX; lane 3, 0.4 nmol of {Delta}nifUS GST-NifB; lane 4, 0.4 nmol of {Delta}nifUS GST-NifB plus 1.7 nmol of NifX; lane 5, 1.7 nmol of NifX; lane 6, 1.7 nmol of NifX plus sub-saturating amounts of NifB-co (4.5 nmol of iron as NifB-co). The positions of GST-NifB in the native gel were determined by immunoblot analysis developed with antibodies to NifB (data not shown). The protein band corresponding to endogenous K. pneumoniae GST protein is marked. A, gel stained with Coomassie to detect proteins. B, gel stained to detect iron.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Dean and co-workers first suggested that NifU and NifS are involved in the maturation or stability of both nitrogenase component proteins, because A. vinelandii nifU or nifS deletion mutants exhibit a 15-fold reduction in NifH activity and a 4-fold reduction in NifDK activity (10). Similar to A. vinelandii, nifS mutants of K. pneumoniae exhibit negligible NifH activity and a 25-fold reduction in NifDK activity (11). In vivo and in vitro evidence have conclusively supported a specific role of NifU and NifS in the assembly and incorporation of the [4Fe-4S] cluster into NifH (12-14).

The exact roles of NifU and NifS in the assembly of the complex [Fe-S] clusters of NifDK are less conclusive. Although their involvement in the assembly of a functional NifDK protein has been established from genetic experiments, finding the one or more specific steps at which NifUS participates in this process has been particularly difficult. Many of the proteins that participate in FeMo-co biosynthesis, such as NifB, NifEN, and NifH, are [Fe-S] proteins, and an impairment in FeMo-co synthesis could initially be attributed to a defect in any of those proteins. The only protein conclusively shown to be involved in the biosyntheses of the P-cluster and FeMo-co is NifH. Two lines of evidence are inconsistent with the possibility that the effect of nifUS mutations on FeMo-co biosynthesis could be mediated by the inability to assemble the [4Fe-4S] cluster of NifH. First, it was shown that a [4Fe-4S] cluster-deficient form of NifH (apoNifH) was fully competent for FeMo-co synthesis and apoNifDK maturation in vitro (43). Second, deletion of the rnf gene clusters of A. vinelandii, which results in deficient incorporation of iron into NifH and a 100-fold decrease in NifH activity, had no effect on the levels of NifDK activity (44).

To investigate whether [Fe-S] clusters assembled by NifU and NifS could serve as substrates for FeMo-co synthesis, we took advantage of the fact that a K. pneumoniae nifN::mu strain (UN1217) accumulates a substantial amount of an isolatable FeMo-co precursor that is synthesized by the activity of NifB (NifB-co). Analyzing the effect of nifU and nifS mutations on the accumulation of NifB-co and on the activity of the NifB protein was necessary to clarify the participation of NifU and NifS in the early steps of FeMo-co biosynthesis, because it excludes the effects mediated by the activities of other [Fe-S] proteins involved in the pathway (e.g. NifEN, NifH, and apoNifDK).

Two domains have been identified in NifB. The N-terminal domain has similarity to SAM-radical proteins and was proposed to contain a [4Fe-4S] center coordinated by three cysteine residues and a molecule of SAM (22). The C-terminal domain is similar to NifX and is shared by a family of proteins that show FeMo-co precursor binding activity (34). NifB proteins purified from K. pneumoniae cells exhibit similar iron contents and UV-visible spectra regardless of the presence or absence of nifUS. Thus, the function of NifUS in the assembly of (at least) some of the [Fe-S]-clusters of NifB can be compensated by other non-Nif machineries for the assembly of simple [Fe-S] clusters. The genome of K. pneumoniae contains genes encoding the components of ISC, SUF, and CSD bacterial systems, all of which are known to participate in the biosynthesis of [Fe-S] clusters.

Other functions of NifUS appeared not to be compensated by the additional [Fe-S] cluster assembly machineries, because a K. pneumoniae {Delta}nifUS nifN::mu mutant strain does not accumulate NifB-co. The 10-fold decrease in NifB polypeptides exhibited by this mutant would contribute to its failure to accumulate NifB-co. A general attenuation of accumulation of nif gene products in K. pneumoniae nifUS mutants has been previously observed (11). The mechanism underlying nifUS requirement for maximal expression of nif genes is not understood. To circumvent this effect, nifB expression was uncoupled from Nif regulation of transcription by using an IPTG-inducible Ptac promoter. When nifB expression was driven by the Ptac promoter, the absence of nifUS had no effect on the levels of NifB polypeptides. However, accumulation of NifB-co activity was still low, suggesting that in vivo NifB-co synthesis was occurring at lower rates in the absence of nifUS.

The data presented in this study suggest that, although a strain lacking nifUS does not accumulate significant levels of NifB-co, it has the capability to assemble a functional NifB protein. The GST-NifB proteins purified from wild-type or {Delta}nifUS genetic backgrounds not only exhibited similar iron contents and UV-visible spectra, but also similar levels of associated NifB-co activity as determined by in vitro FeMo-co synthesis assays. In addition, both the GST-NifB and the {Delta}nifUS GST-NifB proteins seem to be able to transfer NifB-co to NifX, as suggested by the transfer of iron label between the two proteins determined by anoxic native gel electrophoresis. However, the amount of NifB-co activity associated with NifB represents only a small fraction of the cellular NifB-co pool, the size of which was shown to be NifUS-dependent. The main reservoir of NifB-co does not involve the NifB-co-binding proteins NifEN or NifX, because a K. pneumoniae strain lacking the nifENX genes accumulated similar NifB-co activity than UN1217. Whether NifB-co binds to another unidentified NifB-co-binding protein remains uncertain.

Purified preparations of K. pneumoniae GST-NifB supported in vitro FeMo-co synthesis without a requirement for iron, sulfur, or SAM. This result indicates that, as isolated, K. pneumoniae NifB carries a FeMo-co precursor (probably NifB-co). Unlike the purified preparations of K. pneumoniae GST-NifB, the preparations of "as-isolated" A. vinelandii His-NifB protein do not contain NifB-co activity in any of the genetic backgrounds analyzed so far (22, 45) and require addition of iron, sulfur, and SAM to support in vitro FeMo-co synthesis. In addition, GST-NifB purified from K. pneumoniae contains 10 iron atoms per monomer compared with 6 iron atoms per monomer present in His-NifB purified from A. vinelandii. These differences between the as-isolated NifB proteins from K. pneumoniae and A. vinelandii could be caused by the different purification procedures used, or it could actually reflect different properties of both enzymes. Interestingly, when chemically reconstituted, purified A. vinelandii His-NifB protein incorporates three extra iron atoms per monomer and becomes active in vitro. Understanding these differences might provide some insights into the mechanism of the reaction catalyzed by NifB.

Taken together, the results of this work clearly demonstrate the participation of NifU and NifS in NifB-co biosynthesis and hence in FeMo-co biosynthesis. Two different interpretations of the phenotype of a {Delta}nifUS mutant strain and the specific function(s) of NifUS in NifB-co biosynthesis arise from the present study. First, the NifUS system is the major contributor of the overall metabolic flux through NifB by providing [Fe-S] precursors for NifB-co synthesis. The lack of NifUS activities in {Delta}nifUS mutants can be marginally compensated for by the activity of any of the other general machineries for the assembly of simple [Fe-S] clusters present in K. pneumoniae (Fig. 6). Second, NifU and NifS would not be required to assemble the [Fe-S] clusters that are hypothesized to become NifB-co by the SAM radical-dependent activity of NifB. Rather, NifU and NifS would be required to accumulate NifB-co after it has been synthesized by an unknown mechanism that does not involve NifEN or NifX. Although the experimental evidence presented herein strongly supports the first interpretation and is in line with previous genetic information (10, 11), identification and characterization of the putative NifB-co-accumulating mechanism would be necessary to support the second interpretation.


Figure 6
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  		FIGURE 6.
Model for [Fe-S] cluster flow during the early steps of FeMo-co biosynthesis. A, synthesis and fate of NifB-co in a K. pneumoniae strain lacking NifEN. B, residual NifB-co synthesis in a K. pneumoniae strain lacking NifUS. C, proposed role of NifU and NifS as [Fe-S] cluster substrate providers for NifB-co/FeMo-co synthesis in a K. pneumoniae wild-type strain under nitrogen fixing conditions.

 

   FOOTNOTES
 
* This work was supported by NIGMS, National Institutes of Health Grant 35332 (to P. W. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 To whom correspondence should be addressed: Dept. of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720. Tel.: 510-643-3940; Fax: 510-642-4995; E-mail: lrubio{at}nature.berkeley.edu.

2 The abbreviations used are: FeMo-co, iron-molybdenum cofactor; NifB-co, NifB-cofactor; NifDK, MoFe protein or dinitrogenase; NifH, iron protein or dinitrogenase reductase; nif, genes encoding proteins involved in nitrogen fixation; DTH, sodium dithionite; SAM, S-adenosylmethionine; Sarkosyl, n-lauroyl sarcosine; IPTG, isopropyl 1-thio-β-D-galactopyranoside; GST, glutathione S-transferase. Back

3 ApoNifH refers to a form of NifH from which [4Fe-4S] cluster has been removed by chelation. Back

4 A form of apoNifDK that contains the P-clusters but lacks FeMo-co ({Delta}nifB His-tagged apoNifDK) was used for these studies (35). When purified by metal-affinity and ion-exchange chromatography, this form of apoNifDK does not contain the NafY subunit. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Paul Ludden for helpful discussions and support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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