FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2075568922> ?p ?o ?g. }

• W2075568922 endingPage "37025" @default.
• W2075568922 startingPage "37016" @default.
• W2075568922 abstract "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. 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. 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 (α2β2 dinitrogenase) contains the iron-molybdenum cofactor (FeMo-co) 2The abbreviations used are:FeMo-coiron-molybdenum cofactorNifB-coNifB-cofactorNifDKMoFe protein or dinitrogenaseNifHiron protein or dinitrogenase reductasenifgenes encoding proteins involved in nitrogen fixationDTHsodium dithioniteSAMS-adenosylmethionineSarkosyln-lauroyl sarcosineIPTGisopropyl 1-thio-β-d-galactopyranosideGSTglutathione S-transferase. 2The abbreviations used are:FeMo-coiron-molybdenum cofactorNifB-coNifB-cofactorNifDKMoFe protein or dinitrogenaseNifHiron protein or dinitrogenase reductasenifgenes encoding proteins involved in nitrogen fixationDTHsodium dithioniteSAMS-adenosylmethionineSarkosyln-lauroyl sarcosineIPTGisopropyl 1-thio-β-d-galactopyranosideGSTglutathione S-transferase. within the active site in the α-subunit (NifD) and has the [8Fe-7S] P-cluster at the interface of the α- and β-subunits (1Kim J. Rees D.C. Nature. 1992; 360: 553-560Crossref PubMed Scopus (576) Google Scholar). The homodimeric NifH (dinitrogenase reductase) contains a [4Fe-4S] cubane and a site for Mg-ATP binding and hydrolysis (2Georgiadis M.M. Komiya H. Chakrabarti P. Woo D. Kornuc J.J. Rees D.C. Science. 1992; 257: 1653-1659Crossref PubMed Scopus (566) Google Scholar). 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 (3Bulen W.A. LeComte J.R. Proc. Natl. Acad. Sci. U. S. A. 1966; 56: 979-986Crossref PubMed Scopus (210) Google Scholar). 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 (4Chan M.K. Kim J. Rees D.C. Science. 1993; 260: 792-794Crossref PubMed Scopus (486) Google Scholar, 5Einsle O. Tezcan F.A. Andrade S.L. Schmid B. Yoshida M. Howard J.B. Rees D.C. Science. 2002; 297: 1696-1700Crossref PubMed Scopus (881) Google Scholar, 6Yang T.C. Maeser N.K. Laryukhin M. Lee H.I. Dean D.R. Seefeldt L.C. Hoffman B.M. J. Am. Chem. Soc. 2005; 127: 12804-12805Crossref PubMed Scopus (73) Google Scholar). iron-molybdenum cofactor NifB-cofactor MoFe protein or dinitrogenase iron protein or dinitrogenase reductase genes encoding proteins involved in nitrogen fixation sodium dithionite S-adenosylmethionine n-lauroyl sarcosine isopropyl 1-thio-β-d-galactopyranoside glutathione S-transferase. iron-molybdenum cofactor NifB-cofactor MoFe protein or dinitrogenase iron protein or dinitrogenase reductase genes encoding proteins involved in nitrogen fixation sodium dithionite S-adenosylmethionine n-lauroyl sarcosine isopropyl 1-thio-β-d-galactopyranoside glutathione S-transferase. 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. 7Ludden P.W. Rangaraj P. Rubio L.M. Smith B.E. Richards R.L. Newton W.E. Catalysts for Nitrogen Fixation: Nitrogenases, Relevant Chemical Models, and Commercial Processes. 1st Ed. Kluwer Academic Publishers, Dordretch, The Netherlands2004: 219-253Crossref Google Scholar, 8Dos Santos P.C. Dean D.R. Hu Y. Ribbe M.W. Chem. Rev. 2004; 104: 1159-1174Crossref PubMed Scopus (167) Google Scholar, 9Rubio L.M. Ludden P.W. J. Bacteriol. 2005; 187: 405-414Crossref PubMed Scopus (149) Google Scholar 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 (10Jacobson M.R. Cash V.L. Weiss M.C. Laird N.F. Newton W.E. Dean D.R. Mol. Gen. Genet. 1989; 219: 49-57Crossref PubMed Scopus (244) Google Scholar). Similar to A. vinelandii, nifS mutants of K. pneumoniae exhibited negligible NifH activity and a 25-fold reduction in NifDK activity (11Roberts G.P. MacNeil T. MacNeil D. Brill W.J. J. Bacteriol. 1978; 136: 267-279Crossref PubMed Google Scholar). 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 (10Jacobson M.R. Cash V.L. Weiss M.C. Laird N.F. Newton W.E. Dean D.R. Mol. Gen. Genet. 1989; 219: 49-57Crossref PubMed Scopus (244) Google Scholar). 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 (12Dos Santos P.C. Johnson D.C. Ragle B.E. Unciuleac M.C. Dean D.R. J. Bacteriol. 2007; 189: 2854-2862Crossref PubMed Scopus (63) Google Scholar, 13Johnson D.C. Dos Santos P.C. Dean D.R. Biochem. Soc. Trans. 2005; 33: 90-93Crossref PubMed Scopus (33) Google Scholar, 14Dos Santos P.C. Smith A.D. Frazzon J. Cash V.L. Johnson M.K. Dean D.R. J. Biol. Chem. 2004; 279: 19705-19711Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). A series of studies by Johnson and co-workers (reviewed in Ref. 15Johnson D.C. Dean D.R. Smith A.D. Johnson M.K. Annu. Rev. Biochem. 2005; 74: 247-281Crossref PubMed Scopus (1075) Google Scholar) 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 (16Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (498) Google Scholar, 17Zheng L. White R.H. Cash V.L. Dean D.R. Biochemistry. 1994; 33: 4714-4720Crossref PubMed Scopus (352) Google Scholar), and NifU serves as the molecular scaffold for the NifS-directed assembly of [Fe-S] clusters (18Yuvaniyama 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 (271) Google Scholar, 19Smith A.D. Jameson G.N. Dos Santos P.C. Agar J.N. Naik S. Krebs C. Frazzon J. Dean D.R. Huynh B.H. Johnson M.K. Biochemistry. 2005; 44: 12955-12969Crossref PubMed Scopus (111) Google Scholar). Indeed, in vitro experiments showed that NifU could transfer a [4Fe-4S] cluster to apoNifH 3ApoNifH refers to a form of NifH from which [4Fe-4S] cluster has been removed by chelation. 3ApoNifH refers to a form of NifH from which [4Fe-4S] cluster has been removed by chelation. and reconstitute an active holo-NifH (14Dos Santos P.C. Smith A.D. Frazzon J. Cash V.L. Johnson M.K. Dean D.R. J. Biol. Chem. 2004; 279: 19705-19711Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 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 (10Jacobson M.R. Cash V.L. Weiss M.C. Laird N.F. Newton W.E. Dean D.R. Mol. Gen. Genet. 1989; 219: 49-57Crossref PubMed Scopus (244) Google Scholar). 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 (7Ludden P.W. Rangaraj P. Rubio L.M. Smith B.E. Richards R.L. Newton W.E. Catalysts for Nitrogen Fixation: Nitrogenases, Relevant Chemical Models, and Commercial Processes. 1st Ed. Kluwer Academic Publishers, Dordretch, The Netherlands2004: 219-253Crossref Google Scholar, 8Dos Santos P.C. Dean D.R. Hu Y. Ribbe M.W. Chem. Rev. 2004; 104: 1159-1174Crossref PubMed Scopus (167) Google Scholar). 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 (20Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar, 21Allen R.M. Chatterjee R. Ludden P.W. Shah V.K. J. Biol. Chem. 1995; 270: 26890-26896Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 22Curatti L. Ludden P.W. Rubio L.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5297-5301Crossref PubMed Scopus (82) Google Scholar). 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 (20Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar). 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 (21Allen R.M. Chatterjee R. Ludden P.W. Shah V.K. J. Biol. Chem. 1995; 270: 26890-26896Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). 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 (20Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar). 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) (7Ludden P.W. Rangaraj P. Rubio L.M. Smith B.E. Richards R.L. Newton W.E. Catalysts for Nitrogen Fixation: Nitrogenases, Relevant Chemical Models, and Commercial Processes. 1st Ed. Kluwer Academic Publishers, Dordretch, The Netherlands2004: 219-253Crossref Google Scholar, 8Dos Santos P.C. Dean D.R. Hu Y. Ribbe M.W. Chem. Rev. 2004; 104: 1159-1174Crossref PubMed Scopus (167) Google Scholar, 9Rubio L.M. Ludden P.W. J. Bacteriol. 2005; 187: 405-414Crossref PubMed Scopus (149) Google Scholar). 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. K. pneumoniae Strains and Growth Conditions—K. pneumoniae strains UN (wild type) and UN1217 (nifN4536::mu) have been previously described (23MacNeil T. MacNeil D. Roberts G.P. Supiano M.A. Brill W.J. J. Bacteriol. 1978; 136: 253-266Crossref PubMed Google Scholar). 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 (20Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar). 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) (24Nagatani H.H. Shah V.K. Brill W.J. J. Bacteriol. 1974; 120: 697-701Crossref PubMed Google Scholar) was cultivated in 20-liter carboys and derepressed for nitrogenase expression as described before (25Shah V.K. Davis L.C. Brill W.J. Biochim. Biophys. Acta. 1972; 256: 498-511Crossref PubMed Scopus (98) Google Scholar). Escherichia coli DH5α, 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 (26Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). Plasmid Constructions and DNA Manipulations—Plasmid constructions, PCR, and transformation of E. coli were carried out by standard methods (26Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). 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 DNAeasy™ Tissue Kit (Qiagen). Procedures for K. pneumoniae transformation (27Shokolenko I.N. Alexeyev M.F. BioTechniques. 1995; 18: 596-598PubMed Google Scholar), conjugation, and gene replacement (28Walker K.A. Miller V.L. J. Bacteriol. 2004; 186: 4056-4066Crossref PubMed Scopus (56) Google Scholar) 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 (ΔnifU), pRHB289 (ΔnifS), and pRHB290 (ΔnifUS), respectively. In pRHB288 (ΔnifU), 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 (ΔnifS), 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 (ΔnifUS), 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 (ΔnifU), pRHB289 (ΔnifS), or pRHB290 (ΔnifUS) 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 ΔnifU, ΔnifS, and ΔnifUS Mutant Strains—To perform genetic complementation analysis of ΔnifU and ΔnifS mutants, strains UC1, UC2, and UC3 were transformed with plasmid pRHB257 according to (27Shokolenko I.N. Alexeyev M.F. BioTechniques. 1995; 18: 596-598PubMed Google Scholar). 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) (29Hernandez J.A. Igarashi R.Y. Soboh B. Curatti L. Dean D.R. Ludden P.W. Rubio L.M. Mol. Microbiol. 2007; 63: 177-192Crossref PubMed Scopus (47) Google Scholar). 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 (30Philippe N. Alcaraz J.P. Coursange E. Geiselmann J. Schneider D. Plasmid. 2004; 51: 246-255Crossref PubMed Scopus (272) Google Scholar), to generate plasmid pRHB292. Plasmid pRHB292 was transferred to strains UN (wild type), UN1217 (nifN::mu), and UC3 (ΔnifUS 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 (20Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar). 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 (ΔnifB). Plasmid pRHB235 was transferred to strains UN (wild type), UN1217 (nifN::mu), and UC3 (ΔnifUS 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 ΔnifENX 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 ΔnifENX 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 2× 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 × 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 (31Stewart W.P.D. Fitzgerald G.P. Burris R.H. Proc. Natl. Acad. Sci. U. S. A. 1967; 58: 2071-2078Crossref PubMed Scopus (674) Google Scholar). NifDK activity in cell-free extracts was obtained after titration with an excess of the complementary component, NifH, as described (32Shah V.K. Brill W.J. Biochim. Biophys. Acta. 1973; 305: 445-454Crossref PubMed Scopus (105) Google Scholar). 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 (33Shah V.K. Brill W.J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3249-3253Crossref PubMed Scopus (466) Google Scholar) and NifB-co (20Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar) have been described previously. Preparation of crude NifB-co extracts was carried out according to previous study (20Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar) 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 (20Shah V.K. Allen J.R. Spangler N.J. Ludden P.W. J. Biol. Chem. 1994; 269: 1154-1158Abstract Full Text PDF PubMed Google Scholar) 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 (≈3" @default.
• W2075568922 created "2016-06-24" @default.
• W2075568922 creator A5056059561 @default.
• W2075568922 creator A5082473561 @default.
• W2075568922 creator A5083160018 @default.
• W2075568922 date "2007-12-01" @default.
• W2075568922 modified "2023-10-01" @default.
• W2075568922 title "Evidence for nifU and nifS Participation in the Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase" @default.
• W2075568922 cites W1538196126 @default.
• W2075568922 cites W1583466237 @default.
• W2075568922 cites W1600977202 @default.
• W2075568922 cites W1755858263 @default.
• W2075568922 cites W1761113810 @default.
• W2075568922 cites W1978798351 @default.
• W2075568922 cites W1979073262 @default.
• W2075568922 cites W1982670116 @default.
• W2075568922 cites W1984677796 @default.
• W2075568922 cites W1985844569 @default.
• W2075568922 cites W1986546695 @default.
• W2075568922 cites W1995147696 @default.
• W2075568922 cites W1996929743 @default.
• W2075568922 cites W2006594847 @default.
• W2075568922 cites W2010961029 @default.
• W2075568922 cites W2021835639 @default.
• W2075568922 cites W2023884389 @default.
• W2075568922 cites W2027451689 @default.
• W2075568922 cites W2040412213 @default.
• W2075568922 cites W2048600400 @default.
• W2075568922 cites W2051499713 @default.
• W2075568922 cites W2061431807 @default.
• W2075568922 cites W2062424661 @default.
• W2075568922 cites W2065562514 @default.
• W2075568922 cites W2066510550 @default.
• W2075568922 cites W2069005428 @default.
• W2075568922 cites W2078678839 @default.
• W2075568922 cites W2082017984 @default.
• W2075568922 cites W2085243286 @default.
• W2075568922 cites W2085296510 @default.
• W2075568922 cites W2087921651 @default.
• W2075568922 cites W2094926184 @default.
• W2075568922 cites W2096808921 @default.
• W2075568922 cites W2097397685 @default.
• W2075568922 cites W2100837269 @default.
• W2075568922 cites W2116472408 @default.
• W2075568922 cites W2130252251 @default.
• W2075568922 cites W2142756014 @default.
• W2075568922 cites W2152757065 @default.
• W2075568922 cites W2159216181 @default.
• W2075568922 cites W2162956402 @default.
• W2075568922 cites W4232534952 @default.
• W2075568922 doi "https://doi.org/10.1074/jbc.m708097200" @default.
• W2075568922 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17959596" @default.
• W2075568922 hasPublicationYear "2007" @default.
• W2075568922 type Work @default.
• W2075568922 sameAs 2075568922 @default.
• W2075568922 citedByCount "45" @default.
• W2075568922 countsByYear W20755689222012 @default.
• W2075568922 countsByYear W20755689222013 @default.
• W2075568922 countsByYear W20755689222014 @default.
• W2075568922 countsByYear W20755689222015 @default.
• W2075568922 countsByYear W20755689222016 @default.
• W2075568922 countsByYear W20755689222017 @default.
• W2075568922 countsByYear W20755689222018 @default.
• W2075568922 countsByYear W20755689222019 @default.
• W2075568922 countsByYear W20755689222020 @default.
• W2075568922 countsByYear W20755689222021 @default.
• W2075568922 countsByYear W20755689222022 @default.
• W2075568922 countsByYear W20755689222023 @default.
• W2075568922 crossrefType "journal-article" @default.
• W2075568922 hasAuthorship W2075568922A5056059561 @default.
• W2075568922 hasAuthorship W2075568922A5082473561 @default.
• W2075568922 hasAuthorship W2075568922A5083160018 @default.
• W2075568922 hasBestOaLocation W20755689221 @default.
• W2075568922 hasConcept C141280058 @default.
• W2075568922 hasConcept C178790620 @default.
• W2075568922 hasConcept C179104552 @default.
• W2075568922 hasConcept C181199279 @default.
• W2075568922 hasConcept C181440489 @default.
• W2075568922 hasConcept C185592680 @default.
• W2075568922 hasConcept C197957613 @default.
• W2075568922 hasConcept C537208039 @default.
• W2075568922 hasConcept C549387045 @default.
• W2075568922 hasConcept C553450214 @default.
• W2075568922 hasConcept C55493867 @default.
• W2075568922 hasConceptScore W2075568922C141280058 @default.
• W2075568922 hasConceptScore W2075568922C178790620 @default.
• W2075568922 hasConceptScore W2075568922C179104552 @default.
• W2075568922 hasConceptScore W2075568922C181199279 @default.
• W2075568922 hasConceptScore W2075568922C181440489 @default.
• W2075568922 hasConceptScore W2075568922C185592680 @default.
• W2075568922 hasConceptScore W2075568922C197957613 @default.
• W2075568922 hasConceptScore W2075568922C537208039 @default.
• W2075568922 hasConceptScore W2075568922C549387045 @default.
• W2075568922 hasConceptScore W2075568922C553450214 @default.
• W2075568922 hasConceptScore W2075568922C55493867 @default.
• W2075568922 hasIssue "51" @default.
• W2075568922 hasLocation W20755689221 @default.
• W2075568922 hasOpenAccess W2075568922 @default.

• next