Abstract
DNA sequence analysis of a 12236 by fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames. With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins. Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation. Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation). Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe-4S] proteins. Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase. In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase. The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole. Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Arnold W, Pühler A (1988) A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene 70:171–179
Arnold W, Rump A, Klipp W, Priefer UB, Puhler A (1988) Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J Mol Biol 203:715–738
Bali A, Blanco G, Hill S, Kennedy C (1992) Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen. Appl Environ Microbiol 58:1711–1718
Bennett LT, Jacobson MR, Dean DR (1988) Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii. J Biol Chem 263:1364–1369
Cannon W, Charlton W, Buck M (1991) Organization and function of binding sites for the transcriptional activator NifA in the Klebsiella pneumoniae nifE and nifU promoters. J Mol Biol 220:915–931
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Colonna-Romano S, Arnold W, Schlüter A, Boistard P, Pühler A, Priefer UB (1990) An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK. Mol Gen Genet 223:138–147
Dean DR, Jacobson MR (1992) Biochemical genetics of nitrogenase. In: Stacey G, Burris RH, Evans HJ (eds) Biological Nitrogen Fixation. Chapman and Hall, New York, pp 763–834
Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mot Biol 179:125–142
Fonstein M, Zheng S, Haselkorn R (1992) Physical map of the genome of Rhodobacter capsulatus SB 1003. J Bacteriol 174:4070–4077
Foster-Hartnett D, Kranz RG (1992) Analysis of the promoters and upstream sequences of nifAl and nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN. Mot Microbiol 6:1049–1060
Georgiadis MM, Komiya H, Chakrabarti P, Woo D, Kornuc JJ, Rees DC (1992) Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257:1653–1659
Grabau C, Schatt E, Jouanneau Y, Vignais PM (1991) A new [2Fe602-92S] ferredoxin from Rhodobacter capsulatus. Coexpression with a 2[4Fe602-104S] ferredoxin in Escherichia coli. J Biol Chem 266:3294–3299
Haaker H, Laane C, Hellingwerf K, Houwer B, Konings WN, Veeger C (1982) Short-term regulation of the nitrogenase activity in Rhodopseudomonas sphaeroides. Eur J Biochem 127:639–645
Hallenbeck PC, Vignais PM (1981) The effect of electron transport inhibitors on nitrogenase activity in the photosynthetic bacterium, Rhodopseudomonas capsulata. FEMS Microbiol Lett 12:15–18
Hallenbeck PC, Jouanneau Y, Vignais PM (1982) Purification and molecular properties of a soluble ferredoxin from Rhodopseudomonas capsulata. Biochim Biophys Acta 681:168–176
von Heijne G (1986) The distribution of positively charged residues in bacterial inner membrane proteins correlates with the transmembrane topology. EMBO J 5:3021–3027
von Heijne G (1988) Transcending the impenetrable: how proteins come to terms with membranes. Biochim Biophys Acta 947:307–333
Hill S, Kavanagh EP (1980) Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae. J Bacteriol 141:470–475
Hübner P, Willison JC, Vignais PM, Bickle TA (1991) Expression of regulatory nif genes in Rhodobacter capsulatus. J Bacteriol 173:2993–2999
Jacobson MR, Brigle KE, Bennett LT, Setterquist RA, Wilson MS, Cash VL, Beynon J, Newton WE, Dean DR (1989) Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J Bacteriol 171:1017–1027
Joerger RD, Bishop PE (1988) Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii. J Bacteriol 170:1475–1487
Jouanneau Y, Richaud P, Grabau C (1990) The nucleotide sequence of a flavodoxin-like gene which precedes two ferredoxin genes in Rhodobacter capsulatus. Nucleic Acids Res 18:5284
Kim J, Rees DC (1992) Structural models for the metal centers in the nitrogenase molybdenum-iron protein. Science 257:1677–1682
Klein P, Kanehisa M, DeLisi C (1985) The detection and classification of membrane-spanning proteins. Biochim Biophys Acta 815:468–476
Klein G, Klipp W, Jahn A, Steinborn B, Oelze J (1991) The relationship of biomass, polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus. Arch Microbiol 155:477–482
Klein G, Jahn A, Dörffler M, Oelze J (1993) Activity and expression of nitrogenase in Rhodobacter capsulatus under aerobiosis in the dark and in the light. Arch Microbiol 159:233–236
Klipp W (1990) Organization and regulation of nitrogen fixation genes in Rhodobacter capsulatus. In: Gresshoff PM, Roth LE, Stacey G, Newton WE (eds) Nitrogen fixation: Achievements and objectives. Chapman and Hall, New York London, pp 467–474
Klipp W, Masepohl B, Pühler A (1988) Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region. J Bacteriol 170:693–699
Labes M, Pühler A, Simon R (1990) A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for Gram-negative bacteria. Gene 89:37–46
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Martin AE, Burgess BK, Iismaa SE, Smartt CT, Jacobson MR, Dean DR (1989) Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I. J Bacteriol 171:3162–3167
Masepohl B, Klipp W, Pühler A (1988) Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus. Mol Gen Genet 212:27–37
Masepohl B, Angermüller S, Hennecke S, Hübner P, Moreno-Vivian C, Klipp W (1993) Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifU ISVW gene region: possible role of NifW in homocitrate processing. Mot Gen Genet 238:369–382
Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Moreno-Vivian C, Hennecke S, Pühler A, Klipp W (1989a) Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus. J Bacteriol 171:2591–2598
Moreno-Vivian C, Schmehl M, Masepohl B, Arnold W, Klipp W (1989b) DNA sequence and genetic analysis of the Rhodobacter capsulatus nifENX gene region: homology between NifX and NifB suggests involvement of NifX in processing of the ironmolybdenum cofactor. Mol Gen Genet 216:353–363
Morett E, Buck M (1989) In vivo studies on the interaction of RNA polymerase-σ54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. J Mol Biol 210:65–77
Morgan TV, Lundell DJ, Burgess BK (1988) Azotobacter vinelandii ferredoxin I: cloning, sequencing, and mutant analysis. J Biol Chem 263:1370–1375
Nieva-Gomez D, Roberts GP, Klevickis S, Brill WJ (1980) Electron transport to nitrogenase in Klebsiella pneumoniae. Proc Natl Acad Sci USA 77:2555–2558
Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448
Pollock D, Bauer CE, Scolnik PA (1988) Transcription of the Rhodobacter capsulatus nifHDK operon is modulated by the nitrogen source. Construction of plasmid expression vectors based on the nifHDK promoter. Gene 65:269–275
Preker P, Hübner P, Schmehl M, Klipp W, Bickle TA (1992) Mapping and characterization of the promoter elements of the regulatory nif genes rpoN, nifA1 and nifA2 in Rhodobacter capsulatus. Mol Microbiol 6:1035–1047
Prentki P, Krisch HM (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313
Rao JKM, Argos P (1986) A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta 869:197–214
Saeki K, Suetsugu Y, Tokuda K-i, Miyatake Y, Young DA, Marrs BL, Matsubara H (1991) Genetic analysis of functional differences among distinct ferredoxins in Rhodobacter capsulatus. J Biol Chem 266:12889–12895
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schatt E, Jouanneau Y, Vignais PM (1989) Molecular cloning and sequence analysis of the structural gene of ferredoxin I from the photosynthetic bacterium Rhodobacter capsulatus. J Bacteriol 171:6218–6226
Schmidt GW, Matlin KS, Chua N-H (1977) A rapid procedure for selective enrichment of photosynthetic electron transport mutants. Proc Natl Acad Sci USA 74:610–614
Schüddekopf K, Hennecke S, Liese U, Kutsche M, Klipp W (1993) Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus. Mol Microbiol 8:673–684
Shah VK, Stacey G, Brill WJ (1983) Electron transport to nitrogenase. Purification and characterization of pyruvate:flavodoxin oxidoreductase, the nifJ gene product. J Biol Chem 258:12064–12068
Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784–791
Simon R, Quandt J, Klipp W (1989) New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram negative bacteria. Gene 80:161–169
Smith BE, Eady RR (1992) Metalloclusters of the nitrogenases. Eur J Biochem 205:1–15
Staden R (1986) The current status and portability of our sequence handling software. Nucleic Acids Res 14:217–231
Suetsugu Y, Saeki K, Matsubara H (1991) Transcriptional analysis of two Rhodobacter capsulatus ferredoxins by translational fusions to Escherichia coli lacZ. FEBS Lett 292:13–16
Wall JD, Love J, Quinn SP (1984) Spontaneous Nif- mutants of Rhodopseudomonas capsulata. J Bacteriol 159:652–657
Weaver PF, Wall JD, Gest H (1975) Characterization of Rhodopseudomonas capsulata. Arch Microbiol 105:207–216
Willison JC, Vignais PM (1982) The use of metronidazole to isolate Nif− mutants of Rhodopseudomonas capsulata, and the identification of a mutant with altered regulatory properties of nitrogenase. J Gen Microbiol 128:3001–3010
Yakunin AF, Gogotov IN (1983) Properties and regulation of synthesis of two ferredoxins from Rhodopseudomonas capsulata. Biochim Biophys Acta 725:298–308
Author information
Authors and Affiliations
Additional information
Communicated by A. Kondorosi
Rights and permissions
About this article
Cite this article
Schmehl, M., Jahn, A., Meyer zu Vilsendorf, A. et al. Identification of a new class of nitrogen fixation genes in Rhodobacter capsalatus: a putative membrane complex involved in electron transport to nitrogenase. Molec. Gen. Genet. 241, 602–615 (1993). https://doi.org/10.1007/BF00279903
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00279903