Molecular and General Genetics MGG

, Volume 241, Issue 5–6, pp 602–615 | Cite as

Identification of a new class of nitrogen fixation genes in Rhodobacter capsalatus: a putative membrane complex involved in electron transport to nitrogenase

  • Manfred Schmehl
  • Andreas Jahn
  • Andreas Meyer zu Vilsendorf
  • Silke Hennecke
  • Bernd Masepohl
  • Markus Schuppler
  • Martin Marxer
  • Jürgen Oelze
  • Werner Klipp
Original Articles

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.

Key words

Rhodobacter capsulatus rnf genes In vivo and in vitro nitrogenase activities Iron-sulphur proteins Ferredoxins Metronidazole 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Arnold W, Pühler A (1988) A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene 70:171–179Google Scholar
  2. 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 203:715–738Google Scholar
  3. Bali A, Blanco G, Hill S, Kennedy C (1992) Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen. Appl Environ Microbiol 58:1711–1718Google Scholar
  4. Bennett LT, Jacobson MR, Dean DR (1988) Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii. J Biol Chem 263:1364–1369Google Scholar
  5. 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 220:915–931Google Scholar
  6. Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156Google Scholar
  7. 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 223:138–147Google Scholar
  8. 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 763–834Google Scholar
  9. Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mot Biol 179:125–142Google Scholar
  10. Fonstein M, Zheng S, Haselkorn R (1992) Physical map of the genome of Rhodobacter capsulatus SB 1003. J Bacteriol 174:4070–4077Google Scholar
  11. 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 6:1049–1060Google Scholar
  12. Georgiadis MM, Komiya H, Chakrabarti P, Woo D, Kornuc JJ, Rees DC (1992) Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257:1653–1659Google Scholar
  13. 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 266:3294–3299Google Scholar
  14. 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 127:639–645Google Scholar
  15. Hallenbeck PC, Vignais PM (1981) The effect of electron transport inhibitors on nitrogenase activity in the photosynthetic bacterium, Rhodopseudomonas capsulata. FEMS Microbiol Lett 12:15–18Google Scholar
  16. Hallenbeck PC, Jouanneau Y, Vignais PM (1982) Purification and molecular properties of a soluble ferredoxin from Rhodopseudomonas capsulata. Biochim Biophys Acta 681:168–176Google Scholar
  17. von Heijne G (1986) The distribution of positively charged residues in bacterial inner membrane proteins correlates with the transmembrane topology. EMBO J 5:3021–3027Google Scholar
  18. von Heijne G (1988) Transcending the impenetrable: how proteins come to terms with membranes. Biochim Biophys Acta 947:307–333Google Scholar
  19. Hill S, Kavanagh EP (1980) Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae. J Bacteriol 141:470–475Google Scholar
  20. Hübner P, Willison JC, Vignais PM, Bickle TA (1991) Expression of regulatory nif genes in Rhodobacter capsulatus. J Bacteriol 173:2993–2999Google Scholar
  21. 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 171:1017–1027Google Scholar
  22. Joerger RD, Bishop PE (1988) Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii. J Bacteriol 170:1475–1487Google Scholar
  23. 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 18:5284Google Scholar
  24. Kim J, Rees DC (1992) Structural models for the metal centers in the nitrogenase molybdenum-iron protein. Science 257:1677–1682Google Scholar
  25. Klein P, Kanehisa M, DeLisi C (1985) The detection and classification of membrane-spanning proteins. Biochim Biophys Acta 815:468–476Google Scholar
  26. 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 155:477–482Google Scholar
  27. 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 159:233–236Google Scholar
  28. 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 467–474Google Scholar
  29. 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 170:693–699Google Scholar
  30. 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 89:37–46Google Scholar
  31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  32. 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 171:3162–3167Google Scholar
  33. 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 212:27–37Google Scholar
  34. 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 238:369–382Google Scholar
  35. Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560Google Scholar
  36. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  37. 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 171:2591–2598Google Scholar
  38. 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 216:353–363Google Scholar
  39. 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 210:65–77Google Scholar
  40. Morgan TV, Lundell DJ, Burgess BK (1988) Azotobacter vinelandii ferredoxin I: cloning, sequencing, and mutant analysis. J Biol Chem 263:1370–1375Google Scholar
  41. Nieva-Gomez D, Roberts GP, Klevickis S, Brill WJ (1980) Electron transport to nitrogenase in Klebsiella pneumoniae. Proc Natl Acad Sci USA 77:2555–2558Google Scholar
  42. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448Google Scholar
  43. 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 65:269–275Google Scholar
  44. 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 6:1035–1047Google Scholar
  45. Prentki P, Krisch HM (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313Google Scholar
  46. Rao JKM, Argos P (1986) A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta 869:197–214Google Scholar
  47. 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 266:12889–12895Google Scholar
  48. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  49. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  50. 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 171:6218–6226Google Scholar
  51. Schmidt GW, Matlin KS, Chua N-H (1977) A rapid procedure for selective enrichment of photosynthetic electron transport mutants. Proc Natl Acad Sci USA 74:610–614Google Scholar
  52. 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 8:673–684Google Scholar
  53. 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 258:12064–12068Google Scholar
  54. 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 1:784–791Google Scholar
  55. 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 80:161–169Google Scholar
  56. Smith BE, Eady RR (1992) Metalloclusters of the nitrogenases. Eur J Biochem 205:1–15Google Scholar
  57. Staden R (1986) The current status and portability of our sequence handling software. Nucleic Acids Res 14:217–231Google Scholar
  58. Suetsugu Y, Saeki K, Matsubara H (1991) Transcriptional analysis of two Rhodobacter capsulatus ferredoxins by translational fusions to Escherichia coli lacZ. FEBS Lett 292:13–16Google Scholar
  59. Wall JD, Love J, Quinn SP (1984) Spontaneous Nif- mutants of Rhodopseudomonas capsulata. J Bacteriol 159:652–657Google Scholar
  60. Weaver PF, Wall JD, Gest H (1975) Characterization of Rhodopseudomonas capsulata. Arch Microbiol 105:207–216Google Scholar
  61. 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 128:3001–3010Google Scholar
  62. Yakunin AF, Gogotov IN (1983) Properties and regulation of synthesis of two ferredoxins from Rhodopseudomonas capsulata. Biochim Biophys Acta 725:298–308Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Manfred Schmehl
    • 1
  • Andreas Jahn
    • 2
  • Andreas Meyer zu Vilsendorf
    • 1
  • Silke Hennecke
    • 1
  • Bernd Masepohl
    • 1
  • Markus Schuppler
    • 1
  • Martin Marxer
    • 2
  • Jürgen Oelze
    • 2
  • Werner Klipp
    • 1
  1. 1.Lehrstuhl für Genetik, Fakultät für BiologieUniversität BielefeldBielefeldGermany
  2. 2.Institut für Biologie II der Universität FreiburgFreiburg i.Br.Germany

Personalised recommendations