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Archives of Microbiology

, Volume 165, Issue 3, pp 169–178 | Cite as

Bradyrhizobium japonicum possesses two discrete sets of electron transfer flavoprotein genes:fixA, fixB andetfS, etfL

  • Marianne Weidenhaupt
  • Patricia Rossi
  • Christoph Beck
  • Hans-Martin Fischer
  • Hauke Hennecke
Original Paper
  • 161 Downloads

Abstract

A group of four co-regulated genes (fixA, fixB, fixC, fixX) essential for symbiotic nitrogen fixation has been described in several rhizobial species, includingBradyrhizobium japonicum. The complete nucleotide sequence of theB. japonicum fixA, fixB andfixC, genes is reported here. The derived amino acid sequences confirmed the previously noted sequence similarity between FixA and the β-subunit and between FixB and the α-subunit of mammalian andParacoccus denitrificans electron transfer flavoproteins (ETF). Since the classical role of ETF is in β-oxidation of fatty acids, a process unrelated to nitrogen fixation, we rationalized thatB. japonicum ought to contain bona fideetf genes in addition to theetf-like genesfixA andfixB. Therefore, we identified, cloned, sequenced, and transcriptionally analyzed theB. japonicum etfSL genes encoding the β-and α-subunits of ETF. TheetfSL genes, but not thefix genes, are transcribed in aerobically grown cells. An amino acid sequence comparison between all available ETFs and ETF-like proteins revealed the existence of two distinguishable subfamilies. Group I comprises housekeeping ETFs that link acyl-CoA dehydrogenase reactions with the respiratory chain, such as in the fatty acid degradation pathway.B. japonicum EtfS and EtfL clearly belong to this group. Group II contains ETF-like proteins that are synthesized only under certain specific growth conditions and receive electrons from the oxidation of specific substrates. The products of the anaerobically inducedfixA andfixB genes ofB. japonicum are members of that group.B. japonicum is the first example of an organism in which genes for proteins of both groups I and II of the ETF family have been identified.

Key words

Bradyrhizobium japonicum Electron transfer flavoprotein etf Genes fix Genes Nitrogen fixation Phylogenetic tree Protein family 

Abbreviations

ETF

Electron transfer flavoprotein

ETF-QO ETF

ubiquinone oxidoreductase

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References

  1. Arigoni F, Kaminski PA, Hennecke H, Elmerich C (1991) Nucleotide sequence of thefixABC region ofAzorhizobium caulinodans ORS571: similarity of thefixB product with eukaryotic flavoproteins, characterization offixX, and identification ofnifW. Mol Gen Genet 225:514–520PubMedGoogle Scholar
  2. Babst M, Hennecke H, Fischer HM (1996) Two different mechanisms are involved in the heat shock regulation of chaperonin gene expression inBradyrhizobium japonicum. Mol Microbiol (in press)Google Scholar
  3. Bedzyk LA, Escudero KW, Gill RE, Griffin KJ, Frerman FE (1993) Cloning, sequencing and expression of the genes encoding subunits ofParacoccus denitrificans electron transfer flavoprotein. J Biol Chem 268:20211–20217PubMedGoogle Scholar
  4. Bergersen FJ, Turner GL (1990) Bacteriods from soybean root nodules: accumulation of poly-β-hydroxybutyrate during supply of malate and succinate in relation to nitrogen fixation in flow-chamber reactions. Proc R Soc Lond [Biol] 240:39–59Google Scholar
  5. Black PN, DiRusso CC (1994) Molecular and biochemical analysis of fatty acid transport, metabolism, and gene regulation inEscherichia coli. Biochim Biophys Acta 1210:123–145PubMedGoogle Scholar
  6. Brushi M, Guerlesquin F (1988) Structure, function and evolution of bacterial ferredoxins. FEMS Microbiol Rev 54:155–176Google Scholar
  7. Chen D, Swenson RP (1994) Cloning, sequence analysis, and expression of the genes encoding the two subunits of the methylotrophic bacterium W3A1 electron transfer flavoprotein. J Biol Chem 269:32120–32130PubMedGoogle Scholar
  8. Dean DR, Jacobson MR (1992) Biochemical genetics of nitrogenase. In: Stacey G, Burris RH, Evans HJ (ed) Biological nitrogen fixation. Routledge, Chapman & Hall, New York, pp 763–834Google Scholar
  9. Dusha I, Kovalenko S, Banfalvi Z, Kondorosi A (1987)Rhizobium meliloti insertion element ISRm2 and its use for identification of thefixX gene. J Bacteriol 169:1403–1409PubMedGoogle Scholar
  10. Earl CD, Ronson CW, Ausubel FM (1987) Genetic and structural analysis of theRhizobium meliloti fixA, fixB, fixC, andfixX genes. J Bacteriol 169:1127–1136PubMedGoogle Scholar
  11. Eichler K, Bourgis F, Buchet A, Kleber HP, Mandrand-Berthelot MA (1994) Molecular characterization of thecai operon necessary for carnitine metabolism inEscherichia coli. Mol Microbiol 13:775–786PubMedGoogle Scholar
  12. Eichler K, Buchet A, Bourgis F, Kleber HP, Mandrand-Berthelot MA (1995) Thefix Escherichia coli region contains four genes related to carnitine metabolism. J Basic Microbiol 35:217–227PubMedGoogle Scholar
  13. Evans D, Jones R, Woodley P, Robson R (1988) Further analysis of nitrogen fixation (nif) genes inAzotobacter chroococcum: identification and expression inKlebsiella pneumoniae ofnifS, nifV, nifM, andnifB genes and localization ofnifE/N-, nifU-, nifA-andfixABC-like genes. J Gen Microbiol 134:931–942PubMedGoogle Scholar
  14. Feinberg AP, Vogelstein B (1984) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266–267PubMedGoogle Scholar
  15. Finocchiaro G, Ito M, Ikeda Y, Tanaka K (1988) Molecular cloning and nucleotide sequence of cDNAs encoding the α-subunit of human electron transfer flavoprotein. J Biol Chem 263:15773–15780PubMedGoogle Scholar
  16. Finocchiaro G, Colombo I, Garavaglia B, Gellera C, Valdemari G, Garbuglio N, Didonato S (1993) cDNA cloning and mitochondrial import of the β-subunit of the human electron-transfer flavoprotein. Eur J Biochem 213:1003–1008PubMedGoogle Scholar
  17. Fischer H-M (1994) Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev 58:352–386PubMedGoogle Scholar
  18. Fogher C, Dusha I, Barbot P, Elmerich C (1985) Heterologous hybridization ofAzospirillum DNA toRhizobium nod andfix genes. FEMS Microbiol Lett 30:245–249Google Scholar
  19. Frisell WR, MacKenzie CG (1962) Separation and purification of sarcosine dehydrogenase and dimethylglycine dehydrogenase. J Biol Chem 237:94–98PubMedGoogle Scholar
  20. Fuhrmann M, Fischer H-M, Hennecke H (1985) Mapping ofRhiozobium japonicum nifB-, fixBC-, andfixA-like genes and identification of thefixA promoter. Mol Gen Genet 199:315–332Google Scholar
  21. Galimand M, Perroud B, Delorme F, Paquelin A, Vieille C, Bozouklian H, Elmerich C (1989) Identification of DNA regions homologous to nitrogen fixation genesnifE, nifUS andfixABC inAzospirillum brasiliense Sp7. J Gen Microbiol 135:1047–1059PubMedGoogle Scholar
  22. Goodman SI, Axtell KM, Bindoff LA, Beard SE, Gill RE, Frerman FE (1994) Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase. Eur J Biochem 219:277–286PubMedGoogle Scholar
  23. Grönger P, Manian S, Reiländer H, O'Connel M, Priefer UB, Pühler A (1987) Organization and partial sequence of a DNA region of theRhizobium leguminosarum symbiotic plasmid containing the genesfixABC, nifA, nifB and a novel open reading frame. Nucleic Acids Res 15:31–49PubMedGoogle Scholar
  24. Gubler M (1989) Fine-tuning ofnif andfix gene expression by upstream activator sequences inBradyrhizobium japonicum. Mol Microbiol 3:149–159PubMedGoogle Scholar
  25. Gubler M, Hennecke H (1986)fixA, B andC genes are essential for symbiotic and free-living, microaerobic nitrogen fixation. FEBS Lett 200:186–192Google Scholar
  26. Gubler M, Hennecke H (1988) Regulation of thefixA gene andfixBC operon inBradyrhizobium japonicum. J Bacteriol 170: 1205–1214PubMedGoogle Scholar
  27. Gubler M, Zürcher T, Hennecke H (1989) TheBradyrhizobium japonicum fixBCX operon: identification offixX, and a 5′ mRNA region affecting the level of thefixBCX transcript. Mol Microbiol 3:141–148PubMedGoogle Scholar
  28. Hauge JG, Crane FL, Beinert H (1956) On the mechanism of dehydrogenation of fatty acyl derivatives of coenzyme A. J Biol Chem 219:727–733PubMedGoogle Scholar
  29. Husain M, Steenkamp DJ (1985) Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase fromParacoccus denitrificans. J Bacteriol 163: 709–715PubMedGoogle Scholar
  30. Iismaa SE, Watson JM (1987) A gene upstream of theRhizobium trifolii nifA gene encodes a ferredoxin-like protein. Nucleic Acids Res 15:318Google Scholar
  31. Ikeda Y, Tanaka K (1983a) Purification and characterization of isovaleryl coenzyme A dehydrogenase from rat liver mitochondria. J Biol Chem 258:1077–1085PubMedGoogle Scholar
  32. Ikeda Y, Tanaka K (1983b) Purification and properties of 2-methyl branched chain acyl-CoA dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria. J Biol Chem 258:9477–9478PubMedGoogle Scholar
  33. Kaminski PA, Norel F, Desnoues N, Kush A, Salzano G, Elmerich C (1988) Characterization of thefixABC region ofAzorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene. Mol Gen Genet 214:496–502PubMedGoogle Scholar
  34. Kasprazak AA, Steenkamp DJ (1983) Localization of the major dehydrogenases in two methylotrophs by radiochemical labelling. J Bacteriol 156:348–353PubMedGoogle Scholar
  35. Kündig C, Hennecke H, Göttfert M (1993) Correlated physical and genetic map of theBradyrhizobium japonicum 110 genome. J Bacteriol 175:613–622PubMedGoogle Scholar
  36. Leinich AC, Goodman SI (1986) The purification and characterization of glutaryl-CoA dehydrogenase from porcine and human liver. J Biol Chem 261:4090–4096PubMedGoogle Scholar
  37. Loferer H, Bott M, Hennecke H (1993)Bradyrhizobium japonicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochromeaa 3 and development of symbiosis. EMBO J 12:3373–3383PubMedGoogle Scholar
  38. McDermott TR, Griffith SM, Vance CP, Graham PH (1989) Carbon metabolism inBradyrhizobium japonicum bacteroids. FEMS Microbiol Rev 63:327–340Google Scholar
  39. McKie JH, Douglas KT (1991) Evidence for gene duplication forming similar binding folds for NAD(P)H and FAD in pyridine nucleotide-dependent flavoenzymes. FEBS Lett 279:5–8PubMedGoogle Scholar
  40. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78PubMedGoogle Scholar
  41. Michiels J, Vanderleyden J (1993) Cloning and sequence of theRhizobium leguminosarum biovarphaseoli fixA gene. Biochim Biophys Acta 1114:232–233Google Scholar
  42. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  43. Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligonucleotide-directed mutagenesis. Gene 26:101–106PubMedGoogle Scholar
  44. Preisig O, Anthamatten D, Hennecke H (1993) Genes for a microaerobically induced oxidase complex inBradyrhizobium japonicum are essential for a nitrogen-fixing endosymbiosis. Proc Natl Acad Sci USA 90:3309–3313PubMedGoogle Scholar
  45. Pühler A, Aguilar MO, Hynes M, Müller P, Klipp W, Priefer U, Simon R, Weber G (1984) Advances in the genetics of free-living and symbiotic nitrogen fixing bacteria. In: Veeger C, Newton WE (eds) Advances in nitrogen fixation research. Nijhoff, Dordrecht, pp 609–619Google Scholar
  46. Regensburger B, Hennecke H (1983) RNA polymerase fromRhizobium japonicum. Arch Microbiol 135:103–109PubMedGoogle Scholar
  47. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  48. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 83:765–773Google Scholar
  49. Schinzawa K, Inagaki T, Ohishi H, Ichihara C, Tsukagoshi N, Ukada S, Yagi K (1988) Molecular cloning of a cDNA for the α-subunit of rat liver electron transfer flavoprotein. Biochem Biophys Res Commun 155:300–304PubMedGoogle Scholar
  50. Szeto WW, Zimmerman JL, Sundaresan V, Ausubel FM (1984) ARhizobium meliloti symbiotic regulatory gene. Cell 36:1035–1043PubMedGoogle Scholar
  51. Thöny-Meyer L, Preisig O, Zufferey R, Hennecke H (1995) The role of a microaerobically inducedcb-type cytochrome oxidase in symbiotic nitrogen fixation. In: Tikhonovich IA, Provorov NA, Romanov VI, Newton WE (eds) Nitrogen fixation: Fundamentals and applications. Kluwer, Dordrecht, pp 383–388Google Scholar
  52. Tsai MH, Saier MH (1995) Phylogenetic characterization of the ubiquitous electron transfer flavoprotein families ETF-α and ETF-β. Res Microbiol 146:397–404PubMedGoogle Scholar
  53. Watmough NJ, Kiss J, Frerman FE (1992) Structural and redox relationships betweenParacoccus denitrificans, porcine and human electron-transferring flavoproteins. Eur J Biochem 205:1089–1097PubMedGoogle Scholar
  54. Weidenhaupt M (1996) In the search for new NifA-dependent genes and their function inBradyrhizobium japonicum. PhD thesis No. 11411, Federal Institute of Technology, ZürichGoogle Scholar
  55. Yanagi M, Yanmasato K (1993) Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiol Lett 107:115–120PubMedGoogle Scholar
  56. Young JPW, Downer HL, Eardly BD (1991) Phylogeny of the phototrophicRhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J Bacteriol 173:2271–2277PubMedGoogle Scholar
  57. Youngleson JS, Jones DT, Woods DR (1989) Homology between hydroxybutyryl and hydroxyacyl coenzyme A dehydrogenase enzymes fromClostridium acetobutylicum fermentation and vertebrate fatty acid β-oxidation. J Bacteriol 171:6800–6807PubMedGoogle Scholar
  58. Yura T, Mori H, Hagai H, Nagat T, Isihama A, Fujita N, Isono K, Mizobushi K, Nakata A (1992) Systematic sequencing of theEscherichia coli genome: analysis of the 0–2.4 min region. Nucleic Acids Res 20:3305–3308PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • Marianne Weidenhaupt
    • 1
  • Patricia Rossi
    • 1
  • Christoph Beck
    • 1
  • Hans-Martin Fischer
    • 1
  • Hauke Hennecke
    • 1
  1. 1.Mikrobiologisches Institut, Eidgenössische Technische HochschuleETH-ZentrumZürichSwitzerland

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