Archives of Microbiology

, Volume 194, Issue 1, pp 3–11 | Cite as

Phylogenetic perspectives of nitrogen-fixing actinobacteria

  • Maher GtariEmail author
  • Faten Ghodhbane-Gtari
  • Imen Nouioui
  • Nicholas Beauchemin
  • Louis S. Tisa


It was assumed for a long time that the ability to catalyze atmospheric nitrogen (diazotrophy) has a narrow distribution among actinobacteria being limited to the genus Frankia. Recently, the number of nitrogen fixation (nifH) genes identified in other non-Frankia actinobacteria has dramatically increased and has opened investigation on the origin and emergence of diazotrophy among actinobacteria. During the last decade, Mycobacterium flavum, Corynebacterium autotrophicum and a fluorescent Arthrobacter sp. have been reported to have nitrogenase activity, but these studies have not been further verified. Additional reports of nitrogen fixation by Agromyces, Microbacterium, Corynebacterium and Micromonospora isolated from root nodules of leguminous and actinorhizal plants have increased. For several actinobacteria, nitrogen fixation was demonstrated by the ability to grow on nitrogen-free medium, acetylene reduction activity, 15N isotope dilution analysis and identification of a nifH gene via PCR amplification. Moreover, the analyses of draft genome sequences of actinobacteria including Slackia exigua, Rothia mucilaginosa and Gordonibacter pamelaeae have also revealed the presence of nifH-like sequences. Whether these nifH sequences are associated with effective nitrogen fixation in these actinobacteria taxa has not yet been demonstrated. These genes may be vertically or horizontally transferred and be silent sequences. These ideas merit further investigation. This minireview presents a phylogenetic comparison of nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of this catalytic ability among actinobacteria.


Actinobacteria Diazotrophy Phylogeny 



This work was supported in part by the Tunisian Ministry of High Education and Research (MG, FGG and IN). LST and NB were supported in part by NH530 and the College of Life Science and Agriculture, the University of New Hampshire.

Supplementary material

203_2011_733_MOESM1_ESM.pdf (9 kb)
Supplemental Figure 1. Concatenated phylogenetic affiliation of 110 different maximum parsimony trees for amino acid sequences of orthologs among all the genomes. The values at each branch point are the percent number of parsimony trees that displayed the same phylogeny. For each ortholog set, Clustal W (Larkin 2007) was used to align the sequences and a maximum parsimony tree was created using PHYLIP (Felsenstein 1989). (PDF 9 kb)
203_2011_733_MOESM2_ESM.pdf (9 kb)
Supplemental Figure 2. Phylogenetic affiliation of nifH amino acid sequences retrieved from Frankia and non-Frankia genomes used in Supplemental Figure 1. nifH sequences were aligned using Clustal W (Larkin et al., 2007). Maximum parsimony tree was constructed using MEGA version 5 with a 500 Bootstrap test (Tamura et al., 2011). (PDF 9 kb)


  1. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W (1997) Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Baranova NA, Gogotov IN (1974) Fixation of molecular nitrogen by propionic bacteria. Mikrobiologiya 43:791–794 (in Russian)Google Scholar
  3. Benson DR, Silvester WB (1993) Biology of Frankia strains, actinomycete symbionts of actinorhizal plants. Microbiol Rev 57:293–319PubMedGoogle Scholar
  4. Bergan T, Et Kocur M (1982) Stomatococcus mucilaginosus gen. nov., sp. nov., ep. rev., a member of the family Micrococcaceae. Int J Syst Bacteriol 32:374–377CrossRefGoogle Scholar
  5. Berman-Frank I, Lundgren P, Falkowski P (2003) Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res Microbiol 154:157–164PubMedCrossRefGoogle Scholar
  6. Berndt H, Lowe DJ, Yates MG (1978) The nitrogen-fixing system of Corynebacterium autotrophicum. Purification and properties of the nitrogenase components and two ferredoxins. Eur J Biochem 86:133–142PubMedCrossRefGoogle Scholar
  7. Berry AM, Harriott OT, Moreau RA, Osman SF, Benson DR, Jones AD (1993) Hopanoid lipids compose the Frankia vesicle envelope, presumptive barrier of oxygen diffusion to nitrogenase. Proc Natl Acad Sci USA 90:6091–6094PubMedCrossRefGoogle Scholar
  8. Biggins DR, Postgate JR (1969) Nitrogen fixation by cultures and cell-free extracts of Mycobacterium 301. J Gen Microbiol 56:181–193PubMedGoogle Scholar
  9. Biggins DR, Kelly M, Postgate JR (1971) Resolution of nitrogenase of Mycobacterium flavum 301 into two components and cross reaction with nitrogenase components from other bacteria. Europ J Biochem 20:140–143PubMedCrossRefGoogle Scholar
  10. Bowers TH, Reid NM, Lloyd-Jones G (2008) Composition of nifH in a wastewater treatment system reliant on N2 fixation. Appl Microbiol Biotech 79:811–818CrossRefGoogle Scholar
  11. Buckley DH, Huangyutitham V, Hsu SF, Nelson TA (2007) Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil. Appl Environ Microbiol 73:3196–3204PubMedCrossRefGoogle Scholar
  12. Cacciari I, Lippi D, Bordeleau LM (1979) Effect of oxygen on batch and continuous cultures of a nitrogen-fixing Arthrobacter sp. Can J Microbiol 25:746–751PubMedCrossRefGoogle Scholar
  13. Collins MD, Hutson RA, Båverud V, Falsen E (2000) Characterization of a Rothia-like organism from a mouse: description of Rothia nasimurium sp. nov. and reclassification of Stomatococcus mucilaginosus as Rothia mucilaginosa comb. nov. Int J Syst Evol Microbiol 50:1247–1251PubMedCrossRefGoogle Scholar
  14. Dilworth MJ (1966) Acetylene reduction by nitrogen fixing preparations from Clostridium pasteurianum. Biochem Biophys Acta 127:285–294PubMedCrossRefGoogle Scholar
  15. Ding J, Sun H, Su F, Xu Q, Huang Y, Ling P (1981) Studies on nitrogen fixation by actinomycetes. Acta Microbiol Sin 21:424–427Google Scholar
  16. Dixon R, Kahn D (2004) Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol 2:621–631PubMedCrossRefGoogle Scholar
  17. Fani R, Pini F (2006) Molecular characterization of Antarctic nitrogen fixing bacteria. Direct sequence submission to the NCBI GenBank database recordGoogle Scholar
  18. Fani R, Gallo R, Lio P (2000) Molecular evolution of nitrogen fixation: the evolutionary history of the nifD, nifK, nifE, and nifN genes. J Mol Evol 51:1–11PubMedGoogle Scholar
  19. Fedorov MV, Kalininskaya TA (1961) A new species of nitrogen fixing Mycobacterium and its physiological properties (English translation). Mikrobiologiya 30:7–11Google Scholar
  20. Felsenstein J (1989) PHYLIP—phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  21. Fusconi M, Conti C, De Virgilio A, de Vincentiis M (2009) Paucisymptomatic pneumonia due to Rothia mucilaginosa: case report and literature review. Infez Med 17:100–104PubMedGoogle Scholar
  22. Gadkari D, Mörsdorf G, Meyer O (1992) Chemolithoautotrophic assimilation of dinitrogen by Streptomyces thermoautotrophicus UBT1: identification of an unusual N2-fixing system. J Bacteriol 174:6840–6843PubMedGoogle Scholar
  23. Ghodhbane-Gtari F, Essoussi I, Chattaoui M, Chouaia B, Jaouani A, Daffonchio D, Boudabous A, Gtari M (2010) Isolation and characterization of non-Frankia actinobacteria from root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia. Symbiosis 50:51–57CrossRefGoogle Scholar
  24. Giri S, Pati BR (2004) A comparative study on phyllosphere nitrogen fixation by newly isolated Corynebacterium sp. Flavobacterium sp. and their potentialities as biofertilizer. Acta Microbiol Immunol Hung 51:47–56PubMedCrossRefGoogle Scholar
  25. Gonzalez-Ruiz T, Rodriguez-Zaragoza S, Cerritos R, Falcon LI, Souza V (2006) Microbacterium sp., a free living actinobacteria able to aerobically fix nitrogen from a desert soil. Direct sequence submission to the NCBI GenBank database recordGoogle Scholar
  26. Gtari M, Brusetti L, Hassen A, Mora D, Daffonchio D, Boudabous A (2007) Genetic diversity among Elaeagnus compatible Frankia strains and sympatricrelated nitrogen-fixing actinobacteria revealed by nifH sequence analysis. Soil Biol Biochem 39:372–377CrossRefGoogle Scholar
  27. Guillén GM, Valdés M, Liao J, Hirsch AM (1993) Identificacion de actinobacterias aisladas de nodulos de Casuarina, por tecnicas tradicionales y moleculares. Rev Lat Am Microbiol 35:195–200Google Scholar
  28. Harriott OT, Hosted TJ, Benson DR (1995) Sequences of nifX, nifW, nifZ, nifB and two ORF in the Frankia nitrogen fixation gene cluster. Gene 161:63–67PubMedCrossRefGoogle Scholar
  29. Hartmann LS, Barnum SR (2010) Inferring the evolutionary history of Mo-dependent nitrogen fixation from phylogenetic studies of nifK and nifDK. J Mol Evol 71:70–85PubMedCrossRefGoogle Scholar
  30. Hennecke H, Kaluza K, Thöny M, Fuhrmann M, Ludwig W, Stackebrandt E (1985) Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen fixing bacteria. Arch Microbiol 142:342–348CrossRefGoogle Scholar
  31. Hu Y, Fay AW, Lee CC, Ribbe MW (2007) P-cluster maturation on nitrogenase MoFe protein. Proc Natl Acad Sci USA 104:10424–10429PubMedCrossRefGoogle Scholar
  32. Huss-Danell K (1997) Actinorhizal symbioses and their N2 fixation. New Phytol 136:375–405CrossRefGoogle Scholar
  33. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWillam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  34. Lerat E, Daubin V, Ochman H, Moran N (2005) Evolutionary origins of genomic repertoires in bacteria. PLoS Biology 3:807–813CrossRefGoogle Scholar
  35. MacRae IC (1977) Influence of partial pressures of acetylene and nitrogen upon nitrogenase activity of species of Beijerinckia. Aust J Biol Sci 30:593–596PubMedGoogle Scholar
  36. Mahendra S, Alvarez-Cohen L (2005) Pseudonocardia dioxanivorans sp. nov., a novel. Actinomycete that grows on 1,4-dioxane. Int J Syst Evol Microbiol 55:593–598PubMedCrossRefGoogle Scholar
  37. Mahl MC, Wilson PW, Fife MA, Ewing WH (1965) Nitrogen fixation by members of the tribe Klebsielleae. J Bacteriol 89:1482–1487PubMedGoogle Scholar
  38. Martinez-Romero E (2006) Dinitrogen-fixing prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes, vol 1, pp 793–817Google Scholar
  39. Merrick MJ, Edwards RA (1995) Nitrogen control in bacteria. Microbiol Rev 59:604–622PubMedGoogle Scholar
  40. Newton WE (2007) Physiology, biochemistry and molecular biology of nitrogen fixation. In: Bothe H, Ferguson SJ, Newton WE (eds) Biology of the nitrogen cycle. Elsevier, Amsterdam, pp 109–130CrossRefGoogle Scholar
  41. Normand P, Bousquet J (1989) Phylogeny of nitrogenase sequences in Frankia and other nitrogen-fixing microorganisms. J Mol Evol 29:436–447PubMedCrossRefGoogle Scholar
  42. Normand P, Simonet P, Bardin R (1988) Conservation of nif sequences in Frankia. Mol Gen Genet 213:238–246PubMedCrossRefGoogle Scholar
  43. Normand P, Lapierre P, Tisa LS, Gogarten JP, Alloisio N, Cournoyer B, Lavire C, Marechal J, Pujic P, Huang Y, Mastronunzio J, Bickhard D, Bassi C, Rawnsley T, Niemann J, Francino MP, Lapidus A, Martinez M, Goltsman E, Perriere G, Medigue C, Choisne N, Couloux A, Cruveiller S, Labarre L, Rouy Z, Vallenet D, Cong CT, Demange YN, Mullin B, Kopp O, Wang Y, Tomkins J, Berry A, Sellstedt A, Tavares F, Valverde C, Wall L, Benson DR (2007a) Genome characteristics of three facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography. Genome Res 17:7–15PubMedCrossRefGoogle Scholar
  44. Normand P, Queriroux C, Tisa LS, Benson DR, Rouy Z, Médique C (2007b) Exploring the genomes of Frankia. Physiol Plant 130:331–343CrossRefGoogle Scholar
  45. Pankratov TA, Dedysh SN (2009) Cellulolytic Streptomycetes from sphagnum peat bogs and factors controlling their activity. Microbiology 78:227–233CrossRefGoogle Scholar
  46. Perrine-Walker F, Gherbi H, Imanishi L, Hocher V, Ghodhbane-Gtari F, Lavenus J, Benabdoun FM, Nambiar-Veetil M, Svistoonoff S, Laplaze L (2011) Symbiotic signaling in actinorhizal symbioses. Curr Protein Pept Sci 12:156–164PubMedGoogle Scholar
  47. Poco SE Jr, Nakazawa F, Ikeda T, Sato M, Sato T, Hoshino E (1996) Eubacterium exiguum sp. nov., isolated from human oral lesions. Int J Syst Bacteriol 46:1120–1124PubMedCrossRefGoogle Scholar
  48. Postgate JR (1982) The fundamentals of nitrogen fixation. Cambridge University Press, CambridgeGoogle Scholar
  49. Postgate JR, Eady RR (1988) The evolution of biological nitrogen fixation. In: Bothe H, De Bruijn FJ, Newton WE (eds) Nitrogen fixation: hundred years after. Gustav Fischer, Stuttgart, Germany, pp 31–40Google Scholar
  50. Rao VR (1973) Non-symbiotic nitrogen fixation in paddy fields. Doctoral thesis, Academy of Sciences USSR, Moscow. In: Subba Rao GV (ed) Soil microorganisms and plant growth. Mohan Primlani for Oxford & IBH Publishing, New Delhi, pp 82–97Google Scholar
  51. Raymond J, Siefert JL, Staples CR, Blankenship RE (2004) The natural history of nitrogen fixation. Mol Biol Evol 21:541–554PubMedCrossRefGoogle Scholar
  52. Ribbe M, Gadkari D, Meyer O (1997) N2 fixation by Streptomyces thermoautotrophicus involves a molybdenum-dinitrogenase and a manganese-superoxide oxidoreductase that couple N2 reduction to the oxidation of superoxide produced from O2 by a molybdenum-CO dehydrogenase. J Biol Chem 272:26627–26633PubMedCrossRefGoogle Scholar
  53. Rubio LM, Ludden PW (2005) Maturation of nitrogenase: a biochemical puzzle. J Bacteriol 187:405–414PubMedCrossRefGoogle Scholar
  54. Rubio LM, Ludden PW (2008) Biosynthesis of the iron molybdenum cofactor of nitrogenase. Annu Rev Microbiol 62:93–111PubMedCrossRefGoogle Scholar
  55. Ruppel S (1989) Isolation and characterization of dinitrogenfixing bacteria from the rhizosphere of Triticum aestivum and Ammophila arenaria. In: Vancŭra V, Kunc F (eds) Interrelationships between microorganisms and plants in soil. Proceedings of an international symposium. Liblice, Prague, pp 253–262Google Scholar
  56. Silver WS, Postgate JR (1973) Evolution of asymbiotic nitrogen fixation. J Theor Biol 40:1–10PubMedCrossRefGoogle Scholar
  57. Simonet P, Bardin R, Haurat J, Moiroud A, Normand P (1986) Localization of NIF genes on a large plasmid in Frankia sp. strain ULQ0132105009. Mol Gen Genet 204:492–495CrossRefGoogle Scholar
  58. Stackebrandt S, Schumann P (2006) Introduction to the taxonomy of actinobacteria. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes, vol 3, pp 297–321Google Scholar
  59. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kamur S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. doi: 10.1093/molbev/msr121
  60. Trujillo ME, Alonso-Vega P, Rodrıguez R, Carro L, Cerda E, Alonso P, Martınez-Molina E (2010) The genus Micromonospora is widespread in legume root nodules: the example of Lupinus angustifolius. ISME J 4:1282–1289CrossRefGoogle Scholar
  61. Ueda T, Suga Y, Yahiro N, Matsuguchi T (1995) Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. J Bacteriol 177:1414–1417PubMedGoogle Scholar
  62. Valdés M, Pérez N-O, Estrada de los Santos P, Caballero-Mellado J, Peña-Cabriales JJ, Normand P, Hirsch AM (2005) Non-Frankia actinomycetes isolated from surface-sterilized roots of Casuarina equisetifolia fix nitrogen. Appl Environ Microbiol 71:460–466Google Scholar
  63. Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chanter KF, van Sinderen D (2007) Genomics of actinobacteria: tracing the evolution of an ancient phylum. Microbiol Mol Biol Rev 71:495–548PubMedCrossRefGoogle Scholar
  64. Villegas M, Vasquez L, Valdes M (1997) Growth and nitrogenase activity conditions of a new diazotrophic group of filamentous bacteria isolated from Casuarina root nodules. Rev Lat Amer Microbiol 39:65–72Google Scholar
  65. Wade WG (1997) The role of eubacterium species in periodontal disease and other oral infections. Microb Ecol Health Dis 9:367–370CrossRefGoogle Scholar
  66. Wheeler CT, Akkermans ADL, Berry AM (2008) Frankia and actinorhizal plants: a historical perspective. In: Pawlowski K, Newton WE (eds) Nitrogen-fixing actinorhizal symbioses. Springer, Dordrecht, pp 1–24CrossRefGoogle Scholar
  67. Würdemann D, Tindall BJ, Pukall R, Lünsdorf H, Strömpl C, Namuth T, Nahrstedt H, Wos-Oxley M, Ott S, Schreiber S, Timmis KN, Oxley APA (2009) Gordonibacter pamelaeae gen. nov., sp. nov., a new member of the Coriobacteriaceae isolated from a patient with Crohn’s disease, and reclassification of Eggerthella hongkongensis Lau et al. 2006 as Paraeggerthella hongkongensis gen. nov., comb. nov. Int J Syst Evol Microbiol 59:1045–1415CrossRefGoogle Scholar
  68. Young JPW (1992) Phylogenetic classification of nitrogen-fixing organisms. In: Stacey G, Burris RH, Evans HJ (eds) Biological nitrogen fixation. Chapman and Hall, New York, pp 43–48Google Scholar
  69. Young JPW (2005) The phylogeny and evolution of nitrogenases. In: Palacios R, Newton W (eds) Genomes and genomics of nitrogen-fixing organisms. Nitrogen fixation: origins, applications, and research progress, vol 3. Springer, The Netherlands, pp 221–241. doi: 10.1007/1-4020-3054-1_14
  70. Zakhaia F, Jeder H, Willems A, Gillis M, Dreyfus B, de Lajudie P (2006) Diverse bacteria associated with root nodules of spontaneous legumes in Tunisia and first report for nifH-like gene within the genera Microbacterium and Starkeya. Microb Ecol 51:375–393CrossRefGoogle Scholar
  71. Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5:539–554PubMedCrossRefGoogle Scholar
  72. Zheng L, Cash DL, Flint DH, Dean DR (1998) Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBAfdx gene cluster from Azotobacter vinelandii. J Biol Chem 273:13264–13272PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Maher Gtari
    • 1
  • Faten Ghodhbane-Gtari
    • 1
  • Imen Nouioui
    • 1
  • Nicholas Beauchemin
    • 2
  • Louis S. Tisa
    • 2
  1. 1.Laboratoire Microorganismes and Biomolécules ActivesUniversité de Tunis El Manar (FST) et Université de Carthage (ISSTE)TunisTunisia
  2. 2.Department of Cellular, Molecular and Biomedical SciencesUniversity of New HampshireDurhamUSA

Personalised recommendations