Symbiosis

, Volume 50, Issue 1–2, pp 13–26 | Cite as

Wild nodules can be broken: proteomics of Frankia in field-collected root nodules

Article

Abstract

With the genomes of three Frankia strains available, high-throughput proteomics methods can be used to reveal the set of proteins expressed by these bacteria in symbiosis with plants. A question we address is the degree to which the known genomes can be used to study proteomes of uncharacterized frankiae growing in field-collected root nodules. To this end, we have characterized the symbiotic proteomes of Frankia from three plant species, Alnus incana subsp. rugosa, Ceanothus americanus, and Elaeagnus angustifolia. Root nodule proteins were identified using two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC MS/MS) of trypsin-digested protein samples. We identified 1300 Frankia proteins in A. incana nodules using the Frankia alni ACN14a genome and 1100 proteins from E. angustifolia nodules using the EAN1pec genome. In addition, over 100 proteins were identified from C. americanus nodules using a more limited one dimensional LC MS/MS analysis. Many of the most abundant proteins identified are involved in energy and nitrogen metabolism. The enzyme nitrogenase and the nitrogenase iron protein were among the most abundant proteins, reflecting the major process occurring in symbiosis. Several hundred plant proteins were also identified. We highlight the power of proteomics to uncover the physiology of symbiotic Frankia in the environment using heterologous genome information.

Keywords

Frankia Root nodules LC MS/MS Actinorhizal Proteomics Nitrogen fixation 

References

  1. Alloisio N, Felix S, Marechal J, Pujic P, Rouy Z, Vallenet D, Medigue C, Normand P (2007) Frankia alni proteome under nitrogen-fixing and nitrogen-replete conditions. Physiol Plant 130:450–453Google Scholar
  2. Bagnarol E, Popovici J, Marechal J, Alloisio N, Pujic P, Normand P, Fernandez MP (2007) Differential Frankia protein patterns induced by phenolic extracts from Myricaceae seeds. Physiol Plant 130:380-390CrossRefGoogle Scholar
  3. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795CrossRefPubMedGoogle Scholar
  4. Benson DR, Silvester WB (1993) Biology of Frankia strains, actinomycete symbionts of actinorhizal plants. Microbiol Rev 57:293–319PubMedGoogle Scholar
  5. Bickhart D, Gogarten JP, Lapierre P, Tisa LS, Normand P, Benson DR (2009) Insertion sequence content reflects genome plasticity in strains of the root nodule actinobacterium Frankia. BMC Genomics 10:468Google Scholar
  6. Clawson ML, Bourret A, Benson DR (2004) Assessing the phylogeny of Frankia-actinorhizal plant nitrogen-fixing root nodule symbioses with Frankia 16S rRNA and glutamine synthetase gene sequences. Mol Phylogenet Evol 31:131–138CrossRefPubMedGoogle Scholar
  7. Djordjevic MA (2004) Sinorhizobium meliloti metabolism in the root nodule: a proteomic perspective. Proteomics 4:1859–1872CrossRefPubMedGoogle Scholar
  8. Guan C, Ribeiro A, Akkermans AD, Jing Y, van Kammen A, Bisseling T, Pawlowski K (1996) Nitrogen metabolism in actinorhizal nodules of Alnus glutinosa: expression of glutamine synthetase and acetylornithine transaminase. Plant Mol Biol 32:1177–1184CrossRefPubMedGoogle Scholar
  9. Guan C, Akkermans AD, Van Kammen A, Bisseling T, Pawlowski K (1997) ag13 is expressed in Alnus glutinosa nodules in infected cells during endosymbiont degradation and in the nodule pericycle. Physiol Plant 99:601–607CrossRefGoogle Scholar
  10. Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4:1265–1272CrossRefPubMedGoogle Scholar
  11. Kim HB, An CS (2002) Differential expression patterns of an acidic chitinase and a basic chitinase in the root nodule of Elaeagnus umbellata. Mol Plant Microb Interact 15:209–215CrossRefGoogle Scholar
  12. Kim HB, Lee SH, An CS (1999) Isolation and characterization of a cDNA clone encoding asparagine synthetase from root nodules of Elaeagnus umbellata. Plant Sci 149:85–94CrossRefGoogle Scholar
  13. Larrainzar E, Wienkoop S, Weckwerth W, Ladrera R, Arrese-Igor C, González EM (2007) Medicago truncatula Root Nodule Proteome Analysis Reveals Differential Plant and Bacteroid Responses to Drought Stress. Plant Physiol 144:1495–1507CrossRefPubMedGoogle Scholar
  14. Liu J, Miller SS, Graham M et al (2006) Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula. Plant Physiol 141:167–177CrossRefPubMedGoogle Scholar
  15. Mastronunzio JE, Tisa LS, Normand P, Benson DR (2008) Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts. BMC Genomics 9:47CrossRefPubMedGoogle Scholar
  16. Mastronunzio JE, Huang Y, Benson DR (2009) Diminshed exoproteome of Frankia spp. in culture and in symbiosis. Appl Environ Microbiol 75:6721–6728CrossRefPubMedGoogle Scholar
  17. Miettinen JK, Virtanen AI (1952) The free amino acids in the leaves, roots, and root nodules of the alder (Alnus). Physiol Plant 5:540–557CrossRefGoogle Scholar
  18. Normand P, Orso S, Cournoyer B, Jeannin P, Chapelon C, Dawson J, Evtushenko L, Misra AK (1996) Molecular phylogeny of the genus Frankia and related genera and emendation of the family Frankiaceae. Int J Syst Bacteriol 46:1–9PubMedCrossRefGoogle Scholar
  19. Normand P, Lapierre P, Tisa LS et al (2007) Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography. Genome Res 17:7–15CrossRefPubMedGoogle Scholar
  20. Outten FW, Djaman O, Storz G (2004) A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli. Mol Microbiol 52:861–872CrossRefPubMedGoogle Scholar
  21. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567CrossRefPubMedGoogle Scholar
  22. Pruitt KD, Tatusova T, Maglott DR (2007) NCBI Reference Sequence (RefSeq):a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35:D61-5Google Scholar
  23. Reuther J, Wohlleben W (2007) Nitrogen metabolism in Streptomyces coelicolor: transcriptional and post-translational regulation. J Mol Microbiol Biotechnol 12:139–146CrossRefPubMedGoogle Scholar
  24. Sarma AD, Emerich DW (2006) A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicum. Proteomics 6:3008–3028CrossRefPubMedGoogle Scholar
  25. 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 258:12064–12068PubMedGoogle Scholar
  26. Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefPubMedGoogle Scholar
  27. van Ghelue M, Ribeiro A, Solheim B, Akkermans AD, Bisseling T, Pawlowski K (1996) Sucrose synthase and enolase expression in actinorhizal nodules of Alnus glutinosa: comparison with legume nodules. Mol Gen Genet 250:437–446CrossRefPubMedGoogle Scholar
  28. Wheeler CT, Bond G (1970) The amino acids of non-legume root nodules. Phytochemistry 9:705–708CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Department of Molecular and Cell BiologyUniversity of ConnecticutStorrsUSA

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