Marine Biotechnology

, Volume 13, Issue 3, pp 496–504 | Cite as

Shotgun Proteomic Analysis of Emiliania huxleyi, a Marine Phytoplankton Species of Major Biogeochemical Importance

  • Bethan M. Jones
  • Richard J. Edwards
  • Paul J. Skipp
  • C. David O’Connor
  • M. Debora Iglesias-Rodriguez
Original Article

Abstract

Emiliania huxleyi is a unicellular marine phytoplankton species known to play a significant role in global biogeochemistry. Through the dual roles of photosynthesis and production of calcium carbonate (calcification), carbon is transferred from the atmosphere to ocean sediments. Almost nothing is known about the molecular mechanisms that control calcification, a process that is tightly regulated within the cell. To initiate proteomic studies on this important and phylogenetically remote organism, we have devised efficient protein extraction protocols and developed a bioinformatics pipeline that allows the statistically robust assignment of proteins from MS/MS data using preexisting EST sequences. The bioinformatics tool, termed BUDAPEST (Bioinformatics Utility for Data Analysis of Proteomics using ESTs), is fully automated and was used to search against data generated from three strains. BUDAPEST increased the number of identifications over standard protein database searches from 37 to 99 proteins when data were amalgamated. Proteins involved in diverse cellular processes were uncovered. For example, experimental evidence was obtained for a novel type I polyketide synthase and for various photosystem components. The proteomic and bioinformatic approaches developed in this study are of wider applicability, particularly to the oceanographic community where genomic sequence data for species of interest are currently scarce.

Keywords

BUDAPEST Emiliania huxleyi EST analysis Mass spectrometry Shotgun proteomics 

Supplementary material

10126_2010_9320_MOESM1_ESM.doc (60 kb)
ESM Supplementary Table 1Protein identifications from taxonomically restricted protein database (DOC 60.5 kb)
10126_2010_9320_MOESM2_ESM.doc (81 kb)
ESM Supplementary Table 2Consensus protein identifications from BUDAPEST pipeline (DOC 103 kb)
10126_2010_9320_MOESM3_ESM.doc (31 kb)
ESM Supplementary resultsSupplementary results detailing 1) the process and 2) location proteins identified for E. huxleyi strains NZEH, CCMP371 and CCMP1516 with respect to available GO terms (DOC 31 kb)
10126_2010_9320_MOESM4_ESM.zip (1.1 mb)
ESM data 1Trees and alignments generated from taxonomically restricted protein database search dataset (ZIP 1.05 mb)
10126_2010_9320_MOESM5_ESM.zip (4 mb)
ESM data 2Sequences, trees and alignments generated from BUDAPEST analysis (ZIP 4 mb)
10126_2010_9320_MOESM6_ESM.xls (1.7 mb)
ESM data 3Excel worksheets showing raw Mascot results for the searches of MS/MS data against the Emiliania huxley ESTs and taxonomically restricted protein datasets. Also included are peptide, BUDAPEST and taxonomically restricted database worksheets and results (XLS 1.72 mb)

References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acid Res 25:3389–3402CrossRefGoogle Scholar
  2. Araki Y, Gonzalez EL (1998) V- and P-type Ca2+-stimulated ATPases in a calcifying strain of Pleurochrysis sp. (Haptophyceae). J Phycol 34:79–88CrossRefGoogle Scholar
  3. Armbrust EV, Berges JA, Bowler C, Green BR, Martinez D, Putnam NH, Zhou S, Allen AE, Apt KE, Bechner M, Brzezinski MA, Chaal BK, Chiovitti A, Davis AK, Demarest MS, Detter JC, Glavina T, Goodstain D, Hadi MZ, Hellstein U, Hildebrand M, Jenkins BD, Jurka J, Kapitonov VV, Kroger N, Lau WWY, Lane TW, Larimer FW, Lippmeier JC, Lucas S, Medina M, Montsant A, Obornik M, Parker MS, Palenik B, Pazour GJ, Richardson PM, Rynearson TA, Saito MA, Schwartz DC, Thamatrakoln K, Valentin K, Vardi A, Wilkerson FP, Rokhsar DS (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79–86PubMedCrossRefGoogle Scholar
  4. Balch WM, Holligan PM, Ackleson SG, Voss KJ (1991) Biological and optical properties of mesoscale coccolithophore blooms in the Gulf of Maine. Limnol Oceanogr 36:629–643CrossRefGoogle Scholar
  5. Barrell D, Dimmer E, Huntley RP, Binns D, O’donovan C, Apweiler R (2009) The GOA database in 2009—an integrated gene ontology annotation resource. Nucl Acids Res 37:D396–D403PubMedCrossRefGoogle Scholar
  6. Baumann K-H, Böckel B, Frenz M (2004) Coccolith contribution to South Atlantic carbonate sedimentation. In: Thierstein HR, Young JR (eds) Coccolithophores: from molecular processes to global impact. Springer, BerlinGoogle Scholar
  7. Brown CW, Yoder JA (1994) Coccolithophore blooms in the global ocean. J Geophys Res 99:7467–7482CrossRefGoogle Scholar
  8. Contreras L, Ritter A, Dennett G, Boehmwald F, Guitton N, Pineau C, Moenne A, Potin P, Correa JA (2008) Two-dimensional gel electrophoresis analysis of brown algal protein extracts. J Phycol 44:1315–1321CrossRefGoogle Scholar
  9. Cooper B, Neelam A, Campbell KB, Lee J, Liu G, Garrett WM, Scheffler B, Tucker ML (2007) Protein accumulation in the germinating Uromyces appendiculatus uredospore. Mol Plant–Microb Interact 20:857–866CrossRefGoogle Scholar
  10. Corstjens PLAM, Araki Y, Gonzalez EL (2001) A coccolithophorid calcifying vesicle with a vacuolar-type ATPase proton pump: cloning and immunolocalization of the Vo subunit c1. J Phycol 37:71–78CrossRefGoogle Scholar
  11. Crowther R, Pearse B (1981) Assembly and packing of clathrin into coats. J Cell Biol 91:790–797PubMedCrossRefGoogle Scholar
  12. De Vrind-De Jong EW, De Vrind JPM (1997) Algal deposition of carbonates and silicates. Rev Mineral 35:267–307Google Scholar
  13. Dyhrman ST, Haley ST, Birkeland SR, Wurch LL, Cipriano MJ, Mcarthur AG (2006) Long serial analysis of gene expression for gene discovery and transcriptome profiling in the widepread marine coccolithophore Emiliania huxleyi. Appl Environ Microbiol 72:252–260PubMedCrossRefGoogle Scholar
  14. Edwards RJ, Moran N, Devocelle M, Kiernan A, Meade G, Signac W, Foy M, Park SDE, Dunne E, Kenny D, Shields DC (2007) Bioinformatic discovery of novel bioactive peptides. Nat Chem Biol 3:108–112PubMedCrossRefGoogle Scholar
  15. Field CB, Behrenfield MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281:237–240PubMedCrossRefGoogle Scholar
  16. Forgac M (2000) Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases. J Exp Biol 203:71–80PubMedGoogle Scholar
  17. Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrate animals. Plenum, New YorkGoogle Scholar
  18. Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can J Microbiol 8:229–239PubMedCrossRefGoogle Scholar
  19. Holligan PM, Viollier M, Harbour DS, Camus P, Champagne-Philippe M (1983) Satellite and ship studies of coccolithophore production along a continental shelf edge. Nature 304:339–342CrossRefGoogle Scholar
  20. Holligan PM, Fernandez E, Aiken J, Burkill PH, Finch M, Groom SB, Malin G, Muller K, Purdie DA, Robinson C, Trees CC, Turner SM, Van Der Wal P (1993) A biogeochemical study of the coccolithophore, Emiliania huxleyi, in the North Atlantic. Glob Biogeochem Cycles 7:879–900CrossRefGoogle Scholar
  21. Iglesias-Rodriguez MD, Brown CW, Doney SC, Kleypas J, Kolber D, Kolber Z, Hayes PK, Falkowski PG (2002) Representing key phytoplankton functional groups in ocean carbon cycle models: coccolithophorids. Glob Biogeochem Cycles 16:1100CrossRefGoogle Scholar
  22. Isenberg HD, Lavine LS, Weissfellner H (2007) The suppression of mineralization in a coccolithophorid by an inhibitor of carbonic anhydrase. J Eukaryot Microbiol 10:477–479CrossRefGoogle Scholar
  23. John U, Beszteri B, Derella E, Van De Peer Y, Read BR, Moreau H, Cembella A (2008) Novel insights into evolution of protistan polyketide synthases through phylogenomic analysis. Protist 159:21–30PubMedCrossRefGoogle Scholar
  24. Jordan RW, Green JC (1994) A checklist of the extant haptophyta of the world. J Mar Biol Assoc UK 74:149–174CrossRefGoogle Scholar
  25. Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–298PubMedCrossRefGoogle Scholar
  26. Kirchhausen T (2000) Clathrin. Ann Rev Biochem 69:699–727PubMedCrossRefGoogle Scholar
  27. Lewin DA, Mellman I (1998) Sorting out adaptors. Biochim Biophys Acta 1401:129–145PubMedCrossRefGoogle Scholar
  28. Linschooten C, Bleijswijk JDL, Emburg PR, Vrind JPM, Kempers ES, Westbroek P, Jong EWV-D (1991) Role of the light–dark cycle and medium composition on the production of coccoliths by Emiliania huxleyi (Haptophyceae). J Phycol 27:82–86CrossRefGoogle Scholar
  29. Martens L, Hermjakob H, Jones P, Adamski M, Taylor C, States D, Gevaert K, Vandekerckhove J, Apweiler R (2005) PRIDE: the proteomics identifications database. Proteomics 5:3537–3545PubMedCrossRefGoogle Scholar
  30. Milliman JD (1993) Production and accumulation of calcium carbonate in the ocean: budget of a nonsteady state. Glob Biogeochem Cycles 7:927–957CrossRefGoogle Scholar
  31. Nagai K, Yotsukura N, Ikegami H, Kimura H, Morimoto K (2008) Protein extraction for 2-DE from the lamina of Ecklonia kurome (laminariales): recalcitrant tissue containing high levels of viscous polysaccharides. Electrophoresis 29:672–681PubMedCrossRefGoogle Scholar
  32. Nguyen B, Bowers RM, Wahlund TM, Read BR (2005) Suppressive subtractive hybridization of and differences in gene expression content of calcifying and noncalcifying cultures of Emiliania huxleyi strain 1516. Appl Environ Microbiol 71:2564–2575PubMedCrossRefGoogle Scholar
  33. Nunn BL, Aker JR, Shaffer SA, Tsai S, Strzepek RF, Boyd PW, Freeman TL, Brittnacher M, Malmstrom L, Goodlett DR (2009) Deciphering diatom biochemical pathways via whole-cell proteomics. Aquat Microb Ecol 55:241–253PubMedCrossRefGoogle Scholar
  34. Paasche E (2001) A review of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae), with particular reference to growth, coccolith formation and calcification–photosynthesis interactions. Phycologica 40:503–529CrossRefGoogle Scholar
  35. Pearse B (1976) Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Proc Natl Acad Sci USA 73:1255–1259PubMedCrossRefGoogle Scholar
  36. Price MN, Dehal PS, Arkin AP (2009) FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26:1641–1650PubMedCrossRefGoogle Scholar
  37. Quinn P, Bowers RM, Zhang X, Wahlund TM, Fanelli MA, Olszova D, Read BR (2006) cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta). Appl Environ Microbiol 72:5512–5526PubMedCrossRefGoogle Scholar
  38. Raven JA, Beardall J (2003) CO2 acquisition mechanisms in algae: carbon dioxide diffusion and carbon dioxide concentrating mechanisms. In: Larkum A, Raven JA, Douglas S (eds) Photosynthesis in the Algae. Kluwer, DordrechtGoogle Scholar
  39. Riegman R, Stolte W, Noordeloos AAM, Slezak D (2000) Nutrient uptake and alkaline phosphatase (EC 3:1:3:1) activity of Emiliania huxleyi (Prymnesiophyeae) during growth under N and P limitation in continuous cultures. J Phycol 36:87–96CrossRefGoogle Scholar
  40. Robertson JE, Robinson C, Turner DR, Holligan P, Watson AJ, Boyd P, Fernandez E, Finch M (1994) The impact of a coccolithophore bloom on oceanic carbon uptake in the northeast Atlantic during summer 1991. Deep Sea Res I 41:297–314CrossRefGoogle Scholar
  41. Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M (1996) Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 93:14440–14445PubMedCrossRefGoogle Scholar
  42. Skipp P, Robinson J, O’Connor CD, Clarke IN (2005) Shotgun proteomic analysis of Chlamydia trachomatis. Proteomics 5:1558–1573PubMedCrossRefGoogle Scholar
  43. Snyder RV, Gibbs PDL, Palacios A, Abiy L, Dickey R, Lopez JV, Rein KS (2003) Polyketide synthase genes from marine dinoflagellates. Mar Biotechnol 5:1–12PubMedCrossRefGoogle Scholar
  44. Snyder RV, Guerrero MA, Sinigalliano CD, Winshell J, Perez R, Lopez JV, Rein KS (2005) Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis. Phytochemistry 66:1767–1780PubMedCrossRefGoogle Scholar
  45. Staunton J, Weissman K (2001) Polyketide biosynthesis: a millennium review. Nat Prod Rep 18:380–416PubMedCrossRefGoogle Scholar
  46. Tyrrell T, Merico A (2004) Emiliania huxleyi: bloom observations and the conditions that induce them. In: Thierstein HR, Young JR (eds) Coccolithophores: from molecular processes to global impact. Springer, BerlinGoogle Scholar
  47. Uniprot (2008) The universal protein resource (UniProt). Nucleic Acids Res 36:D190–D195CrossRefGoogle Scholar
  48. Wahlund TM, Hadaegh AR, Clark R, Nguyen B, Fanelli M, Read BR (2004a) Analysis of expressed sequence tags from calcifying cells of marine coccolithophorid (Emiliania huxleyi). Mar Biotechnol 6:278–290PubMedCrossRefGoogle Scholar
  49. Wahlund TM, Zhang X, Read BA (2004b) Expressed sequence tag profiles from calcifying and non-calcifying cultures of Emiliania huxleyi. Micropaleontology 50:145–155CrossRefGoogle Scholar
  50. Wang D-Z, Lin L, Hong H-S (2009) Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis. HarmAlgae 8:685–691Google Scholar
  51. Westbroek P, Young J, Linschooten K (1989) Coccolith production (biomineralization) in the marine alga Emiliania huxleyi. J Protozool 36:368–373Google Scholar
  52. Westbroek P, Brown CW, Van Bleijswijk J, Brownlee C, Brummer GJ, Conte M, Egge J, Fernandez E, Jordan R, Knappertsbusch M, Stefels J, Veldhuis M, Van Der Wal P, Young J (1993) A model system approach to biological climate forcing. The example of Emiliania huxleyi. Global Planet Change 8:27–46CrossRefGoogle Scholar
  53. Wong P-F, Tan L-J, Nawi H, Abubakar S (2006) Proteomics of the red alga, Gracilaria changii (Gracilariales, Rhodophyta). J Phycol 42:113–120CrossRefGoogle Scholar
  54. Young JR, Henriksen K (2003) Biomineralization within vesicles: the calcite of coccoliths. Rev Mineral Geochem 54:189–215CrossRefGoogle Scholar
  55. Zhu G, Lagier MJ, Stejskal F, Millership JJ, Cai X, Keithly JS (2002) Cryptosporidium parvum: the first protist known to encode a putative polyketide synthase. Gene 298:79–89PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Bethan M. Jones
    • 1
  • Richard J. Edwards
    • 2
  • Paul J. Skipp
    • 2
    • 3
  • C. David O’Connor
    • 2
    • 3
  • M. Debora Iglesias-Rodriguez
    • 1
  1. 1.School of Ocean and Earth Science, National Oceanography Centre, SouthamptonUniversity of SouthamptonSouthamptonUK
  2. 2.School of Biological SciencesUniversity of SouthamptonSouthamptonUK
  3. 3.Centre for Proteomic ResearchUniversity of SouthamptonSouthamptonUK

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