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.
Similar content being viewed by others
References
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 25:3389–3402
Araki Y, Gonzalez EL (1998) V- and P-type Ca2+-stimulated ATPases in a calcifying strain of Pleurochrysis sp. (Haptophyceae). J Phycol 34:79–88
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 306:79–86
Balch WM, Holligan PM, Ackleson SG, Voss KJ (1991) Biological and optical properties of mesoscale coccolithophore blooms in the Gulf of Maine. Limnol Oceanogr 36:629–643
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–D403
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, Berlin
Brown CW, Yoder JA (1994) Coccolithophore blooms in the global ocean. J Geophys Res 99:7467–7482
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 44:1315–1321
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 20:857–866
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 37:71–78
Crowther R, Pearse B (1981) Assembly and packing of clathrin into coats. J Cell Biol 91:790–797
De Vrind-De Jong EW, De Vrind JPM (1997) Algal deposition of carbonates and silicates. Rev Mineral 35:267–307
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 72:252–260
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 3:108–112
Field CB, Behrenfield MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281:237–240
Forgac M (2000) Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases. J Exp Biol 203:71–80
Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrate animals. Plenum, New York
Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can J Microbiol 8:229–239
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 304:339–342
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 7:879–900
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 16:1100
Isenberg HD, Lavine LS, Weissfellner H (2007) The suppression of mineralization in a coccolithophorid by an inhibitor of carbonic anhydrase. J Eukaryot Microbiol 10:477–479
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 159:21–30
Jordan RW, Green JC (1994) A checklist of the extant haptophyta of the world. J Mar Biol Assoc UK 74:149–174
Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–298
Kirchhausen T (2000) Clathrin. Ann Rev Biochem 69:699–727
Lewin DA, Mellman I (1998) Sorting out adaptors. Biochim Biophys Acta 1401:129–145
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 27:82–86
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 5:3537–3545
Milliman JD (1993) Production and accumulation of calcium carbonate in the ocean: budget of a nonsteady state. Glob Biogeochem Cycles 7:927–957
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 29:672–681
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 71:2564–2575
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 55:241–253
Paasche E (2001) A review of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae), with particular reference to growth, coccolith formation and calcification–photosynthesis interactions. Phycologica 40:503–529
Pearse B (1976) Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Proc Natl Acad Sci USA 73:1255–1259
Price MN, Dehal PS, Arkin AP (2009) FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26:1641–1650
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 72:5512–5526
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, Dordrecht
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 36:87–96
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 41:297–314
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 93:14440–14445
Skipp P, Robinson J, O’Connor CD, Clarke IN (2005) Shotgun proteomic analysis of Chlamydia trachomatis. Proteomics 5:1558–1573
Snyder RV, Gibbs PDL, Palacios A, Abiy L, Dickey R, Lopez JV, Rein KS (2003) Polyketide synthase genes from marine dinoflagellates. Mar Biotechnol 5:1–12
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 66:1767–1780
Staunton J, Weissman K (2001) Polyketide biosynthesis: a millennium review. Nat Prod Rep 18:380–416
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, Berlin
Uniprot (2008) The universal protein resource (UniProt). Nucleic Acids Res 36:D190–D195
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 6:278–290
Wahlund TM, Zhang X, Read BA (2004b) Expressed sequence tag profiles from calcifying and non-calcifying cultures of Emiliania huxleyi. Micropaleontology 50:145–155
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 8:685–691
Westbroek P, Young J, Linschooten K (1989) Coccolith production (biomineralization) in the marine alga Emiliania huxleyi. J Protozool 36:368–373
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 8:27–46
Wong P-F, Tan L-J, Nawi H, Abubakar S (2006) Proteomics of the red alga, Gracilaria changii (Gracilariales, Rhodophyta). J Phycol 42:113–120
Young JR, Henriksen K (2003) Biomineralization within vesicles: the calcite of coccoliths. Rev Mineral Geochem 54:189–215
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 298:79–89
Acknowledgements
BMJ was supported by a Natural Environment Research Council (NERC) PhD studentship (NER/S/A2006/14211). This work is a contribution to the “European Project on Ocean Acidification” (EPOCA) which received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 211384. We gratefully acknowledge the technical support of Dr John Gittins, Miss Shanon Pead and Mrs Therese Cavassa. We would also like to thank two anonymous reviewers for their comments on this manuscript.
Author information
Authors and Affiliations
Corresponding author
Additional information
All programmes, datasets and result files are available from: http://www.personal.soton.ac.uk/re1u06/research/ehux/.
Bethan M. Jones and Richard J. Edwards contributed equally to this work.
Electronic Supplementary Materials
Below is the link to the electronic supplementary material.
ESM Supplementary Table 1
Protein identifications from taxonomically restricted protein database (DOC 60.5 kb)
ESM Supplementary Table 2
Consensus protein identifications from BUDAPEST pipeline (DOC 103 kb)
ESM Supplementary results
Supplementary 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)
ESM data 1
Trees and alignments generated from taxonomically restricted protein database search dataset (ZIP 1.05 mb)
ESM data 2
Sequences, trees and alignments generated from BUDAPEST analysis (ZIP 4 mb)
ESM data 3
Excel 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)
Rights and permissions
About this article
Cite this article
Jones, B.M., Edwards, R.J., Skipp, P.J. et al. Shotgun Proteomic Analysis of Emiliania huxleyi, a Marine Phytoplankton Species of Major Biogeochemical Importance. Mar Biotechnol 13, 496–504 (2011). https://doi.org/10.1007/s10126-010-9320-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10126-010-9320-0