Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

BMC Molecular Biology

David E Housman²  Deborah A Hogan⁵  Jerry Pelletier³  Sean Milton⁴  Deborah L Robertson¹  Stephen S Bates⁷  Brooks M Henningsen⁶  Katie Rose Boissonneault² 

[1] Biology Department, Clark University, 950 Main Street, Worcester, MA 01610, USA;Koch Institute, Massachusetts Institute of Technology, 76-553, 77 Massachusetts Avenue, Cambridge, MA 02139, USA;Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada;Present address: Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA;Department of Microbiology and Immunology, Vail Building Room 208, Dartmouth Medical School, Hanover, NH 03755, USA;Present address: Mascoma Corporation, 67 Etna Road Suite 300, Lebanon, NH 03766, USA;Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton, New Brunswick E1C 9B6, Canada

关键词: Diatom;    Bacillariophyceae;    Pseudo-nitzschia multiseries;    Domoic acid;    Reference gene;    Normalization;    RT-qPCR;    cDNA microarray;    Gene regulation;    Gene expression;   

Others  :  1090579
DOI  :  10.1186/1471-2199-14-25

 received in 2013-06-12, accepted in 2013-10-18,  发布年份 2013

【 摘 要 】

Background

Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions.

Results

Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions.

Conclusion

Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.

【 授权许可】

   
2013 Boissonneault et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Bates SS, Bird CJ, de Freitas ASW, Foxall R, Gilgan M, Hanic LA, Johnson GR, McCulloch AW, Odense P, Pocklington R, Quilliam MA, Sim PG, Smith JC, Subba Rao DV, Todd ECD, Walter JA, Wright JLC: Pennate diatom Nitzschia pungens as the primary source of domoic acid, a toxin in shellfish from eastern Prince Edward Island, Canada. Can J Fish Aquat Sci 1989, 46:1203-1215.
• [2]Douglas DJ, Bates SS: Production of domoic acid, a neurotoxic amino acid, by an axenic culture of the marine diatom Nitzschia pungens f. multiseries Hasle. Can J Fish Aquat Sci 1992, 49:85-90.
• [3]Douglas DJ, Ramsey UP, Walter JA, Wright JLC: Biosynthesis of the neurotoxin domoic acid by the marine diatom Nitzschia pungens forma multiseries, determined with [13C]-labelled precursors and nuclear magnetic resonance. J Chem Soc Chem Commun 1992, 1992:714-7156.
• [4]Wright JLC, Boyd RK, de Freitas ASW, Falk M, Foxall RA, Jamieson WD, Laycock MV, McCulloch AW, McInnes AG, Odense P, Pathak VP, Quilliam MA, Ragan MA, Sim PG, Thibault P, Walter JA, Gilgan M, Richard DJA, Dewar D: Identification of domoic acid, a neuroexcitatory amino acid, in toxic mussels from eastern Prince Edward Island. Can J Chem 1989, 67:714-716.
• [5]Takemoto T, Daigo K: Constituents of Chondria armata. Chem Pharm Bull 1958, 6:578-580.
• [6]Ramsdell JS: The molecular and integrative basis to domoic acid toxicity. In Phycotoxins: Chemistry and Biochemistry. Edited by Botana L. Cambridge, MA: Blackwell Publishing Professional; 2007:223-250.
• [7]Lelong A, Hégaret H, Soudant P, Bates SS: Pseudo-nitzschia (Bacillariophyceae) species, domoic acid and amnesic shellfish poisoning: revisiting previous paradigms. Phycologia 2012, 51:168-216.
• [8]Trainer VL, Bates SS, Lundholm N, Thessen AE, Cochlan WP, Adams NG, Trick CG: Pseudo-nitzschia physiological ecology, phylogeny, toxicity, monitoring and impacts on ecosystem health. Harmful Algae 2012, 14:271-300.
• [9]Bates SS: Ecophysiology and metabolism of ASP toxin production. In Physiological Ecology of Harmful Algal Blooms. Edited by Anderson DM, Cembella AD, Hallegraeff GM. Heidelberg: Springer-Verlag; 1998:405-426.
• [10]Bates SS, Garrison DL, Horner RA: Bloom dynamics and physiology of domoic-acid-producing Pseudo-nitzschia species. In Physiological Ecology of Harmful Algal Blooms. Edited by Anderson DM, Cembella AD, Hallegraeff GM, Anderson DM, Cembella AD, Hallegraeff GM. Heidelberg: Springer-Verlag; 1998:267-292.
• [11]Bates SS, Trainer VL: The ecology of harmful diatoms. In Ecology of Harmful Algae Ecological Studies. Volume 189. Edited by Granéli E, Turner J. Heidleberg: Springer-Verlag; 2006:81-93.
• [12]Ramsey UP, Douglas DJ, Walter JA, Wright JLC: Biosynthesis of domoic acid by the diatom Pseudo-nitzschia multiseries. Nat Toxins 1998, 6:137-146.
• [13]Savage TJ, Smith GJ, Clark AT, Saucedo PN: Condensation of the isoprenoid and amino precursors in the biosynthesis of domoic acid. Toxicon 2012, 59:25-33.
• [14]Pan Y, Bates SS, Cembella AD: Environmental stress and domoic acid production by Pseudo-nitzschia: a physiological perspective. Nat Toxins 1998, 6:127-135.
• [15]Bates SS, Douglas DJ, Doucette GJ, Léger C: Enhancement of domoic acid production by reintroducing bacteria to axenic cultures of the diatom Pseudo-nitzschia multiseries. Nat Toxins 1995, 3:428-435.
• [16]Osada M, Stewart JE: Gluconic acid/gluconolactone: physiological influences on domoic acid production by bacteria associated with Pseudo-nitzschia multiseries. Aquat Microb Ecol 1997, 12:203-209.
• [17]Stewart JE: Bacterial involvement in determining domoic acid levels apparent in Pseudo-nitzschia multiseries cultures. Aquat Microb Ecol 2008, 50:135-144.
• [18]Kotaki Y, Koike K, Yoshida M, Thuoc CV, Huyen NTM, Hoi NC, Fukuyo Y, Kodama M: Domoic acid production in Nitzschia sp. isolated from a shrimp-culture pond in Do Son, Vietnam. J Phycol 2000, 36:1057-1060.
• [19]Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J: qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 2007, 8:R19. BioMed Central Full Text
• [20]Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:1-12.
• [21]Gunjan A, Paik J, Verreault A: Regulation of histone synthesis and nucleosome assembly. Biochimie 2005, 87:625-635.
• [22]Armbrust EV, Parker MS, Rocap G, Jenkins B, Bates SS, US Department of Energy Joint Genome Institute: Pseudo-nitzschia multiseries CLN-47 genome sequence, Assembly v1. 2011. [http://genome.jgi.doe.gov/Psemu1/Psemu1.home.html webcite
• [23]Cui B, Liu Y, Gorovsky M: Deposition and function of histone H3 variants in Tetrahymena thermophila. Mol Cell Biol 2006, 26:7719-7730.
• [24]Elsaesser S, Goldberg A, Allis C: New functions for an old variant: no substitute for histone H3.3. Curr Opin Genet Dev 2010, 20:110-117.
• [25]Jin J, Cai Y, Li B, Conaway R, Workman J, Conaway J, Kusch T: In and out: histone variant exchange in chromatin. Trends Biochem Sci 2005, 30:680-687.
• [26]Yu L, Gorovsky M: Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila. Mol Cell Biol 1997, 17:6303-6310.
• [27]Anju V, Kapros T, Waterborg J: Identification of a replication-independent replacement histone H3 in the basidiomycete Ustilago maydis. J Biol Chem 2011, 286:25790-25800.
• [28]Siaut M, Heijde M, Mangogna M, Montsant A, Coesel S, Allen A, Manfredonia A, Falciatore A, Bowler C: Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 2007, 406:23-35.
• [29]Pan Y, Subba Rao DV, Mann KH, Brown RG, Pocklington R: Effects of silicate limitation on production of domoic acid, a neurotoxin, by the diatom Pseudo-nitzschia multiseries (Hasle). I. Batch culture studies. Mar Ecol Prog Ser 1996, 131:225-233.
• [30]Valenzuela J, Mazurie A, Carlson RP, Gerlach R, Cooksey KE, Peyton BM, Fields MW: Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum. Biotechnol Biofuels 2012, 5:40. BioMed Central Full Text
• [31]Corstjens PLAM, González EL: Effects of nitrogen and phosphorus availability on the expression of the coccolith-vesicle V-ATPase (subunit c) of Pleurochrysis (Haptophyta). J Phycol 2004, 40:82-87.
• [32]Dyhrman ST, Haley ST, Birkeland SR, Wurch LL, Cipriano MJ, McArthur AG: Long serial analysis of gene expression for gene discovery and transcriptome profiling in the widespread marine coccolithophore Emiliania huxleyi. Appl Environ Microbiol 2006, 72:252-260.
• [33]Reinbothe S, Reinbothe C: The regulation of enzymes involved in chlorophyll biosynthesis. Eur J Biochem 1996, 237:323-343.
• [34]Kajander T, Merckel M, Thompson A, Deacon A, Mazur P, Kozarich J, Goldman A: The structure of Neurospora crassa 3-carboxy-cis, cis-muconate lactonizing enzyme, a beta propeller cycloisomerase. Structure 2002, 10:483-492.
• [35]Mazur P, Henzel W, Mattoo S, Kozarich J: 3-Carboxy-cis, cis-muconate lactonizing enzyme from Neurospora crassa: an alternate cycloisomerase motif. J Bacteriol 1994, 176:18-28.
• [36]Mazur P, Pieken W, Budihas S, Williams S, Wong S, Kozarich J: Cis, cis-muconate lactonizing enzyme from Trichosporon cutaneum: evidence for a novel class of cycloisomerases in eucaryotes. Biochemistry 1994, 33:1961-1970.
• [37]Thomason L, Court D, Datta A, Khanna R, Rosner J: Identification of the Escherichia coli K-12 ybhE gene as pgl, encoding 6-phosphogluconolactonase. J Bacteriol 2004, 186:8248-8253.
• [38]Zimenkov D, Gulevich A, Skorokhodova A, Biriukova I, Kozlov Y, Mashko S: Escherichia coli ORF ybhE is pgl gene encoding 6-phosphogluconolactonase (EC 3.1.1.31) that has no homology with known 6PGLs from other organisms. FEMS Microbiol Lett 2005, 224:275-280.
• [39]Henningsen B: Bioinformatic and Gene Expression Analysis of an SLC6 Homolog in the Toxin-Producing Marine Diatom Pseudo-nitzschia multiseries. MS thesis. Plymouth State University, Biological Sciences; 2012.
• [40]Beuve N, Rispail N, Laine P, Cliquet J-B, Ourry A, Le Deunff E: Putative role of gamma-aminobutyric acid (GABA) as a long-distance signal in up-regulation of nitrate uptake in Brassica napus L. Plant Cell Environ 2004, 27:1035-1046.
• [41]Chevrot R, Rosen R, Haudecoeur E, Cirou A, Shelp B, Ron E, Faure D: GABA controls the level of quorum-sensing signal in Agrobacterium tumefaciens. Proc Natl Acad Sci USA 2006, 103:7460-7464.
• [42]Palanivelu R, Brass L, Edlund AF, Preuss D: Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels. Cell 2003, 114:47-59.
• [43]Johnston M, Gallacher S, Smith EA, Glover LA: Detection of N-acyl homoserine lactones in marine bacteria associated with production and biotransformation of sodium channel blocking toxins and the microflora of toxin-producing phytoplankton. In Harmful Algal Blooms. Edited by Hallegraeff GM, Blackburn SI, Bolch CJ, Lewis RJ. Paris: Intergovernmental Oceanographic Commission of UNESCO; 2001:375-378.
• [44]Amin S, Parker M, Armbrust E: Interactions between diatoms and bacteria. Microbiol Mol Biol R 2012, 76:667-684.
• [45]Maldonado MT, Hughes MP, Rue EL, Wells ML: The effect of Fe and Cu on growth and domoic acid production by Pseudo-nitzschia multiseries and Pseudo-nitzschia australis. Limnol Oceanogr 2002, 47:515-526.
• [46]Rue E, Bruland K: Domoic acid binds iron and copper: a possible role for the toxin produced by the marine diatom Pseudo-nitzschia. Mar Chem 2001, 76:127-134.
• [47]Wells ML, Trick CG, Cochlan WP, Hughes MP, Trainer VL: Domoic acid: the synergy of iron, copper, and the toxicity of diatoms. Limnol Oceanogr 2005, 50:1908-1917.
• [48]Carnal N, Black C: Phosphofructokinase activities in photosynthetic organisms: the occurrence of pyrophosphate-dependent 6-phosphofructokinase in plants and algae. Plant Physiol 1983, 71:150-155.
• [49]Lea PJ, Chen ZH, Leegood RC, Walker RP: Does phosphoenolpyruvate carboxykinase have a role in both amino acid and carbohydrate metabolism? Amino Acids 2001, 20:225-241.
• [50]Lehmann T, Ratajczak L: The pivotal role of glutamate dehydrogenase (GDH) in the mobilization of N and C from storage material to asparagine in germinating seeds of yellow lupine. J Plant Physiol 2008, 165:149-158.
• [51]Qui X, Wie W, Lian X, Zhang Q: Molecular analyses of the rice glutamate dehydronase gene family and their response to nitrogen and phosphorous deprivation. Plant Cell Rep 2009, 28:1115-1126.
• [52]Parrish CC, de Freitas ASW, Bodennec G, MacPherson EJ, Ackman RG: Lipid composition of the toxic marine diatom, Nitzschia pungens. Phytochemistry 1991, 30:113-116.
• [53]Black P, DiRusso C: Yeast acyl-CoA synthetases at the crossroads of fatty acid metabolism and regulation. Biochim Biophys Acta 2007, 1771:286-298.
• [54]Shockey J, Browse J: Genome-level and biochemical diversity of the acyl-activating enzyme superfamily in plants. Plant J 2011, 66:143-160.
• [55]Guillard RRL, Ryther JH: Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can J Microbiol 1962, 8:229-239.
• [56]Pocklington R, Milley JE, Bates SS, Bird CJ, de Freitas ASW, Quilliam MA: Trace determination of domoic acid in seawater and phytoplankton by high-performance liquid chromatography of the fluorenylmethoxycarbonyl (FMOC) derivative. Internat J Environ Anal Chem 1990, 38:351-368.
• [57]Garthwaite I, Ross K, Miles C, Hansen R, Foster D, Wilkins A, Towers N: Polyclonal antibodies to domoic acid, and their use in immunoassays for domoic acid in sea water and shellfish. Nat Toxins 1998, 6:93-104.
• [58]Clesceri LS, Greenberg AE, Trussell RR (Eds): Standard Methods for the Examination of Water and Wastewater. 17th edition. Washington, D.C: American Public Health Association; 1989.
• [59]Mullin JB, Riley JP: The colorimetric determination of silicate with special reference to sea and natural waters. Anal Chim Acta 1955, 12:162.
• [60]Strickland JDH, Parsons TR: A Manual of Seawater Analysis. Fisheries Research Board of Canada 1965.
• [61]Eaton AD, Clesceri LS, Greenberg AE (Eds): Standard Methods for the Examination of Water and Wastewater. 19th edition. Washington, D.C: American Public Health Association; 1995.
• [62]Murphy J, Riley JP: A modified single solution method for determination of phosphate in natural waters. Anal Chim Acta 1962, 27:31-36.
• [63]Bendschneider K, Robinson R: A new spectrophotometic method for the determination of nitrite in seawater. J Mar Res 1952, 11:87-96.
• [64]Wood E, Armstrong F, Richards F: Determination of nitrate in sea water by cadmium copper reduction to nitrite. J Mar Biol Assoc UK 1967, 47:23-31.
• [65]Das M, Harvey H, Chu LL, Sinha M, Pelletier J: Full-length cDNAs: more than just reaching the ends. Physiol Genomics 2001, 6:57-80.
• [66]Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
• [67]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410.
• [68]Conesa A, Götz S, García-Gómez J, Terol J, Talón M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics (Oxford, England) 2005, 21:3674-3676.
• [69]The Open Reading Frame Finder. http://www.ncbi.nlm.nih.gov/gorf/gorf.html webcite
• [70]Emanuelsson O, Brunak S, Von Heijne G, Nielsen H: Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2007, 2:953-971.
• [71]Apt KE, Zaslavkaia L, Lippmeier JC, Lang LC, Kilian O, Wetherbee R, Grossman AR, Kroth PG: In vivo characterization of diatom multipartite plastid targeting signals. J Cell Sci 2002, 115:4061-4069.
• [72]Gruber A, Vugrinec S, Hempel F, Gould SB, Maier U-G, Kroth PG: Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif. Plant Mol Biol 2007, 64:519-530.
• [73]Kilian O, Kroth PG: Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids. Plant J 2005, 41:175-183.
• [74]Edgar R, Barrett T: NCBI GEO standards and services for microarray data. Nat Biotechnol 2006, 24:1471-1472.
• [75]Park T, Yi SG, Kang SH, Lee S, Lee YS, Simon R: Evaluation of normalization methods for microarray data. BMC Bioinformatics 2003, 4:33. BioMed Central Full Text
• [76]Quackenbush J: Microarray data normalization and transformation. Nat Genet 2002, 32(Suppl):496-501.
• [77]Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 2001, 98:5116-5121.
• [78]Hong W, Tibshirani R, Chu G: Local false discovery rate facilitates comparison of different microarray experiments. Nucleic Acids Res 2009, 37:7483-7497.
• [79]Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C, Aach J, Ansorge W, CA B, Causton H, et al.: Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet 2001, 4:365-371.
• [80]Vandesompele J, Kubista M, Pfalffl MW: Reference gene validation software for improved normalization. In Real-time PCR: Current Technology and Application. Edited by Logan J, Edwards K, Saunders N. Norfolk: Caister Academic Press; 2009:47-64.
• [81]Rieu I, Powers SJ: Real-time quantitative RT-PCR: design, calculations, and statistics. Plant Cell 2009, 21:1031-1033.
• [82]Bustin S, Beaulieu J-F, Huggett J, Jaggi R, Kibenge F, Olsvik P, Penning L, Toegel S: MIQE precis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Molec Biol 2010, 11:74. BioMed Central Full Text
• [83]Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT: The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009, 55:611-622.