Diatoms perform about 20% of photosynthesis on earth, play a crucial role in forming the base of the marine food web, and sequester anthropogenic carbon into the deep ocean through the biological pump. In some areas of the ocean, diatom growth is limited by the organic micronutrient cobalamin (vitamin B12). Cobalamin affects phytoplankton growth rate and biomass yield, though the consequences of cobalamin limitation are not well understood on the biochemical level. In a controlled laboratory setting, we grew two diatoms, Thalassiosira pseudonana and Navicula pelliculosa, under cobalamin limited and replete conditions to elucidate changes in metabolite pools under cobalamin limitation. Using targeted and untargeted metabolomics, we show that the cobalamin-dependent T. pseudonana experienced a metabolic cascade under cobalamin limitation that a ects the central methionine cycle, the transsulfuration pathway, and the composition of its osmolyte pools. N. pelliculosa, a cobalamin-independent diatom, also showed evidence for changes in the methionine cycle when grown without cobalamin, though to a lesser extent than what we observed in T. pseudonana. In both diatoms the concentration of 5'-methylthioadenosine decreased when limited for cobalamin, suggesting a disruption in the diatoms' polyamine biosynthesis. Furthermore, two acylcarnitines and adenosyl cobalamin accumulated in T. pseudonana under cobalamin limitation, suggesting the use of an adenosylcobalamin-dependent enzyme, methylmalonyl CoA mutase. Overall, these changes in metabolite pools yield insight into the metabolic consequences of cobalamin limitation in diatoms and suggest that cobalamin starvation may have consequences for microbial interactions that are based on metabolite production by phytoplankton.
Thalassiosira pseudonana protocols and data are reported in the current study MTBLS703.
Navicula pelliculosa protocols and data are reported in MTBLS708.
", "submissionDate": "2018-07-06", "publicReleaseDate": "2019-06-04", "people": [{"comments": [], "firstName": "Katherine", "lastName": "Heal", "email": "kheal@uw.edu", "affiliation": "University of Washington", "address": "", "fax": "", "midInitials": "R", "phone": "6126164840", "roles": [{"comments": [], "annotationValue": "Postdoctoral Scholar", "termSource": {"comments": [], "name": "NCIT", "file": "http://data.bioontology.org/ontologies/NCIT", "version": "44", "description": "National Cancer Institute Thesaurus"}, "termAccession": "http://ncicb.nci.nih.gov/xml/owl/EVS/Thesaurus.owl"}]}, {"comments": [], "firstName": "Katherine R", "lastName": "Heal", "email": "", "affiliation": "School of Oceanography, University of Washington, Seattle, WA 98195, USA.", "address": "", "fax": "", "midInitials": "", "phone": "", "roles": [{"comments": [], "annotationValue": "Author", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": "http://purl.obolibrary.org/obo/NCIT_C42781"}]}, {"comments": [], "firstName": "Natalie A", "lastName": "Kellogg", "email": "", "affiliation": "School of Oceanography, University of Washington, Seattle, WA 98195, USA.", "address": "", "fax": "", "midInitials": "", "phone": "", "roles": [{"comments": [], "annotationValue": "Author", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": "http://purl.obolibrary.org/obo/NCIT_C42781"}]}, {"comments": [], "firstName": "Laura T", "lastName": "Carlson", "email": "", "affiliation": "School of Oceanography, University of Washington, Seattle, WA 98195, USA.", "address": "", "fax": "", "midInitials": "", "phone": "", "roles": [{"comments": [], "annotationValue": "Author", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": "http://purl.obolibrary.org/obo/NCIT_C42781"}]}, {"comments": [], "firstName": "Regina M", "lastName": "Lionheart", "email": "", "affiliation": "School of Oceanography, University of Washington, Seattle, WA 98195, USA.", "address": "", "fax": "", "midInitials": "", "phone": "", "roles": [{"comments": [], "annotationValue": "Author", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": "http://purl.obolibrary.org/obo/NCIT_C42781"}]}, {"comments": [], "firstName": "Anitra E", "lastName": "Ingalls", "email": "", "affiliation": "School of Oceanography, University of Washington, Seattle, WA 98195, USA. 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Using a combusted glass apparatus and gentle vacuum filtration, we harvested two 35 ml cultures onto one 47 mm 0.2 \u03bcm PTFE filters when the cultures were in exponential phase.", "uri": "", "version": "", "parameters": [{"comments": [], "parameterName": {"comments": [], "annotationValue": "", "termSource": null, "termAccession": ""}}], "components": []}, {"comments": [], "name": "Extraction", "protocolType": {"comments": [], "annotationValue": "Extraction", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}, "description": "Filters of samples and media blanks were extracted as previously described[1]. Briefly, polar and nonpolar metabolites were extracted using a modified Bligh\u2013Dyer extraction[2][3] using 1:1 methanol/water (aqueous phase) and dichloromethane (organic phase). Methodological blanks were extracted and analyzed along with each sample set. To aid in normalization, we added a cocktail of internal standards (listed in Table S7 in the paper associated with this study) before and after extraction. Ref: [1] Boysen AK, Heal KR, Carlson LT, Ingalls AE. Best-Matched Internal Standard Normalization in Liquid Chromatography-Mass Spectrometry Metabolomics Applied to Environmental Samples. Anal Chem. 2018 Jan 16;90(2):1363-1369. doi:10.1021/acs.analchem.7b04400. PMID:29239170 [2] Bligh EG and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911-7. doi:10.1139/o59-099. PMID:13671378 [3] Canelas AB, ten Pierick A, Ras C, Seifar RM, van Dam JC, van Gulik WM, Heijnen JJ. Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics. Anal Chem. 2009 Sep 1;81(17):7379-89. doi:10.1021/ac900999t. PMID:19653633", "uri": "", "version": "", "parameters": [{"comments": [], "parameterName": {"comments": [], "annotationValue": "Derivatization", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}}, {"comments": [], "parameterName": {"comments": [], "annotationValue": "Post Extraction", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}}], "components": []}, {"comments": [], "name": "Chromatography", "protocolType": {"comments": [], "annotationValue": "Chromatography", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}, "description": "To obtain metabolomic data, we separated compounds using reverse phase (RP, for the organic and aqueous fractions) and hydrophilic interaction liquid chromatography (HILIC, for aqueous fraction only), using the exact specifications as previously described[1]. After extraction, samples were run within 24 h for HILIC and within 96 h for RP; between extraction and analysis, samples were stored at -80 \u00b0C. For reversed phase (RP) chromatography, a Waters Acquity UPLC HSS Cyano column (1.8 \u03bcm particle size, 2.1 mm x 100 mm) equipped with a Waters Acquity UPLC HSS Cyano guard column (1.8 \u03bcm particle size, 2.1 mm x 5 mm) was used with 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) at a flow rate of 0.4 ml/min. The column was held at 5% B for 2 min, ramped to 100% B over 16 min, held at 100% B for 2 min, and equilibrated at 5% B for 5 min (25 min total). The column was maintained at 35 \u00b0C. The injection volume was 15 \u03bcl. When starting a batch, the column was equilibrated at the starting conditions for at least 10 min; several water blanks were run before and throughout a batch. After a batch, the column was flushed with 100% acetonitrile for 20 to 30 min. For HILIC chromatography, a SeQuant ZIC-pHILIC column (5 mm particle size, 2.1 mm x 150 mm, from Millipore) was used with 10 mM ammonium carbonate in 85:15 acetonitrile to water (Solvent A) and 10 mM ammonium carbonate in 60:40 water to acetonitrile (Solvent B) at a flow rate of 0.15 ml/min. The column was held at 100% A for 2 min, ramped to 100% B over 18 min, held at 100% B for 5 min, and equilibrated at 100% A for 25 min (50 min total). The column was maintained at 30 \u00b0C. The injection volume was 2 \u03bcl. Starting a batch, the column was equilibrated at the starting conditions for at least 30 min. To improve the performance of the HILIC column, we maintained the same injection volume, kept the instrument running water blanks between samples as necessary, and injected standards in a representative matrix in addition to standards in water. After each batch, the column was flushed with 10 mM ammonium carbonate in 85:15 water to acetonitrile for 20 to 30 min. Ref: [1] Boysen AK, Heal KR, Carlson LT, Ingalls AE. Best-Matched Internal Standard Normalization in Liquid Chromatography-Mass Spectrometry Metabolomics Applied to Environmental Samples. Anal Chem. 2018 Jan 16;90(2):1363-1369. doi:10.1021/acs.analchem.7b04400. 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Full scan (non-fragmented mass to charge (m/z)) data acquisition from the QE has been published previously[1]. Throughout the run, we ran a pooled sample several times in order to monitor signal stability and train normalization. For untargeted metaboloics analysis, we collected fragmentation spectra (MS2) on our pooled samples using data-dependent acquisition (DDA). For samples separated via HILIC, separate injections were analyzed in positive and negative ion modes. MS2 spectra were collected from 50-500 m/z at resolution of 30,000 at 200 m/z. For each MS scan, DDA was set to perform on the top 5 most abundant ions with dynamic exclusion of 20 s using a normalized collision induced dissociation at 35 V. For RP, samples were only run in positive mode; DDA was performed in the same manner but with a dynamic exclusion time of 10 s due to narrower peaks than in HILIC. Ref: [1] Boysen AK, Heal KR, Carlson LT, Ingalls AE. Best-Matched Internal Standard Normalization in Liquid Chromatography-Mass Spectrometry Metabolomics Applied to Environmental Samples. Anal Chem. 2018 Jan 16;90(2):1363-1369. doi:10.1021/acs.analchem.7b04400. PMID:29239170", "uri": "", "version": "", "parameters": [{"comments": [], "parameterName": {"comments": [], "annotationValue": "Scan polarity", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}}, {"comments": [], "parameterName": {"comments": [], "annotationValue": "Mass analyzer", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}}, {"comments": [], "parameterName": {"comments": [], "annotationValue": "Ion source", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}}, {"comments": [], "parameterName": {"comments": [], "annotationValue": "Instrument", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}}, {"comments": [], "parameterName": {"comments": [], "annotationValue": "Scan m/z range", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}}], "components": []}, {"comments": [], "name": "Data transformation", "protocolType": {"comments": [], "annotationValue": "Data transformation", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}, "description": "Data collected for MS and MS2 from the QE mass spectrometer were first converted to .mzxml using MSConvert[1] with positive and negative scans processed separately. Ref: [1] Chambers, MC et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol. 2012 Oct;30(10):918-20. doi: 0.1038/nbt.2377. PMID:23051804", "uri": "", "version": "", "parameters": [{"comments": [], "parameterName": {"comments": [], "annotationValue": "", "termSource": null, "termAccession": ""}}], "components": []}, {"comments": [], "name": "Metabolite identification", "protocolType": {"comments": [], "annotationValue": "Metabolite identification", "termSource": {"comments": [], "name": "", "file": "http://purl.obolibrary.org/obo/ncit.owl", "version": "18.10e", "description": "NCI Thesaurus OBO Edition"}, "termAccession": ""}, "description": "For mass features without a standard match but with MS2, we searched against the publicly available LC/MS MS2 spectral databases, including MassBank[1], Global Natural Products Social Molecular Networking[2], MetiTree[3], RIKEN tandem mass spectral database[4], and the Human Metabolome Database[5] (all downloaded from http://mona.fiehnlab.ucdavis.edu/downloads on November 17, 2017). We searched for spectra with matching parent m/z and a cosine similarity > 0.8 from an ESI spectrum, using the same algorithms as MassBank[1]. When a compound in the MassBank database matched m/z and MS2, we assigned a putative identification (confidence level 2). For some of these compounds, we later obtained standards and upgraded their identification to confidence level 1. For mass features without confidence levels 1 or 2 identification, we searched for possible matches to compounds in the KEGG database based only on m/z, these identifications are considered possible identification (confidence level 3). Our extraction analysis resulted in both a highly polar aqueous and a more non-polar organic fraction, which were analyzed separately. Although there are many more mass features in the organic fraction (see Table S8 in the paper associated with this study), most of the primary metabolites are extracted into the aqueous fraction[6], and we focused our untargeted analysis on this fraction to understand the primary metabolic response to cobalamin limitation. For many mass features, the aforementioned identification attempts were not fruitful, so we attempted to identify quality mass features that showed a univariate response to cobalamin limitation from our aqueous extraction, using a manual approach with the larger, but not publicly available, Metlin database[7]. We searched mass features by m/z (assuming M+H, M+NH4, or M+Na for positive ionization, M-H, M+Cl for negative ionization, at a 5 ppm tolerance) and compared MS2 when obtained by DDA and available in the database. We assigned putative identifications using the same confidence levels as discussed above. Ref: [1] Horai H et al. MassBank: a public repository for sharing mass spectral data for life sciences. J Mass Spectrom. 2010 Jul;45(7):703-14. doi:10.1002/jms.1777. PMID:20623627 [2] Wang M et al. Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nat Biotechnol. 2016 Aug 9;34(8):828-837. doi:10.1038/nbt.3597. PMID:27504778 [3] Vaniya A and Fiehn O. 2015. Using fragmentation trees and mass spectral trees for identifying unknown compounds in metabolomics. Trends Analyt Chem. 2015 Jun 1;69:52-61. doi:10.1016/j.trac.2015.04.002. PMID:26213431 [4] Sawada Y, Nakabayashi R, Yamada Y, Suzuki M, Sato M, Sakata A, Akiyama K, Sakurai T, Matsuda F, Aoki T, Hirai MY, Saito K. RIKEN tandem mass spectral database (ReSpect) for phytochemicals: a plant-specific MS/MS-based data resource and database. Phytochemistry. 2012 Oct;82:38-45. doi:10.1016/j.phytochem.2012.07.007. PMID:22867903 [5] Wishart DS et al. HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35(Database issue):D521-6. doi:10.1093/nar/gkl923. PMID:17202168 [6] Boysen AK, Heal KR, Carlson LT, Ingalls AE. Best-Matched Internal Standard Normalization in Liquid Chromatography-Mass Spectrometry Metabolomics Applied to Environmental Samples. Anal Chem. 2018 Jan 16;90(2):1363-1369. doi:10.1021/acs.analchem.7b04400. PMID:29239170 [7] Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem. 2006 Feb 1;78(3):779-87. doi:10.1021/ac051437y. 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