Computational protocol: Globally Important Haptophyte Algae Use Exogenous Pyrimidine Compounds More Efficiently than Thiamin

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Protocol publication

[…] 18S rRNA gene sequences were retrieved from the MMETSP (https://imicrobe.us/project/view/104) metadata files and aligned using ClustalW. Positions with gaps were masked. Maximum likelihood phylogenetic trees were computed using the general time-reversible model in MEGA6 (). A discrete gamma distribution was used to model evolutionary rate differences among sites, and the rate variation model allowed for some sites to be evolutionarily variable. There were a total of 1,651 positions in the final data set. Data were bootstrapped using 100 replicates. Based on the topology of the 18S rRNA gene tree, each of the 28 MMETSP species was assigned to one of six established taxonomic orders. Subsequent reporting of thiamin pathway analysis uses this phylogeny to establish taxonomic grouping. A phylogenetic analysis was also performed on TENA_E and TENA_C protein sequences retrieved from the MMETSP peptide files. These were aligned using ClustalW, and gap positions were masked. Maximum likelihood phylogenetic trees were computed using the Whelan and Goldman +Freq. model in MEGA6 (). A discrete gamma distribution was used to model evolutionary rate differences among sites. There were a total of 193 positions in the final data set. Data were bootstrapped using 100 replicates. [...] Fifty-eight haptophyte transcriptomes from 28 species were obtained from the MMETSP database, as were the genomes of Emiliania huxleyi CCMP1516 () and Chrysochromulina tobin CCMP291 (). Peptide files predicted for all 58 MMETSP haptophyte transcriptomes were used to construct a searchable database and queried using Sequenceserver 0.8.0. Searches were performed for thiG, THI4, THIM, THIC, THID, THIE, THIL, TPK, and TENA using initial query sequences recovered from the genomes of E. huxleyi CCMP1516, Chrysochromulina sp. strain CCMP291, Guillardia theta CCMP2712, Paulinella chromatophora M0880, and Phaeodactylum tricornutum CCAP1055/1 () (). Sequences acquired using BLASTP and TBLASTN searches were further used as queries in iterative BLASTs against the haptophyte MMETSP data set (E value of ≤10−15). Identity assignment for the recovered sequences was based on subsequent BLASTP and BLASTX searches against the NCBI nr database as well as the Pfam-A database with a cutoff E value of ≤10−15. Since lack of presence in a transcriptome-predicted proteome cannot be interpreted as proof of absence of a protein from the genome, results were grouped into a higher level of taxonomic organization to increase the robustness of the conclusions. Note that when a protein was not detected in the predicted protein set, we also searched the transcript assemblies using TBLASTN prior to categorizing the protein as “not found.” Predicted TENA proteins were further characterized by active site residues as defined in Zea mays and A. thaliana (). A cysteine residue at the active site resulted in categorization as TENA_C. If the cysteine residue was lacking but two conserved glutamate residues were present, it was termed TENA_E.We also performed searches for thiG, THI4, THIC, THI, THIE, and TENA in 138 predicted protein sets representing 22 marine phyla in MMETSP as well as a broader set of prasinophyte algae. This was done using a database queried using Sequenceserver 0.8.0 with query protein sequences retrieved from the genomes of P. tricornutum CCAP1055/1, A. thaliana, and Saccharomyces cerevisiae (). [...] Differences in growth rates and cellular quotas between thiamin and pyrimidine analog treatments were tested using one-way analysis of variance (ANOVA) and Holm-Sidak tests for post hoc analysis using SigmaPlot 13 (Systat Software, Inc., USA). Minimum cellular quotas were calculated from maximum cell densities in thiamin and pyrimidine analog treatments based on regression analysis. […]

Pipeline specifications

Software tools Clustal W, MEGA, SequenceServer, BLASTP, TBLASTN, BLASTX, SigmaPlot
Databases Pfam iMicrobe MMETSP
Applications Miscellaneous, Phylogenetics, Amino acid sequence alignment
Organisms Emiliania huxleyi
Chemicals Thiamine