Influence of cobalamin scarcity on diatom molecular physiology and identification of a cobalamin acquisition protein

Abstract

Diatoms are responsible for ~40% of marine primary production and are key players in global carbon cycling. There is mounting evidence that diatom growth is influenced by cobalamin (vitamin B(12)) availability. This cobalt-containing micronutrient is only produced by some bacteria and archaea but is required by many diatoms and other eukaryotic phytoplankton. Despite its potential importance, little is known about mechanisms of cobalamin acquisition in diatoms or the impact of cobalamin scarcity on diatom molecular physiology. Proteomic profiling and RNA-sequencing transcriptomic analysis of the cultured diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana revealed three distinct strategies used by diatoms to cope with low cobalamin: increased cobalamin acquisition machinery, decreased cobalamin demand, and management of reduced methionine synthase activity through changes in folate and S-adenosyl methionine metabolism. One previously uncharacterized protein, cobalamin acquisition protein 1 (CBA1), was up to 160-fold more abundant under low cobalamin availability in both diatoms. Autologous overexpression of CBA1 revealed association with the outside of the cell and likely endoplasmic reticulum localization. Cobalamin uptake rates were elevated in strains overexpressing CBA1, directly linking this protein to cobalamin acquisition. CBA1 is unlike characterized cobalamin acquisition proteins and is the only currently identified algal protein known to be implicated in cobalamin uptake. The abundance and widespread distribution of transcripts encoding CBA1 in environmental samples suggests that cobalamin is an important nutritional factor for phytoplankton. Future study of CBA1 and other molecular signatures of cobalamin scarcity identified here will yield insight into the evolution of cobalamin utilization and facilitate monitoring of cobalamin starvation in oceanic diatom communities.

Fig. 1.

Effect of low cobalamin and iron availability on growth and protein expression in two diatoms. Cell density over time for T. pseudonana (A) and P. tricornutum (B) grown under four different nutrient regimes: low vitamin B₁₂, low Fe, low vitamin B₁₂ with low Fe, and replete. Values shown are means of triplicate cultures, and error bars are 1 SD. Arrows indicate where samples for proteomic and transcriptomic analyses were taken. Low cobalamin availability had a much larger impact on T. pseudonana growth than on the growth of P. tricornutum, likely attributable to P. tricornutum’s use of MetE as an alternative to the vitamin B₁₂-requiring MetH. Because of the low iron concentrations used, iron limitation had a more severe impact on growth than low vitamin B₁₂ in this the experiment. (C–F) Limitation scenarios were verified by resupplying cultures with cobalamin and iron. Fluorescence over time is shown as means of single measurements of triplicate cultures, with error bars representing 1 SD. Each culture in A and B (at arrows) was split in four and resupplied with nothing (control), vitamin B₁₂, iron, or vitamin B₁₂ and iron together. (C) Growth in low vitamin B₁₂ with low iron cultures of T. pseudonana was only rescued by the addition of both vitamin B₁₂ and iron together; fluorescence in +Fe cultures and +vitamin B₁₂ and Fe cultures was significantly different (Student’s paired t test, P = 0.0004; time = 8 d). (E) Iron addition alone rescued growth in low vitamin B₁₂ and iron P. tricornutum, and coaddition of vitamin B₁₂ and Fe together further enhanced growth; fluorescence in +Fe cultures and + vitamin B₁₂ and Fe cultures was significantly different (Student’s paired t test, P = 0.0007; time = 4 d). Shotgun MS analyses of T. pseudonana (G) in the low vitamin B₁₂ vs. replete treatment and P. tricornutum (H) in the low vitamin B₁₂ with low iron treatment vs. low iron treatment reveal vitamin B₁₂-responsive proteins. Each point is an identified protein, with the mean of its technical triplicate abundance scores in one treatment plotted against the mean of abundance scores in another treatment. The solid line is 1:1 abundance, and the dashed lines denote 2:1 and 1:2 abundances. Protein CBA1, Tp11697 and Pt 48322, is highlighted in pink, and MetE is highlighted in blue. Proteins in green are considered differentially abundant (Fisher exact test, P < 0.01).

Fig. 2.

Transcriptomic analyses reveal additional patterns in cobalamin-responsive gene products. (A and B) Comparative proteome and transcriptome responses to cobalamin deprivation are shown. All gene products for which there was both protein- and transcript-based quantitative information are displayed. The fold change (log₂) between the transcript abundance (RPKM value) in the cobalamin-starved and replete treatments is shown on the y axis, and the fold change (log₂) between the protein abundance (spectral counting score) in the cobalamin-starved and replete treatments is shown on the x axis. For the protein data, any null values were replaced with a spectral counting score of 0.33, the lowest measurable value in our experiments, to facilitate the computation. Generally, coherence between the proteome and transcriptome responses is limited to specific proteins that display enhanced abundance under cobalamin starvation in both the transcript and protein pools. These include CBA1, MetE, ThiC, and cytosolic SHMT, as noted by color and identified in the key. (C) Heat map displays select T. pseudonana transcript responses to cobalamin and iron starvation. Fold change RPKM values are shown for the low iron vs. replete, low vitamin B₁₂ with low Fe vs. low Fe, low vitamin B₁₂ with low Fe vs. replete, and low vitamin B₁₂ vs. replete treatments, with up-regulation denoted in red and down-regulation denoted in green. The genes were selected by high-to-low ordering of the log₂-transformed fold change RPKM values and sorted by the comparison between low vitamin B₁₂ vs. replete treatments. Gene products highlighted in A (ThiC, CBA1) are also highlighted in C.

Fig. 3.

CBA1 is much more abundant under low vitamin B₁₂ availability via three independent quantitative analyses. (A) Bars are means of spectral counting abundance scores for protein CBA1 in four treatments in both diatoms as measured via shotgun ion trap MS. Error bars represent 1 SD about the mean of technical triplicate measurements. (B) Bars are means of transcript RPKM abundance scores for CBA1 sequences in four treatments in both diatoms in RNA-seq transcriptomic analyses. Error bars represent 1 SD about the mean of biological duplicate measurements. (C) Absolute abundance of two peptides from CBA1 in P. tricornutum measured via the highly sensitive and quantitative technique SRM MS in two low vitamin B₁₂ and two replete cultures. Error bars are 1 SD about the mean of technical triplicate measurements.

Fig. 4.

Protein CBA1 is directly implicated in cobalamin acquisition. Protein CBA1 appears to be localized to the outside of the cell and likely the ER, and is directly implicated in cobalamin acquisition. Epifluorescent (A) and confocal (B) micrographs of protein CBA1 fused to YFP and overexpressed in P. tricornutum. YFP fluorescence is false-colored green, whereas chlorophyll a fluorescence is false-colored red. The side panels of the confocal image show the fluorescence distribution in the cross-sections of the central image indicated by the light yellow lines. (C) Cobalamin uptake rates by wt P. tricornutum and transgenic P. tricornutum cell lines overexpressing CBA1 (CBA1-OE1, CBA1-OE2) or Urease (Urease-OE1) measured over 24 h in exponential growth phase under vitamin B₁₂-replete conditions (n = 3). The growth rate over the 24-h experiment for the wt (WT) was 0.72 ± 0.07; for Urease-OE1, it was 1.01 ± 0.02; for CBA1-OE2, it was 1.10 ± 0.03; and for CBA1-OE1, it was 1.08 ± 0.03, given as mean of measurements on biological triplicate cultures ± 1 SD.

Fig. 5.

Transcripts encoding CBA1 are expressed in diverse marine environments. Phylogenetic tree containing CBA1 sequences from 454 metatranscriptomic (cDNA) libraries from the Ross Sea of the Southern Ocean, Monterey Bay, Puget Sound, and North Pacific Ocean. Reference sequences from P. tricornutum, F. cylindrus, T. pseudonana, A. anophagefferens, and E. siliculosus genomes were used to construct these trees (37) and are shown in black. CBA1-like sequences from environmental samples are shown in color, as described in the key. CBA1 transcripts were detectable in diverse marine environments, suggesting that cobalamin acquisition is an important component of diatom molecular physiology.

Fig. 6.

RNA-seq analysis reveals coregulation of MetE and a two-component sensor. RNA-seq coverage for an 11-kb region of the P. tricornutum genome is shown. Individual tracks are shown for each treatment, cobalamin and iron starvation, cobalamin starvation, iron starvation, and the replete control. The x axis shows the position in the genome, and the y axis (gray shading) shows the relative coverage of transcript data. Vertical color lines represent areas in the coverage mapping where there were mismatches of the reads to the reference genome (A = green, C = blue, G = yellow, T = red). The bottom track shows the gene models from the Joint Genome Institute 2.0 genome project. Transcripts mapping to cobalamin-independent methionine synthase (metE) are much more abundant under cobalamin scarcity and with low cobalamin and low iron. In addition, a two-component histidine kinase sensor appears to be coregulated with metE, and may thus play a role in the P. tricornutum response to cobalamin starvation.

Fig. 7.

Interconnections between methionine, folate, PLP metabolism, and cobalamin availability. (A) Schematic diagram describes the connections between PLP, folate (THF), methionine, and thiamine metabolism in two diatom species, displayed with supporting protein abundance data. The gene products involved in these pathways and their responses to cobalamin scarcity are shown for each diatom, as denoted in the key. The behavior of both transcripts and proteins are shown, with Pt indicating P. tricornutum (Left) and Tp indicating T. pseudonana (Right). Dark blue indicates that the gene product is more abundant under −vitamin B₁₂ vs. replete conditions and −vitamin B₁₂Fe vs. −Fe conditions, and lighter blue indicates that the gene product was more abundant under one of those conditions. Black denotes that there was no change observed between these conditions, and white indicates that the product was not detected. AHCase, adenosylhomocysteinase; cSHMT, cytosolic serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase. (B–E) Abundance patterns for select proteins included in the schematic above are displayed. Bar graphs of spectral counting abundance scores for proteins of interest are given for each of four treatments in both diatoms, where bars are means of technical triplicate measurements and error bars are 1 SD about the mean. Overall, these patterns suggest that there are interconnections between methionine, folate, PLP, and thiamine metabolism and cobalamin availability in diatoms.

Fig. 8.

Diatoms display three primary responses to cobalamin scarcity. Schematic representation of the three primary responses to cobalamin starvation in two diatoms. Both diatoms enhanced CBA1 production, likely in an effort to enhance cobalamin acquisition. The magnitude of the increase in CBA1 protein and transcripts was larger for T. pseudonana, likely because it has an absolute cobalamin requirement. P. tricornutum enhanced MetE production to reduce cobalamin demand. Both diatoms also appeared to conduct cellular rearrangements to cope with reduced methionine synthase activity, including enhanced cytosolic SHMT, MetK, and radical AdoMet (SAM) enzyme ThiC abundance under low cobalamin availability.

Fig. P1.

CBA1 is directly implicated in cobalamin acquisition. (A) Protein abundance scores show that CBA1 is much more abundant under low cobalamin availability (-B12, -B12Fe) compared with growth under replete conditions (Replete) or low iron (-Fe). (B) Measurements of cobalamin uptake rates by wild type (WT) P. tricornutum and two transgenic P. tricornutum cell lines overexpressing CBA1 or the unrelated protein urease as a control show that CBA1 is involved in cobalamin acquisition. Epifluorescent (C) and confocal (D) micrographs of CBA1 fused to YFP, overexpressed in P. tricornutum, suggest that CBA1 is a secreted protein localized to the cell periphery and is likely exported via the endoplasmic reticulum (white arrow). YFP fluorescence is false-colored green, and chlorophyll fluorescence is false-colored red. (E and F) Schematic representation of the diatom response to cobalamin scarcity: CBA1 (orange) possibly binds cobalamin and facilitates its uptake via additional, unidentified proteins (blue). Low cobalamin results in the use of MetE over MetH and enhanced MetK (S-adenosylmethionine synthase) and ThiC (thiamine biosynthesis protein) abundance.