Research ArticleMICROBIOLOGY

Endocytosis-mediated siderophore uptake as a strategy for Fe acquisition in diatoms

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Science Advances  16 May 2018:
Vol. 4, no. 5, eaar4536
DOI: 10.1126/sciadv.aar4536
  • Fig. 1 Iron uptake from different iron sources by T. oceanica, P. tricornutum, and T. pseudonana.

    The figure shows specificity of siderophore uptake in three species of diatoms. The cells were precultured in iron-deficient medium, harvested at mid-exponential growth, and washed once with iron-free medium. The cells were then resuspended in growth medium containing one of the following 55Fe-labeled sources supplied at 1 μM: ferric EDTA (black), FCH (red), FOB (blue), or ENT (green). The cells were washed on filters at intervals with the washing buffer, and 55Fe associated to the washed cells was counted by scintillation. Data are means ± SD from four experiments (biological replicates).

  • Fig. 2 P. tricornutum ISIP1 knockdown cells are unable to grow on FOB as the sole source of iron.

    (A) P. tricornutum is able to grow on 15 pM Fe′ (black line) and 10 nM FOB (red line) as sole Fe sources in Aquil media. The ISIP1 knockdown (k/d) line shows reduced growth on Fe′ (blue line) and no growth on FOB (green). Error bars denote SDs from three biological replicates. Before the start of the experiment, cultures were depleted of Fe through a 10-day preculture on low-Fe Aquil. (B) Comparison of siderophore uptake by P. tricornutum wild-type (WT) cells (rhombus markers) versus ISIP1 (square) and ISIP2a (triangle) knockdown cell lines.

  • Fig. 3 Iron uptake from siderophores and from ferric EDTA is not regulated in the same way.

    The figure shows rates of iron uptake from ferric EDTA (A) and FOB (B) as well as ferrireductase activity (C) as a function of time in wild-type (black) and ISIP1 knockdown cell lines (red) grown in iron-rich (closed symbols) or iron-deficient medium (open symbols). The cells were grown for 1 week in iron-rich medium (1 μM ferric citrate in Mf medium), washed twice with iron-free Mf medium, and re-inoculated at 2 × 106 cells/ml in iron-free (open symbols) or iron-rich medium (1 μM ferric citrate; closed symbols). The cultures were diluted every 2 days with the same media to maintain cell density at 2 × 106 cells/ml for 2 weeks. For ferrireductase activity, initial rates of uptake (first 30 min of kinetics) are plotted against the day since the start of the experiment. Data are means ± SD from four experiments (two biological replicates with two technical replicates). ISIP1 knockdown cell lines show a marked descrease in Fe uptake from FOB, but not from ferric EDTA, and a higher ferrireductase activity than wild-type cells. Maximal induction of FOB uptake activity under iron-deficient condition occurred before maximal induction of ferric EDTA uptake activity.

  • Fig. 4 Siderophores are taken up into the cell by endocytosis.

    (A) Cells of the wild-type (top) and ISIP1 knockdown line (bottom) were grown for 5 days in iron-deficient medium, concentrated to about 5 × 107 cells/ml, and then incubated for 15 hours with 1 μM of the fluorescent conjugate of desferrioxamine B complexed with gallium (Ga-DFOB-NBD). Intracellular accumulation of the fluorescent siderophore analog was only observed in wild-type cells. Scale bar, 5 μm. (B) DAPI (4′,6-diamidino-2-phenylindole) staining of the cells (scale bar, 5 μm) to reveal that nuclear localization confirmed siderophore localization in a vesicle close to the chloroplast (red, chloroplast; blue, nucleus; green, fluorescent siderophore). (C) Iron uptake from 1 μM FOB (left) and from 1 μM ferric EDTA (right) by wild-type (black circles) and ISIP1 knockdown cells (red squares) in the presence (closed symbols) or absence (open symbols) of 10 μM of the endocytosis inhibitor dynasore. Dynasore inhibited iron uptake from FOB and enhanced iron uptake from ferric EDTA. (D) Effect of other endocytosis inhibitors on the rate of iron uptake from FOB and ferric EDTA (1 μM). Cells were preincubated for 10 min with the following inhibitors before adding 55Fe: dynasore (10 μM), dansylcadaverine (100 μM), phenylarsine oxide (10 μM), dimethyl amiloride (50 μM), chlorpromazine (50 μM), or methyl-β-cyclodextrin (1 mM) [added from 1000× stock solutions in dimethyl sulfoxide (DMSO); pure DMSO was added as a control].

  • Fig. 5 Siderophores are taken into the chloroplast.

    Effect of FOB coupled to protoporphyrinogen oxidase inhibitors on the growth and chlorophyll content of wild-type and ISIP1 knockdown cells shows that siderophores taken up by the cells can reach the chloroplast. In these experiments, chemical derivatives of the siderophore FOB were used in a Trojan horse approach to reach (or not) the target of the inhibitors coupled to FOB (inhibitors of protoporphyrinogen oxidase, one of the last enzymes of heme and chlorophyll biosynthesis). Wild-type and ISIP1 knockdown cells were inoculated at about 5 × 105 cells/ml in the Mf medium containing 1 to 5 μM of FOB-DPE (with a 10% excess iron, that is, an Fe/ligand ratio of 1.1 Fe for 1 DFOB) coupled to diphenyl-ether (FOB-DPE) (A and B) or to acifluorfen (FOB-AF) (C). Cell growth (top; black circles, wild-type; red squares, ISIP1 knockdown) and cell chlorophyll content (bottom; black circles, wild type; red squares, ISIP1 knockdown) were monitored by flow cytometry (see Materials and Methods; chlorophyll fluorescence is expressed in 105× FL3 units). (D) Wild-type cells were grown in the presence of 5 μM of the unconjugated forms of AF (circles) or DPE (squares). In control experiments (E), wild-type (black circles) and ISIP1 knockdown cells (red squares) were grown in the presence of 1 μM FOB (uncoupled, with a 10% excess iron; closed symbols), and wild-type cells were grown with 1 μM FOB-DPE plus an excess of FOB (10 μM, uncoupled; open circles). Data are means ± SD from four experiments (biological replicates).

  • Fig. 6 ISIP1 presence in eukaryotes and distribution/expression in the global ocean.

    (A) Heatmap showing the presence of homologs of P. tricornutum ISIP1, identified by reciprocal BLAST best-hit search with a threshold e value of 1 × 10−5, against different eukaryotic species contained in a curated library consisting of decontaminated sequences from MMETSP, supplemented with a further 79 eukaryotic genome and transcriptome libraries (table S1) (41). Cells in color correspond to the presence of a homologous sequence in a particular strain; gray cells indicate species queried where no homolog was detected. Lineages are shown per the consensus topologies obtained from recent phylogenomic studies of nuclear genomes and are shaded by origin. Two transfer events, labeled with curved red arrows, demonstrate (A) a proposed plastid-associated gene transfer event that occurred from a common ancestor of pelagophytes and dictyochophytes into a common ancestor of the haptophyte lineage (41), and (B) the inferred origin point of the haptophyte-derived plastids found in fucoxanthin-containing dinoflagellates. (B) Analysis of abundance of ISIP1 sequences in the Tara Oceans metagenome and metatranscriptome data set, determined for seven ocean provinces [North Pacific Ocean (NPO), Southern Ocean (SO), South Pacific Ocean (SPO), North Atlantic Ocean (NAO), South Atlantic Ocean (SAO), Mediterranean Sea (MS), and Indian Ocean (IO)], and given as RPKM (indicated by size of circles; normalized against a set of housekeeping genes and transcripts; see Materials and Methods). The position of the circles on the diatom 18S phylogeny tree reflects the degree of taxonomic assignation from the sequence data.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaar4536/DC1

    fig. S1. Siderophore uptake in P. tricornutum fits simple Michaelis-Menten kinetics.

    fig. S2. Construction of an ISIP1 knockdown in P. tricornutum and expression of ISIP1 transcript and protein.

    fig. S3. Siderophore uptake involves a binding step.

    fig. S4. Siderophore uptake involves endocytosis.

    fig. S5. ISIP1 knockdown lines are defective in endocytosis.

    fig. S6. ISIP1-YFP localization and abundance under different Fe supplementation regimes.

    fig. S7. ISIP1 predicted protein sequence and domain features.

    fig. S8. ISIP1 phylogenetic tree.

    movie S1. Live cell imaging of the uptake of NBD conjugate of FOB (FOB-NBD) by P. tricornutum.

    table S1. Data used to construct Fig. 6 and fig. S8.

    Reference (61)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Siderophore uptake in P. tricornutum fits simple Michaelis-Menten kinetics.
    • fig. S2. Construction of an ISIP1 knockdown in P. tricornutum and expression of ISIP1 transcript and protein.
    • fig. S3. Siderophore uptake involves a binding step.
    • fig. S4. Siderophore uptake involves endocytosis.
    • fig. S5. ISIP1 knockdown lines are defective in endocytosis.
    • fig. S6. ISIP1-YFP localization and abundance under different Fe supplementation regimes.
    • fig. S7. ISIP1 predicted protein sequence and domain features.
    • fig. S8. ISIP1 phylogenetic tree.
    • Legend for movie S1
    • Reference (61)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mov format). Live cell imaging of the uptake of NBD conjugate of FOB (FOB-NBD) by P. tricornutum.
    • table S1 (Microsoft Excel format). Data used to construct Fig. 6 and fig. S8.

    Files in this Data Supplement:

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