Abstract
Iron uptake by diatoms is a biochemical process with global biogeochemical implications. In large regions of the surface ocean diatoms are both responsible for the majority of primary production and frequently experiencing iron limitation of growth. The strategies used by these phytoplankton to extract iron from seawater constrain carbon flux into higher trophic levels and sequestration into sediments. In this study we use reverse genetic techniques to target putative iron-acquisition genes in the model pennate diatom Phaeodactylum tricornutum We describe components of a reduction-dependent siderophore acquisition pathway that relies on a bacterial-derived receptor protein and provides a viable alternative to inorganic iron uptake under certain conditions. This form of iron uptake entails a close association between diatoms and siderophore-producing organisms during low-iron conditions. Homologs of these proteins are found distributed across diatom lineages, suggesting the significance of siderophore utilization by diatoms in the marine environment. Evaluation of specific proteins enables us to confirm independent iron-acquisition pathways in diatoms and characterize their preferred substrates. These findings refine our mechanistic understanding of the multiple iron-uptake systems used by diatoms and help us better predict the influence of iron speciation on taxa-specific iron bioavailability.
Highlights
Detailed descriptions of cell lines, culturing conditions, analytical techniques, field work, and phylogenetic analyses are presented in SI Appendix
All cell lines used in this study were created from P. tricornutum strain CCMP 632 [46], and knockout cell lines were created using CRISPR/Cas9 and TALEN [90] technology, and selection conducted using the phleomycin antibiotic
Iron uptake rates were determined in short-term uptake assays using 59Fe-labeled substrates, and gallium uptake was measured in cell digests using inductively coupled plasma-mass spectrometry
Summary
Detailed descriptions of cell lines, culturing conditions, analytical techniques, field work, and phylogenetic analyses are presented in SI Appendix. All cell lines used in this study were created from P. tricornutum strain CCMP 632 [46], and knockout cell lines were created using CRISPR/Cas and TALEN [90] technology, and selection conducted using the phleomycin antibiotic. FBP1 complementation constructs contained the NAT gene for nourseothricin resistance and were selected on agar plates containing phleomycin and nourseothricin. All culturing was conducted using trace metal clean techniques, as was collection and preparation of natural seawater media. Iron uptake rates were determined in short-term uptake assays using 59Fe-labeled substrates, and gallium uptake was measured in cell digests using inductively coupled plasma-mass spectrometry. Ferric reductase activity was determined using a BPDS method [59]
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