Investigations of phytoplankton responses to iron stress in seawater are complicated by the fact that iron concentrations do not necessarily reflect bioavailability. Most studies to date have been based on single species or field samples and are problematic to interpret. Here, we report results from an experimental cocultivation model system that enabled us to evaluate interspecific competition as a function of iron content and form, and to study the effect of nutritional conditions on the proteomic profiles of individual species. Our study revealed that the dinoflagellate Amphidinium carterae was able to utilize iron from a hydroxamate siderophore, a strategy that could provide an ecological advantage in environments where siderophores present an important source of iron. Additionally, proteomic analysis allowed us to identify a potential candidate protein involved in iron acquisition from hydroxamate siderophores, a strategy that is largely unknown in eukaryotic phytoplankton.

• Klíčová slova
• (s)PLS-DA, (sparse) partial least squares discriminant analysis, AUC, area under curve, Amphidinium carterae, AtpE, ATP synthase, BCS, bathocuproinedisulfonic acid disodium salt, CREG1, cellular repressor of E1A stimulated genes 1, DFOB, desferrioxamine B, EDTA, ethylenediaminetetraacetic acid, ENT, enterobactin, FACS, fluorescence-activated cell sorting, FBAI, fructose-bisphosphate aldolase I, FBAII, fructose-bisphosphate aldolase II, FBP1, putative ferrichrome-binding protein, FOB, ferrioxamine B, Flow cytometry, ISIP, iron starvation induced protein, Iron, LHCX, light-harvesting complex subunits, LL, long-term iron limitation, LR, iron enrichment, Marine microalgae, NBD, nitrobenz-2-oxa-1,3-diazole, NPQ, nonphotochemical quenching, PAGE, polyacrylamide gel electrophoresis, PSI, photosystem I, PSII, photosystem II, PetA, cytochrome b6/f, Proteomics, PsaC, photosystem I iron-sulfur center, PsaD, photosystem I reaction center subunit II, PsaE, photosystem I reaction center subunit IV, PsaL, photosystem I reaction center subunit XI, PsbC, photosystem II CP43 reaction center protein, PsbV, cytochrome c-550, RR, long-term iron sufficiency, SOD1, superoxide dismutase [Cu-Zn], Siderophores,
• Publikační typ
• časopisecké články MeSH

UNLABELLED: The model pennate diatom Phaeodactylum tricornutum is able to assimilate a range of iron sources. It therefore provides a platform to study different mechanisms of iron processing concomitantly in the same cell. In this study, we follow the localization of three iron starvation induced proteins (ISIPs) in vivo, driven by their native promoters and tagged by fluorophores in an engineered line of P. tricornutum. We find that the localization patterns of ISIPs are dynamic and variable depending on the overall iron status of the cell and the source of iron it is exposed to. Notwithstanding, a shared destination of the three ISIPs both under ferric iron and siderophore-bound iron supplementation is a globular compartment in the vicinity of the chloroplast. In a proteomic analysis, we identify that the cell engages endocytosis machinery involved in the vesicular trafficking as a response to siderophore molecules, even when these are not bound to iron. Our results suggest that there may be a direct vesicle traffic connection between the diatom cell membrane and the periplastidial compartment (PPC) that co-opts clathrin-mediated endocytosis and the "cytoplasm to vacuole" (Cvt) pathway, for proteins involved in iron assimilation. Proteomics data are available via ProteomeXchange with identifier PXD021172. HIGHLIGHT: The marine diatom P. tricornutum engages a vesicular network to traffic siderophores and phytotransferrin from the cell membrane directly to a putative iron processing site in the vicinity of the chloroplast.

• Klíčová slova
• P. tricornutum, diatoms, fluorescent proteins, iron, iron starvation induced proteins, proteome, siderophores,
• Publikační typ
• časopisecké články MeSH

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed; however, proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

• Klíčová slova
• APEX2, chloroplast, diatom, infectious disease, iron, metal trafficking, microbiology, phytotransferrin, plant biology,
• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• chloroplasty metabolismus   MeSH
• multigenová rodina MeSH
• proteomika metody   MeSH
• rozsivky genetika   metabolismus   MeSH
• transferin metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• transferin MeSH
• železo MeSH

Oceanic phytoplankton species have highly efficient mechanisms of iron acquisition, as they can take up iron from environments in which it is present at subnanomolar concentrations. In eukaryotes, three main models were proposed for iron transport into the cells by first studying the kinetics of iron uptake in different algal species and then, more recently, by using modern biological techniques on the model diatom Phaeodactylum tricornutum. In the first model, the rate of uptake is dependent on the concentration of unchelated Fe species, and is thus limited thermodynamically. Iron is transported by endocytosis after carbonate-dependent binding of Fe(III)' (inorganic soluble ferric species) to phytotransferrin at the cell surface. In this strategy the cells are able to take up iron from very low iron concentration. In an alternative model, kinetically limited for iron acquisition, the extracellular reduction of all iron species (including Fe') is a prerequisite for iron acquisition. This strategy allows the cells to take up iron from a great variety of ferric species. In a third model, hydroxamate siderophores can be transported by endocytosis (dependent on ISIP1) after binding to the FBP1 protein, and iron is released from the siderophores by FRE2-dependent reduction. In prokaryotes, one mechanism of iron uptake is based on the use of siderophores excreted by the cells. Iron-loaded siderophores are transported across the cell outer membrane via a TonB-dependent transporter (TBDT), and are then transported into the cells by an ABC transporter. Open ocean cyanobacteria do not excrete siderophores but can probably use siderophores produced by other organisms. In an alternative model, inorganic ferric species are transported through the outer membrane by TBDT or by porins, and are taken up by the ABC transporter system FutABC. Alternatively, ferric iron of the periplasmic space can be reduced by the alternative respiratory terminal oxidase (ARTO) and the ferrous ions can be transported by divalent metal transporters (FeoB or ZIP). After reoxidation, iron can be taken up by the high-affinity permease Ftr1.

• Klíčová slova
• iron, iron uptake, micro-algae, ocean, phytoplankton,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

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.

• Klíčová slova
• diatom, ferric reductase, iron acquisition, phytoplankton, siderophore,
• MeSH
• biologická dostupnost MeSH
• biologický transport MeSH
• CRISPR-Cas systémy MeSH
• druhová specificita MeSH
• FMN-reduktasa genetika   metabolismus   MeSH
• fylogeneze MeSH
• galium metabolismus   MeSH
• genový knockout MeSH
• klimatické změny MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• mikrobiota MeSH
• mořská voda chemie   MeSH
• oxidace-redukce MeSH
• proteiny vnější bakteriální membrány metabolismus   MeSH
• receptory buněčného povrchu metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• rozsivky genetika   růst a vývoj   metabolismus   MeSH
• siderofory metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• ferric citrate iron reductase MeSH Prohlížeč
• FMN-reduktasa MeSH
• galium MeSH
• membránové transportní proteiny MeSH
• proteiny vnější bakteriální membrány MeSH
• receptory buněčného povrchu MeSH
• rekombinantní fúzní proteiny MeSH
• siderofory MeSH
• siderophore receptors MeSH Prohlížeč
• železo MeSH

Phytoplankton growth is limited in vast oceanic regions by the low bioavailability of iron. Iron fertilization often results in diatom blooms, yet the physiological underpinnings for how diatoms survive in chronically iron-limited waters and outcompete other phytoplankton when iron becomes available are unresolved. We show that some diatoms can use siderophore-bound iron, and exhibit a species-specific recognition for siderophore types. In Phaeodactylum tricornutum, hydroxamate siderophores are taken up without previous reduction by a high-affinity mechanism that involves binding to the cell surface followed by endocytosis-mediated uptake and delivery to the chloroplast. The affinity recorded is the highest ever described for an iron transport system in any eukaryotic cell. Collectively, our observations suggest that there are likely a variety of iron uptake mechanisms in diatoms besides the well-established reductive mechanism. We show that iron starvation-induced protein 1 (ISIP1) plays an important role in the uptake of siderophores, and through bioinformatics analyses we deduce that this protein is largely diatom-specific. We quantify expression of ISIP1 in the global ocean by querying the Tara Oceans atlas of eukaryotic genes and show a link between the abundance and distribution of diatom-associated ISIP1 with ocean provinces defined by chronic iron starvation.

• MeSH
• chloroplasty metabolismus   MeSH
• druhová specificita MeSH
• endocytóza * MeSH
• genový knockdown MeSH
• rozsivky fyziologie   MeSH
• siderofory metabolismus   MeSH
• transport proteinů MeSH
• vodní organismy metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• siderofory MeSH
• železo MeSH

In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton. Although most dissolved iron in the marine environment is complexed with organic molecules, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts, including the ferric iron-concentrating protein ISIP2A, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine environmental genomic datasets, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

• MeSH
• biologický transport MeSH
• endocytóza MeSH
• fytoplankton klasifikace   genetika   metabolismus   MeSH
• genom genetika   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• molekulární evoluce MeSH
• mořská voda chemie   MeSH
• rozsivky genetika   metabolismus   MeSH
• transferin metabolismus   MeSH
• uhličitany metabolismus   MeSH
• vodní organismy klasifikace   genetika   metabolismus   MeSH
• železo metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• transferin MeSH
• uhličitany MeSH
• železo MeSH

Microbial biodegradation and biotransformation reactions are essential to most bioremediation processes, yet the specific organisms, genes, and mechanisms involved are often not well understood. Stable isotope probing (SIP) enables researchers to directly link microbial metabolic capability to phylogenetic and metagenomic information within a community context by tracking isotopically labeled substances into phylogenetically and functionally informative biomarkers. SIP is thus applicable as a tool for the identification of active members of the microbial community and associated genes integral to the community functional potential, such as biodegradative processes. The rapid evolution of SIP over the last decade and integration with metagenomics provide researchers with a much deeper insight into potential biodegradative genes, processes, and applications, thereby enabling an improved mechanistic understanding that can facilitate advances in the field of bioremediation.

• MeSH
• biodegradace * MeSH
• fylogeneze MeSH
• izotopové značení metody   MeSH
• látky znečišťující životní prostředí metabolismus   MeSH
• metagenomika metody   trendy   MeSH
• uhlík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• látky znečišťující životní prostředí MeSH
• uhlík MeSH

We investigated iron uptake mechanisms in five marine microalgae from different ecologically important phyla: the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, the prasinophyceae Ostreococcus tauri and Micromonas pusilla, and the coccolithophore Emiliania huxleyi. Among these species, only the two diatoms were clearly able to reduce iron, via an inducible (P. tricornutum) or constitutive (T. pseudonana) ferrireductase system displaying characteristics similar to the yeast (Saccharomyces cerevisiae) flavohemoproteins proteins. Iron uptake mechanisms probably involve very different components according to the species, but the species we studied shared common features. Regardless of the presence and/or induction of a ferrireductase system, all the species were able to take up both ferric and ferrous iron, and iron reduction was not a prerequisite for uptake. Iron uptake decreased with increasing the affinity constants of iron-ligand complexes and with increasing ligand-iron ratios. Therefore, at least one step of the iron uptake mechanism involves a thermodynamically controlled process. Another step escapes to simple thermodynamic rules and involves specific and strong binding of ferric as well as ferrous iron at the cell surface before uptake of iron. Binding was paradoxically increased in iron-rich conditions, whereas uptake per se was induced in all species only after prolonged iron deprivation. We sought cell proteins loaded with iron following iron uptake. One such protein in O. tauri may be ferritin, and in P. tricornutum, Isip1 may be involved. We conclude that the species we studied have uptake systems for both ferric and ferrous iron, both involving specific iron binding at the cell surface.

• MeSH
• autoradiografie MeSH
• biologické modely MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• chelátory železa farmakologie   MeSH
• FMN-reduktasa metabolismus   MeSH
• fylogeneze MeSH
• kinetika MeSH
• ligandy MeSH
• mikrořasy účinky léků   enzymologie   růst a vývoj   metabolismus   MeSH
• oxidace-redukce účinky léků   MeSH
• Saccharomyces cerevisiae účinky léků   metabolismus   MeSH
• transport elektronů účinky léků   MeSH
• vodní organismy růst a vývoj   metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• chelátory železa MeSH
• ferric citrate iron reductase MeSH Prohlížeč
• FMN-reduktasa MeSH
• ligandy MeSH
• železo MeSH