Periodic Reporting for period 1 - IRONCOMM (Investigating the role of bacteria-produced siderophores in satisfying diatom Fe requirements.) Reporting period: 2017-07-01 to 2019-06-30 Summary of the context and overall objectives of the project The greatest impact of climate change due to rising atmospheric CO2 has been and willcontinue to be exerted on ocean biomes. Ocean warming dominates the increase in energystored in the climate system, whilst uptake of CO2 drives the decrease in pH of seawater.An ensuing change will be to the speciation and solubility of divalent metals. The primary aim of IRONCOMM is to shed light on how marine microbial communities will respond to changing pools of iron in a progressively acidifying and warming Ocean. The focus is on diatoms in particular, which are ubiquitous in ocean waters and are responsible for an estimated 20% of the total primary production on Earth. They are key players in microbial marine ecosystems, which means that knowledge gained in diatom model systems is immediately globally scalable. The research of IRONCOMM is concerned with Fe in particular, since this micronutrient has been shown to play a regulating role in the growth dynamics of marine phytoplankton.There were three main objectives to the project:1. To identify whether diatoms are able to uptake and use siderophores, which are organic chelators of iron. It is unclear whether siderophore bound iron is bioavailable to diatoms. 2. To develop laboratory co-culture systems for exploring the hypothesis for mutualism between siderophore-producing bacteria and diatoms. 3. To assess the extent of siderophore uptake by photosynthetic eukaryotes and possible implicated interactions with bacterioplankton in the global ocean based on metatranscriptome data catalogued as part of the Tara Oceans project.IRONCOMM takes an interdisciplinary approach that marries molecular studies of laboratory model systems to global scale analyses of environmentally-derived metatranscriptomic data. Conclusions: We were successful in meeting all three of the objectives of IRONCOMM. We experimentally verified that diatoms are capable of siderophore uptake and use. In published results, we showed that a) diatoms have a preference for the type of siderophore they uptake, b) the uptake mechanism relies on endocytosis (a eukaryote-specific adaptation), c) we identified some of the molecular components involved in the process, notably the Iron Starvation Induced Protein 1 (ISIP1), which is necessary for this function. We conducted phylogenetic analyses and showed that ISIP1 is a diatom-specific protein, suggesting that this may be a diatom adaptation, which has led to the dominance of diatoms in present day oceans. Further, to meet objective 3 we mined the Tara Oceans dataset and were able to show that ISIP1 is ubiquitously expressed in ocean waters. We proposed the use of ISIP1 as a global biomarker for siderophore uptake and use – an important component of global ocean models used in climate change research. Finally, we met our second objective, by isolating bacteria associated with diatoms and identifying siderophore producers. In ongoing work we are investigating the partnerships between siderophore producers and diatoms. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Initial analyses focused on three model diatoms: Phaeodactylum tricornutum, Thalassiosira pseudonana and Thalassiosira oceanica. We studied the uptake of different iron sources (by using radiolabeled iron) in iron-starved cultures. We showed preferential uptake of hydroxamate siderophores by P. tricornutum, and catecholate siderophores by T. oceanica. T. pseudonana was unable to uptake siderophores. Next, we generated mutants of P. tricornutum, exploring the roles of three iron starvation induced proteins: ISIP1, ISIP2a and ISIP3. The physiology of knock-down lines revealed that ISIP1 was necessary for the uptake of siderophores into the diatom cell, and microscopy revealed that the siderophore complex was being targeted to the chloroplast. We generated a trichromatic line of P. tricornutum, tagging the three ISIPs to fluorophores. In collaboration with Charles University of Prague (where the Marie Curie candidate undertook a placement), we tracked the ISIPs in a single cell using cutting-edge fluorescence microscopy techniques. Through this as yet unpublished work work we were able to identify a (putative) novel iron-processing compartment in diatoms. To complement our laboratory based studies, we conducted a range of in-silico investigations using the largest ‘omics dataset for the global ocean – the Tara ocean metatrascriptome and metagenome catalogues. Our main results quantified the expression of ISIP1 in the global ocean, demonstrating that it is highly expressed and correlated with low availability of soluble iron. This indicates that the mechanism of iron uptake we identified in our laboratory experiments is a key physiological function of diatoms in the wild. We have disseminated our findings in a publication in Science Advances, as well as through presentations in global conferences. All the data in the publication is available to the scientific community. Further, we have proposed the use of ISIP1 as a biomarker for siderophore uptake by diatoms for use in global ocean models, which predict phytoplankton responses to ongoing climate change. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our work has uncovered a novel mechanism for iron acquisition in the diatoms, opening the field to a range of follow-up investigations. The socio-economic implications are largely to the discipline of climate change science. We are beginning to open up the “black box” of the biological phytoplankton response to climate change, which is largely ignored in ocean models. We have also made significant advances in understanding diatom physiology and ecology – providing new avenues for research in cell biology, which may be transferrable. In our own work, there are two further publications which are being prepared. The first is summarizing our microscopy results of fluorescently tagged ISIP proteins in a diatom cells. The findings here are striking physiologically, but importantly they also demonstrate state of the art imaging techniques. What we have been able to do with three fluorescent proteins in a single cell is unprecedented in our field. The second publication will focus on our in silico bioinformatics analyses. We are investigating the production of siderophores by mining ‘whole pathways’ rather than genes in the Tara Oceans metatrasncriptomics atlas. This final publication from the project will push the boundaries of what is possible in the ‘omics world, as well as uncover active processes that are shaping our planet’s ocean microbiome. Iron uptake from different iron sources by T. oceanica, P. tricornutum, and T. pseudonana.
