Biology subjects

Doenier, J.

Publications and source records attributed to Doenier, J..

4 recordsLinked to original sources

bioRxiv2025

A genome-wide CRISPR screen reveals how diatoms thrive in dynamic light

Diatoms are a highly diverse group of phytoplankton that have a large impact on global primary production and carbon sequestration in the ocean1,2. However, they are evolutionarily divergent from model phototrophs of the green lineage, and limited screening tools have hampered discovery of unique diatom biology. To address this challenge, we developed a genome-wide CRISPR/Cas9 screen in the model marine diatom, Phaeodactylum tricornutum. The screen was applied to identify genes required for survival in different light regimes, including both high light and fluctuating light. We identified a broad set of uncharacterized genes, providing the foundation for mechanistic studies of diatom adaptation to dynamic light. Among these genes, we demonstrated that the red lineage-exclusive gene STROBE1 is a new potentiator of cyclic electron flow (CEF) required for CEF to generate a trans-thylakoid proton gradient3,4. As dynamic light conditions are common in marine environments, STROBE1 and other genes identified in this screen may contribute to the broad ecological success of diatoms5,6. This genome-wide genetic screen in P. tricornutum will accelerate the unbiased discovery of novel gene functions in these ecologically important organisms.

microbiology↗

bioRxiv2024

Genomes of nitrogen-fixing eukaryotes reveal a non-canonical model of organellogenesis

Endosymbiotic gene transfer and import of host-encoded proteins are considered hallmarks of organelles necessary for stable integration of two cells. However, newer endosymbiotic models have challenged the origin and timing of such genetic integration during organellogenesis. Epithemia diatoms contain diazoplasts, obligate endosymbionts that are closely related to recently-described nitrogen-fixing organelles and share similar function as integral cell compartments. We report genomic analyses of two species which are highly divergent but share a common ancestor at the origin of the endosymbiosis. We found minimal evidence of genetic integration in E.clementina: nonfunctional diazoplast-to-nucleus DNA transfers and 6 host-encoded proteins of unknown function in the diazoplast proteome, far fewer than detected in other recently-acquired endosymbionts designated organelles. Epithemia diazoplasts are a valuable counterpoint to existing organellogenesis models, demonstrating that endosymbionts can function as integral compartments absent significant genetic integration. The minimal genetic integration makes diazoplasts valuable blueprints for bioengineering endosymbiotic compartments de novo.

evolutionary biology↗

bioRxiv2023

Transition from free-living cyanobacterium to endosymbiont enables adaptations favoring nitrogen fixation in a photosynthetic eukaryote.

Epithemia spp. diatoms contain obligate, nitrogen-fixing endosymbionts, or "diazoplasts", derived from cyanobacteria. These algae are a rare example of photosynthetic eukaryotes that have successfully coupled oxygenic photosynthesis with oxygen-sensitive nitrogenase activity. Here, we report a newly-isolated species, E. clementina, as a model to investigate endosymbiotic acquisition of nitrogen fixation. To detect the metabolic changes associated with endosymbiotic specialization, we compared nitrogen fixation, associated carbon and nitrogen metabolism, and their regulatory pathways in the Epithemia diazoplast with its close, free-living cyanobacterial relative, Crocosphaera subtropica. Unlike C. subtropica, we show that nitrogenase activity in the diazoplast is concurrent with, and even dependent on, host photosynthesis and no longer associated with cyanobacterial glycogen storage suggesting carbohydrates are imported from the host diatom. Carbohydrate catabolism in the diazoplast indicates that the oxidative pentose pathway and oxidative phosphorylation, in concert, generates reducing equivalents and ATP and consumes oxygen to support nitrogenase activity. In contrast to expanded nitrogenase activity, the diazoplast has diminished ability to utilize alternative nitrogen sources. Upon ammonium repletion, negative feedback regulation of nitrogen fixation was conserved, however ammonia assimilation showed paradoxical responses in the diazoplast compared with C. subtropica. The altered nitrogen regulation likely favors nitrogen transfer to the host. Our results suggest that the diazoplast is specialized for endosymbiotic nitrogen fixation. Altogether, we establish a new model for studying endosymbiosis, perform the first functional characterization of this diazotroph endosymbiosis, and identify metabolic adaptations for endosymbiotic acquisition of a critical biological function.

cell biology↗

bioRxiv2021

Tethered NOS-3, a nematode Nanos RNA-binding protein, enhances reporter expression and mRNA stability

Robust methods are critical for testing the in vivo regulatory mechanism of RNA binding proteins. Here we report improvement of a protein-mRNA tethering assay to probe the function of an RNA binding protein in its natural context within the C. elegans adult germline. The assay relies on a dual reporter expressing two mRNAs from a single promoter and resolved by trans-splicing. The gfp reporter 3UTR harbors functional binding elements for {lambda}N22 peptide, while the mCherry reporter 3UTR carries mutated nonfunctional elements. This strategy enables internally controlled quantitation of reporter protein by immunofluorescence and mRNA by smFISH. To test the new system, we analyzed a C. elegans Nanos protein, NOS-3, which serves as a post-transcriptional regulator of germ cell fate. Unexpectedly, tethered NOS-3 enhanced reporter expression. We confirmed this enhancement activity with a second reporter engineered at an endogenous germline gene. NOS-3 enhancement of reporter expression was associated with its N-terminal intrinsically disordered region, not its C-terminal zinc fingers. RNA quantitation revealed that tethered NOS-3 enhances stability of the reporter mRNA. We suggest that this direct NOS-3 enhancement activity may explain a paradox: classically Nanos proteins are expected to repress RNA, but nos-3 had been found to promote gld-1 expression, an effect that could be direct. Regardless, the new dual reporter dramatically improves in situ quantitation of reporter expression after RBP tethering to determine its molecular mechanism in a multicellular tissue.

developmental biology↗