Biology subjects

Riekhof, W. R.

Publications and source records attributed to Riekhof, W. R..

9 recordsLinked to original sources

bioRxiv2025

Crusty-Pinky: A Novel Polyextremotolerant Fungus and its Methylobacterium Symbionts Could be an Essential Symbiosis for the Biological Soil Crust Consortium

JF2 08-2F Crusty is a novel melanized polyextremotolerant fungus isolated from a biological soil crust, which we believe harbors Methylobacterium spp. endosymbionts, called Pinky. Crusty is capable of utilizing many sources of carbon and nitrogen and is resistant to multiple metals and UV-C due to its melanized cell wall. We were unable to recover a Pinky-free culture of Crusty via usage of antibiotics. However, when exposed to antibiotics that kill or stop the growth of the Pinky, growth of Crusty is significantly stunted, implying that actively growing Pinky symbionts are needed for Crustys optimal growth. The Crusty-Pinky symbiosis also seems to be able to perform active metabolism in carbonless and nitrogenless medium, which we believe is due to Pinkys ability to perform aerobic anoxygenic photosynthesis. Finally, Pinky was identified as being capable of growth stimulation of the algae Chlorella sorokiniana, indicating that Pinky likely produces cytokinins or auxins which Methylobacterium are known for. Features of this symbiosis provide us insight into the ecological roles of these microbes within the biological soil crust.

microbiology↗

bioRxiv2025

A chemical genomic screen identifies novel genetic interactors of the methylation-dependent de novo pathway of phosphatidylcholine biosynthesis in Saccharomyces cerevisiae.

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in most eukaryotic cell and organelle membranes, and cells must regulate its synthesis and interorganelle transport to maintain correct membrane compositions and biophysical properties. Our knowledge of genes encoding enzymes of PtdCho biosynthesis is largely complete, and a great deal is understood about their localization and regulation, however our understanding of molecular mechanisms regulating PtdCho biosynthesis and trafficking remains incomplete. To identify genes that show epistatic relationships with the methylation pathway of PtdCho biosynthesis, we performed a chemical-genomic screen of a Saccharomyces cerevisiae gene-deletion mutant collection using a phosphatidylethanolamine (PtdEtn) methyltransferase inhibitor, 2-hydroxyethylhydrazine (HEH). HEH functions by selectively inhibiting the PtdEtn methyltransferase enzymes Pem1p/Cho2p and Pem2p/Opi3p. We demonstrate that the addition of exogenous choline or lyso-phosphatidylcholine can recover HEH-mediated growth inhibition, and used this finding to design a functional-genomic screen to identify genes which, when deleted, render the strain unable to grow when the methylation pathway is partially inhibited. We now report the identification of 410 S. cerevisiae gene deletion mutants that exhibit HEH hypersensitivity, and identify among those a core set of 21 genes that are known to epistatically interact with genes encoding enzymes of the PtdEtn methylation pathway. This gene set was enriched in functions relating to glycerolipid and sterol biosynthesis and their regulation, the high-osmolarity glycerol (HOG) pathway, and genes involved in chromatin remodeling and transcriptional regulation. These results demonstrate that PtdCho produced by any one of the Kennedy pathway, methylation pathway, or acyltransferase pathway can maintain necessary cellular PtdCho compositions, but that disruption of any one of these pathways leads to epistatic interactions with non-overlapping subsets of genes, thus providing new insights on the specific functions of these pathways. The design and implementation of this screening strategy establishes HEH as a useful tool for specific inhibition of the methylation pathway in high-throughput functional-gnomic screens, which will facilitate further studies on the synthesis, transport, and function of PtdCho.

cell biology↗

bioRxiv2023

Nitrogen starvation leads to TOR kinase-mediated downregulation of fatty acid synthesis in the algae Chlorella sorokiniana and Chlamydomonas reinhardtii

The accumulation of triacylglycerol (TAG) as a storage compound in eukaryotic algae has been the subject of extensive studies over the last 50 years. The model industrial alga Chlorella sorokiniana accumulates TAG and other storage compounds under nitrogen (N)-limited growth. Previously we used transcriptomics to explore the regulation of TAG synthesis in C. sorokiniana. Surprisingly, our analysis showed that the expression of several key genes encoding enzymes involved in plastidic fatty acid synthesis are significantly repressed. Metabolic labeling with radiolabeled acetate showed that de novo fatty acid synthesis is indeed downregulated under N-limitation. Likewise, inhibition of the Target of Rapamycin kinase (TOR), a key regulator of metabolism and growth, decreased fatty acid synthesis. We compared the changes in proteins and phosphoprotein abundance using a proteomics and phosphoproteomics approach in C. sorokiniana cells under N-limitation or TOR inhibition and found extensive overlap between the N-limited and TOR-inhibited conditions. We also identified changes in the phosphorylation levels of TOR complex proteins, TOR-kinase and RAPTOR, under N-limitation, indicating that TOR signaling is altered. Our results indicate that under N-limitation there is significant metabolic remodeling, including fatty acid synthesis, mediated by TOR signaling. We find that TOR-mediated metabolic remodeling of fatty acid synthesis under N-limitation is conserved in the chlorophyte algae Chlorella sorokiniana and Chlamydomonas reinhardtii.

plant biology↗

bioRxiv2021

Deciphering the potential niche of novel black yeast fungal isolates in a biological soil crust based on genomes, phenotyping, and melanin regulation

Black yeasts are polyextremotolerant fungi that contain high amounts of melanin in their cell wall and maintain a primarily yeast form. These fungi grow in xeric, nutrient deplete environments which implies that they require highly flexible metabolisms and have been suggested to contain the ability to form lichen-like mutualisms with nearby algae and bacteria. However, the exact ecological niche and interactions between these fungi and their surrounding community is not well understood. We have isolated two novel black yeasts from the genus Exophiala that were recovered from dryland biological soil crusts. Despite notable differences in colony and cellular morphology, both fungi appear to be members of the same species, which has been named Exophiala viscosa (i.e., E. viscosa JF 03-3 Goopy and E. viscosa JF 03-4F Slimy). A combination of whole genome sequencing, phenotypic experiments, and melanin regulation experiments have been performed on these isolates to fully characterize these fungi and help decipher their fundamental niches within the biological soil crust consortium. Our results reveal that E. viscosa is capable of utilizing a wide variety of carbon and nitrogen sources potentially derived from symbiotic microbes, can withstand many forms of abiotic stresses, and excrete melanin that can potentially provide UV resistance to the biological soil crust community. Besides the identification of a novel species within the genus Exophiala, our study also provides new insight into the regulation of melanin production in polyextremotolerant fungi.

microbiology↗

bioRxiv2021

Micafungin-induced Cell Wall Damage Stimulates Microcycle Conidiation in Aspergillus nidulans

Fungal cell wall receptors relay messages about the state of the cell wall to the nucleus through the Cell Wall Integrity Signaling (CWIS) pathway. The ultimate role of the CWIS pathway is to coordinate repair of cell wall damage and to restore normal hyphal growth. Echinocandins such as micafungin represent a class of antifungals that trigger cell wall damage by affecting synthesis of {beta}-glucans, filamentous fungis response to these antifungals are fundamentally unknown. To obtain a better understanding of the dynamics of the CWIS response and its multiple effects, we have coupled dynamic transcriptome analysis with morphological studies of Aspergillus nidulans hyphae responding to micafungin. Our results reveal that expression of the master regulator of asexual development, BrlA, is induced by micafungin exposure. Further study showed that micafungin elicits microcycle conidiation in a BrlA-dependent manner, and that this response is abolished in the absence of MpkA. Our results suggest that microcycle conidiation may be a general response to cell wall perturbation which in some cases would enable fungi to tolerate or survive otherwise lethal damage.

molecular biology↗

bioRxiv2021

Reactive oxygen species mediate thylakoid membrane remodeling and triacylglycerol synthesis under nitrogen starvation in the alga Chlorella sorokiniana

Many microbes accumulate energy storage molecules such as triglycerides and starch during nutrient limitation. In eukaryotic green algae grown under nitrogen limiting conditions, triglyceride accumulation is coupled with chlorosis and growth arrest. In this study we show that accumulation of reactive oxygen species (ROS) under nitrogen limitation in the microalga Chlorella sorokiniana is involved in thylakoid membrane remodeling, leading to chlorosis. We show that ROS accumulation under nitrogen limitation is an active process involving downregulation of expression of ROS-quenching enzymes, such as superoxide dismutases, catalase, peroxiredoxin, and glutathione peroxidase-like, and upregulation of enzymes involved in generating ROS, such as NADPH oxidase, xanthine oxidase and amine oxidases. Expression of enzymes involved in ascorbate and glutathione metabolism are also affected under these conditions. Quenching ROS under nitrogen limitation reduces TAG accumulation, adding additional evidence for the role of ROS signaling in the process.

plant biology↗

bioRxiv2020

A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents.

The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the alkylphosphocholines exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of Saccharomyces cerevisiae titratable-essential gene and haploid non-essential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). 58 strains out of ~5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum (ER) and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1{Delta}), an oxysterol binding protein homolog (osh2{Delta}), a number of ER resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

genetics↗

bioRxiv2017

Synthesis, secretion, and perception of abscisic acid regulates stress responses in Chlorella sorokiniana

Abscisic acid (ABA) is a phytohormone that has been extensively characterized in higher plants for its roles in seed and bud dormancy, leaf abscission, and stress responses. Genomic studies have identified orthologs for ABA-related genes throughout the Viridiplantae, including in unicellular algae; however, the role of ABA in algal physiology has not been characterized, and the existence of such a role has been a matter of dispute. In this study, we demonstrate that ABA is involved in regulating algal stress responses. Chlorella sorokiniana strain UTEX 1230 contains genes orthologous to those of higher plants which are essential for ABA biosynthesis, sensing, and degradation. RNAseq-based transcriptomic studies reveal that treatment with ABA induces dramatic changes in gene expression profiles, including the induction of a subset of genes involved in DNA replication and repair, a phenomenon which has been demonstrated in higher plants. Pretreatment of C. sorokiniana cultures with ABA exerts a protective effect on cell viability in response to ultraviolet radiation. Additionally, C. sorokiniana produces and secretes biologically relevant amounts of both ABA and the oxylipin 12-oxo-phytodienoic acid (OPDA) into the growth medium in response to abiotic stressors. Taken together, these phenomena suggest that ABA signaling evolved as an intercellular stress response signaling molecule in eukaryotic microalgae prior to the evolution of multicellularity and colonization of land.

plant biology↗

bioRxiv2017

Molecular machinery of auxin synthesis, secretion, and perception in the unicellular chlorophyte alga Chlorella sorokiniana UTEX 1230

Indole-3-acetic acid is a ubiquitous small molecule found in all domains of life. It is the predominant and most active auxin in seed plants, where it coordinates a variety of complex growth and development processes. The potential origin of auxin signaling in algae remains a matter of some controversy. In order to clarify the evolutionary context of algal auxin signaling, we undertook a genomic survey to assess whether auxin acts as a signaling molecule in the emerging model chlorophyte Chlorella sorokiniana UTEX 1230. C. sorokiniana produces the auxin indole-3-acetic acid (IAA), which was present in both the cell pellet and in the supernatant at a concentration of ~ 1 nM, and its genome encodes orthologs of genes related to auxin synthesis, transport, and signaling in higher plants. Candidate orthologs for the canonical AUX/IAA signaling pathway were not found; however, auxin-binding protein 1 (ABP1), an alternate auxin receptor, is present and highly conserved at essential auxin binding and zinc coordinating residues. Additionally, candidate orthologs for PIN proteins, responsible for intercellular, vectorial auxin transport in higher plants, were not found, but PILs (PIN-Like) proteins, a recently discovered family that mediates intracellular auxin transport, were identified. The distribution of auxin related gene in this unicellular chlorophyte demonstrates that a core suite of auxin signaling components was present early in the evolution of plants. Understanding the simplified auxin signaling pathways in chlorophytes will aid in understanding phytohormone signaling and crosstalk in seed plants, and in understanding the diversification and integration of developmental signals during the evolution of multicellular plants.

plant biology↗