real-time-metabolic-detection-living-cells-using-hyperpolarized-13c

Real-Time Metabolic Detection in Living Cells Using Hyperpolarized <sup>13</sup>C NMR

The protocol presents a method to integrate an in-house-built bioreactor system with hyperpolarized 13C nuclear magnetic resonance (NMR) spectroscopy to ...

Osaka Metropolitan University

Scleractinian corals form symbiotic relationships with a variety of microorganisms, including endosymbiotic dinoflagellates of the family Symbiodiniaceae, and with bacteria, which are collectively termed coral holobionts. Interactions between hosts and their symbionts are critical to the physiological status of corals. Coral-microorganism interactions have been studied extensively, but dinoflagellate-bacterial interactions remain largely unexplored. Here, we developed a microbiome manipulation method employing KAS-antibiotic treatment (kanamycin, ampicillin, and streptomycin) to favor pigmented bacteria residing on cultured  and , major endosymbionts of corals, and isolated several carotenoid-producing bacteria from cell surfaces of the microalgae. Following KAS-antibiotic treatment of  sp. strain NIES-4077, pigmented bacteria increased 8-fold based on colony-forming assays from the parental strain, and 100% of bacterial sequences retrieved through 16S rRNA amplicon sequencing were affiliated with the genus . Microbiome manipulation enabled host microalgae to maintain higher maximum quantum yield of photosystem II (variable fluorescence divided by maximum fluorescence [/]) under light-stress conditions, compared to the parental strain. Furthermore, by combining culture-dependent and -independent techniques, we demonstrated that species of the family Symbiodiniaceae and pigmented bacteria form strong interactions. Dinoflagellates protected bacteria from antibiotics, while pigmented bacteria protected microalgal cells from light stress via carotenoid production. Here, we describe for the first time a symbiotic relationship in which dinoflagellates and bacteria mutually reduce environmental stress. Investigations of microalgal-bacterial interactions further document bacterial contributions to coral holobionts and may facilitate development of novel techniques for microbiome-mediated coral reef conservation.  Coral reefs cover less than 0.1% of the ocean floor, but about 25% of all marine species depend on coral reefs at some point in their life cycles. However, rising ocean temperatures associated with global climate change are a serious threat to coral reefs, causing dysfunction of the photosynthetic apparatus of endosymbiotic microalgae of corals, and overproducing reactive oxygen species harmful to corals. We manipulated the microbiome using an antibiotic treatment to favor pigmented bacteria, enabling their symbiotic microalgal partners to maintain higher photosynthetic function under insolation stress. Furthermore, we investigated mechanisms underlying microalgal-bacterial interactions, describing for the first time a symbiotic relationship in which the two symbionts mutually reduce environmental stress. Our findings extend current insights about microalgal-bacterial interactions, enabling better understanding of bacterial contributions to coral holobionts under stressful conditions and offering hope of reducing the adverse impacts of global warming on coral reefs.

Mutualistic Interactions between Dinoflagellates and Pigmented Bacteria Mitigate Environmental Stress.

Spatial reorganization of metabolic enzymes to form the "metabolic enzymes transiently assembling (META) body" is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27-42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed "META body-forming sequences (METAfos)." METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.

Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies.

Under hypoxia, Saccharomyces cerevisiae forms cytoplasmic condensates composed of proteins, including glycolytic enzymes, that are thought to regulate cellular metabolism. However, the hypoxic conditions required for condensate formation remain unclear. In this study, we developed a 300-mL-scale culture method to produce condensate-forming cells by precisely controlling the dissolved oxygen (DO) level in the media. Using enolase as a model, a foci formation rate of more than 50% was achieved at ∼0.1% DO, and the results showed that the DO level affected the foci formation rate. The foci formation rates of the previously reported foci-deficient strains and strains with single amino acid substitutions in the endogenous enolase were examined, and the effect of these amino acid substitutions on glucose consumption and ethanol and glycerol production under hypoxia was evaluated. The results of this study contribute to the investigation of the mechanisms that regulate biomacromolecular condensates under hypoxia.

Large-scale preparation of yeast strains expressing condensates derived from a glycolytic enzyme via controlled dissolved oxygen levels under hypoxia.

Dissolution dynamic nuclear polarization (-DNP) has enabled applications such as the real-time monitoring of chemical reactions. Such applications are mainly for C and N spins with long spin-lattice relaxation times in the molecules of interest. However, the only applications for phosphorus using -DNP are pH imaging and nucleation during crystallization due to the short relaxation times. Here we show that it is possible to observe enzyme reactions using -DNP with phosphorus. Hyperpolarized P spins in pyrophosphate were obtained using bullet-DNP, which requires less dilution of highly polarized solid samples. Real-time monitoring of the hydrolysis reaction of pyrophosphate by inorganic pyrophosphatase from baker's yeast at physiological pH and was successfully achieved and the reaction rate was determined. This is an important reaction for a wide range of applications related to medicine, agriculture, and quantum life science.

Real-Time Monitoring of Hydrolysis Reactions of Pyrophosphates with Dissolution Dynamic Nuclear Polarization.

Coral reefs are at risk of bleaching due to various environmental and anthropogenic stressors such as global warming and chemical pollutants. However, there is little understanding of stressor-specific mechanisms that cause coral bleaching. Therefore, conducting accurate ecotoxicological risk assessments and deciphering modes of action of potentially deleterious ultraviolet (UV) filters (sunscreen compounds) are crucial issues. In this study, we evaluated the toxicity and bleaching effect of benzophenone-3 (BP-3), which is widely used in sunscreen products, on the reef-building coral Acropora tenuis. Furthermore, to understand differences in UV filter- and temperature-induced adverse effects, a comparative ecotoxicogenomic approach using RNA-seq was integrated into a toxicity test to clarify differences in gene expression changes induced by BP-3 and heat stress (31 °C). The lethal concentration 50 % (LC50) was calculated as 3.9 mg/L, indicating that the aquatic environmental risk on corals posed by BP-3 was low based on the risk assessment in this study. Differentially expressed genes related to oxidative stress and extracellular matrix organization were involved in coral responses to both BP-3 and heat stress, but their patterns differed. Whereas immune and heat-shock responses were activated in response to heat stress, activation of a drug metabolism pathway and several signal transduction pathways were identified in BP-3 treatment groups. Our study enhances understanding of stress responses in corals induced by UV filters and thermal stress. Using potential gene markers identified in this study for eco-epidemiological surveys of stressed corals, we urgently need to develop effective countermeasures.

Deciphering mechanisms of UV filter (benzophenone-3)- and high temperature-induced adverse effects in the coral Acropora tenuis, using ecotoxicogenomics.

Compartmentalization of multiple enzymes in cellulo and in vitro is a means of controlling the cascade reaction of metabolic enzymes. The compartmentation of enzymes through liquid-liquid phase separation may facilitate the reversible control of biocatalytic cascade reactions, thereby reducing the transcriptional and translational burden. This has attracted attention as a potential application in bioproduction. Recent research has demonstrated the existence and regulatory mechanisms of various enzyme compartments within cells. Mounting evidence suggests that enzyme compartmentation allows in vitro and in vivo regulation of cellular metabolism. However, the comprehensive regulatory mechanisms of enzyme condensates in cells and ideal organization of cellular systems remain unknown. This review provides an overview of the recent progress in multiple enzyme compartmentation in cells and summarizes strategies to reconstruct multiple enzyme assemblies in vitro and in cellulo. By examining parallel examples, we have evaluated the consensus and future perspectives of enzyme condensation.

Article	Year
Mutualistic Interactions between Dinoflagellates and Pigmented Bacteria Mitigate Environmental Stress. Microbiology spectrum\| PubMed ID: 36651852	2023
Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies. PloS one\| PubMed ID: 37053166	2023
Large-scale preparation of yeast strains expressing condensates derived from a glycolytic enzyme via controlled dissolved oxygen levels under hypoxia. Letters in applied microbiology\| PubMed ID: 37587018	2023
Real-Time Monitoring of Hydrolysis Reactions of Pyrophosphates with Dissolution Dynamic Nuclear Polarization. The journal of physical chemistry letters\| PubMed ID: 38980118	2024
Deciphering mechanisms of UV filter (benzophenone-3)- and high temperature-induced adverse effects in the coral Acropora tenuis, using ecotoxicogenomics. The Science of the total environment\| PubMed ID: 39278489	2024
Compartmentation of multiple metabolic enzymes and their preparation in vitro and in cellulo. Biochimica et biophysica acta. General subjects\| PubMed ID: 40058614	2025

Natsuko Miura

Biography

JoVE Journal Publications

Other Publications