Partitioning of coumarin 152 (C152) in phosphatidylcholine vesicles was quantified using time-resolved fluorescence emission. Phospholipid vesicles were comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine (12:0 DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0 DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (16:0 DPPC). C152 fluorescence emission decays were fit to three lifetimes, corresponding to C152 solvated by the aqueous buffer, embedded in polar lipid headgroups, and surrounded by the nonpolar lipid membrane core. Partitioning was measured as a function of sample temperature and vesicle composition. C152 in all three lipid systems showed qualitatively similar partitioning behavior. Partitioning into a gel phase membrane was thermoneutral and slightly entropically favored. Partitioning of C152 near the lipid membrane headgroups was entropically driven and endothermic. Well above the melting temperature, exsolvation of C152 from the membrane back into the aqueous buffer was enthalpically driven but entropically unfavorable. Regardless of solution temperature, relatively little (<15%) C152 partitions into the hydrophobic core of the membrane. The magnitudes of the forces driving C152 partitioning systematically increased with alkyl chain length (DLPC < DMPC < DPPC). Differences in partitioning between the three vesicle systems are attributed to differences in surface area per lipid as membrane phase changes from the gel to liquid-crystalline state.

Time resolved fluorescence emission was used to quantify coumarin 152 (C152) partitioning into a model lipid vesicle membrane. For these studies, the lipid vesicles were composed of the symmetric, saturated phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0 DMPC). C152 fluorescence lifetimes were measured as a function of sample temperature, and changes in the relative contributions of these lifetimes (corrected for quantum yield) to the overall emission decay data were attributed to changes in the distribution of C152 solutes between the aqueous buffer, the polar vesicle headgroup region, and the hydrophobic interior of the vesicle bilayer. When the bilayer was in its more rigid, gel state, C152 remained predominantly in the aqueous buffer. Upon melting to its liquid crystalline state, each bilayer showed evidence of accommodating more C152 into a polar region associated with the lipid headgroups. At no temperature did C152 show strong affinity for the bilayer's hydrophobic interior. Above 50 °C, this behavior reversed itself with C152 moving back out of the vesicle membrane and into the buffer. All observed changes in partitioning behavior were reversible. The interesting temperature dependence of C152 partitioning suggests that C152 solvation within the lipid headgroup region represents a sensitive balance between enthalpic and entropic contributions with C152 accommodation by the bilayer being exothermic but entropically unfavorable.

Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of methyl-coenzyme M reductase subunit A (mcrA) gene amplicons demonstrated a wide diversity of Mcr-encoding archaea inhabit geothermal features with differing physicochemical regimes across YNP. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represent minor fractions of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in the stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. We revealed that diverse Mcr-encoding archaea with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP and can be enriched under methanogenic conditions. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.

Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated the methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of gene amplicons of methyl-coenzyme M reductase subunit A (mcrA) indicated a wide diversity of Mcr-encoding archaea across geothermal features with differing physicochemical regimes. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, or hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represented a minor fraction of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. Altogether, we revealed that diverse Mcr-encoding populations with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.

Time-resolved fluorescence and differential scanning calorimetry were used to determine the partitioning of coumarin 152 (C152) into large unilamellar vesicles composed of binary mixtures of two phosphatidylcholines (12:0/12:0 DLPC and 14:0/14:0 DMPC) and vesicles composed of binary mixtures of a phosphatidylcholine and a phosphatidylethanolamine (14:0/14:0 DMPC and 14:0/14:0 DMPE). Differential scanning calorimetry showed that both DLPC/DMPC and DMPC/DMPE are miscible in lipid vesicles. Time-resolved fluorescence indicated that C152 partitioning into DLPC/DMPC mixtures showed nearly ideal behavior that was described with weighted contributions from C152 partitioning into pure DLPC and pure DMPC vesicles. In contrast, C152 partitioning into DMPC/DMPE mixtures was distinctly nonideal. For DMPC/DMPE lipid vesicles having DMPC mole fractions between 10 and 80%, C152 partitioning into the bilayer was measurably enhanced near the melting temperature, relative to expectations based simply on weighted contributions from C152 partitioning into vesicles comprised of pure lipids. The origin of this behavior remains uncertain. For vesicles comprised of pure DMPE, C152 shows almost no partitioning into the membrane, with ≥80% of the solute remaining in the buffer solution at temperatures between 10 and 50 °C.