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1. Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply

   https://doi.org/10.3389/fgene.2022.812764  

   Crouse, Matthew S. ; Caton, Joel S. ; Claycombe-Larson, Kate J. ; Diniz, Wellison J. ; Lindholm-Perry, Amanda K. ; Reynolds, Lawrence P. ; Dahlen, Carl R. ; Borowicz, Pawel P. ; Ward, Alison K. ( February 2022 , Frontiers in Genetics)

   Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B 12 ) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine–folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle’s minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine–folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest ( p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism. 

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2. A comprehensive time‐course metabolite profiling of the model cyanobacterium Synechocystis sp. PCC 6803 under diurnal light:dark cycles

   https://doi.org/10.1111/tpj.14320  

   Werner, Allison ; Broeckling, Corey D. ; Prasad, Ashok ; Peebles, Christie A. M. ( April 2019 , The Plant Journal)

   Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking, yet has implications for improved yield from plants, algae and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites—including carbohydrates, lipids, amino acids, pigments, cofactors, nucleic acids and polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light:dark cycles was developed and applied. A custom photobioreactor and multi‐platform mass spectrometry workflow enabled metabolite profiling every 30–120 min across a 24‐h diurnal sinusoidal LD (‘sinLD’) cycle peaking at 1600 μmol photons m⁻²sec⁻¹. We report widespread oscillations across the sinLD cycle with 90%, 94% and 40% of the identified polar/semi‐polar, non‐polar and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation and cell division phases of growth. During the lag phase, amino acids and nucleic acids accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day‐to‐night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially relevant strain design.

    

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3. Immediate Transcriptional Response to a Temperature Pulse under a Fluctuating Thermal Regime

   https://doi.org/10.1093/icb/icz096  

   Melicher, Dacotah ; Torson, Alex S ; Anderson, Tanner J ; Yocum, George D ; Rinehart, Joseph P ; Bowsher, Julia H ( June 2019 , Integrative and Comparative Biology)

   Abstract The response of ectotherms to temperature stress is complex, non-linear, and is influenced by life stage and previous thermal exposure. Mortality is higher under constant low temperatures than under a fluctuating thermal regime (FTR) that maintains the same low temperature but adds a brief, daily pulse of increased temperature. Long term exposure to FTR has been shown to increase transcription of genes involved in oxidative stress, immune function, and metabolic pathways, which may aid in recovery from chill injury and oxidative damage. Previous research suggests the transcriptional response that protects against sub-lethal damage occurs rapidly under exposure to fluctuating temperatures. However, existing studies have only examined gene expression after a week or over many months. Here we characterize gene expression during a single temperature cycle under FTR. Development of pupating alfalfa leafcutting bees (Megachile rotundata) was interrupted at the red-eye stage and were transferred to 6°C with a 1-h pulse to 20°C and returned to 6°C. RNA was collected before, during, and after the temperature pulse and compared to pupae maintained at a static 6°C. The warm pulse is sufficient to cause expression of transcripts that repair cell membrane damage, modify membrane composition, produce antifreeze proteins, restore ion homeostasis, and respond to oxidative stress. This pattern of expression indicates that even brief exposure to warm temperatures has significant protective effects on insects exposed to stressful cold temperatures that persist beyond the warm pulse. Megachile rotundata’s sensitivity to temperature fluctuations indicates that short exposures to temperature changes affect development and physiology. Genes associated with developmental patterning are expressed after the warm pulse, suggesting that 1 h at 20°C was enough to resume development in the pupae. The greatest difference in gene expression occurred between pupae collected after the warm pulse and at constant low temperatures. Although both were collected at the same time and temperature, the transcriptional response to one FTR cycle included multiple transcripts previously identified under long-term FTR exposure associated with recovery from chill injury, indicating that the effects of FTR occur rapidly and are persistent. 

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4. Light availability modulates the effects of warming in a marine N₂ fixer

   https://doi.org/10.5194/bg-17-1169-2020  

   Yi, Xiangqi ; Fu, Fei-Xue ; Hutchins, David A. ; Gao, Kunshan ( January 2020 , Biogeosciences)

   Abstract. Trichodesmium species, as a group of photosynthetic N2 fixers(diazotrophs), play an important role in the marine biogeochemical cycles ofnitrogen and carbon, especially in oligotrophic waters. How ongoing oceanwarming may interact with light availability to affect Trichodesmium is not yet clear. Wegrew Trichodesmium erythraeum IMS 101 at three temperature levels of 23, 27, and 31∘C undergrowth-limiting and growth-saturating light levels of 50 and 160 µmol quanta m−2 s−1, respectively, for at least 10 generations and thenmeasured physiological performance, including the specific growth rate, N2fixation rate, and photosynthesis. Light availability significantly modulatedthe growth response of Trichodesmium to temperature, with the specific growth ratepeaking at ∼27∘C under the light-saturatingconditions, while growth of light-limited cultures was non-responsive acrossthe tested temperatures (23, 27, and 31∘C). Short-term thermalresponses for N2 fixation indicated that both high growth temperatureand light intensity increased the optimum temperature (Topt) forN2 fixation and decreased its susceptibility to supra-optimaltemperatures (deactivation energy – Eh). Simultaneously, alllight-limited cultures with low Topt and high Eh were unable tosustain N2 fixation during short-term exposure to high temperatures (33–34∘C) that are not lethal for the cells grown underlight-saturating conditions. Our results imply that Trichodesmium spp. growing under lowlight levels while distributed deep in the euphotic zone or under cloudyweather conditions might be less sensitive to long-term temperature changesthat occur on the timescale of multiple generations but are more susceptible toabrupt (less than one generation time span) temperature changes, such asthose induced by cyclones and heat waves. 

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5. Evaluation of Genomic Sequence-Based Growth Rate Methods for Synchronized Synechococcus Cultures

   https://doi.org/10.1128/AEM.01743-21  

   Carroll, Julia ; Van Oostende, Nicolas ; Ward, Bess B. ( January 2022 , Applied and Environmental Microbiology)

   Glass, Jennifer B. (Ed.)

   ABSTRACT Standard methods for calculating microbial growth rates (μ) through the use of proxies, such as in situ fluorescence, cell cycle, or cell counts, are critical for determining the magnitude of the role bacteria play in marine carbon (C) and nitrogen (N) cycles. Taxon-specific growth rates in mixed assemblages would be useful for attributing biogeochemical processes to individual species and understanding niche differentiation among related clades, such as found in Synechococcus and Prochlorococcus . We tested three novel DNA sequencing-based methods (iRep, bPTR, and GRiD) for evaluating the growth of light-synchronized Synechococcus cultures under different light intensities and temperatures. In vivo fluorescence and cell cycle analysis were used to obtain standard estimates of growth rate for comparison with those of the sequence-based methods (SBM). None of the SBM values were correlated with growth rates calculated by standard techniques despite the fact that all three SBM were correlated with the percentage of cells in S phase (DNA replication) over the diel cycle. Inaccuracy in determining the time of maximum DNA replication is unlikely to account entirely for the absence of a relationship between SBM and growth rate, but the fact that most microbes in the surface ocean exhibit some degree of diel cyclicity is a caution for application of these methods. SBM correlate with DNA replication but cannot be interpreted quantitatively in terms of growth rate. IMPORTANCE Small but abundant, cyanobacterial strains such as the photosynthetic Synechococcus spp. are important because they contribute significantly to primary productivity in the ocean. These bacteria generate oxygen and provide biologically available carbon, which is essential for organisms at higher trophic levels. The small size and diversity of natural microbial assemblages mean that taxon-specific activities (e.g., growth rate) are difficult to obtain in the field. It has been suggested that sequence-based methods (SBM) may be able to solve this problem. We find, however, that SBM can detect DNA replication and are correlated with phases of the cell cycle but cannot be interpreted in terms of absolute growth rate for Synechococcus cultures growing under a day-night cycle, like that experienced in the ocean. 

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