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Item Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum(2012-06) Valenzuela, Jacob J.; Mazurie, Aurélien J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Fields, Matthew W.BACKGROUND: Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome hasbeen sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis hasbeen reported under different growth conditions. To elucidate P. tricornutum gene expression profiles duringnutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P)and whole-genome transcripts were monitored over time via RNA-sequence determination.RESULTS: The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, theincrease in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphatewas depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be anearly trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genesassociated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulationafter growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbonreduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbonassimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon(DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed(2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth.Alternative pathways that could utilize HCO-3 were also suggested by the gene expression profiles (e.g., putativepropionyl-CoA and methylmalonyl-CoA decarboxylases).CONCLUSION: The results indicate that P. tricornutum continued carbon dioxide reduction when population growthwas arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels.Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up ofprecursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actualenzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellularresponses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.Item In situ gene expression profiling of the thermoacidophilic alga C yanidioschyzon in relation to visible and ultraviolet irradiance(2014-06) Skorupa, Dana J.; Castenholz, R. W.; Mazurie, Aurélien J.; Carey, Charles C.; Rosenzweig, F.; McDermott, Timothy R.Ultraviolet and high-intensity visible radiation generate reactive intermediates that damage phototrophic microorganisms. In Yellowstone National Park, the thermoacidophilic alga Cyanidioschyzon exhibits an annual seasonal biomass fluctuation referred to as 'mat decline', where algal viability decreases as ultraviolet and visible irradiances increase during summer. We examined the role irradiance might play in mat decline using irradiance filters that uncouple ultraviolet and visible effects along with custom microarrays to study gene expression in situ. Of the 6,507 genes, 88% showed no response to ultraviolet or visible, implying that at the biomolecular level, these algae inhabit a chemostat-like environment and is consistent with the near constant aqueous chemistry measured. The remaining genes exhibited expression changes linked to ultraviolet exposure, to increased visible radiation, or to the apparent combined effects of ultraviolet and visible. Expression of DNA repetitive elements was synchronized, being repressed by visible but also influenced by ultraviolet. At highest irradiance levels, these algae reduced transcription of genes encoding functions involved with DNA replication, photosynthesis and cell cycle progression but exhibited an uptick in activities related to repairing DNA damage. This corroborates known physiological responses to ultraviolet and visible radiation, and leads us to provisionally conclude that mat decline is linked to photoinhibition.Item Real-Time Digitization of Metabolomics Patterns from a Living System Using Mass Spectrometry(2014-10) Heinemann, Joshua; Noon, Brigit; Mohigmi, Mohammad J.; Mazurie, Aurélien J.; Dickensheets, David L.; Bothner, BrianThe real-time quantification of changes in intracellular metabolic activities has the potential to vastly improve upon traditional transcriptomics and metabolomics assays for the prediction of current and future cellular phenotypes. This is in part because intracellular processes reveal themselves as specific temporal patterns of variation in metabolite abundance that can be detected with existing signal processing algorithms. Although metabolite abundance levels can be quantified by mass spectrometry (MS), large-scale real-time monitoring of metabolite abundance has yet to be realized because of technological limitations for fast extraction of metabolites from cells and biological fluids. To address this issue, we have designed a microfluidic-based inline small molecule extraction system, which allows for continuous metabolomic analysis of living systems using MS. The system requires minimal supervision, and has been successful at real-time monitoring of bacteria and blood. Feature-based pattern analysis of Escherichia coli growth and stress revealed cyclic patterns and forecastable metabolic trajectories. Using these trajectories, future phenotypes could be inferred as they exhibit predictable transitions in both growth and stress related changes. Herein, we describe an interface for tracking metabolic changes directly from blood or cell suspension in real-time.