No abstract

Taurine (2-aminoethanesulfonic acid) is an amino acid-like compound widely distributed in animals and an essential nutrient in some species. Targeted metabolomics of marine and freshwater microalgae combined with medium supplementation identified biosynthetic pathway intermediates and necessary catalytic activities. Genomic analysis was then used to predict the first taurine biosynthetic pathway in these organisms. MRM-based electrospray ionization (ESI) LC-MS/MS analysis demonstrated that taurine is synthesized using a carbon backbone from L-serine combined with sulfur derived from sulfate. Metabolite analysis showed a nonuniform pattern in levels of pathway intermediates that were both species and supplement dependent. While increased culture salinity raised taurine levels modestly in marine alga, taurine levels were strongly induced in a freshwater species implicating taurine as an organic osmolyte. Conservation of the synthetic pathway in algae and metazoans together with a pattern of intermittent distribution in other lineages suggests that it arose early in 0 1 5 ) 2 eukaryotic evolution. Elevated levels of cell-associated taurine in algae could offer a new and biorenewable source of this unusual bioactive compound.

The relative effects of three precise nitrogen limitation regimes on green micro-algae were assessed using the Trebouxiophycean alga Coccomyxa subellipsoidea grown in a chemostatic bioreactor system. The data provides further evidence that growth and triglyceride (TAG) accumulation are concurrent and independently proportional to the degree of nitrogen limitation in algae. Additionally, TAG accumulation was observed to proceed via oscillations with respect to time and percent dry weight quantity. The predator-prey model was applied to fit the experimental data and to obtain the physiological significance of these oscillations. The results determine the conditions of maximum neutral lipid productivity with respect to nitrate stress and highlight an area of potential future research.

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching.

Electrochemical characteristics of immobilized double-stranded DNA (dsDNA) on a Au electrode were studied as a function of coverage using a home-built optoelectrochemical method. The method allows probing of local redox processes on a 6 μm spot by measuring both differential reflectivity (SEED-R) and interferometry (SEED-I). The former is sensitive to redox ions that tend to adsorb to the electrode, while SEED-I is sensitive to nonadsorbing ions. The redox reaction maxima, R max and Δmax from SEED-R and SEED-I, respectively, are linearly proportional to amperometric peak current, I max. The DNA binding is measured by a redox active dye, methylene blue, that intercalates in dsDNA, leading to an R max. Concomitantly, the absence of Δmax for [Fe­(CN)6]4–/3– by SEED-I ensures that there is no leakage current from voids/defects in the alkanethiol passivation layer at the same spot of measurement. The binding was regulated electrochemically to obtain the binding fraction, f, ranging about three orders of magnitude. A remarkably sharp transition, f = f T = 1.25 × 10–3, was observed. Below f T, dsDNA molecules behaved as individual single-molecule nanoelectrodes. Above the crossover transition, R max, per dsDNA molecule dropped rapidly as f –1/2 toward a planar-like monolayer. The SEED-R peak at f ∼ 3.3 × 10–4 (∼270 dsDNA molecules) was (statistically) robust, corresponding to a responsivity of ∼0.45 zeptomoles of dsDNA/spot. Differential pulse voltammetry in the single-molecule regime estimated that the current per dsDNA molecule was ∼4.1 fA. Compared with published amperometric results, the reported semilogarithmic dependence on target concentration is in the f > f T regime.

Circulating microRNA are promising diagnostic and prognostic biomarkers of disease in quantitative blood tests. A label-free, PCR-free, electrochemical microarray technology on a monolith electrode is described, with 10 attomolar (aM) sensitivity and responsiveness to binding of <1 zeptomole of target to immobilized ssDNA probes with zero background. Specificity is 100% in a mixture with five nonspecific miRNA each with a 103-fold higher concentration. Direct measurement on plasma-derived miRNA without cDNA conversion and PCR demonstrated multiplexing and near-ideal quantitative correlation with an equivalent pure sample. The dynamic range is a target concentration ranging from 10–2 to 103 femtomolar (fM). This PCR-free novel technology can be applied as a test for cancer diagnosis/prognosis to detect 103 copies of a miRNA sequence in RNA extracted from 100 μL of plasma.

Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H 2) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H 2 production limit. The study generated a kinetic model explaining H 2 overproduction and predicted a continuous fermentation system. A Leudking-Piret equationbased model predicted that H 2 production by Tma200 (0.217 mol-H 2 g-1-biomass) and Tma100 (0.147 mol-H 2 g-1-biomass) were higher than wild type (0.096 mol-H 2 g-1-biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h-1) and Tma100 (0.495 h-1) are compared with the wild type (0.663 h-1). A higher 1 digitalcommons.unl.edu

Algae are often promoted as feedstock organisms to produce a sustainable petroleum fossil fuel alternative. However, to induce lipid accumulation most often requires a severe stress that is difficult to induce in large batch cultures. The objective of this study is to analyze and mathematically model heat stress on growth, chlorophyll content, triacylglyceride, and starch synthesis in algae. We initially screened 30 algal species for the most pronounced induction of lipid droplets from heat stress using confocal microscopy and mass spectroscopy techniques. One species, Coccomyxa subellipsoidea C169, was selected and subjected to further biochemical analyses using a jacketed bioreactor amended with 1% CO2 at 25°C, 30°C, 32°C, 33°C, 34°C, 35°C, and 36°C. Lipid and starch accumulation was less extreme than N stress. Growth was reduced above 25°C, but heat stress induced lipid droplet synthesis was negatively correlated with growth only past a demonstrated threshold temperature above 32°C. The optimal temperature for lipid accumulation was 35°C, which led to 6% of dry weight triglyceride content and a 72% reduction from optimal growth after 5 days. Fatty acid influx rates into triglycerides and 15N labeling of amino acids and proteins indicate that heat stress is mechanistically distinct from N stress. Thus, this study lends support to a novel hypothesis that lipid droplet triglycerides result from a redistribution of carbon flux as fatty acids to neutral storage lipids over membrane or other lipids.