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Qian, X, Kumaraswamy GK, Zhang S, Gates C, Ananyev GM, Bryant DA, Dismukes GC.  2015.  Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.. Biotechnol Bioeng. 113(5):979-988. Abstract
To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations-both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))-were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.
Qian, X, Kim M K, Kumaraswamy KG, Agarwal A, Lun DS, Dismukes CG.  2016.  Flux balance analysis of photoautotrophic metabolism: Uncovering new biological details of subsystems involved in cyanobacterial photosynthesis. Biochimica et Biophysica Acta (BBA) - Bioenergetics. :-. AbstractWebsite
We have constructed and experimentally tested a comprehensive genome-scale model of photoautotrophic growth, denoted iSyp821, for the cyanobacterium Synechococcus sp. PCC 7002. iSyp821 incorporates a variable biomass objective function (vBOF), in which stoichiometries of the major biomass components vary according to light intensity. The vBOF was constrained to fit the measured cellular carbohydrate/protein content under different light intensities. iSyp821 provides rigorous agreement with experimentally measured cell growth rates and inorganic carbon uptake rates as a function of light intensity. iSyp821 predicts two observed metabolic transitions that occur as light intensity increases: 1) from PSI-cyclic to linear electron flow (greater redox energy), and 2) from carbon allocation as proteins (growth) to carbohydrates (energy storage) mode. iSyp821 predicts photoautotrophic carbon flux into 1) a hybrid gluconeogenesis-pentose phosphate (PP) pathway that produces glycogen by an alternative pathway than conventional gluconeogenesis, and 2) the photorespiration pathway to synthesize the essential amino acid, glycine. Quantitative fluxes through both pathways were verified experimentally by following the kinetics of formation of 13C metabolites from 13CO2 fixation. iSyp821 was modified to include changes in gene products (enzymes) from experimentally measured transcriptomic data and applied to estimate changes in concentrations of metabolites arising from nutrient stress. Using this strategy, we found that iSyp821 correctly predicts the observed redistribution pattern of carbon products under nitrogen depletion, including decreased rates of CO2 uptake, amino acid synthesis, and increased rates of glycogen and lipid synthesis.
Qin, H, Burnette DT, Bae Y-K, Forscher P, Barr MM, Rosenbaum JL.  2005.  Intraflagellar Transport is Required for the Vectorial Movement of TRPV Channels in the Ciliary Membrane. Curr Biol. 15:1695-1699. Abstract
The membranes of all eukaryotic motile (9 + 2) and immotile primary (9 + 0) cilia harbor channels and receptors involved in sensory transduction (reviewed by). These membrane proteins are transported from the cytoplasm onto the ciliary membrane by vesicles targeted for exocytosis at a point adjacent to the ciliary basal body. Here, we use time-lapse fluorescence microscopy to demonstrate that select GFP-tagged sensory receptors undergo rapid vectorial transport along the entire length of the cilia of Caenorhabditis elegans sensory neurons. Transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 move in ciliary membranes at rates comparable to the intraflagellar transport (IFT) machinery located between the membrane and the underlying axonemal microtubules. OSM-9 motility is disrupted in certain IFT mutant backgrounds. Surprisingly, motility of transient receptor potential polycystin (TRPP) channel PKD-2 (polycystic kidney disease-2), a mechano-receptor, was not detected. Our study demonstrates that IFT, previously shown to be necessary for transport of axonemal components, is also involved in the motility of TRPV membrane protein movement along cilia of C. elegans sensory cells.