Search

Nitrate removal from culture media is widely used to enhance autofermentative hydrogen production in cyanobacteria during dark anaerobiosis. Here we have performed a systematic inventory of carbon and nitrogen metabolites, redox pools, and excreted product fluxes which show that addition of nitrate to cultures of Synechococcus sp. PCC 7002 has no influence on glycogen catabolic rate, but shifts the distribution of excreted products from predominantly lactate and H 2 to predominantly CO 2 and nitrite, while increasing the total consumption of intracellular reducing equivalents (mainly glycogen) by 3-fold. Together with LC–MS derived metabolite pool sizes these data show that glycogen catabolism is redirected from the upper-glycolytic (EMP) pathway to the oxidative pentose phosphate (OPP) pathway upon nitrate addition. This metabolic switch in carbon catabolism is shown to temporally correlate with the pyridine nucleotide redox-poise (NAD(P)H/NAD(P) + ) and demonstrates the reductant availability controls H 2 evolution in cyanobacteria.

ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.

We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultrasensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted 1H and 13C NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 µM in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous (1H2O) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5 × longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of 13C-labeled versus unlabeled metabolites were determined from integral ratios of 1H peaks that exhibit 13C1H J-couplings and independently confirmed by direct measurement of areas of corresponding 13C resonances. 13C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima. Copyright © 2009 John Wiley & Sons, Ltd.

Mossbauer studies of [{μ-S(CH2C(CH3)2CH2S}(μ-CO)FeIIFeI(PMe3)2(CO)3]PF6 (1OX), a model complex for the oxidized state of the [FeFe] hydrogenases, and the parent FeIFeI derivative are reported. The paramagnetic 1OX is part of a series featuring a dimethylpropanedithiolate bridge, introducing steric hindrance with profound impact on the electronic structure of the diiron complex. Well-resolved spectra of 1OX allow determination of the magnetic hyperfine couplings for the low-spin distal FeI (\( {\text{Fe}}^{\text{I}} _{\text{ D}} \)) site, Ax,y,z = [−24 (6), −12 (2), 20 (2)] MHz, and the detection of significant internal fields (approximately 2.3 T) at the low-spin ferrous site, confirmed by density functional theory (DFT) calculations. Mossbauer spectra of 1OX show nonequivalent sites and no evidence of delocalization up to 200 K. Insight from the experimental hyperfine tensors of the FeI site is used in correlation with DFT to reveal the spatial distribution of metal orbitals. The Fe–Fe bond in [Fe2{μ-S(CH2C(CH3)2CH2S}(PMe3)2(CO)4] (1) involving two \( d_{{z^{2} }} \)-type orbitals is crucial in keeping the structure intact in the presence of strain. On oxidation, the distal iron site is not restricted by the Fe–Fe bond, and thus the more stable isomer results from inversion of the square pyramid, rotating the \( d_{{z^{2} }} \) orbital of \( {\text{Fe}}^{\text{I}} _{\text{ D}} \). DFT calculations imply that the Mossbauer properties can be traced to this \( d_{{z^{2} }} \) orbital. The structure of the magnetic hyperfine coupling tensor, A, of the low-spin FeI in 1OX is discussed in the context of the known A tensors for the oxidized states of the [FeFe] hydrogenases.

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry ( F v / F m ). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H 2 production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H 2 production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H 2 revealed two temporally resolved phases of H 2 production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD + ratio revealed that the reductant generated by PFOR contributing to the first phase of H 2 production was not in equilibrium with bulk NADH/NAD + and that the second phase corresponded to the equilibrium NADH-mediated process.