ADP-Dependent Phosphofructokinases in Mesophilic and Thermophilic Methanogenic Archaea

Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PP(i)-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order Thermococcales, including Pyrococcus and Thermococcus spp., have recently been demonstrated to possess a unique ADP-dependent PFK (ADP-PFK) that appears to be phylogenetically distinct. Here, we report the presence of ADP-PFKs in glycogen-producing members of the orders Methanococcales and Methanosarcinales, including both mesophilic and thermophilic representatives. To verify the substrate specificities of the methanogenic kinases, the gene encoding the ADP-PFK from Methanococcus jannaschii was functionally expressed in Escherichia coli, and the produced enzyme was purified and characterized in detail. Compared to its counterparts from the two members of the order Thermococcales, the M. jannaschii ADP-PFK has an extremely low K(m) for fructose 6-phosphate (9.6 microM), and it accepts both ADP and acetyl-phosphate as phosphoryl donors. Phylogenetic analysis of the ADP-PFK reveals it to be a key enzyme of the modified Embden-Meyerhof pathway of heterotrophic and chemolithoautotrophic archaea. Interestingly, uncharacterized homologs of this unusual kinase are present in several eucarya.     

F-actin in mitotic spindles of synchronized suspension culture cells of tobacco visualized by confocal laser scanning microscopy

TRITC-labelled phalloidin was used to visualize F-actin distribution during mitosis in Nicotiana tabacum BY-2 suspension cells. Aphidicolin was used to synchronize cell suspensions, which enabled sufficient numbers of mitotic cells to be obtained. F-actin was present in the spindle, and its orientation seemed to correlate with the known microtubular arrays. The use of confocal microscopy greatly reduced background fluorescence, and therefore fine actin filaments could be observed in spindles previously thought to be devoid of actin.     

Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.     

Development and cellular organization ofPinus sylvesfris pollen tubes

The organization ofPinus sylvestris pollen tubes during growth was studied by video microscopy of living cells and by electron microscopy after freeze-fixation and freeze-substitution (FF-FS). Pollen germinated and the tubes grew slowly for a total period of about 7 days. Some of the grains formed two tubes, while 10–50% of the tubes ramified. These features are in accordance with development in vivo. The cytoplasmic hyaline cap at the tip disappeared during the 2nd or 3rd day of culture. Aggregates of starch grains progressively migrated from the grain into the tube and later into the branches. Vacuoles first appeared at day 2 and eventually filled large parts of the tube. The tube nucleus was located at variable distances from the tip. Some of the organelles showed linear movements in a mostly circulatory pattern, but the majority of the organelles showed brownian-like movements. Rhodamine-phalloidin-stained actin filaments had a gross axial orientation and were found throughout the tube including at the tip. The ultrastructure of pollen tubes was well preserved after FF-FS, but signs of shrinkage were visible. The secretory vesicles in growing tips were not organized in a vesicle cone, and coated pits had a low density with only local accumulations, which is in accordance with slow growth. The mitochondria contained small cristae and a darkly stained matrix and were located more towards the periphery of the tube, indicating low respiratory activity and low oxygen levels. The dictyosomes carried typical trans-Golgi networks, but some contained less than the normal number of cisternae. Other elements of the cytoplasm were irregularly spaced rough endoplasmic reticulum, many multivesicular bodies, lipid droplets and two types of vacuoles. The typical organization associated with tip growth in angiosperm pollen tubes, e.g.Nicotiana tabacum, was not present inP. sylvestris pollen tubes. The different morphology may relate to the growth rate and not to the type of growth.     

The role of ethanol oxidation during carboxydotrophic growth of Clostridium autoethanogenum

The Wood–Ljungdahl pathway is an ancient metabolic route used by acetogenic carboxydotrophs to convert CO into acetate, and some cases ethanol. When produced, ethanol is generally seen as an end product of acetogenic metabolism, but here we show that it acts as an important intermediate and co-substrate during carboxydotrophic growth of Clostridium autoethanogenum. Depending on CO availability, C. autoethanogenum is able to rapidly switch between ethanol production and utilization, hereby optimizing its carboxydotrophic growth. The importance of the aldehyde ferredoxin:oxidoreductase (AOR) route for ethanol production in carboxydotrophic acetogens is known; however, the role of the bifunctional alcohol dehydrogenase AdhE (Ald–Adh) route in ethanol metabolism remains largely unclear. We show that the mutant strain C. autoethanogenum ∆adhE1a, lacking the Ald subunit of the main bifunctional aldehyde/alcohol dehydrogenase (AdhE, CAETHG_3747), has poor ethanol oxidation capabilities, with a negative impact on biomass yield. This indicates that the Adh–Ald route plays a major role in ethanol oxidation during carboxydotrophic growth, enabling subsequent energy conservation via substrate-level phosphorylation using acetate kinase. Subsequent chemostat experiments with C. autoethanogenum show that the wild type, in contrast to ∆adhE1a, is more resilient to sudden changes in CO supply and utilizes ethanol as a temporary storage for reduction equivalents and energy during CO-abundant conditions, reserving these ‘stored assets’ for more CO-limited conditions. This shows that the direction of the ethanol metabolism is very dynamic during carboxydotrophic acetogenesis and opens new insights in the central metabolism of C. autoethanogenum and similar acetogens.     

Reductive dechlorination of tetrachloroethene to cis-1, 2-dichloroethene by a thermophilic anaerobic enrichment culture.

Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas. Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to cis-1, 2-dichloroethene at the optimum temperature of 60 to 65 degrees C. After several transfers, methanogens were eliminated from the culture. Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H2, formate, or acetate. Fumarate and L-malate led to the highest dechlorination rate. In the absence of PCE, fumarate was fermented to acetate, H2, CO2, and succinate. With PCE, less H2 was formed, suggesting that PCE competed for the reducing equivalents leading to H2. PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor. At the optimum dissolved PCE concentration of approximately 60 microM, a high dechlorination rate of 1.1 micromol h-1 mg-1 (dry weight) was found, which indicates that the dechlorination is not a cometabolic activity. Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod. Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives. The highest similarity was found with the genus Dehalobacter (90%), represented by the halorespiring organism Dehalobacter restrictus, and with the genus Desulfotomaculum (86%).     

Anaerobic Microbial Reductive Dehalogenation of Chlorinated Ethenes

The current knowledge on microbial reductive dechlorination of chlorinated ethenes (CEs) and its application are discussed. Physiological studies on CEs dechlorinating microorganisms indicate that a distinction can be made between cometabolic dechlorination and halorespiration. Whereas cometabolic dechlorination is a coincidental and nonspecific side reaction, catalyzed by several methanogenic and acetogenic bacteria, halorespiration is a specific enzymatic reaction from which metabolic energy can be gained. In contrast to the well-studied biological dechlorination of PCE to cis-DCE, little is known about the biology of the further dechlorination from cis-DCE to ethene. Bacteria performing the latter reaction have not yet been isolated. Microbial reductive dechlorination can be applied to the in situ bioremediation of CEs contaminated sites. From laboratory and field studies, it has become clear that the dechlorination of tetrachloroethene (PCE) to cis-clichloroethene (cis-DCE) occurs rapidly and can be stimulated relatively easily. However, complete reduction to ethene appears to be a slower process that is more difficult to achieve.     

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (K_m, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the “threonine dehydrogenase and related Zn-dependent dehydrogenases” (COG1063). Metal analysis confirmed the presence of 2 Zn²⁺ atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys₃₇, His₇₀, and Glu₇₁, while the structural zinc site is probably composed of Cys₁₀₀, Cys₁₀₃, Cys₁₀₆, and Cys₁₁₄. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.     

Genetic and biochemical characterization of a short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.