is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H + CO or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 × 10 A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase (), resulting in a Δ uracil-auxotrophic strain, TKV002. Reintroduction of on a plasmid or insertion of into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in The gene (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using as a selective marker via two single homologous recombination events. The resulting Δ strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium. Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for synthesis of carbon compounds such as acetate, butyrate, or ethanol from H + CO or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer to produce fuels and chemicals.
Children appear to be more susceptible to orthostatic stress than adults. Therefore, tilt protocols commonly used in adults lack specificity in teenage patients. A specificity > 85% may be obtained by performing the tilt test at 60 degrees or 70 degrees for no longer than 10 min.
Summary Acetogenic bacteria recently attracted attention because they reduce carbon dioxide (CO2) with hydrogen (H2) to acetate or to other products such as ethanol. Besides gases, acetogens use a broad range of substrates, but conversion of the sugar alcohol mannitol has rarely been reported. We found that the thermophilic acetogenic bacterium Thermoanaerobacter kivui grew on mannitol with a specific growth rate of 0.33 h−1 to a final optical density (OD600) of 2.2. Acetate was the major product formed. A lag phase was observed only in cultures pre‐grown on glucose, not in those pre‐grown on mannitol, indicating that mannitol metabolism is regulated. Mannitol‐1‐phosphate dehydrogenase (MtlD) activity was observed in cell‐free extracts of cells grown on mannitol only. A gene cluster (TKV_c02830–TKV_c02860) for mannitol uptake and conversion was identified in the T. kivui genome, and its involvement was confirmed by deleting the mtlD gene (TKV_c02860) encoding the key enzyme MtlD. Finally, we overexpressed mtlD, and the recombinant MtlD carried out the reduction of fructose‐6‐phosphate with NADH, at a high VMAX of 1235 U mg−1 at 65°C. The enzyme was thermostable for 40 min at 75°C, thereby representing the first characterized MtlD from a thermophile.
Ultraviolet irradiation of 11I acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) produces a loss of trytophan fluorescence which is best descri as the sum of two separable first-order processes, one much more rapid than the other. In addition, the enzyme undergoes an all-or-none inactivation that is monotonically first order. Simultaneous with activity loss, photoscission takes place and results in a molecular weight drop of 1 X 105; this decrease is first order with a rate constant identical to that for enzymatic inactivation. These processes are accompanied by apparent conformational changes, as shown by circular dichroic and difference absorption spectra. The relative photochemical inactivation efficiency of incident light is unity when corrected for the wavelength dependence of fluorescence excitation, which is consistent with an efficient F6rster resonance. transfer of energy among the aromatic chromophores. The extreme sensitivity of acetylcholinesterase to photodestruction upon photon absorption and the several events that follow it not only suggest that these findings might be a basis for a useful molecular probe of the structure of'this enzyme, but also indicate that additional care should be taken when conducting spectroscopic studies in the UV region.Recent studies on acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) have used extrinsic fluorescent probes to determine active-site geometry (1-3). However, little work has been done on the protein's intrinsic fluorescence. During such a study on llS acetylcholinesterase, we observed that the enzyme was denatured by incident UV irradiation. Accordingly, we performed some preliminary characterizations of this process, and we report here our initial findings.MATERIALS AND METHODS Homogenous 11S acetylcholinesterase was prepared from Electrophorus electricus and its activity was assayed as described (4). Irradiation and fluorescence measurements were done at 250C in 1 X 1 cm-quartz cells with a thermostatted Perkin-Elmer MPF-44B fluorometer operated in the quantum corrected mode. Circular dichroic spectra were obtained at room temperature (--200C) with a JASCO (Tokyo, Japan) J-500 A recording spectropolarimeter standardized with D-10-camphorsulfonic acid (5). Difference absorption measurements were made at 250C with a Beckman model 25 spectrophotometer. Molecular weight determinations were carried out as before at SOC and 14,000 rpm in 4-mm solution columns (6) with an initial A2w of 0.2. A iv of 0.731 cm3/g was used for control and irradiated enzyme.Theoretical calculations of concentration, C, as a function of radial distance for model systems were made as follows:[2] in which A = (1 -ip) w2/2RT (the symbols having their customary meanings), a and b are meniscus and solution base radius, respectively, Mt is the molecular weight of the ith species, Co,j, the starting concentration of the ith species, and Ca., and Cj are the concentrations of the ith species at the meniscus and radius positions a and r, respectively. Th...
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