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Properties of phosphoribulokinase from Thiobacillus neapolitanus 总被引:3,自引:1,他引:3
Partially purified preparations of ribulose-5-phosphate kinase (specific activity, 50 to 125 mumoles per min per mg of protein) were employed in a series of kinetic experiments in the presence of several concentrations of H(+), Mg(2+), adenosine triphosphate (ATP), and phosphoenolpyruvate (PEP). The pH optimum of the enzyme was found to be 7.9; at this pH and above, response of the enzyme to variations in ATP concentration was hyperbolic, exhibiting a K(m) of 7 x 10(-4)m ATP. At pH values below the optimum the response to ATP was sigmoidal, as it was throughout the entire pH range in the presence of PEP at a concentration greater than 5 x 10(-4)m. In the presence of PEP the pH optimum shifted to pH 8.4. In contrast, phosphoribulokinase from spinach exhibited hyperbolic responses throughout its pH range with no inhibition caused by PEP. Thiobacillus neapolitanus phosphoribulokinase was inhibited by PEP in a sigmoidal manner; however, in the presence of suboptimal concentrations of Mg(2+) the addition of PEP caused significant stimulation of activity. It is postulated that the enzyme consists of interacting subunits with several sites on the enzyme for binding ATP and with several separate sites binding PEP. It is suggested that PEP functions as a regulator of CO(2) fixation when the organism is under conditions of unlimited concentrations of substrate and CO(2). 相似文献
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D. P. Kelly 《Archives of microbiology》1970,73(2):177-192
Summary A comparative study has been made of the metabolism in several strains of Thiobacillus neapolitanus of formate, acetate, propionate, butyrate, valerate and pyruvate. Conflicting reports in the literature concerning the mechanism of pyruvate assimilation in thiobacilli have been resolved. Pyruvate undergoes decarboxylation to yield acetyl coenzyme A, which is converted to glutamate, proline and arginine via reactions of the incomplete Krebs' cycle of this organism. Pyruvate is converted also to alanine, valine, isoleucine, leucine and lysine by mechanisms like those in heterotrophs. No aspartate is formed from the C-3 of pyruvate. Removal of the C-1 of pyruvate yields carbon dioxide, which is refixed into all cell constituents. Formate is not produced by this scission reaction, as formate itself is incorporated almost exclusively into purines. Aspartate can be synthesized by the activities of phosphoenolpyruvate carboxylase and oxaloacetate-glutamate transamination. Carbon from propionate is converted principally to lipids, although some amino acid production occurs with the same distinctive labelling pattern as is found after acetate assimilation by T. neapolitanus strains C and X. Butyrate and valerate also showed some distinctive patterns of incorporation into cell constituents. Fluoropyruvate and fluoropropionate inhibited the growth of T. neapolitanus and the mechanisms of this poisoning are discussed.Generally these compounds contributed only small proportions of the total cell carbon and tended to be converted to limited numbers of cell components. The thiobacilli thus tend to conserve carbon from these compounds and not to degrade them to carbon dioxide on a large scale when growing in an otherwise autotrophic medium. 相似文献
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A Sk?odowska 《Acta microbiologica Polonica》1985,34(3-4):271-276
Different bacterial cell fractions of Thiobacillus neapolitanus were examined in order to localize the active sites for thiosulphate and sulphite oxidation. Difference spectra of the fractions were made to determine the level at which electrons from sulphite and thiosulphate enter the respiratory chain. Active sites for thiosulphate are probably strictly connected with cell membranes. Thiosulphate and sulphite reduced cytochome b and c in bacterial cell extracts. It has been found that thiosulphate oxidation is accompanied by production of tetrathionate and trithionate. 相似文献
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Yolande A. Holthuijzen Jan F. L. van Breemen J. Gijs Kuenen Wil N. Konings 《Archives of microbiology》1986,144(4):398-404
For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO
ribulose-1,5-bisphosphate carboxylase
- PMSF
phenylmethylsulfonyl fluoride
- PAA
gelectrophoresis, polyacrylamide gelelectrophoresis
- SDS
sodium dodecyl sulphate
- CIE
crossed immunoelectrophoresis
- IEF
isoelectric focusing 相似文献
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Growth Kinetics and Yield Coefficients of the Extreme Thermophile Thermothrix thiopara in Continuous Culture 总被引:1,自引:1,他引:1
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Thermothrix thiopara did not appear to be stressed at high temperature (72°C). Both the actual and theoretical yields were higher than those of analogous mesophilic sulfur bacteria, and the specific growth rate (μmax) was more rapid than that of most autotrophs. The specific growth rate (0.58 h−1), specific maintenance rate (0.11 h−1), actual molar growth yield at μmax (Ymax = 16 g mol−1), and theoretical molar growth yield (YG = 24 g mol−1) were all higher for T. thiopara (72°C) than for mesophilic (25 to 30°C) Thiobacillus spp. The growth efficiencies for T. thiopara at 70 and 75°C (0.84 and 0.78) were significantly higher than at 65°C (0.47). Corresponding specific maintenance rates were highest at 65°C (0.41 h−1) and lowest at 70 and 75°C (0.11 and 0.15 h−1, respectively). Growth efficiencies of metabolically similar mesophiles were generally higher than for T. thiopara. However, the actual yields at μmax were higher for T. thiopara because its theoretical yield was higher. Thus, at 70°C, T. thiopara was capable of deriving more metabolically useful energy from thiosulfate than were mesophilic sulfur bacteria at 25 and 30°C. The low growth efficiency of T. thiopara reflected higher maintenance expenditures. T. thiopara had higher maintenance rates than Thiobacillus ferroxidans or Thiobacillus denitrificans, but also attained higher molar growth yields. It is concluded that sulfur metabolism may be more efficient overall at extremely high temperatures due to increased theoretical yields despite increased maintenance requirements. 相似文献
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Yolande A. Holthuijzen Jan F. L. van Breemen Wil N. Konings Ernst F. J. van Bruggen 《Archives of microbiology》1986,144(3):258-262
The outer part of the carboxysomes of Thiobacillus neapolitanus was examined by electron microscopy using negatively stained, cryo-treated, frozen hydrated and freeze dried specimens. From stereo-micrographs of freeze dried and fixated carboxysomes the three dimensional structure of the carboxysomes was elucidated. The carboxysomes always appear as hexagonal bodies, which possess twelve pentameric planes. This indicates that carboxysomes have the form of a pentagonal dodecahedron. Inside the carboxysomes the ribulose-1,5-bisphosphate carboxylase molecules are arranged in rows and concentric rings. Negatively stained and cryo-treated carboxysomes do not differ significantly in size. The mean size of these carboxysomes is 117.3±6.9 nm (n=782) 相似文献
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Enzymes of Intermediary Carbohydrate Metabolism in the Obligate Autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus 总被引:1,自引:1,他引:1
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Levels of enzymes operative in the Embden-Meyerhof-Parnas (glycolytic) pathway, pentose phosphate cycle, citric acid cycle, and certain other phases of intermediary carbohydrate metabolism have been compared in Thiobacillus thioparus and T. neapolitanus. All enzymes of the glycolytic pathway except phosphofructokinase were demonstrated in both organisms. There were some striking quantitative differences between the two organisms with respect to the activities of the individual enzymes of the glycolytic pathway and the citric acid cycle. Qualitative differences were also found: the isocitrate dehydrogenase activity of T. thioparus is strictly nicotinamide adenine dinucleotide phosphate (NADP)-dependent, whereas that of T. neapolitanus is primarily nicotinamide adenine dinucleotide-dependent, activity with NADP being low; the glucose-6-phosphate dehydrogenase of T. thioparus is particulate, whereas that of T. neapolitanus is partly soluble and partly particulate; the 6-phosphogluconate dehydrogenase of T. thioparus is soluble, that of T. neapolitanus is partly soluble and partly particulate. All enzymes which function in the carbon reduction cycle were present at very high levels. In contrast, enzymes which operate exclusively in cycles other than the carbon reduction cycle were present at low levels. Of the enzymes not operative in the carbon reduction cycle that were examined, isocitric dehydrogenase had the highest specific activity. Both organisms possessed reduced nicotinamide adenine dinucleotide dehydrogenase activity. The qualitative and quantitative aspects of the data are discussed in relation to possible biochemical explanations of obligate autotrophy. 相似文献
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Two cloning vectors designed for use in Escherichia coli and the thiobacilli were constructed by combining a Thiobacillus intermedius plasmid replicon with a multicloning site, lacZ(prm1), and either a kanamycin or a streptomycin resistance gene. Conditions necessary for the introduction of DNA into T. intermedius and T. neapolitanus via electroporation were examined and optimized. By using optimal electroporation conditions, the gene encoding a carboxysome shell protein, csoS1A, was insertionally inactivated in T. neapolitanus. The mutant showed a reduced number of carboxysomes and an increased level of CO(inf2) necessary for growth. 相似文献
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RP1, a broad-host-range incompatibility group P1 plasmid specifying multiple drug resistances, has been transferred into the chemolithotrophic bacterium Thiobacillus neapolitanus. The ability of T. neapolitanus to receive, express, and transmit RP1-encoded antibiotic resistances was examined. The data show that this obligate chemolithotroph can accept, replicate, and express heterologous plasmid DNA from a heterotrophic bacterium. 相似文献
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Assimilation and Metabolism of Exogenous Organic Compounds by the Strict Autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus 总被引:2,自引:2,他引:2
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The assimilation and utilization of the individual carbon atoms of pyruvate and acetate by cells of Thiobacillus thioparus and T. neapolitanus, in the presence and absence of an energy source, were studied by use of radioactive substrates. Both organisms produced 14CO2 from 14C-labeled pyruvate, but more came from carbon 1 than from carbons 2 or 3. The conversion of the carbons of acetate to CO2 by both organisms was much less than that from any of the pyruvate carbons. When labeled pyruvate and acetate were incubated with these organisms, small amounts of radioactivity were found in the tricholoacetic acid-soluble material, nucleic acids, and lipids, and larger amounts were found in the protein fraction. The composition of the incubation medium affected the amount of utilization and incorporation of labeled substrates by both organisms. The presence of an exogenous energy source (Na2S2O3) suppressed incorporation of the labeled substrates into various cellular components by T. thioparus, but enhanced incorporation by T. neapolitanus. When 14C-pyruvate was used as a substrate, as many as 12 radioactive compounds were found in the water-soluble fraction in the experiments with T. neapolitanus, whereas no more than three radioactive compounds were detected in this fraction in the experiments with T. thioparus. Of the total 14C activity found in the water-soluble fractions, malic acid contained the highest percentage. These findings are discussed in light of the overall metabolism of these two sulfur-oxidizing obligate chemoautotrophs, as well as in relation to the biochemical basis of chemoautotrophy. 相似文献
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