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  • 1
    ISSN: 1432-184X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The formation of microbial mats by thermophilic organisms on submerged rocks in radioactive thermal springs was followed quantitatively in situ as well as under experimental conditions, by determining the change in dry weight and organic matter as a function of time. Furthermore, the decay of the rock occurring in the springs could be shown to be directly related to the microbial colonization. Early in that process the formation of silicious gels, facilitating the settling of the organisms, could be observed. Simultaneously, this was accompanied by the leaching of silica from the underlying rock. This resulted in the destruction of the rock, which had been altered to a fine-grained dust underneath the colonizing mats; the microorganisms were found to move further downward within this layer. From the heavy metals present in the rock—iron (Fe), copper (Cu), manganese (Mn), uranium (U)— the leaching of uranium could be demonstrated, leading to the acquisition of this metal in the microbial mats in concentrations up to 15.34μg/mg dry weight. Direct evidence for the leaching of Si (silicon) and U could be obtained by measurement of these elements after their release from ground rock chips in cultures with microorganisms from the hot springs at 50°C. X-ray analysis of the biomats strongly suggested that Cu, Mn, and Fe are also accumulated.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 129 (1981), S. 127-128 
    ISSN: 1432-072X
    Keywords: Continuous culture ; Adaptation ; Simulation of hot springs ; Boiling point ; Caldoactive bacteria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Bacillus caldolyticus, a caldoactive bacterium originating from a hot spring at Yellowstone Park, was grown in a defined medium, whose composition resembled that of the pool water. Using a chemostat culture, which simulated the natural conditions, the organism could be adapted to grow at 100°C at a reasonable rate. Under increased pressure growth occurred also at 105° C.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 130 (1981), S. 159-164 
    ISSN: 1432-072X
    Keywords: Adaptation ; Boiling point ; Caldoactive enzymes ; Stabilization ; Thermostability ; Thermal characteristics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In chemostat cultures of Bacillus caldolyticus, adaptation in a single step from 70–100°C was followed under aerobic and oxygen-limited conditions and was found to proceed more smoothly under the latter circumstances. Variations of the medium (e.g. yeast extract or silicate concentrations) showed that growth at 100°C is in all respects similar to that of cultures at moderate temperatures. Enzyme preparations derived from cultures at 5°C intervals between 70 and 100°C were used to determine the temperature range. For all nine enzymes tested, the optimum temperature was found to be 67°C; the latter was independent of the growth temperature. Differences were found, however, with respect to the maximum temperature of individual enzymes, and three groups, with maxima between 70 and 80°C, 80 and 90°C and 90 and 100°C can be distinguished. Again, there was no correlation with the growth temperature. Stability experiments also revealed that enzymes from the same organism can have different thermal properties: Some were found to be quite thermolabile (e.g. the pyruvate kinase), while others (e.g. hexokinase and glutamate-pyruvate transaminase) exhibited a high thermostability. These properties were not related to the growth temperature within the 70–100°C range, too. Six of the enzymes tested could be stabilized by their respective substrates, but the degree of protection varied for individual enzymes. Three enzymes (acetate kinase, glutamate dehydrogenase and myokinase) could not be stabilized by their substrates. Comparative experiments with the hexokinase suggested, that the thermal integrity of the enzymes is better protected within the cell as compared to the stability of the enzyme preparations.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 91 (1973), S. 241-254 
    ISSN: 1432-072X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Bacillus caldolyticus, grown at 70°C, produces a highly active extracellular amylase and protease. Both enzymes are formed either within the membrane, or at its inner surface. The activity of both extracellular enzymes was found to decline drastically when brain-heart infusion was omitted from the medium. A simultaneous increase of both enzymes inside the cell was observed. The shifting in extra- and intra-cellular activity was caused by changes in membrane composition due to the increase of anteiso-odd and n-even, and the decrease of iso-odd fatty acids. Membrane composition and enzymic activity could be influenced by the addition of either leucine or iso-leucine as precursors for the synthesis of branched-chain fatty acids: In presence of leucine the anteiso-odd and n-even fatty acids returned to their normal level, while the iso-odd fatty acids increased. Simultaneously the extracellular protease activity increased, and the intracellular activity declined. Growth in amylose-medium supplied with leucine lead to a decrease of both the intra- and extracellular amylase, and changes in the fatty acid composition of the membrane which could not be restored by transfer of the organism to complete media. Addition of iso-leucine first lead to a sharp decrease of extracellular protease and a drastic increase of intracellular protease activity, accompanied by an increase of anteiso-odd and n-even fatty acids, and a decrease of iso-odd compounds. After the second growth in presence of iso-leucine the intra- and extra-cellular protease activity was reversed, and thus showed a return to the starting situation. The reversal is accompanied by the preferential incorporation of fatty acids with a higher melting point into the membrane. Extracellular amylase activity was found to increase after the first growth with iso-leucine, and to decline sharply after the second culture with iso-leucine, together with a very high intracellular amylase activity at that point. Extra- and intra-cellular amylase activity both declined upon growth in complete medium, while the fatty acid distribution remained different from the initial composition.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 112 (1977), S. 103-107 
    ISSN: 1432-072X
    Keywords: Alkaline phosphatase ; Adaptation ; Derepression ; Repression ; Phosphite ; Hypophosphite
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract When Escherichia coli cells were grown in media containing either phosphite or hypophosphite as the sole source of phosphorus, they responded to this situation primarily in the same way as phosphatelimited cultures: The activity of alkaline phosphatase increased drastically, which under natural conditions would enable the cells to compklensatae for the shortage increased drastically, which under natural conditions would enable the cells to compensate for the shortage of phosphate. Subsequent transfers, however, resulted in a quite different response: While the phosphatase activity of phosphate-limited cells stays at a high derepressed level, its increase was followed by a gradual decline in organisms grown on phosphite or hypophosphite. After eight to ten transfers on these P-compounds, phosphatase activity was back to its initial, repressed, low level, indicating that the cells were fully adapted to these substrates. Adaptation to either PO 3 3- or PO 2 3- was completely abolished if the cells were again grown with PO 4 3- as P-source, whereafter the entire process of adaptation had to be repeated. The observed adaptation pattern, reflected by the alterations of phosphatase activity, was qualitatively equal with PO 3 3- and PO 2 3- , but quantitatively different, because the response to hypophosphite gave much higher values than the increase obtained with phosphite. Phosphite-adapted cells are not simultaneously adapted to hypophosphite, but their response to the latter was less intense than observed after direct transfers from PO 4 3- to PO 2 3- . Adaptation to hypophosphite, however, led simultaneously to phosphite adaptation, so that these cells can utilize both P-compounds as a substitute for phosphate.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 90 (1973), S. 223-232 
    ISSN: 1432-072X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium. Direct concentration and separation from liquid media, omitting ammonium sulfate treatment, was also found to be possible, although prolonged flushing with buffer was necessary to obtain satisfactory separation. During purification from the ammonium sulfate fractions, amylase activity was found to decrease but could be restored by Ca-ions. At 70°C, a final concentration of 0.5 mM CaCl2, was sufficient for full restoration, while three times that amount was necessary at 80°C. Determination of the K m-values for Ca at different temperatures resulted in an asymptotically increasing curve at temperatures beyond 75°C. Addition of Ca had a pronounced effect on the stability of the amylase at 80°C but not at 90°C. Protease activity and stability was not affected by Ca-ions.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 106 (1975), S. 201-207 
    ISSN: 1432-072X
    Keywords: Thermophilic bacteria ; Thermostable amylase ; Bacillus caldolyticus ; Protoplasts of B. caldolyticus ; Ca-ions and MW of amylase ; Amylase activity and Ca-ions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract One of the typical properties of the extracellular amylase produced by Bacillus caldolyticus is the tendency to disintegrate into subunits with a molecular weight (MW) of less than 10 000, when the enzyme is subjected to ultrafiltration. Disintegration is due to a loss of Ca-ions, leading to nonactive subunits. Activity can be fully restored by addition of Ca-ions. Reactivation occurs also spontaneously if the low MW fraction is stored in glassware. Comparative ultrafiltration experiments with the subunit fraction with or without a supply of Ca revealed that in oresence of this divalen cation the subunit reaggregated to the active enzyme. The different distribution patterns obtained in absence or presence of Ca showed that reactivation is directly linked to the formation of a high MW form of the protein. Substitution of Ca by other divalent cations also led to reaggregation. These aggregates are, however, inactive. The enzyme was found to be formed intracellularly in its low MW form. Experiments with protoplasts revealed that these are capable to produce and release the amylase. When the production of the enzyme by protoplasts declined, full restoration could be achieved by a recovery treatment. Normally, the enzyme released by the protoplasts consisted of equal portions of the high, medium, and low MW form of the amylase. If the cells were, however, depleted as a result of continued incubations, the extruded enzyme consisted increasingly of the low MW form, which could finally represent more than 80%. This trend could be completely reversed by the supply of carbon and nitrogen sources during the recovery treatment, whereafter the enzyme consisted again of the initially observed equal amounts of the three MW forms. Vesicles prepared from the protoplasts were also found to release amylase, but on a lower level, and only for a very limited time, with no possibility to regain activity by a recovery treatment. Subunit formation was also observed during column chromatography, which could be counteracted by a sufficient supply with Ca-ions.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 95 (1974), S. 267-274 
    ISSN: 1432-072X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Bacillus caldolyticus can utilize phosphorus either as phosphate, phosphite, or hypophosphite. When cultures are supplied with PO2 as the sole source of phosphorus, the hypophosphite is oxidized to phosphate, which accumulates in the medium prior to the beginning of the log phase, and is then metabolised during growth. Resting cell suspensions also have the ability to oxidise PO2 to PO4. The reaction is specific for hypophosphite: PO3 is not oxidised to PO4, regardless of whether the cells are grown in PO3- or PO2-medium. The hypophosphite oxidase works optimally between pH 7.0 to 7.5, with a temperature optimum at 75°C; theK m for NaH2PO2 is 320 μM. Sonication of cells, followed by high-speed centrifugation and ammonium sulfate fractionation of the cell-free extract showed that the PO2 oxidation, which is accompanied by the formation of NADH, requires at least three components: An ammonium sulfate fraction of the cell-free extract, the residue fraction containing the respiratory chain, and NAD as cofactor. Most probably a second cofactor, so far not characterized, is required to accomplish full activity.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 134 (1983), S. 247-250 
    ISSN: 1432-072X
    Keywords: Thermophilic actinomycete ; Enzyme stability ; Fatty acids ; Temperature change ; Streptomyces
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The thermostability of several enzymes from the facultative thermophilic actinomycete Streptomyces sp., derived from cells grown in the temperature range from 37°C to 60°C, has been examined. A correlation between the growth temperature of the cultures and the heat stability of the enzymes could be demonstrated for alanine dehydrogenase, acetate kinase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, myokinase and pyruvate kinase. Except for the isocitrate dehydrogenase, which showed a linear increase of its stability throughout the entire temperature range, all enzymes exhibited a steep increase of the heat stability up to about 50°C, but no further increase in the higher growth range, suggesting, that from 50°C upward a shift to the exclusive production of thermostable protein occurs. Furthermore, the stability of alanine dehydrogenase and pyruvate kinase was found to increase substantially in presence of their substrates. In contrast, substrate-mediated stabilization was very weak with glucose-6-phosphate dehydrogenase and totally absent with acetate kinase, isocitrate dehydrogenase and myokinase. A comparison with previous observations with the same enzymes from Bacillus flavothermus showed, that enzymes from different organisms can have different thermal properties. Determination of the fatty acid composition of Streptomyces sp. cell, grown at different temperatures, showed relatively small alterations, with the main changes occurring between 37°C and 40°C.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 95 (1974), S. 67-78 
    ISSN: 1432-072X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A mesophilic bacterium,Proteus mirabilis, which was known to be able to accumulate monomer silicate ions, a thermophilic bacterium,Bacillus caldolyticus, originating from a habitat with high silica concentrations, and a silicautilizing plant,Equisetum arvense, were all found to produce monomer silica from its polymer. The monomer silica, resulting from the mineralysis of either experimentally polymerized silica, or from quartz, is taken up byP. mirabilis cells, and also byEquisetum, which then deposits the silica again as a polymer in its stem and leaves. WithB. caldolyticus, which does not utilize the depolymerized product under the given conditions, we found that the intensity of the mineralysis depends on the growth rate of the organism.
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