In vivo and In vitro function of the intracellular proteolytic apparatus in nongrowing bacillus megaterium under heat stress

In Bacillus megaterium sporulating at 35 degreesC, up to 90% of 10-min pulse-labeled proteins were degraded. Degradation proceeded in two waves. Short-lived proteins, i.e., intrinsically labile proteins and proteins made short-lived because of starvation, were mostly degraded during the reversible sporulation phase. Their amount corresponded to 20% or slightly more during 2 h. The second wave of protein degradation, which followed during the irreversible sporulation phase at 35 degreesC, increased the amount of total degradable pulse-labeled proteins to about 90%. This wave was absent in the isogenic asporogenic mutant 27-36 or in the wild strain, whose sporulation was inhibited by increased temperature. The proportion of degradable proteins was thus reduced to less than 40% in the asporogenic mutant incubated at 35 degreesC and to 46% in the wild strain whose sporulation was suppressed by the temperature of 47 degreesC. Unlike sporulating cells, these cells were thus capable of degrading short-lived and denatured proteins, but were not able to degrade most of other proteins. The in vitro protein degradation was substantially enhanced by increasing the Ca2+ concentration, suggesting a role of Ca2+-dependent proteinase(s) in the process.

Cell viability and protein turnover in nongrowing Bacillus megaterium at sporulation suppressing temperature.

In Bacillus megaterium, a temperature that suppresses sporulation (43 degrees C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40-41 degrees C). Here we show that, when cells grown at 35 degrees C and transferred to a sporulation medium, were subjected to shifts between 35 degrees C and the sporulation suppressing temperature (SST, 43 degrees C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35 degrees C for 2-3 h (T2-T3), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T4-T5. Protein turnover rate increased again after T2 and up to T8 60-70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T3 no asymmetric septa were observed and the amount of heat-resistant spores at T8 was by 4-5 orders lower than at 35 degrees C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T3 but then its velocity sharply dropped and the amount of degraded protein at T8 corresponded to slightly more than one-half of that found at 35 degrees C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the amount of degraded protein was slightly lower than at 35 degrees C. A later (T4) shift to SST had no effect on the sporulation process.

Asporogenic Bacillus megaterium mutant 27-36 degrades intrinsically short-lived proteins but fails to convert most of other proteins to a short-lived fraction.

Asporogenic mutant blocked in the 0-II sporulation stage degraded pulse-labelled proteins in the sporulation medium at the same rate as the parental strain for the first two hours. The degraded fraction was mostly composed of intrinsically short-lived proteins which were degraded even after enriching the medium with amino acids and growth resumption. Proteins accessible to degradation because of nutritional shift down formed a lesser proportion of this fraction. The acceleration of protein turnover in the parent strain during the irreversible sporulation phase was not developed in the mutant. A first order kinetic model of protein degradation was used for parameter estimation. Ca(2+)-dependent intracellular serine proteinase was synthesized in an inactive form, which was activated by increasing Ca2+ concentration to 30 mM.

Determination of platinum in plants by emission spectrometry after preconcentration on modified silicagel.

Silicagel Separon SGX C18 (particle size 7 microm) was suitable for the preconcentration of 2-20 microg of Pt from 0.1M hydrochloric acid in the presence of cationic surfactants especially dimethyllaurylbenzylammonium bromide, with subsequent elution with 96% ethanol. The recovery was 86-110% for 2 microg of Pt. The sample matrix corresponding to 2.5 g of average plant ash does not interfere. The final emission spectrometry of platinum was carried out in 15 A dc-arc at Pt I 265.942 nm in the presence of Au as internal standard (Au I 267.595 nm). RSD was 6.3% in average.

Effect of actinomycin D on viability, sporulation and nucleotide pool of Bacillus megaterium.

A transient 7-fold rise of ppGpp concentration, 2-3-fold increase of pppGpp concentration and 50% drop of the concentration of GTP in Bacillus megaterium cells immediately after their transfer to the sporulation medium were observed. Actinomycin D, in concentrations inhibiting RNA synthesis by 95%, blocked the rise of the (p)ppGpp pool and caused an instant several-fold increase of the GTP level. When the cells were exposed to actinomycin D in the sporulation medium for a 1-h period (time 0-1 h, 1-2 h or 2.20-3.20-h), they were able to form colonies on nutrient agar after being kept, in addition for 1-2 h in the sporulation medium free of the antibiotic. The ability of sporulation was, however, markedly limited. The share of cells that could sporulate increased when the irreversible sporulation phase was reached.

Regulation of extracellular proteins and alpha-amylase secretion by temperature in Bacillus subtilis.

alpha-Amylase was found to be the main protein secreted by Bacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45 degrees C, respectively. A change in temperature can affect the pattern of proteins secreted as detected by gel electrophoresis. 14C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30 degrees C than that at 40 or 45 degrees C. The effect of temperature on alpha-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.

Plasmid pIMI38--the pBR322 derivative with increased stability in E. coli cells.

Plasmid pIM138 which had been characterized by the higher resistance of its DNA replication to the action of clorobiocin in comparison with the progenitor plasmid, was tested for its stability in host cells in the absence of the antibiotic. Growing without selective pressure, pIM138 was better maintained in cells than pBR322. The stability in the presence and in the absence of clorobiocin can be unanimously assigned to the plasmid itself, but some influence of host cells cannot be excluded.

Effect of temperature on alpha-amylase formation and DNA replication in Bacillus subtilis.

The effect of temperature on extracellular alpha-amylase synthesis and chromosomal and plasmid DNA replication in Bacillus subtilis A18 carrying plasmid pMI10 was studied. The specific growth rate mu increased with elevated temperature up to 42.5 degrees C, while the activities of alpha-amylase per population dry mass decreased. No obvious quantitative changes of 14C-thymidine incorporation per dry mass increase and no basic differences in plasmid copy number in the range of temperatures from 25 to 40 degrees C were found.

Turnover of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae: differences between in vivo and in vitro degradation.

Degradation of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae in vivo was compared with that in cell-free extracts. Protein degradation in vivo, when the cells were labelled with 14C-leucine during growth in the presence of ethionine, was affected by the concentration of the analogue used. Proteins synthesized in the presence of 0.2-1 mM ethionine were degraded most rapidly in both organisms. The proteolytic enzyme system of yeast degraded the analogue-containing proteins in vitro faster than the normal proteins. This holds also for proteins synthesized in the presence of 5 mM ethionine, whose degradation in vivo was impaired. The proteolytic system of B. megaterium, on the other hand, was unable in vitro to differentiate between normal and abnormal proteins. Denatured proteins underwent preferential degradation over normal and ethionine-containing proteins.

[Succinate dehydrogenase activity in the mesonephros in the process of development of chick embryos]. / Aktivnost' suktsinatdegidrogenazy v mezonefrose v protsesse razvitiia émbrionov tsypliat.

The activity of succinate dehydrogenase in the mesonephros tissue of chick embryos is rather high on the 6th day of embryonal development and is not subjected to significant changes up to the 17th day. The absence of wide variations in the activity of this enzyme during the period of degeneration of the mesonephros may be due to a parallel decrease in the organ's mass and in the number of its mitochondria.

Synthesis of exocellular proteins during the exponential and stationary phase of growth of Bacillus megaterium.

Synthesis of exocellular metalloprotease and cellular and exocellular proteins in the sporogenic strain Bacillus megaterium J-27 and asporogenic strain KM 1 was investigated. Both organisms excrete the enzyme into the medium during growth and during the stationary phase. In the asporogenic strain the excretion decreases at the end of the exponential phase. In the sporogenic strain it continues during the transition to the stationary phase at the original rate and proteolytic activity in the medium increases two to three times during 2 h after the end of the exponential phase. Both organisms synthesize relatively more exocellular proteins during the exponential phase than during the stationary phase. The proportion of exocellular protein synthesized during the exponential phase does not exceed 3 % of total proteins, during the stationary phase this proportion usually decreases to less than 1 %.

Protein turnover in growing cultures of Bacillus megaterium.

A growing population of Bacillus megaterium, prelabelled with 14C-amino acids displays protein turnover, the rate of which is influenced by the growth rate. The size of the pool of short-lived proteins, degraded with a half-life of less than 1 h, is directly proportional to the mass doubling time during subsequent growth of the population. However, their degradation constant is almost the same under all conditions. The degradation constant of residual long-lived proteins is influenced by the growth rate. The labile fraction can be replenished by a combination of shift-up and shift-down treatments. Tetracycline decreases the size of the labile (short-lived) fraction and almost stops the degradation of long-lived proteins. Correlation of protein turnover and intracellular proteolytic activity indicates that other factors in addition to the proteinase are necessary for the degradation of short-lived, as well as long-lived proteins.

Turnover of proteins in asporogenic Bacillus megaterium. Evidence for a gradual decrease of the turnover rate.

The rate of protein turnover in asporogenic Bacillus megaterium decreases continuously during incubation in a sporulation medium. The capability of equilibration of external amino acids with amino acids in the metabolic pool of non-growing cells was retained for at least 5 h. Leucine, while repressing the synthesis of the exocellular protease, does not significantly influence the course of protein degradation in vivo. Transfer of non-growing cells after 4 h to a fresh sporulation medium does not influence the rate of protein degradation. The gradual decrease of the rate of protein turnover in non-growing cells of the asporogenic variant is thus not an artifact caused by a decreased uptake of amino acids by cells or by conditions under which the protein turnover is determined.

Relationships between intracellular proteolytic activity and protein turnover in Bacillus megaterium.

When incubated in a sporulation medium, the sporogenous strains of Bacillus megaterium degrade proteins at a rate of 4-10% X h-1. The maximal rate of protein turnover is reached after 3-4 hrs at the time of development of forespores and then decreases again. The rate of protein turnover in the asporogenous strain decreases steadily under similar conditions from 3-8% X h-1 at the beginning of incubation to 1% X h-1 after 5-6 hrs in the sporulation medium. The rate of degradation of proteins in vitro in protoplast lysates is similar or higher than the rate of protein turnover. The exocellular, as well as periplasmic proteolytic activity, is suppressed by amino acids more severely than the activity in protoplasts. Mutants devoid of the exocellular proteolytic enzyme contain also less proteolytic activity in the periplasm than in the protoplasts, in contrast to the wild strain. However, their rate of protein turnover, as well as the degradation of abnormal proteins is similar to that in the wild strain. This supports a view that the proteolytic system in protoplasts is involved in intracellular protein catabolism. The periplasmic enzyme can be considered as a kind of the exocellular proteinase.

Intracellular proteolytic activity during sporulation of Bacillus megaterium.

Intracellular proteolytic activity increased during incubation of the sporogenic strain of Bacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respect to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3-5 h of incubation. After 7 h 20-50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteins in vitro at a rate up to 150 mug mg-1 h-1 and native proteins at a rate up to 70 mug mg-1 h-1. Degradation of proteins in vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 mug mg-1 h-1 and the inactivation of beta-galactosidase at a rate of 70 mug mg-1 h-1. The intracellular proteolytic activity was inhibited to 65-88% by EDTA, to 23-76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50-65% of the activity were localized in protoplasts. Another strain of Bacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain of Bacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3-4 h. The intracellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.

The effect of substitution of diaminopimelic acid by 4-hydroxy-diaminopimelic acid on the synthesis and degradation of murein in Escherichia coli 173-25.

Defects in the formation of the septum and gradually autolysis of cells occur when the dap-dependent mutant of Escherichia coli is grown in a medium with 4-hydroxy-diaminopimelic acid. When the culture grown in the presence of the labelled analogue is supplemented with the non-radioactive diaminopimelic acid a portion of the TCA-soluble radioactivity is released from the cells during 20 min after the addition of diaminopimelic acid. During this time interval the elongated forms formed in the presence of the analogue divide, however, only on the condition that the above forms are not irreversibly damaged. The increased concentration of the analogue in the medium substantially suppresses the irregularities in the development of the septum as well as the degradation of analogue containing cell wall. However, the growth rate in the presence of the analogue is always slightly lower than that in the presence of diaminopimelic acid. The cell wall pulse-labelled with diaminopimelic acid or its analogue for a time interval shorter than 1/4 of the generation time exhibits the same or only slightly higher rate of diaminopimelic acid is probably utilized less effectively for the synthesis of murein than diaminopimelic acid. However, its incorporation into the wall does not result in pronounced damage of the cell.