M. Strnadová

External factors involved in the regulation of synthesis of an extracellular proteinase in Bacillus megaterium: effect of temperature

SummaryWe studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35–38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24–28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26–34° C) when the rate of temperature decrease was 0.2–0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32–34° C. The optimum temperature decrease was 0.8° C/h.

Temperature shifts and sporulation of Bacillus megaterium

Summary: The permissive temperature for sporulation of Bacillus megaterium 27 (up to 42 °C) was found to be 4–5 °C lower than that for growth. The non-permissive temperature suppressed the initial phases of sporulation characterized by the synthesis of an extracellular proteinase but the cells retained the ability to sporulate for several hours. Neither growth at supraoptimal temperatures nor heat shock applied at the end of the growth phase increased the permissive sporulation temperature. The organism synthesized at least ten heat-shock proteins, the dominant one being HSP 69. These proteins were also found in cells after 3 h of incubation at 43·5 °C but their presence did not ensure the ability to sporulate at this temperature. The rise of temperature provoked an imbalance between synthesis and degradation of cellular proteins, whose role in suppression of sporulation is discussed.

Turnover kinetics of proteins labeled in different sporulation phases ofBacillus megaterium

Bacillus megaterium was labeled by 10-min pulses of14C-leucine at the end of the growth phase or at 1, 3.5 and 5 h after transfer to a sporulation medium. Proteins labeled during growth or reversible sporulation phase were degraded in two-phase kinetics,i.e. a decreasing degradation rate was followed by its substantial increase. Proteins labeled during the irreversible sporulation phase were degraded at a continuously decreasing degradation rate only. However, when the amount of degraded proteins was expressed as a portion of proteins degradable during the whole sporulation cycle, the degradation was rapid and followed similar kinetics irrespective of the time of labeling. The degradation constants fluctuated in this case between 0.207/h and 0.275/h. The protein fraction insensitive to turnover increased with the time of incubation in the sporulation medium in parallel to the amount of proteins appearing in spores.

Dual effect of amino acids on the development of intracellular proteolytic activity in the irreversible sporulation phase ofBacillus megaterium

Amino acids added to a population ofBacillus megaterium immediately after its transfer to a sporulation medium stimulated growth, delayed sporulation by 1 h, and delayed the development of intracellular cytoplasmic serine proteinase (ISP) activity. However, the ISP activity in late sporulation stages exceeded twice that of the control population. Amino acids supplemented at T3, i.e., at the time when engulfed forespores were developing, caused a decrease of specific ISP activity. The course of the phenylmethane sulfonyl fluoride (PMSF)-resistant activity in the cytoplasm was not affected by amino acids. Intracellular degradation of proteins prelabeled at the end of the growth phase was decreased by amino acids during the reversible sporulation phase but was only slightly affected later.

External factors involved in the regulation of an extracellular proteinase synthesis in Bacillus megaterium

SummaryA mathematical model was formulated to describe the kinetics and stoichiometry of growth and proteinase production in Bacillus megaterium. Synthesis of the extracellular proteinase in a batch culture is repressed by amino acids. The specific rate of formation of the enzyme (rE) can be described by the formula {ie373-1}, where k2 and k3 stand for the non-repressible and repressible part of enzyme synthesis respectively, kS2 is a repression coefficient and S2 indicates the concentration of amono acids; the values of k2 and kS2 depend on the composition of the mixture of amino acids. Even in a high concentration, a single amino acid is less effective than a mixture of amino acids. The dependence of the proteinase repression on the concentration of an external amino acid (leucine) follows the same course as its rate of incorporation into proteins, approaching saturation at concentrations higher than 50 μM (half saturation approximately 10 μM). However, the total uptake of leucine did not exhibit any saturation even at 500 μM external concentration.

Elevated content of hsp 70 does not induce tolerance of sporulation to higher temperature

The temperature permissive for sporulation (up to 42°C) inBacillus megaterium is by 4–5°C lower than that for its growth (up to 46–47°C). The ability ofB. megaterium cells to synthesize and degrade stress proteins under incubation in the sporulation medium was therefore investigated. The higher level of hsp 70, a typical stress protein induced by a temperature shock in postexponential growth phase, did not increase the permissive temperature of sporulation. The hsp 70 protein did not undergo a rapid turnover and its portion in the soluble protein fraction did not drop for at least 6 h at a temperature that was nonpermissive for sporulation (43.5°C). On the other hand, the elevated level of hsp 70 could not bring about the inhibition of sporulation as it was retained in the cells even after a shift of the temperature to 35°C, permitting sporulation of the culture.

Regulation of extracellular proteins and α-amylase secretion by temperature inBacillus subtilis

Abstractα-Amylase was found to be the main protein secreted byBacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45°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°C than that at 40 or 45°C. The effect of temperature on α-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.

Effect of temperature on α-amylase formation and DNA replication inBacillus subtilis

The effect of temperature on extracellular α-amylase synthesis and chromosomal and plasrnid DNA replication inBacillus subtilis A18 carrying plasmid pMI 10 was studied. The specific growth rate μ increased with elevated temperature up to 42.5°C, while the activities of α-amylase per population dry mass decreased. No obvious quantitative changes of14C-thymidine incorporation per dry mass increase and no basic differences in plasmid copy number in the range of temperatures from 25 to 40°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.

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

A transient 7-fold rise of ppGpp concentration, 2-3-fold increase of pppGpp concentration and 50 % drop of the concentration of GTP inBacillus 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.