J. Chaloupka

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus 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 respeot 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 proteinsin vitro at a rate up to 150 μg mg-1 h-1 and native proteins at a rate up to 70 μg mg-1 h-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg-1 h-1 and the inactivation of beta-galactosidase at a rate of 70 μg 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 ofBacillus 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 ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.

A proteolytic system in growing and non-growing cells ofEscherichia coli

Summary(1)Escherichia coli cells contain a protease system splitting its own proteins as well as casein labelled with131I at a slightly alkaline pH. The rate of proteolysis is not markedly influenced by ethylenediaminotetraacetic acid or by iodoacetic acid, p-chlormercuribenzoate diminishes the rate of hydrolysis of131I casein but its effect cannot be reversed by cysteine.(2)The proteolytic system is not present in the cells in the form of an enzymogen and its activity is not diminished even after 55 passages on a synthetic medium containing ammonium chloride as the sole nitrogen source.(3)In living cells proteins labelled with36S methionine are degraded at a rate not exceeding 0.2%/hr. at 29–30°, both in a growing culture and in the normal stationary phase. Similarly, the protease activity in cell-free extracts does not fundamentally change in the course of development of the culture.(4)Proteins in extracts are split at a rate of 1.8–2.3%/hr. at 32° according to results based on colorimetric estimations with tyrosine, or at a rate of 1.1–1.3%/hr, at 32° as shown by the course of degradation of35S proteins. No resynthesis of proteins from liberated amino acids took place in the extracts under the described experimental conditions.(5)The decomposition of labelled proteins in cell-free systems was stimulated by dinitrophenol and sodium azide.Abstract(1)Escherichia титр клетки содер жат протеазы Система разделения собственных белков в качестве равно как казеин с символикой (вс131) Я на несколько ще лочного рН. Темпы про теолиза не заметно влияют ethylenediaminotetraacetic кислоту или iodoacetic кисло та, п-chlormercuribenzoate снижает темп ы гидролиза (su131) Я казеи н, но ее эффект не може т быть отменено цист еина.(2)Протеолитической с истеме нет в клетках в виде enzymogen и ее деятельн ость не уменьшилась даже после 55 проходов по синтетическим ср едних хлористого ам мония, содержащий в качестве единственн ым источником азота.(3)В живых клетках белк ов с символикой (su36)S мет ионин являются дегр адировавших в разме ре, не превышает 0, 2% / час. на 29-30 °, как в растущей к ультуре и в нормальн ых стационарного эт апа. Точно так же, прот еазы деятельность в камере без выдержки не в корне изменить ход развитие культуры.(4)Белки в выписках дел ятся на темпы 1.8-2.3% / час. на 32 ° по на основе резуль татов оценки колори метрических с тироз ин, или по курсу 1.1-1.3% / ч, пр и 32 °, как показано на Конечно деградации (su35)S белков. Нет resynthesis белко в из освобожденных а мино кислот проходи л в соответствии с эк страктами описание экспериментальных условиях.(5)Разложение называю т белки в клетке своб одных систем стимул ируется dinitrophenol и натрия азид.

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.

Protease activity in cells ofBacillus megaterium during derepression

A proteolytic activity hydrolyzing denatured proteins ofBacillus megaterium labelled with35S or14C amino acids was detected in cells of the asporogenic strain ofBacillus megaterium. The substrate is hydrolyzed by the enzyme or enzymes at optimum pH around 7, their activity being almost completely inhibited by EDTA ando-phenanthroline. PMSF, the inhibitor of serine proteases, is slightly inhibitory. Gel filtration on a Sephadex column separated the protease activity to two or three fractions.The protease activity in cells with the repressed synthesis of protease corresponds to 5–20 μg of substrate degraded per hour by 1 mg of protein at 37°C. It increases five to ten-fold during the derepression. When the intracellular protease activity increases the extracellular enzyme begins to be excreted into the medium. The intracellular protease activity rapidly decreases after the addition of chloramphenicol or of a mixture of amino acids to the derepressed culture.Half or even more of the protease activity is released from the cells during their conversion to protoplasts by means of lysozyme. This “periplasmic” activity remains mostly in the supernatant also after mesosomes have been centrifuged down from the periplasm. A portion of the activity bound in protoplasts sediments together with membrane fraction after their lysis.

Turnover of mucopeptide during the life cycle ofBacillus megaterium

Sporogenic and asporogenicBacillus megaterium strains, as well asBacillus cereus degraded the murein component of the cell wall labelled with14C-diaminopimelic acid to TCA-soluble compounds during growth. The rate of murein turnover was about 15% during one generation in all three cases. The addition of chloramphenicol instantaneously markedly decreased the degradation rate, whereas in the presence of penicillin the degradation proceeded at the beginning at a rate comparable with that in the control and decreased only after a certain time interval. The cell wall degradation was considerably or completely stopped during the stationary phase of growth. In sporogenic strains ofBacillus megaterium andBacillus cereus the release of mature spores was associated with a new wave of the wall degradation, during which the wall of the sporangial cell was completely digested to TCA-soluble fragments. Free spores contained practically no mucopeptide component (cortex or spore wall) originating from the wall of the vegetative cell. A possible existence of a stable fraction of the cell wall not subject to turnover was investigated by measuring the3H/14C ratio in cells labelled simultaneously with3H (or14C)-diaminopimelic acid and14C (or3H)-leucine. The ratio changed during five generations, remaining constant later. This indicates that a certain portion of murein could be stable. The murein degradation during growth was not associated with secretion or release of a significant quantity of autolytic enzymes into the medium. The wall was apparently attacked from the inside. On the other hand, the release of the spore was accompanied by an increasing autolytic activity in the medium. This latter activity reached values corresponding to 3–8 μg lysozyme/ml.

Exocellular proteases ofSerratia marcescens and their toxicity to larvae ofGalleria mellonella

Out of 18 strains ofSerratia marcescens producing exocellular proteases the strainSerratia marcescens CCEB 415 was selected according to preliminary experiments. It could be shown that the train exhibits proteolytic activity reaching up to 10 TU per 1 ml of the culture filtrate in a medium with gelatine and peptone. Two proteolytic enzymes could be demonstrated by means of specific inhibitors EDTA and diisopropyfluorophosphate: metaloprotease with optimum activity at pH 7.5 and serine protease with pH optimum of 10.9. The enzymes were purified on Sephadex and DEAE cellulose columns and by means of gel electrophoresis. However, it was not possible to separate them. The optimum temperature for activity of the mixture of the two enzymes was 50 ° C, molecular weight varied around 37000 (according to gel filtration); certain kinetic characteristics of their activity were determined. Excess subtrate (casein) inhibited activity of the enzyme mixture. Toxicity of proteases expressed as LD50 units equals 78. 10−3 TU per larva ofGalleria mellonella.

DEGRADATION AND TURNOVER OF BACTERIAL CELL WALL MUCOPEPTIDES IN GROWING BACTERIA.

The mucopeptide layer of the cell wall ofBacillus megaterium is broken down into separate components during growth of the cells. The released diaminopimelic acid is partly decarboxylated to lysine, which is incorporated in the proteins and partly used for cell wall resynthesis. The smaller portion of the degraded mucopeptide is released into the medium in the form of non-utilized fragments. The rate of the mucopeptide turnover is a function of the rate of growth of the culture. About 15–20% of the rigid layer of the cell wall is degraded during on cell division. The sensitivity ofBacillus megaterium to lysozyme and the rate of its conversion to protoplasts is also proportionate to the rate of growth of the culture. There is no measurable mucopeptide turnover in non-growing cells, either in the stationary phase of the culture or in starvation in nitrogen-free medium. The resistance of the cell wall to lysozyme also increases during the stationary phase. The rigid component of the cell wall is probably also broken down during growth ofBacillus cereus andEscherichia coli cultures.AbstractСлой мукопептидов в клеточной оболочке Bacillus megaterium в течение роста клеток расщепляется на отдельные составные части. Освобождающаяся диаминопимеловая кислота частично декарбоксируется на лизин, который включается в белки. Меньшая часть расщепленного мукопептида выделяется в среду в форме неиспользованных осколков. Скорость «turnover» мукопептидов зависит от скорости роста культуры. В течение одного клеточного деления деградируется около 15–20% плотного слоя клеточной оболочки. Чувствительность Bacillus megaterium к лизоциму и скорость его превращения в протопласты также пропорциональна скорости роста культуры. В нерастущих клетках—как в стационарной фазе культуры, так в при ее голодании в безазотной среде—«turnover” мукопептидов не осуществляется. В то же время в стационарной фазе повышается устойчивость клеточных оболочек к лизоциму. Расщепление плотного компонента клеточной оболочки имеет место и в ходе размножения культур Bacillus cereus и Escherichia coli.

Mutants ofBacillus megaterium with altered synthesis of an exocellular neutral proteinase

Germinated spores ofBacillus megaterium were mutagenized with ethyl methanesulphonate and spread on test agar with caseinate. Colonies with altered proteolytic zones or morphology were isolated and tested in liquid media. The mutants can be divided into four groups: A) those producing more proteinase in both growth and sporulation media, B) those producing the same amount of the enzyme in growth medium but higher amount in sporulation medium, C) those producing less proteinase in the growth medium and more in the sporulation one, D) those producing less or no enzyme. Clones of the first three groups were phenotypically asporogenic. All mutants producing more enzyme during growth retained their sensitivity to repression by amino acids. Isolation of mutants of types B) and C) supports the idea of differences in the control of proteinase synthesis during growth and during sporulation.

Functional half-life of the exocellular protease mRNA ofBacillus megaterium

Functional half-life of the exocellular protease mRNA was determined in exponentially growing and stationary cells of the asporogenic strain ofBacillus megaterium, KM and in the sporogenic strain ofB. megaterium 27 during sporulation. No reserve of the protease mRNA could be detected in the cells and the half-lives were determined to be 6–7 min in the exponential and stationary cells ofB. megaterium KM and 7.5 – 8.5 min inB. megaterium 27. The mean half-life of mRNA for cell proteins was determined to be 3.5–4.5 min. Thus, as compared with the mean half-life of mRNA for cell proteins that of mRNA for the exocellular protease is slightly longer.

Turnover of murein in cellular and filamentous populations ofBacillus megaterium

Bacillus megaterium grows in the form of filaments at temperatures above 45°C. The rate of turnover of the cell wall begins to decrease gradually under these conditions. At the same time sensitivity of the filamentous forms to lysozyme decreases. Filaments outgrown at 48°C retain the decreased rate of turnover of the cell wall for a certain time after transfer to 30°C, in spite of the fact that septa are formed and filaments are converted to cells. However, a population incubated longer than 2 h at 48°C often ceases to grow and the growth is not restored even after transfer to 30°C. Three clones of the asporogenic strainBacillus megaterium KM differing somewhat in their ability to form filaments at 35°C differ mutually also in the rate of turnover of the cell wall. However, the decreased rate of the turnover cannot be unambiguously correlated with the increased tendency to form filaments.