Localization of proteases in cells ofEscherichia coli andBacillus megaterium

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   Изучалася локализация протеаз в клетках Bacillus megaterium и Escherichia coli. B. megaterium—это типичный протеолитический микроорганизм, тогда как E. coli вообще не выделяет протеазы в среду.     
   
   

Protease activity in cells ofBacillus megaterium during derepression

A proteolytic activity hydrolyzing denatured proteins of Bacillus megaterium labelled with 35S or 14C amino acids was detected in cells of the asporogenic strain of Bacillus megaterium. The substrate is hydrolyzed by the enzyme or enzymes at optimum pH around 7, their activity being almost completely inhibited by EDTA and o-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 mug of substrate degraded per hour by 1 mg of protein at 37 degrees 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.     

Observation of nuclear fusion in living cells ofBacillus megaterium

Summary Fusion of nuclear bodies was observed by phase contrast microscopy in living cells ofBacillus megaterium grown on nutrient agar with added sodium monofluoroacetate. The nuclear fusion is perhaps carried out in the stage of the resting nucleus. The resting nuclear bodies in adjoining cells are fused in about twenty minutes. A part of this work was published in the Japanese Journal of Genetics34, 88, 1959 (in Japanese).     

Sensitivity of spores and growing cells ofBacillus thuringiensis varisraeliensis andBacillus sphaericus to osmotic variations

EntomopathogenicBacillus thuringiensis var.israelensis (Bti) andBacillus sphaericus (Bf) strains species were studied in relation to their capacity to resist osmotic and nutritional shifts. Their behavior was compared with other bacilli,B. subtilis (Bs) andB. megaterium (Bm). In contrast to these reference strains, vegetative cultures of both species presented a dramatic sensitivity to hyperosmotic shock, independent of the growth period assayed. Subjected to an osmotic and nutritional shift-down (one hundredth dilution in water), Bti cultures resisted it, divided, and sporulated, as did Bm strains, whereas Bf and Bs cultures lysed or died. Spores from these toxic species were of less quality regarding resistance to heat or osmotic strength; but a nontoxic Bti derivative produced spores of better quality. Spore germination was also followed in these strains. The poor spore quality of these species correlated well with their poor survival in field experiments.     

Tensile strength of cell walls of living cells

A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered.     

The growth of cells ofBacillus megaterium in a hypertonic phosphate buffer

The morphological changes in cells grown in a phosphate medium were described. The synthesis of certain macromolecules under these conditions was characterized quantitatively and simultaneous structural changes in the cells demonstrated. It was shown that structural alterations in the cell wall resulting in striking changes of the cell shape were not caused by an altered rate of synthesis of the mucopolymers.     

Characteristics of intracellular proteolytic activities ofBacillus megaterium

Intracellular proteolytic activities ofB. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called C_I was stable, had a relative molecular mass ofM _r=105000 (M=105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated C_II, was labile and had a relative molecular mass ofM _r=46000 (M=46 kg/mol). Because of its lability it could not be characterized in detail. In the “periplasm” only a single proteolytic enzyme P (M _r=28000;M=28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12 that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the cytoplasmic proteinase C_I, could be demonstrated.     

Lethal germination of spores ofBacillus megaterium 9885

Washed spore suspensions germinated promptly without prior heat shock in a basal germination solution containingl-leucine.Germination was inhibited by dipicolinic acid. The inhibition was reversed by eitherl-leucine or phosphate.Phosphate accelerated the rate and increased the extent of germination, which was accompanied by an uncommonly large fall in the optical density of the suspension, but phosphate also caused a massive lysis after germination. This was accompanied by a sudden shedding of the spore coats. The suspensions consisted of shrivelled, cellular walls and membranes attached to the empty spore coats.Lysis of the germinated cells was prevented by fairly high concentrations of Ca or Mg.During germination, exogenous Ca we used Ca⁴⁵ was absorbed by the cells. Both cells and sonically disrupted cellular particles firmly retained the calcium, and evidence suggested that much of the Ca was bound in the cytoplasmic membranes.The cations contained in plain agar enabled spores which germinated on tryptone soya agar plates to develop into colonies; in the corresponding broth medium these spores lysed upon germination.Hypertonic sucrose delayed but did not prevent 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. The results published here were presented at the 2nd Harden Conference “Cell walls and cell membranes”, Wye, Kent, England in September 1970.     

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.     

Characterization of degradation products of the cell wall released during growth and sporulation ofBacillus megaterium

Asporogenic and sporogenic strains ofBacillus megaterium KM release during growth heterogeneous fragments of the cell wall into the medium the non-dialyzable fraction representing 50–90% by the total. During lysis of sporangia the non-dialyzable fraction represents only 30% of the soluble fraction of autolyzed walls. Gel filtration on Sephadex permits to separate the non-dialyzable fragments of the cell wall released during growth into two fractions contaning simultaneously peptidoglycan and phosphorus. The two fractions contain peptidoglycan components in the same ratio as in the cell wall. Only one peptidoglycan macromolecular fraction, smaller than the fractions released during growth, was detected by gel filtration in the material released during lysis of sporangia.     

Degradation and replenishment of the labile protein fraction in non-growing cells of the asporogenic mutant ofBacillus megaterium

Kinetics of degradation of labelled proteins was followed in two asporogenic mutants ofBacillus megaterium during incubation in a sporulation medium. Both the mutant producing exocellular protease (KM 1prn ⁺) and the mutant not producing the enzyme (KM 12prn) were found to contain a labile protein fraction, whose proportion decreases with prolonged time of labelling and whose half-life is about 1 h. Most proteins were relatively stable and were degraded at a rate of 1 %/h and 2 %/h in strains KM 1 and KM 12, respectively (half life 70–80 h and 35–40 h in strains KM 1 and KM 12, respectively). The intracellular proteolytic activity of the KM 12 mutant remains practically the same during incubation in the sporulation medium or slowly increases. The labile protein fraction practically disappears from the cells after a 3.5-h incubation. When such a culture is then subjected to a shift-up and transferred again to the sporulation medium, the rate of protein turnover temporarily increases. The temporary increase of the turnover rate is caused by a partial replenishment of the labile protein fraction rather than by an accelerated degradation of the relatively stable fraction. The intracellular proteolytic activity does not increase under these conditions. The wild sporogenic strain ofB. megaterium also contains the labile protein fraction. Its half protein life is 1 h or less. However, the second protein fraction is degraded much more rapidly than in the asporogenic mutants and its half life is 6–7 h.     

Mating-induced shedding of cell walls, removal of walls from vegetative cells, and osmotic stress induce presumed cell wall genes in Chlamydomonas

The first step in sexual differentiation of the unicellular green alga Chlamydomonas reinhardtii is the formation of gametes. Three genes, GAS28, GAS30, and GAS31, encoding Hyp-rich glycoproteins that presumably are cell wall constituents, are expressed in the late phase of gametogenesis. These genes, in addition, are activated by zygote formation and cell wall removal and by the application of osmotic stress. The induction by zygote formation could be traced to cell wall shedding prior to gamete fusion since it was seen in mutants defective in cell fusion. However, it was absent in mutants defective in the initial steps of mating, i.e. in flagellar agglutination and in accumulation of adenosine 3',5'-cyclic monophosphate in response to this agglutination. Induction of the three GAS genes was also observed when cultures were exposed to hypoosmotic or hyperosmotic stress. To address the question whether the induction seen upon cell wall removal from both gametes and vegetative cells was elicited by osmotic stress, cell wall removal was performed under isosmotic conditions. Also under such conditions an activation of the genes was observed, suggesting that the signaling pathway(s) is (are) activated by wall removal itself.     

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.