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.     

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.     

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

The rate of protein turnover in asporogenicBacillus 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 degradationin 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.     

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

Synthesis of exocellular metalloprotease and cellular and exocellular proteins in the sporogenic strainBacillus 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 exooellular 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 %.     

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.     

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.     

Purification, characterization, and role of nucleases and serine proteases in Streptomyces differentiation. Analogies with the biochemical processes described in late steps of eukaryotic apoptosis.

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.     

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.     

Cell viability and protein turnover in nongrowingBacillus megaterium at sporulation suppressing temperature

InBacillus megaterium, a temperature that suppresses sporulation (43°C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40–41°C). Here we show that, when cells grown at 35°C and transferred to a sporulation medium, were subjected to shifts between 35°C and the sporulation suppressing temperature (SST, 43°C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35°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°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°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 (T_2.5), 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°C. A later (T₄) shift to SST had no effect on the sporulation process.     

Protein turnover in exponential and stationary phase Vibrio cells

Vibrio strain 14 supports phage alpha 3a growth in standing stationary phase cells but not in shaking (aerated) stationary phase cells. In exponential cells, protein was turned over at 1.8% h-1, and the rate was increased by starvation or inhibition of protein synthesis. In shaking stationary phase cells the rate of protein turnover was low (1.0% h-1) for proteins synthesised during growth but high (20% h-1) for recently synthesised proteins. In contrast recently synthesised proteins in standing stationary phase cells were stable over 60 min and proteins synthesised during growth were turned over at 2.9% h-1. ppGpp and pppGpp were detected in exponential cells, but were not detected in stationary phase cells.     

Sporulation in Bacillus subtilis. The role of exoprotease

1. Intracellular turnover of protein was measured in wild-type Bacillus subtilis, which produces exoprotease at stage I in the sporulation process. Protein is degraded at a rate of 8–10%/hr. 2. As a result of this turnover, the proteins of the mother cell are extensively degraded and resynthesized by about 6hr., so that the later stages of spore formation occur in a cytoplasm containing mainly `new' protein. 3. The same protease appears to be responsible both for the intracellular turnover of protein and for extracellular proteolytic activity. In mutants that have lost the exoenzyme the intracellular protein is stable for many hours. In addition, these mutants fail to produce antibiotic and are asporogenous. When the exoprotease is regained as a result of back-mutation all the lost capacities of the cell are restored together. 4. Protease activity also accounts for the change in antigenic pattern of extracts of cells sampled during sporulation. Immunoelectrophoresis shows that, in the wild-type, the antigens characteristic of the vegetative cell have largely disappeared after a few hours; in the proteaseless mutants the vegetative-cell pattern is conserved. Apart from changing the protein pattern of the cell the protease could also have the function of removing protein inhibitors of sporulation. Other possible interpretations of the results are discussed.     

Natural variability of Streptomyces spheroides--a producer of extracellular proteolytic enzymes possessing fibrinolytic action

Streptomyces spheroides M 8-2 was obtained using UV irradiation of spores taken from the parent culture. It shows a high activity of exocellular proteases which are capable of fibrin hydrolysis and degradation of blood clots. On certain media, a population of S. spheroides M8-2 was shown to yield three variants differing in their morphologo-biochemical properties. During submerged cultivation, these variants produce different quantities of exocellular proteases (3 to 19 units per 1 mg of biomass) with the fibrinolytic activity. Variants I and III are most active; their proteolytic activity is 10-14 units per 1 mg of biomass, on the average, and can reach 18-19 units per 1 mg of biomass. Variants with a low activity (variant II) are accumulated when the actinomycets is kept as spores on a solid medium with corn extract and on a solid medium with fibrin. The high proteolytic activity of the strain can be preserved by selection on a diagnostic medium with fibrin taking account of the diameter of hydrolytic zones around the colonies and selecting solely the colonies of variants I and III.     

Proteinase activities of Saccharomyces cerevisiae during sporulation.

Sporulation in Saccharomyces cerevisiae occurs in the absence of a exogenous nitrogen source. Thus, the internal amino acid pool and the supply of nitrogen compounds from protein and nucleic acid turnover must be sufficient for new protein synthesis. Since sporulation involves an increased rate of protein turnover, an investigation was conducted of the changes in the specific activity of various proteinases. A minimum of 30% of the vegetative proteins was turned over during the course of sporulation. There was a 10- to 25-fold increase in specific activity of various proteinases, with a maximum activity around 20 h after transfer into the sporulation medium. The increase in activities was due to de novo synthesis since inhibition of protein synthesis by cycloheximide blocks both an increase in proteinase activities and sporulation. There was no increase observed in proteinase activities of nonsporogenic cultures (a and alpha/alpha strains) inoculated into the sporulation medium, suggesting that the increase in proteinase activities is "sporulation specific" and not a consequence of step-down conditions. The elution patterns through diethylaminoethyl-Sephadex chromatography of various proteinases extracted from T0 and T18 cells were similar, and no new species was observed.     

In Vivo and In Vitro Function of the Intracellular Proteolytic Apparatus in Nongrowing Bacillus megaterium Under Heat Stress

In Bacillus megaterium sporulating at 35°C, 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°C, 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°C and to 46% in the wild strain whose sporulation was suppressed by the temperature of 47°C. 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. Received: 23 July 1998 / Accepted: 19 August 1998     

Protein synthesis and breakdown in the mother-cell and forespore compartments during spore morphogenesis in Bacillus megaterium

Recently developed techniques for isolating forespores from bacilli at all stages of spore morphogenesis have been exploited to investigate the contribution of each of the two compartments of the sporulating cell to the overall pattern of protein synthesis and degradation during sporulation in Bacillus megaterium. These studies have shown: (1) that protein synthesis continues in both compartments throughout spore morphogenesis; (2) that the degradation of proteins made at all times during vegetative growth and sporulation is confined to the mother-cell compartment; (3) that proteins synthesized in the mother-cell compartment during sporulation are subsequently degraded more rapidly than proteins synthesized during vegetative growth. This rate of degradation increases the later the proteins are synthesized in the sporulation sequence. Mature spores were disrupted, and the percentage of the total protein in soluble and particulate fractions was determined. Pulse-labelling experiments were performed to investigate the extent to which the proteins of these two fractions are newly synthesized during sporulation. These data were used to calculate the extent of capture of vegetative cell protein at the time of formation of the forespore septum. The value obtained is consistent with evidence from electron micrographs and supports a model for the origin of spore protein in which there is no protein turnover in the developing forespore.     

Proteolytic stability of insecticidal toxins expressed in recombinant bacilli

The production of the vegetative mosquitocidal toxin Mtx1 from Bacillus sphaericus was redirected to the sporulation phase by replacement of its weak, native promoter with the strong sporulation promoter of the bin genes. Recombinant bacilli developed toxicity during early sporulation, but this declined rapidly in later stages, indicating the proteolytic instability of the toxin. Inhibition studies indicated the action of a serine proteinase, and similar degradation was also seen with the purified B. sphaericus enzyme sphericase. Following the identification of the initial cleavage site involved in this degradation, mutant Mtx1 proteins were expressed in an attempt to overcome destructive cleavage while remaining capable of proteolytic activation. However, the apparently broad specificity of sphericase seems to make this impossible. The stability of a further vegetative toxin, Mtx2, was also found to be low when it was exposed to sphericase or conditioned medium. Random mutation of the receptor binding loops of the Bacillus thuringiensis Cry1Aa toxin did, in contrast, allow production of significant levels of spore-associated protein in the form of parasporal crystals. The exploitation of vegetative toxins may, therefore, be greatly limited by their susceptibility to proteinases produced by the host bacteria, whereas the sequestration of sporulation-associated toxins into crystals may make them more amenable to use in strain improvement.     

Demonstration of a cell-associated, inactive precursor of an exocellular protease produced by Pseudomonas aeruginosa

The enzymatically active form of protease 1, the major exocellular protein produced by Pseudomonas aeruginosa strain 34362, has been shown to exist exclusively exocellularly with no significant cell-associated activity. However, the presence of a cell-associated, enzymatically inactive protein which is serologically cross-reactive with, and convertible to, active enzyme has been demonstrated. One method of conversion of "precursor" to active enzyme is via limited proteolysis. Two assay systems for precursor were developed, one a radioimmune assay, and the other a proteolytic activation procedure. Localization studies suggest that the association while more tenacious than classical periplasmic enzymes is still an ionic rather than a covalent one. Kinetics of production studies showed to precursor to be synthesized early in the growth cycle and to accumulate prior to the rapid release of the active enzyme. Molecular weight studies showed only slight changes produced upon activation.     

Genomic sequence-based discovery of novel angucyclinone antibiotics from marine Streptomyces sp. W007

Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B.?sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B.?sphaericus is dependent on sporulation initiation.     

Netropsin suppresses the rise of activity of an intracellular proteolytic system in sporulatingBacillus megaterium

Intracellular catabolism of proteins labeled at the end of the exponential growth proceeded in two phases during sporulation. The first phase was induced by starvation and took place also in cells whose sporulation was inhibited by netropsin. The second phase of degradation, which was triggered at the onset of the irreversible sporulation phase, was inhibited by netropsin. Intracellular proteolytic activity determined in disintegrated cells, i.e., primarily the activity of the cytoplasmic Ca²⁺-dependent serine proteinase(s) at the first place, was increasing throughout the sporulation process and reached its maximum during the irreversible sporulation phase. Its increase was suppressed by netropsin. Fractionation of the cell sap by HPLC revealed a similar distribution of proteolytic activities in the extract from control and netropsin-inhibited cells. The antibiotic thus probably affected the activation, not the formation of the cytoplasmic serine proteinase(s). Netropsin also inhibited an increase of proteolytic activity in the membrane fraction, probably owing to the presence of two different proteolytic enzymes.