Regulation of the formation of protease inBacillus megaterium

During the growth of the asporogenous variant ofBacillus megaterium KM in medium containing NO₃ ⁻ as nitrogen source, the relative rate of extracellular protease synthesis is higher than in the presence of NH₄ ⁺. It approaches the relative rate of enzyme synthesis at the incubation of cells in nitrogen-free medium with glucose. This supports the suggestion that even amino acids which are synthesized endogenously slow down the protease production. In the postlogarithmic or stationary phase the protease production stops. The interruption of enzyme production does not appear as a result of insufficient aeration in a dense suspension, or of accumulation of amino acids or their metabolites in cells. The non-growing cells retain their ability to renew the enzyme synthesis when transferred into a fresh medium, even into a medium without nitrogen source. In the same way it is possible to “induce” the protease production, if Ca²⁺ is added to cells in the stationary phase when the population was grown in the Ca²⁺ free medium. The amount of enzyme produced at the expense of protein turnover by the non-growing populations is sufficient for the fast hydrolysis of exogenous protein in the medium and for assuring the influx of a sufficient amount of peptides into the cells. In such a case the growth of the culture is therefore very quickly renewed.     

Regulation of the formation of protease inBacillus megaterium

Protease is synthesized by the cultures growing in a glucose-containing mineral medium. However, it is formed even during incubation of the washed cells in a nitrogen free medium. The enzyme synthesis is decreased substantially by the addition of the individual amino acids or their mixture. Threonine, isoleucine, leucine and valine are the most inhibitory. Arginine, cysteine, glycine, lysine and tryptophan in concentrations of 10³ m do not inhibit the production of protease. The growth of the culture is also somewhat inhibited by threonine and isoleucine, the repression of protease being, however, much higher. Concentrations of 10³ m inhibit its synthesis by 80–90%. However, the enzyme activity is not influenced. The inhibition is caused byl,-isomers. Repression of the enzyme synthesis after the addition of threonine into the medium is much greater in a growing culture than in a culture starving in a nitrogen-free medium. However the level of free threonine in the pool is roughly the same in both growing and non-growing cultures. A mixture of 13 amino acids, which themselves are little inhibitory, suppresses the synthesis of protease much more than threonine or isoleucine. The inhibitory effect of the individual amino acids on the enzyme formation is apparently additive.     

Regulation of formation of proteases inBacillus megaterium

Protease was formed only at the end of the growth phase and its synthesis continued in the stationary phase during the growth of the sporulatingBacillus megaterium KM Sp⁺ in complex media with amino acids or peptone and glucose. The enzyme was also not formed during the growth phase in the glucose containing mineral medium and was detected only later during the stationary phase, smaller quantities being observed than those formed in the complex medium. The addition of glucose at the beginning of the synthesis of protease inhibited the production of the enzyme. On the other hand, the addition of the mixture of amino acids under the same conditions stimulated its formation several fold. Cysteine blocked the synthesis of the enzyme unlike other amino acids. Within a certain range the stimulatory effect of amino acids was related to their concentration, being manifested only after a lag period of several hours. The ability to form protease dissappeared after the formation of refractile spores in sporangial cells. Preliminary communication published in Biochem. biophys. Res. Commun. 37: 233, 1969.     

Derepressive Formation of l-Leucine-Pyruvate Transaminase under Nitrogen-Free Conditions in Gluconobacter suboxydans

l-Leucine-pyruvate transaminase activity increased 6- to 20-fold in 3 hr when Gluconobacter suboxydans cells grown on yeast extract-medium were transferred to and incubated in a nitrogen-free medium. The increase in enzyme activity was influenced remarkably by the age and concentration of cells used. The phenomenon depended upon de novo synthesis of enzyme protein.

The enzyme activity in cell-free extracts of cells incubated under a nitrogen-free condition decreased remarkably after heat treatment at 50°C (pH 6.0) or after freezing and thawing. The level of such enzyme inactivation was high in extracts of cells in the early stages of induction and low in later stages.     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.     

The production of protease by dense suspensions ofBacillus megaterium KM Sp−

The synthesis and secretion of extracellular protease was demonstrated during the incubation of dense susponsions of the asporogenicBacillus megaterium KM. The overall production of the enzyme by cells incubated with glucose in a nitrogen-free medium was found to be only slightly lower than that in the presence of an inorganic nitrogen source. The capacity to form protease decreased exponentially with increasing density of the bacterial suspension. The synthesis of the enzyme was interrupted after the exhaustion of glucose. A repeated exchange of the medium made it possible to reach relatively high and continuous production of protease for several hours. The total amount of extracellular proteins synthesized during incubation of the dense suspension in media with or without a nitrogen source was less than 2% of a total of newly formed proteins. The amount of these extracellular proteins was slightly lower in the absence of Ca²⁺ being considerably decreased when the dense suspension was incubated with chloramphenicol.     

The phosphorylation of ribosomal proteins L14 and S3 in Krebs II ascites cells.

Krebs II ascites cells incubated in Earle's saline (lacking glucose and amino acids) contain ribosomes with proteins S6 and Lgamma phosphorylated, as do ascites cells grown in the peritonea of mice or hamster fibroblasts grown in Eagle's medium. When ascites cells were incubated in Eagle's medium (containing glucose and amino acids) there was extensive glycolysis, producing very acidic conditions, and ribosomal proteins S3 and L14 became phosphorylated whereas Lgamma became dephosphorylated. This altered pattern of phosphorylation could not be produced merely by incubating ascites cells in Earle's saline at a decreased pH, but a rather similar pattern was produced when Earle's saline was supplemented with amino acids (but with glucose still omitted). These results suggest that depriving ascites cells of glucose may induce the synthesis of a protein (or proteins), necessary for alteration of the pattern of phosphorylation of the ribosomal proteins.     

Effect of nitrate,ammonia and nitrogen starvation on the regulation of nitrate reductase in Cyanidium caldarium

Summary Cells of Cyanidium caldarium grown with ammonia or ammonium nitrate as nitrogen source do not contain appreciable nitrate reductase activity. The alga develops the capacity to synthesize the enzyme when it is transferred from the ammonium medium to a nitrogen-free medium. Nitrate is not needed as an inducer and no enhancement in the rate of enzyme synthesis is observed when it is present. By contrast, whereas the synthesis of the enzyme in nitrogen-free medium proceeds at an increasing rate, in the nitrate medium it attains a stationary level after a short time.Nitrate grown cells possess variable amount of inactive nitrate reductase (from 9 to 60%) whereas in nitrogen-free medium the enzyme occurs principally in a fully active form. Addition of ammonia inactivates reversibly the preexisting enzyme. The inactive enzyme is measurable in the crude extract after activation by heating.It is suggested that in Cyanidium the inactivating effect of ammonia, which is the end product of nitrate reduction, in association with the repression of enzyme controls the level of nitrate reductase activity.     

Carbon source regulation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase in Neurospora crassa: induction and repression of enzyme synthesis

Synthesis and release of NAD(P)ase by Neurospora crassa wild type was studied in experiments in which mycelia grown in Vogel minimal medium were transferred to media containing protein as the only carbon source. Several results are presented suggesting that the NAD(P)ase may be induced by the presence of protein in the culture medium. Low concentrations of sucrose or glucose (0.1%), Casamino acids or some amino acids such as methionine, cysteine, phenylalanine and tryptophan strongly repressed the enzyme synthesis. Under induction conditions NAD(P)ase and alkaline protease appeared together in the culture medium. It would appear that NAD(P)ase and alkaline protease are coordinately regulated by a common control mechanism related to carbon catabolism.     

Enzyme biosynthesis in Escherichia coli

Escherichia coli B synthesized beta-galactosidase and an enzyme system for D-xylose when exposed to lactose and xylose respectively in nitrogen-free media. The amount of beta-galactosidase formed in the absence of external nitrogen depended upon the nature of the medium in which the cells had originally been grown. Half as much of this enzyme was synthesized without exogenous nitrogen by cells taken from a nitrogen-rich medium as was formed by cells under favorable conditions with an external supply of nitrogen. Escherichia coli B contained a pool of nitrogen compounds soluble in 80 per cent ethanol and made up of several ninhydrin-positive components. One of these was identified chromatographically as glycine using an authentic radioactive sample. Another substance behaved like serine on the chromatograms. The internal pool of amino acids and peptides was large enough to account for the beta-galactosidase synthesized by cells exposed to lactose in a medium free of nitrogen. Some degree of interaction of the syntheses of the beta-galactosidase and xylose enzyme systems was observed in nitrogen-free media. This interaction produced a greater effect on the formation of beta-galactosidase and was attributed to a limiting factor(s) in the internal nitrogenous pool or to a limiting intermediate in enzyme synthesis.     

Characteristics of Protease Production by Cephalosporium sp

We investigated protease formation by Cephalosporium sp. strain KM388, which produced trypsin inhibitor in the same cultures, in medium containing polypeptone, meat extract, and glucose (natural medium) and in medium containing NaNO₃, glucose, and yeast extract (semisynthetic medium). In natural medium, protease was secreted into the culture broth after cessation of growth caused by consumption of the polypeptone, the growth-limiting substrate. Enzyme formation in the stationary growth phase was due to de novo and so-called preferential synthesis, because cycloheximide immediately inhibited enzyme formation. In semisynthetic medium, protease was produced in parallel with mycelial growth, but production was repressed by the addition of polypeptone to the medium; protease production began after the added polypeptone was consumed. On the other hand, if glucose was eliminated from natural medium, the lag period of initiation of enzyme production was reduced until the late exponential phase. The addition of phosphate up to a concentration of 1.0% to natural medium also shortened the lag period and damped the pH change of the broth during cultivation.     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.     

Metabolic network analysis of Bacillus clausii on minimal and semirich medium using (13)C-labeled glucose

Using (13)C-labeled glucose fed to the facultative alkalophilic Bacillus clausii producing the alkaline serine protease Savinase, the intracellular fluxes were quantified in continuous cultivation and in batch cultivation on a minimal medium. The flux through the pentose phosphate pathway was found to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis. Two futile cycles in the pyruvate metabolism were included in the metabolic network. A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose. The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch cultivations. It was found that leucine, isoleucine, and phenylalanine were taken up from the medium and not synthesized de novo from glucose. In contrast, serine and threonine were completely synthesized from other metabolites and not taken up from the medium. Valine, proline, and lysine were partly taken up from the medium and partly synthesized from glucose. The metabolic network analysis was extended to include analysis of growth on the semirich medium containing amino acids, and the metabolic flux distribution on this medium was estimated and compared with growth on minimal medium.     

Effect of exogenous factors on extracellular alkaline ribonuclease synthesis in Bacillus mesentericus

Bacillus mesentericus was found to assimilate nucleic acids as a source of nitrogen and phosphorus. Nucleic acids added to the medium as a source of nitrogen or phosphorus stimulated synthesis of ribonuclease. When washed bacterial cells were incubated for a short period of time in a fresh nutrient medium containing RNA, synthesis of RNAase was also induced. Synthesis of the enzyme was inhibited by high concentrations of chloramphenicol and actinomycin D, and stimulated by low concentrations of actinomycin D. Therefore, alkaline RNAase is an inducible enzyme which participates in the nutrition processes of bacteria.     

The incorporation of glycerol and lysine into the lipid fraction of Staphylococcus aureus

1. Incubation of washed cells of Staphylococcus aureus with [1-¹⁴C]glycerol results in the incorporation of glycerol into the lipid fraction of the cells. The rate of incorporation is increased by the presence of glucose and amino acids. The presence of amino acids increases incorporation into the fraction containing O-amino acid esters of phosphatidylglycerol. 2. Glycerol, incorporated into washed cells by incubation with glycerol, glucose and amino acids, is rapidly released from the lipid fraction when cells are incubated at low suspension densities in buffer. 3. Of nine amino acids tested, only lysine is significantly incorporated into the lipid fraction. The incorporation is increased by the presence of glycerol, glucose and other amino acids, especially aspartate and glutamate. 4. The incorporation of lysine is increased by the addition of puromycin at concentrations that inhibit protein synthesis. Chloramphenicol does not increase the incorporation of lysine but abolishes the enhancing effect of puromycin. 5. The enhancing effect of puromycin is accompanied by a similar increase in the incorporation of lysine into the fraction soluble in hot trichloroacetic acid. 6. Lysine is incorporated into the lipid fraction that contains O-amino acid esters of phosphatidylglycerol and corresponds in properties to phosphatidylglyceryl-lysine. 7. Lysine is rapidly released from the lipid of cells incubated in buffer only at low suspension densities. 8. Incubation of cells with the phosphatidylglyceryl-lysine fraction does not lead to the appearance of free lysine or to incorporation into the fraction insoluble in hot trichloroacetic acid.     

Regulation of exocellular protease in Neurospora crassa: induction and repression under conditions of nitrogen starvation.

Exponential-phase cells of Neurospora crassa require the continued presence of a protein inducer and nitrogen starvation to induce exocellular protease under conditions where protein is the sole nitrogen source. The nature of the protein inducer appears relatively unimportant, since both soluble proteins (e.g., myoglobin) and insoluble proteins (e.g., corn zein) will effect induction. Nonstarved cells of N. crassa appear to have small nitrogen pools, since nitrogen starvation of exponential cells prior to transfer into a medium where protein is the sole nitrogen source effects starvation-time-dependent decreases in protease biosynthesis. Ammonium ion represses protease synthesis, with apparent specificity at low concentrations. The amino acids arginine, tryptophan, and threonine effect repression of protease biosynthesis under conditions of nitrogen starvation. Under conditions of sulfur starvation, the amino acids cysteine, methionine, and cystine repress protease biosynthesis. In carbon-starved cells, all of the above amino acids, plus histidine, isoleucine, leucine, lysine, phenylalanine, and valine, effect repression. Examination of amino acid pools formed when cells are grown on protein as the sole nitrogen source demonstrated that the amino acids which repress protease biosynthesis under conditions where protein is the sole carbon source accumulate in significant amounts during the course of protease induction, with kinetics consonant with the induction process.     

Synthesis of the extracellular protease byBacillus pumilus

Bacillus pumilus synthesizes an oxtracellular protease during the stationary phase of growth when the intracellular protease level is rather low. The formation of the enzyme is blocked by chloramphenicol. A shift from batch to continuous cultivation is accompanied by a decreased enzyme level in the medium. The original concentration of the enzyme in the medium can be attained only after reverting to batch cultivation. The protease is not synthesized during growth in a mineral medium with glucose. Glutamic acid, arginine and ornithine are among the amino acids which stimulate the enzyme formation. Yeast extract enhances the formation of the protease, the active component of the extract being only the brown pigment with attached Fe³⁺ ions. The B-vitamins and other essential growth factors contained in the yeast extract are without effect on the protease formation. Free ferric ions also induce protease formation, the level being roughly proportional to the concentration of Fe³⁺ in the medium. The synthesis is also enhanced by Mn²⁺. Growth of the culture under oxygen limitation results in the suppression of protease formation.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

C oleman , G.S. & H all , F.J. 1984. The uptake and utilization of Entodinium caudatum , bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa. Journal of Applied Bacteriology 56 , 283–294.
Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum , ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus mega-terium, Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bouis , although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and i>Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro , although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa . Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

Factors influencing the formation and stability of D-glucoside 3-dehydrogenase activity in cultures of Agrobacterium tumefaciens.

D-glucoside 3-dehydrogenase specific activity in Agrobacterium tumefaciens was maximal towards the end of the exponential growth phase of batch cultures; over 90% of the activity disappeared within the next 15 h. Manganese ions, although essential for growth of the organism, strongly repressed D-glucoside 3-dehydrogenase synthesis in sucrose medium but had little effect when the carbon source was methyl alpha-D-glucoside. D-Glucoside 3-dehydrogenase activity increased linearly with increasing specific growth rate in chemostat cultures limited by carbon, nitrogen, phosphate or manganese when methyl alpha-D-glucoside was the carbon source. High enzyme activity was found with sucrose as carbon source only when the growth medium was manganese-limited. D-Glucoside 3-dehydrogenase activity disappeared from A. tumefaciens incubated in carbon- and nitrogen-free medium or in nitrogen-free medium containing succinate, but on continued incubation the activity returned and was then stable. The recovery of activity could be prevented by chloramphenicol or erythromycin. Bacteria containing the recovered dehydrogenase activity could not convert sucrose to 3-ketosucrose when oxygen acted as the terminal electron acceptor, but produced 3-ketosucrose at the normal rate in the presence of ferricyanide. D-Glucoside 3-dehydrogenase activity disappeared irreversibly from bacteria incubated in nitrogen-free medium containing sucrose. Loss of activity followed first order kinetics in bacteria taken from nitrogen-, phosphate- or manganese-limited chemostat steady states; an accelerating rate of decay occurred in cells grown under carbon-limitation. 8-Hydroxyquinoline, chloramphenicol, erythromycin, 2,4-dinitrophenol and manganese ions could reduce the rate of decay.     

Regulation of invertase in Aspergillus nidulans: effect of different carbon sources

Aspergillus nidulans produces an extracellular beta-D-fructofuranoside fructohydrolase (invertase) when grown on a medium containing the beta-fructofuranosides sucrose or raffinose, indicating that synthesis is subject to induction by the substrate. On a growth medium containing sucrose, production was maximal at 15 h in cultures incubated at 28 C degrees. After this time the level of detectable invertase in the cultures declined. A proportion of the enzyme was secreted during the linear growth phase of the fungus. Various sugars were investigated for induction of invertase, but only the two beta-fructofuranosides induced high production levels; with the other sugars, the enzyme was produced only at a low constitutive level. Mycelium grown under repressive conditions (1% glucose), rapidly produced invertase when transferred to sucrose-containing medium. After 80 min the invertase level in these cultures was 26-fold higher than the constitutive level. The repressive effect of other sugars, e.g. glucose and xylose, on invertase production was also demonstrated in this experimental system.