A continuous culture study of the regulation of extracellular protease production inVibrio SA1

During growth ofVibrio SA1 in a lactate-limited chemostat in the presence of 2mm phenylalanine as an inducer, the rate of production of two proteolytic enzymes, namely an endopeptidase and an aminopeptidase, was dependent upon the dilution rate. An optimum in the rate of synthesis of both proteases was observed at a dilution rate of 0.23 h^-1 and enzyme production only occurred between dilution rates of 0.06 and 0.45 h^-1. Without inducer a low rate of aminopeptidase production was found with an optimum at 0.19 h^-1, but only trace amounts of endopeptidase were detectable in the culture. In the presence of inducer the rate of enzyme production increased with increasing dilution rates over the range 0.06 to 0.23 h^-1 which was explained by an increase in saturation of inducer sites. The progressive decrease in the rate of protease production at higher dilution rates was ascribed to an increasing effect of catabolite repression by the increasing concentration of the growth substrate. It was shown that 5mm cyclic AMP could not relieve catabolite repression caused by glucose or lactate. Repression of protease production also occurred in the presence of higher concentrations (5mm) phenylalanine and other amino acids and by ammonium ions. It is suggested that the energy-status of the cell may play an important role in the regulation of protease synthesis inVibrio SA1.This study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Studies on cellulase production by a Bacillus subtilis

Bacillus subtilis AU-1 was found to produce carboxymethylcellulase (CMCase) and Avicelase activities in the culture supernatant when grown on a variety of carbohydrates as major carbon source. Maximum CMCase production was obtained in a liquid medium containing 0.2% D (+) raffinose as inducer, 0.5% each of yeast extract, casamino acids and proteose peptone at 50 °C and at an initial pH of 6.0. CMCase activity was detected at early log phase of growth, and reached the maximum level at early stationary phase of growth which occurred at the 10th hour of cultivation. The optimal temperature for CMCase activity was 65 °C, and the enzyme was highly stable up to 60 °C. CMCase synthesis was subjected to catabolite repression by glucose and cellobiose.     

Induction and repression of the collagenase synthesis in Acinetobacter sp.]

The synthesis of collagenase in Acinetobacter sp. was found to be inducible by denatured collagen and by its high molecular weight fragments. The presence in the inducer of part of the tertiary structure appear to be indispensable. On the other hand, an addition of Casamino acids, meat protein hydrolysate, or a mixture of amino acids with a similar composition to gelatin does not stimulate collagenase synthesis. Enzyme production was severely repressed in the early phase of growth by glucose, arabinose, and ribose, single amino acids, proline, hydroxyproline, alanine, glutamic acid or casein acid hydrolysate. A mechanism of repression similar to catabolite repression was involved in the phenomenon caused by carbohydrates. However, the fact that cyclic adenosine 3'5-monophosphate did not overcome the repression caused by amino acids or Casamino acids, in contrast to classical catabolite repression, suggests that these two forms of repression may be distinct.     

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.     

Control of 3T3 cell proliferation by calcium

Summary When a population of 3T3 mouse cells was subcultured regularly at confluency, the original epitheliodid or stellate cells disappeared and, by the ninth passage, they had been replaced by spindle-shaped cells. The original cells proliferated only when the extracellular calcium concentration exceeded 0.1mm, and their proliferative activity became maximum only when the calcium concentration was 0.5mm. The spindle-shaped cells were much more sensitive to proliferative stimulation by calcium. Although these cells also could not proliferate without extracellular ionic calcium, they proliferated maximally in the presence of as little as 0.05mm calcium. Thus, calcium is a major regulator of the proliferation of 3T3 mouse cells. Moreover, it appears that the sensitivity of the proliferative machinery to the calcium ion can vary greatly within an established cell line.     

Pectinase Production by Bacteria Associated with Improved Preservative Permeability in Sitka Spruce: Synthesis and Secretion of Polygalacturonate Lyase by Cytophaga johnsonii

Cytophaga johnsonii synthesized a polygalacturonate lyase which produced random cleavage of galacturonic acid polymers. No pectin methyl-esterase or hydrolytic pectinase activities could be detected in cultures of the organism. Polygalacturonate lyase synthesis was inducible and also subject to repression by glucose and other compounds. Galacturonic acid was the most effective inducer; lower activities were obtained with citrus pectin, polygalacturonic and polypectic acids. Glucose repression of lyase synthesis was not alleviated by 5 mM-adenosine-3'.5'-cyclic-monophosphate. Enzyme production was growth-linked and ceased when batch cultures entered the stationary phase. In steady-state chemostat cultures lyase activity was maximal at a dilution rate ( D ) of 0.19 h^-1. Polygalacturonate lyase was both cell-bound and free in the supernatant medium. The proportion of free enzyme increased throughout the batch growth cycle and in chemostat cultures over 70% of the activity was cell free at dilution rates below 0.05 h^-1.     

Cultural conditions for the induction of chondroitinase ABC in Proteus vulgaris cells

The conditions for the induction of chondroitinase ABC by Proteus vulgaris cells were studied to obtain cells with high chondroitinase ABC activity. The activity of the enzyme was found to increase when the cells were incubated in an induction medium containing chondroitin sulfate C as an inducer. The induction was most effective at pH 8.0, 25°C and the inducer was depolymerized in association with the increase in enzyme activity. For maximal induction, the addition of yeast extract, peptone and casamino acid was required. The increase in activity was inhibited by the presence of such antibiotics as chloramphenicol and actinomycine D. The induction was also catabolically repressed by the presence of glucose, glycerol or tricarboxylic acids.     

Inhibition of active strontium transport from erythrocyte ghosts by internal calcium: Evidence for a specificity controlling site

Summary The inhibition of strontium transport from erythrocyte ghosts by internal calcium was investigated. When active strontium transport was measured in the presence of increasing levels of internal calcium it was found that the inhibition of strontium transport started at an internal calcium level of 0.3mm and was virtually complete when this concentration reached 1.0mm. It was also noted that calcium transport was virtually constant between concentrations of 0.3 and 1.0mm. This experiment indicated that calcium did not inhibit strontium transport by competing for the active site of the transport system. This inhibition was partially reversed by increasing the internal magnesium concentration from 1 to 4mm. A higher level of magnesium at the time of lysis and during incubation enhanced strontium transport. However, the inhibition remained noncompetitive with respect to calcium. Manganese was also found to support calcium and strontium transport. However, it could not reverse the inhibition of strontium transport by internal calcium at any concentration tested. In fact, manganese restored the inhibition of strontium transport by calcium in ghosts that were prepared and incubated in solutions that had high magnesium levels.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20C and pH 69 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

Enzyme induction and repression in Arthrobacter crystallopoietes

Summary Catabolic effects which exert control over the inducible synthesis of three enzymes in Arthrobacter crystallopoietes involve at least three different mechanisms: interference with inducer transport, severe catabolite repression, and transient repression. The rate of histidase induction by histidine is reduced by incubation of the cells with succinate or glucose. The maximum effect of succinate, 67% reduction in histidase production, occurs only after 100 min of incubation with succinate. At least 3h of incubation are required for the maximum effect of glucose (31% reduction in enzyme induction). Both succinate and glucose inhibit histidine transport. Cyclic adenosine 3,5-monophosphate (cyclic AMP), at 10^-7 M, slightly stimulates the induction of histidase in cultures both with or without succinate. No conditions were found in which cyclic AMP abolishes the effect of succinate. Induction of l-serine dehydratase by glycine is severely and permanently repressed by glucose and to a lesser extent by citrate. Glucose does not affect glycine uptake. Succinate, fumarate, and aspartate, which are all better substrates than glucose or citrate for growth of A. crystallopoietes, have no effect on l-serine dehydratase induction. Induction and repression of l-serine dehydratase are not affected by cyclic AMP. Synthesis of isocitrate lyase after addition of acetate is unaffected by glucose but is severely repressed by succinate or fumarate. Aspartate and glutamate cause a transient repression of enzyme synthesis after which synthesis proceeds at the control rate. The ability to transport acetate is inducible. Development of this capacity in the presence of acetate is not affected by succinate or glutamate. Cyclic AMP has no effect on enzyme production or repression. A. crystallopoietes takes up radioactive cyclic AMP and has at least one of the enzymes of cyclic AMP metabolism, adenyl cyclase.     

Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport

Summary The mechanisms by which glucose regulates calcium fluxes in pancreatic endocrine cells were investigated by monitoring the efflux of⁴⁵Ca from prelabeled and perifused rat pancreatic islets. In the absence of both extracellular calcium and glucose, partial or total removal of extracellular sodium decreases the efflux of⁴⁵Ca from prelabeled islets. Glucose also reduces the efflux of⁴⁵Ca from islets perifused in the absence of extracellular calcium. This inhibitory effect of glucose on⁴⁵Ca efflux is decreased by half when the extracellular concentration of sodium is lowered to 24mm. In the absence of extracellular calcium but presence of glucose, partial or even total removal of extracellular sodium fails to decrease the efflux of⁴⁵Ca. At normal extracellular calcium concentration (1mm) partial removal of extracellular sodium dramatically increases⁴⁵Ca efflux from pancreatic islets. This increase in⁴⁵Ca efflux is partially but not totally suppressed by either 16.7mm glucose or cobalt. It is totally suppressed by 4.4mm glucose or by the combination of 16.7mm glucose and cobalt. At normal extracellular calcium concentration, glucose initially reduces and subsequently increases⁴⁵Ca efflux. The initial fall is unaffected by tetrodotoxin but decreased by 50% at low extracellular sodium concentration (24mm). The present results suggest the existence in pancreatic endocrine cells of a glucose-sensitive process of sodium-calcium counter-transport. By inhibiting such a process, glucose may decrease the efflux of calcium from islet cells. The effect of glucose is not mediated by an increase in intracellular sodium concentration. It could contribute to the intracellular accumulation of calcium which is thought to trigger insulin release.This paper is the IVth in a series.     

Induction and carbon catabolite repression of isoamylase production in Rhizopus oryzae PR7

The growth and the extracellular isoamylase production by Rhizopus oryzae PR7 MTCC 9642 were studied in a stationary culture at 28°C, with maximum isoamylase production obtained after 72 hours. Glycogen was found to be the best inducer for isoamylase synthesis, followed by maltose and dextrin. The enzyme was found to be repressed by glucose and this repression was not overcome by the addition of cGMP. The abrupt reduction in enzyme synthesis after the addition of exogenous glucose in a glycogen-induced culture medium confirmed the repressive action of glucose. An almost similar rate of repression was found to be exerted by α- and β-cyclodextrins. The inhibition of enzyme production after the addition of cycloheximide, a translation blocker, indicated the existence of de novo synthesis of the enzyme.     

Synthesis and regulation of extracellular beta (1-3) glucanase and protease by cytophaga sp. In batch and continuous culture

Lytic enzyme systems with the ability to break whole cells of yeast are a mixture of several enzymes and virtually all contain beta(1-3)glucanases and some protease. It appears that the presence of these two enzyme activities is necessary to break the two layers of the rigid cell wall. The enzyme system of Cytophaga NCIB 9497 has a high activity towards the walls of yeast and also of bacteria. This article describes the production of this extracellular lytic enzyme system in batch and continuous culture-it was found to be inducible. The synthesis and regulation of the two main constituent enzymes, beta(1-3)glucanase and protease, have been investigated. The synthesis of beta(1-3)glucanase is regulated by bothinduction (by an unknown inducer) and catabolite repression. Highbeta(1-3)glucanase activities were obtained in continuous culture at low dilution rates over a narrow range (0.05-0.10 h(-1)), and there is evidence of the presence of more than one glucanase enzyme. Proteolytic activity appears subject to catabolite repression and made up of the activities of more than one protease enzyme. Productivity and enzyme concentration were increased several fold in continuous culture when compared to batch culture.     

Effect of glucose and cyclic adenosine 3',5'-monophosphate on the synthesis of succinate dehydrogenase and isocitrate lyase in Escherichia coli.

Repressed respiration of Escherichia coli cells grown in the presence of 2% glucose was derepressed when the cells were incubated in a buffer containing casamino acids. The glucose-repressed cells were deficient in succinate dehydrogenase [EC 1.3.99.1] and isocitrate lyase [EC 4.1.3.1] activities, which increased during the incubation. The increases in respiratory activity and enzyme activity on incubation were repressed by glucose, but except for isocitrate lyase these repressions could be restored by the addition of cyclic adenosine 3',5'-monophosphate. Inhibitors of protein synthesis blocked the increase of enzyme activity on incubation without glucose, or with glucose and the cyclic nucleotide.     

Effect of glucose on sporulation and extracellular amylase production byClostridium perfringens type A in a defined medium

The effect of glucose and other sugars on sporulation and extracellular amylase production byClostridium perfringens NCTC 8679 type A in a defined medium was studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity, while disaccharides such as lactose, maltose, and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10mm except for fructose (25mm). Levels of heat-resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in this strain ofC. perfringens.     

Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a45Ca exchange technique in the presence of magnesium,phosphate, and ATP at 37°C

Summary The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37°C and in the presence of 2mm inorganic phosphate, 3mm ATP, 0.05 or 1.1mm free magnesium and a calcium buffer, nitrilotriacetic acid, was investigated using a⁴⁵Ca exchange technique. The amounts of⁴⁰Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of⁴⁵Ca exchange. At 0.05mm free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 m, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 m. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The⁴⁵Ca exchange data were found to be consistent with a system in which two compartments of exchangeable calcium are associated with the mitochondria. Changes in the concentration of inorganic phosphate did not significantly affect the⁴⁵Ca exchange curves, whereas an increase in the concentration of free magnesium inhibited exchange. The maximum rate of calcium outflow from the mitochondria was estimated to be 1.7 nmol/min per mg of protein, and the value ofK _0.5 for intramitochondrial exchangeable calcium to be about 1.6 nmol per mg of protein. Ruthenium Red decreased the fractional transfer rate for calcium inflow to the mitochondria while nupercaine affected principally the fractional transfer rates for the transfer of calcium between the two mitochondrial compartments. The use of the incubation conditions and⁴⁵Ca exchange technique described in this report for studies of the effects of agents which may alter mitochondrial calcium uptake or release (e.g., the pre-treatment of cells with hormones) is briefly discussed.     

Glucose inhibition of galactose-induced synthesis of β-galactosidase in Streptomyces violaceus

Various carbon compounds inhibited galactose induced synthesis of a -galactosidase activity in Streptomyces violaceus. Glucose and 2-deoxyglucose, but not methyl--d-glucose, caused inhibition of galactose uptake activity. In addition, glucose, or one of its metabolites, inhibited the synthesis of the glactose uptake system. Therefore it is concluded that the main inhibitory activity of glucose on galactose induced enzyme synthesis is exerted through inducer exclusion. Other carbon sources, such as d-ribose, d-gluconate, cellobiose or dl--glycerophosphate, did not inhibit uptake of the inducer galactose and may exert their effect through catabolite repression, inactivation or direct enzyme inhibition.     

Some aspects of the regulation of the production of extracellular proteolytic enzymes by a marine bacterium

A highly proteolytic Gram-negative, rod-shaped bacterium was isolated from the gills of fresh plaice and the effect of culture conditions on the production of proteolytic enzymes was investigated. When the organism, strain SA 1, was grown in the presence of complex mixtures of proteins and amino acids, both endopeptidase and aminopeptidase activity was demonstrated in the cell-free culture medium. However, synthesis of these enzymes was not observed when the organism was grown in a mineral medium with lactate or succinate as the only carbon and energy source. Synthesis of both endopeptidase and aminopeptidase was induced by the presence of amino acids in the medium. Of the amino acids tested, l-phenylalanine was found to be the best single inducer for the production of endopeptidase. When in addition one or more different amino acids were added, endopeptidase production was found to increase with increasing complexity of the mixture, up to a maximum which was obtained with five different amino acids. Production of the aminopeptidase was optimal when l-glutamic acid was used as a single inducer. For this enzyme the amount of enzyme activity released in the medium decreased with increasing complexity of the amino acid mixture. Endopeptidase as well as aminopeptidase activity was found to accumulate in the medium at the end of the logarithmic growth phase, when the culture was no longer growing exponentially. When the stationary phase was reached, enzyme production stopped. Production of both enzymes was immediately halted upon addition of chloramphenicol and was found to be repressed by glucose and lactate. These results suggest that synthesis of proteolytic extracellular enzymes by the organism studied is controlled by an efficient regulatory mechanism, in which growth rate is an important parameter.