Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

Regulation of Staphylococcal Enterotoxin B

Several factors influenced the formation of enterotoxin B by Staphylococcus aureus strain S-6. In the standard casein hydrolysate medium, toxin was not produced in detectable quantities during exponential growth; it was produced during the post-exponential phase when total protein synthesis was arithmetic. The rate of toxin synthesis was much greater than the rate of total protein synthesis. The appearance of enterotoxin was inhibited by chloramphenicol; thus, the presence of toxin was dependent on de novo protein synthesis. When low concentrations of glucose (<0.30%) were added to the casein hydrolysate medium, growth was diauxic; glucose was completely metabolized during the first growth period. During the second growth period, enterotoxin was synthesized. In unbuffered casein hydrolysate medium containing excess glucose, toxin synthesis was completely repressed. The absence of toxin production under such conditions might be explained by the low (4.6) pH resulting from the acid end products of glucose metabolism. At pH <5.0, little or no toxin was produced. Toxin synthesis was initiated in the presence of glucose when the medium were buffered at any pH above 5.6. In such media, the differential rates of toxin synthesis, with respect to the rates of total protein synthesis, were lower than the differential rates in casein hydrolysate medium alone. Addition of glucose to a culture synthesizing toxin resulted in an immediate decrease in the differential rate without any change in pH. Thus, toxin synthesis appeared to be regulated by catabolite repression.     

Regulation of Staphylococcal Enterotoxin B: Effect of Thiamine Starvation

During the transition between the exponential and stationary phases of growth, there was a rapid accumulation of both cell-associated and extracellular enterotoxin B. Extracellular enterotoxin was synthesized until the cells entered the stationary phase during which cell-bound toxin was not detected. The differential rate of toxin synthesis relative to that of total protein synthesis was greater at pH 7.7 than at 6.0. Addition of glucose decreased the differential rate of toxin synthesis. This decrease was greater at pH 7.7 than at 6.0. Addition of pyruvate decreased the differential rate at pH 7.7 but not at 6.0. Analysis of the nongaseous end products of glucose and pyruvate metabolism showed that conditions which favor the oxidative decarboxylation of pyruvate also favor the repression of toxin synthesis. Elimination of thiamine from the medium prevented the oxidative decarboxylation of pyruvate by Staphylococcus aureus S-6 and partially or completely reversed the repression of toxin synthesis by glucose and pyruvate. In the absence of an added energy source, thiamine starvation caused a decrease in protein synthesis but an increased differential rate of toxin synthesis which was greater at pH 7.7 than at 6.0. In the absence of thiamine, pyruvate was not metabolized but caused a decrease in the rate of protein synthesis. This resulted in a twofold increase in the differential rate of toxin synthesis. Thus, conditions which altered the oxidative decarboxylation of pyruvate or decreased the rate of protein synthesis increased the rate of enterotoxin B synthesis.     

Joint culturing of Streptococcus lactis, strain MGU with Proteus vulgaris and Bacillus mesentericus

Nisin synthesis by Streptococcus lactis, strain MGU, grown as a combined culture together with Proteus vulgaris and Bacillus mesentericus under stationary conditions or with stirring does not depend on the quantity of inoculated associated cells. Nisin synthesis in the combined culture drops down by 10-20% at the initial pH 7.5 of the growth medium which is unfavourable for S. lactis producing nisin. The level of nisin biosynthesis does not rise when the pH of the medium is adjusted (either naturally or artificially) to 6.6-6.8 in the presence of glucose and yeast autolysate. S. lactis inhibits the growth of B. mesentericus when grown together with it whereas P. vulgaris inhibits the growth of S. lactis in their combined culture.     

Defined conditions for synthesis of Bacillus cereus enterotoxin by fermenter-grown cultures.

A strain of Bacillus cereus produced high levels of enterotoxin when grown in a semidefined medium in a laboratory scale fermenter. The optimum conditions for enterotoxin synthesis by cultures grown in this medium, which contained Casamino Acids and yeast extract, were found to be: inoculation of vigorously gorwing culture at the 1% level, addition of glucose at a concentration of 1%, control of culture pH at 8.0, incubation at 32 degrees C, use of a moderate stirring rate, and addition of air at low flow rates to minimize foaming. The enterotoxin yield in fermenter-grown cultures was approximately 20 to 50 times higher than the yield obtained in shake flask cultures.     

Defined conditions for synthesis of Bacillus cereus enterotoxin by fermenter-grown cultures.

A strain of Bacillus cereus produced high levels of enterotoxin when grown in a semidefined medium in a laboratory scale fermenter. The optimum conditions for enterotoxin synthesis by cultures grown in this medium, which contained Casamino Acids and yeast extract, were found to be: inoculation of vigorously gorwing culture at the 1% level, addition of glucose at a concentration of 1%, control of culture pH at 8.0, incubation at 32 degrees C, use of a moderate stirring rate, and addition of air at low flow rates to minimize foaming. The enterotoxin yield in fermenter-grown cultures was approximately 20 to 50 times higher than the yield obtained in shake flask cultures.     

Methanogens in the human intestinal tract and oral cavity

Curvularia lunata var.aeria was grown in YPD (yeast extract, peptone, and dextrose) medium (pH 6.5) at 28°C with varying concentrations (10–40 g/L) of glucose for the production of rifamycin oxidase. Enzyme activity and glucose concentration were found to be indirectly related to the production of black intracellular pigment by the organism. Depletion of glucose level and rise of culture pH initiate the synthesis of pigment. Carboxymethylcellulose (CMC) was used as a carbon source to improve the enzyme yield, but utilization of the substrate in the reactor was much less. Compared with 10 g/L of CMC in the medium, low or high concentrations of CMC did not yield any better result. Addition of glucose in YPC (yeast extract, peptone, and CMC) medium did not increase the enzyme activity, and glucose was rapidly utilized byC. lunata, forming pellets rather than mycelia.     

Fractionation of the beta-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were beta-linked as shown by H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

Biosynthesis and regulation of fructose-1,6-bisphosphatase and phosphofructokinase in Saccharomyces cerevisiae grown in the presence of glucose and gluconeogenic carbon sources.

The mode of synthesis and the regulation of fructose-1,6-bisphosphatase (Fbpase), a gluconeogenic enzyme, and phosphofructokinase (PFK), a glycolytic enzyme, were investigated in Saccharomyces cerevisiae after growth in the presence of different concentrations of glucose or various gluconeogenic carbon sources. The activity of FBPase appeared in the cells after the complete disappearance of glucose from the growth medium with a concomitant increase of the pH and no significant change in the levels of accumulated ethanol. The appearance of FBPase activity following glucose depletion was dependent upon the synthesis of protein. The FBPase PFK were present in glucose-, ethanol-, glycerol-, lactate-, or pyruvate-grown cells; however, the time of appearance and the levels of both these enzymes varied. The FBPase activity was always higher in 1% glucose-grown cells than in cells grown in the presence of gluconeogenic carbon sources. Phosphoglucose isomerase activity did not vary significantly. Addition of glucose to an FBPase and PFK synthesizing culture resulted in a complete loss, followed by a reappearance, of PFK activity. In the presence of cycloheximide the disappearance of glucose and the changes in the levels of FBPase and PFK were decreased significantly. It is concluded that S. cerevisiae exhibits a more efficient synthesis of FBPase after the exhaustion of glucose compared to the activity present in cells grown in the presence of exogenous gluconeogenic carbon sources. Two metabolically antagonistic enzymes, FBPase and PFK, are present during the transition phase, but not during the exponential phase, of growth, and the decay or inactivation of these enzymes in vivo may be dependent upon a glucose-induced protease activity.     

Factors influencing biohydrogenation and conjugated linoleic acid production by mixed rumen fungi

The objective of this study was to evaluate the effect of soluble carbohydrates (glucose, cellobiose), pH (6.0, 6.5, 7.0), and rumen microbial growth factors (VFA, vitamins) on biohydrogenation of linoleic acid (LA) by mixed rumen fungi. Addition of glucose or cellobiose to culture media slowed the rate of biohydrogenation;only 35-40% of LA was converted to conjugated linoleic acid (CLA) or vaccenic acid (VA) within 24 h of incubation, whereas in the control treatment, 100% of LA was converted within 24 h. Addition of VFA or vitamins did not affect biohydrogenation activity or CLA production. Culturing rumen fungi at pH 6.0 slowed biohydrogenation compared with pH 6.5 or 7.0. CLA production was reduced by pH 6.0 compared with control (pH 6.5), but was higher with pH 7.0. Biohydrogenation of LA to VA was complete within 72 h at pH 6.0, 24 h at pH 6.5, and 48 h at pH 7.0. It is concluded that optimum conditions for biohydrogenation of LA and for CLA production by rumen fungi were provided without addition of soluble carbohydrates, VFA or vitamins to the culture medium; optimum pH was 6.5 for biohydrogenation and 7.0 for CLA production.     

Dependence of activities of polysaccharide hydrolases and oxidases from Cerrena unicolor on the source of carbon and aromatic acids in culture media

The activities of carboxymethylcellulase and xylanase in the higher basidial fungus Cerrena unicolor grown in avicel-containing medium reached 1.95 and 1.50 units per mg protein, respectively, whereas in mannitol-containing medium they ranged from 0.02 to 0.05 units per mg protein. The activity of fungal beta-glucosidase depended on the carbon source in the culture medium and ranged from 2.1 units per mg protein in the presence of mannitol to 17.3 units per mg protein in the presence of avicel. In contrast to polysaccharides, easily metabolizable substrates (cellobiose, mannitol, and glucose) provided the highest rates of secretion of laccase (52.7-123.5 ncat per mg protein) and ligninase (22-106 units per mg protein). The addition of tangerine pomace, a substrate enriched with aromatic compounds, to the culture medium caused an increase in the rate of bio-synthesis of laccase and ligninase to 862 ncat per ml and 557 units per ml, respectively. Aromatic compounds such as p-xylidine and veratric aldehyde increased the laccase activity of C. unicolor IBB 62 from 7.9 to 23.6 and 18.3 ncat per mg protein, respectively. Veratryl alcohol caused a sevenfold increase in the activity of Mn-dependent peroxidase in the culture medium.     

Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.     

Regulation of breakdown and synthesis of L-glutamate decarboxylase in Clostridium perfringens.

L-Glutamate decarboxylase (GAD) activity of Clostridium perfringens (ATCC 8009) cells grown in various culture conditions was investigated. Remarkable variations of GAD level occur during the growth cycle in thioglycollate broth. These changes are affected by the pH of the culture medium. Addition of alkali to the culture media results in decrease of cell GAD activity, whereas increase of enzyme level occurs only in cells growing in unbuffered media. The results indicate that the mechanism regulating the GAD levels is sensitive to the changes of pH (or buffering substances) rather than to the steady pH values. Neither repression by glucose nor induction by L-glutamate was observed. Moreover, high concentrations of the free amino acid substrate in the culture media considerably decrease cell GAD activity, owing to the buffering effect of the amino acid. The molecular mechanism supporting the variations of GAD activity during the growth cycle of the cells were investigated and tentatively related to the structural and functional properties of the pure enzyme. It is shown that the drop of GAD activity during the lag phase is due to protein breakdown. Evidence is presented suggesting a control of protein degradation by its quaternary structure. Data are also reported supporting de novo synthesis of GAD during the late logarithmic phase of cell growth. Finally, the possible role of GAD as part of the pH regulation system of C. perfringens cells is discussed in relation both to physiologic conditions of the bacterial cell and to the molecular mechanisms regulating the GAD activity in vivo.     

Fractionation of the β-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were β-linked as shown by ¹H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

Effect of sugars, hydrogen ion concentration and ammonium nitrate on the formation of citric acid by Aspergillus niger.

Aspergillus niger Mulder strain when grown on a synthetic medium containing urea as the sole source of nitrogen at pH 5.2, formed a mixture of citric and gluconic acids. On growing the organism at pH 2.0 the gluconic acid content was reduced but citric acid yield remained low. Addition of NH4NO3 to the medium lowered the gluconic acid yields to undetectable levels with a simultaneous increase in the citric acid content. Of the sugars used for the production of citric acid, sucrose in an unautoclaved medium was found to be the best carbon source. Sucrose medium if autoclaved at pH 2.0, or a mixture of glucose and fructose instead of sucrose gave lower yields of citric acid. Under optimum conditions only citric acid was produced and the yield was 66-68 per litre after a growth period of about 10 days.     

Synthesis and degradation of tyrosinase in cultured melanoma cells

The tyrosinase (EC 1.14.18.1) activity of cultured B-16 mouse melanoma cells (C2M) in the stationary phase depends greatly on whether the culture medium contains glucose or galactose. The activity in medium containing galactose was about ten times that in medium containing glucose at pH 7.2. This difference in tyrosinase activity was concluded to be due to a shift of balance between synthesis and degradation of the enzyme. Experiments were conducted with stationary phase cultures in the presence of cycloheximide. The melanoma cells did not synthesize tyrosinase in medium containing glucose in the stationary phase. But when they were cultured under identical conditions, except that glucose was replaced by galactose, they continued to synthesize tyrosinase. The rate of synthesis in medium containing galactose at pH 6.3 was one third of that in the same medium at about pH 7, in which the increase in specific activity of tyrosinase per day was about 30 nmoles/mg cell protein per hr. The rate of degradation of the enzyme was practically the same in medium containing glucose as in medium containing galactose, and largely depended on the pH of the culture medium. At pH 6.3, the half-life was about one third of that at pH 7.2, where it was about 1.8 days. The degradation at acidic pH values was much reduced by ammonium salt and was strongly inhibited by the protease inhibitor, leupeptin.     

Kinetic control of ethanol production by Zymomonas mobilis

Summary Zymomonas mobilis UQM 2716 was grown anaerobically in continuous culture (D = 0.1/h; 30° C) 3nder glucose or nitrogen limitation at pH 6.5 or 4.0. The rates of glucose consumption and ethanol production were lowest during glucose-limited growth at pH 6.5, but increased during growth at pH 4.0 or under nitrogen limitation, and were highest during nitrogen-limited growth at pH 4.0. The uncoupling agent CCCP substantially increased the rate of glucose consumption by glucose-limited cultures at pH 6.5, but had much less effect at pH 4.0. Washed cells also metabolised glucose rapidly, irrespective of the conditions under which the original cultures were grown, and the rates were variably increased by low pH and CCCP. Broken cells exhibited substantial ATPase activity, which was increased by growth at low pH. It was concluded that the fermentation rates of cultures growing under glucose or nitrogen limitation at pH 6.5, or under glucose limitation at pH 4.0, are determined by the rate at which energy is dissipated by various cellular activities (including growth, ATP-dependent proton extrusion for maintenance of the protonmotive force and the intracellular pH, and an essentially constitutive ATP-wasting reaction that only operates in the presence of excess glucose). During growth under nitrogen limitation at pH 4.0 the rate of energy dissipation is sufficiently high for the fermentation rate to be determined by the inherent catalytic activity of the catabolic pathway.Abbreviations CCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - qG rate of glucose consumption (g glucose/g dry wt cells/h) - qE rate of ethanol production (g ethanol/g dry wt cells/h) - Y growth yield (g dry wt cells/g glucose) - D dilution rate Offprint requests to: C. W. Jones     

Concentration-dependent repression of the soluble and membrane components of the Streptococcus mutans phosphoenolpyruvate: sugar phosphotransferase system by glucose.

Growth of Streptococcus mutans Ingbritt in continuous culture (pH 7.0, dilution rate of 0.1 h-1) at medium glucose concentrations above 2.6 mM resulted in repression of the sugar-specific membrane components, enzyme IIGlc (EIIGlc) and EIIMan, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In one experiment, significant repression (27-fold) was observed with 73 mM glucose when the glycolytic capacity of the cells was reduced by only 2-fold and when the culture was still glucose limited. In a more comprehensive experiment in which cells were grown in continuous culture at eight glucose concentrations from 2.6 to 304 mM, in addition to repression of specific EII activities for glucose, mannose, 2-deoxyglucose, and fructose, synthesis of the general protein, EI, was repressed at all glucose levels above 2.6 mM to a maximum of 4-fold at 304 mM glucose when the culture was growing with excess glucose (i.e., nitrogen limited). The other PTS general protein, HPr, was less sensitive to the exogenous glucose level but was nevertheless repressed fourfold under glucose-excess conditions. The Km for glucose for EIIGlc increased from 0.22 mM during growth at 3.6 mM glucose (glucose limited) to 0.48 mM at 271 mM glucose (glucose excess). The shift from heterofermentation to homofermentation during growth with increasing glucose levels suggests the involvement of glycolytic intermediates, ATP, or another high-energy phosphate metabolite in regulation of the synthesis of the PTS components in S. mutans.     

The Effect of Carbohydrate and Nitrogen Concentration on Phenol Synthesis in Paul's Scarlet Rose Cells Grown in Tissue Culture

The effects of exogenous concentrations of glucose and nitrate on total ethanol extractable phenols and leucoanthocyanins were studied in Paul's Scarlet Rose cells grown in either liquid suspension or solid culture. Aliquots of liquid suspension cultured cells were harvested during logarithmic, early stationary, and late stationary periods of growth for determination of fresh weight, dry weight, total ethanol extractable phenols and leucoanthocyanins. Cells produced phenols during all phases of growth, but at stationary phase, the production was greatest. Increasing concentrations of exogenous glucose in the culture medium resulted in an increased synthesis of total phenols in logarithmic cells, and an increased synthesis of total phenols and leucoanthocyanin in stationary cells. Addition of increased concentrations of exogenous nitrate to the stationary cells grown in suspension culture markedly reduced synthesis of leucoanthocyanins although total phenol synthesis was not significantly affected. Similar observations were made in cells cultured on solid medium in respect to exogenous glucose concentration, however these cells differed from the suspension cultured cells by having increased amounts of total phenol synthesis and decreased synthesis of leucoanthocyanins in the presence of increasing concentrations of exogenous nitrate in the culture medium.     

Glucose repression of enterotoxins A, B and C and other extracellular proteins in staphlyococci in batch and continuous culture.

The production of enterotoxins, lipase and total extracellular protein by four strains of Staphylococcus aureus grown in batch culture at a controlled pH of 6.5 in a completely defined medium was markedly reduced by glucose or glycerol constantly maintained at 0.I M. A concomitant increase in the production of deoxyribonuclease, up to 13-fold, showed however that not all extracellular proteins are under the same control mechanism. The presence of glucose and glycerol in the medium also resulted in a rapid increase in the specific growth rate. However, growth of S. aureus s6 in Mgilimited continuous culture showed that glucose repression of enterotoxin B when the growth rate was held constant was more than twice that in batch culture. Therefore glucose repression can occur independently of an increase in growth rate. The specific rate of production of enterotoxin B, lipase, deoxyribonuclease, beta-haemolysin and total extracellular protein by S. aureus s6 increased as the growth rate increased from 0.07 to 0.24 h-1. Non-replicating cells grown in the absence of glucose produced considerable amounts of enterotoxin, and production was not repressed by the presence of glucose in the resuspension medium. In contrast, no enterotoxin B or C was obtained from nonreplicating cells grown in the presence of glucose. Chloramphenicol completely inhibited enterotoxin production by non-replicating cells, indicating that synthesis of new protein was required.