Catabolite repression in Streptomyces venezuelae. Induction of beta-galactosidase, chloramphenicol production, and intracellular cyclic adenosine 3',5'-monophosphate concentrations

Chloramphenicol production was studied in cultures of Streptomyces venezuelae growing in a simple buffered medium with ammonia as the nitrogen source and glucose, lactose, or a glucose-lactose mixture as the sole source of carbon. With each carbon source the antibiotic was formed during growth. In the glucose-lactose medium, the production pattern was biphasic; a marked decrease in the rate of synthesis was associated with depletion of glucose from the medium and a corresponding diauxie pause in growth. Cells of S. venezuelae contained an inducible beta-galactosidase. Induction by lactose was suppressed by glucose. Measurement of the concentration of intracellular adenosine 3',5'-cyclic monophosphate during growth of cultures with glucose or a glucose-lactose mixture as the source of carbon showed no appreciable changes coinciding with depletion of glucose or the onset of chloramphenicol biosynthesis. It is concluded that the cyclic nucleotide does not mediate selective nutrient utilization or control antibiotic biosynthesis in this organism.     

Role of the carbon source in regulating chloramphenicol production by Streptomyces venezuelae: studies in batch and continuous cultures

Both carbon- and nitrogen-limited media that supported a biphasic pattern of growth and chloramphenicol biosynthesis were devised for batch cultures of Streptomyces venezuelae. Where onset of the idiophase was associated with nitrogen depletion, a sharp peak of arylamine synthetase activity coincided with the onset of antibiotic production. The specific activity of the enzyme was highest when the carbon source in the medium was also near depletion at the trophophase-idiophase boundary. In media providing a substantial excess of carbon source through the idiophase, the peak specific activity was reduced by 75%, although the timing of enzyme synthesis was unaltered. Moreover, chemostat cultures in which the growth rate was limited by the glucose concentration in the input medium failed to show a decrease in specific production of chloramphenicol as the steady-state intracellular glucose concentration was increased. The results suggest that a form of "carbon catabolite repression" regulates synthesis of chloramphenicol biosynthetic enzymes during a trophophase-idiophase transition induced by nitrogen starvation. However, this regulatory mechanism does not establish the timing of antibiotic biosynthesis and does not function during nitrogen-sufficient growth in the presence of excess glucose.     

Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae Cavara. III. Multiplicity of beta-glucosidase, and purification and properties of a second component.

To determine the relationship between the induction patterns of three components of beta-glucosidase of Pyricularia oryzae and carbon sources in the growth medium, various culture conditions were examined. Avicel, hydroxyethylcellulose and methyl-beta-D-glucoside as the carbon source induced both beta-glucosidase components, GB-1 and GB-2, whereas cellobiose and gentiobiose induced only one component, GB-1. Thus, these two components were induced independently and hence thought to be isozymes. The GB-2 was purified to homogeneity by ion exchange and gel filtration chromatographies from two different cultures on methyl-beta-D-glucoside and Avicel. The specific activity of GB-2 when salicin was used as substrate was approximately 5.9 mg glucose/min/mg protein. GB-2 was found to be an oligomeric glycoprotein, which consisted of two subunits with molecular weight of approximately 120,000, comprising a relatively large number of acidic amino acids and mannose, as is the case with GB-1. These two isozymes were clearly different in thermostability, GB-2 being more thermolabile than GB-1. However, the same carboxyl group (pKa 4.2--4.8) was found to be strongly implicated in the formation and dissociation of the enzyme-substrate complex for both of the enzymes, from the analysis of kinetic parameters as a function of pH.     

A Fourth Escherichia Coli Gene System with the Potential to Evolve β-Glucoside Utilization

Escherichia coli K12 is being used to study the potential for adaptive evolution that is present in the genome of a single organism. Wild-type E. coli K12 do not utilize any of the beta-glucoside sugars arbutin, salicin or cellobiose. It has been shown that mutations at three cryptic loci allow utilization of these sugars. Mutations in the bgl operon allow inducible growth on arbutin and salicin while cel mutations allow constitutive utilization of cellobiose as well as arbutin and salicin. Mutations in a third cryptic locus, arbT, allow the transport of arbutin. A salicin+ arbutin+ cellobiose+ mutant has been isolated from a strain which is deleted for the both the bgl and cel operons. Because the mutant utilized salicin and cellobiose as well as arbutin, it is unlikely it is the result of a mutation in arbT. A second step mutant exhibited enhanced growth on salicin and a third step mutant showed better growth on cellobiose. A fourfold level of induction in response to arbutin and a twofold level of induction in response to salicin was observed when these mutants were assayed on the artificial substrate p-nitrophenyl-beta-D-glucoside. Although growth on cellobiose minimal medium can be detected after prolonged periods of time, these strains are severely inhibited by cellobiose in liquid medium. This system has been cloned and does not hybridize to either bgl or cel specific probes. We have designated this gene system the sac locus. The sac locus is a fourth set of genes with the potential for evolving to provide beta-glucoside utilization.     

Kinetic mechanism of beta-glucosidase from Trichoderma reesei QM 9414

beta-Glucosidase is a key enzyme in the hydrolysis of cellulose to D-glucose. beta-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl beta-glucosides. The double-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl beta-D-glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 +/- 0.2 mM and V = 2.09 +/- 0.52 mumol.min-1.mg-1 for salicin; K = 1.22 +/- 0.3 mM and V = 1.14 +/- 0.21 mumol.min-1.mg-1 for cellobiose; K = 0.19 +/- 0.02 mM and V = 29.67 +/- 3.25 mumol.min-1.mg-1 for p-nitrophenyl beta-D-glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by beta-glucosidase on p-nitrophenyl beta-D-glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl beta-D-glucopyranoside hydrolysis.     

Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum.

Clostridium thermohydrosulfuricum consumed glucose in preference to cellobiose as an energy source for growth. The rates of substrate uptake in glucose- and cellobiose-grown cell suspensions were 45 and 24 nmol/min per mg (dry weight), respectively, at 65 degrees C. The molar growth yields (i.e., grams of cells per mole of glucose equivalents) were similar on cellobiose and glucose (19 and 16, respectively). Both glucose- and cellobiose-grown cells contained a glucose permease activity and high levels of hexokinase (greater 0.34 mumol/min per mg of protein at 40 degrees C). Growth on cellobiose was associated with induction of a cellobiose permease activity. In contrast, Clostridium thermocellum metabolized cellobiose in preference to glucose as an energy source and displayed lower growth rates on both substrates. The substrate uptake rates in cellobiose- and glucose-grown cell suspensions were 18 and 17 nmol/min per mg (dry weight), respectively. The molar yields were 38 on cellobiose and 20 on glucose. Extracts of glucose- and cellobiose-grown cells both contained cellobiose phosphorylase and phosphoglucomutase activities, whereas only glucose-grown cells contained detectable levels of glucose permease and hexokinase activities. The general catalytic and kinetic properties of the glucose- and cellobiose-catabolizing enzymes in the two species are described, and a model is proposed to distinguish differential saccharide metabolism by these thermophilic ethanologens.     

Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli

DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar.     

Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli.

DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar.     

Induction of α-amylase by maltooligosaccharides in Thermomonospora curvata

The action pattern of the α-amylase produced by Thermomonospora curvata is unique. Maltooligosaccharides (maltose to maltopentaose) were tested individually for their ability to induce α-amylase in this thermophilic actinomycete. Maltotetraose was the most inductive followed by maltotriose. Maltose was a good inducer of amylase production when used as sole carbon source, but had relatively little inductive capacity in the presence of either glucose or cellobiose. When cellobiose was added during exponential growth on maltose, maltose utilization and extracellular α-amylase accumulation were transiently inhibited. With maltotriose as the initial carbon source, addition of cellobiose did not inhibit the utilization of the trisaccharide; however, cellobiose, whether added during exponential growth or stationary phase, resulted in the rapid degradation of amylase when maltotriose was depleted from the medium. This inactivation did not appear to be a growth phase-induced phenomenon because stationary phase cells in the absence of cellobiose maintained their peak extracellular amylase level. This cellobiose-mediated α-amylase inactivation would be particularly important during production of the enzyme on a complex lignocellulosic substrate.     

Cellobiose dehydrogenase influences the production of S. microspora β-glucosidase

BglG, a Stachybotrys microspora β-glucosidase produced in the presence of glucose and cellobiose, was used individually as sole carbon source. The time course synthesis of BglG showed two aspects: (1) an exponential curve, observed in glucose Mandels medium, and (2) a cloche curve, observed in cellobiose containing cultures. A decrease was observed in bglG production at the 6th, 8th and 10th days during mycelium growth in cellobiose Mandels medium, which allowed for the assumption that the anabolism of a bglG inhibitor factor was produced with cellobiose but not with glucose. Cellobiose dehydrogenases (CDH) activity was, on another hand detected in cellobiose grown cultures but not in glucose containing ones. The aliquots, withdrawn at the time course of bglG production in the presence of cellobiose, gave rise to an inhibitory effect on bglG activity. This result was obtained with and without the heat treatment (5 min at 100°C) of the aliquots, which supported the non-proteinaceous nature of the inhibitor factor. Furthermore, sugar chromatographic analyses revealed the appearance of a secondary metabolite in the cellobiose Mandels medium and indicated that the factor behind the bglG activity cloche curve was a δ-gluconolactone. Seeing that the latter follows a strong inhibitory effect on bglG activity, it is speculated that the decrease in bglG activity during the time course of bglG synthesis in cellobiose Mandels medium is assigned to the release of δ-gluconolactone. This paper presents and validates an explanatory model for this hypothesis.     

Excretion of alpha-keto acids by strains of Streptomyces venezuelae

Cultures of Streptomyces venezuelae released acidic metabolites during nitrogen-limited growth on glucose. The main products were pyruvic acid and alpha-ketoglutaric acid. Variation in the extent of acid production was observed; spores of the parental strain 13s gave approximately 10% of low-producing colonies when plated on acid-base indicator medium. Examination of one low producer, strain PC 51-5, showed that differences in acid production became apparent only in low-glucose media containing manganese. In both strains PC 51-5 and 13s, uptake of alpha-keto-[5-14C]glutaric acid occurred by diffusion and no marked differences in permeability to alpha-ketoglutarate were detected. However, differences were observed in the activity of alpha-ketoglutarate dehydrogenase. In cultures of strain PC 51-5, the specific activity of the enzyme increased throughout growth, whereas in the parental strain activity decreased and could not be detected in older mycelium. Loss of enzyme activity was accompanied by excretion of alpha-ketoglutaric acid and failure to assimilate the product after glucose exhaustion. The results suggest that accumulation of pyruvic and alpha-ketoglutaric acids in S. venezuelae cultures grown in glucose-containing media may be due to regulatory suppression of the dehydrogenases by this carbon source.     

Studies on Thermoactinomyces sp. TM9208. I. Description of the strain

A strain of Thermoactinomyces sp., TM9208, isolated from a soil sample in the Taipei area showed antagonistic activity to Gram positive bacteria but not to the Gram negative on the potato extract agar plate by the cross streaking method. The strain showed strong starch hydrolysis, beta type of hemolysis, brownish yellow growth on the potato extract agar and green on the nutrient agar. Its aerial mycelium, white to grayish white, was long and straight with short branches. The spores are single and have smooth surface. It is a good utilizer of starch, maltose and cellobiose as carbon source, but utilizes dulcitol, salicin, sucrose, rhamnose and sorbitol poorly.     

Biochemistry and genetics of chloramphenicol production

In Streptomyces venezuelae, chloramphenicol is derived by an unusual diversion of chorismate, the branchpoint intermediate of the pathway involved in the biosynthesis of aromatic amino acids. In the chloramphenicol-producing organism, the DAHP synthetase was neither feedback inhibited nor repressed. Chorismate mutase was not repressed or inhibited by the intermediates or end-products of the shikimate-chorismate pathway. However, anthranilate synthetase and prephenate dehydratase are feedback inhibited by tryptophan and phenylalanine, respectively. During growth, when primary metabolism is not perfectly coordinated, decreasing demand for aromatic amino acids results in shunting of chorismate towards chloramphenicol biosynthesis.The endogenous synthesis of chloramphenicol produced by Streptomyces venezuelae is inhibited by the increasing concentration of chloramphenicol in the medium. Arylamine synthetase, the first enzyme involved in chloramphenicol biosynthesis, is repressed by the secreted chloramphenicol, by dl-p-aminophenylalanine and l-threo-p-aminophenylserinol. The excess intracellular chorismate pool is diverted to other aromatic shunt metabolites if biosynthesis of chloramphenicol is inhibited. There appears to be a glutamine binding protein subunit which is shared by several enzymes involved in amination of the aromatic ring of chorismate.Chloramphenicol producing organism also inactivated intracellular chloramphenicol. However, the resistance of the streptomycetes is due to inducible impermeability of the organism to chloramphenicol during antibiotic production. Streptomyces venezuelae is sensitive to chloramphenicol when it is not engaged in antibiotic production. The resistance to and production of chloramphenicol are induced simultaneously.A linkage map for 17 marker loci using Streptomyces venezuelae has been constructed. Restriction enzyme map of a plasmid from the chloramphenicol-producing streptomycetes has also been developed. The role of the plasmid in chloramphenicol biosynthesis and the life-cycle of the Streptomyces venezuelae is not yet understood.     

Effects of addition of chloramphenicol on the growth and ultrastructure of Streptomyces venezuelae

The effects of adding chloramphenicol before inoculation and during exponential growth of Streptomyces venezuelae (3022a) in fermentors were studied. The responses of the organism during synthesis of chloramphenicol (in a glycerol-serine-lactate medium) were compared with those in media supporting less (glycerol-nutrient broth-yeast extract) or no synthesis (glucosemineral salts). In systems where little or no synthesis of the chloramphenicol occurred, addition of the antibiotic induced micromorphological and ultrastructural abnormalities similar to those reported for sensitive bacteria. There was also an increase in the frequency of mesosomes and electron-light areas. It was suggested that the former may be associated with activity of chloramphenicol hydrolase and the latter with storage and/or excretion of the breakdown product; N-acetyl p-nitro-phenylserinol. When chloramphenicol synthesis occurred, addition of the antibiotic had less effect on the micromorphology or ultrastructure of S. venezuelae as permeability barriers to external chloramphenicol had been established. Electron-light areas were frequent, possibly being associated with storage and excretion of precursors of chloramphenicol.     

Xylose and Glucose Utilization by Bacteroides xylanolyticus X5-1 Cells Grown in Batch and Continuous Culture

During cultivation on a mixture of xylose and glucose, Bacteroides xylanolyticus X5-1 showed neither diauxic growth nor a substrate preference. Xylose-limited continuous-culture cells were able to consume xylose and glucose both as single substrates and as mixed substrates without any lag phase. When glucose was the growth-limiting substrate, the microorganism was unable to consume xylose. However, in the presence of a small amount of glucose or pyruvate, xylose was utilized after a short lag phase. In glucose-limited cells, xylose isomerase was present at low activity but xylulose kinase activity could not be detected. On addition of a mixture of xylose and glucose, xylose isomerase was induced immediately and xylulose kinase was induced after about 30 min. The induction of the two enzymes was sensitive to chloramphenicol, showing de novo synthesis. Xylose uptake in glucose-grown cells was very low, but the uptake rate could be increased when incubated with a xylose-glucose mixture. The increase in the uptake rate was not affected by chloramphenicol, indicating that a constitutive uptake system had to be activated. The inability of B. xylanolyticus X5-1 cells undergoing glucose-limited continuous culture to induce the xylose catabolic pathway after the addition of only xylose probably was caused by energy limitation.     

Characterization of cellobiose fermentations to ethanol by yeasts

Twenty-two different yeasts were screened for their ability to ferment both glucose and cellobiose. The fermentation characteristics of Candida lusitaniae (NRRL Y-5394) and C. wickerhamii (NRRL Y-2563) were selected for further study because their initial rate of ethanol production from cellobiose was faster than the other test cultures. C. lusitaniae produced 44 g/L ethanol from 90 g/L cellobiose after 5-7 days. When higher carbohydrate concentrations were employed, fermentation ceased when the ethanol concentration reached 45-60 g/L. C. lusitaniae exhibited barely detectable levels of beta-glucosidase, even though the culture actively fermented cellobiose. C. wickerhamii produced ethanol from cellobiose at a rate equivalent to C. lusitaniae; however, once the ethanol concentration reached 20 g/L, fermentation ceased. Using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as substrate, beta-glucosidase (3-5 U/mL) was detected when C. wickerhamii was grown anaerobically on glucose or cellobiose. About 35% of the beta-glucosidase activity was excreted into the medium. The cell-associated activity was highest against pNPG and salicin. Approximately 100-fold less activity was detected with cellobiose as substrate. When empolying these organisms in a simultaneous saccharification-fermentation of avicel, using Trichoderma reesei cellulase as the saccharifying agent, 10-30% more ethanol was produced by the two yeasts capable of fermenting cellobiose than by the control, Saccharomyces cerevisiae.     

Kinetics of the utilization of different C sources and the cellulose formation by Acetobacter xylinum

Different methylated glucose derivatives and cellobiose were examined as the carbon sources for growth and cellulose formation by Acetobacter xylinum. HPLC studies were carried out to gain information about the kinetics of the utilization of the C sources used. The type and yields of the synthesized cellulose were described. Besides glucose, cellobiose was a substrate for the synthesis of this polysaccharide by the bacteria. Other methylated derivatives of glucose were not accepted for a comparable synthesis of this polymer. An estimation of citrate in an unmodified culture liquid (SH medium) showed utilization in a late phase of cultivation. The influence of this organic acid on the pH value, cellulose synthesis and growth is described. By the application of citric acid as a sole carbon source “gel-like” forms of cellulose were formed generally.