Pathways of the synthesis of glutamic acid by cephalosporin C-producing Acremonium chrysogenum

There were observed two pathways of glutamic acid formation in two strains of Acremonium chrysogenum differing in the production levels of cephalosporin C. The pathway involving glutamate dehydrogenase is known. The other pathway involved amination catalyzed by glutamine synthetase. Activity of both the enzymes during intensive synthesis of the antibiotic was higher in the highly productive strain. Under conditions of limited nitrogen content in the medium production of glutamate during the antibiotic biosynthesis depended on glutamine synthetase. When there was an excess of nitrogen in the medium the main role in production of glutamic acid at the phase of cephalosporin synthesis was played by the other enzyme i. e. glutamate dehydrogenase. By the dynamics the curve of the glutamate dehydrogenase activity correlated with that of the antibiotic production.     

Relationship between sugar consumption and tricarboxylic acid cycle enzyme activity in a high bialaphos-producing strain

The biosynthesis of bialaphos [a herbicide, 2-amino-4(hydroxy)(methyl)-phosphinoylbutyryl-alanylalanine] produced by Streptomyces hygroscopicus, proceeds through the degradation of glucose to phosphoenol pyruvate, the formation of a CP bond, acetic addition by acetyl-CoA and alanine addition. Therefore, bialaphos formation is considered to be closely related to glucose metabolism. Based on this hypothesis, sugar consumption and the activities of the tricarboxylic acid cycle enzymes were examined using a highly productive strain and a strain of the lower productivity. It was clear that the highly productive strain has a lower sugar consumption rate and lower yield of cells, compared with the lower productivity strain. The activities of the tricarboxylic acid cycle enzymes of the highly productive strain were lower than those of the lower productivity strain, while the activities of the glyoxylic acid cycle enzymes of the highly productive strain were higher. From these findings, it is suggested that the highly productive strain suppresses the flow from acetyl-CoA and pyruvate (as substrates of bialaphos) to the tricarboxylic acid cycle, and efficiently directs these substrates to the secondary metabolism by activation of the glyoxylic acid cycle, resulting in a high rate of bialaphos production in this strain.     

Regulation of biosynthesis of secondary metabolites. I. Biosynthesis of chlortetracycline and tricarboxylic acid cycle activity

In the course of submerged cultivation of low-production and industrial production strains of Streptomyces aureofaciens, the activity of enzymes of the tricurboxylic acid cycle was studied. The activities of citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37) were estimated spectrophotometrically in cell-free preparations. In the growth phase, mainly the initial reactions of the cycle were active with both strains. In production-phase, the activities of enzymes in the low-production strain were 2–5 × higher than in the production strain. Benzylthioeyanate, at a concentration of 5 × l0^?5M, stimulated chlortetracycline production of both strains with accompanying decrease in activity of the enzymes of the tricarboxylic acid cycle. The role of the tricarboxylic acid cycle in control of chlortetracycline biosynthesis is discussed.     

Relationship between intracellular pH and antibiotic biosynthesis in Fusidium coccineum

A correlation between the value of the intracellular pH and the biosynthesis of fusidic acid was studied by ³¹P-NMR spectroscopy in two strains of the fungus Fusidium coccineum. One of the strains was highly active and the other strain had low activity with respect to the antibiotic production. The position of the orthophosphate resonance in the ³¹P-NMR spectra was considered as a measure of the intracellular pH. In the cells of the highly active strain pH was in the range 7.0–7.5 in the cytoplasm and 6.1–6.25 in the vacuoles. In the cells of the strain with low activity was in the range 7.3–7.9 in the cytoplasm and 6.0–6.2 in the vacuoles. During high antibiotic productivity, the intracellular pH in the highly active strain full sharply, while in the less active strain it effectively did not change. This suggested that the change in the intracellular pH was responsible for the action of the enzymes in the cells and could be a factor defining the function of the cyanide-resistant respiration pathway and consequently the synthesis of fusidic acid in F. coccineum. *** DIRECT SUPPORT *** AG903062 00009     

High-molecular polyphosphates and pyrophosphate in cyclosporin- producing Tolypocladium sp. and their role in the processes of growth and antibiotic synthesis

The contents of high-energy phosphorous compounds, i.e. three fractions of polyphosphates, pyrophosphate, and ATP were determined in isogenic strains of Tolypocladium sp. differing in cyclosporine production levels. The content of polyphosphates was 1 to 2 orders of magnitude greater than that of pyrophosphate or ATP and did not depend on the strain productivity. During the period of the mycelial intensive growth and at the beginning of antibiotic synthesis, the level of polyphosphates lowered 2-3-fold and the content of pyrophosphate markedly decreased as well. The activities of polyphosphatase and pyrophosphatase during the culture growth and cyclosporine biosynthesis was higher in the highly productive strain.     

Mutagenic effect of mitomycin C on different strains of oleandomycin producer Streptomyces antibioticus

Mitomycin C, a DNA-tropic antibiotic, was shown to have a lethal effect on spore sprouts of two strains of Streptomyces antibioticus, an organism producing oleandomycin. When the time of exposure to the antibiotic increased there was an almost equal decrease in the survival rate. The mutagen action on the morphological variation and antibiotic production of the two closely related strains were diverse due to their genetic differences. The strain isolated after the culture treatment with a chemical mutagen and subjected to a more prolonged maintaining selection showed lower variation with respect to its colony morphology. The other strain isolated after treatment of the culture with high concentrations of its own antibiotic showed lower variation with respect to its antibiotic production property. The shift in the antibiotic production in the direction of the low active variants was characteristic of the both highly productive strains.     

Characteristics of the oxidative metabolism in strains with varying levels of fucidin biosynthesis]

Oxidative capacity of the fusidin-producing strains with various biosynthetic activity was studied comparatively. The studies showed that by their capacity to oxidize pyruvate and some metabolites of the tricarboxylic acid cycle (acetate, succinate, malate) the strains were arranged in the order reverse to their antibiotic activity. Such regularity was observed during the whole fermentation process and was most pronounced by the 3rd and 4th days (beginning of the idiophase). The rate of glucose oxidation was higher in more active strains. The same regularity was noted in the 2nd phase of the strain development associated with beginning of fusidin biosynthesis. In the 1st phase (the 1st and 2nd days) the strains almost did not differ by their capacity to oxidize glucose. By oxidation of phosphorylated ethers of carbohydrates (glucose-6-phosphate and fructoso-6-phosphate) the strains did not differ. Various fusidin-producing strains oxidized NAD-N and NADP-N approximately with the same rate. It is supposed that mutations leading to increased antibiotic production are associated with changes in acetate metabolism in the direction of more intensive biosynthesis of isoprenoid compounds, potential precursors of the fusidin molecule.     

Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation.

Mutants of Escherichia coli K-12 constitutive for the synthesis of the enzymes of fatty acid degradation (fadR) have elevated levels of the glyoxylate shunt enzymes, isocitrate lyase and malate synthase. A temperature-sensitive fadR strain has high levels of glyoxylate shunt enzymes when grown at elevated temperatures but has low, inducible levels of glyoxylate shunt enzymes when grown at low temperatures. The increased activity of glyoxylate shunt enzymes did not appear to be due to the degradation of intracellular fatty acids in fadR strains or differences in allosteric effectors in fadR versus fadR+ strains. These studies suggest that the fadR gene product may be involved in the regulation of the glyoxylate operon.     

Developmental cycle and the cytomorphological characteristics of the oleandomycin producer Streptomyces antibioticus

The developmental cycle and cytomorphological features of the industrial strain OL-1 and its variant 0968 of the oleandomycin-producing organism were studied. Variant 0968 was obtained as a result of exposure of the spores of strain OL-1 to UV light. When grown under submerged conditions in flasks with the rich medium, the strains were characterized by a complete developmental cycle consisting of three generations of the hyphae. Every generation had a tendency for formation of submerged spores. The UV-induced variant differed from the industrial strain by higher levels of the antibiotic accumulation which correlated with higher rates of the spore germination. The strains were characterized by polymorphism of the mycelium and formation of submerged spores during their cultivation which is likely to prolong the antibiotic synthesis from 120 to 216 hours from the inoculation moment. The long-term selection of the oleandomycin-producing organism on the rich medium markedly changed the culture genotype and resulted in significant changes in the developmental cycle under submerged cultivation conditions. The data may be used for the microscopic control of the process of oleandomycin production.     

Adenylate level in the mycelium of Penicillium nigricans Thom. grown on various carbon sources]

Concentrations of ATP and ADP and their dynamics during cultivation (2, 5, 9 and 13 days) of a highly productive strains of P. nigricans on a mineral medium in the presence of various carbon sources, such as glucose, succinate or acetate were studied. It was shown that the levels of ATP and ADP in the mycelium depended on the carbon source: the maximum and minimum ATP concentrations were found on the glucose and acetate media respectively, the maximum and minimum ADP concentrations showed inverse dependence. The concentrations of both adenylates on the same carbon source depended on the strain. The dynamics of the adenylates levels during cultivation showed an analogous dependence on the carbon source and the strain. Thus, the highly productive strain was characterized by a constant ATP level on glucose and succinate and variation on acetate, while the ADP level was characterized by a decrease by the 9th day of cultivation on any of the carbon sources. The low productive strain was characterized by variations in the level of ATP in any media used, stability of the ADP level by the 2nd--9th day of cultivation on the glucose medium and by the 2nd--5th day of cultivation on the succinate medium and a decrease by the 9th day of cultivation on the acetate and succinate media. The ratio of ATP/ADP at the phase of griseofulvin biosynthesis (9 days) markedly increased in both the strains when cultivated on the media with any of the carbon sources.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

Regulation of sulphur amino acids metabolic enzymes in Cephalosporium acremonium strains differing in antibiotic production

Three strains of Cephalosporium acremonium with different potential of cephalosporin C production show differences in the levels of sulphur amino acid metabolic enzymes. The regulation of these enzymes is also different in the strains. In the superior producing strain two enzymes directly involved in cysteine synthesis are the least susceptible to repression by methionine. In this strain cystathionine metabolizing enzymes seem to favour cysteine synthesis. Antibiotic producing strains, in contrast to the non-producing strain, are highly sensitive to chromate.     

Proteomic analysis of Candida magnoliae strains by two-dimensional gel electrophoresis and mass spectrometry

Candida magnoliae which has been newly isolated from honey comb is an osmotolerant yeast to produce erythritol as a major product. Erythritol is a noncariogenic, low calorie sweetener and safe for diabetics. Strain development by chemical mutation to obtain the improved erythritol yield and productivity relative to the parental strain made it necessary to elucidate the physiological differences between the wild and mutant strains. Proteomic analyses of C. magnoliae wild and mutant strains with two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry were carried out to identify intracellular proteins and to estimate the effects of newly characterized metabolic enzymes on the yeast cell growth and erythritol production. Most of the molecular mass of intracellular proteins were distributed in the range of pI 4-8 and molecular mass of approximately 130 kDa. Six out of nine protein spots expressed at different levels between the wild and mutant strains were analyzed with nanoelectrospray tandem mass spectrometry and identified by comparing amino acid sequences with the National Center for Biotechnology Information and Saccharomyces Genome Databases. Except for Ygr086cp, these proteins were believed to be the metabolic enzymes involved in the citric acid cycle (citrate synthase, succinyl-CoA ligase and fumarase) and the glycolysis pathway (pyruvate decarboxylase and enolase). Up-regulated enzymes in the citric acid cycle could explain high growth of the C. magnoliae mutant strain owing to the increased NADH and ATP formation. Down-regulated enolase and up-regulated fumarase in the mutant strain seemed to play a role in the improved bioconversion of erythrose-4-phosphate to erythritol compared with the wild strain.     

Histamine production by Klebsiella pneumoniae and an incident of scombroid fish poisoning.

A histamine-producing strain of Klebsiella pneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges-Proskauer tests.     

Histamine production by Klebsiella pneumoniae and an incident of scombroid fish poisoning.

A histamine-producing strain of Klebsiella pneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges-Proskauer tests.     

Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization

AIMS: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry. METHODS AND RESULTS: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65 degrees C and pH 4.5, respectively. Activity remained stable after incubation at 60 degrees C and pH 4.5 for 3 h. CONCLUSIONS: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications.     

Effect of oxygen partial pressure on bialaphos synthesis,sugar metabolism and the activity of tricarboxylic acid cycle enzymes in submerged culture of Streptomyces hygroscopicus

Bialaphos [a herbicide, 2-amino-4-(hydroxy)(methyl)-phospinoylbutyryl-alanyl-alanine] produced by Streptomyces hygroscopicus increased significantly as the oxygen supply decreased during both growth and production phases of batch culture. Under low oxygen partial pressure, there were decreases in sugar consumption by the cells, cell concentration and the activity of tricarboxylic acid cycle enzymes in the cells. On the other hand, the activity of glyoxylic acid cycle enzymes in the cells increased. These phenomena were more prominent in high producing than in low producing strains. From these results, it is suggested that in high bialaphos producing strains, low oxygen partial pressure suppresses the activity of tricarboxylic acid enzymes so that both acetyl-CoA and pyruvate (the substrates for bialaphos synthesis) are used mainly for secondary metabolism. This results in a high production of bialaphos.     

Peripheral metabolism of Pseudomonal putida transconjugants degrading chloro- and methylaromatic compounds

Peripheral metabolism was studied in the Pseudomonas putida 37cc transconjugant. In the strain grown on benzoate, pyrocatechase (PC) I with a low activity to chlorocatechols was induced, whereas PCII actively decomposing chlorocatechols was induced during its growth on 3-chlorobenzoic acid. The P. putida 37cc transconjugant grown on alpha-methylstyrene (MS) exerted the activity of both metapyrocatechase (MPC) and PC, whereas in the parent strain P. putida R-1 only MPC was involved in the degradation of alpha-MS. The substrate specificity of the enzymes involved in the ring cleavage by P. putida 37cc was compared to show that, apparently, MPC of the transconjugant was similar to this enzyme in the strain R-1 while PC decomposing chlorocatechols was similar to PC of the P. putida 87 donor. The regulation of the enzymes mediating the ring cleavage was studied in the parent strains and transconjugants.     

Combinations of Clavulanic Acid and other β-lactam Antibiotics against Strains of Pseudomonas aeruginosa, Serratia marcescens and other Bacteria

Combinations of clavulanic acid, a new β-lactamase inhibitor, with five cephalosporins and one cephamycin were tested against cell-free β-lactamases obtained from Serratia marcescens, Pseudomonas aeruginosa and an Enterobacter strain, 265A. Cefotaxime was the most resistant antibiotic and cephalothin the most sensitive antibiotic to β-lactamases. Low concentrations of clavulanic acid gave some protection against the Serratia and Pseudomonas enzymes. The most active source of β-lactamase was the 265A strain, against which only cefotaxime was highly resistant. Clavulanic acid had only a slight inhibitory effect on this enzyme, which was confirmed by an agar method, and potentiated slightly the activity of cephalothin and cefoxitin against two β-lactamase producing strains of Staphylococcus aureus. Lysis by cephalothin of one strain of S. marcescens was potentiated in the presence of clavulanic acid.     

Initial steps in the anaerobic degradation of 3,4,5-trihydroxybenzoate by Eubacterium oxidoreducens: characterization of mutants and role of 1,2,3,5-tetrahydroxybenzene.

Chemical mutagenesis and antibiotic enrichment techniques were used to isolate five mutant strains of the obligate anaerobe Eubacterium oxidoreducens that were unable to grow on 3,4,5-trihydroxybenzoate (gallate). Two strains could not transform gallate and showed no detectable gallate decarboxylase activity. Two other strains transformed gallate to pyrogallol and dihydrophloroglucinol but lacked the hydrolase activity responsible for ring cleavage. A fifth strain accumulated pyrogallol, although it contained adequate levels of the enzymes proposed for the complete transformation of gallate to the ring cleavage product. The conversion of pyrogallol to phloroglucinol by cell extract of the wild-type strain was dependent on the addition of 1,2,3,5-tetrahydroxybenzene or dimethyl sulfoxide. This activity was induced by growth on gallate, while the other enzymes involved in the initial reactions of gallate catabolism were constitutively expressed during growth on crotonate. The results confirm the initial steps in the pathway previously proposed for the metabolism of gallate by E. oxidoreducens, except for the conversion of pyrogallol to phloroglucinol.