Use of analogs of primary metabolites in the selection of the producer of polymyxin B]

A number of amino acids were found to have effects on the growth of the polymyxin B-producing culture and biosynthesis of the antibiotic by it. Of special importance was the stimulating effect by methionine. Four selection stages were carried out with using structural analogs of purines and amino acids as selective factors. There were no stable variants with increased antibiotic productivity among the mutants resistant to the analogs of purines and leucine. The levels of polymyxin B accumulation by the variants resistant to 4-fluorophenylalanine were 30 to 50 per cent higher than those in the controls and the variants were characterized by low morphological and antibiotic production variation in the subcultures. The mechanisms of the methionine physiological effect and the prospects of using analogs of the primary metabolites in improvement of the culture producing polymyxin B are discussed.     

Effect of phosphorus on polymyxin B biosynthesis by B. polymyxa 1538 on media of varying makeup]

The effect of phosphorus on biosynthesis of polymyxin B by B. polymyxa 1538 was studied and its optimal concentration in the synthetic and two complex media was determined. Correlation between the culture growth and consumption of the main components, on the one hand, and concentration of phosphorus in the medium, on the other hand, was found. It was shown that the effect of phosphorus on biosynthesis of polymyxin B did not depend on the carbon source in the medium and aeration conditions.     

Importance of corn extract components for the biosynthesis of polymyxin B by B. polymyxa strain 1538]

The effect of some components of corn-steep liquor, such as biotin, organic nitrogen, inorganic phosphorus and other mineral elements on biosynthesis of polymyxin B by B. polymyxa, strain 1538 was studied. It was found that biotin and organic nitrogen had the most significant effect on the antibiotic accumulation. The effect of inorganic phosphorus and other mineral elements on accumulation of polymyxin by B. polymyxa, strain 1538 was less significant.     

Enzyme activity of Bacillus polymyxa 153, the producer of polymyxin B, during sporulation and biosynthesis

Metabolic properties of Bacillus polymyxa 153 were studied during vegetative growth, polymyxin B biosynthesis and active sporulation. In the cell extracts there was detected activity of exoproteases, endoproteases, tricarboxylic acid cycle dehydrogenases and pyruvate dehydrogenase. The enzymes activity in the cells growing into spores was higher than that in the cells of the vegetative developmental type. The activity of the enzymes depended on the culture age.     

Mechanism of Polymyxin B Resistance in Proteus mirabilis

The lipids from three types of organisms-a Proteus mirabilis wild type highly resistant to polymyxin B, a polymyxin B-sensitive mutant derived from the wild type, and the wild type grown in the presence of sulfadiazine resulting in phenotypic conversion to polymyxin B sensitivity-were examined to determine the nature of polymyxin B resistance. The phospholipid compositions were nearly identical; each organism contained similar small amounts of N-methyl phosphatidylethanolamine in addition to comparable quantities of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. the fatty acid compositions were similar in the exponential phase of growth; in the stationary phase, sulfadiazine markedly inhibited the synthesis of cyclopropane fatty acids. Liposomes prepared from the dried lipids of the three types of organisms were extensively and similarly disrupted by the polymyxin. These findings suggest that polymyxin B resistance in P. mirabilis is determined by the cell envelope which prevents access of the antibiotic to the susceptible lipid target sites.     

Effect of vitamins and various amino acids on glucose isomerase biosynthesis by Streptomyces albogriseolus culture

The effect of vitamins (B1, B2, B3, B6, B12, H, PP and folic acid) and amino acids (glutamic and aspartic acids) on glucose isomerase biosynthesis was studied in Streptomyces albogriseolus. These compounds were added either alone or in combinations to different growth media (synthetic and complex). The results were processed using mathematical methods, and the following mixture of vitamins and amino acids was proposed to be added to the complex fermentation medium: B2, 10 micrograms/L; B6, 10 micrograms/L; H, 1 microgram/L; aspartic acid, 0.01 microM. The production of glucose isomerase rose more than 1.5 times after such additions.     

Effect of L-malic and citric acids metabolism on the essential amino acid requirements for Oenococcus oeni growth

AIMS: The purpose of this work was to study the effect of L-malic and/or citric acids on Oenococcus oeni m growth in deficient nutritional conditions, and their roles as possible biosynthetic precursors of the essential amino acids. METHODS AND RESULTS: Bacterial cultures were performed in synthetic media. Bacterial growth rate was reduced or annulled when one amino acid was omitted from basal medium, especially for members of aspartate family, except lysine. The organic acids increased or restored the growth rates to the respective reference values. In each medium deficient in one essential amino acid, the L-malic acid utilization was accompanied by an increase of L-lactic acid concentration and accounted for approximately 100%l-malic acid consumed. D-lactic acid formation from glucose decreased in the medium without cysteine. Except for tyrosine, the recovery of glucose-citrate as D-lactic acid was lower than in the complete medium when asparagine, isoleucine or cysteine were excluded. The ethanol and acetate production was not modified. CONCLUSIONS: L-malic and citric acids favoured Oenococcus oeni m growth in nutritional stress conditions. Specifically citric acid was involved in the biosynthesis of the aspartate-derived essential amino acids and glucose in the cysteine biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Such beneficial effect of l-malic and citric acids on amino acids requirements of Oenococcus oeni m have great significance considering the low amino acids concentration in wine.     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E.

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental study of the organotropic side-effect properties of polymyxin B]

Toxicity of Soviet polymyxin B and its effect on the central peripheral nervous system, blood circulation, respiration, smooth muscles, functional liver and kidney state, growth and development of young animals, the picture of the peripheral blood were studied in acute and chronic experiments on various species of animals. It was found that polymyxin B had a suppressing effect on the peripheral n-cholinoreactive systems of the neuromuscle synapses of the skeletal muscles and ganglia of the sympathic and parasympathic innervation and deprimizing effect on the central nervous system. Caffeine, adrenaline and calcium chloride proved to be the antagonists of the neurotoxic effects of polymyxin B. The chronic experiments revealed that polymyxin B induced disorders in the kidney function and morphological changes in the glomeruli after its repeated administration. No significant effect of polymyxin B on the growth and development of the young animals, the functional state of the liver and the picture of the peripheral blood was observed when the drug was used in doses corresponding to the therapeutic ones in clinics.     

Biosynthesis of polymyxin E by a cell-free enzyme system. IV. Acylation of enzyme-bound L-2,4-diaminobutyric acid

An enzyme fraction, which catalyzes the ATP-PPi exchange reaction dependent on the three constituent amino acids of polymyxin E, was partially purified from crude extracts of Aerobacillus polyaerogenes. The approximate molecular weight was estimated to be 640,000 by Sepharose 4B gel filtration. Incubation of the enzyme with octanoyl coenzyme A and diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yielded octanoyldiaminobutyric acid thioesterified to the enzyme protein. On mild alkali treatment, octanoyldiaminobutyric acid, identified by paper chromatography, was released from the enzyme protein. From its acid hydrolyzate, diaminobutyric acid and octanoic acid were recovered in a molar ratio of 1 to 0.7. An ammonium sulfate fraction was required as the source of an acyltransferase for acylation of the enzyme-bound diaminobutyric acid. When [14C]-threonine was incubated with L-2,4-diaminobutyric acid in the presence of octanoyl coenzyme A, octanoyldiaminobutyrylthreonine bound to the enzyme protein was formed. These results suggest that acyldiaminobutyric acid bound to the enzyme protein is a possible initiation complex in the biosynthesis of polymyxin E.     

Effect of inorganic phosphorus on the biosynthesis of polymyxin B]

A synthetic medium containing optimal levels of the sources of carbon, nitrogen and inorganic phosphorus and providing satisfactory yields of polymyxin B was developed for 2 strains of Bac. polymyxa 933 and VK-153. The consumption of phosphorus in the medium by the strains and the antibiotic biosynthesis levels depended on the form of phosphorus added to the medium. Optimal biosynthesis of polymyxin B was observed at lower concentration levels of soluble soluble phosphorus in the medium than the bacterial growth.     

Sensitivity of Sherman's propionic acid bacilli to antibacterial preparations and vitamin B12 synthesis

Sherman propionic acid bacilli were sensitive to benzylpenicillin, ampicillin, ceporin, tetracyclines, oleandomycin, oletetrin, tetraolean, sigmamycin, levomycetin and furadonine. Methicillin, oxacillin, monomycin, kanamycin, polymyxin and furazolidone had an insignificant effect on the above organism. The subbacteriostatic concentrations of methicillin, oxacillin, streptomycin, monomycin, kanamycin, neomycin, tetraolean, sigmamycin, polymyxin M and ristomycin increased the biosynthesis of vitamin B12 by Sherman propionic acid bacilli, while benzylpenicillin, ampicillin, tetracyclines, oleandomycin, oletetrin, levomycetin and furadonine in the subbacteriostatic concentrations inhibited this process.     

影响多粘菌素B对绿脓杆菌杀菌效力的主要环境因素的研究

本文通过改变温度,水活度,气体条件和营养含量等影响绿脓杆菌生长的主要环境因素,测定多粘菌素B对绿脓杆菌的最小杀菌浓度(MBC)。结果表明环境因素导致或显著影响绿脓杆菌对抗生素的生态耐受性。实验表明多粘菌素B对绿脓杆菌的杀菌效力,除药物对细菌特有的药理学作用外,还取决于细菌的生长环境。结合冷休克率试验表明,环境影响细菌群体处于分裂状态的菌数。若分裂状态菌数下降表明生长速度减慢。提示了多粘菌素B对绿脓杆菌的效力指数,定量分析可以作为其综合效力作用的表现。以同步培养法确定在单个细胞周期中的抗生素敏感阶段。同时以冷休克率试验资料证明细菌处于分裂状态和幼龄期是其敏感阶段。初步阐述了生长速度缓慢与药物的生态耐受性密切相关。     

Amino Acid Requirements for the Production of Enterotoxin B by Staphylococcus aureus S-6 in a Chemically Defined Medium

A minimal chemically defined medium has been developed for growth (approximately 25 Klett units) and production of detectable enterotoxin B (approximately 5-6 mug/ml) by Staphylococcus aureus S-6. This medium contains monosodium glutamate as a source of carbon, nitrogen, and energy, three additional amino acids (arginine, cystine, and phenylalanine), six inorganic salts, and four vitamins. Increasing the concentrations of several amino acids in a series of defined media gave no increase in enterotoxin production. Apparently the limiting factor for growth and enterotoxin production in these media is the biosynthesis of one or more missing amino acids, rather than the concentration of the amino acids present in the media. An additional requirement for proline and valine was observed when glucose was added as the primary source of energy. When compared to complex media, our results indicated that the inhibitory effect of glucose on enterotoxin synthesis in defined media was less evident or totally absent.     

Possible explanation for the metabolic radioprotective effect of cysteine on Escherichia coli B

The growth of logarithmic-phase cultures of E. coli B cells on glucose-mineral medium was partially inhibited by 0.2 mM L-cysteine-HCl. Twenty other amino acids failed to show a similar inhibiting effect even at a concentration of 10 mM. After incubation of the log-phase culture in the presence of 2.0 mM cysteine for 30 minutes (inhibition about 70 %) the cells were centrifuged, resuspended, and diluted 200-fold in cysteine-free phosphate buffer. These cells exhibited increased resistance to the effect of X-rays as measured by the number of colony-forming units (DRF = 2.1). Both the growth-inhibiting and the radioprotective effects of cysteine could be diminished by the presence of 0.5 mg of casein hydrolyzate per milliliter during the 30-minute preirradiation culture period. This "anti-cysteine" effect was caused mainly by L-leucine, L-isoleucine, L-valine, and L-threonine from among the amino acids of casein hydrolyzate. It is suggested that cysteine inhibits the biosynthesis of some amino acids, thereby blocking protein synthesis, which may result in increased radioresistance.     

Isolation,structural characterization,and properties of mattacin (polymyxin M), a cyclic peptide antibiotic produced by Paenibacillus kobensis M

Mattacin is a nonribosomally synthesized, decapeptide antibiotic produced by Paenibacillus kobensis M. The producing strain was isolated from a soil/manure sample and identified using 16 S rRNA sequence homology along with chemical and morphological characterization. An efficient production and isolation procedure was developed to afford pure mattacin. Structure elucidation using a combination of chemical degradation, multidimensional NMR studies (COSY, HMBC, HMQC, ROESY), and mass spectrometric (MALDI MS/MS) analyses showed that mattacin is identical to polymyxin M, an uncommon antibiotic reported previously in certain Bacillus species by Russian investigators. Mattacin (polymyxin M) is cyclic and possesses an amide linkage between the C-terminal threonine and the side chain amino group of the diaminobutyric acid residue at position 4. It contains an (S)-6-methyloctanoic acid moiety attached as an amide at the N-terminal amino group, one D-leucine, six L-alpha,gamma-diaminobutyric acid, and three L-threonine residues. Transfer NOE experiments on the conformational preferences of mattacin when bound to lipid A and microcalorimetry studies on binding to lipopolysaccharide showed that its behavior was very similar to that observed in previous studies of polymyxin B (a commercial antibiotic), suggesting an identical mechanism of action. It was capable of inhibiting the growth of a wide variety of Gram-positive and Gram-negative bacteria, including several human and plant pathogens with activity comparable with purified polymyxin B. The biosynthesis of mattacin was also examined briefly using transpositional mutagenesis by which 10 production mutants were obtained, revealing a set of genes involved in production.     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.     

The action of combinations of the nonapeptide polymyxin B with antibiotics on gram-negative bacteria

Activity of polymyxin B nonapeptide alone and in combination with other antibiotics against clinical strains of Pseudomonas and enteric bacteria was studied. It was shown that nonapeptide was highly active against Pseudomonas and moderately active against enteric bacteria. In combination with rifampicin, fusidic acid or erythromycin the nonapeptide had a potentiating effect on the tested strains.