J. Cudlín

How many genes are required for the synthesis of chlortetracycline

When contemplating the applicability of the theory of polygenic heredity to the development of views on the genetic regulation of the biosynthesis of secondary metabolites one encounters two extreme concepts. The first assumes that with increasing knowledge of the molecular basis of the biosynthesis of these compounds the views of polygenic heredity (formulated at the beginning of this century) cannot fundamentally contribute to the interpretation of new experimental data and to the formulation of a new working hypothesis in accord with the present state of our knowledge. The second concept proceeds from the simple fact that there is no unified theory of genetics at the present time to account satisfactorily for the heredity of complicated quantitative characters, such as the number of eggs laid, milk production, body weight, the production of various pigments etc. Most geneticists studying the improvement of industrial strains producing antibiotics are of the opinion that an increase in the production of these substances represents a very complicated matter and, basically, possesses all the features of polygenic heredity.

Biological activity of hydroxyanthraquinones and their glucosides toward microorganisms.

Five mono- and dihydroxyanthraquinones as well as 12 of their glucosides (both free and acetylated) were tested with six different microbial species using the plate-diffusion method. None of the tested substances was active againstEscherichia coli, 15 of the 17 substances displayed an activity towardBacillus subtilis, Bacillus cereus, Candida albicans, Saccharomyces cerevisiae andStreptomyces aurecfaciens. Relationships between the substance type and biological activity are discussed.

Very-long-chain fatty acids from lower organism

The qualitative occurrence and quantitative proportion of very-long-chain fatty acids (above C22). mainly in lower organisms and briefly in higher plants and animals is described.

Production of quinomycin A inStreptomyces lasaliensis

In addition to lasalocid, an oligoether coccidiostatic compound, other compounds are synthesized byStreptomyces lasaliensis. Mutants producing either of two antibiotics, lasalocid A or quinomycin A (an antibiotic of quinoxaline character), were obtained by natural selection and by mutagenesis. Methods of isolation, purification and estimation of both compounds were established.

The immunoinhibitory and immunostimulatory effects of hydroxyanthra- and hydroxynaphthoquinone derivatives.

The immunostimulatory and immunoinhibitory effects of 44 hydroxyanthra- and hydroxynaphthoquinone derivatives in tissue culture were investigated. In the test system used, the final effect,i.e. production of antibodies against sheep red blood cells is a result of cooperation between macrophages, T lymphocytes and B lymphocytes. It was found when testing selected 20 derivatives that methylation, acetylation or substitution of the hydroxy group by the amino group led to the loss of the inhibitory activity and, on the contrary, pronounced immunostimulatory effects could be observed in other derivatives. Naphthoquinones (juglone, lawsone) were more effective as immunoinhibitors than anthraquinones (alizarin, quinizarin) and their derivatives. In both groups of compounds glucosidation results in a substantial increase of the immunoinhibitory effect. The present work is a part of a more extensive study concerning modification of the molecules of various compounds and its relationship with the effect on immunological reactions.

Regulation of biosynthesis of secondary metabolites. IV. Inhibition of citrate synthase in Streptomyces aureofaciens by adenosine triphosphate.

Partially purified citrate synthase (EC 4.1.3.7) was isolated from submerged mycelium ofStreptomyces aureofaciens RIA 57, a strain producing chlortetracycline. Enzymatic activity was determined spectrophotometrically by means of 5,5′-dithio-bis-(2-nitrobenzoic acid). Citrate synthase was inhibited by ATP, inhibition being competitive with respect to acetyl coenzyme A. In 5mm concentration, ATP caused 55% inhibition of the enzyme. ADP, in the same concentration, caused 30% inhibition, while AMP caused none. Other natural nucleoside di- and triphosphates, in 1–5mm concentration, did not significantly affect citrate synthase inStreptomyces aureofaciens. Mg2+ inhibited the activity of the enzyme, but also reduced the negative effect of ATP. The role of ATP in regulation of the metabolic paths of acetyl CoA is discussed.

Partial purification and properties of glucosyltransferase fromStreptomyces aureofaciens

Differential centrifugation, precipitation with ammonium sulphate and chromatography on DEAE-cellulose led to a twenty-fold purification of glucosyltransferase fromStreptomyces aureofaciens B 96. The Michaelis constants for glucosyluridyl diphosphate (UDP-glucose) was 10.8 μm, for 1,2-dihydroxy-9, 10-anthraquinone (alizarin) 110 μm; the maximum rate of glucosylation reaction was 5.32 μmol per s per mg protein. The pH optimum was at 7.1; the flat temperature optimum was at 30 °C. Using some hydroxy derivatives of 9,10-anthraquinone it was found that the production of glucosides from aglycones with α-hydroxyl groups was about 1/8 of the values obtained with β-hydroxyl substrates. In both types of aglycones the presence of another hydroxyl group led to a higher glucoside production.

Isolation of glucosyltransferase fromStreptomyces aureofaciens

Streptomyces aureofaciens B 96 grown on a synthetic medium glucosylated exogenous 1,2-dihydroxy-9, 10-anthraquinone (alizarin). The glucosylation was inhibited by 2,4-dinitrophenol added to the cultivation medium. A cell-free preparation was obtained from the mycelium isolated after 16 h of growth and was found to catalyze the transfer of glucose from glucosyluridyl diphosphate to 1,2-dihydroxy-9, 10-anthraquinone, giving rise to l-hydroxy-2-(β-D-glueopyranosyloxy)-9,10-anthraquinone.

Microbial Glucosidation of Dihydroxyanthraquinones. General Properties of the Glucosidation System

Corresponding mono-β-d-glucosides were obtained by fermentation ofStreptomyces aureofaciens B 96 with four isomeric dihydroxyanthraquinones (alizarin, quinizarin, chrysazin and anthraflavin). The effect of some factors (sugar source, concentration of substrates, pH) on biosynthesis of 1-hydroxy-2-(β-d-glucopyranosyloxy)anthraquinone was studied and optimum conditions for the microbial glucosidation were determined.

Regulation of biosynthesis of secondary metabolites. XV. Isolation and characterization of auxotrophic mutants in Streptomyces aureofaciens

Auxotrophic mutants ofStreptomyces aureofaciens, necessary for a further genetic analysis, were obtained by treating standard strains and their mutants (differing by their biosynthetic activities) with UV-radiation and N-methyl-N′-nitro-N-nitrosoguanidine (MNG). Mean frequencies of induced mutations varied within 0.001–0.18% depending on the strain and experimental conditions. Doses of UV radiation and MNG yielding 0.1–0.9% and 0.001–0.0009% survival, respectively, were found to be roost effective. The technique of subsequent enrichment was found to be more useful for the detection of mutants than the technique of total isolation. Most isolated auxotrophs required arginine. Among other growth factors, methionine, purine bases and serine (or glycine) were frequently required. A new modification of the enrichment technique utilizing a synthetic arginine-free medium was worked out for the exclusion of thearg mutants. The nutritional deficiency was in most auxotrophs associated with a decrease or a complete loss of the ability to produce secondary metabolites. Difficulties caused by unequal mutation patterns and high unstability of mutants as well as presumable loci of certain genetic blocks leading to auxotrophy are discussed.