Z. Hošťálek

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.

Effect of inorganic phosphate and benzyl thiocyanate on the activity of anhydrotetracycline oxygenase inStreptomyces aureofaciens

The level of anhydrotetracycline oxygenase (an enzyme catalyzing the penultimate reaction in the biosynthesis of tetracycline) inStreptomyces aureofaciens was substantially influenced by the amount of inorganic phosphate and by the presence of benzyl thiocyanate in the cultivation medium. Phosphate decreased the specific activity of the enzyme, particularly when added to a growing culture. On the other hand, benzyl thiocyanate increased the specific activity of the enzyme. Its effect was most conspicuous in the growth phase. The effect of benzyl thiocyanate was more pronounced in the low-production strain than in the producing variant. Inorganic phosphate and benzyl thiocyanate did not influence the enzyme activityin vitro. Phosphate added to the growing cultures was readily absorbed by the cells. During this time the enzyme synthesis was repressed, derepression occurred only after exhaustion of phosphate from the medium. The stimulatory efect of benzyl thiocyanate on the enzyme synthesis was not reversed by the inorganic phosphate added.

Catabolite regulation of antibiotic biosynthesis

ConclusionIn view of the possible mediation of carbon catabolite repression of antibiotic biosynthesis by phosphorylated substances, the concept of the role of phosphorus in the regulation of secondary metabolism should be re-evaluated. Many conclusions are based on an analogy with the effect of phosphorus in animal or plant cells (for review cf. Martin 1977). However, in contrast with plant and animal physiology, the production physiology of actinomycetes, typical soil microorganisms, was studied under nonphysiological conditions of nutrient over-supply in a submerged culture. This is analogous e.g. to space biology which studies the physiology of organisms in the state of weightlessness. Both disciplines have an extremely high significance but the elucidation of basic biological regularities is to be carried out back on Earth.In conclusion we may state that a critical evaluation of our contemporary knowledge seem to support the hypothesis that the antibiotic biosynthesis in actinomycetes, as well as the spore formation in bacilli — both regulated by the mechanism of catabolite repression — has its ecological significance, i.e. makes it possible for the organism to survive under conditions when vegetative growth is limited. For this reason this aotivity was preserved in the course of evolution as a protective mechanism of microbial populations and remained a part of the genome of many species.

Role of ATP-glucokinase and polyphosphate glucokinase inStreptomyces aureofaciens

The activity of ATP-glucokinase and of polyphosphate glucokinase was examined during growth of the actinomyceteStreptomyces aureofaciens 8425 under conditions of intense chlortetracycline (CTC) synthesis. ATP-glucokinase was active in the strain only during the logarithmic phase of culture growth; the activity of polyphosphate glucokinase appears only at the end of the logarithmic phase of growth and rises in parallel with the rate of CTC biosynthesis in the stationary phase. During the rise of activity of polyphosphate glucokinase and of CTC biosynthesis the cells accumulate sugar phosphates, mainly glucose-6-phosphate. It appears that the biosynthesis of CTC inStreptomyces aureofaciens takes place at the expense of glycolysis, using up the high-energy phosphate of high-molecular polyphosphates.

Role of phosphatases in the biosynthesis of chlortetracycline inStreptomyces aureofaciens

ATP diphosphohydrolase activity and inorganic pyrophosphatase reached two maxima during cultivation of the low- and high-producing variant ofStreptomyces aureofaciens under conditions of phosphate limitation,i.e. after 30 and 70 h of cultivation. Increased levels of inorganic phosphate in a medium inhibitory to biosynthesis of chlortetracycline markedly decreased the levels of both enzymes. The ATP diphosphohydrolase activity was detected both in the supernatant and membrane fractions of the cell-free preparation of the mycelium.

Intra- and interspecific cosynthetic activity of mutants ofStreptomyces coeruleorubidus andStreptomyces galilaeus impaired in the biosynthesis of anthracyclines

Cosynthesis of anthracycline compounds was followed in five phenotypic groups of mutants ofStreptomyces coeruleorubidus (A - E), blocked in the biosynthesis of the daunomycine complex, and in two mutant types ofStreptomyces galilaeus (F, G) blooked in the biosynthesis of glycosides of ε-pyrromy-cinone and aklavinone. Glycosides of daunomycinone and 13-dihydrodaunomycinone were produced in combinations A + B, A + C, A + D, A + E and A + F, ε-rhodomycinone was synthesized in combinations A + E, A + F, B + E and B + F. During the cultivation of types B - E with type G or F non-anthra-cycline compounds, typical ofS. galilaeus, were cosynthesized. No cosynthesis could be observed in other combinations of the mutant types. Negative results were also obtained with combinations of mutants of the same group and during cultivation of all mutant types with streptomycetes not producing anthracyclines. A scheme illustrating metabolic pathways leading to the biosynthesis of daunomycinone, aklavinone, ε-pyrromycinone and ε-rhodomycinone inS. coeruleorubidus andS. galilaeus was constructed.

Properties of apyrase and inorganic pyrophosphatase inStreptomyces aureofaciens

Apyrase (ATP-diphosphohydrolase, EC 3.6.1.5) and inorganic pyrophosphatase (EC 3.6.1.1) were partially purified fromS. aureofaciens RIA 57 and characterized. Apyrase degrades, in addition to ATP, other nucleoside triphosphates and nucleoside diphosphates, diphosphate, thiamine diphosphate, phosphoenolpyruvate and oligophosphates of chain lengthn ≦ 90. The apyrase activity was detected in the membrane and supernatant fractions. Its properties (substrate specificity, effect of inhibitors, pH optimum and effect of Mg2+ ions) were similar in both fractions except for the effect of oligomycin that inhibited only the membrane fraction. Pyrophosphatase exhibited a strict substrate specificity, substrates other than diphosphate being degraded relatively slowly. Of other enzymes exhibiting the phosphatase activity acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1), trimetaphosphatase (EC 3.6.1.2) and exopolyphosphatase (EC 3.6.1.11) degrading oligophosphates of chain lengthn = 15, 40 and 60, were detected.

Synthesis and degradation of proteins and DNA inStreptomyces aureofaciens

The rate of protein synthesis inStreptomyces aureofaciens, measured by incorporation ofU-14C-l-leucine into cells, fluctuated during the production phase in the range of 10–15 % of the values determined in the phase of intensive growth. Tetracycline partially inhibited the protein synthesis during the growth phase only. The proteins synthesized between the 6th and 18th hour of growth, were 75 % degraded by the 48th hour. The DNA synthesis, measured by means of incorporation of 2-14C-thymine into the mycelium, occurred predominantly during the first 24 h of cultivation. Similarly, DNA synthesized between the 6th and 12th hour of cultivation was degraded by 75 % after 48 h. The turnover of culture proteins is thus caused largely by degradation of old cells and growth of new ones which are more resistant to tetracycline. The activity of alanine aminotransferase and aspartate aminotransferase increase substantially towards the end of fermentation.

Developmental mutants ofStreptomyces coeruleorubidus, a producer of anthracyclines: Isolation and preliminary characterization

AbstbactThe wild strainStreptomyces coeruleorubidus JA 10092 was found to segregate into two spontaneous morphological variants (spo-1 andbld-1) with a different ability to form aerial mycelium in media with glucose as the main carbon source. Six new types of developmental mutants were obtained from the bald variantbld-1 after treatment with mutagens (UV light, γ radiation, nitrous acid) and after natural selection Formation of the aerial mycelium was fully suppressed in thebld-2 type growing on media both with glucose and with starch. The other types were bald only on starch media, forming the aerial mycelium on media with glucose; typesspo-2, spo-3, spo-4 andspo-5 differed in size, shape and surface structure of spores, the typewhi formed asporogenous aerial hyphae.

Electron-cytochemical demonstration of polyphosphates and the appropriate phosphatases in the glycocalyx ofStreptomyces aureofaciens

Electron-cytochemical stainings based on Gomori type lead precipitation techniques showed that both polyphosphates (by energy dispersive analysis of X-rays) and appropriate phosphatases were present in the capsular fibrils covering the mycelium ofStreptomyces aureofaciens, producing low quantities of chlortetracycline. In light of the known differences in polyphosphate contents and glycocalyx secretion in low- and high-producing strains, our results point to new aspects of compartmentation of actinomycetal cells with respect to secondary metabolism and to the role of microbial glycocalyx in the process.