I. Málek

The study of mutagenesis inMycobacterium phlei

Results obtained when studying mutagenesis inMycobacterium phlei are summarized in this work. It was the aim of this paper to obtain an overall summary of mutation properties of this model in the selected genetic markers. Therefore, auxotrophic mutants, STM and INH resistant mutants and mutants with changed pigmentation induced by UV-radiation, ethyl methanesulfonate, nitrosoguanidine, nitrous acid, hydroxylamine and acriflavine were evaluated both qualitatively and quantitatively. Oceasional changes of morphology of rods and colonies and inactivating effects of the used mutagens were also considered.

The mutagenic effect of nitrosoguanidine onMycobacterium phlei PA

The effect of nitrosoguanidine on the induction of three types of mutagenic changes inMycobacterium phlei PA was studied. The mutagenic changes included: change of prototrophy to auxotrophy, conversion of sensitivity to isoniazide to resistańce and sensitivity to streptomycin to resistance. The induction of auxotrophic mutants was successful especially when using NTG at a concentration of 1000 μg/ml. A total of 100 auxotrophs was obtained out of which only 13 were sufficiently stable to be used in further studies. Amino acid requirements especially the glycine−(serine−) type characterized more than half of all auxotrophic mutants obtained. A group of mutants requiring purines also included a high number of mutants. A considerable spontaneous reversion frequency was found in both groups of auxotrophs. The kinetics of the induction of INH-resistant mutants by nitrosoguanidine at a concentration of 1000 μg/ml was studied and a high induction of these mutants, particularly at high lethal effect of the mutagen found. The mutability of the STMr marker was relatively low in the present model microorganism as compared with the two markers mentioned earlier.

Metabolites ofStreptomyces noursei

Production of cycloheximide and fungicidin by the producing strainStreptomyces noursei 52/152 was shown to be coupled. Cycloheximide added to the culture medium increased the production of fungicidin, addition of fungicidin caused an increase of the production of cycloheximide. Antibiotic compounds added were degraded in the course of cultivation approximately to the same amount as if produced in control cultures. Cycloheximide added to the culture medium was not transformed to actiphenol. It was suggested that the synthesis of secondary metabolites ofStreptomyces noursei is controlled by mechanisms analogous to repression or allosteric inhibition.AbstractБыло доказано, что образование циклогексимида и фунгицидина у штамма Str. noursei 52/152 взаимно связаны. Прибавление к культивационной среде циклогексимида вызывало повышение продукции фунгицидина; прибавление фунгицидина повышало продукцию циклогексимида. Прибавляемые антибиотические вещества в течение культивирования деградируются приблизительно до такого же количества, какое образуется в контрольных культурах. Циклогексимид, внесенный в культивационную среду, не превращался в актифенол. —Высказывается предположение, что синтез вторичных метаболитов у Str. noursei управляется механизмами, аналогичными подавлению или аллостерическому угнетению.

Theory of semicontinuous and continuous cultivation applied to the yeastTorula utilis

SummaryMathematical relationships between the dilution and growth rates were derived for semicontinuous cultivation of microorganisms. It was shown that the fully continuous cultivation process represents a special limiting case of semicontinuous processes and makes it possible to utilize the maximum amount of substrate per unit time and fermentor volume.In studying the optimum conditions of reproduction of the yeastTorula utilis it was shown experimentally that the continuous process is, at the same time, the most economical form of fermentation making the full utilization of the substrate and the capacity of the apparatus possible.It was demonstrated that in cases when the yeast utilizes a single source of carbon or where different sources of carbon are utilized simultaneously and at equal rates, a single-stage process of fermentation is more economical than a multi-stage one.AbstractБыли выведены математические зависимости между скоростью разведения и скоростью роста в условиях полунепрерывной культивации микроорганизмов. Было доказано, что вполне непрерывный способ культивации представляет особыи—крайний—случай полунепрерывных процессов и позволяет обработку максимального количества субстрата на единицу времени и объема ферментора.При изучении оптимальных условий размножения дрожжей Torula utilis было экспериментально доказано, что непрерывный процесс представляет одновременно наиболее удобный способ осуществления ферментации, позволяющий полное использование субстрата и мощности оборудования.Было доказано, что в случаях, когда дрожжи используют один источник углерода или же различные истичники углерода одновременно и с одинаковой скоростью, одноступенчатый способ ферментации более выгоден, чем многоступенчатый процесс.

The mutagenic effect of ethyl methanesulfonate on a non-acid-fast strain ofMycobacterium phlei

Ethyl methanesulfonate was used for the induction of three types of mutants in a non-acidfast strain ofMycobacterium phlei. A total of 20 auxotrophie mutants was isolated. The mutants were isolated mostly when using doses yielding higher survival of the cells of the basic suspension. The auxotrophic mutants isolated required mostly amino acids, two mutants required purines and three mutants required vitamins. By determining the frequency of spontaneous reversions, it was found that 9 auxotrophic mutants could be used for further genetic studies. These included the following phenotypes: isoleucine−, leucine−, lysine−, nicotinic acid−, pyridoxine−, and xanthine−. Seven scotochromogenic mutants were isolated after ethyl methanesulfonate treatment. One was ochre, the remaining six were orange. Six achromogenic mutants were detected. Spontaneous auxotrophic mutants, scotochromogenic and achromogenic mutants were not isolated. The treatment with 0.2m ethyl methanesulfonate resulted in an increase in the frequency of STM-resistant mutants, the maximum phenotypic expression taking place after 72 hours cultivation in a liquid medium without the drug. The frequency of induced STM-resistant mutants varied within the range of 8.6.10−5–1.0.10−4 as compared with the frequency of spontaneous mutants 5.8.10−6–8.8.10−6.

The induction of pigmentation change in a non-acid-fast strain ofMycobacterium phlei by ultraviolet radiation

Five scotochromogenic mutants and 11 achromogenic mutants were induced by UV irradiation of the non-acid-fast photochromogenic PN strain ofMycobacterium phlei. Spontaneous scotochromogenic and achromogenic mutants were not obtained. Colonies of the scotochromogenic mutants are orange, except for one mutant which is ochre. Three mutants are resistant to STM. Out of 11 achromogenic mutants 3 were induced by UV treatment of the original photochromogenic strain, 8 were prepared from the scotochromogenic mutant. No significant differences in the sensitivity to UV rays were found among the scotochromogenic mutant, achromogenic mutant and the photochromogenic PN strain ofMycobacterium phlei under the given experimental conditions. Scotochromogenic mutants and most achromogenic mutants are stable and suitable for further genetic investigation. Pigmentation changes can be used as genetic marker in mutation studies.

The mutagenic effect of hydroxylamine onMycobacterium phlei strain PA

The mutagenic effect of 0.05m and 1m HA onMycobacterium phlei PA was investigated. To establish the mutagenic effect the inactivating effect was studied under the same experimental conditions. Hydroxylamine at a higher concentration (1m) exhibited relatively high mutagenic effect. This was indicated by about 100-fold and 10-fold higher frequency of INHr and STMr mutants, respectively (as compared with spontaneous mutations) and induction of auxotrophic mutants. On the other hand, the mutagenic effect of 0.05m hydroxylamine was low under the same experimental conditions.The inactivating effect of a higher HA concentration (1m under given experimental conditions) was considerably higher when using the given model microorganism than that of the lower one (0.05m under the same experimental conditions). This finding does not agree with literature data obtained in other experimental models.

The comparison of a new mutant ofMycobacterium phlei with some strains of rapidly growing mycobacteria

A red, scotochromogenic mutant of the non-acid-fast strain ofMycobacterium phlei was obtained. Mutual relationship between this mutant and rapidly growing mycobacteria was studied using complex morphological, cultivation, biochemical and serological analyses as well as determination of the base composition in DNA. Taxonomical aspects of individual analyses are discussed.

Metabolites of Streptomyces noursei IV. Formation of secondary metabolites by producing mutants.

The production variability of secondary metabolites was studied after treatment with mutagenic factors. 852 isolates were examined, out of which 308 after X-irradiation, 277 after u. v.—irradiation and 267 after treatment with nitrogen mustard. According to the production of individual secondary metabolites examined it was possible to divide the strains obtained into 4 groups.AbstractИзучалась изменчивость продукции вторичных метаболитов у Streptomyces noursei после применения мутагенных факторов. Были подвергнуты оценке 852 изолята, в том числе 308 изолятов после Х-облучения, 277—после УФ-облучения и 267 изолятов—после применения азотистого иприта. С точки зрения продукции отдельных исследуемых вторичных метаболитов можно было разделить полученные штаммы на 4 группы.

The use of nitrosoguanidine in the study of mutagenesis in a non-acid-fast strain ofMycobacterium phlei

N-methyl-N-nitroso-N′-nitroguanidine was used for the induction of two types of mutations in the PN strain ofMycobacterium phlei. Nineteen auxotrophic, 136 scotochromogenic and 50 achromogenic mutants were isolated after of treatment with nitrosoguanidine at a concentration of 1,000 μg/ml. Auxotrophic mutants required primarily amino acids and vitamins and half of them may be used for further genetic work due to their low frequency of spontaneous reversions. Colonies of scotochromogenic mutants were orange with the exception of one, which was strawberry red. Most scotochromogenic mutants were detected on a streptomycin containing medium. Roughly two thirds of the scotochromogenic mutants and one half of achromogenic mutants did not revert to the original photochromogenic character.