The replication map of the chromosome ofMycobacterium phlei

It was the aim of the present work to construct the replication map of the chromosome ofMycobacterium phlei. The method of mutagenesis of the replication point by N-methyl-N-nitroso-N’-nitroguanidine in synchronously dividing populations and the method of analysis of gene frequency were applied. The order of replication of 19 genes on the chromosome was determined by means of induction of back mutations and forward mutations in auxotrophic mutants PAleu and PAmet and in double auxotrophic mutants with methionine as a reference marker.     

Electronmicroscopic comparison of the acid-fast and non-acid-fast strain ofMycobacterium phlei

Ultrastructure of the acid-fast PA strain ofMycobacterium phlei and its non-acid-fast PN mutant was studied in an electron microscope. Results of electronmicroscopic studies were supplemented with observation under an optical microscope, cultivation and chemical analysis of the wall mucopeptide. The effect of lysozyme and glycine on ultrastructure of cells of these strains was also followed. Electron microscopy revealed main differences in thickness and structure of cell walls, in intracellular membraneous system and in shape of rods of the studied strains.     

Comparison of pigments produced by several strains ofMycobacterium phlei

Pigments of three genetically closely related strains ofMycobacterium phlei were studied. It was found that individual strains produced various quantities of coloured metabolites when cultivated in the dark. They differ from each other also in the quality of the produced pigments. Extracted pigments were separated by thin-layer chromatography and identified as carotenoids. High quantities of lycopene were found in a red non-acid-fast mutant. Biogenetic aspects of the pigments found in the studied strains ofMycobacterium phlei are discussed.     

Mutagenic effect of ultraviolet radiation on the non-acid-fast strain ofMycobacterium phlei

The mutability of the PN strain ofMycobacterium phlei was examined after induction of auxotrophic mutants and of STM and VM-resistant mutants, by UV irradiation. A total of 30 auxotrophic mutants were isolated, most of them amino acid-dependent five purine-dependent, and one uracil-dependent. To induce the mutants higher UV doses had to be used so that the survival of cells in the original suspension would not exceed a few per cent. For further genetic work use can be made of 8 auxotrophic mutants (PN try^?ura^?, PN arg^?ura^?, PN ileu^?val^?, PN ileu^?, PN leu^?, PN lys^?, PN lys^?-VM^r, PN val^?), these showing a low frequency of spontaneous reversions. No spontaneous auxotrophic mutants have been found. The frequency of STM and VM-resistant mutants is increased upon UV irradiation, a post-irradiation incubation in a liquid medium without the drug being essential for their phenotypic expression. The highest increase of the number of these mutants is attained after 48 h of post-irradiation incubation and it has been found that, within a certain experimental scatter, the same frequency increase is found on using a complete or a minimal liquid medium. The frequency of spontaneous STM-resistant mutants lies within 5.8×10^?6–8.8×10^?6, of those VM-resistant between 3.1×10^?5 and 4.1×10^?5. The highest frequency of induced STM-resistant mutants lies between 3.0×10^?5 and 9.3×10^?5 and of VM-resistant mutants between 1.1×10^?4 and 2.2×10^?4     

Carotenoids synthesized by UV-induced mutants of a non-acid-fast strain ofMycobacterium phlei

The composition of the cell pigment of three mutants (PN₁, PN₂, PN₃) of a non-acid-fast strain ofMycobacterium phlei (PN) induced with UV-radiation was investigated. It was found that the mutants contain carotenes typical for the original parent strain, but that the quantity of the colourless precursor, phytoene, is substantially lower, and, on the contrary, the amount of lycopene is increased (especially in mutants PN₂ and PN₃). In addition, all mutants synthesized xanthins in considerable quantities: the PN₁ mutant above all with the β-carotene carbon skeleton, whereas for the PN₂ and PN₃ mutants xanthins with the lycopene structure were typical. The hit of the genome of the original strain caused by the mutagen is thus manifested even in the synthesis of secondary metabolites.     

Mapping of the chromosome ofMycobacterium phlei by means of mutagenesis of the replication point

The aim of the present work was to construct a replication map of the chromosome ofMycobacterium phlei. The method of mutagenesis of the replication point by means of nitrosoguanidine was applied to synchronously multiplying populations. Back mutations and forward mutations were induced m auxotrophic mutants PAmet and PAleu as well as in double auxotrophic mutants with methlomne as the reference marker and the following order of replication of eleven genes on the chromosome was thus established:leu-Eth, Res-Stm, Oyk-pur-met, arg, Cyk-Bac-inl     

The effect of daylight on the biosynthesis of carotenoids by non-acid-fast strains ofMycobacterium phlei

The changes of the cell pigment composition of two non-acid-fast forms ofMycobacterium phlei (PN, PNR), caused by exposure to daylight, were studied. Though during cultivation in the dark carotenes were mostly produced, cultures exposed to the light mostly formed their oxygen-containing derivatives. Xanthins derived from lycopene, the main carotenoid formed by the PNR strain, were obtained from the mycelium of this mycobacterium after it was exposed to daylight. The exposure of the PN culture to daylight results in the formation of xanthins and in the stimulation of lycopene synthesis.     

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 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.     

The occurrence of geometrical isomers of lycopene in the modification of the UV-induced mutant ofMycobacterium phlei

During transfers of the UV-induced mutant of the non-acid-fast strain ofMycobacterium phlei (PN₃), its somewhat differently orange-red coloured modification was found. The analysis of the cell pigment showed that unlike the PN₃ mutant which synthesizes the whole range of carotenes and xanthins, in the modification approximately half of the total amount of carotenoids is represented by chromatographically heterogeneous lycopene, the second half by γ-carotene. The further study of the lycopene fractions led to the conclusion that their difference is caused by the geometrical isomerism of the double bonds. The origin of the describedcis-trans-isomers of lycopene is discussed.     

The induction of mutants in a non-acid-fast strain ofMycobacterium phlei with nitrous acid

The inactivation and mutagenic effets of nitrous acid on a non-acid-fast strain ofMycobacterium phlei were studied. It was found that 0.017m NaNO₂ at pH 4.4 may be used for the induction of auxotrophic mutants, scotochromogenic and achromogenic mutants and STM-resistant mutants. Three doubly auxotrophic mutants, three mutants requiring amino acids and three mutants depending on vitamins were obtained. One mutant was not classified. Eighteen scotochromogenic mutants were isolated, seventeen of them were orange. Only ten achromogenic mutants were isolated. Twelve scotochromogenic and eight achromogenic mutants could be used in further genetic studies as they did not revert spontaneously to photochromogeny. Six auxotrophic mutants could be used due to their low frequency of spontaneous reversions. The frequency of STM-resistant mutants increased on an average seven-fold after the mutagenic treatment as compared with the spontaneous frequency.     

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 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.     

Relationship between the culture medium and the fatty acid composition of diphtheria and non-pathogenic corynebacteria]

The gasochromatic method was applied to the study of the cellular fatty acids composition in diphtheria and nonpathogenic corynebacteria (diphtheroids and psendo diptheria bacillus). Marked differences in the content of unsaturated fatty acids were revealed in them. Thus, palmito leic acid served the preponderant unsaturated fatty acid in Corynebacteria diphtheriae, and unsaturated fatty acids with 18 carbon atoms (octadeconoic and linoleic)--in nonpathogenic corynebacteria. The mentioned changes permit use this sign as differential. When grown on Loeffler's medium all the corynebacteria under study had a similar fatty acid composition characterized by the prevalence of unsaturated fatty acids with 18 carbon atoms. On the basis of studying the fatty acid spectrum of the nutrient media used it is supposed that one of the factors determining the revealed dependence of the corynebacterial fatty acid composition on the culture medium was the fatty acid composition of the latter.     

Relationship between bovine serum composition and its capacity to stimulate cell culture growth

A relationship has been studied between the growth-stimulating activity and chemical composition of bovine serum (BS) that is widely used for cell culture growth and virus replication. A direct correlation was shown between the growth-promoting activity of bovine serum, manifested on the model of chick embryo fibroblasts, and the contents of phospholipids, total lipids, proteins, and calcium in this serum. A significant inverse association has also been revealed between the growth-promoting activity of the serum and the amounts of cholesterol and glucose in this.     

Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.