V. Rytíř

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.

The mutation range ofBrevibacterium sp. M27

The mutation range was studied inBrevibacterium sp. M27 after UV irradiation and after treatment with N-methyl-N’-nitro-N-nitrosoguanidine. The induction of auxotrophic mutants and mutants resistant to streptomycin and tetracycline was investigated. A collection of auxotrophic mutants for the studies of genetic transfer in this model was prepared.

Genetic transformation and transfection ofBacillus subtilis spheroplasts

Genetic transformation and transfection of lysozyme-treatedBacillus subtilis spheroplasts 168M ind occurs only if they are stabilized with 0.5m phosphate buffer and not if they are stabilized with 0.5m sucrose. Spheroplasts prepared from maximally competent cells give maximum transformation and transfection results. The results indicate that the DNA receptors must also be intact in the spheroplasts.

Modification of the cell wall inBrevibacterium sp. M 27

Osmotically fragile cells ofBrevibacterium sp. M 27 were obtained after treatment with lysozyme and penicillin. These forms were detected by optical and electron microscopy.

A study on the effect of certain compounds during elimination of plasmids inEscherichia coli

Of several chemicals tested on the elimination of plasmids fromEscherichia coli K-12, the compound designated ICR-170 was most effective, applied at 100 μg/ml, the effect being comparable to that of acriflavin. It had no effect on the elimination of the R1 plasmid fromEscherichia coli JC 5455.

Genetic transfers in Brevibacterium sp.

A positive genetic transfer by protoplast fusion was obtained in auxotrophic mutantsBrevibacterium sp. M27his andBrevibacterium sp. M27arg. Transformation and protoplast fusion with liposomes (as genetic transfers in intact cells and their protoplasts by both the chromosomal and plasmid DNA) did not lead to transfer of the markers followed.

The use of vancomycin for the isolation of auxotrophic mutants inMycobacterium smegmatis

A method using vancomycin for the accumulation of auxotrophic mutants ofMycobacterium smegmatis M54/81 induced by N-methyl-N′-nitro-N-nitrosoguanidine was developed. As compared with the simple replication technique the yield of auxotrophic mutants was twenty-fold.

The attempt to transform streptomycin-resistance inMycobacterium phlei

Acid-fast and non-acid-fast strains ofMycobacterium phlei were used for the induction of streptomycin-resistance by isolated DNA. Biological activity of a transforming principle isolated from the cells homogenized in a Hughes press was confirmed. No reproducible positive results were obtained under given experimental conditions. Possible reasons for the lack of positive results are discussed.

Optimal conditions for induction of certain mutation types inBrevibacterium flavum by N-Methyl-N’-nitro-N-nitrosoguanidine

Conditions under which it is possible to induce auxotrophic mutants and DL-selenomethionine-resistant mutants inB. flavum by N-methyl-N’-nitro-N-nitrosoguanidine were determined. The yield of auxotrophic mutants was increased to 3% during mutagenesis in the first stage and to 1.5% in the second stage when using the enrichment-selective method with vancomycin. The optimal vancomycin concentration for inactivation of prototrophic cells growing in a minimal medium was 200 mg/L and the optimal time of treatment was 8 h. When testing the effect of three amino acid analogues (dl-ethionine,dl-selenomethionine and L-methionine sulfoximine) it was found thatB. flavum is sensitive todl-selenomethionine present in the minimal cultivation medium. Mutants resistant to 1 mg/mL of selenomethionine were isolated. Both isotope studies and measurement of growth indicate thatdl-ethionine also entersB. flavum cells, although its competition with endogenously synthesized methionine is not significant.

Induction of mutants resistant to antibiotics in Brevibacterium flavum.

The optimum eonditions for the induction of mutants resistant to antibiotics inBrevibacterium flavum ATCC 14067 were determined. UV irradiation at the energy fluence of 6.5 kJ/m2 and N-methyl-N’-nitro-N-nitrosoguanidine (1 mg/mL) at pH 6.0 were used for the induction of mutants. Mutant strains resistant to rifampicin, oleandomycin, streptomycin and erythromycin were prepared.