J. Koníček

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.

Production and characteristics of the exocellular polysaccharide in mutant strains ofXanthomonas fuscans

Production of the exocellular polysaccharide of the phytopathogenic bacteriumXanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes containing lactose. Six stablelac+ mutants were obtained after the treatment withN-methyl-N′-nitroso-N′-nitroguanidine. The mutants were compared with the parent strain. Morphological and cultivation characteristics, as well as production of the exooellular polysaccharide were compared. The production was found to be maximal during the stationary phase of growth in strains cultivated under submerged conditions. Gas chromatography revealed that the polysaccharide of the parent strain is formed by α- and β-D-glucose and α- and β-d-mannose with a small amount ofd-ribose and 6-deoxy-l-mannose. Composition of the polysaccharides produced by the mutant strains (lac+) does not qualitatively differ from that of the parent strain. However, they were found to contain a higher quantity ofd-mannose, which is favourable for their industrial utilization.

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 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.

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

Use of whey for production of exocellular polysaccharide by a mutant strain of Xanthomonas campestris

Growth and kinetics of the production of exocellular polysaccharide was studied in a mutant strain ofXanthomonas campestris lac+ during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1.4%. The results were comparable with those obtained during cultivation on a lactose medium.

Effect of Tween 80 and dimethyl sulfoxide on biosynthesis of L-lysine in regulatory mutants of Corynebacterium glutamicum.

By using dimethyl sulfoxide or Tween 80 (1 or 0.2 %), the production ofl-lysine was increased by 20–28 and 23–25%, respectively, in regulatory mutant strains ofCorynebacterium glutamicum. The stimulation observed is supposed to be caused by influencing cellular surface structures.

Genetic transfers in mycobacteria

Mycobacteria hold a special position among other bacterial genera, having a different composition of the cell wall, highly variable morphology of cells and colonies, specific growth properties, production of secondary metabolites etc. Mycobacteria are a group of microorganisms with a widerange of pathogenicity, varying from saprophytic to highly pathogenic species. Mycobacteria are a somewhat difficult model for studying genetic transfers, but they are important from the practical point of view as agents of serious human and animal diseases. In addition to slow growth, the main obstacle to genetic transfer studies in pathogenic mycobacteria is their cell wall. It is a barrier complicating the penetration of genetic material in both directions and its gentle disruption that would preserve the integrity of bacterial DNA is still a problem. Studies of genetic problems in mycobacteria started in the sixties when geneticists concentrated mainly on transformation, transduction, lysogeny and mutagenesis, in agreement with trends of work with other bacterial models. In spite of considerable effort knowledge of the genetics of mycobacteria is delayed as compared with other microbial models. However, even in mycobacteria a series of more or less successful attemps at genetic transfer between varieties of the same species or betwoen two representatives of different species have boon described. Nevertheless, moShods of genetic transfer have not yet been satisfactorily mastered and mutagenesis remains the main reliable means of at least a rough characterization of the myeobacterial genome. In the present communication we summarize the results obtained SO fa r .

Possibilities of the conjugation process in mycobacteria

Results obtained when studying conjugation in mycobacteria by means of different methods are summarized. The method of conjugation on surface of a solid complete medium was tested with different auxotrophic mutants of different strains ofMycobacterium smegmatis. It was not possible to obtain positive results even by means of the above method. This was probably due to unsuitability of the chosen strains ofMycobacterium smegmatis. Preparation of the donor strain by transfer of the F factor fromEscherichia coli F’ORF 1ade+ lac+ pro+ toMycobacterium phlei PA adeStmr by means of sexduction is described. Frequency of the phenotype PAade+ Stmr increased in the average by two and a half orders of magnitude with respect to the control, however, a further transfer from cultures of the cellsade+ Stmr to cells ade could not be demonstrated. Experiments aimed at transferring the R factor from strainsEscherichia coli K-12 toMycobacterium phlei were unsuccessful.