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The type and level of resistance of Bac. licheniformis 1001 to the antibiotic produced by it, i. e. bacitracin and the effect of bacitracin and some other drugs on the culture variation with respect to the feature of the antibiotic production were studied. It was found that strain 1001 possessed an inducible resistance to its own antibiotic. In addition, a cross inducible resistance to bacitracin and penicillin was observed. The mutations of the resistance to bacitracin, as well as streptomycin, rifampicin and sulfaethidole had no effect on bacitracin production by strain 1001.

Recombinants between B. subtilis and B. licheniformis were prepared by fusion of the bacterial protoplasts. Genetically marked strains SB25 trp C hisH and 168 ade-met-leu- of B. subtilis and 1001 ura-thr- and 1001 met- of B. licheniformis were used as the parent strains. The recombinants were selected with the indirect method followed by analysis of their nutrient requirements and cultural and morphological features. All the hybrids acquired the specific properties of B. subtilis. Apparently, their formation was based on the whole chromosome of B. subtilis and recombination of separate fragments of B. licheniformis with it. Hybrids with prototrophic properties with respect to one, two or three markers of the initial strains were detected independent of the genotype of the B. subtilis parent strains. Moreover, the protoplast fusion resulted in formation of hybrids which were prototrophic with respect to the amino acid markers of B. subtilis and deficient with respect to homoserine and thiamine or only thiamine, whereas the initial strains were not auxotrophic with respect to homoserine and thiamine. Thi-Hom- and a number of the prototrophic recombinants were characterized by the capacity for increased synthesis of riboflavin lacking in the initial cultures. Homologous and heterologous transformation appeared to be possible in the recombinants of the Thi-Hom- phenotype, while transformation of the initial strain SB25 by the intergenocytic markers was possible in reciprocal crossings. It is concluded that contrary to transformation of isolated DNA, protoplast fusion may result in formation of interspecies recombinants of B. subtilis and B. licheniformis with respect to different operones of amino acid synthesis.

Production of bacitracin by Bac. licheniformis 1001 and its spontaneous autolysin resistant variants was studied. It was found that the antibiotic activity of some variants was 1.5--2 times higher than that of the initial strain. No differences in the activity of serine exoprotease in the initial strain and resistant variants were observed. The latter variants lost their resistance to autolysis in 2--3 subcultures on solid and liquid nutrient media. their antibiotic activity in these cases decreased to the control level. The study indicates that there is a phenomenologic relation between the autolytic and antibiotic activities of Bac. licheniformis. The nature of the relation is not known yet. Possibly, it is due to changes in the specific metabolic steps connected with regulation of bacitracin synthesis.

As a result of fusion of the protoplasts of B. subtilis and B. licheniformis the majority of the prototrophic hybrids, as well as the auxotrophs with the Thi-Hom-phenotype or the Thi-phenotype acquired capacity for over-production of riboflavin lacking in the initial parent strains. When grown on the minimal Spizizen medium with aeration at 37 degrees C the auxotrophic recombinants accumulated 10-60 micrograms/ml of riboflavin for 2 days, while the prototrophic recombinants accumulated up to 90 micrograms/ml of riboflavin. The respective figures for their cultivation in the sucrose fermentation medium were 100-330 and 600 micrograms/ml. For mapping 3 random variants of them with different phenotypes, i.e. SL-7 (prototroph), SL-15 Thi-and SL-52 Thi-Hom-were used. Localization of the mutation on the chromosome of B. subtilis was based on transformation experiments with three marker crossing, where hybrid strains SL-7, S1-15 and SL-52 were used as the DNA donor and strain lys-rib-of B. subtilis was used as the recipient. The analysis showed that the required mutation designated as R1 was localized on the chromosome of B. subtilis in the regulatory region rib0 of the riboflavin operon.

The bacitracin production levels in some auxotrophic mutants of a highly active strain 1001 and strain ATCC 10716 were studied. It was shown that auxotrophic mutations in the genomes of the above strains resulted in decreasing of bacitracin production. In double auxotrophic mutants the synthesis of bacitracin was sometimes at the level of the initial strain. It was found that various amino acids and bases added to the cultivation medium in concentrations of 1 to 10 mg/ml either increased or decreased the production of bacitracin. It is suggested that some amino acids may play the decisive role in the control of the bacitracin synthesis.

Analysis of phagolysates in production of fodder grisin provided determination of 3 serologically and morphologically different actinophages, 2 of which, i. e. Pg 81 and Pg 100 affected the sensitive culture as virulent phages and 1, i. e. Pg 2 affected it as a moderate phage. Investigation of the fermentation broth of strain 15 of Act. griseus did not confirm the supposition that this strain was polylysogenic and actinophages Pg 81 and Pg 100 were virulent mutants of moderate phages present in the cells. A phenomenon of limitation and modification controlled by the host cell was noted in the sensitive culture infected with actinophage Pg 81. The actinomycete mutants which did not limit but modified the phage were isolated. Under the effect of chemical mutagens the actinomycete mutants stable to virulent phages were selected. Treatment of the phage-stable mutants with ethylene imine and UV-light provided an increase in their antibiotic production levels by 75 to 80 per cent.