Population heterogeneity of plasmid-bearing and plasmid-freeBacillus subtilis strains under different environmental conditions

The population heterogeneity of recombinant and plasmid-freeBacillus subtilis strains introduced into aquatic microcosms was studied. After introduction, the population of the plasmid-free strainB. subtilis 2335 in microcosms has long been represented by both vegetative cells and spores, whereas, already ten days after introduction, the population of the recombinant strainB. subtilis 2335/105 (Km^rInf⁺) was represented only by spores. The number of plasmid copies in the spore isolates of the recombinant strain was the same as before introduction, but the plasmid abundance in the vegetative isolates of this strain decreased. The isolates ofB. subtilis 2335/105 obtained from microcosms and the variants of this strain obtained by ten successive subcultures on M9 and 0. I× M9 media with and without kanamycin (Km) differed in the number of plasmid copies, Km resistance, and maximum biomass yield during batch cultivation. Irrespective of the presence of Km, more than 50% of the variants subcultured on M9 medium showed reduced plasmid abundance. At the same time, about 70% of the variants subcultured on 0.1 × M9 medium with Km and 90% of the variants subcultured on the same medium without Km retained the initial number of plasmid copies. The variants subcultured on media with Km retained the initial biomass level. In more than 70% of the variants isolated from media without Km, the biomass yield increased.