A RAPID AND SIMPLE METHOD FOR BACILLUS-SUBTILIS TRANSFORMATION ON SOLID MEDIA

Cells of Bacillus subtilis strains 168 and W23 deprived of an amino ac id or a base on a given solid medium were found to develop competence. We describe a rapid and simple method of genetic transformation of th is organism consisting in spreading a sample containing 1 mu g DNA and 10(7) exponentially growing cells of an auxotrophic mutant onto plate s devoid of the required amino acid or base. After overnight incubatio n, about 100-200 prototrophic transformants per plate were obtained, i .e. a frequency of about 10(-5), as compared to 10(-4) routinely obtai ned by the method of transformation in liquid medium with frozen compe tent cells. Plasmids and other chromosomal or plasmid-borne markers, w hich cannot be directly selected for, were transferred by congression. The dependence of the transformation efficiency on cell density, medi um richness, incubation time and the nature of transforming DNA was in vestigated. We conclude that the development of competence accompanies amino acid or base starvation of cells under appropriate physiologica l conditions.