Transformation of Bacillus subtilis in chocolate milk: evidence for low frequency of establishment of cells transformed under non-selective conditions

Abstract

Transformation of naturally competent Bacillus subtilis with plasmid was carried out in chocolate milk without antibiotics. Transformed cells were enumerated during the entire growth phase in chocolate milk. When DNA was added to aliquots of a batch culture after different times of incubation, transformation events were detected at all different growth stages. When DNA was added to a batch culture together with the inoculum, transformed cells were detected at the onset of exponential growth. However, apparently no or only limited growth of these transformed cells was observed. To clarify, whether the limitation of growth was due to suppression by non-transformed cells, different proportions of B. subtilis cells either carrying or not carrying the plasmid were mixed and inoculated into chocolate milk without antibiotic. Our results indicate that suppression appears to be of minor importance. Instead, plasmid-bearing cells appear to suffer from a prolonged lag-phase. However, the failure to exhibit significant growth of cells which had taken up the plasmid in chocolate milk appears to be due to failure of these cells to establish themselves as permanently transformed under non-selective conditions.