Bacillus subtilis 168 genetic transformation mediated by outgrowing spores: necessity for cell contact

Transforming activity released in sequential genetic order during the first synchronous cycle of DNA replication during outgrowth of spores of Bacillus subtilis 168 was investigated. A transformation assay was used consisting of outgrowing spores as DNA donors and multiply marked competent cells as recipients. DNA synthesis inhibitors known to stop DNA release were used during and subsequent to DNA transfer to recipient cells. The released DNA sedimented with the outgrowing cells after low-speed centrifugation, and it was discovered that markers released both early and late were resistant to up to 500 microgram of deoxyribonuclease per ml under conditions in which the transforming capacity of purified DNA was eliminated by 5 microgram of the nuclease per ml. Inaccessibility to deoxyribonuclease was increased and maintained during the transformation event while detergents and proteolytic attack did not expose the released chromosome to nuclease action. The results indicate that tight physical contact between outgrowing spores and competent cells is required for transformation in this system.