Genetic analysis of Bacillus subtilis stable L-forms obtained via long-term cultivation

Various bacteria can change to a spherical cell-wall-deficient state, called L-from, in the presence of antibiotics that inhibit cell wall synthesis. L-forms are classified into two types: unstable and stable L-forms. Unstable L-forms revert to a normal walled state in the absence of antibiotics, while stable L-forms remain in their wall-deficient state. The conversion from unstable to stable L-forms has been often observed during long-term cultivation. However, the genetic cause for this conversion is not yet fully understood. Here, we obtained stable Bacillus subtilis L-form strains from unstable L-form strains via three independent long-term culturing experiments. The whole genome sequencing of the long-cultured strains identified many mutations, and some mutations were commonly found in all three long-cultured strains. The knockout strain of one of the commonly mutated genes, tagF, in the ancestral strain lost the ability to revert to walled state (rod shape), supporting that eliminating the function of tagF gene is one of the possible methods to convert unstable L-forms to a stable state.