Conversion of Protoplasts of Colistin-producing Bacillus colistinus to L-forms and Bacillary Forms

Abstract

The conversion of Bacillus colistinus protoplasts to L-forms was stimulated by adding bovine serum albumin or heat-inactivated horse serum to the conversion medium. In RGB2 and R2 soft agar medium (0.5% agar), CaCl₂ as an osmotic stabilizer promoted the conversion of protoplasts to L-forms at a high frequency (13~83%), but not to bacillary forms, although the L-forms were nearly all disrupted 9 days after initiation of the culture. When protoplasts were plated on R2 soft agar medium containing 0.5 m succinate as another osmotic stabilizer, L-colonies were obtained at a frequency of .4.7 ~ 7.2% and the L-forms stably propagated. Electron microscopic observation showed L-forms having fragments of cell walls together with many wall-less L-forms. The L-forms could not revert to bacillary forms even 3 months after cultivation and remained as wall-less forms in repeated sub-cultures. However, protoplasts and L-forms plated on R hard agar medium (2.5% agarose) changed to bacillary forms. Bacillary conversion was enhanced when the concentration of succinate in the hard agar medium was lowered to 0.35 m.