Expression and maintenance of plasmid resistance in regenerating protoplasts of Bacillus subtilis

Expression of plasmid-encoded resistances in regenerating protoplasts of Bacillus subtilis occurs only after wall synthesis has been resumed. This is observed for protoplasts obtained from cells already containing plasmids (pC194, pT127, pK545) or for plasmid-bearing cells coming from a PEG-mediated transformation. Recovery of expression needs a 2-h incubation of protoplasts, previously washed to get rid of their lysozyme content, in rich hypertonic medium (SMMP). A longer incubation (24-h) results in the obtention of regenerants; however, most of them have lost their resistant phenotype in contrast to those obtained from the usual solid regeneration plates. This finding suggests either a high curing effect or some kind of gene inactivation phenomenon. Discussion is focused on the critical points that have to be considered when polyethylenglycol-mediated transformation of protoplasts is applied to recombinant DNA technology.