Protoplast Transformation of Recalcitrant Alkaliphilic Bacillus sp. with Methylated Plasmid DNA and a Developed Hard Agar Regeneration Medium

Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×105 transformants per µg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.