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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 1.1×10 5  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.

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