NATURAL AND ELECTROPORATION-MEDIATED TRANSFORMATION OF METHANOCOCCUS-VOLTAE PROTOPLASTS

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per mu g of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per mu g of vector DNA. However, a 380-fold improvement (3,417 transformants per mu g of DNA) over the frequency of natural transformation with whole cells aas achieved by electroporation of protoplasts,vith linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.