NATURAL AND ELECTROPORATION-MEDIATED TRANSFORMATION OF METHANOCOCCUS-VOLTAE PROTOPLASTS

The lack of high-efficiency transformation systems has severely impede d genetic research on methanogenic members of the kingdom Archaeobacte ria. By using protoplasts of Methanococcus voltae and an integration v ector, Mip1, previously shown to impart puromycin resistance, we obtai ned natural transformation frequencies that were about 80-fold higher (705 transformants per mu g of transforming DNA) than that reported wi th whole cells. Electroporation-mediated transformation of M. voltae p rotoplasts 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 achi eved by electroporation of protoplasts,vith linearized DNA. This gener al approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to pr otoplasts as a result of their tendency to lyse in hypotonic solutions .