Broad-Host-Range Plasmid by Electroporation

Methylotrophic bacteria able to grow on one-carbon compounds, such as methanol and monomethylamine, as sole carbon sources have received considerable attention because of their potential commercial applications, including the production of single-cell protein, amino acids, pyrroloquinoline quinone, and poly-3-hydroxybutyrate. The utility of methylotrophs should be increased by the use of recombinant DNA technology. In genetic studies with methylotrophic bacteria as the host organisms, the conjugative transfer system has been used exclusively for introducing DNA molecules into the cells owing to the lack of an effective transformation system. However, this system is not only technically cumbersome and time-consuming; it is also limited in the number and type of plasmids used because the participation of plasmids carrying the gene transfer function is essential. The electroporation technique has been widely used to transfer DNA into mammalian cells (4), plant protoplasts (8), and yeast cells (11). Recently, this technique has also been applied to transforming a variety of prokaryotic cells with plasmid DNAs (5, 7, 16). However, electric transformation of methylotrophic bacteria has not yet been reported (6). In order to develop a transformation system for methylotrophic bacteria, we here used the electroporation technique with a pink-pigmented facultative methylotrophic bacterium, Methylobacterium extorquens NR-2, and a 21-kb broad-hostrange plasmid, pLA2917, as a model test system. The optimal electric conditions for transformation efficiency are described in this article.