Electroporation of Pink-Pigmented methylotrophic bacteria

Electroporation conditions were determined for electroporating the broad-host-range plasmid pHX200V-47-ml (22.4 kb) into four pink-pigmented methylotrophic strains (Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1, Methylobacterium organophilum XX, and Pseudomonas sp. M27). For these methylotrophs, a high electric-field strength (15 kV/cm) and high DNA concentration (18.75 ng/~L competent cells) were necessary to produce plasmid-containing cells. The addition of 30% polyethylene glycol (PEG) to the electrocompetent cells resulted in a fiveto six-fold increase in electroporation efficiency in strains M. methylotrophus AS1, M. extorquens AM1, and M. organophilum XX, and also allowed successful electroporation of Pseudomonas sp. M27 (no electroporants were obtained without PEG). The maximum electroporation efficiencies for M. methylotrophus AS1, M. extorquens AM1, M. organophilum XX, and Pseudomonas sp. M27 were 1.31 • 10 3, 2.57 x 103, 5.25 • 10 3, and 1.2 • 104 electroporants/(~g DNA, respectively). This, along with our previous article (1), are the first reports of a plasmid being successfully electroporated into these strains. Index Entries: Electroporation; methylotrophs.