Gene transfer in Gram-positive bacteria can be based on natural competence (in rare in­stances), conjugation, transformation of protoplasts, or, most commonly, electrotransfor­mation (ET) (58). Examples of gene transfer to Gram-positive obligate anaerobic ther — mophiles are limited to Thermoanaerobacterium species (59, 60) and a preliminary report of protoplast transformation of C. thermocellum, where the transformation efficiency was relatively low, e. g., ~103/^g ofDNA (61).

Tyurin and coworkers (58) conducted ET of several strains of C. thermocellum using plasmid pIKm1, developed by Mai and Wiegel (59, 60), as a transformation vector. A custom-built pulse generator was used to apply a square 10-ms pulse to an ET cuvette. Optimizations, including optimized periods of incubation both before and after electric pulse application, subculture in the presence ofisoniacin prior to electric pulse application, use of a custom-built cuvette embedded in an ice block during pulse application, and high (25-kV/cm) field strength, increased transformation efficiencies of C. thermocellum strain DSM 1313 from <1 to 2.2 x 105 transformants/^g of DNA. The protocol and pre­ferred conditions for strain DSM 1313 resulted in transformation efficiencies of 5.0 x 104 transformants/^g of DNA for C. thermocellum strain ATCC 27405 and approximately 1 x 103 transformants/^g of DNA for C. thermocellum strains DSM 4150 and 7072.

An interesting phenomenon was that current oscillations at about 24 MHz was observed during ET of Gram-positive bacteria C. thermocellum ATCC 27405, C. thermocellum DSM 1313, and T. saccharolyticum YS 485 using a pulse gated by a square signal generated by a custom generator (62). Oscillations were not observed for controls — including E. coli cells and a water blank. When a passive electrical filter consisting of an inductor and resistor in parallel was added to the system to prevent the development of oscillations, ET efficiencies were reduced dramatically for all three strains at all field strengths tested.