Role of Spontaneous Current Oscillations during High-Efficiency Electrotransformation of Thermophilic Anaerobes

摘要

Current oscillations at about 24 MHz were observed during electrotransformation (ET) of the thermophilic anaerobes Clostridium thermocellum ATCC 27405, C. thermocellum DSM 1313, and Thermoanaerobacterium saccharolyticum YS 485, using a pulse gated by a square signal generated by a custom generator. In experiments in which only the field strength was varied, all three of these strains resulted in a one-to-one correspondence between the appearance of current oscillations and successful ET. Oscillations accompanied ET of both C. thermocellum strains only at field strengths of ≥12 kV/cm, and ET was only observed above the same threshold. Similarly, for T. saccharolyticum, oscillations were only observed at field strengths of ≥10 kV/cm, and ET was only observed above the same threshold. 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. The maximum tested field strength, 25 kV/cm, resulted in the maximum measured transformation efficiency for all three strains. At this field strength, the efficiency of ET in the absence of oscillations was decreased compared to that observed in the presence of oscillations by 500-fold for C. thermocellum ATCC 27405, 2,500-fold for C. thermocellum DSM 1313, and 280-fold for T. saccharolyticum. Controls using the same apparatus with Escherichia coli cells or a resistor with a value representative of the direct current resistance of typical cell samples did not develop oscillations, and ET efficiencies obtained with E. coli were the same with or without the electrical filter included in the pulse generator circuit. The results are interpreted to indicate that spontaneously arising oscillations have a large beneficial effect on transformation efficiency in the system employed here and that the development of oscillations in this system is affected by the cell species present.