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

Abstract

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.

Development of pyrF- Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant

“…In contrast to the original protocol (37,38), the generation of competent cells has been simplified such that the isoniacin incubation, cellobiose solution wash, and custom-built electroporation cuvettes are no longer required. Best results are obtained with a custom pulse generator, but success can be achieved with a commercial device (the latter being the scope of a separate study).…”

“…The last two organisms have either an unusually high transformation frequency or feasibility for acquiring DNA from E. coli via conjugation, allowing the use of suicide plasmids (1,16,19). By comparison, the recently reported method of C. thermocellum transformation consists of a complex and cumbersome electroporation protocol using a custom pulse delivery system (37,38). In our hands, efficiency of the C. thermocellum electrotransformation system does not compare with that of typical model organisms and does not enable the use of nonreplicating plasmids as a means of gene manipulation.…”

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

“…Transformation of T. saccharolyticum was performed as previously described (14,16) with modifications during selection on erythromycin. Cells transformed with pSGD8E were allowed to recover at 48°C for 4 h and subsequently plated on solid medium at pH 6.0 containing erythromycin at 5 g/ml and incubated at 48°C for 4 days.…”

“…Extensive efforts using classical mutagenesis techniques to obtain stable strains exhibiting high-ethanol yields over a range of conditions have not been successful (13). Genetic systems suitable for engineering thermophiles have long limited strain development, but have started to emerge (14)(15)(16) along with the first reports of metabolic engineering in thermophilic, saccharolytic hosts (17,18). Here, we report engineering T. saccharolyticum JW/SL-YS485 to produce ethanol as the only significant organic product.…”

Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

“…Genetic engineering is likely necessary for commercial application of Thermanaerobacter or Thermoanaerobacterium species (26,27,44). As genetic systems for these bacteria have emerged (28,45), increased product yields have been demonstrated by gene knockout of L-lactate dehydrogenase (9,14), phosphotransacetylase and acetate kinase (40), and hydrogenase (39). Despite this recent progress, genetic transformation is still considered the greatest barrier for engineering these organisms (44).…”