Electrotransformation of Clostridium acetobutylicum ATCC 824 using high-voltage radio frequency modulated square pulses

Abstract

Molecular biological improvement of industrial solventogenic clostridia could be enhanced by a higher efficiency of electrotransformation. In this research, we used a new approach to determine the frequency spontaneously generated by Clostridium acetobutylicum ATCC 824 cells during the application of a square high-voltage pulse. Once the frequency of 100 kHz was determined we transformed clostridial cells with pSOS84 plasmid DNA using radio-frequency modulated high-voltage square pulses (electric field strength 12 kVcm −1 ; pulse duration 22·5 ms; frequency of pulse modulation 100 kHz) to reach an efficiency exceeding 10 6 transformants mg −1 of plasmid DNA. We propose a possible role for cellular membrane structures in affecting the transformation yield.

Electrotransformation of Clostridium thermocellum

“…The development and application of gene transfer methods has been pursued most extensively among gram-positive obligate anaerobes in the case of Clostridium acetobutylicum (11,19,22,27,41) and is also well established among several other mesophilic noncellulolytic Clostridium species (20,27,47). An ET protocol developed for the cellulolytic mesophile Clostridium cellulolyticum (8,38) has been used to express pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis (6).…”

“…Described protocols for gene transfer in gram-positive bacteria are based on natural competence (in rare instances) (43), conjugation (conjugative mobilization) (1,8,24,43,45), transformation of partial or complete protoplasts (11,31,37,46), or, most commonly, electrotransformation (ET) (5,7,10,15,16,22,23,27,34,41,43,47). The presence of a restriction system(s) is an important property of recipient strains in the context of developing methods for gene transfer (9,25), and specific methylation has been required to protect transforming DNA from attack by restriction endonucleases and to achieve high transformation frequencies in several Clostridium species (4,8,18).…”

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

“…In addition, no current oscillations were detected when the cell sample was replaced with its resistive equivalent or with HPLC-grade water. Thus, we can conclude that the electrical characteristics of the cell samples play an important role in the appearance of In previous work using the same pulse generator employed herein, Tyurin et al (18) reported oscillations of C. acetobutylicum ATCC 824 at about 100 kHz. When the circuit was modified to apply a range of selected frequencies to the sample, the ET efficiency peaked at 100 kHz.…”

“…Many gram-positive bacteria have a complex intracellular morphology. In particular, bacteria of the genus Clostridium feature prespores in most vegetative cells, as well as additional intracellular membrane structures (2,18). E. coli, which does not form spores, has a relatively simple intracellular morphology in comparison.…”

“…Spontaneous (i.e., not applied externally) current oscillations at much lower frequencies have been observed in prior studies using the same circuit and cuvette design, with ϳ100-kHz oscillations observed for Clostrid- ium acetobutylicum ATCC 824 (18) and ϳ17-kHz oscillations observed for Corynebacterium glutamicum ATCC 13032 (19). Externally applied oscillatory electrical pulses were shown to be effective for ET of C. acetobutylicum, with the ET efficiency being a strong function of the frequency (18). Externally applied oscillatory pulses have also been used for ET of other microorganisms (14) and mammalian cells (3,23).…”