Electrotransformation of Clostridium thermosaccharolyticum

Abstract

Transformation of the thermophile Clostridium thermosaccharolyticum ATCC 31960 was achieved using plasmid pCTC1 and electroporation. Evidence supporting transformation was provided by Southern blots, detection of the plasmid in 10 out of 10 erythromycin-resistant clones, retransformation of E. coli and C. thermosaccharolyticum with plasmid DNA isolated from C. thermossccharolyticum, and a proportional relationship between the number of transformants and the amount of DNA added. Transformation efficiencies were very low for plasmid DNA prepared from E. coli (0.6 transformants mg-1 DNA), although somewhat higher for plasmid DNA prepared from C. thermosaccharolyticum (52 transformants mg-1 DNA). Transformation- dependent erythromycin resistance indicates that an adenosine methylase gene originating from Enterococcus faecalis, a mesophile, is expressed in C. thermosaccharolyricum. The plasmid pCTC1 appears to be replicated independently of the chromosome, as indicated by visualization of recovered plasmid on gels, and retransformation using recovered plasmid. pCTC1 is maintained in C. thermosaccharolyticum at both 45 and 60°C. Restriction analysis showed little or no rearrangement occurred upon passage through the thermophile.