Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824

The copy number and stability of several plasmid vectors in Clostridium acetobutylicum ATCC 824 were determined. The protocols were modified from the traditional ones to overcome the problems associated with unusual behavior of C. acetobutylicum cells on solid medium. The plasmid copy numbers of pSYL2, pFNK1, pFNK3, and pFNK5 in strain ATCC 824 were 14, 8, 6, and 6, respectively. pSYL2 and pFNK1 were segregationally stable, since the fractions of plasmid-carrying cells after 60 generations of growth without antibiotic (erythromycin) were 73% and 77%, respectively. Vector pFNK1 carrying fermentative genes was found to be rather unstable. The observed instability seemed to be due to the complex host-plasmid interactions by amplified expression of enzymes involved in the tightly regulated primary metabolism of C. acetobutylicum.