PARAMETERS AFFECTING PLASMID STABILITY IN BACILLUS-SUBTILIS

For the analysis of parameters affecting plasmid stability in Bacillus subtilis, we used a pUB110-derived shuttle plasmid containing direct and inverted nucleotide sequence repeats (DRs and IRs). Deletions of up to 6 kb were found to occur between DRs of 7 to 16 bp. IRs as small as 43 or 58 bp were shown to stimulate the formation of these deletions in their neighbourhood. However, these structural features (DRs and IRs) per se were not responsible for plasmid instability. The unstable recombinant plasmids, but not their deletion-carrying (DELTA) derivatives, were found to impair the growth of the host and to accumulate high amounts of linear plasmid multimers [high mol. wt. (hmw) DNA]. We propose that the accumulation of hmw DNA may be the major reason for the selective pressure against recombinant plasmids, and the enrichment of DELTA-plasmids. Host mutations and other parameters increasing the stability of recombinant plasmids in B. subtilis are described.