Plasmid instabilities of single and three-plasmid systems in Escherichia coli during continuous cultivation.

Plasmid instabilities in E. coli JM103 carrying three plasmids (pRK248cI, pMTC48, pEcoR4) and a single plasmid system (pTG206) for the production of fusion EcoRI (SPA::EcoRI) and catechol 2,3-dioxygenase, respectively, were investigated in continuous cultures under selective and non-selective conditions. In a three-plasmid system, pRK248cI was lost gradually together with pMTC48 from the host under non-selective conditions. The selective pressure against pRK248cI stabilized the pMTC48. This indicates that the loss of pMTC48 under non-selective conditions was caused by the loss of cI857 gene (coded by pRK248cI) which resulted in the overproduction of the toxic gene product (coded by pMTC48). In the case of single plasmid (pTG206) system, the plasmid lost from the host under non-selective conditions. This plasmid was stabilized in the host growing under selective conditions. During this period we obtained some ampicillin resistant colonies which gave low levels of enzyme activities compared to the normal plasmid bearing cells. Plasmid analysis from the above cells showed that the plasmid has undergone structural instability. Further, restriction analysis of this plasmid exhibited an additional PvuII site in a 0.9 kbp fragment that was integrated near the tet promoter which controls the expression of the xyl E gene, thereby resulting low levels of enzyme activities. Our results indicate that some of the IS elements which are present in the host chromosome were responsible for such instabilities to turn off the synthesis by inserting into the tet promoter region to lower the protein formation during the bioprocess.