THE EXPRESSION OF A PLASMID-SPECIFIED EXPORTED PROTEIN CAUSES STRUCTURAL PLASMID INSTABILITY IN BACILLUS-SUBTILIS

The rolling-circle plasmid pGP1 was used to study the effects of the e xpression of a plasmid-specified exported protein on structural plasmi d stability in Bacillus subtilis. pGP1 contains a fusion between the B acillus licheniformis penP gene, encoding a C-terminally truncated pen icillinase, and the Escherichia coli beta-galactosidase (lacZ) gene. T wo processes affected the accumulation of pGP1 variants with deletions in the penP-lacZ region. First, divergent transcription from genes up stream of penP-lacZ increased pGP1 deletion frequencies up to about 10 -fold. Second, the removal of the PenP signal peptide resulted in comp letely stable plasmids, indicating that the entry of the PenP fragment into the protein export pathway is an important factor in the instabi lity of pGP1. On the basis of these results, we propose a model in whi ch the temporary anchoring of the plasmid to the membrane through the cotranscriptional and cotranslational entry of PenP into the protein e xport pathway creates domains of local hypersupercoiling, which we ass ume to be targets for deletion formation.