p CGR 2 copy number depends on the par locus that forms a P ar C – P ar B – DNA partition complex in C orynebacterium glutamicum

Aims To characterize the par system of C orynebacterium glutamicum p CGR 2 and to manipulate the par components to effectively manipulate plasmid copy number. Methods and Results P ar B binds sequence specifically to centromere‐binding sites around the par AB operon and serves as an autorepressor. A small ORF ( orf4 , later named par C ) downstream of par AB encodes a protein with 23·7% sequence identity with P ar B . P ar B is also implicated in the repression of par C transcription. Nonetheless, this P ar C protein does not bind to centromere‐binding sites and is not essential for plasmid stability. Introduction of a frameshift mutation within P ar C implicated the protein in regulation of both par AB and par C . E lectrophoretic M obility S hift A ssay confirmed a previously unreported P ar C – P ar B – par S partition complex. P ar C also interacts directly with P ar B without the mediation of the centromere sites. Deletion of the par components resulted in different plasmid copy numbers. Conclusions A previously unreported P ar C – P ar B – par S partition complex is formed in pCGR 2, where interaction of P ar C with P ar B – par S may affect the level of repression by P ar B . Modifying the par components and antisense RNA enables manipulation of plasmid copy number to varying degrees. Significance and Impact of Study Genetically manipulating the par components, in combination with deactivation of antisense RNA , is a novel approach to artificially elevate plasmid copy number. This approach can be applied for development of new genetic engineering tools.