To characterize the par system of Corynebacterium glutamicum pCGR2 and to manipulate the par components to effectively manipulate plasmid copy number.Methods and Results
ParB binds sequence specifically to centromere-binding sites around the parAB operon and serves as an autorepressor. A small ORF (orf4, later named parC) downstream of parAB encodes a protein with 23·7% sequence identity with ParB. ParB is also implicated in the repression of parC transcription. Nonetheless, this ParC protein does not bind to centromere-binding sites and is not essential for plasmid stability. Introduction of a frameshift mutation within ParC implicated the protein in regulation of both parAB and parC. Electrophoretic Mobility Shift Assay confirmed a previously unreported ParC–ParB–parS partition complex. ParC also interacts directly with ParB without the mediation of the centromere sites. Deletion of the par components resulted in different plasmid copy numbers.Conclusions
A previously unreported ParC–ParB–parS partition complex is formed in pCGR2, where interaction of ParC with ParB–parS may affect the level of repression by ParB. 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.