pCGR2 copy number depends on the par locus that forms a ParC-ParB-DNA partition complex in Corynebacterium glutamicum

Abstract

Aims: 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.

Keywords: Corynebacterium glutamicum; antisense RNA; pCGR2; partition; plasmid copy number.