pCGR2 copy number depends on the par locus that forms a ParC–ParB–DNA partition complex in Corynebacterium glutamicum
Article first published online: 7 JUN 2013
© 2013 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 115, Issue 2, pages 495–508, August 2013
How to Cite
Okibe, N., Suzuki, N., Inui, M. and Yukawa, H. (2013), pCGR2 copy number depends on the par locus that forms a ParC–ParB–DNA partition complex in Corynebacterium glutamicum. Journal of Applied Microbiology, 115: 495–508. doi: 10.1111/jam.12257
- Issue published online: 17 JUL 2013
- Article first published online: 7 JUN 2013
- Accepted manuscript online: 18 MAY 2013 08:55AM EST
- Manuscript Accepted: 29 APR 2013
- Manuscript Revised: 12 APR 2013
- Manuscript Received: 19 FEB 2013
- New Energy and Industrial Technology Development Organization (NEDO)
- antisense RNA;
- Corynebacterium glutamicum ;
- plasmid copy number
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.
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.