|Characterization of a 24-kb plasmid pCGR2 newly isolated from Corynebacterium glutamicum.
Appl. Microbiol. Biotechnol. 87: 1855-1866. 2010.
Y. Tsuchida, S. Kimura, N. Suzuki, M. Inui and H. Yukawa.
A 24-kb plasmid with 21 open reading frames (ORFs) was newly isolated from
Corynebacterium glutamicum ATCC 14997 and named pCGR2. Three of its ORFs were indispensable for stable
autonomous replication of pCGR2 in C. glutamicum: in the absence of selective pressure, deletion derivatives of pCGR2 containing the three ORFs showed stability in C. glutamicum for over 50 generations. The first of these ORFs encoded replicase repA whose gene product revealed high amino acid sequence similarity to corresponding
gene products of C. glutamicum pCG1-family plasmids in general, and to that of pTET3 plasmid repA in particular. The other two ORFs were located upstream of repA and exhibited high sequence similarity to pTET3 parA and parB, respectively. Interestingly, plasmids based on the pCGR2 were compatible
not only with those based on different family plasmids (pBL1, pCASE1) but
also with those based on pCG1-family plasmid. Plasmids comprising pCGR2
repA showed a copy number of four in C. glutamicum. The number increased to 240 upon introduction of a mutation within the repA origin of the putative promoter for counter-transcribed RNA. This 60-fold
increase in copy number should immensely contribute towards enhanced expression
of desired genes in C. glutamicum.