High copy number mutants derived from Corynebacterium glutamicum cryptic plasmid pAM330 and copy number control

A high copy number mutant plasmid, designated pVC7H1, was isolated from an Escherichia coli–Corynebacterium glutamicum shuttle vector pVC7N derived from cryptic plasmid pAM330 that was originally found in Brevibacterium lactofermentum 2256 (formally C. glutamicum ATCC 13869). The copy number of pVC7N was estimated to be about 11 per chromosome, whereas pVC7H1 displayed a copy number of 112 per chromosome in C. glutamicum. The mutation (designated copA1) was in a region between long inverted repeats (designated the copA1 region) and was identified as a single base conversion of cytosine to adenine. By introduction of a cytosine to guanine mutation (designated copA2) at the same site as copA1, a further high copy number mutant (>300 copies of the plasmid per chromosome) was generated. Through genetic and RNA-Seq analyses of the copA1 region, it was determined that a small RNA (designated sRNA1) is produced from the upstream region of repA, a gene encoding a possible replication initiator protein, and sRNA1 is a possible regulator of the copy number of pAM330-replicon-contaning plasmids. Determination of the precise transcription start sites of sRNA1 and repA-mRNA suggested that sRNA1 could sequester a presumed ribosome binding site of repA-mRNA from ribosomes by an antisense RNA-mediated mechanism. Our data also indicate that the secondary-structure of sRNA1 is crucial for its function in plasmid copy number control.