Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum.
pCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72&emsp14;bp, CrrI; and 73&emsp14;bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem-loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.
Okibe
Naoko
N
Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
Suzuki
Nobuaki
N
Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
Inui
Masayuki
M
Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
Yukawa
Hideaki
H
Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D048230:Corynebacterium glutamicum / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial