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 bp, CrrI; and 73 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.
The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrAB Ent genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28 %) isolates were positive for ccrAB Ent genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrA Ent probe (n=76) and partial DNA sequencing of ccrA Ent and ccrB Ent genes (n=38). ccrAB Ent genes were present in Enterococcus faecium (58/216, 27 %), Enterococcus durans (31/38, 82 %), Enterococcus hirae (27/52, 50 %), Enterococcus casseliflavus (1/4, 25 %) and Enterococcus gallinarum (1/2, 50 %). In the eight other species tested, including Enterococcus faecalis (n=94), ccrAB Ent genes were not found. Thirty-eight sequenced ccrAB Ent genes from five different enterococcal species showed 94–100 % nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrAB Ent flanking chromosomal genes. Expression analysis of ccrAB Ent genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrAB Ent mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrAB Ent genes were detected in both hospital-related (10/29, 34 %) and non-hospital (4/10, 40 %) strains of E. faecium. Various sequence types were represented by both ccrAB Ent positive and negative isolates, suggesting acquisition or loss of ccrAB Ent in E. faecium. In summary, ccrAB Ent genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups.