Molecular analysis of the replication region of the pCIZ2 plasmid from the multiple bacteriocin producer strain Enterococcus faecium L50

The sequence analysis of the 7383 bp plasmid pCIZ2 from Enterococcus faecium L50 enabled the identification of a DNA region involved in its replication. The structural organization of the pCIZ2 replication region is highly similar to those of well-known theta-replicating plasmids. It contains an untranslated region, the putative replication origin (ori), constituted by two sets of direct repeats of 12 and 22 bp (iterons), and followed by three open-reading frames (orf8 to orf10). orf8 encodes the replication initiation protein (RepE). The transcriptional start site of the replication locus was identified 13 nucleotides upstream of the repE start codon. A two-dimensional agarose gel electrophoresis analysis revealed pCIZ2 intermediates profile typical of the theta-type replication mechanism. Subcloning of different DNA fragments of the pCIZ2 replication region in Escherichia coli and, subsequently, in the plasmidless E. faecium L50/14-2 allowed the determination of the minimal replicon on a 1.2 kb DNA fragment containing only the overall ori and repE which also act in trans. The involvement of orf9 in the plasmid copy number and in the plasmid stability was investigated. The pCIZ2 recombinant plasmids constitute narrow-host range shuttle cloning vectors (E. coli-E. faecium) that could be very useful for enterococcal genes studies, allowing an easy identification due to their histochemical recognition.