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Characterization of the theta-type plasmid pCD3.4 from Carnobacterium divergens, and modulation of its host range by RepA mutation

Marco J. van Belkum

Michael E. Stiles

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5

Correspondence
Michael E. Stiles
michael.stiles{at}telusplanet.net

The complete nucleotide sequence of the 3475 bp plasmid pCD3.4 from Carnobacterium divergens LV13, which encodes the bacteriocin divergicin A, was determined. Nucleotide sequence, deletion and complementation analyses revealed the presence of a trans-acting replication protein, RepA, and DNA sequences involved in plasmid replication and copy-number control. The DNA region preceding the repA gene probably contains the origin of replication. This sequence includes four and a half direct repeats (iterons) of 22 bp, to which RepA is thought to bind, and an AT-rich region containing a 12 bp repeat, at which initiation of DNA might occur. Further upstream of this sequence resides a fifth iteron required for optimal plasmid replication. The RepA protein shows homology to replication proteins of the pUCL287 subfamily of theta-type replicons. Two ORFs were found downstream of the repA gene that could be deleted without affecting replication and stability of the plasmid. pCD3.4 has a narrow host range, and could only be maintained in Carnobacterium spp.; however, a mutant of the plasmid was obtained that enabled the pCD3.4 replicon to replicate in Enterococcus faecium, but not in Carnobacterium spp. The mutation was located in the C-terminal region of the RepA protein, changing a proline into a serine. This is believed to be the first example of such plasmid-host-range modulation in Gram-positive bacteria.

The GenBank/EMBL/DDBJ accession number for the pCD3.4 nucleotide sequence reported in this paper is DQ087597.

Present address: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.

Present address: CanBiocin Inc., 1015, 8308-114 Street, Edmonton, Alberta, Canada T6G 2E1.