Nucleotide sequence and analysis of pWC1, a pC194-type rolling circle replicon in Lactococcus lactis

A 2.8-kb cryptic plasmid showing no homology to either pFX3 (rolling circle, pE194-type) or pCI305 (theta-type) lactococcal replicons was identified in Lactococcus lactis subsp. cremoris 2204. The plasmid, pWC1, was compatible with both pCI3340 (a pCI305 derivative) and pFX3 in L. lactis subsp. cremoris 2204. Sequence analysis of pWC1 showed one major ORF encoding a protein with a deduced size of 316 amino acids (aa). Database comparisons showed that the protein was distinct from the pFX- and pCI-type replication proteins (less than 21% aa identity), but shared significant homology (up to 57% aa identity) with the replication proteins from a different group of rolling circle plasmids (pC194-type) commonly found in gram-positive bacteria. A pC194-type rolling circle plasmid has not been previously described in L. lactis. Further sequence analysis showed a conserved double-stranded origin of replication in pWC1 preceded by a large (118-bp) direct repeat. The chloramphenicol-resistance gene from pC194 was inserted into a nonessential region of pWC1 to give pCP12. The host range of pCP12 included Streptococcus thermophilus, Enterococcus faecalis, and Staphylococcus aureus, but not Escherichia coli. Both pCP12 and to a lesser extent pWC1 generated single-stranded DNA (ssDNA) in L. lactis. A possible single-stranded origin of replication was identified by sequence analysis of pWC1 and by comparing levels of ssDNA produced by pCP12 deletion derivatives. The pWC1 replicon may be a useful addition to other replicons currently available for vector construction.