Nucleotide Sequence and Analysis of pWC1, A pC194-Type Rolling Circle Replicon inLactococcus lactis

Abstract

A 2.8-kb cryptic plasmid showing no homology to either pFX3 (rolling circle, pE194-type) or pCI305 (theta-type) lactococcal replicons was identified inLactococcus lactissubsp.cremoris2204. The plasmid, pWC1, was compatible with both pCI3340 (a pCI305 derivative) and pFX3 inL. lactissubsp.cremoris2204. 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 inL. 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 includedStreptococcus thermophilus, Enterococcus faecalis,andStaphylococcus aureus,but notEscherichia coli.Both pCP12 and to a lesser extent pWC1 generated single-stranded DNA (ssDNA) inL. 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.