NUCLEOTIDE-SEQUENCE AND ANALYSIS OF PWC1, A PC194-TYPE ROLLING CIRCLEREPLICON IN LACTOCOCCUS-LACTIS

A 2.8-kb cryptic plasmid showing no homology to either pFX3 (rolling c ircle, pE194-type) or pCI305 (theta-type) lactococcal replicons was id entified 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 ma jor ORF encoding a protein with a deduced size of 316 amino acids (aa) . Database comparisons showed that the protein was distinct from the p FX- and pCI-type replication proteins (less than 21% aa identity), but shared significant homology (up to 57% aa identity) with the replicat ion proteins from a different group of rolling circle plasmids (pC194- type) commonly found in gram-positive bacteria. A pC194-type rolling c ircle plasmid has not been previously described in L. lactis. Further sequence analysis showed a conserved double-stranded origin of replica tion in pWC1 preceded by a large (118-bp) direct repeat. The chloramph enicol-resistance gene from pC194 was inserted into a nonessential reg ion of pWC1 to give pCP12. The host range of pCP12 included Streptococ cus thermophilus, Enterococcus faecalis, and Staphylococcus aureus, bu t not Escherichia coli. Both pCP12 and to a lesser extent pWC1 generat ed single-stranded DNA (ssDNA) in L. lactis. A possible single-strande d origin of replication was identified by sequence analysis of pWC1 an d by comparing levels of ssDNA produced by pCP12 deletion derivatives. The pWC1 replicon may be a useful addition to other replicons current ly available for vector construction. (C) 1996 Academic Press, Inc.