Cj. Pillidge et al., NUCLEOTIDE-SEQUENCE AND ANALYSIS OF PWC1, A PC194-TYPE ROLLING CIRCLEREPLICON IN LACTOCOCCUS-LACTIS, Plasmid, 35(2), 1996, pp. 131-140
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.