Ml. Pedersen et al., GENETIC-ANALYSIS OF THE MINIMAL REPLICON OF THE LACTOCOCCUS-LACTIS SUBSP LACTIS BIOVAR DIACETYLACTIS CITRATE PLASMID, MGG. Molecular & general genetics, 244(4), 1994, pp. 374-382
Using a combination of mutagenesis with the transposon gamma delta and
polymerase chain reaction subcloning, the essential elements of the r
eplication region of the Lactococcus lactis subsp. lactis biovar diace
tylactis citrate plasmid have been identified. An open reading frame,
coding for a protein with homology to Rep proteins from other Lactococ
cus plasmids, is essential. This protein is tl ans-acting and could no
t be replaced by the Rep protein from another Lactococcus plasmid. A s
econd open reading frame immediately downstream from the first could b
e removed or inactivated with no apparent effect on plasmid replicatio
n. A region containing two 10 bp direct repeats and three tandem repea
ts of a 22 bp sequence, immediately upstream of the essential open rea
ding frame, is also essential and probably includes the origin of repl
ication. A 181-bp DNA fragment containing this region was sufficient t
o allow replication in Lactococcus if the trans-acting protein was pro
vided on another replicon. Single-stranded replication intermediates c
ould not be detected, suggesting that the citrate plasmid uses theta r
eplication rather than rolling-circle replication.