GENETIC-ANALYSIS OF THE MINIMAL REPLICON OF THE LACTOCOCCUS-LACTIS SUBSP LACTIS BIOVAR DIACETYLACTIS CITRATE PLASMID

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.