Identification of the origin and direction of replication of the broad-host-range plasmid pLS1

The replication origin of the fully sequenced broad-host-range streptococcal plasmid pLS1 has been determined by the use of an in vitro replication system prepared from Escherichia coli, a host in which the plasmid can be established. Replicative intermediates were isolated from reaction mixtures that contained dideoxythymidine triphosphate, thus limiting the average extent of in vitro synthesis. Analysis of HinfI-cleaved intermediates demonstrated that the origin of replication is included within a 443-bp fragment. Replication proceeds unidirectionally in the same direction as transcription of plasmid mRNAs. Isolation of deletion derivatives allowed us to define the replication origin of pLS1 within a region of 284 bp. Replication of pLS1 occurs through single-stranded intermediates by a rolling circle mechanism. Cleavage of supercoiled plasmid DNAs with endonuclease S1 followed by restriction mapping, allowed the positioning of three major specific S1 sites in regions of high potential to form secondary structures. One of these inverted repeats is located in the region where the origin of replication of pLS1 has been defined.