The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n'-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.
Copyright 1999 Academic Press.