Replication control of a small cryptic plasmid of Escherichia coli

The role of the RepA initiator protein in replication and copy-number contr ol of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. Th e identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n '-pasH, which were esse ntial for efficient plasmid replication. The cop region also harbors a regi on of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, P-rep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding pri marily as a monomer or dimer to BD-l to initiate RepA transcription and pla smid replication and as higher oligomers to BD-2 tio autoregulate repA tran scription, the balance being reflected in plasmid copy number. An active in tegration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding gene s himA and himD. Low concentrations of MF predisposed the cop region to Rep A binding, although when highly expressed in trans RepA effectively displac ed bound II-Il; and it overcame IHF dependency. Incompatibility was shown t o be due to the titration of RepA at the cop locus but could be easily over ridden by excess RepA. Both RepA binding sites were required to maintain in compatibility and effective pKL1 replication. Neither antisense RNA nor ite rons were found to be involved in pKL1 regulation, thus pKL1 is a novel exa mple of autoregulation of DNA replication. When produced in excess from a h elper plasmid, RepA induced pKL1 replication to unusually high levels (>250 0 copies/cell). In addition, pKL1 replication could be artificially modulat ed and a wide range of copy numbers maintained. (C) 1999 Academic Press.