Molecular analysis of the replication elements of the broad-host-range RepA/C replicon

RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1. The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number. The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa protein (ORF2) (Llanes et al., 1994b). In this work, using an in vitro transcription/translation system, we detected a polypeptide whose size corresponded well to that of the deduced product of ORF1. Deletion and insertion mutation analysis showed that ORF1 is essential for replication; it encodes an initiator protein (called RepA). ORF2 was not essential for replication in Escherichia coli and its function remains to be determined. Using complementation experiments, the replication origin (ori) of RepA/C was defined. The ori was located in a 600-bp fragment downstream from repA, containing 10 direct repeats. To study the control of repA expression, a transcriptional fusion PrepA::lacZ was constructed. Its analysis showed that repA is transcriptionally autoregulated as are most repA genes of replicons controlled by iterons.