Molecular Analysis of the Replication Elements of the Broad-Host-Range RepA/C Replicon

Abstract

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) (Llaneset al.,1994b). In this work, using anin vitrotranscription/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 inEscherichia coliand its function remains to be determined. Using complementation experiments, the replication origin (ori) of RepA/C was defined. Theoriwas located in a 600-bp fragment downstream fromrepA,containing 10 direct repeats. To study the control ofrepAexpression, a transcriptional fusionPrepA::lacZwas constructed. Its analysis showed thatrepAis transcriptionally autoregulated as are mostrepAgenes of replicons controlled by iterons.