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 p lasmids and was isolated recently from plasmid RA1. The sequence of th is autoreplicative region was established; it contains 13 repeats? sug gesting that the replicon uses iterons to control its copy number. The sequence contains two ORFs, one potentially coding for a 33-kDa prote in (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 mu tation analysis showed that ORF1 is essential for replication; it enco des an initiator protein (called RepA). ORF2 was not essential for rep lication in Escherichia coli and its function remains to be determined . Using complementation experiments, the replication origin (ori) of R epA/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 m ost repA genes of replicons controlled by iterons. (C) 1996 Academic P ress, Inc.