Deciphering the Regulatory Circuits of RA3 Replication Module - Mechanisms of the Copy Number Control.

The RA3 plasmid, the archetype of IncU incompatibility group, represents a mosaic-modular genome of 45.9 kb. The replication module encompasses and (initiator) surrounded by two long repetitive sequences DR1 and DR2 of unknown function. Here, we mapped the origin of replication to the 3' end of and showed that was activated by the transcription coming from in the adjacent stability module. Using various in vivo and in vitro methods we demonstrated that the expression proceeded either from located in the intergenic region or from the upstream strong that was autoregulated by RepA. Additionally, the activity was modulated by the transcription from the overlapping, divergently oriented . Both mRNA (antisense for mRNA) and its small polypeptide product, RepX, were strong incompatibility determinants. Hence, we showed that the sophisticated RA3 copy number control combined the multivalent regulation of expression, RepB titration by DR1, and transcriptional activation of , dependent on the RA3 global regulatory network. Similarly organized replicons have been found in diverse bacterial species confirming the significance of these mechanisms in establishing the IncU plasmids in a broad spectrum of hosts.