IncP-9 replication initiator protein binds to multiple DNA sequences in oriV and recruits host DnaA protein

The minimal replicon from IncP-9 plasmid pM3, consisting of oriV and rep, is able to replicate in Pseudomonas putida but not in Escherichia coli, unless production of Rep protein is increased. The Rep protein, at 20 kDa, is the smallest replication protein so far identified for a theta replicating plasmid. Rep was purified and shown to bind in three blocks across the oriV region that do not correlate with a single unique binding sequence. The block closest to rep is not necessary for oriV function. Rep forms at least two types of complex—one rendering the DNA entirely resistant to cleavage, the other occupying one side of the helix. No short segment of oriV showed the same affinity for Rep as the whole of oriV. The oriV region did not bind purified DnaA from E. coli, P. putida or P. aeruginosa but when Rep was present also, super-shifts were found with DnaA in a sequence-specific manner. Scrambling of the primary candidate DnaA box did not inactivate oriV but did increase the level of Rep required to activate oriV. The general pattern of Rep-DNA recognition sequences in oriV indicates that the IncP-9 system falls outside of the paradigms of model plasmids that have been well-studied to date.