A broad host range replicon with different requirements for replication initiation in three bacterial species

Plasmid RK2 is unusual in its ability to replicate stably in a wide range o f Gram-negative bacteria. The replication origin (oriV) and a plasmid-encod ed initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essentia l for RK2 replication. To examine initiation events in bacteria unrelated t o Escherichia coli, the genes encoding the replicative helicase, DnaB, of P seudomonas putida and Pseudomonas aeruginosa were isolated and used to cons truct protein expression vectors. The purified proteins were tested for act ivity along with E. coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P. putida DnaB was active with either TrfA-33 or TrfA-44, while P. aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E,coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an AT Pase accessory protein. Thus, a DnaC-like accessory ATPase is not universal ly required for loading the essential replicative helicase at a replication origin.