Abstract
Replication initiation of the broad host range plasmid RK2 requires binding of the host-encoded DnaA protein to specific sequences (DnaA boxes) at its replication origin (oriV). In contrast to a chromosomal replication origin, which functionally interacts only with the native DnaA protein of the organism, the ability of RK2 to replicate in a wide range of Gram-negative bacterial hosts requires the interaction of oriV with many different DnaA proteins. In this study we compared the interactions of oriV with five different DnaA proteins. DNase I footprint, gel mobility shift, and surface plasmon resonance analyses showed that the DnaA proteins from Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa bind to the DnaA boxes at oriV and are capable of inducing open complex formation, the first step in the replication initiation process. However, DnaA proteins from two Gram-positive bacteria, Bacillus subtilis and Streptomyces lividans, while capable of specifically interacting with the DnaA box sequences at oriV, do not bind stably and fail to induce open complex formation. These results suggest that the inability of the DnaA protein of a host bacterium to form a stable and functional complex with the DnaA boxes at oriV is a limiting step for plasmid host range.
Highlights
DnaA proteins are the universal initiators of replication from the chromosomal replication origin in bacteria
Sequence Comparison of the Different DnaA Proteins—Three of the five dnaA genes encoding the proteins used in this study have been sequenced and published (26 –28)
The mechanism by which binding of the DnaA protein to DnaA boxes at bacterial replication origins promotes unwinding of the AT-rich region is not understood, but some insight to this process can be gained by studying the interaction of DnaA proteins with the DnaA box sequences of different organisms
Summary
DnaA proteins are the universal initiators of replication from the chromosomal replication origin (oriC) in bacteria. A minimal functional origin derived from this plasmid, which retains this broad host range replication activity, consists of the following elements: four DnaA boxes, five repeats of highly conserved 17-mer sequences (iterons), and an AT-rich region containing four 13-mer sequences. Replication from this origin (oriV) requires a plasmid encoded replication protein, TrfA, which binds to the iterons [8, 9]. It has been shown recently that box A4 plays a important role at least in E. coli in that it directs the binding of the DnaA protein to the other three boxes [10]
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