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

Abstract

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity 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 ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.

Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

“…The protocol and bacterial strain used for the purification of the E. coli β-clamp were kindly provided by D. Bastia (Medical University of South Carolina, Charleston, SC). E. coli proteins DnaA-His6, DnaB-His6, DnaC, and ClpX were purified as described (43,(47)(48)(49). All TrfA preparations used in the tests were N-terminally histidine-tagged 33-kDa versions of TrfA.…”

A Specific Region in the N Terminus of a Replication Initiation Protein of Plasmid RK2 Is Required for Recruitment of Pseudomonas aeruginosa DnaB Helicase to the Plasmid Origin

“…Construction of trfA Mutations-Plasmid pGC1 (20) was used to express His6TrfA-44(M98L/G254D/S267L), a variant of TrfA-44 that has six histidine residues inserted between the first amino acid (Met) and the second amino acid (Asn) of the native protein to allow for ease of purification, the M98L amino acid substitution that replaces the native methionine start of TrfA-33 with a leucine and thus eliminates TrfA-33 expression, and the G254D and S267L changes that result in a primarily monomeric form of the protein. A previous study had shown that although wild type TrfA protein is primarily a dimer in solution, it was the monomeric form that was essential for replication initiation activity (26).…”

“…Plasmids expressing the TrfA-44 N-terminal mutants were transferred into E. coli strain JP313 to overexpress and purify the proteins as described previously for TrfA-44 (20).…”

“…The simplicity of this system has allowed for a direct comparison of the mechanism for DNA replication initiation in different bacterial species (20,21).…”

“…Since it has been shown that the open complex formed on a supercoiled oriV template is indistinguishable when either TrfA-33 or TrfA-44 proteins are used with the DnaA proteins of E. coli, P. putida, or P. aeruginosa (20), the observed differences in activity of the two forms of the TrfA initiation protein in P. aeruginosa are likely due to a difference in their ability to interact with the P. aeruginosa DnaB helicase. This suggested a role for the N-terminal 97 amino acids, which are unique to TrfA-44, in DnaB recruitment in Pseudomonas.…”