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 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.
Caspi
R
R
Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0322, USA.
Pacek
M
M
Consiglieri
G
G
Helinski
D R
DR
Toukdarian
A
A
Konieczny
I
I
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D018741:Replication Origin; D012093:Replicon; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; C067405:TrfA protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases