Interactions of DnaA proteins from distantly related bacteria with the replication origin of the broad host range plasmid RK2.
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.
Caspi
R
R
Department of Biology, University of California, San Diego, La Jolla, California 92093-0322, USA.
Helinski
D R
DR
Pacek
M
M
Konieczny
I
I
D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000378:metabolism*; D018983:DNA Footprinting; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053488:DnaB Helicases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006639:Histidine; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D018741:Replication Origin; D012984:Software; D013302:Streptomyces
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D010455:Peptides; C067405:TrfA protein, E coli; C033223:polyhistidine; D006639:Histidine; D004265:DNA Helicases; D053488:DnaB Helicases