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.
Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.
Zhong
Zhenping
Z
Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0322, USA.
Helinski
Donald
D
Toukdarian
Aresa
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002845:Chromatography; D004265:DNA Helicases / Q000235:genetics*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D013997:Time Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C067405:TrfA protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases