The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations.
The minimal replicon of the broad-host-range plasmid RK2 consists of the origin of vegetative replication (oriV) and a gene (trfA) encoding an essential replication protein that binds to short repeats in oriV. We report here the results of a DNA sequence analysis of seven unique mutants that are temperature sensitive for replication in Escherichia coli. The mutations (designated rts) were distributed throughout 40% of the downstream part of the trfA gene. Spontaneous revertants of the rts mutants were isolated, and further analysis of four such revertants demonstrated that the new phenotypes resulted from intragenic second-site copy up (cop) mutations. Subcloning experiments showed that all tested intragenic combinations of rts and cop mutations resulted in elimination or strong reduction of the temperature sensitivity of replication. This suppression was also observed under conditions where the mutant TrfA protein was provided in trans with respect to oriV, indicating that the reduction in temperature sensitivity could not be a TrfA protein dosage effect. The phenotypes of two of the cop mutants in Pseudomonas aeruginosa were analyzed; the results demonstrated that the mutants were either not functional or poorly functional in this host. The rts mutant plasmids were also reduced in their ability to replicate in P. aeruginosa, and the intragenic cop mutations did not improve the functionality of these mutants. The significance of the results is discussed in relation to current models of the mechanism of action of the TrfA protein.
Haugan
K
K
UNIGEN Center for Molecular Biology, University of Trondheim, Norway.
Karunakaran
P
P
Blatny
J M
JM
Valla
S
S
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics*; D005809:Genes, Regulator / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012093:Replicon / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C067405:TrfA protein, E coli