This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Haugan, K Articles by Valla, S Search for Related Content PubMed PubMed Citation Articles by Haugan, K Articles by Valla, S

research-article

The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations.

UNIGEN Center for Molecular Biology, University of Trondheim, Norway.

ABSTRACT

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.

This article has been cited by other articles:

• Wild, J., Hradecna, Z., Szybalski, W. (2002). Conditionally Amplifiable BACs: Switching From Single-Copy to High-Copy Vectors and Genomic Clones. Genome Res. 12: 1434-1444 [Abstract] [Full Text]  
• Karunakaran, P., Blatny, J. M., Ertesvåg, H., Valla, S. (1998). Species-Dependent Phenotypes of Replication-Temperature-Sensitive trfA Mutants of Plasmid RK2: a Codon-Neutral Base Substitution Stimulates Temperature Sensitivity by Leading to Reduced Levels of trfA Expression. J. Bacteriol. 180: 3793-3798 [Abstract] [Full Text]  
• del Solar, G., Giraldo, R., Ruiz-Echevarria, M. J., Espinosa, M., Diaz-Orejas, R. (1998). Replication and Control of Circular Bacterial Plasmids. Microbiol. Mol. Biol. Rev. 62: 434-464 [Abstract] [Full Text]