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Journal of Bacteriology, May 2003, p. 3060-3067, Vol. 185, No. 10
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.10.3060-3067.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of the Double-Partitioning Modules of R27: Correlating Plasmid Stability with Plasmid Localization

Trevor D. Lawley1 and Diane E. Taylor1,2*

Departments of Biological Sciences,1 Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2R32

Received 7 November 2002/ Accepted 10 March 2003

Plasmid R27 contains two independent partitioning modules, designated Par1 and Par2, within transfer region 2. Par1 is member of the type I partitioning family (Walker-type ATPase), and Par2 is a member of the type II partitioning family (actin-type ATPase). Stability tests of cloned Par1 and Par2 and insertional disruptions of Par1 and Par2 within R27 demonstrated that Par1 is the major stability determinant whereas Par2 is the minor stability determinant. Creation of double-partitioning mutants resulted in R27 integrating into the chromosome, suggesting that at least one partitioning module is required for R27 to exist in the extrachromosomal form. Using the lacO/LacI-green fluorescent protein (GFP) system, we labeled and visualized R27 and R27 partitioning mutants (Par1- and Par2-) under different growth conditions in live Escherichia coli cells. Plasmid R27 was visualized as the discrete GFP foci present at the mid- and quarter-cell regions in >99% of the cells. Time lapse experiments demonstrated that an increase in R27 plasmid foci resulted from focus duplication in either the mid- or quarter-cell regions of E. coli. Both R27 Par- variants gave a high percentage of plasmidless cells, as suggested by a uniform GFP signal, and cells with GFP patterns scattered throughout the entire cell, suggesting that plasmid molecules are randomly distributed throughout the cytoplasm. Those cells that did contain R27 Par- with one or two discrete foci had localization patterns that were statistically different from those formed with wild-type R27. Therefore, these results suggest that partitioning-impaired plasmids are characterized by individual and clustered plasmids that are randomly located within the host cytoplasm.

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, 1-28 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2R3, Canada. Phone: 1 780 492 4777. Fax: 1 780 492 7521. E-mail: diane.taylor{at}ualberta.ca.

Journal of Bacteriology, May 2003, p. 3060-3067, Vol. 185, No. 10
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.10.3060-3067.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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