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Journal of Bacteriology, March 2000, p. 1757-1760, Vol. 182, No. 6
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Isolation of an Inner Membrane-Derived Subfraction That Supports In Vitro Replication of a Mini-RK2 Plasmid in Escherichia coli

Peter D. Kim and William Firshein*

Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459

Received 8 November 1999/Accepted 22 December 1999

Previous results have demonstrated that the inner, but not the outer, membrane fraction of Escherichia coli is the site of membrane-associated DNA replication of plasmid RK2, a broad-host-range plasmid capable of replication in a wide variety of gram-negative hosts (K. Michaels, J. Mei, and W. Firshein, Plasmid 32:19-31, 1994). To resolve the inner membrane replication site further, the procedure of Ishidate et al. (K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, G. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) was used to separate the inner membrane into a number of subfractions, of which only one, a small subfraction containing only 10% of the entire membrane, was found to synthesize DNA inhibited by antibody prepared against the plasmid-encoded initiation protein TrfA. This is the same subfraction that was also found to bind oriV and TrfA to the greatest extent in filter binding assays (J. Mei, S. Benashski, and W. Firshein, J. Bacteriol. 177:6766-6772, 1995).

* Corresponding author. Mailing address: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459. Phone: (860) 685-2432. Fax: (860) 685-2141. E-mail: wfirshein{at}wesleyan.edu.

Journal of Bacteriology, March 2000, p. 1757-1760, Vol. 182, No. 6
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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