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Journal of Bacteriology, May 2000, p. 2604-2610, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

DnaA, the Initiator of Escherichia coli Chromosomal Replication, Is Located at the Cell Membrane

Gillian Newman and Elliott Crooke*

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007

Received 2 November 1999/Accepted 18 January 2000

Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd., NW, Washington, DC 20007. Phone: (202) 687-1644. Fax: (202) 687-7186. E-mail: crooke{at}bc.georgetown.edu.

Journal of Bacteriology, May 2000, p. 2604-2610, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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