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Journal of Bacteriology, February 2005, p. 1227-1237, Vol. 187, No. 4
0021-9193/05/$08.00+0     doi:10.1128/JB.187.4.1227-1237.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of a Novel Allele of Escherichia coli dnaB Helicase That Compromises the Stability of Plasmid P1

Roderick A. Slavcev and Barbara E. Funnell*

Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada

Received 12 September 2004/ Accepted 8 November 2004

Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34°C. Shifting dnaB277 cells to 42°C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.

* Corresponding author. Mailing address: Department of Medical Genetics and Microbiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8. Phone: (416) 978-1665. Fax: (416) 978-6885. E-mail: b.funnell{at}utoronto.ca.

Journal of Bacteriology, February 2005, p. 1227-1237, Vol. 187, No. 4
0021-9193/05/$08.00+0     doi:10.1128/JB.187.4.1227-1237.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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