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J. Bacteriol. doi:10.1128/JB.00846-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Stabilization of pSW100 from Pantoea stewartii by F conjugation system

Molecular Genetics Laboratory, Department of Microbiology and Immunology, Chang-Gung University, Kwei-Shan, Taoyuan, 333, Taiwan

^* To whom correspondence should be addressed. Email: cgliu{at}mail.cgu.edu.tw.

	Abstract

Plasmid pSW100 is one of the 13 plasmids from Pantoea stewartii subsp. stewartii SW2, which has a replicon that resembles that of ColE1. This work uses a pSW100 derivative, pSW140K, to study how the pSW100 replicon is stably maintained in its hosts. Our results indicate that although pSW140K is stable in E. coli HB101, the plasmid is rapidly lost in another E. coli strain, DH5, indicating that the genetic background of an E. coli strain affects the stability of pSW140K. Mutagenesis of E. coli HB101 with EZ::TN<DHFR-1> revealed that a mutation in traC, traF, traG, traN, and traV, which encode the components of the sex pilus assembly, reduces plasmid stability. Furthermore, this work identified that a 38-bp region, which is located immediately upstream of the RNAII promoter, is critical to the maintenance of plasmid stability in E. coli HB101. Not only does TraC bind to the region but also deleting the region destabilizes the plasmid. Furthermore, inserting this 38-bp fragment into a plasmid that contains the minimal replicon from pSW200 stabilizes the plasmid in E. coli HB101. Fluorescence in situ hybridization and immunofluorescence staining also revealed that a derivative of pSW100, pSW128A and TraC are colocalized in the cell, suggesting that pSW100 may use the sex pilus assembly as a partition apparatus to ensure the even distribution of the plasmid during cell division and thus maintain the plasmid stability.