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Journal of Bacteriology, September 1998, p. 4922-4928, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The Bacteroides fragilis BtgA Mobilization Protein Binds to the oriT Region of pBFTM10

Leonid A. Sitailo,1 Alexander M. Zagariya,1,dagger Patrick J. Arnold,1 Gayatri Vedantam,1 and David W. Hecht1,2,*

Department of Medicine, VA Hospital, Hines, Illinois 60141,1 and Department of Medicine, Microbiology and Immunology, Program in Molecular Biology, Loyola University Chicago, Maywood, Illinois 601532

Received 15 January 1998/Accepted 13 July 1998

The Bacteroides fragilis conjugal plasmid pBFTM10 contains two genes, btgA and btgB, and a putative oriT region necessary for transfer in Bacteroides fragilis and Escherichia coli. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of btgA revealed three sets of inverted repeats, potentially locating the oriT region. A 304-bp DNA fragment comprising this putative oriT region was cloned and confirmed to be the functional pBFTM10 oriT by bacterial conjugation experiments using E. coli and B. fragilis. btgA was cloned and overexpressed in E. coli, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognate oriT. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to the oriT-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.

* Corresponding author. Mailing address: Loyola University Chicago, Bldg. 54, Room 101, 2160 S. First Ave., Maywood, IL 60153. Phone: (708) 216-2792. Fax: (708) 216-2269. E-mail: dhecht{at}luc.edu.

dagger Present address: Division of Neonatology, Department of Pediatrics, University of Illinois at Chicago, Chicago, IL 60616.

Journal of Bacteriology, September 1998, p. 4922-4928, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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