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Applied and Environmental Microbiology, December 2007, p. 7506-7514, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01084-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Expanding the Helicobacter pylori Genetic Toolbox: Modification of an Endogenous Plasmid for Use as a Transcriptional Reporter and Complementation Vector

Beth M. Carpenter,¹ Timothy K. McDaniel,² Jeannette M. Whitmire,¹ Hanan Gancz,¹ Silvia Guidotti,³ Stefano Censini,³ and D. Scott Merrell^1*

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, Maryland 20814,¹ Illumina, Inc., 9885 Towne Centre Dr., San Diego, California 92121-1975,² Novartis Vaccines and Diagnostics S.r.l., Via Fiorentina 1, 53100 Siena, Italy³

Received 15 May 2007/ Accepted 25 September 2007

Helicobacter pylori is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous H. pylori plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an Escherichia coli origin of replication, a kanamycin resistance cassette, a promoterless gfpmut3 gene, and a functional multiple cloning site to form pTM117. The promoters of amiE and pfr, two well-characterized Fur-regulated promoters, were fused to the promoterless gfpmut3, and green fluorescent protein (GFP) expression of the fusions in wild-type and fur strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing amiE, pfr, and fur expression in wild-type and fur strains carrying a wild-type copy of fur on the plasmid. Proper regulation of these genes was restored in the fur background under high- and low-iron conditions, signifying complementation of both iron-bound and apo Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of H. pylori.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Uniformed Services University of the Heath Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814. Phone: (301) 295-1584. Fax: (301) 295-3773. E-mail: dmerrell{at}usuhs.mil

Published ahead of print on 5 October 2007.

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Applied and Environmental Microbiology, December 2007, p. 7506-7514, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01084-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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