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Applied and Environmental Microbiology, April 2008, p. 2095-2102, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.01348-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori{triangledown} ,{dagger}

Ivo G. Boneca,1* Chantal Ecobichon,1 Catherine Chaput,1,{ddagger} Aurélie Mathieu,1,3 Stéphanie Guadagnini,2 Marie-Christine Prévost,2 Frédéric Colland,4 Agnès Labigne,1 and Hilde de Reuse1

Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Department of Microbiology, Paris, France,1 Institut Pasteur, Plate-forme de Microscopie Electronique, Department of Cell Biology and Infection, Paris, France,2 UMR 217 CNRS/CEA, Department of Radiobiology and Radiopathology, Commissariat à l'Energie Atomique, Fontenay aux Roses, France,3 Hybrigenics SA, Paris, France4

Received 18 June 2007/ Accepted 25 January 2008

The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-D-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.

* Corresponding author. Mailing address: Groupe Biologie et Génétique de la Paroi Bactérienne, Unité de Pathogénie Bactérienne des Muqueuses, Department of Microbiology, Institut Pasteur, 75724 Paris, France. Phone: 33-1-4438-9516. Fax: 33-1-4061-3640. E-mail: bonecai{at}pasteur.fr

{triangledown} Published ahead of print on 1 February 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Department of Cellular Microbiology, Max Planck Institute, Charitéplatz 1, D-10117 Berlin, Germany.

Applied and Environmental Microbiology, April 2008, p. 2095-2102, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.01348-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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