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Infection and Immunity, June 2000, p. 3242-3250, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Listeriolysin O as a Reporter To Identify Constitutive and In Vivo-Inducible Promoters in the Pathogen Listeria monocytogenes

Iharilalao Dubail, Patrick Berche, The European Listeria Genome Consortium, and Alain Charbit^*

Laboratoire de Microbiologie, INSERM U-411, Faculté de Médecine Necker, 75730 Paris Cedex 15, France

Received 3 February 2000/Returned for modification 8 March 2000/Accepted 16 March 2000

Listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chromosomal fragments upstream of the promoterless hly gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the hly-based system as an in vivo expression technology tool, nine in vivo-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative N-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in L. monocytogenes.

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^* Corresponding author. Mailing address: INSERM U411, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France. Phone: 33 1-40 61 53 76. Fax: 33 1-40 61 55 92. E-mail: charbit{at}necker.fr.

The European Listeria Genome Consortium is composed of Philippe Glaser, Alexandra Amend, Fernando Baquero-Mochales, Patrick Berche, Helmut Bloecker, Petra Brandt, Carmen Buchrieser, Trinad Chakraborty, Alain Charbit, Elisabeth Couvé, Antoine de Daruvar, Pierre Dehoux, Eugen Domann, Gustavo Dominguez-Bernal, Lionel Durant, Karl-Dieter Entian, Lionel Frangeul, Hafida Fsihi, Francisco Garcia del Portillo, Patricia Garrido, Werner Goebel, Nuria Gomez-Lopez, Torsten Hain, Joerg Hauf, David Jackson, Jurgen Kreft, Frank Kunst, Jorge Mata-Vicente, Eva Ng, Gabriele Nordsiek, Jose Claudio Perez-Diaz, Bettina Remmel, Matthias Rose, Christophe Rusniok, Thomas Schlueter, Jose-Antonio Vazquez-Boland, Hartmut Voss, Jurgen Wehland, and Pascale Cossart.

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Infection and Immunity, June 2000, p. 3242-3250, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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