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Journal of Bacteriology, January 2006, p. 556-568, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.556-568.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening{dagger}

Biju Joseph,1,{ddagger} Karin Przybilla,1,§,{ddagger} Claudia Stühler,1 Kristina Schauer,2 Jörg Slaghuis,1 Thilo M. Fuchs,2 and Werner Goebel1*

Theodor-Boveri-Institut (Biozentrum), Lehrstuhl für Mikrobiologie, Universität Würzburg, D-97074 Würzburg, Germany,1 Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie, Technische Universität München, D-85350 Freising, Germany2

Received 16 June 2005/ Accepted 16 October 2005

A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.

* Corresponding author. Mailing address: Theodor-Boveri-Institut (Biozentrum), Lehrstuhl für Mikrobiologie, Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany. Phone: 49-931-8884400. Fax: 49-931-8884402. E-mail: goebel{at}biozentrum.uni-wuerzburg.de.

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

{ddagger} These two authors contributed equally to this work.

§ Present address: Merckle GmbH, Ludwig-Merckle-Str. 3, 89143 Blaubeuren-Weiler, Germany.

Journal of Bacteriology, January 2006, p. 556-568, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.556-568.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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