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Applied and Environmental Microbiology, April 2006, p. 2876-2884, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2876-2884.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression in Listeria monocytogenes

Peter A. Bron,1,{dagger} Ian R. Monk,1,2,{dagger} Sinéad C. Corr,1,2 Colin Hill,1,2* and Cormac G. M. Gahan1,2,3

Alimentary Pharmabiotic Centre,1 Department of Microbiology,2 School of Pharmacy, University College Cork, Cork, Ireland3

Received 25 August 2005/ Accepted 16 January 2006

In this paper we describe construction of a luciferase-based vector, pPL2lux, and use of this vector to study gene expression in Listeria monocytogenes. pPL2lux is a derivative of the listerial integration vector pPL2 and harbors a synthetic luxABCDE operon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2lux derivatives in which the secA and hlyA promoters were translationally fused to luxABCDE and integrated as a single copy into the chromosome of L. monocytogenes EGD-e. Growth experiments revealed that hlyA was expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereas secA expression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2lux as a quantitative reporter system for real-time, noninvasive evaluation of gene expression in L. monocytogenes.

* Corresponding author. Mailing address: Alimentary Pharmabiotic Centre, University College Cork, Western Rd., Cork, Ireland. Phone: 353 214 901 373. Fax: 353 214 903 101. E-mail: c.hill{at}ucc.ie.

{dagger} P.A.B. and I.R.M. contributed equally to this work.

Applied and Environmental Microbiology, April 2006, p. 2876-2884, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2876-2884.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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