Novel luciferase reporter system for in vitro and organ-specific monitoring of differential gene expression in Listeria monocytogenes.
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.
Bron
Peter A
PA
Alimentary Pharmabiotic Centre, University College Cork, Western Road, Cork, Ireland.
Monk
Ian R
IR
Corr
Sinéad C
SC
Hill
Colin
C
Gahan
Cormac G M
CG
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018938:Caco-2 Cells; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000382:microbiology; D008089:Listeria monocytogenes / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity; D008088:Listeriosis / Q000382:microbiology; D008099:Liver / Q000382:microbiology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009928:Organ Specificity; D010957:Plasmids / Q000235:genetics; D013154:Spleen / Q000382:microbiology
D001426:Bacterial Proteins; D003470:Culture Media; D006460:Hemolysin Proteins; D008156:Luciferases