title: Listeriolysin O as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen Listeria monocytogenes.
abstract: 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.
authors.affiliation: Laboratoire de Microbiologie, INSERM U-411, Faculté de Médecine Necker, 75730 Paris Cedex 15, France.
authors.forename: I
authors.initials: I
authors.lastname: Dubail
authors.forename: P
authors.initials: P
authors.lastname: Berche
authors.forename: A
authors.initials: A
authors.lastname: Charbit
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D015698:Genomic Library; D006360:Heat-Shock Proteins / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics*; D006461:Hemolysis; D008089:Listeria monocytogenes / Q000235:genetics* / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes