title: LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O:3 gene expression in vivo.
abstract: It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-Poc::luxAB and YeO3-Pop1::luxAB, respectively. In vitro, at 37°C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22°C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice.
authors.affiliation: Istanbul University, Faculty of Science, Department of Biology, Basic and Industrial Microbiology Section, Istanbul, Turkey.|Ege University, Faculty of Science, Department of Biology, Basic and Industrial Microbiology Section, Izmir, Turkey.|Department of Bacteriology and Immunology, Medicum, and Research Programs Unit, Immunobiology, University of Helsinki, Helsinki, Finland.
authors.forename: Elif
authors.initials: E
authors.lastname: Bozcal
authors.affiliation: Ege University, Faculty of Science, Department of Biology, General Biology Section, Izmir, Turkey.|Ege University, Center for Drug Research and Development and Pharmacokinetic Applications, Izmir, Turkey.
authors.forename: Melih
authors.initials: M
authors.lastname: Dagdeviren
authors.affiliation: Ege University, Faculty of Science, Department of Biology, Basic and Industrial Microbiology Section, Izmir, Turkey.
authors.forename: Atac
authors.initials: A
authors.lastname: Uzel
authors.affiliation: Department of Bacteriology and Immunology, Medicum, and Research Programs Unit, Immunobiology, University of Helsinki, Helsinki, Finland.|Division of Clinical Microbiology, Helsinki University Hospital, HUSLAB, Helsinki, Finland.
authors.forename: Mikael
authors.identifier: http://orcid.org/0000-0001-8791-9260
authors.initials: M
authors.lastname: Skurnik
mesh_terms: D000818:Animals; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D016680:Genome, Bacterial; D008070:Lipopolysaccharides / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D005810:Multigene Family; D009154:Mutation; D019081:O Antigens / Q000378:metabolism; D009876:Operon; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D015009:Yersinia Infections / Q000382:microbiology*; D015008:Yersinia enterocolitica / Q000235:genetics*
chemical_list: D008070:Lipopolysaccharides; D019081:O Antigens