title: Green fluorescent protein as a new expression marker in mycobacteria.
abstract: This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM-11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM-11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co-factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free-living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.
authors.affiliation: Laboratoire de Microbiologie Génétique et Moléculaire, INSERM CJF9109, Institut Pasteur de Lille, France.
authors.forename: L
authors.initials: L
authors.lastname: Kremer
authors.forename: A
authors.initials: A
authors.lastname: Baulard
authors.forename: J
authors.initials: J
authors.lastname: Estaquier
authors.forename: O
authors.initials: O
authors.lastname: Poulain-Godefroy
authors.forename: C
authors.initials: C
authors.lastname: Locht
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D018834:Chaperonin 60 / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D009163:Mycobacterium bovis / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis
chemical_list: D000995:Antitubercular Agents; D018834:Chaperonin 60; D017931:DNA Primers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins