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Green fluorescent protein as a new expression marker in mycobacteria
Author(s) -
Kremer Laurent,
Baulard Alain,
Estaquier Jérôme,
PoulainGodefroy Odile,
Locht Camille
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.mmi_17050913.x
Subject(s) - green fluorescent protein , biology , mycobacterium smegmatis , aequorea victoria , reporter gene , recombinant dna , microbiology and biotechnology , fluorescence microscope , shuttle vector , flow cytometry , fluorescence , gene , gene expression , vector (molecular biology) , mycobacterium tuberculosis , biochemistry , medicine , tuberculosis , physics , pathology , quantum mechanics
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.