Green fluorescent protein as a new expression marker in mycobacteria

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.