GREEN FLUORESCENT PROTEIN AS A NEW EXPRESSION MARKER IN MYCOBACTERIA

This study describes the use and the advantages of the green fluoresce nt protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 prom oter in the shuttle vector pGFM-11. The gfp expression in the recombin ant Mycobacterium smegmatis and BCG was readily detected on agar plate s by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 deriv ative that lacks the hsp60 promoter, no fluorescence was observed. How ever, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacte ria harbouring pGFM-11 and grown in liquid media could be quantified b y spectrofluorimetry. This allowed for easy assessment of drug suscept ibility. As GFP does not require the addition of substrates or co-fact ors, the green fluorescent bacilli could be directly observed within i nfected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or fre e-living recombinant mycobacteria could also be analysed by flow cytom etry. The GFP thus appears to be a convenient reporter for mycobacteri a, allowing tracing of recombinant mycobacteria, isolation of promoter s with interesting properties, in vivo drug testing and the developmen t of new diagnostic tools.