GREEN FLUORESCENT PROTEIN AS A MARKER FOR GENE-EXPRESSION AND CELL BIOLOGY OF MYCOBACTERIAL INTERACTIONS WITH MACROPHAGES

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluore scence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shutt le-plasmid vector carrying gfp cDNA was constructed and used to genera te transcriptional fusions with promoters of interest and to examine t heir expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters stud led w ere: (i) ahpCfrom Mycoosis and Mycobacterium leprae, a gene encoding a lkyl hydroperoxide reductase which, along with the divergently transcr ibed regulator oxyR, are homologues of corresponding stress-response s ystems in enteric bacteria and play a role in isoniazid sensitivity; ( ii) mtrA, an M. tuberculosis response regulator belonging to the super family of bacterial two-component signal-transduction systems; (iii) h sp60 a previously characterized heat-shock gene from M. bovis; and (iv ) tbprc3, a newly isolated promoter from M. tuberculosis. Expression o f these promoters in mycobacteria was analysed using epifluorescence m icroscopy, laser scanning confocal microscopy, fluorescence spectrosco py, and flow cytometry. These approaches permitted assessment of fluor escence prior to and after macrophage infection, and analyses of promo ter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting fro m cells harbouring the vector used to construct the fusion. In additio n, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitate d localization and isolation of phagocytic vesicles containing mycobac teria. The experiments presented here suggest that GFP will be a usefu l tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophag es.