THE IDENTIFICATION OF MYCOBACTERIUM-MARINUM GENES DIFFERENTIALLY EXPRESSED IN MACROPHAGE PHAGOSOMES USING PROMOTER FUSIONS TO GREEN FLUORESCENT PROTEIN

Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow-grow ing pathogenic mycobacteria that is able to survive and replicate in m acrophages. Using the promoter-capture Vector pFPV27, we have construc ted a library of 200-1000 bp fragments of M. marinum genomic DNA inser ted upstream of a promoterless green fluorescent protein (GFP) gene. O nly those plasmids that contain an active promoter will express GFP. M acrophages were infected with this fusion library, and phagosomes cont aining fluorescent bacteria were isolated. Promoter constructs that we re more active intracellularly were isolated with a fluorescence-activ ated cell sorter, and inserts were partially sequenced. The promoter f usions expressed intracellularly exhibited homology to mycobacterial g enes encoding, among others, membrane proteins and biosynthetic enzyme s. Intracellular expression of GFP was 2-20 times that of the same clo nes grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tub erculosis. These constructs were positive for GFP expression in all my cobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabl ed us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.