Isolation of strong expression signals of Mycobacterium tuberculosis

The natural fluorescence of the Aequoria victoria green fluorescent protein was exploited to isolate strong expression signals of Mycobacterium tuberc ulosis. Mycobacterium bovis bacille Calmette-Guerin harbouring M. tuberculo sis fragments driving high levels of gfp expression were isolated by fluore scence-activated cell sorting (FACS), DNA sequencing and subsequent compari son with the M, tuberculosis genome sequence revealed that a total of nine postulated promoters had been identified, The majority of the promoters dis played activity that was greater than or equal to the Mycobacterium fortuit um beta -lactamase promoter, one of the strongest mycobacterial promoters c haracterized to date, Two of the promoters corresponded to proteins predict ed to be involved in calcium and magnesium utilization, the importance of s uch functions for cell physiology suggesting why these two genes are contro lled by strong transcription signals. The seven other promoters corresponde d to genes encoding proteins of unknown function. Promoter activity was mai ntained after prolonged incubation within macrophages, implying that these promoters could be used to drive sustained foreign gene expression in vivo, The strength of these expression signals identified could be employed for the overexpression of foreign genes in mycobacteria to aid protein purifica tion and vaccine vector development. Furthermore, this study demonstrated t hat FAGS provides a sensitive and efficient technique to measure and select strong mycobacterial expression signals.