Activity of mycobacterial promoters during intracellular and extracellular growth

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Abstract
PUS933, a bifunctionalMycobacterium-Escherichia coli translationalfusion vector containing an amino-terminally truncatedE. coli lacZreporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of theMycobacterium bovisBCGhsp60gene, theMycobacterium leprae28 kDa gene and theM. leprae18 kDa gene were constructed and introduced intoE. coli, Mycobacterium smegmatisandM. bovisBCG. -Galactosidase activity was measured for mycobacteria grown in liquid culture. Primerextension analysis was used to determine the transcriptional start point for the 18 kDa promoter inM. smegmatis.Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCGhsp60gene promoter and theM. leprae28 kDa gene promoter gave high levels of -galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression inM. smegmatisand BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival ofM. lepraewithin macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.