Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector

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Abstract
We have constructed a promoter selection vector for mycobacteria to analyze the sequences involved in mycobacterial transcriptional regulation. The vector pSD7 contains extrachromosomal origins of replication from Escherichia coli as well as from Mycobacterium fortuitum and a kanamycin resistance gene for positive selection in mycobacteria. The promoterless chloramphenicol acetyltransferase (CAT) reporter gene has been used to detect mycobacterial promoter elements in a homologous environment and to quantify their relative strengths. Using pSD7, we have isolated 125 promoter clones from the slowly growing pathogen Mycobacterium tuberculosis H37Rv and 350 clones from the fast-growing saprophyte Mycobacterium smegmatis. The promoters exhibited a wide range of strengths, as indicated by their corresponding CAT reporter activities (5 to 2,500 nmol/min/mg of protein). However, while most of the M. smegmatis promoters supported relatively higher CAT activities ranging from 100 to 2,500 nmol/min/mg of protein, a majority of those from M. tuberculosis supported CAT activities ranging from 5 to only about 100 nmol/min/mg of protein. Our results indicate that stronger promoters occur less frequently in the case of M. tuberculosis compared with M. smegmatis. To assess the extent of divergence of mycobacterial promoters vis-à-vis those of E. coli, the CAT activities supported by the promoters in E. coli were measured and compared with their corresponding activities in mycobacteria. Most of the mycobacterial promoter elements functioned poorly in E. coli. The homologous selection system that we have developed has thus enabled the identification of mycobacterial promoters that apparently function optimally only in a native environment.