CLONING AND ASSESSMENT OF MYCOBACTERIAL PROMOTERS BY USING A PLASMID SHUTTLE VECTOR

We have constructed a promoter selection vector for mycobacteria to an alyze the sequences involved in mycobacterial transcriptional regulati on. The vector pSD7 contains extrachromosomal origins of replication f rom Escherichia coli as well as from Mycobacterium fortuitum and a kan amycin resistance gene for positive selection in mycobacteria. The pro moterless chloramphenicol acetyltransferase (CAT) reporter gene has be en used to detect mycobacterial promoter elements in a homologous envi ronment and to quantify their relative strengths. Using pSD7, we have isolated 125 promoter clones from the slowly growing pathogen Mycobact erium tuberculosis H37Rv and 350 clones from the fast-growing saprophy te Mycobacterium smegmatis. The promoters exhibited a wide range of st rengths, as indicated by their corresponding CAT reporter activities ( 5 to 2,500 nmol/min/mg of protein). However, while most of the M. smeg matis promoters supported relatively higher CAT activities ranging fro m 100 to 2,500 amol/min/mg of protein, a majority of those from M. tub erculosis supported CAT activities ranging from 5 to only about 100 nm ol/min/mg of protein. Our results indicate that stronger promoters occ ur less frequently in the case of M. tuberculosis compared with M. sme gmatis. To assess the extent of divergence of mycobacterial promoters vis-a-vis those of E. coli, the CAT activities supported by the promot ers in E. coli were measured and compared with their corresponding act ivities in mycobacteria. Most of the mycobacterial promoter elements f unctioned poorly in E. coli. The homologous selection system that we h ave developed has thus enabled the identification of mycobacterial pro moters that apparently function optimally only in a native environment .