Sk. Dasgupta et al., CLONING AND ASSESSMENT OF MYCOBACTERIAL PROMOTERS BY USING A PLASMID SHUTTLE VECTOR, Journal of bacteriology, 175(16), 1993, pp. 5186-5192
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
.