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

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-a-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

Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria

“…Selection of promoters based on lacZ (βGal) expression by pSD5B offers an advantage in that cloning of a wide range of promoters is possible without incorporating any bias towards the promoters of a certain range as can occur in vectors using antibiotic resistance-encoding genes as a basis for promoter selection (DasGupta et al, 1993). Besides, the mycobacterial promoters isolated in pSD5B can be directly assessed for their transcriptional strength in E. coli.…”

“…5A and B, lane 2). To examine that all necessary features of pSD5C function appropriately, a fragment encoding CAT (gene cat, containing its own ribosome-binding site and start codon without the promoter region) derived from pSD7 (DasGupta et al, 1993) was cloned in the PstI site of pSD5C to obtain pSD5C-CAT and the expression of cat was analysed in E. coli and M. smegmatis by 0.1% SDS-10% PAGE and Western blotting. Since cat was cloned in a reading frame different from malE, it was not expressed as an MBP fusion protein.…”

Isolation of strong expression signals of Mycobacterium tuberculosis

“…By using FACS to isolate recombinant bacteria displaying strong fluorescence, we significantly limit the number of colonies required to be screened by initially eliminating the majority of bacteria not displaying promoter activity, and can also place a limit on the level of promoter strength preferred during the cell sorting process. Such advantages are not seen with systems based on the β-galactosidase and chloramphenicol acetyltransferase reporter proteins which have previously been employed to isolate promoters of M. tuberculosis (Timm et al, 1994a ;Das Gupta et al, 1993). Apart from providing information on the genes of M. tuberculosis that are strongly expressed by the bacterium, the promoters identified in this study may prove useful in the overexpression of foreign genes in mycobacteria to aid protein purification and vaccine vector development.…”

“…Alternatively, more generalized approaches have been used to identify a broad range of promoters, including those of a constitutive nature, through the use of reporter proteins and direct analysis of individual recombinant clones. These latter types of studies are hindered by the large number of recombinant colonies that need to be screened as the frequency of clones carrying true promoters is typically quite low (Timm et al, 1994a ;Das Gupta et al, 1993). While the majority of studies analysing mycobacterial transcription regulation have focused on promoters whose activity varies in response to defined environmental signals, it is of interest to selectively define strong constitutive expression signals of M. tuberculosis.…”

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

“…Initial experiments examined the functional activity of a range of previously characterized mycobacterial promoters in three mycobacterial cloning hosts and in E. coli. The promoters used in the present study have been employed by other workers for the development of mycobacterial expression vectors or in the study of gene regulation in mycobacteria (Murray et al, 1992 ;Das Gupta et al, 1993 ;Yasutomi et al, 1993 ;Dellagostin et al, 1995). Yet, to our knowledge no study has compared the functional activities of these promoters when cloned in a unique expression system and there is no information on their ability to regulate gene expression in M. vaccae.…”