A study of mycobacterial transcriptional apparatus: identification of novel features in promoter elements

Abstract

Our earlier studies on transcriptional signals of mycobacteria had revealed that (i) strong promoters occur less frequently in the slowly growing pathogen Mycobacterium tuberculosis H37Rv than in the fast-growing saprophyte M. smegmatis and (ii) mycobacterial promoters function poorly in Escherichia coli. We now present evidence that RNA polymerases of M. smegmatis, M. tuberculosis, and M. bovis BCG recognize promoter elements with comparable efficiencies. Analysis of these randomly isolated mycobacterial promoters by DNA sequencing, primer extension, and deletion experiments revealed that their ؊10 regions are highly similar to those of E. coli promoters, in contrast to their ؊35 regions, which can tolerate a greater variety of sequences, owing presumably to the presence of multiple sigma factors with different or overlapping specificities for ؊35 regions, as reported earlier for the Streptomyces promoters. A comparison of the ؊10 and ؊35 binding domains of MysA, HrdB, and RpoD (the principal sigma factors of M. smegmatis, Streptomyces aureofaciens, and E. coli, respectively) showed that all three sigma factors have nearly identical ؊10 binding domains. However, the ؊35 binding domains of the principal mycobacterial and streptomycete sigma factors, although nearly identical to each other, are vastly different from the corresponding region of the sigma factor of E. coli. Thus, the transcriptional signals of mycobacteria have features in common with Streptomyces promoters but differ from those of E. coli because of major differences in the ؊35 regions of the promoters and the corresponding binding domain in the sigma factor.The genus Mycobacterium includes pathogens that cause the highest number of bacterial infections and human deaths every year (2). Because of the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (7) and the increasing incidence of mycobacterial infections in human immunodeficiency virus-positive cases (4), tuberculosis has emerged as a major global threat to health. Despite their highly pathogenic nature, progress towards an understanding of gene structure and gene expression in mycobacteria has been slow. The lack of information on mycobacterial transcriptional signals has particularly impeded our understanding of the regulation of gene expression in these organisms.Only a few mycobacterial promoters have been studied. These include promoters for the 16S rRNA genes of M. smegmatis ( (21); and the three promoters responsible for transcribing the repressor-like gp71 protein of mycobacteriophage L5 (22). It is, however, difficult to assess the nature of transcriptional signals in mycobacteria by comparison of these promoter sequences, as many of these belong to specifically regulated genes and thus may not contribute towards the elucidation of constitutive gene expression in mycobacteria. The purpose of an unbiased study of mycobacterial transcriptional signals will be best achieved by analysis of randomly isolated promoters by using a promoter probe vector. We had earlier r...

Cloning and Characterization of the Promoters of Temperate Mycobacteriophage L1

“…1). The pSD5S30 vector was constructed by cloning a mycobacteria-specific promoter S30 into the multiple-cloning site of pSD5 (Bashyam et al, 1996;Jain et al, 1997). This newly-constructed promoter probe vector, pLG, can replicate in both E. coli and M. smegmatis, and showed very low lacZ activity in both species (Table 1).…”

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

“…In designing the vector pSD5C for expression of genes in mycobacteria, we have taken advantage of the fact that the majority of E. coli promoters function in mycobacteria albeit with efficiencies different from those in E. coli (Bashyam et al, 1996). In pSD5C, gene expression is under the transcriptional control of the Ptac promoter.…”

“…Protein samples for SDS-PAGE were prepared by addition of 4 × loading buffer to a final concentration of 1 × (50 mM Tris · HCl pH 6.8/l% SDS/1% β-mercaptoethanol/10% glycerol/0.1% bromophenol blue), boiled for 10 min, centrifuged at 12 000 × g for 10 min at room temperature, subjected to 0.1% SDS-7.5% PAGE and transferred to nitrocellulose membrane (Schleicher&Schuell, Keene, NH, USA) for probing with Ab. bovis BCG were separately transformed with pSD5C and pSD5C-CAT as described previously (Bashyam et al, 1996) and transformants were selected on Middlebrook 7H10…”

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

“…1). Mycobacterial promoters usually have well‐conserved ‐10 regions, while ‐35 are less conserved and hardly recognizable, probably because of the high number of sigma factors operating in these bacteria (Bashyam et al ., 1996; Newton‐Foot and Gey van Pittius, 2013; Manganelli, 2014). Moreover, several mycobacterial promoters present extended ‐10 regions (containing a TGn sequence at the 5′‐end) which increase their potency, and it was proposed that these promoters do not require the presence of a ‐35 sequence (Kenney and Churchward, 1996).…”