Characterization of the Two Mycobacterium tuberculosis recA Promoters

Abstract

The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli 70 ؊35 elements but located much closer to the ؊10 element is important for optimal expression of P1, whereas the sequence at the ؊35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.The protein RecA is very important for a number of processes related to DNA metabolism and has been best characterized in Escherichia coli. It is a central component of recombination, whether by the RecBCD pathway or the RecF pathway (20). In either case, RecA forms a nucleoprotein filament on regions of single-stranded DNA and by its ability to simultaneously bind double-stranded DNA performs the search for homologous sequences. Strand exchange then initiates the actual process of recombination. RecA also has a key regulatory role in the response to DNA damage, owing to the ability of the nucleoprotein filament formed on regions of single-stranded DNA to stimulate the cleavage of the repressor protein LexA (15,25). Under normal conditions, LexA binds to a specific sequence upstream of the genes it regulates, which include recA and lexA themselves, and inhibits their expression (4, 26); however, following cleavage, the fragments of LexA formed do not effectively bind to this site (3), permitting increased transcription. As well as regulating the expression of other genes following DNA damage, RecA has a direct role in recombinational DNA repair which is particularly important for replication restart following the collapse of a replication fork, for example, when it reaches a damaged segment of DNA (7). Although the majority of studies on RecA function have been conducted with the E. coli protein, RecA is highly conserved across bacterial species (18, 34). Thus, it appears most likely that it performs equivalent functions in all bacteria.In many species of bacteria, expression of RecA is induced following DNA damage. In the majority of these cases this process is regulated by a homolog of LexA, as in E. coli (12), although the sequence to which the LexA homolog binds varies between species. In both E. coli and the well-studied grampositive bacterium Bacillus subtilis, recA is expressed from a single promoter (6, 39). It was recently ...

Genetics ofStreptomyces rimosus, the Oxytetracycline Producer

“…Ahel and collaborators (2) proposed a Ϫ35 box (TTGTCA) and Ϫ10 box (TAGCGT) that are only 11 bp apart. They noticed that regulatory sequences of this putative promoter and a part of the spacer region are almost identical to those of the DNA damage-inducible promoter of the Mycobacterium tuberculosis recA gene (162), which has been well characterized (76). This sequence is perfectly conserved in all four recA genes analyzed from Streptomyces to date (4,153,166,236).…”

Expression of the Mycobacterium tuberculosis acr -Coregulated Genes from the DevR (DosR) Regulon Is Controlled by Multiple Levels of Regulation

“…It has been noted by others that the distance between the Ϫ10 and Ϫ35 regions is not critical in mycobacterial promoters (24) and varies between 13 and 24 bp (12,27,35). In contrast, the spacing between the Ϫ10 and Ϫ35 regions in most E. coli promoters is 17 Ϯ 1 bp (16).…”

The Mycobacterium-Specific Gene Rv2719c Is DNA Damage Inducible Independently of RecA

“…Comparison of the data in Fig. 4b and 4c suggests that the activity of this promoter is enhanced in the presence of additional DNA from upstream of the gene, as has been found previously for the M. tuberculosis recA promoters (12). Nevertheless, the contribution made by such a promoter to the expression of Rv2719c appears to be small under DNA-damaging conditions.…”

“…However, in this case there was a stronger signal for the smaller band; thus, it is possible that the changes intro- Promoter element identification using transcriptional fusions. Upstream of the inducible transcription start site, there are motifs similar to those originally identified at the recA P1 promoter (12), which is also DNA damage inducible independently of RecA (5), and subsequently found upstream of other M. tuberculosis DNA damage-inducible genes (10). Although the smaller primer extension product appeared most likely to be an artifact from the above analyses, inspection of the DNA sequence upstream of this potential start site identified motifs resembling the E. coli 70 promoter elements (Fig.…”