Identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress-response sigma factor σF

Abstract

A previously optimized Escherichia coli two-plasmid system was used to identify Mycobacterium tuberculosis promoters recognized by RNA polymerase containing the M. tuberculosis stress response sigma factor sigma(F). The method allowed the identification of five new sigma(F)-dependent promoters. Transcriptional start points of the promoters were determined by high-resolution S1-nuclease mapping using RNA prepared from E. coli containing the two-plasmid system. The promoters were confirmed by an in vitro transcription assay. The Mycobacterium smegmatis and Mycobacterium tuberculosis core RNA polymerases, after complementation with sigma(F), were able to recognize all the five promoters. All the promoters contained sequences highly similar to the sequence of the previously identified M. tuberculosis sigma(F)-dependent promoter, usfXp1. Comparison of the promoters revealed a sigma(F) consensus sequence GtTtga-N(14-18)-GGGTAT. The sigma(F)-dependent promoters may govern expression of genes encoding a transcription regulator homologous to the response regulators of bacterial two-component signal transduction systems and proteins with unknown function.

Mycobacterium tuberculosis SigF Regulates Genes Encoding Cell Wall-Associated Proteins and Directly Regulates the Transcriptional Regulatory Gene phoY1

“…In addition, we detected significant upregulation of Rv2884, which was shown biochemically to have a sigF-dependent promoter (15). Induction of sigF also resulted in significantly increased expression of a putative operon containing Rv1823, Rv1824, Rv1825, and Rv1826 that has been shown to be under the control of the SigF-dependent Rv1823 promoter (15). Finally, the sigF-inducible strain showed increased expression of phoY1 (Rv3301c) and Rv3300c, which appear to be members of an operon.…”

“…This study provides the first evidence that the Rv2884 gene and the Rv1823 operon, which have sigF-dependent promoters (15), are, in fact, positively regulated by SigF in vivo. The Rv2884 gene encodes a probable transcriptional regulator with significant sequence similarity to many response regulators of bacterial two-component signal transduction systems.…”

“…Among these genes were members of the mycobacterial membrane large (mmpL) family, including mmpL2, mmpL5, and mmpL11, which encode proteins involved in transmembrane transport, and which are virulence factors promoting in vivo survival of M. tuberculosis (5,19). In addition, we detected significant upregulation of Rv2884, which was shown biochemically to have a sigF-dependent promoter (15). Induction of sigF also resulted in significantly increased expression of a putative operon containing Rv1823, Rv1824, Rv1825, and Rv1826 that has been shown to be under the control of the SigF-dependent Rv1823 promoter (15).…”

“…Therefore, the SigF-dependent promoter sequence of phoY1 appears to be GGATTG-N 16 -GGGTAT. We identified a total of four genes that have been shown to be transcribed by SigF in vitro, including usfX (2), Rv1823, Rv2884 (15), and phoY1 (this study), which were also found to be more highly expressed in vivo following overexpression of sigF (this study). Based on knowledge of the usfX promoter sequence (2), we examined the 5Ј untranslated regions of the other three genes for sequence similarity in order to discern a sigF promoter consensus sequence.…”

“…A list of 51 such genes was identified and then compared to the list of genes found to be significantly regulated at least at two consecutive time points by microarray analysis following sigF induction. This comparison yielded a group of nine genes, including Rv1823, which previously was shown to have a SigFdependent promoter (15), as well as mmpL11 and phoY1. Because expression of phoY1 was markedly increased following sigF induction, in vitro transcriptional analysis was used to determine if SigF directly regulates phoY1.…”