Molecular Characterization of the eis Promoter of Mycobacterium tuberculosis

Abstract

To further understand Mycobacterium tuberculosis pathogenesis, the regulation of potential virulence genes needs to be investigated. The eis gene of M. tuberculosis H37Rv enhances the intracellular survival of Mycobacterium smegmatis , which does not contain eis , within macrophages (J. Wei, J. L. Dahl, J. W. Moulder, E. A. Roberts, P. O'Gaora, D. B. Young, and R. L. Friedman, J. Bacteriol. 182: 377-384, 2000). Experiments were done to characterize the eis promoter in M. smegmatis and M. tuberculosis H37Ra. The putative −10 and −35 regions matched the Escherichia coli σ ⁷⁰ consensus 67 and 83%, respectively, making it a group A/ SigA -like mycobacterial promoter. Expression of site-directed variants of the core promoter region, determined by flow cytometry using gfp as a reporter, showed that the putative −10 region is essential for eis expression. In addition, site-directed alteration of the eis promoter to the consensus E. coli σ ⁷⁰ promoter elements increased gfp transcription to levels similar to that driven by the heat shock promoter, p hsp60 , of Mycobacterium bovis BCG. Upstream promoter deletion analysis showed that a 200- and 412-bp region of the promoter was necessary for maximum expression of gfp in M. smegmatis and M. tuberculosis H37Ra, respectively. Random mutagenesis of the 412-bp eis promoter, using a catechol 2,3-dioxygenase screen and activity assay, defined nucleotides upstream of the core promoter region that are essential to eis expression in both M. smegmatis and M. tuberculosis H37Ra, including a region homologous to a DinR cis element.