Functional Analysis of Promoters, mRNA Cleavage, and mRNA Secondary Structure on esxB-esxA in Mycolicibacterium smegmatis

The ESX-1 secretion system is critical for the virulence of <i>Mycobacterium tuberculosis</i> as well as for conjugation in the saprophytic model <i>Mycolicibacterium smegmatis</i>. EsxB (CFP-10) and EsxA (ESAT-6) are secreted effectors required for the function of ESX-1 systems. While some transcription factors regulating the expression of <i>esxB</i> and <i>esxA</i> have been identified, little work has addressed their promoter structures or other determinants of their expression. Here, we defined two promoters, one located two genes upstream of <i>esxB</i> and one located immediately upstream, that contribute substantially to the expression of <i>esxB</i> and <i>esxA</i>. We also defined an mRNA cleavage site within the <i>esxB</i> 5' untranslated region (UTR) and found that a single-nucleotide substitution reprogramed the position of this cleavage event without impacting <i>esxB</i>-<i>esxA</i> transcript abundance. We furthermore investigated the impact of a double stem-loop structure in the <i>esxB</i> 5' UTR and found that it does not confer stability on a reporter gene transcript. Consistent with this, there was no detectable correlation between mRNA half-life and secondary structure near the 5' ends of 5' UTRs on a transcriptome-wide basis. Collectively, these data shed light on the determinants of <i>esxB-esxA</i> expression in <i>M. smegmatis</i> as well as provide broader insight into the determinants of mRNA cleavage in mycobacteria and the relationship between 5' UTR secondary structure and mRNA stability.