Figure 2. Control of gene expression by a C. acetobutylicum M-box RNA.

The Ca_c0685 and Ca_c3329 riboswitches were fused downstream of a constitutive promoter (PrpsD) and upstream of the yellow fluorescent reporter gene (yfp) to determine whether they could control heterologous gene expression in a divalent cation-dependent manner. Control strains either lacking the yfp reporter construct or containing a constitutive PrpsD-yfp fusion were included in this study. Cells were cultured to mid-logarithmic growth phase in 2xYT rich medium supplemented with 50 µM MgCl₂ (-), then incubated with 2 mM chelating agent, EDTA, or incubated in the presence of excess magnesium (2 mM) for one hour. Total RNA was assessed by staining of rRNA bands. Abundance of the yfp gene and of a zinc-responsive control transcript were monitored by S1 mapping. Radiolabeled DNA probes (Table S2) were used for S1 mapping of the yfp transcript.