Synthetic riboswitches that induce gene expression in diverse bacterial species

Abstract

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.

FIG. 1.

(A) Bacterial phylogeny. The species studied here are shown in the ovals. (B) Activation ratios and expression levels for riboswitches A to E in each organism. Right axes: expression levels in the absence of theophylline (open circles) and in the presence of theophylline (2 mM; closed circles). Measurements are of β-galactosidase activity in Miller units (20) for all organisms, except for S. pyogenes (β-glucuronidase [GUS] activity in GUS units [14]) and M. smegmatis (normalized fluorescence of GFP [8]). Errors are smaller than the symbol size. Left axes: activation ratios of the riboswitches, which were determined by dividing expression levels in the presence of theophylline by expression levels in the absence of theophylline.

FIG. 2.

Images of riboswitches B and C controlling the expression of a MamK-GFP fusion in M. magneticum in the presence of theophylline (1 mM) as a function of time. Left panels are phase-contrast images; right panels monitor GFP fluorescence emission (2-μm scale bar). All fluorescent images were exposed for 6 s, and the cells were visualized with a 100× objective.