A Riboswitch-Based Inducible Gene Expression System for Mycobacteria

Jessica C Seeliger,Justin P Gallivan,Kimberly M Sogi,Carolyn R Bertozzi,Mary L Previti,Shana Topp,Olivier Neyrolles

doi:10.1371/journal.pone.0029266

Abstract

Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

Highlights

Tools for manipulating gene expression are fundamental to genetic studies
We have recently reported a series of theophylline-responsive riboswitches that can control gene expression in a range of Gramnegative and Gram-positive bacteria, including M. smegmatis (Msmeg), a fastgrowing, non-pathogenic species that is a widely used model system for Mycobacterium tuberculosis (Mtb) [21]
We further determined that the riboswitch functions as a titratable system, like the Tn10 tetracycline repressor-based (TetR) repressor, rather than as a bistable switch, like the nitrile-inducible system [14]

Summary

Introduction

Tools for manipulating gene expression are fundamental to genetic studies. Usually under the control of a small molecule, are useful because they permit exquisite experimental control over both the dose and timing of gene expression. Inducible promoters are widely used to silence genes via direct transcriptional control or antisense methodologies and to overexpress proteins for biochemical and structural studies [1]. Several inducible expression systems exist for Gramnegative bacteria, adaptation to distantly related bacteria has proven difficult. Species that lack diverse regulated expression tools include the mycobacteria, among them Mycobacterium tuberculosis (Mtb), which causes tuberculosis in humans, and species that are commonly used as models for Mtb such as the fish and amphibian pathogen M. marinum, the non-pathogenic M. smegmatis (Msmeg), and the vaccine strain M. bovis BCG [5]. Unique challenges inherent to the biology of these medically relevant organisms, such as their pathogenesis, slow growth rate, and inefficient DNA uptake, have significantly hindered molecular genetics studies [6]

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