A Riboswitch-Based Inducible Gene Expression System for Mycobacteria

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

Linderos sociales y culturales para salud mental comunitaria

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R. Brunner, R. Kausen & M. Titze (Eds.): Worterbuch der Individualpsychologie (R. L.) 58.- Centro de Rehabilitación de Alcohólicos (Ed.). Memorias del III Congreso Iberoamericano sobre Alcohol y Alcoholismo (C. Sogi) 59.- T. Crooks, R. Bartus, S. Ferris & S. Gershon (Eds.): Treatment Development strategies for Alzheimer's Disease (A. Arregui) 59.- Ch. Desrouesne: Practique Neurologique (L. Trelles) 60.- U. Geuter (Ed.): Daten zur geschichte der Deutschen Psychologie (Vol. 1: Psychologische Institute, Fachgesellschaften, Fachzeitschriften und Serien, Biographien, Emigranten, 1879-1945) (R. Leon) 60. - Roger Guerra·Garcia (Ed.). Problemas poblacionales peruanas II. (c. Sogi) 61.- L. J. Pongralz: Problemgeschichte der Psychologie (R. Leon) 62.- Humberto Roselli: La Locura de Epifanio y otros ensayos (J. Mariategui) 63.- D. J. Weatherall, J. C. Ledingham & D. A. Warrell (Eds.). Oxford Textbook of Medicine (L Trelles) 64

Theophylline controls endogenous KatG expression and restores sensitivity to isoniazid.

<p>(A) A single recombination event between the <i>Msmeg</i> chromosome and a plasmid containing the promoter-riboswitch combination and 500 bp of KatG yields the RiboS-<i>katG</i> strain containing a single full-length copy of <i>katG</i> under riboswitch control. The positive control for wild-type (<b>1</b>) and RiboS-katG (<b>3</b>) corresponds to the first 777 bp of <i>katG</i>. A primer specific to the promoter-riboswitch yields the predicted 1065-bp product from RiboS-<i>katG</i> (<b>4</b>), but not the wild type (<b>2</b>), confirming the recombination. (B) The isoniazid EC₅₀ for <i>Msmeg</i> wild type (open circles) and RiboS-<i>katG</i> (filled squares) was measured in response to 0–10 mM theophylline. Data are presented as mean ± SEM of three independent experiments. (<i>inset</i>) The anti-KatG immunoblot for <i>Msmeg</i> wild type and RiboS-<i>katG</i> shows the response to 0–5 mM theophylline after 6 h. The GroEL immunoblot serves as a loading control, and data are representative of two independent experiments.</p

Theophylline riboswitch-controlled gene induction is reversible.

<p>(A) GFP fluorescence as a function of time in 0 mM (open) or 2 mM (filled) theophylline for <i>Msmeg</i> (circles) and <i>Mtb</i> (squares) harboring ribo-gfp. <i>Msmeg</i> vector and <i>Mtb</i> wild -type controls are shown as triangles and diamonds. Doubling times for <i>Msmeg</i> and <i>Mtb</i> are approximately 3 and 24 h, respectively. Data are presented as mean ± SEM of three independent experiments. GFP fluorescence from <i>Msmeg</i>::ribo-gfp and vector control strains was (B) monitored over time and (C) analyzed by flow cytometry after incubation with (+) or without (−) 2 mM theophylline. Theophylline was maintained or removed by media exchange after 1.3 doubling times (4 h; arrow). Kinetic data are presented as the mean ± SEM of eight replicates for each sample and are representative of three independent experiments. (D) Immunoblot analysis shows GFP induction in <i>Mtb</i> whole-cell lysates after incubation in 2 mM theophylline for one and two days (<i>top</i>). On day 2, theophylline was maintained (+) or removed by media exchange (−) and grown for an additional two days (<i>bottom</i>). Band intensities were corrected for background, and GFP signal was normalized against the GroEL loading control.</p

Theophylline induces riboswitch-mediated gene expression in <i>Msmeg</i> and <i>Mtb</i>.

<p>(A) Riboswitch-controlled GFP fluorescence in <i>Msmeg</i> (filled circles) and <i>Mtb</i> (filled squares) and β-galactosidase activity in <i>Msmeg</i> (filled triangles) in response to incubation in 0–5 mM theophylline for 6 h. Empty vector negative controls for GFP fluorescence and β-galactosidase activity are shown as open circles and triangles. Data are presented as relative fluorescence (RFU) for GFP and in Miller units for β-galactosidase, and as the mean ± SEM of three independent experiments. (B) Flow cytometry analysis of riboswitch-controlled GFP expression in <i>Msmeg</i> treated for 6 h with varying concentrations of theophylline. The empty vector control is shown in black. Results are representative of three or more independent experiments. (C) Immunoblot analysis of whole-cell lysates from <i>Mtb</i> harboring ribo-gfp, empty vector, or Phsp60-gfp positive control constructs. Band intensities were corrected for background, and GFP signal was normalized against the GroEL loading control.</p