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

Biosynthesis and Regulation of Sulfomenaquinone, a Metabolite Associated with Virulence in <i>Mycobacterium tuberculosis</i>

Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the <i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase <i>stf3</i>, includes a putative cytochrome P450 gene (<i>cyp128</i>) and a gene of unknown function, <i>rv2269c</i>. We demonstrate that <i>cyp128</i> and <i>stf3</i> are sufficient for the biosynthesis of SMK from menaquinone and <i>rv2269c</i> exhibits promoter activity in <i>M. tuberculosis</i>. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in <i>M. tuberculosis</i>. Finally, we showed in a mouse model of infection that the loss of <i>cyp128</i> exhibits a hypervirulent phenotype similar to that in previous studies of the <i>stf3</i> mutant. These findings provide a platform for defining the molecular basis of SMK’s role in <i>M. tuberculosis</i> pathogenesis

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-controlled <i>Mtb</i> gene expression in a macrophage infection model.

<p>Murine macrophage-like RAW 264.7 cells infected with (A) <i>Mtb</i> wild type or (B) <i>Mtb</i>::ribo-gfp were induced with 0 mM or 0.5 mM theophylline for 24 h. Overlaid fluorescence signals from DAPI and GFP channels show nuclei (blue) and GFP-expressing bacteria (green). Panels on right show additional DIC light microscopy overlay. Scale bar represents 10 µm. Images are representative of three independent experiments for each condition.</p

A theophylline-responsive riboswitch variant exerts translational control of gene expression.

<p>A synthetic theophylline-responsive riboswitch variant adopts a fold that sequesters the ribosome binding site (RBS) in the mRNA transcript. In the presence of theophylline, the riboswitch adopts a conformation in which the aptamer is bound to theophylline. The RBS is then released and able to promote protein translation. (The sequence for riboswitch E′ from ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029266#pone.0029266-Topp1" target="_blank">[21]</a> is depicted.)</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

The reaction catalyzed by Rv3406.

<p>The alpha carbon of an alkyl sulfate is oxidized by Rv3406 in the presence of α-ketoglutarate and spontaneously collapses to an aldehyde and sulfate, liberating CO₂ and succinate. The product formation was monitored using a coupled assay using LADH to reduce the aldehyde in a NADH dependent manner.</p

Protein alignment of alkyl sulfatase enzymes with taurine dioxygenase enzymes.

<p><b>Enzymes in bold have been biochemically characterized.</b> (A) Alignment of disordered loop 1 where the red boxes are indicating the taurine binding residues in taurine dioxygenases and the analogous amino acids in alkyl sulfatase enzymes. (B) Alignment of disordered loop 2 where the red box is indicating the conserved phenylalanine in taurine dioxygenases and the analogous tyrosine in alkyl sulfate enzymes.</p

Rv3406 is essential in Mtb for growth on 2-EHS as the sole sulfur source.

<p>(A) Growth of Mtb strains using either 2-EHS alone or 2-EHS with sodium sulfate. (B) Growth of Mtb strains on <i>n</i>-heptyl sulfate or SDS. Data represents three biological replicates and error bars denote standard deviation. Asterisk indicates a p value of less than 0.005.</p

Biochemical characterization of Rv3406.

<p>(A) Rv3406 is an αKG and ascorbate dependent sulfatase. Black squares are the complete assay with Rv3406, 2-EHS, αKG, ascorbate and iron. Red triangles are without 2-EHS. Blue circles are without αKG. (B) Rv3406 is an iron dependent enzyme. (C) The rate of Rv3406 accelerates with the addition of ascorbate up to 1 mM. Rv3406 enzyme concentration was between 0.5 and 0.75 µM for all experiments.</p