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

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 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

Compartment-Specific Labeling of Bacterial Periplasmic Proteins by Peroxidase-Mediated Biotinylation

The study of the bacterial periplasm requires techniques with sufficient spatial resolution and sensitivity to resolve the components and processes within this subcellular compartment. Peroxidase-mediated biotinylation has enabled targeted labeling of proteins within subcellular compartments of mammalian cells. We investigated whether this methodology could be applied to the bacterial periplasm. In this study, we demonstrated that peroxidase-mediated biotinylation can be performed in mycobacteria and <i>Escherichia coli</i>. To eliminate detection artifacts from natively biotinylated mycobacterial proteins, we validated two alternative labeling substrates, tyramide azide and tyramide alkyne, which enable biotin-independent detection of labeled proteins. We also targeted peroxidase expression to the periplasm, resulting in compartment-specific labeling of periplasmic versus cytoplasmic proteins in mycobacteria. Finally, we showed that this method can be used to validate protein relocalization to the cytoplasm upon removal of a secretion signal. This novel application of peroxidase-mediated protein labeling will advance efforts to characterize the role of the periplasm in bacterial physiology and pathogenesis

A Screen for Protein–Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A

Outer membrane lipids in pathogenic mycobacteria are important for virulence and survival. Although the biosynthesis of these lipids has been extensively studied, mechanisms responsible for their assembly in the outer membrane are not understood. In the study of Gram-negative outer membrane assembly, protein–protein interactions define transport mechanisms, but analogous interactions have not been explored in mycobacteria. Here we identified interactions with the lipid transport protein LprG. Using site-specific photo-cross-linking in live mycobacteria, we mapped three major interaction interfaces within LprG. We went on to identify proteins that cross-link at the entrance to the lipid binding pocket, an area likely relevant to LprG transport function. We verified LprG site-specific interactions with two hits, the conserved lipoproteins LppK and LppI. We further showed that LprG interacts physically and functionally with the mycolyltransferase Ag85A, as loss of either protein leads to similar defects in cell growth and mycolylation. Overall, our results support a model in which protein interactions coordinate multiple pathways in outer membrane biogenesis and connect lipid biosynthesis to transport

A Screen for Protein–Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A

Outer membrane lipids in pathogenic mycobacteria are important for virulence and survival. Although the biosynthesis of these lipids has been extensively studied, mechanisms responsible for their assembly in the outer membrane are not understood. In the study of Gram-negative outer membrane assembly, protein–protein interactions define transport mechanisms, but analogous interactions have not been explored in mycobacteria. Here we identified interactions with the lipid transport protein LprG. Using site-specific photo-cross-linking in live mycobacteria, we mapped three major interaction interfaces within LprG. We went on to identify proteins that cross-link at the entrance to the lipid binding pocket, an area likely relevant to LprG transport function. We verified LprG site-specific interactions with two hits, the conserved lipoproteins LppK and LppI. We further showed that LprG interacts physically and functionally with the mycolyltransferase Ag85A, as loss of either protein leads to similar defects in cell growth and mycolylation. Overall, our results support a model in which protein interactions coordinate multiple pathways in outer membrane biogenesis and connect lipid biosynthesis to transport

Rv1410 function is necessary for growth of <i>Mtb</i> in mice with immune deficiencies, indicating that loss of virulence is intrinsic to the bacterium.

<p>Colony forming units (cfu) of WT (H37Rv, black) and <i>rv1410</i>::Tn (Mut1, red) from spleen and lungs of mice infected with a 3:1 (Mut1:WT) mixture. (A) C57/Bl6 (B) <i>nos2-/-</i> (C) <i>phox-/-</i> (D) <i>phox-/-</i> fed aminoguanidine <i>ad libitum</i>, and (E) <i>inf-γ -/-</i> mice. Cfu were measured in lung and spleen at 1, 7, and 30 days post infection. Error bars show mean +/- SD of three to five animals per time point.</p

Model for the role of LprG-Rv1410-mediated TAG transport in regulating the growth rate of <i>Mtb</i>.

<p>(A) In the presence of a functional LprG-Rv1410, FasI makes acyl primers (acyl-CoA) for cell wall lipid synthesis (including TAG via TAG synthases, <i>Tgs)</i>. TAG can then be 1) incorporated into the <i>Mtb</i> cell wall via the action of LprG-Rv1410 or 2) hydrolyzed by lipases. Acetyl-CoA for anapleurosis of the TCA cycle is generated via β-oxidation of free fatty acids (FFA) liberated from TAG. (B) Loss of LprG-Rv1410 function results in increased intracellular TAG due to lack of transport into the cell wall. The presence of accumulated intracellular TAG may inhibit growth by a currently unknown mechanism. Decreasing lipolysis via lipase inhibitors further increases intracellular TAG levels. Addition of acetate, glycerol, or FFA bypasses the need for lipolysis by providing acetyl-coA that feeds into the TCA cycle and relieves growth arrest.</p