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

The rv1184c Locus Encodes Chp2, an Acyltransferase in Mycobacterium tuberculosis Polyacyltrehalose Lipid Biosynthesis

Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates

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 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 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-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

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

ABSTRACT Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but also by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mycobacterium tuberculosis (Mtb) genes. We explored the role of codon pair bias by recoding Mtb genes (rpoB, mmpL3, and ndh) and assessing their expression in the closely related and tractable model organism M. smegmatis. To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which, in turn, led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation. IMPORTANCE Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites

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