Benzoic Acid-Inducible Gene Expression in Mycobacteria

<div><p>Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the <i>Pseudomonas putida</i>-derived positively regulated XylS/<i>Pm</i> expression system to control inducible gene expression in <i>Mycobacterium smegmatis</i> and <i>Mycobacterium tuberculosis</i>, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/<i>Pm-</i>regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer <i>m</i>-toluate, we achieve a robust, time- and dose-dependent reversible induction of <i>Pm</i>-mediated expression in mycobacteria, with low background expression levels. XylS/<i>Pm</i> is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.</p></div

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Benzoic acid-inducible expression system, XylS/<i>Pm</i>, for regulation of genes in mycobacteria.

<p>The inducible <i>Pm</i> promoter and its activator XylS regulate the expression of “your favorite gene” (YFG). XylS is constitutively expressed under control of <i>Ptet</i> in the absence of anhydro-tetracycline (atc) and binds the <i>Pm</i> promoter in the presence of the inducer <i>m</i>-toluate. This facilitates expression of YFG and leaves the expression system ON (upper panel). In the absence of <i>m</i>-toluate, XylS is not activated, leaving the expression system OFF, as expression from <i>Pm</i> is not induced (middle panel). Reverse TetR is constitutively expressed by <i>Psmyc</i>, and binds the operator in <i>Ptet</i> in the presence of atc blocking transcription of <i>xylS</i>. Addition of atc leaves the system in a more fully OFF mode as potential basal <i>Pm</i>-mediated transcription caused by excessive levels of XylS is abolished (lower panel).</p

<i>Pm</i>–mediated basal expression is low and compares favorably to <i>Ptet-</i>mediated basal expression in <i>Mtb</i>.

<p>(A-B) <i>Mtb</i> transformed with pMDX-luc or pUV15tetORm::luciferase was diluted to OD₆₀₀ 0.005 in the presence or absence of m-toluate (1.5 mM) or atc (200 ng/ml), respectively. Samples were grown in triplicates and monitored for 7 days registering OD₆₀₀ at 2, 4 and 7 days. Basal expression from <i>Pm</i> and <i>Ptet</i> is presented by level of luciferase produced by <i>Mtb</i> pMDX-luc and <i>Mtb</i> pUV15tetORm::luciferase. (A) shows the luciferase expression over time in induced sample and (B) shows the basal expression from uninduced samples over time. The results represent two independent experiments.</p

<i>Pm</i>–mediated basal expression is low and compares favorably to <i>Ptet</i>-mediated basal expression in <i>Msmeg</i>.

<p>(A-B) <i>Msmeg</i> transformed with pMDX-zeo or pTET-zeo was diluted to OD₆₀₀ 0.005 in the presence or absence of m-toluate (1.5 mM) or atc (200 ng/ml), respectively, and increasing amounts of zeocin (0, 0.5, 2.5, 5, 7.5, 10, 15, 25, 50, 100, 150, 200, 250 or 500 μg/ml). Samples were grown in triplicates and monitored for 120 hours by a Bioscreen, registering OD₆₀₀ every other hour. Basal expression from <i>Pm</i> and <i>Ptet</i> is presented by growth of <i>Msmeg</i> pMDX-zeo and <i>Msmeg</i> pTET-zeo in increasing concentrations of zeocin, when the respective sample grown in the <i>absence</i> of zeocin reached mid log phase (A) or stationary phase (B). (C) Induced and uninduced samples of pMDX-zeo strain in late log phase. (D) Induced and uninduced samples of pTET-zeo strain in mid log phase. The results represent two independent experiments.</p

Induction of <i>Pm</i> with <i>m</i>-toluate is robust, time- and dose-dependent.

<p><i>Msmeg</i> transformed with the expression vectors pMDX-luc or the empty vector pMDX (no reporter gene) treated with increasing concentrations of <i>m</i>-toluate (induced) or ethanol carrier (uninduced). Cells were incubated at 30°C, and luciferase expression was determined at 2.5, 5.5, 11, 23, 31 and 49 hours after addition of <i>m</i>-toluate. (A) Fold induction of RLU in induced samples compared to uninduced samples. (B) Growth of uninduced and induced samples of pMDX-luc corresponding to samples in (A). (C) Time course of luciferase induction from pMDX-luc with 1.5 mM <i>m</i>-toluate or ethanol carrier. (D) Maximal induction of pMH109 and pMDX-luc-transformed <i>Msmeg</i> induced with 2 mM <i>m</i>-toluate. (E) Amount of luciferase produced as determined by the activity of known luciferase concentrations (0, 0.1, 0.2, 0.4 0.6, 0.8 and 1 μg/ml luciferase) in mid log phase or (F) stationary phase. Luciferase fraction of total bacterial protein shown in brackets. RLUs were normalized to the OD₆₀₀ of the samples before luciferase assay. All results are representative of two or more independent experiments.</p

Regulation of the zeocin resistance gene in the presence of zeocin.

<p>(A) <i>Msmeg</i> transformed with pMDX-zeo was grown to OD₆₀₀ 0.05–0.1 before addition of 1.5 mM <i>m</i>-toluate (induced) or ethanol carrier (uninduced) was added. Cells were then incubated for 5 hours at 30°C. The cells were normalized by OD₆₀₀, serially diluted, and spotted on plates containing increasing amounts of zeocin and 1.5 mM <i>m</i>-toluate (induced) or ethanol (uninduced) and incubated at 30°C for 2 days. (B) <i>Msmeg</i> transformed with pMDX-zeo was pre-induced for 5 hours as described above, then diluted to OD₆₀₀ 0.005 and grown in triplicates in micro-plate wells in the presence of increasing concentrations of zeocin (0, 0.5, 2.5, 5, 7.5, 10, 15, 25, 50, 100, 150, 200 and 250 μg/ml) and 1.5 mM <i>m</i>-toluate (induced) or ethanol carrier (uninduced) shaking at 37°C. Growth was monitored by Bioscreen, registering OD₆₀₀ every other hour. The samples are presented by the OD₆₀₀ of uninduced or induced <i>Msmeg</i> pMDX-zeo in increasing concentrations of zeocin, when the respective sample grown in the <i>absence</i> of zeocin reached mid log phase. Error bars represent standard deviations and the results represent three independent experiments.</p

Induction of <i>Pm</i> in <i>Mtb</i>.

<p><i>Mtb</i> transformed with the expression vector pMDX-luc or the empty vector pMDX were induced with indicated concentrations of <i>m</i>-toluate (induced), or ethanol carrier (uninduced). Samples were maintained rolling at 37°C and analyzed for luciferase expression levels at 2, 3, 4 and 6 days after induction. (A) Fold induction of RLU of induced to uninduced samples normalized for OD₆₀₀. (B) Growth of uninduced and induced samples containing pMDX-luc corresponding to samples in (A). The results are representative for two independent experiments.</p