Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</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

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

<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

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

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

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

Phosphorylation regulates the rate of cell elongation.

<p><b>(A)</b> Phospho-transfer profiling with the kinase domains of the major serine-threonine protein kinases of <i>M</i>. <i>tuberculosis</i> (<i>Mtb</i> Pkn) reveals that PknB efficiently phosphorylates <i>Mtb</i> MBP-PonA1_cyto. <b>(B)</b><i>M</i>. <i>smegmatis</i> cells that express a T50A allele of <i>ponA1</i> (134 cells; approximate p-value = 0.0145 by the Kolmogorov-Smirnov test) are longer than isogenic wildtype cells (219 cells), while cells that express a T50D allele (139 cells; approximate p-value = 0.0082 by the Kolmogorov-Smirnov test) are shorter than isogenic wildtype, suggesting PonA1’s phosphorylation regulates cell elongation or division. <b>(C)</b> Timelapse microscopy revealed that cells that expressed a T50A (127 cells; approximate p-value = 0.0002 by the Kolmogorov-Smirnov test) allele elongated faster than isogenic wildtype cells (174 cells), which was phenocopied by a truncation of the cytoplasmic tail (<i>Δ</i>cyto; 202 cells; approximate p-value < 0.0001 by the Kolmogorov-Smirnov test). These data suggest that PonA1’s phosphorylation negatively regulates cell elongation. <b>(D)</b> Similarly, phosphorylation status of PonA1 affects total cell length in <i>M</i>. <i>tuberculosis</i>. Cells that expressed a T34A allele (211 cells; approximate p-value = 0.0066 by the Kolmogorov-Smirnov test) exhibited an average cell length 5% longer than isogenic wildtype (202 cells), and cells that expressed a T34D allele (207 cells; approximate p-value < 0.0001 by the Kolmogorov-Smirnov test) were 11% shorter than isogenic wildtype. This suggests that PonA1’s unusual phosphorylation negatively regulates cell elongation in <i>M</i>. <i>tuberculosis</i> as in <i>M</i>. <i>smegmatis</i>. <b>(E)</b><i>Msm</i> cells that encode a T50A,TP- allele of PonA1 are defective for normal cell separation. These cells form short chains of cells with multiple septa (white arrows). These data suggest that PonA1’s phosphorylation may regulate PonA1 TG activity, the remaining functional catalytic activity for this allele, and that alterations to PonA1’s TG activity impact the cell’s peptidoglycan and consequent cleavage of that peptidoglycan.</p

A model for how PonA1 promotes cell elongation in mycobacteria.

<p><b>(A)</b> We propose a model wherein PonA1 localizes early to the growth tip and promotes pole elongation through PG synthesis. PonA1 may recruit other factors to form the elongation complex or they are recruited independently of PonA1 (factors not shown). Together with these factors, polar elongation proceeds (orange cells). PonA1 furthers cell elongation through its synthesis of PG. PonA1’s synthesis of glycan strands (colored subunits, panels 1–3) and its crosslinking of those glycan strands (black lines, panels 2–3) promote normal cell length. These catalytic activities are potentially modulated through PonA1’s phosphorylation (arrow), which acts to regulate the rate of cell elongation. <b>(B)</b> Excess PonA1 stimulates ectopic cell elongation (orange cells). The frequency of ectopic pole formation increases with imbalanced PG crosslinking and may result from changes in local peptidoglycan architecture (panels 1–2, factors removed from panel 2 for clarity). These architectural changes may act like a sink that ‘recruits’ additional protein-interactors of PonA1 or additional elongation complex components to spur cell elongation at ectopic sites (panel 3, dark gray factors).</p