Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome

Silencing Essential Protein Secretion in Mycobacterium smegmatis by Using Tetracycline Repressors▿

Many processes that are essential for mycobacterial growth are poorly understood. To facilitate genetic analyses of such processes in mycobacteria, we and others have developed regulated expression systems that are repressed by a tetracycline repressor (TetR) and induced with tetracyclines, permitting the construction of conditional mutants of essential genes. A disadvantage of these systems is that tetracyclines function as transcriptional inducers and have to be removed to initiate gene silencing. Recently, reverse TetR mutants were identified that require tetracyclines as corepressors. Here, we report that one of these mutants, TetR r1.7, allows efficient repression of lacZ expression in Mycobacterium smegmatis in the presence but not the absence of anhydrotetracycline (atc). TetR and TetR r1.7 also allowed efficient silencing of the essential secA1 gene, as demonstrated by inhibition of the growth of a conditional mutant and dose-dependent depletion of the SecA1 protein after the removal or addition, respectively, of atc. The kinetics of SecA1 depletion were similar with TetR and TetR r1.7. To test whether silencing of secA1 could help identify substrates of the general secretion pathway, we analyzed the main porin of M. smegmatis, MspA. This showed that the amount of cell envelope-associated MspA decreased more than 90-fold after secA1 silencing. We thus demonstrated that TetR r1.7 allows the construction of conditional mycobacterial mutants in which the expression of an essential gene can be efficiently silenced by the addition of atc and that gene silencing permits the identification of candidate substrates of mycobacterial secretion systems

Disruption of an <i>M. tuberculosis</i> Membrane Protein Causes a Magnesium-dependent Cell Division Defect and Failure to Persist in Mice

<div><p>The identification of <i>Mycobacterium tuberculosis</i> genes necessary for persistence <i>in vivo</i> provides insight into bacterial biology as well as host defense strategies. We show that disruption of <i>M. tuberculosis</i> membrane protein PerM (Rv0955) resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant’s near normal growth during acute infection. The <i>perM</i> mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the <i>perM</i> mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in <i>M. smegmatis</i>. The mutant was acutely sensitive to β-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives <i>M. tuberculosis</i> of magnesium.</p></div

<i>PerM</i>::tn induces a hyperinflammatory cytokine response in mouse macrophages.

<p>(<b>A</b>-<b>D</b>) Cytokine content in cell culture supernatant of wt BMDM exposed for 24 hours to: (<b>A</b>) live Mtb at multiplicity of infection (MOI) 10; (<b>B</b>) live wt, <i>perM</i>::tn, or an equal mix of wt and <i>perM</i>::tn at total MOI of 10; (<b>C</b>) formalin-killed Mtb at MOI 20; (<b>D</b>) sterile, Mtb-conditioned media from 8 day old cultures. Cytokines/chemokines were not detectable in uninfected BMDM. (<b>E</b>) TNF production of a NOD1/NOD2^-/- cell line derived from B6 mice (NOD) or TLR2^-/- BMDM (TLR2) infected with wt or <i>perM</i>::tn at MOI 20. Protein concentration measured by ELISA. Data are means ± SD of three biological replicates. * P< 0.05, ** P< 0.01, *** P< 0.005.</p

PerM is necessary for Mtb persistence <i>in vivo</i> in an IFN-γ-dependent manner.

<p>(<b>A</b>) Colony-forming units (CFU) in lungs (left) and spleens (right) from C57BL/6 mice infected by aerosol with H37Rv (wt), the <i>perM</i> transposon mutant (<i>perM</i>::tn), or the genetically complemented mutant (comp). Data are means ± SD of 4 mice and representative of three independent experiments. (<b>B</b>) Hematoxylin and eosin-stained lung sections of infected mice showing progression of lesions. (<b>C</b>) CFU in lungs of IFN-γ^-/- mice infected by aerosol with wt or <i>perM</i>::tn. Data are means ± SD of 4 mice.</p

<i>PerM</i>::tn is highly sensitive to β-lactam antibiotics.

<p><i>PerM</i>::tn is highly sensitive to β-lactam antibiotics.</p

PerM is required for replication and survival in reduced magnesium.

<p>(<b>A</b>) Growth measured by optical density (OD₅₈₀) of strains grown in Sauton’s minimal media with 0.05% Tween-80. (<b>B</b>) Macroscopic appearance of liquid cultures. Cultures were grown standing in Sauton’s media, then agitated carefully to visualize the disrupted film in <i>perM</i>::tn cultures. (<b>C</b>) Replication, measured by OD (above), and survival, measured by CFU (below), of Mtb grown at the indicated concentrations of Mg²⁺ added to nominally Mg²⁺-free Sauton’s minimal media. (<b>D</b>) TNF concentration in cell culture supernatant of bone marrow-derived mouse macrophages exposed for 24 hours to live Mtb (MOI 10), following 6 days of Mtb culture in 500 or 2000 μM Mg²⁺. Data are means ± SD of three replicates and representative of two independent experiments.</p

PerM accumulates at the septum during cell division.

<p>Representative image series of replicating <i>M</i>. <i>smegmatis</i> expressing PerM-GFP. Arrows point to septa. Inset corresponds to septum indicated by a dashed arrow. Numbers indicate time in hours. Scale bar, 4 μm.</p

Transcriptome comparisons of wt and <i>perM</i>::tn in reduced Mg²⁺.

<p>Transcriptome comparisons of wt and <i>perM</i>::tn in reduced Mg²⁺.</p