Plasmids used in this study.

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

Bacterial luciferase encoding vectors used in this study.

<p>Bacterial luciferase encoding vectors used in this study.</p

<i>Gaussia</i> luciferase encoding vectors used in this study.

<p><i>Gaussia</i> luciferase encoding vectors used in this study.</p

Bioluminescence correlates with cell density during exponential growth <i>in vitro</i>.

<p>Cultures of <i>M. smegmatis</i> pMVhsp+FFluc (<b>a</b>), pMVhsp+Gluc (<b>b</b>) and pMVhsp+Lux (<b>c</b>) were inoculated to an optical density (OD) at 600 nm of 0.1 and the OD and the luminescence [given as relative light units (RLUs)] measured over 28 h. The luminescence was measured with integration times of 5, 0.1, and 10 s respectively, and substrate concentrations of 470 µM luciferin for FFluc and 40 µM coelenterazine for Gluc. The values represented correspond to the means of two independent cultures measured in triplicate. The error bars indicate standard deviations. A near linear relationship was found between bioluminescence [given as RLUs] and colony counts (given as colony forming units [CFU]) for mid-log cultures of <i>M. smegmatis</i> pMVhsp+FFluc (<b>d</b>), pMVhsp+Gluc (<b>e</b>) and pMVhsp+Lux (<b>f</b>) using a plate luminometer.</p

Firefly luciferase encoding vectors used in this study.

<p>Firefly luciferase encoding vectors used in this study.</p

Primers used in this study.

a<p>In italics, sequence added to include restriction sites (underlined) for cloning procedures, and optimized Shine-Dalgarno sequence (in bold) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010777#pone.0010777-LeDantec1" target="_blank">[45]</a>.</p

Kinetics of FFluc activity in <i>M. smegmatis</i> infected mice after intranasal administration of luciferin.

<p>Mice were endotracheally inoculated with 6.6×10⁶ CFU of <i>M. smegmatis</i> pMV306hsp+FFlucWT [two representative mice (M1 and M2) out of four are shown] or with 6.8×10⁶ CFU of <i>M. smegmatis</i> pMV306hsp as a control (one representative mouse out of two is shown). 20 µl of 15 mg ml⁻¹ or 30mg ml⁻¹ luciferin intranasal was administered 24 h post-inoculation and mice were imaged 0, 5, 10, 15, 30, 60, 120 and 180 min after. (<b>a</b>) Images were obtained using an IVIS Spectrum and are displayed as pseudocolour images of peak bioluminescence (given as photons s⁻¹ cm⁻² sr⁻¹). Red represents the most intense light emission while blue correspond to the weakest signal. The colour bar indicates relative signal intensity. Mice were imaged with an integration time of 30 s. Three representative time points are shown. (<b>b</b>) Signal intensity (given as photons s⁻¹) in the lungs was quantified for each time point using the region of interest tool in the Living Image software program.</p

BLI of <i>gluc</i>-expressing <i>M. smegmatis</i>.

<p>Mice were endotracheally inoculated with 3.32×10⁶ CFU of <i>M. smegmatis</i> pMV306hsp+Gluc [two representative mice (M1 and M2) out of three are shown,) or with 1.58×10⁷ CFU of <i>M. smegmatis</i> pMV306hsp as a control (one out of two mice is shown). 10 µg of coelenterazine intranasal was administered 24 h post-inoculation and mice were imaged at time points 0, 5, 10, 15, 30, 60, 120 and 180 min. (<b>a</b>) Images were obtained using an IVIS Spectrum and are displayed as pseudocolour images of peak bioluminescence (given as photons s⁻¹ cm⁻² sr⁻¹), with variations in colour representing light intensity at a given location. Integration time was 5 min. (<b>b</b>) Bioluminescence (given as photons s⁻¹) was quantified using the Living image software. (C) 10 µg of coelenterazine was given intraperitoneally to the same mice 5 h post-intranasal coelenterazine. Mice were imaged 0, 5, 10, 15, 20, 25 and 30 min post-intraperitoneal coelenterazine with integration times of 3 min.</p

Bioluminescence correlates with substrate concentration at low concentrations.

<p>Luminescence (given as relative light units [RLUs]) of <i>M. smegmatis</i> pMVhsp+FFluc (<b>a</b>) and <i>M. smegmatis</i> pMVhsp+Gluc (<b>b</b>) was measured with integration times of 5 s and 0.1 s respectively. The substrate concentrations assayed ranged from 20 to 4710 µM luciferin for FFluc, and from 0.05 to 400 µM coelenterazine for Gluc. Means and standard deviations (smaller than symbols) of six replicates are shown.</p

Bioluminescence levels in <i>M. tuberculosis</i> and <i>M. smegmatis</i> are comparable <i>in vitro</i>.

<p>Relative light units (RLUs) were measured in 10 <i>M. smegmatis</i> and 10 <i>M. tuberculosis</i> clones transformed with pMV306hsp+FFluc (<b>a</b>), pMV306hsp+Gluc (<b>b</b>) or pMV306hsp+Lux (<b>c</b>). Results are corrected for the background. Statistical significance was evaluated by the Mann-Whitney non-parametric test for Lux, and by unpaired t test for FFluc and Gluc (data normality passed) and those found to be significant (p&lt;0.05) are indicated with *.</p