Plasmids used in this study.

<p>Plasmids used in this study.</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

<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

<i>Gaussia</i> luciferase is secreted from mycobacterial cells.

<p>Luminescence (given as relative light units [RLUs]) was measured in culture, supernatant and cell samples of <i>M. smegmatis</i> producing Gluc <i>Mycobacterium</i> optimised with (GlucSS) or without (Gluc) signal peptide, Gluc wild-type with (GlucWT+SS) or without (GlucWT-SS) signal peptide, and FFluc as control. Assays were performed with three independent cultures and each culture was measured in duplicate. As the data was not normally distributed, median values are displayed (bar) with inter-quartile ranges (box), and highest and lowest values (whiskers).</p

BLI of <i>ffluc</i>-expressing <i>M. tuberculosis</i>.

<p>Mice were inoculated endotracheally with 5×10⁶ CFU of either wild-type <i>M. tuberculosis</i> (control) or FFluc-producing <i>M. tuberculosis</i>. 20 µl of 30 mg ml⁻¹ luciferin was administered intranasally and mice were imaged 5–10 min after. Mice were contained in a large air-tight box for safety considerations. The image was obtained using an IVIS Spectrum and is displayed as a pseudocolour image 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 1 min.</p

Bacterial luciferase encoding vectors used in this study.

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

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