Complementation of <i>mas</i>::<i>tn</i> in the Δ<i>eccC</i>_<i>5</i> mutant by replacement of the integrated pMV vector.

<p>Indicated strains were electroporated with the input DNA shown on the left. Input DNA consisted of the pMV-361-<i>hyg</i> plasmid containing the indicated constructs. Valid insertion of input DNA was scored as + or-, similarly as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005190#pgen.1005190.t001" target="_blank">Table 1</a>. Results are representative data of three independent experiments.</p><p>Complementation of <i>mas</i>::<i>tn</i> in the Δ<i>eccC</i>_<i>5</i> mutant by replacement of the integrated pMV vector.</p

Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.

<p>* The <i>eccC</i>_<i>5</i> NBD mutants appear to have a dominant negative effect on the functioning of endogenous EccC₅.</p><p>^$ Colonies showed a strong growth defect, i.e. colonies were visible only after 17 days, compared to 10 days for the wild-type strain.</p><p>Replacement of pMV-<i>eccBC</i>_<i>5</i> by the input DNA was scored. Input DNA consisted of the pMV-361-<i>hyg</i> plasmid containing the indicated constructs. “+” indicates that more than 100 colonies were detected after electroporation with the indicated vector. “–” indicates between 0–20 colonies were found after electroporation. These latter colonies were shown by PCR to still contain the original vector, indicating illegitimate recombination or spontaneous antibiotic resistance. Results are representative data of three independent experiments.</p><p>Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.</p

ESX-5 is involved in fatty acid uptake.

<p>A) Growth of indicated <i>M</i>. <i>marinum</i> strains on Tween-80 as a sole carbon source was assessed by measuring optical density at different time points. Depicted is the average of three biological replicates. Error bars indicate standard deviations. B) Uptake of a fluorescently labeled fatty acid after 72 hours of hypoxic growth was measured by FACS analysis. 20.000 events gated for similar size were acquired for WT::<i>mspA</i> (black), Δ<i>mycP5</i>::<i>mspA</i> (light grey) or Δ<i>mycP5-C</i>::<i>mspA</i> (dark grey). C) Quantification of FACS analysis. Mean fluorescent intensity of three experiments per strain was acquired by FACS. Background staining, quantified by adding the fluorescent fatty acid to an unstained culture one hour before washing the cells, was deducted from the measured values. Error bars indicate the standard deviations and One-way ANOVA showed a statistical difference between the samples of <i>p</i> = 0.010. D) Uptake of the fluorescently labeled fatty acid and formation of lipid bodies was confirmed by confocal microscopy.</p

Expression of MycP5 is essential for growth of <i>M</i>. <i>bovis</i> BCG.

<p>A, B) The BCG-Pasteur c-<i>mycP5</i>-tet-on (A) and c-<i>mycP5</i>-tet-off (B) mutants were grown for 21 days on Middlebrook 7H10 agar plates containing the indicated ATc concentrations. Full growth of c-<i>mycP5</i>-tet-on was only observed at 10 μg/ml ATc, whereas this concentration of ATc did not completely abolish colony growth of c-<i>mycP5</i>-tet-off. C, D) Resazurin reduction is dependent on ATc-induced expression/repression of <i>mycP5</i>. Cells of the BCG-Pasteur c-<i>mycP5</i>-tet-on (C), or c-<i>mycP5-</i>tet-off (D) mutants were grown as liquid cultures in 96-well microtiter plates for 6 days at 37°C at the indicated ATc concentrations, after which 10% Alamar Blue was added and fluorescence (585 nm) was measured after 16 h incubation to determine metabolic activity as a correlate of growth. Values are means of triplicates; error bars represent the standard deviation.</p

Role of NBDs domains of EccC5 in ESX-5 dependent secretion and membrane complex assembly.

<p>A) Predicted transmembrane domains (dark grey), and NBD (light grey) of EccC₅ are indicated. The positions of relevant residues are depicted with a black bar. The numbers represent the position in amino acids. B) Secretion of PE_PGRS proteins in the different EccC₅ mutant strains was analyzed by immunoblot of supernatants and cell pellets of wild-type (WT) <i>M</i>. <i>marinum</i> and the <i>eccC5</i> deletion strain <i>(</i>Δ<i>eccC5</i>) complemented with various <i>eccC5</i> mutated genes. GroEL2 staining was used as a control for lysis and equal loading. C) Immunoblot analysis of EccC₅ expression in isolated membranes of indicated strains. D) Blue native PAGE and immunoblot analysis using an anti-EccD₅ antibody of the ESX-5 membrane of <i>M</i>. <i>marinum</i> Δ<i>eccC5</i>::<i>mspA</i>, complemented either with an empty vector (-) or with various <i>eccC5</i> mutated genes. For all samples that contained EccC₅ variants the characteristic pattern of ESX-5 membrane complexes was observed [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005190#pgen.1005190.ref028" target="_blank">28</a>], consisting of the largest ~1.5 MDa complex and two additional smaller subcomplexes (indicated by the three arrowheads).</p

Secretion analysis of ESX-5 mutant strains.

<p>A) A schematic representation of the ESX-5 region of <i>M</i>. <i>marinum</i> with the different ESX-5 mutations used in this study. Bars above the gene cluster indicate regions deleted by targeted knock-out mutagenesis. Arrows below indicate position and orientation of transposons (named LA1 to LA12) in mutants of the parental strain <i>M</i>. <i>marinum</i>::<i>mspA</i> defective in ESX-5 dependent secretion. B) Secretion analysis of <i>M</i>.<i>marinum</i>::<i>mspA (</i>WT::<i>mspA)</i>, a <i>mycP5</i> transposon mutant (<i>mycP5</i>::<i>tn</i>, corresponding to LA9 in (A)) and the complemented version of this strain (<i>mycP5</i>::<i>tn</i>-C). Secreted proteins (S) were separated from bacterial cells (P) by centrifugation. In addition, surface-associated proteins were enriched from the bacterial cells by extraction with 0.5% Genapol X-080 (GS) and separated from non-solubilized proteins (GP) by centrifugation. All fractions were analyzed for the presence of PE_PGRS proteins by immunoblotting. GroEL2 staining was used as a loading and lysis control. C) Expression of EccB₅ and EspG₅ was analyzed by immunoblotting of total cell lysates of wild-type <i>M</i>. <i>marinum</i> (WT), the <i>Δesx-5</i>::<i>mspA</i> mutant and the complemented <i>Δesx-5</i>::<i>esx-5tub</i> strain. D) The same strains as under (C) were analyzed for their ability to express and secrete PE_PGRS proteins following the same procedure as under (B).</p

Model for the essentiality of ESX-5.

<p>A) Presented is the working hypothesis, in which the ESX-5 system is responsible for the insertion of several channel- or pore-forming protein (indicated by the question marks) that mediates the uptake of essential nutrients and/or other metabolites. B) The lack of PDIMs (left) or the presence of MspA-like porins (right) increases the permeability of the outer membrane, allowing the passive diffusion of the hypothesized essential nutrient(s), thereby circumventing the essentiality of ESX-5.</p

<i>mas</i> mutation or introduction of <i>mspA</i> lead to increased outer membrane permeability.

<p>A, B) Sensitivity to a combination of ampicillin and clavulanic acid of different <i>M</i>. <i>marinum</i> strains was measured by performing a disc diffusion assay on the indicated strains and measuring the surface of the growth inhibition zone. The <i>mas</i> transposon (<i>mas</i>::<i>tn)</i> mutants exhibit increased sensitivity, independent of the presence of an intact copy of <i>eccC5</i> (A). Similarly, introduction of <i>pSMT3</i>::<i>mspA</i> also leads to an increase in sensitivity independent on the presence of <i>mycP5</i> (B). Values are the means of triplicates; error bars indicate the standard deviation. C) Uptake of EtBr, measured by flow cytometric analysis. <i>M</i>. <i>marinum</i> wild-type (dark grey) and <i>M</i>. <i>marinum</i>::<i>mspA</i> (black) were incubated with 20 μM EtBr for 60 min and 20.000 events were analyzed for their fluorescence intensity at 585/540 nm. Light-grey lines indicate unstained samples. All measurements are depicted in duplicates and are representatives of three independent experiments. <i>ΔeccC5</i>-C and <i>ΔmycP5</i>-C refer to the complementation strains of the <i>M</i>. <i>marinum ΔeccC5</i> or the <i>ΔmycP5</i> mutants complemented with pMV::<i>eccBC5</i> or pMV::<i>mycP5</i> respectively.</p

<i>M</i>. <i>marinum</i> genes with enriched numbers of transposon insertions in <i>M</i>. <i>marinum</i> supplemented with MspA.

<p>Depicted are the number of transposon insertions detected by TraDIS in wild-type <i>M</i>. <i>marinum</i> or in a strain expressing MspA. The detected insertions are normalized for the total amount of reads per sample and genes are ranked based on the fold change between <i>M</i>.<i>marinum</i>::<i>mspA</i> divided by wild type <i>M</i>.<i>marinum</i>. The top ten hits are depicted. ESX-5 components are in bold.</p><p><i>M</i>. <i>marinum</i> genes with enriched numbers of transposon insertions in <i>M</i>. <i>marinum</i> supplemented with MspA.</p