Analysis of the <i>M. tuberculosis</i> secretome identifies a high proportion of Tat substrates enriched in the <i>M. tuberculosis phoP</i> mutant.

<p><b>A.</b> Heat map displaying values from in-depth proteomic quantifications of the secreted protein fraction. Values for EsxA, EsxB, EspA and EspC are displayed as internal controls since these proteins are barely detectable in the <i>M. tuberculosis phoP</i> mutant. The other proteins are representative examples of putative Tat-secreted substrates according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004183#ppat.1004183-SaintJoanis1" target="_blank">[24]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004183#ppat.1004183-Marrichi1" target="_blank">[26]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004183#ppat.1004183-Dilks1" target="_blank">[27]</a>. Note the opposite trend between EsxA/EsxB/EspA/EspC and Tat-secreted proteins. <b>B.</b> Western blot analysis of Ag85C, Rv2525c and EsxA in <i>M. tuberculosis</i> wild type, <i>phoP</i> mutant and complemented strains. Results are representative of three independent experiments. <b>C.</b> Proposed regulatory network involving PhoP, Mcr7 and <i>tatC</i>. PhoP controls transcription of Mcr7 (red transcript), which anneals to the 5′-end of the <i>tatC</i> mRNA (blue transcript) occluding the RBS (boxed) and consequently impairing translation of TatC. <b>D.</b> Northern blot analysis of Mcr7 in wild type, <i>phoP</i> mutant and <i>mcr7</i>-complemented strains of <i>M. tuberculosis</i>. Introduction of <i>mcr7</i> under the 16S rRNA promoter in a <i>phoP</i> mutant efficiently restores production of this molecule. Expression of the 5S rRNA is used as a control of RNA loaded in each lane. <b>E.</b> Analysis of TatC expression by Western blot in wild type, <i>phoP</i> mutant, <i>phoP</i>-complemented mutant and <i>mcr7</i>-complemented strains of <i>M. tuberculosis</i>. Note the higher expression levels of TatC in the <i>phoP</i> mutant compared to the wild type. Complementation of a <i>phoP</i> mutant with <i>phoP</i> or <i>mcr7</i> restored wild type levels of TatC. GroEL2 is used as a control of protein loaded in each lane. Signal intensity was quantified from three independent experiments and plotted in the graphs below the Western blot images.