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).