Fig. 2.

Non-cognate EssC variants support secretion of EsxA but not EsxC. (a, b) Strain RN6390 or the isogenic essC deletion strain carrying pRAB11 (empty) or pRAB11 encoding the indicated essC variant was subcultured into TSB medium supplemented with 1 µM haemin [34] and either 25 ng ml⁻¹ (RN6390 ΔessC/pEssC_RN6390) or 100 ng ml⁻¹ (RN6390 ΔessC/pEssC_MRSA252/pEssC_ST398/pEssC_EMRSA15) anhydrotetracycline (ATC) to induce plasmid-encoded gene expression. The strains were grown aerobically until an OD₆₀₀ of 2 was reached, after which (a) 10 µl of OD₆₀₀ 1 adjusted cells were separated on an 8 % bis-Tris acrylamide gel and analysed by Western blotting using anti-EssC antisera [20], or (b) the cultures were separated into supernatant and whole-cell fractions and the equivalent of 200 µl of culture supernatant (sn) and 10 µl of resuspended cell sample adjusted to an OD₆₀₀=1 were separated on a 15 % bis-Tris gel and immunoblotted using the antiserum raised against EsxA [20], EsxC [20] or the cytosolic control, TrxA [35].