(A) Schematic representation of the ESS gene cluster. Proteins with defined functions are indicated as follows: secreted proteins (red), FtsK SpoIIIE-like ATPase (yellow), proposed secretion machinery (grey), not known (white). The locus is annotated for strain USA300. Acronyms Esx and Ess refer to ESAT-6 like secretion extracellular and ESAT-6 like secretion system. Genes previously referred as ESAT-6 secretion accessory (EsaC, EsaD) have been renamed to better reflect their contribution to the secretion system. (B) Bacterial cultures of S. aureus USA300 (WT) or isogenic mutants, esxA, esxB, esxC, esxD and essA, essB, essB, essC were fractionated into cells and medium. Proteins in all fractions were precipitated with trichloroacetic acid, separated by SDS-PAGE and detected by immunoblotting with antibodies specific for secreted substrates (α-EsxA, α-EsxB, α-EsxC, α-EsxD) and cytosolic and secreted protein controls (α-L6, α-Hla).