Identification and characterization of EssB. (A) S. aureus USA300 (WT) or isogenic mutant essB were examined for production (T: total culture extracts) and subcellular localization of EssB (C: cell extracts followed by 100,000 x g sedimentation and separation of soluble, S and insoluble I proteins; M: medium). Proteins in each fraction were precipitated with trichloroacetic acid, separated by SDS-PAGE and detected by immunoblotting with specific antibodies [α-EssB, as well as α-L6, α-Hla, α-SrtA, as cytoplasmic, secreted and membrane protein controls, respectively]. (B) Plasmid complementation analysis of bacterial cultures separated between cells (C) and medium (M). S. aureus USA300 (WT) or essB mutants harboring or not a complementing plasmid (p essB ) were examined for their ability to secrete EsxA in the culture medium. Samples were analyzed as in panel A.