Complementation and dominant negative activity of truncated EssB variants. (A-B) Complementation studies. S. aureus USA300 lacking functional essB was transformed with vector carrying either no insert, or various truncated variants of EssB or full length EssB. (A) The subcellular localization of EssB immune reactive species was assessed by subjecting cell lysates to ultracentrifugation to separate soluble (S) and (I) insoluble proteins and proteins in both extracts were resolved by SDS-PAGE followed by immunoblotting with specific antibodies (α-SrtA is used for subcelluar fractionation control of an insoluble membrane protein). (B) Cultures were examined for production and secretion of EsxA. Cultures were spun to separate proteins in cells (C) from secreted protein in the medium (M). α-L6 is used for fractionation control of a cytosolic protein. (C-D) Dominant negative studies. Truncated variants of EssB were examined for protein localization (C) and EsxA secretion (D) as described in panel A. All plasmids were transformed in wild-type strain USA300 (WT). All truncated variants with the exception of EssBΔM lacking PTMD prevented secretion of EsxA. The data for a duplicate of three independent experiments are shown. Arrows indicate proteins with correct mass found in reduced abundance (white arrow: EssBN; red arrow: EssBNM; blue and purple arrows: endogenous EssB). Protein products with aberrant mass are depicted with asterisks.