FIG 2.

essH is dispensable for S. aureus growth and required for Esx protein secretion. (A) Overnight cultures of bacteria were normalized to an A₆₀₀ of 5, diluted 1:100 in fresh medium, and grown at 37°C. Growth was monitored as increased absorbance (A₆₀₀) over 24 h. (B) To assess protein secretion, cultures of S. aureus USA300 LAC* (wild type [WT]) or its isogenic essB, essH, essH(vector), and essH(pessH) variants, were grown to an A₆₀₀ of 3.0 and centrifuged to separate proteins in the extracellular medium fraction (medium) from staphylococci in the sediment. Bacteria were suspended in PBS and lysed with lysostaphin to release their cellular proteins (cell). Proteins were precipitated with trichloroacetic acid (TCA) and resuspended in buffer such that medium fractions were 25 times more concentrated than cell fractions. Extracts were separated by SDS-PAGE and electrotransferred to PVDF membranes for immunoblot analyses with rabbit polyclonal antibodies specific for EsxA (α-EsxA), EsxC (α-EsxC), α-hemolysin (α-Hla), and ribosomal protein L6 (α-L6). Numbers (in kilodaltons) indicate the migratory positions of molecular weight markers by SDS-PAGE. (C) Cultures of S. aureus USA300 LAC* (WT) or its isogenic essB, essH, essH(vector), and essH(pessH_STREP) variants were grown to an A₆₀₀ of 3.0, fractionated, and analyzed as described above with the addition of rabbit polyclonal antibodies specific for EssH (α-EssH).