Secretion status of PopN, Pcr1, Pcr2, Pcr3, and Pcr4. popN (A), pcr1 (B), pcr2 (C), pcr3 (D), and pcr4 (E) mutant strains harboring an empty vector (pUCP19 or pYAN0636) or a vector containing the corresponding genes fused to a Flag tag at the C terminus were grown in L broth with (+) or without (−) EGTA. Culture supernatants were subjected to Western blotting to detect ExoS (upper panels) or Flag-tagged proteins (lower panels). Vector control plasmid pYAN0636 is a product of fusion of pDN19 and pCTC-Flag. Plasmids pYAN0632, pYAN0633, pYAN0634, and pYAN0635 contained Pcr1-Flag, Pcr2-Flag, Pcr3-Flag, and Pcr4-Flag, respectively.