(A)To identify the Pla cleavage sites in Y. pestis YapE, YapE point mutants that altered OmpT cleavage of YapE were expressed in Y. pestis and Western blots were performed with anti-YapE serum. V = Vector only (pMWO-034); Yp = WT Y. pestis YapE (pMBL313); K338M = K338M pt. mutant (pLOU021); R343P = R343P pt. mutant (pLOU022); T = YapEΔ34-236 (pLOU109). As a reference, V and Yp samples expressed in E. coli are also included. (B) Anti-YapE Western blots of Y. pestis YapE expressed in Y. pestis (Yp), Y. pestis Δpla, or Y. pestis with catalytically inactive Pla (D206A). V = vector control. (C)Whole cell proteins and outer membrane enriched samples from Y. pestis stained with Flamingo fluorescent stain. Yp = Y. pestis YapE (pMBL313); V = Vector control (pMWO-034). (D) Alignment of YapE proteins between amino acids 235 and 250. Yp = Y. pestis YapE, Yt = Y. pseudotuberculosis YapE; Ye = Y. enterocolitica YapE; underlined region represent the N-terminal amino acids of the Pla cleaved YapE peptide. Gray arrowheads represent full length YapE, white arrowheads represent YapE OmpT cleavage site/product, and black arrowheads represent primary YapE Pla cleavage site/product.