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. Author manuscript; available in PMC: 2014 Jul 1.

Published in final edited form as: Mol Microbiol. 2013 Jun 10;89(2):276–287. doi: 10.1111/mmi.12273

To identify the OmpT cleavage site in Y. pestis YapE, we constructed chimeric proteins consisting of the N-terminal portion of the Y. enterocolitica protein and the C-terminal portion of the Y. pestis protein. These chimeric proteins allowed for the identification of regions where the OmpT cleavage was located. Once the region was identified, K and R residues were identified in this region that differed between the two Yersinia orthologs and site directed mutagenesis was used to identify residues required for cleavage. (A) Schematic of the chimeric proteins. White and black boxes represent Y. enterocolitica and Y. pestis sequences, respectively. (B) Alignment of YapE proteins highlighting K and R residues (*) that differ between Y. pestis and Y. enterocolitica. Yp = Y. pestis YapE, Yt = Y. pseudotuberculosis YapE; Ye = Y. enterocolitica YapE; * = residues mutated in Y. pestis YapE pt mutants. (C) Settling assay with E. coli expressing YapE chimeric proteins. ● = Vector only (pMWO-034); ○ = Y. pestis YapE (pMBL313); ▼ = Y. enterocolitica YapE (pMBL311); ■ = Chimera 1 (pMBL326); □ = Chimera 2 (pLOU013). (D) Settling assay with E. coli expressing Y. pestis YapE point mutations. ● = Vector only (pMWO-034); ○ = Y. pestis YapE (pMWO-034); ∇ = K338M (pLOU021); = R343P (pLOU022); ■ = K375D (pLOU023); □ = K416R (pLOU024). For settling assays, YapE otholog expression was induced for 2 hrs and then samples were harvest from static cultures at a specific depth at 1 hr intervals. The absorbance (OD₆₀₀) of these samples were determined and compared to the OD₆₀₀ at T=0. Three independent biological replicates were performed for each bacterial strain and each assay was repeated three times (n=9). Each symbol represents the mean percent initial absorbance ± the standard deviation; ** = P≤0.002; *** = P≤0.0002. (E) Western blot analysis of YapE cleavage from strains in C and D, respectively. V = Vector only; Yp = WT Y. pestis YapE; C1 = Chimera 1; C2 = Chimera 2; Ye = Y. enterocolitica YapE; 1 = K338M; 2 = R343P; 3 = K375D; 4 = K416R. White arrowheads represent YapE OmpT cleavage site/product.

Inline graphic — To identify the OmpT cleavage site in Y. pestis YapE, we constructed chimeric proteins consisting of the N-terminal portion of the Y. enterocolitica protein and the C-terminal portion of the Y. pestis protein. These chimeric proteins allowed for the identification of regions where the OmpT cleavage was located. Once the region was identified, K and R residues were identified in this region that differed between the two Yersinia orthologs and site directed mutagenesis was used to identify residues required for cleavage. (A) Schematic of the chimeric proteins. White and black boxes represent Y. enterocolitica and Y. pestis sequences, respectively. (B) Alignment of YapE proteins highlighting K and R residues (*) that differ between Y. pestis and Y. enterocolitica. Yp = Y. pestis YapE, Yt = Y. pseudotuberculosis YapE; Ye = Y. enterocolitica YapE; * = residues mutated in Y. pestis YapE pt mutants. (C) Settling assay with E. coli expressing YapE chimeric proteins. ● = Vector only (pMWO-034); ○ = Y. pestis YapE (pMBL313); ▼ = Y. enterocolitica YapE (pMBL311); ■ = Chimera 1 (pMBL326); □ = Chimera 2 (pLOU013). (D) Settling assay with E. coli expressing Y. pestis YapE point mutations. ● = Vector only (pMWO-034); ○ = Y. pestis YapE (pMWO-034); ∇ = K338M (pLOU021); = R343P (pLOU022); ■ = K375D (pLOU023); □ = K416R (pLOU024). For settling assays, YapE otholog expression was induced for 2 hrs and then samples were harvest from static cultures at a specific depth at 1 hr intervals. The absorbance (OD₆₀₀) of these samples were determined and compared to the OD₆₀₀ at T=0. Three independent biological replicates were performed for each bacterial strain and each assay was repeated three times (n=9). Each symbol represents the mean percent initial absorbance ± the standard deviation; ** = P≤0.002; *** = P≤0.0002. (E) Western blot analysis of YapE cleavage from strains in C and D, respectively. V = Vector only; Yp = WT Y. pestis YapE; C1 = Chimera 1; C2 = Chimera 2; Ye = Y. enterocolitica YapE; 1 = K338M; 2 = R343P; 3 = K375D; 4 = K416R. White arrowheads represent YapE OmpT cleavage site/product.