FIG. 3.

Analysis of Yop synthesis and secretion by lcrV mutants of Y. pseudotuberculosis. Bacteria were grown in BHI medium, with (+) or without (−) Ca²⁺. Proteins were separated by SDS-PAGE and identified by immunoblot analysis, using polyclonal rabbit anti-YopH, anti-YopB, anti-LcrV, anti-YopD, and anti-YopE antisera or an antiserum recognizing all secreted Yops (α-Yop). Bacterium-associated protein levels were determined using pelleted bacteria (P). Total sample (T) refers to a mixture of proteins secreted into the culture medium and contained within intact bacteria, while supernatant samples (S) contained only secreted proteins. The experiment was repeated at least two times, with reproducible results. (A to C) Yop synthesis and secretion by lcrV mutants still containing the negative regulatory element LcrQ. Lanes: a and b, YPIII/pIB102 (parent); c and d, YPIII/pIB10201 (FS+1); e and f, YPIII/pIB10202 (FS−1); g and h, YPIII/pIB10203 (Scramble); i and j, YPIII/pIB19 (ΔlcrV null mutant lacking codons 10 to 313); k and l, YPIII/pIB10204 (V1 [Δ2-20]); m and n, YPIII/pIB10205 (V2 [Δ3-20]); o and p, YPIII/pIB10206 (V3 [Δ5-20]); q and r, YPIII/pIB10207 (V4 [Δ7-20]); s and t, YPIII/pIB10208 (V5 [Δ9-20]); u and v, YPIII/pIB10209 (V6 [Δ11-20]); x and y, YPIII/pIB10210 (V7 [Δ13-20]); z and å, YPIII/pIB10211 (V8 [Δ15-20]); ä and ö, YPIII/pIB10212 (V9 [Δ17-20]); aa and bb, YPIII/pIB10213 (V10 [Δ19-20]); cc and dd, YPIII/pIB10214 (V11 [Δ25-40]); ee and ff, YPIII/pIB10215 (V12 [Δ2-4]); gg and hh, YPIII/pIB10216 (V13 [Δ5-7]); ii and jj, YPIII/pIB10217 (V14 [Δ8-10]); kk and ll, YPIII/pIB10218 (V15 [Δ11-13]); mm and nn, YPIII/pIB10219 (V16 [Δ14-16]); oo and pp, YPIII/pIB10220 (V17 [Δ17-18]). (D) Proteins secreted from the equivalent strains differing only by the disruption of lcrQ via allelic exchange with an sp-sm resistance cartridge.