Figure 2.

Analysis of the Yop proteins secreted by different yscU mutants. (A) Coomassie-stained 12% SDS–PAGE of Yops secreted by wt, ΔyscP, ΔyscU mutant bacteria, ΔyscU mutant bacteria overexpressing mutated yscU alleles in trans from the pBAD promoter, or bacteria carrying the yscU_N263A allele at the yscU locus (natural yscU promoter). (B) Expression and export of LcrV and YopE in Y. enterocolitica E40 wt and yscU_N263A mutant bacteria. Total cell (TC) and supernatant fractions (SN) were analyzed by immunoblotting using anti-LcrV or anti-YopE antibodies. Shown are samples from two independent experiments. (C) Expression and export of LcrV after overexpression of lcrV in trans from the pBAD promoter in Y. enterocolitica wt and yscU_N263A mutant bacteria. L-arabinose concentrations ranging from 0.02 to 0.5% were used to induce LcrV synthesis when bacteria were shifted to 37°C and again 2 h later. TC and SN were analyzed by immunoblotting using anti-LcrV antibodies. Strains and plasmids used: wt (pYV40); ΔyscP (pLJ4036); ΔyscU (pLY4001); yscU⁺⁺⁺ (pLY7); yscU_N263A⁺⁺⁺ (pSTW7); yscU_P264A⁺⁺⁺ (pSTW8); yscU_T265A⁺⁺⁺ (pSTW9); yscU_N263A (pISO4007); lcrV⁺⁺⁺ (pPB42).