In addition, the tagged proteins accumulated both in standard LB and in LB supplemented with zinc in zur deleted strains, confirming that zin T and znu A are negatively regulated by Zur, as already observed in other bacteria in previous studies [4, 12, 18, 31, 32]. Figure 2 ZinT and ZnuA accumulation in zur wild type and in zur deleted strains. RG-F116 (zin T::3xFLAG- kan), RG-F117 (znu A::3xFLAG-

kan), RG-F118 (Δ zur :: cat zin T::3xFLAG- kan) and RG-F119 (Δ zur :: cat znu A::3xFLAG- kan) strains were grown for 4 h in LB medium in presence or absence of 0.2 mM ZnSO4, 0.5 mM EDTA or 0.2 mM CdSO4 as indicated. The extracts were analyzed by Western blot. To evaluate the specificity of the response of zin T and znu A to metal ions, the accumulation of the two proteins

was analyzed in modM9 supplemented LY2109761 mouse with 5 μM ZnSO4, FeSO4, CuSO4 or MnCl2. The expression of both genes was repressed by zinc (Figure 3) whereas, in contrast to the results obtained with S. enterica [17], znu A and, to a lesser extent, zin T expression was partially inhibited by copper. Small differences in the regulation of the Zur-regulated genes between E. coli O157:H7 and S. enterica (PP134 and SA140) were also suggested by a titration of protein accumulation in response to external zinc (Figure 4). In E. coli O157:H7 strains the two genes were similarly expressed, with a slightly higher ZinT accumulation in presence of 0.5 μM ZnSO4. In contrast, in S. enterica only ZnuA was detectable at this zinc concentration. Figure 3 Influence of metals on ZinT and ZnuA accumulation. MK-4827 RG-F116 (zin T::3xFLAG- kan) and RG-F117 (znu A::3xFLAG- kan) strains were grown for 16 h in modM9 (lanes 1 and 6) in presence of ZnSO4 (lanes 2 and 7), FeSO4 (lanes 3 and 8), CuSO4 (lanes 4 and 9)

or MnCl2 (lanes 5 and 10). Metal concentration was 5 μM. The extracts were analyzed by Western blot. Figure 4 Zinc-dependent ZinT and ZnuA accumulation in E. coli O157:H7 and S. enterica strains. RG-F116 (zin T::3xFLAG- kan), RG-F117 (znu A::3xFLAG- kan) E. coli O157:H7 strains or PP134 (zin T::3xFLAG- kan) and SA140 (znu A::3xFLAG- kan ilv I::Tn10dTac- ca t:: Amoxicillin 3xFLAG- kan) S. enterica strains were grown for 16 h in modM9 supplemented or not with various concentrations of ZnSO4, as indicated. The extracts were analyzed by Western blot. In SA140 strain the chloramphenicol acetyltransferase (CAT) was used as an internal standard. The accumulation of the tagged-proteins was analyzed also in this website mutant strains deleted of zin T (RG-F120) or of znu A (RG-F121). Figure 5 shows that ZnuA accumulation in the strain lacking a functional zin T was comparable to that observed in the wild type strain in the same conditions (see Figure 2). In contrast, ZinT was expressed by the RG-F121 strain either in LB, where it was normally absent (Figure 5), or in modM9 supplemented with zinc (Figure 6).