Additional file 3 : Figure S3 shows that E. coli O157:H7 secretes only a very limited number of proteins in modM9 and that there is not an evident release of intracellular proteins. In an attempt to identify a role for extracellular ZinT, we investigated the possibility that secreted ZinT could rebind to the bacterial cell. Cultures of RG-F120 strain, bearing a gene encoding a tagged-ZnuA and a deletion in zin T, were incubated for 4 h with extracellular tagged-ZinT obtained from the supernatant culture of RG-F116 strain grown in modM9 for 6 h. Subsequently, cellular extracts were analyzed by Western blot

to examine the fate of ZinT, using tagged ZnuA as positive control. As shown in Figure 8, when RG-F120 was grown in LB or in LB with 0.5 mM EDTA in presence of check details 25 μg of extracellular ZinT the protein was not found in association with the bacterial cell. Unexpectedly, we observed that extracellular ZinT induced the accumulation of ZnuA in LB (Figure 8 lane 3), where this protein was hardly detectable (see Figure 2). Such induction of znu A gene was not observed (Figure 8 lane 6) in bacteria incubated in presence of a hundredfold lower amount of extracellular ZinT (0.25 μg), suggesting that ZnuA accumulation could be due to the ability of extracellular ZinT to

sequester external zinc. To verify this possibility, the experiment was repeated using either apo- or zinc-containing ZinT. Epothilone B (EPO906, Patupilone) ZnuA accumulation appeared in LB only when the apo-form (data not shown) was used, showing the similar expression pattern obtained find more with the extracellular ZinT produced in modM9. These results indicated that apo-ZinT sequesters environmental zinc thus inducing the zur regulon, and that extracellular ZinT released by bacteria grown in modM9 is mainly in the zinc-free form, as already indicated by results described in Figure 7. Figure 8 Influence of extracellular ZinT on z nu A expression. RG-F120 (Δ zin T:: cat znu A::3xFLAG- kan) strain was grown in LB medium (lanes 2, 3, 5 and 6) or LB supplemented with 0.5 mM EDTA (lanes

4 and 7) in presence of 25 μg (lanes 2, 3 and 4) or 0.25 μg (lanes 5, 6 and 7) extracellular ZinT. The extracts, analyzed by Western blot, were prepared after a 4 h growth (lanes 3, 4, 6 and 7), or immediately after the addition of extracellular ZinT (lanes 2 and 5), as negative control. 25 μg of extracellular ZinT was loaded in lane 1 as positive control. In order to obtain strains unable to secrete ZinT we used the RG-F116 strain to delete etp C (RG-F122) or etp D (RG-F123), the first two genes of the operon of T2SS [33]. Contrary to our expectations, tagged-ZinT was detected in the supernatant of these mutants grown in LB supplemented with 0.5 mM EDTA and its accumulation was comparable to that observed in the wild type strain (data not shown).