Resolved proteins were transferred to polyvinylidene difluoride membranes (Thermo Scientific), and detected with mouse anti-FLAG Ab M2 (1 : 5000) and alkaline phosphatase-conjugated goat anti-mouse IgG (1 : 10 000) as described previously (Uzzau et al., 2001). Salmonella Typhi strains were grown overnight in LB broth at 37 °C with shaking. Aliquots of these cultures were subcultured in LB broth and supplemented with chloramphenicol Vincristine when required. To induce STY1365 expression, isopropyl-β-d-thio-galactoside (IPTG, Sigma) was added

at a final concentration of 1 mM. Cultures were incubated with shaking at 37 °C and growth was measured at OD600 nm. Salmonella Typhi strains were grown overnight in LB broth at 37 °C with shaking. Subcultures of strains were grown in LB broth and supplemented with chloramphenicol and IPTG when required. At an OD600 nm of 0.2, bacteria were harvested by centrifugation, and extraction of outer-membrane proteins was performed as described above. Proteins

were quantified using the Pierce® BCA Protein Assay Kit (Thermo Scientific). Twenty micrograms of proteins were resolved by SDS-PAGE (12.5%) as described by Lobos & Mora (1991). The intensity of bands was analyzed using imagemeter software (Adobe) Exposure of bacteria to crystal violet (1.5 μg mL−1) was performed by the method described previously (Onufryk et al., 2005). The efficiencies of plating were calculated by dividing the number of CFUs of a given strain on supplemented LB plates by

the number of CFUs of the same strain on LB plates. below Assays for each strain were performed Alectinib in duplicate and repeated three times with three independent isolates. All results are expressed as the means±SD of an individual experiment performed in triplicate. P-values were calculated according to Student’s t-test, and a value of P<0.05 was considered to be statistically significant. According to S. Typhi CT18 genome, STY1365 corresponds to a sequence interrupted by a premature stop codon (TGA). This observation was correlated to the data obtained by the STY1365 sequencing in S. Typhi STH2370 (Rodas et al., 2010). To find sequences with identity to STY1365 and flanking regions a blast algorithm was used. We found two sequences both with identity to a prophage-like element of S. Typhimurium DT104 (Saitoh et al., 2005; Fig. 1a). One sequence corresponds to artA and the other sequence to artB. Although no putative ORFs were annotated downstream of artB, our analysis showed that this region has 89% of identity to STY1365, suggesting that this is probably a prophage-like element (Fig. 1a). No dual-start motif was found in the predicted amino acid sequence of STY1365, but our blast searches revealed that the closest homologues of STY1365 amino acid sequence (57 residues) are all putative holins of different E. coli strains and the phage ΦP27. STY1365 showed 76% identity (e=6e−12) to EcolTa2 holin of E. coli TA271, 78% identity (e=4e–11) to ECDG_01257 holin of E.