The assays were performed as in Antunez-Lamas et al. (2009). Briefly, three medium A plates (0.3% agar) were inoculated in the centre with a sterile toothpick and incubated at 28 °C. Motility was then assessed qualitatively by measuring the radial growth formed by the bacterial cells migrating away from the point of inoculation. Assays were performed as above, but in 0.7%

agar medium A plates. selleck chemicals llc The means were compared in one-way using Fisher’s protected least significant difference (LSD) method (P=0.05). To test Cu sensitivity, D. dadantii 3937 cells grown in KB medium were collected and resuspended to OD600 nm≈0.1 in fresh KB medium with various CuCl2 concentrations. Cell growth was estimated by monitoring OD600 nm using a microbiology growth reader (Bioscreen C, Labsystems). The highest OD600 nm values were reached by the wild-type strain after 18 h. Dickeya dadantii 3937 cells were grown in KB medium and then collected and resuspended to OD600 nm≈0.1 in fresh KB medium containing the iron chelators 2′2- dipyridyl or ethylenediamine-N,N′-bis-2-hydroxy-phenylacetic acid (EDDHA) at different concentrations. The means were compared in one-way using Fisher’s

protected LSD method (P=0.05). The effect of tat mutation on virulence was tested in witloof chicory leaves (Cichorium intybus) and in potato tubers (Solanum tuberosum cv. Monalisa). Virulence assays on chicory leaves were performed as described

previously (Bauer HSP tumor et al., 1994). Briefly, 20 chicory leaves were pair-inoculated with 10 μL of a suspension containing 105 CFU of D. dadantii 3937 wild-type or derivative strains in 10 mM MgCl2. The virulence was estimated by the macerated area after incubation diglyceride in a moist chamber at 28 °C for 18 h. The differences between the wild-type and the mutant strains were statistically assessed using a paired Student’s t-test. To test virulence in potato tubers, each tuber was inoculated at two locations with 50 μL containing 5 × 105 bacterial cells and incubated at 28 °C for 40 h (Lopez-Solanilla et al., 1998). Macerated tissues of 20 potato tubers were analysed as in chicory leaves. All plants used in these assays were purchased from a local supermarket. A search of the D. dadantii 3937 protein database with two software programs for Tat signal peptide identification, tatfind1.4 (Rose et al., 2002) and tatp (Bendtsen et al., 2005), revealed the presence of 44 potential substrates (see Table 1). Fourteen proteins were predicted by both programs, 15 only by tatfind1.4 and 15 only by tatp. All these proteins (Table 1) included in their signal peptide a pair of arginines present in the Tat consensus motif S/TRRxFLK (Berks, 1996).