salmonis

proteins, the amino acid sequences of Ps-Tox and PS-Antox were analysed using the protparam tool of the Expasy Proteomic Server. The molecular weight of the Ps-Antox protein was 8.9 kDa and its theoretical pI is 4.79. The predicted molecular weight of Ps-Tox was 15.9 kDa with a pI of 6.7. In order to characterize this pair of predicted proteins, we cloned the ps-Antox and ps-Tox genes into the pET27b+ expression vector, either individually or together and attempted to express the genes in E. coli BL21 DE3. After the IPTG induction, the expression of the recombinants proteins was checked Entinostat every 1 h for a 3-h period, finding that the greatest amount of the two proteins is obtained 2-h post-IPTG induction. The expression level of Ps-Antox was much lower than that of its partner Ps-Tox when expressed alone (Fig. 2). When the two proteins were expressed simultaneously in a bicistronic operon the expression level of both proteins was similar, not showing a significant

polar effect (Fig. 2). To determine the toxic proprieties of the P. salmonis Dabrafenib chemical structure toxin, we made cultures of the E. coli transformant cells in the presence of IPTG. The growth of the E. coli carrying the pET27b+ vector that contains the ps-Tox gene was minimal in the presence of IPTG during 8 h of growth kinetics (Fig. 3a). In contrast, the growth of E. coli strains that contained the ps-Antox and ps-Tox-Antox in the pET27b+ was normal compared with the host that had the vector without insertion (Fig. 3a). All the transformants strains grew normally in the find more absence of IPTG, including the strain with the ps-Tox gene in the pET27b+ vector (Fig. 3b). When the strains were streaked out on LB agar plates supplemented with IPTG, the results were the same as those obtained in LB broth (data not

shown). The model constructed is presented in Fig. 4b and c. In general, the secondary structure is conserved compared with that of the M. tuberculosis VapC-5 toxin. Some amino acids implicated in the toxin function are conserved, in particular, three of the four acidic amino acids present in the PIN domains that are related with an exonuclease activity (Miallau et al., 2008), VapC-5: D26, E57, D115, D135, and Ps-Tox: D6, E44, D100, and E121, as can be seen Fig. 4a (see Table S1). In order to determine whether the newly described toxin behaves in the same way as most described toxins, we tested Ps-Tox for putative RNAse activity. When P. salmonis RNA was treated with a crude extract of E. coli containing the recombinant Ps-Tox protein, a significant degradation was observed compared with that of the untreated sample (Fig. 5, lanes 2 and 6, respectively). The same effect was observed in the corresponding extract containing Ps-Antox and Ps-Tox-Antox proteins (Fig. 5, lanes 1 and 3, respectively). The RNA degradation produced by the protein extract that contains Ps-Antox and Ps-Tox-Antox could also have been produced by E.

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