) and incubated for a further 3 h at the same culture conditions as before. An aliquot of 200 μL was taken at the end of every hour and centrifuged at 15 500 g for 10 min, the resultant pellets were resuspended in 100 μL of Laemmli sample buffer. The expression of the recombinant Ps-Tox, Ps-Antox, and Ps-Tox-Antox proteins was visualized on an 18% Tris-tricine urea sodium dodecyl sulphate
polyacrylamide gel electrophoresis stained with Coomassie Blue R-250 (Winkler Ltd.). In order to determine the potential toxic effect of the Ps-Tox protein of P. salmonis, we evaluated the growth rate of E. coli cells. The E. coli strains that contain the ps-Tox, ps-Antox, and ps-Tox-Antox genes were grown on LB broth, in 96-well plates, supplemented with 50 μg mL−1 kanamycin and 1 mM IPTG and incubated at 37 °C for 8 h in constant shaking (200 r.p.m.). Absorbance (OD600 nm) was measured every hour to determine the growth level Forskolin concentration of the cells. As an experimental
Bcl-2 inhibitor control, we used the same E. coli with the P. salmonis TA genes, which were grown on LB without IPTG in the same conditions described above. Additionally, the E. coli transformant cells were streaked out on agar plates supplemented with 50 μg mL−1 of kanamycin and 1 mM of IPTG. The plates were incubated at 37 °C overnight and the growth level was evaluated. Based upon the recently determined structure of the VapBC complex of Mycobacterium tuberculosis (Miallau et al., 2008) (PDB ID: 3DBO), we performed a homology model of the Ps-Tox protein. We used the Ergoloid Swiss Model server (Schwede et al., 2003; Arnold et al., 2006), and constructed the model with an alignment of the Mycobacterium VapC-5 toxin and the P. salmonis Ps-Tox toxin. The antitoxin sequence has a 20% identity (%ID) and the toxin sequence has 24% ID. The alignment between Ps-Tox and VapC-5 was made with jalview (Clamp et al., 2004) and the figures were made with the vmd software (Humphrey et al., 1996). In order to determine the putative target of the Ps-Tox
protein, it was tested for RNase activity based on the presence of a PIN domain. Piscirickettsia salmonis was grown on 5 mL of MC5 medium under the same conditions described above. Two-day-old cultures were centrifuged at 6000 g for 20 min at 4 °C. The RNA was extracted from the bacterial pellet with Trizol® LS reagent (Invitrogen), according to the manufacturer’s instructions. The RNA concentration was measured by spectrophotometry. The RNA was kept at −80 °C until use. The recombinant proteins Ps-Tox, Ps-Antox, and Ps-Tox-Antox were expressed on E. coli. Frozen vials of E. coli BL21 (DE3) bearing the Ps-Tox, Ps-Antox, and Ps-Tox-Antox containing plasmid were used to inoculate 5 mL of LB broth supplemented with 50 μg mL−1 of kanamycin. The culture was grown overnight at 37 °C and 250 r.p.m. Then, 2 mL of these cultures was added to 50 mL of LB broth supplemented with 50 μg mL−1 of kanamycin and the cultures were incubated 250 r.p.m.
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