Our data indicate that only the loss of the plp gene has a significant effect on hemolysis of fish erythrocytes by V. anguillarum culture supernatant, while the loss of rtxA and/or vah1 has little effect. Further,
supernatant from the hemolysin triple mutant XM90 (vah1 rtxA plp) exhibits no hemolytic Tofacitinib activity on fish blood compared to M93Sm (Table 2), indicating that Vah1, RtxA, and Plp are responsible for all secreted hemolytic activity by V. anguillarum. Finally, complementation of any plp mutant with plp (in trans) restores hemolytic activity to V. anguillarum culture supernatant (Table 2). Conclusion V. anguillarum Plp is a secreted hemolysin with phosphatidylcholine-specific phospholipase A2 activity. The ability of Plp to digest the abundant phosphatidylcholine Selleck PU-H71 found in the membrane of fish erythrocytes causes their lysis. The three hemolysins, Plp, Vah1 and RtxA, account for all hemolytic activity in V. anguillarum culture supernatant under the experiment conditions described in this study. Finally, infection studies in rainbow trout demonstrate that the plp and vah1 genes are not required for virulence. Methods Bacterial strains, plasmids, and growth conditions All bacterial strains and plasmids used Selleckchem ARN-509 in this report are listed in Table 1. V. anguillarum strains were routinely
grown in Luria-Bertani broth plus 2% NaCl (LB20) , supplemented with the appropriate antibiotic, in a shaking water bath at 27°C. E. coli strains were routinely grown in Luria-Bertani broth plus 1% NaCl (LB10). Antibiotics were used at the following concentrations: streptomycin, 200 μg/ml; ampicillin, 100 μg/ml (Ap100); chloramphenicol, 20 μg/ml (Cm20) for E. coli and 5 μg/ml (Cm5) for V. anguillarum; kanamycin, 50 μg/ml (Km50) for E. coli and 80 μg/ml (Km80) for V. anguillarum; tetracycline, 15 Amine dehydrogenase μg/ml (Tc15) for E. coli, 1 μg/ml (Tc1) for V. anguillarum grown in liquid medium, and 2 μg/ml (Tc2) for V. anguillarum
grown on agar plates. Insertional mutagenesis Insertional mutations were made by using a modification of the procedure described by Milton et al.. Briefly, primers (Table 3) were designed based on the target gene sequence of M93Sm. Then a 200–300 bp DNA fragment of the target gene was PCR amplified and ligated into the suicide vector pNQ705-1 (GenBank accession no. KC795685) after digestion with SacI and XbaI. The ligation mixture was introduced into E. coli Sm10 by electroporation using BioRad Gene Pulser II (BioRad, Hercules, CA). Transformants were selected on LB10 Cm20 agar plates. The construction of the recombinant pNQ705 was confirmed by both PCR amplification and restriction analysis.