FIG 9.

Toxicity for zebrafish of material secreted by the T9SS. Wild-type, mutant, and complemented cells were cultured to mid-log phase (Klett units, 50 to 60) in TYES (A) or modified Shieh medium (B). Cells were removed by centrifugation, followed by filtration. Zebrafish (strain Eckwill crossed with strain Tupfel Long-fin; 6 fish per bacterial strain per trial) were exposed to cell-free spent medium (100 ml) for 5 to 7 h at 28°C, and mortalities were recorded every 30 min. Three independent trials were conducted for each strain. No mortalities occurred for control fish exposed to TYES or modified Shieh medium that had not been previously inoculated with F. columnare, or for fish exposed to cell-free culture fluids from the ΔgldN mutants or ΔporV mutant. (A) Toxicity of cell-free spent media from cultures of strains derived from F. columnare IA-S-4. Strains used were wild-type F. columnare IA-S-4, the ΔgldN_IA-S-4 mutant, and the ΔgldN_IA-S-4 mutant complemented with plasmid pLN5. (B) Toxicity of cell-free spent media from cultures of strains derived from F. columnare C#2. Strains used were wild-type F. columnare C#2, the ΔgldN_C#2 mutant, the ΔgldN_C#2 mutant complemented with plasmid pLN8, the ΔporV_C#2 mutant, and the ΔporV_C#2 mutant complemented with plasmid pLN11.