FIG 6.

RpoX positively regulates motility, biofilm formation, and hemolytic activities. (A) Motility assays of WT, ΔrpoX, and rpoX⁺ strains. Diluted cultures were spotted onto swimming and swarming plates (containing 0.3% and 1.5% agar, respectively) and incubated for 48 h or 12 h at 42°C. Three independent cultures were used for each strain, and a representative result is displayed. (B) Assays of biofilm formation by different strains. For WT, ΔN646_4611, ΔrpoX, and rpoX⁺ strains, biofilm formation in glass tubes containing LBS medium after 48 h of culturing was assayed. N646_4611^OE/ΔN646_4611 and N646_4611^OE/ΔrpoX strains were cultured in LBS medium with 0.04% l-arabinose for 48 h. The results are presented as the means ± SD (n = 3). **, P < 0.01; ***, P < 0.001; NS, not significant (by t test). (C) Hemolytic activities of WT, ΔrpoX, ΔrpoE, rpoX⁺, and ΔluxR strains grown on sheep blood agar plates at 30°C (top) and at 42°C (bottom). (D) qRT-PCR analysis of the transcripts of the selected genes. Total RNA was isolated from the ΔrpoX and rpoX⁺ strains after 12 h of growth in liquid culture. The results are presented as the means ± SD (n = 3). (E) Median lethal dose (LD₅₀) of WT, ΔrpoX, and ΔrpoE strains in zebrafish. Series of dilutions of WT, ΔrpoX, and ΔrpoE strains were intramuscularly inoculated into fish that were acclimated at 30°C for 4 weeks. A total of 30 fish were used for each of the dilutions. The infected fish were cultivated at 30°C and monitored for 7 days. The results are presented as the means ± SD (n = 3). *, P < 0.05 (by t test).