Fig. 1. Growth analysis of S. iniae, E. coli, B. subtilis, E. piscicida. The absorbance and viable count were measured every 12 hours. The absorbance was measured at OD600, and the number of viable cells was determined by counting the colonies formed in the BHI agar plate after serial dilution with 0.85% NaCl solution.(A)S. iniae FP5228, (B) E. coli DH5a, (C) B. subtilis subsp. subtilis, (D) E. piscicida CK41; dark gray line : OD600, gray line : viable count (CFU/ml).
Fig. 2. Construction of overexpression vector of toxin and antitoxin in S. iniae FP5228. (A) pBP1140 vector construction process. (B) Confirmation of pBP1140 vector using PCR. M : marker, lane 1 ; antitoxin ε : 287 bp, lane 2; pBAD :: toxin ζ : 2643bp, lane 3 ; toxin ζ : 864 bp.
Fig. 3. Growth analysis of toxin antitoxin overexpressed E. coli. The growth rate of the arabinose overexpressing strain at the early stage of growth was slower than the other conditions. (A) Growth curves measured using absorbance (OD 600nm). (B) Growth curves measured by viable count. (log CFU/ml/1,000,000), ◆ : control, ■ : IPTG induction, ▲ : Arabinose induction, × : IPTG & arabinose induction.
Fig. 4. Morphological changes of E. coli BL21 overexpressing Toxin. Overexpression of toxin in E. coli BL21 confirmed that the cell length was prolonged. (Gram staining, ×400) ; (A) control (not induction), (B) IPTG induction, (C) Arabionse induction, (D) IPTG & arabinose induction.
Fig. 5. Characteristic difference between wild type S. iniae (FP 5228) and plasmid-cured S. iniae (CK287). (A) Growth curves and viable counts of S. iniae FP5228 and CK287. It was confirmed that the decrease in viable cells was slightly improved in CK287.; ◆ OD600 of FP5228, ▲: OD600 of CK287, ■: CFU/ml of FP5228, ×: CFU/ml of CK287. (B) Plasmid preparation of S. iniae FP5228 and CK287.; lane 1 : FP5228, lane 2 : CK287. Plasmid size: 13,781 bp.
Fig. 6. Comparison of biofilm formation between S. iniae FP5228 and CK287.
Fig. 7. MTT assay between S. iniae FP5228 and CK287. In all cases, wild type S. iniae showed lower value than CK287. (n=3) (*p< 0.1).
Fig. 8. Virulence of S. iniae strains in zebrafish. To confirm the virulence, different concentrations of bacteria were inoculated. Survival rate was measured for 12 days. ◆, FP5228 108 CFU/fish; ■, FP5228 107 CFU/fish; ▲, FP5228 106 CFU/fish; Χ, CK287 108 CFU/fish; Ж, CK287 107 CFU/fish; ●, CK287 106 CFU/fish.
Table 1. PCR primers used in this study
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