Figure 1

(A) Cloning of exsA gene. M: DL2000 marker. Lane 1-4: The 861 bp fragment amplified from genomic DNAs of HY9901 using primer pairs of ExsA-F/ExsA-R. (B)exsA gene nucleotide and its encoded amino acid sequence.
Figure 2

Construction and confirmation of the knockout mutant strain HY9901ΔexsA. M: DL2000 marker, Lane 1. A fragment of 1,811 bp is obtained for HY9901 using primer pairs of exsA-A1-F/exsA-A2-R. Lane 2. A fragment of 11,019 bp is obtained for HY9901ΔesxA using primer pairs of exsA-A1-F/exsA-A2-R.
Figure 3

Characteristics of different strains. (A) Genetic stability detection of HY9901ΔexsA deletion mutantt. M: DL2000 marker; Lane 1. The 861 bp fragment amplified from genomic DNAs of HY9901 using primer pairs of ExsA-F/ExsA-R; Lane 2. The negative result amplified from genomic DNAs of ΔexsA using primer pairs of ExsA-F/ExsA-R. (B) Growth rates of HY9901ΔexsA and wild-type strain HY9901 (wt). Aliquots of cell culture were obtained at various time points and measured for cell density at OD600. (C) Activity of extracellaluar proteases. The extracellular protease activity was measured at OD442. (D) Measurement of biofilm by LSCM. (a) HY9901 2.5d diagram. (b)HY9901ΔexsA 2.5d diagram. (c) HY9901 2d diagram. (d) HY9901ΔexsA 2d diagram; HY9901 Biofilm thickness: 60 ± 10 μm, HY9901ΔexsA Biofilm thickness:90 ± 20 μm. (E) Swarming diameters were measured after 24 h incubation on TSA containing 0.3% agar plates. **p < 0.01.
Figure 4

(A) Differential analysis volcano map. (B) The most enriched GO terms of differentially expressed genes.
Figure 5

Comparison of qRT-PCR and RNA-Seq.
Figure 6

hop gene expression analysis. (A) Expression of hop genes by DMEM between HY9901 wild type and HY9901ΔexsA. (B) Expression of the LacZ reporter gene between HY9901 wild type and HY9901ΔexsA.
Figure 7

Propagation of HY9901ΔexsA in grouper liver (A) and spleen (B) following i.m. injection with 5 μL 1 × 105 cfu mL−1 ΔexsA. Control fish were i.m. injection with 5 μL sterile PBS. The number of viable bacteria was shown as the mean ± standard of three samples.
Figure 8

Survival in groups vaccinated with HY9901ΔexsA and PBS following challenge with V. alginolyticus HY9901.
Figure 9

Comparative analysis of the expression of immune-related genes in liver (A) and spleen (B) of zebrafish given the live attenuated vaccine and unvaccinated zebrafish. The liver and spleen of zebrafish were sampled at 1 day before challenge, and the mRNA level of each immune-related gene was normalized to that of β-actin. Bars represent the mean relative expression of three biological replicates and error bars represent standard deviation.
Figure 10

The pathological changes of vaccinated zebrafish. Zebrafish liver tissue section, 400× magnification (A1: injected with PBS A2: injected with HY9901 A3: injected with ΔexsA). Zebrafish spleen tissue section, 200 × magnification (B1: injected with PBS B2: injected with HY9901 B3: injected with ΔexsA). The triangle (▴) represents hyperemia, and the arrow (↗) represents the blurred boundary of lymphocytes in the figure.
Acknowledgments
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