The wild-type V. splendidus strain was dissociated from the focus of sea cucumbers with SUS and preserved in our laboratory. Zhang et al. tested the strain’s pathogenicity in sea cucumbers by means of a reinfection experiment (27). The bacterial cultures of the wild-type strain and indicated mutant were shaken (180 rpm) at 28°C in Zobell’s 2216E medium (5 g·L⁻¹ tryptone, 1 g·L⁻¹ yeast extract in filtered natural seawater). Escherichia coli S17λpir and DH5α (Takara, China) were grown in Luria–Bertani (LB) broth or agar at 37°C. The antimicrobial agent was purchased from a commercial source Sangon (Shanghai, China). Antibiotics were added to the medium at the following concentrations: kanamycin (Kn) 50 μg·ml⁻¹, gentamicin (Gm) 100 μg·ml⁻¹, and ampicillin (Ap) 100 μg·ml⁻¹. The vectors used in the experiment were pMD19-T, which was purchased from Takara (Beijing, China); vector pBBR1MCS-5, which was purchased from Fenghui Biotechnology (Changsha, China); and vectors pET28a and pBT3, which were kept in our laboratory. Restriction endonucleases were purchased from New England Biolabs and used according to the manufacturer’s instructions.

The primers used in the present study were designed according to the genomic DNA of the V. splendidus strain LGP32 with accession number FM954973.2 and listed in Table 1. MTVs was constructed as described previously (28, 29). Plasmid pET28aVsfur containing constitutive promoter P_Trc was generated to construct the MTVs with the reverse Vsfur sequence. The antisense RNA fragment of Vsfur was amplified with the forward primer VsfurF (Xho I) and reverse primer VsfurR (BamH I) and ligated into pMD19-T for subsequent restriction digestion. Plasmid pBT3Vsfur was constructed by ligating Vsfur into pBT3, between the BamH I/Xho I sites. The reverse Vsfur fragment fusing Trc promoter was obtained after pBT3Vsfur was digested with Swa I. The P_Trc-Vsfur fragment was inserted into the blunt end site EcoR V of pET28a and then transformed into S17λπ to construct S17λπ/pET28aVsfur. To transform the recombinant plasmid pET28aVsfur into wild-type V. splendidus, bacterial conjugation was conducted as described previously (30). The conjugants were placed on 2216E agar together with Kn and Ap, and the positive conjugants were picked out and confirmed by PCR (T7/T7ter) and sequencing.

To complement the fur deletion mutant of V. splendidus, we designed a pair of primers, fur^CF/R, to amplify the promoter region and complete ORF of the Vsfur gene. The fragment was ligated into pMD19-T and digested with BamH I and Xho I. Subsequently, fragments with BamH I and Xho I cleavage sites were cloned into the wide-host vector pBBR1MCS-5, which had different replicon and antibiotic resistance from pET28a. The recombinant vector pBBR1MCS-5Vsfur was transferred into MTVs by bacterial conjugation. The positive conjugants were selected on 2216E agar plates amended with Kn and Gm. Finally, the complemented strain fur^C was confirmed by PCR (pBBR1MCS-5F/R) and used for further research.

Vibrio splendidus strains, including WTVs, MTVs and fur^C were coated to 2216E agar at 28°C for 24 h. Single colonies were transferred into 10 ml of fresh 2216E broth and allowed to reach the optical density at 600 nm (OD₆₀₀) of 1.0. The culture was diluted proportionally to the same concentration. One hundred microlitre aliquots of WTVs, MTVs, and fur^C were reinoculated into flasks with 100 ml of fresh 2216E broth, 2216E broth supplemented with 100 μM iron chelator 2,2′-dipyridyl (DP), and 2216E broth supplemented with 50 μM FeCl₃ and grown at 28°C with agitation at 180 rpm. The OD₆₀₀ was recorded at different time points with an ultraviolet spectrophotometer (Mapada Instruments Co. Ltd., Shanghai, China). The experiment was repeated three times for each sample.

Total RNA was isolated from WTVs and MTVs cells at different OD₆₀₀ values. Data were standardized with the endogenous reference gene 16S rDNA (933F and 16SRTR1). The PrimeScript RT kit (Takara, Japan) and the reagent for removing genomic DNA were used for reverse transcription. Real-time polymerase chain reaction (RT-PCR) was performed in an ABI 7500 RT-PCR detection system (Applied Biosystems) with a total volume of 20 μl containing 0.4 μl of Dye-II (ROX), 0.8 μl of each of the forward and reverse primers (10 μM), 8 μl of diluted cDNA template, and 10 μl of 2 × SYBR Green Mix. The experimental procedure was set as follows: 95°C for 5 min to activate the polymerase, 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s. Fluorescent signals were collected at the extension stage of each cycle. The primers for RT-PCR are listed in Table 1. The fold changes of gene expression were determined using the 2^−ΔΔCT method (31).

The sea cucumber artificial infection experiments proceeded as described by Liang et al. (32). Healthy sea cucumbers (weight, 3 ± 1 g) were temporarily reared for 2–3 days before the experiment. Then, 105 sea cucumbers were randomly divided into seven groups. Each group was placed in an aquarium containing 10 ml of aerated natural seawater (salinity, 28 psu), and the temperature was maintained at approximately 16°C. For microbial infection experiments, the WTVs and MTVs strains were prepared as described in culture conditions. The cells were collected when OD₆₀₀ reached 1.0 by centrifugation at 8000 × g, washed three times with PBS, and resuspended in seawater. Three groups were continuously immersed in 1 × 10⁷, 1 × 10⁶, and 1 × 10⁵ CFU·ml⁻¹ WTVs and the other three groups were immersed in 1 × 10⁷, 1 × 10⁶, and 1 × 10⁵ CFU·ml⁻¹ MTVs. At the same time, the negative control group was inoculated with sterile PBS. The symptoms of infected A. japonicus were observed and the daily mortality was recorded.

The distributions of V. splendidus in the different A. japonicus tissues were determined as described previously (32). Sea cucumbers in the same growth state were equally divided into two groups, and each group was infected by WTVs and MTVs in a final concentration of 10⁷ CFU·ml⁻¹. After A. japonicus was subjected to immersion infection for 48 h, the body wall, muscle, respiratory tree, intestine, and tentacle of A. japonicus were collected using sterilized scissors and tweezers. Three tissue samples from three sea cucumbers were collected and mixed together; this was repeated three times. The specimens were supplemented with sterile PBS to 2 ml. Subsequently, the mixtures were homogenized by a homogenizer. One hundred microlitre gradient diluted mixtures were spread on 2216E agar plates supplemented with Ap (100 μg·ml⁻¹). Meanwhile, the coelomic fluids were filtered through a 200-mesh and spread on 2216E agar plates supplemented with Ap. Single colonies from each plate were identified by 16S rDNA sequencing analysis and the number of colonies was counted after incubation at 28°C for 24 h.

Swarming motility was assayed as described previously (33). WTVs, MTVs, and fur^C strains were separately grown in fresh 2216E broth at 28°C with agitation at 180 rpm, and OD₆₀₀ was adjusted to 1.0. Then, 2 μl bacterial cell suspension was dropped on the center of 2216E, 2216E supplemented with 200 μM iron chelator DP, and 2216E supplemented with 100 μM FeCl₃ plates containing 0.3% agar. The 2216E agar plates were then incubated at 28°C for 3 days to observe the diameters of the swarming halos. The iron treatment with different concentrations was repeated three times.

Biofilm formation assays for WTVs, MTVs, and fur^C were conducted as described by Luo et al. (34). Briefly, the WTVs, MTVs, and fur^C strains were prepared as described in culture conditions. The cell suspensions were adjusted to OD₆₀₀ = 1.0 with fresh 2216E broth. Then, 2 μl of WTVs, MTVs, and fur^C strains were transferred into 198 μl of 2216E broth, 2216E broth supplemented with 200 μM DP, and 2216E broth supplemented with 100 μM FeCl₃ in a 96-well plate and allowed to grow at 28°C for 48 h. Subsequently, the plate was washed three times with sterile PBS. Biofilms were stained with 200 μl of crystal violet (1%) for 30 min and washed with sterile PBS. The culture plate was then placed upside down on the absorbent paper. The adsorbed biofilm was dissolved with 200 μl of ethanol and the OD₅₇₀ was measured. The iron treatment with different concentrations was conducted in five independent biological replicates.

Promoter prediction analyses were conducted with online prediction tools at http://www.softberry.com. The 500-bp upstream sequences of virulence-related genes from the initial codon site were used to search the Fur binding box. All data are expressed as the mean ± SD of at least three sets of independent experiments. Statistical significance was determined by one-way Analysis of Variance (ANOVA) with a Dunnett’s test. The significance level was defined as *p < 0.05, **p < 0.01.

The growth curves of WTVs, MTVs, and fur^C were measured under different iron concentration culture conditions. The OD₆₀₀ was measured at 3, 6, 9, 12, 21, 24, 45, and 48 h. Although iron chelator DP inhibited the growth of three strains within 24 h, the growth curves of the WTVs, MTVs, and fur^C strains were almost the same in 2216E broth (Figure 1A), 2216E broth supplemented with 50 μM FeCl₃ (Figure 1B), and 2216E broth supplemented with 100 μM DP (Figure 1C). In this study, Vsfur was knocked down by antisense RNA interference to construct MTVs. Our previous research demonstrated that V. splendidus showed resistance to ampicillin, which is a β-lactam antibiotic, but V. splendidus was sensitive to kanamycin (35). Thus, plasmid pET28a with kanamycin resistance, which can replicate in V. splendidus, was used as a carrier vector to introduce an exogenous DNA fragment containing P_Trc-Vsfur. Meanwhile, constitutive promoter P_Trc could regulate the mRNA expression constant to a certain extent, improving the interference efficiency. Single colonies of WTVs and MTVs were inoculated into tubes with 10 ml of fresh 2216E medium supplemented with Ap and Kn cultured at 28°C overnight. No bacteria grew in the blank control and WTVs group, and the bacteria of MTVs could grow in the 2216E medium supplemented with Ap and Kn (Figure 2A). These results suggest that the recombinant plasmid pET28aVsfur was successfully transformed into V. splendidus (MTVs) and had no obvious influence on the growth rate of V. splendidus.

The WTVs and MTVs cells were collected at the OD₆₀₀ of 0.6, 1.0, and 1.5 to determine the temporal expressions of Vsfur and virulence-related genes. The expression levels of Vsfur, Vshppd, Vsm, and Vsflic from MTVs were not significantly affected in the early log phase at the OD₆₀₀ of 0.6. The expression of the Vsfur mRNA level was significantly downregulated 0.44- and 0.43-fold (p < 0.05) in MTVs at the OD₆₀₀ of 1.0 and 1.5 compared with the WTVs (Figure 2B). Compared with the WTVs, the significant increases in the transcription of Vshppd mRNA were 3.54- and 7.33-fold (p < 0.01) in MTVs at the OD₆₀₀ of 1.0 and 1.5 (Figure 3A). Meanwhile, the expression of Vsm mRNA was extremely significantly upregulated at the OD₆₀₀ of 1.0 and 1.5 compared with the OD₆₀₀ of 0.6. Compared with the WTVs, the significant increases in the transcription of Vsm mRNA were 2.10- and 15.92-fold (p < 0.01) in MTVs at the OD₆₀₀ of 1.0 and 1.5 (Figure 3B). Under the same conditions, the mRNA level of Vsflic was downregulated 0.56-fold (p < 0.05) in MTVs at the OD₆₀₀ of 1.0 compared with the WTVs (Figure 3C). These results suggest that the expression of the Vsfur gene was knocked down successfully in the mid and stationary log phase. Moreover, the downregulation of Vsfur, a global transcription factor that affects a number of virulence-related genes in V. splendidus, including upregulated genes in the MTVs for iron acquisition Vshppd (hemolysin) and Vsm (metalloprotease) and downregulated genes such as Vsflic (flagella C) related to flagellum biosynthesis. In summary, Vsfur has both positive and negative regulatory functions.

Promoter sequence analyses from the virulence-related genes of Vshppd, Vsm, and Vsflic were performed to predict the putative Fur binding boxes in the V. splendidus strain. The promoter regions before the ATG initial cordon of the three virulence-related genes were searched in the genomic DNA database. BPROM¹ prediction proved that the upstream of each virulence-related gene contained a typical promoter containing-35 and-10 regions (Figure 4). The Fur binding site was also searched and predicted near the-35 and-10 regions. These putative Fur binding boxes of Vshppd, Vsm, and Vsflic showed 52.63, 63.16, and 52.63% homology to the consensus Fur binding site sequence (5′-GATAATGATAATGATTATC-3′), respectively (Figure 4D). This sequence alignment suggests that Fur might bind directly to the promoter and regulate the expression of Vshppd, Vsm, and Vsflic.

In the artificial infection experiment, WTVs showed stronger pathogenicity than MTVs. Compared with the WTVs group, the A. japonicus infected with the MTVs strain showed a decrease in mortality and an obvious delay in the time of death (Figure 5A). The first mortalities of WTVs were observed 4 days post-infection with 1 × 10⁷ CFU·ml⁻¹ WTVs, and the first mortalities of MTVs were observed 4 days post-infection with 1 × 10⁶ CFU·ml⁻¹ MTVs. The total number of deaths in the WTVs group in 18 days were 7, 6, and 2 post-infection with 1 × 10⁷, 1 × 10⁶, and 1 × 10⁵ CFU·ml⁻¹ WTVs, respectively. The total number of deaths in the MTVs group in 18 days were 1, 2, and 0 post-infection with 1 × 10⁷, 1 × 10⁶, and 1 × 10⁵ CFU·ml⁻¹ MTVs, respectively. The A. japonicus infected with WTVs showed obvious characteristics of SUS, such as head shaking and skin ulceration. However, the A. japonicus group infected with MTVs did not show obvious characteristics of SUS (Figure 5B). No death occurred in the negative control group during the whole experiment. The median lethal doses (LD₅₀) of WTVs and MTVs calculated by SPSS were 9.116 × 10⁶ and 1.658 × 10¹¹ CFU·ml⁻¹, respectively. These results indicate that Vsfur is crucial to the pathogenicity of V. splendidus.

The quantitative detection of cell colonization in various tissues of A. japonicus after immersion infection with WTVs and MTVs is shown in Figure 6. The colonization quantities of WTVs to the body wall, muscle, respiratory tree, intestine, and tentacle tissues were 9.53 × 10⁷, 5.51 × 10⁸, 7.77 × 10⁷, 1.24 × 10⁸, and 2.42 × 10⁸ CFU·g⁻¹, respectively. The colonization amount of WTVs to the coelomic fluid was 2.95 × 10⁷ CFU·ml⁻¹. The colonization quantities of MTVs to all tissues except the body wall and respiratory tree decreased. The colonization quantities of MTVs to the body wall, muscle, respiratory tree, intestine, and tentacle tissues were 1.71 × 10⁷, 8.92 × 10⁷, 4.78 × 10⁷, 2.70 × 10⁷, and 6.71 × 10⁷ CFU·g⁻¹, respectively. The colonization amount of MTVs to the coelomic fluid was 1.02 × 10⁷ CFU·ml⁻¹.

The motility of WTVs, MTVs, and fur^C strains was measured in 2216E agar plates, and the results demonstrated an obvious difference in the swarming motility of WTVs and MTVs (Figure 7A). The swarming halo diameter of WTVs was 3–4 mm more than that of MTVs at each time point and the diameter of MTVs was 79–81% (p < 0.01) of that of WTVs in normal conditions (Figure 7B). Meanwhile, the swarming motility of MTVs was reduced by approximately 0.68-fold compared with that of WTVs in iron-replete conditions (Figure 7C). Under the iron-starved conditions, the motility of WTVs and MTVs was restricted and could not be spread normally (Figure 7D). These results suggest that Vsfur contributes to the swarming motility of V. splendidus.

The biofilm formation was determined using a crystal violet (CV) staining assay. The biofilms of WTVs, MTVs, and fur^C that attached to 96-well plates were analyzed in normal 2216E medium. Consistent with the results of swarming motility, the biofilm formation of MTVs on the surface of a 96-well plate was 24% (p < 0.01) and 22% of WTVs and fur^C, respectively (Figure 8A). The biofilm formation of MTVs was 81% (p < 0.05) of WTVs in iron-replete conditions. Under the iron-starved conditions, the biofilm formation of WTVs and MTVs showed no significant difference (Figure 8B). Taken together, these results indicate that deletion of Vsfur results in decreased biofilm formation, which depends on the presence of Fe³⁺.