The bacterial strains and plasmids used in this study, and their relevant characteristics are described in Table 1. R. anatipestifer strains were grown at 37°C in tryptic soy broth (TSB) (Difco, Detroit, USA) in an atmosphere of 5% CO₂, E. coli strains were cultured at 37°C in Luria Bertani broth (Sigma-Aldrich, St. Louis, USA). Both R. anatipestifer strains and E. coli strains included in this study were obtained from laboratory stocks of the Department of Veterinary Microbiology and Immunology of Huazhong Agricultural University, China. Where necessary, the following antibiotics were added in to the selection media: ampicillin (Amp), 100 mg/mL; spectinomycin (Spc), 100 mg/mL; kanamycin (Kan), 100 mg/mL; and medium was supplemented with 2, 6-diaminopimelic acid (DAP), 100 mg/mL; 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal), 20 mg/mL; Isopropylβ-D-1-thiogalactopyranoside (IPTG), 20 mg/mL; 4-chloro-DL-phenylalanine (cPhe), 0.2% (w/v).

For the construction of suicide vector pRE-lacZ-mpheS-spc, a 3.9 kb fragment of pRE was amplified from suicide vector pRE112 using primer S1L (Table 1) (introducing BamHI, SalI, and NdeI site) and S2R (Table 1) (introducing XhoI and BamHI site); then digested with BamHI enzyme and ligated to generate circular pRE which contained the essential components of the conjugational transfer. As RA cannot catabolize sucrose, the selected marker SacB was removed. The mutated pheS gene (mpheS) and the sequence of the multiple cloning sites were engineered into pUC57 to generate pUC57-mpheS and pUC57-MCS by the GenScript Corporation (Nanjing, China). This pUC57-mpheS vector contained the mutated R. anatipestifer pheS gene with altered DNA sequences (Supplemental Figure 1), which was driven by an upstream PS12 promoter of the R. anatipestifer rpsL gene. The 1.1 kb spc cassette was amplified from plasmid pIC333 using primers spcL and spcR (Table 1), mpheS was amplified from plasmid pUC57-mpheS using primers pheS1and pheS2 (Table 1). The spc cassette was then fused with the mpheS fragment using overlap PCR (introducing KpnI and XbaI site). The fragment of mpheS-spc was inserted into pMD18T to generate pMD18T-mpheS-spc. The pMD18T-mpheS-spc was digested with KpnI and XbaI enzymes, and 2.2 kb PS12-mpheS-spc fragment was ligated into pUC57-MCS and digested with KpnI and XbaI to obtain pUC57-MCS-mpheS-spc. The pRE and pUC57-MCS-mpheS-spc were digested with XhoI and XbaI and ligated to generate pRE-mpheS-spc. The next step was to amplify a 3.3 kb fragment of lacZ from E. coli BL21 genome using primer lacZR and lacZL (Table 1) (introducing a BamHI site). A 135 bp fragment of PS12 promoter was amplified from RA-YM using primer rpsL-LacZ (introducing a XhoI site) and rpsR-LacZ (Table 1) and fused to lacZ fragment by overlap PCR using rpsL-LacZ and lacZR primers. pRE-mpheS-spc and lacZ fragments were digested with BamHI and XhoI and ligated to obtain 9 kb pRE-lacZ-mpheS-spc.

The shuttle plasmid pRES-JXrep-spc was constructed in several steps including the amplification of 2.5 kb fragment of replicon region and replicase gene (Genebank: KY806579) with primers rep1 and rep2 (Table 1) (introducing a XhoI site). The wild type plasmid of R. anatipestifer strain RA-JX was used as a template. A 1.1 kb spc cassette was amplified from plasmid pIC333 using primers spcL1 and spcR1 (Table 1), thereby introducing a BamHI site. The replicon region of RA-JX was joined with the spc cassette using overlap PCR. The JXrep-spc fragment was inserted into pMD18T to obtain pMD18T-JXrep-spc. In the next step, plasmid pRE and pMD18T-JXrep-spc were digested with XhoI and BamHI and ligated to generate the shuttle plasmid pRES-JXrep-spc.

To obtain the suicide vector pRE-lacZ-mpheS-spc-fur for the deletion of whole fur gene from R. anatipestifer RA-YM strain, upstream (738 bp) and downstream (802 bp) DNA fragments were amplified using primers Fur-L1 and Fur-L2 (Table 1) (introducing SphI site), Fur-R1 and Fur-R2 (Table 1) (introducing KpnI site), respectively. The two fragments were joined together by overlap PCR. The LR fragment and pRE-lacZ-mpheS-spc were digested with SphI and KpnI; 1.5 kb LR fragment was inserted into pRE-lacZ-mpheS-spc to generate the suicide vector pRE-lacZ-mpheS-spc-fur. E. coli strain x7213 was used as a donor in conjugation step to introduce the suicide vector pRE-lacZ-mpheS-spc-fur into RA-YM strain as described previously (Hu et al., 2011). For phenotypic detection of mutant strains, conjugation filters were plated on tryptic soya agar (TSA) containing 100 μg/mL Spc. Colonies were then grown on TSA containing cPhe (0.2%) and X-gal (40 μg/mL). Appearance of white colonies confirmed successful construction of deletion mutant strains. For identification of recombinants carrying the chromosomal fur gene deletion, colonies were analyzed using PCR primers FurL1 and FurR2 to determine presence of wild-type or mutant allele at the target locus. The wild-type and deleted alleles could be differentiated on the basis of size of amplicon by agarose gel electrophoresis.

Similarly, for generation of complemented mutant strain, shuttle vector pRES-JXrep-spc-fur was constructed by amplification of the promoter sequence (171 bp) and the coding sequence (486 bp) of fur gene. The promoter sequence and the coding sequence were amplified using primers Promoter-fur1 and Promoter-fur2 (introducing KpnI site, Table 1), primers Fur-inL and Fur-inR (introducing SphI site, Table 1). The two fragments were joined together by overlap PCR. The plasmid pRES-JXrep-spc and fur gene fragment were digested with SphI and KpnI, then the fragment of 657 bp was inserted into plasmid pRES-JXrep-spc to obtain shuttle vector pRES-JXrep-spc-fur. The E. coli strain x7213 was used as donor in conjugation transfer of shuttle vector into the RA-YM Δfur strain (Hu et al., 2011). The phenotypic identification of complemented mutant strain was conducted on TSA plates containing Spc 100 μg/mL. Furthermore, PCR reaction was performed using primers Fur inL and Fur inR to ensure that recombinant strains were harboring shuttle vectors.

The colonies of the wild-type and the RA-YM Δfur mutant were suspended into tryptic soya broth (TSB) and incubated overnight with shaking at 37°C to an OD₆₀₀ of 0.2. FeCl₃ (Sigma-Aldrich) or 2, 2-Dipyridyl (2, 2-DP, Sigma-Aldrich) was added to the bacterial suspension to produce a final concentration of 200 and 30 μM, as iron restricted and iron rich conditions, respectively and incubated at 37°C until the OD₆₀₀ reached 0.8. Total RNA was extracted from bacteria solution using Bacterial RNA Kit (OMEGA, Norcross, USA) following the guidelines. Extracted RNA was purified with RNase-free DNase (Promega, Wisconsin, USA) at 37°C for 30 min to remove impurities of DNA, the DNA-free purified RNA was examined by 1% agarose gel electrophoresis. Purified RNA (23S rRNA and 16S rRNA) was sent to Huada Gene Center (Shenzhen, China) for RNA sequencing. All RNA samples were performed in two independent biological replicates (BioProject: SRP106941).

RT-qPCR was performed to quantify the expression of genes regulated by Fur. Primers were designed with Primer 5.0 software. RNA was extracted from wild-type and RA-YM Δfur strains grown in iron-restricted and iron-rich medium. RNA was reverse transcribed to cDNA using PrimerScript RT regent Kit with gDNA Eraser (Takara, Dalian, China). Real-time PCR reaction was performed using SYBR Premix (Takara, Dalian, China). Each reaction was performed in triplicate. Relative quantification of gene expression was calculated according to 2^−ΔΔCt method, RA-YM 16S rRNA was used as reference gene for normalized expression for each RNA sample.

The fragment of fur gene was amplified using the primers Fur1 and Fur2 (introducing the BamHI and XhoI sites); the fragment and vector pET-28a were digested with BamHI and XhoI and restricted fragment was ligated into the expression vector pET-28a to generate the expression vector pET-28a-fur. The expression plasmid was then transformed into competent cells of E. coli BL21 (DE3). Then the Fur protein was purified with an ÄKTA Purifier (GE His Trap FF, USA).

EMSA was performed with the Lightshift Chemiluminescent EMSA Kit (Thermo fisher scientific, Waltham, USA). The reaction was incubated at 30°C for 1 h, then loaded into 6% non-denaturing polyacrylamide gel electrophoretic and exposured. The reaction mixture (20 μL) contained 1 μg biotin labeled DNA fragment, 2 μL binding buffer, 1 μL KCl, 1 μL MgCl₂, 1 μL glycerol, 1 μL NP-40 and 1 μL Poly(dI-dC) and desired concentration of Fur protein, the final concentration of Fur protein were 0, 0.1, 1, and 10 μg in four lanes. 16S rDNA was used as a negative control. DNA fragments to be identified were amplified by biotin labeled primer (Sangon, Shanghai, China). The length of DNA fragments ranged from 350 to 420 bp.

One-day-old Cherry Valley ducklings obtained from the Wuhan Duck Farm (Wuhan, China) housed in cages under 12-h light/dark cycle, at controlled temperature (28–30°C) and free access to food and water during the whole course of this study. Care and maintenance of all animals were in line with the standards of Institutional Animal Care. This experiment was approved by the Institutional Animal Experimental Committee of the Veterinary Faculty of Huazhong Agricultural University.

To determine the role of fur in virulence, the median lethal dose (LD₅₀) of the deletion mutant RA-YM Δfur strain, the complemented mutant RA-YM Δfur strain and the wild-type RA-YM was measured using the Reed–Muench method (Reed and Muench, 1938). For each wild type, mutant and complemented strains, 12-day-old ducklings were evenly divided into five groups (10 ducklings/group). All five groups were injected intramuscularly with 1.0 × 10⁴, 1.0 × 10⁵, 1.0 × 10⁶, 1.0 × 10⁷, and 1.0 × 10⁸ colony forming units (CFU) of wild type strain, respectively. Similarly, mutant and complemented strains were injected to respective groups of ducklings for the evaluation of LD₅₀. Moribund ducklings were killed humanely and counted as dead. Dead ducklings were identified for the presence of RA. Mortality of the ducklings was recorded daily for a period of 10 days.

A comparative analysis of bacterial load in the blood of ducklings infected with mutants and wild type was made. Blood and target organs (brain, liver, heart and spleen) were collected at 24 and 48 h post-inoculation (five ducklings per group at each time-point). The target organs were homogenized with PBS to obtain supernatant. Blood and supernatant were plated on TSB agar plates for bacterial count with a 10-fold dilution method. In addition, the degree of lesions developing on the liver, spleen, heart and brain by the wild-type and the Δfur mutant strains were also recorded. For pathological investigations, all tissues were immersed in 10% formalin solution, embedded in paraffin section and stained with hematoxylin and eosin (H E). The pathological findings of the wild-type and the RA-YM Δfur mutant were compared.

As homologous recombination follows a two-step procedure, the selection of the R. anatipestifer Δfur mutant was carried out in two steps (Stibitz, 1994). The selection of mutant with Spc resistance was initially carried out, followed by the expression of mpheS gene. The function of lacZ gene could directly confirm whether the plasmid had been excised. The pheS gene was engineered by substituting alternative bases at numerous positions. The sequence similarity rate between wild-type pheS and mpheS was 71%. However, no difference was observed in amino acid sequence with the exception of the A301G mutation. In this study, the mpheS gene was driven by the promoter of RA rpsL gene. The first process was obtained by growing RA-YM strains on TSA medium containing Spc resistance. The first process obtained the merodiploid strains, which harbored the suicide vector. Then, the merodiploid strains were screened on TSA medium containing 0.2% cPhe and X-gal to obtain the deletion mutant. The merodiploid strains grew on the plate containing Spc but had no growth on the agar plate with 0.2% cPhe. The wild strains could be grown on the plate contains cPhe (Figures 1A2,A3). In addition, the merodiploid strains appeared as blue colonies while the wild type strain was of a white color on the plate containing X-gal (Figures 1A4,A5). This finding demonstrated the effectiveness of the counter-selectable markers pheS in RA. A suicide vector pRE-lacZ-mpheS-spc-fur, containing mutated pheS as a counter-selectable marker (Figure 1A1) was constructed and successfully transformed into RA-YM strain to generate RA-YM Δfur strain.

To determine whether the plasmid replicated and deleted fur gene after homologous recombination, no PCR amplification of fur gene and smaller LR fragment size (1,540 bp) from RA-YM Δfur strain as compared to larger LR fragment size (2,008 bp) from RA-YM strain (Figure 1C) confirmed the plasmid activity and recombination. Similarly, development of a recombinant RA-YM Δfur complemented mutant strain was confirmed by PCR amplification of fur and spc genes as shown in Figure 1D.

A comparison of gene expression regulated by iron and/or Fur in wild type (WT) and mutant (Δfur) strains grown in iron rich (+Fe) and iron restricted condition(−Fe) was exclusively established and comparison of RA-YM Δfur deletion mutant strain with wild type RA-YM was also performed. In our experiments, the significance of differentially expressed genes was estimated by the false discovery rate (FDR) and was considered significant if FDR < 0.001 and the |log₂Ratio| >1(Ernst et al., 2005a; Ledala et al., 2010). In total, 25 genes were downregulated and 45 genes were upregulated by iron when grown in iron-restricted conditions, in both parent and mutant. Seventeen genes were directly regulated by Fur. The expression of eight genes randomly selected regulated by iron and Fur was confirmed by real-time PCR in RA-YM and the Δfur mutant (Figure 7). The real-time PCR result was in accordance to the transcriptional data.

The putative Fur binding sequence and the distance from the start condon of the genes regulated by Fur is shown in Table 5. Promoter sequences were analyzed using software RegPredict and ClustalW for identification of putative binding sequence of Fur protein which was 19 bp long and sequence was predicted as 5′-ATTTAGAATTATTCTAAAT-3′ (Figure 8A). Therefore, the Fur binding sequence might be located within 100 bp of the translation initiation codon of the regulated genes (Table 5). To verify the putative role of the Fur-box sequence, the promoters of hmuR, sprT, RAYM_01847, RAYM_09824 were selected for electrophoretic mobility shift assay (EMSA). Our findings illustrated that purified Fur protein could bind to the DNA fragment containing the putative Fur-box (Figure 8B).

The LD₅₀ values of RA-YM, RA-YM Δfur deletion mutant and RA-YM Δfur complemented strain were recorded as 2.0 × 10⁶ CFU, 1.6 × 10⁸ CFU, 1.2 × 10⁷CFU, respectively. LD₅₀ counted for wild type as compared to RA-YM Δfur deletion mutant was approximately 80 times higher, whereas no significant difference was observed as in the case of wild type in comparison to RA-YM Δfur complemented mutant that was six times higher. Due to slight difference between wild type and RA-YM Δfur complemented mutant, a further comparison was established only between wild type and RA-YM Δfur deletion mutant strains. As the fur gene was disrupted in RA-YM Δfur deletion mutant strains resulted in attenuation of virulence of RA. However, virulence to ducklings was partially restored when the mutant was complemented with the plasmid pRES-JXrep-spc.

Microbiological analysis of heart, brain, liver and spleen showed that bacterial load was higher in wild type RA-YM strain compared to RA-YM Δfur deletion mutant strain at 24 and 48 h post-infection collection, a detailed comparison is shown in Figures 2A,B. Correspondingly, the pathological investigations illustrated that lesions were more significant in wild type as compared to mutant strains. The epicardial tissue of ducklings infected with wild type bacteria consisted of a higher degree of fibrinous exudate and inflammatory cell infiltration as compared to infection with mutant pathogens after 24 and 48 h (Figure 3). The lesions in brain tissue after both 24 and 48 h, the subarachnoid space was examined where mild inflammatory cell infiltration was noted in case of RA-YM Δfur deletion mutant strain as compared to wild type RA-YM strains (Figure 4). Similarly, a large number of hepatocytes expressed fatty degeneration and slight fibrotic effusion when infected with wild type RA-YM bacteria, whereas, such lesions were hardly observed in ducklings infected with RA-YM Δfur mutant bacteria after 24 h inoculation. A higher degree of fibrotic effusions in liver tissue was observed after 48 h of infection with wild type pathogens, in comparison to mutant pathogens. Collectively, severe hepatic congestion was noticed in ducklings infected with wild type RA-YM strains (Figure 5). Likely, both splenomegaly and congestion of spleen was observed in ducklings after 24 and 48 h in case of infection with wild type pathogens, whereas only splenomegaly was noticed in case of infection with mutant pathogens (Figure 6). Conclusively, all groups of ducklings, inoculated with wild type RA-YM strains, were severely infected in comparison to those inoculated with RA-YM Δfur deletion mutant strains. The control group of ducklings showed no significant pathological lesions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.