Construction of the suicide plasmid was performed as previously reported (14). Specifically, for B. canis RM6/66 mucR gene mutant, a 433-bp upstream fragment and a 500-bp downstream fragment of mucR gene were amplified using D-mucR-UF/UR and D-mucR-DF/DR primers, respectively (Supplementary Table 1). The two fragments were then used as templates for the second round of overlap polymerase chain reaction (PCR). The purified PCR product was digested with KpnI and HindIII restriction enzymes and cloned into the puc19-sacB plasmid, which was verified using PCR and sequencing.

The B. canis RM6/66 mucR gene unmarked in-frame deletion mutant was constructed with allelic replacement using a two-step method (14). The suicide plasmid was transformed into the wild-type B. canis RM6/66 strain by electro transformation. Single crossover integrates were then selected on tryptic soy agar (TSA) supplemented with ampicillin (100 μg/ml). The second crossover selection was conducted by plating the bacteria on TSA containing 5% sucrose. Ampicillin sensitive colonies were selected and verified by PCR and sequencing.

The mucR fragment, including the promoter sequence (500-bp of the intergenic region upstream of mucR start codon), was amplified with C-mucR-F/R primers (Supplementary Table 1). The PCR products were ligated to pMR11 plasmid, which was then referred to as the complementation plasmid pMR-mucR. To construct the complemented strain (CmucR), pMR-mucR was electroporated into mucR deletion mutant and then the cells were plated onto TSA containing ampicillin to detect the presence of pMR-mucR in the complemented strain. The resulting strains were verified by PCR and sequencing. For determination of minimum inhibitory concentrations (MICs) of antibiotics, the complemented strain (CmucR1) with chloramphenicol resistance was constructed using pBBR1MCS-1 plasmid using the same primers (C-mucR-F/R) as previously reported (15).

For RNA-seq assay, Brucella strains (RM6/66 and ΔmucR) were grown in three different tubes with TSB at 37°C from a single colony until the log phase was reached. Total RNA was isolated using TRIzol according to the manufacturer's instructions. Residual DNA in the samples was removed using DNase I. RNA concentration and purity were determined spectrophotometrically using an ND 1000 spectrophotometer (Thermo Scientific, Wilmington, USA).

The sequencing library of each RNA sample was prepared using NEB Next Ultra Directional RNA Library Prep Kit for Illumina as recommended by the manufacturer. Briefly, RNA fragments were reverse-transcribed and amplified to double-stranded cDNA and then ligated with an adaptor. The amplified cDNA was purified using the magnetic bead-based method and the molar concentration was determined for each cDNA library. The HiSeq X Ten platform was used to perform transcriptome sequencing. Sequencing and subsequent bioinformatics analysis were completed at Novel Bioinformatics Co., Ltd (Shanghai, China).

qRT-PCR was performed as previously described (16). Differentially expressed genes (DEGs) were verified by qRT-PCR using different samples like that in RNA-seq. Samples were amplified in a 20-μl reaction containing 10-μl 2 × SYBR® Premix Ex TaqTMII (TAKARA), 100 nM forward and reverse primers and 1-μl 10-fold diluted cDNA sample. 16S rRNA, which is constantly transcribed in bacteria, was chosen as the housekeeping gene. For each gene, qRT-PCR was performed in triplicate and relative transcription levels were determined using the 2^−ΔΔCt method. Primers used for qRT-PCR are provided in Supplementary Table 1.

The multiplication of B. canis RM6/66 and its derived strains in RAW264.7 macrophages were evaluated to determine the ability of ΔmucR mutant strain to survive intracellularly. The assays were performed as previously described (14). Briefly, in a permissive culture condition, B. canis strains were cultured in a flask with 0.2 μM vent cap for 12 h. RAW264.7 cells were seeded in 24-well plates (Corning, NY, USA) and then infected with Brucella strains at a multiplicity of infection (MOI) of 100. After 1 h of incubation, cells were washed thrice with phosphate buffer saline (PBS) and incubated in Dulbecco's Modified Eagle Medium (DMEM) containing gentamycin (50 μg/ml) to eliminate extracellular bacteria. At 1, 24, and 48 h post-infection (hpi), cells were washed and lysed with 500 μl 0.1% (v/v) Triton X-100 water solution, and then the bacteria count was determined by plating on TSA. The assays were run in triplicate and repeated at least twice.

Virulence assay was performed using BALB/c mice as previously reported (14) with some modifications. Mice were inoculated intraperitoneally with 0.1 ml (1 ×10⁷ CFU) of B. canis RM6/66 and its derived strains. On the 7th and 28th day post-infection (dpi), mice were euthanized via asphyxiation (n = 4 per group), after which the spleens were removed, weighed, and homogenized in 1 ml of PBS. The bacterial colonies were counted by the TSA plate method and CFUs per spleen were calculated.

To test the susceptibility of Brucella strains under different stress conditions, modified stress assays were performed as previously reported (9, 15). Brucella strains (with an initial density of 1 ×10⁶ CFU/mL) were, respectively, cultured in an acidic tryptic soy broth (TSB) medium (pH 4.5) for 2 h. Afterward, the concentration of bacteria was measured by plate count and the survival rate (%) calculated as surviving bacteria relative to the TSB control.

To determine the sensitivity of ΔmucR mutant to heat shock stress and hypertonic environments, 5 μl gradient dilution of bacterial cultures (1 ×10⁹ CFU/mL) were cultured on TSA medium at 42°C or TSA medium containing 200 mM sodium chloride (NaCl) at 37°C for 72 h, respectively.

To compare the growth of B. canis RM6/66, ΔmucR and the complemented strain in low iron medium, the TSB medium was limited in iron by adding different concentrations of the iron chelator, 2,2-dipyridyl (2.5, 5, and 10 mM). The bacteria were cultured in this medium at the same initial density (1 ×10⁶ CFU/ml) and the CFUs were determined at 48 h for each strain.

MIC values of the antibiotics from both β-lactam (Ampicillin, Amoxycillin, and Carbenicillin) and non-β-lactam (Ciprofloxacin, Gentamicin, and Tetracycline) were determined as previously reported (16).

Differences between the means of the experimental and the control groups were analyzed using the independent-samples t-test using SPSS 17.0 program. The differences were considered to be significant at p < 0.05.

B. canis RM6/66 mucR deletion mutant was constructed via a double recombination event and confirmed by PCR (Figure 1A). mucR complement strain CmucR was constructed by restoring the mucR gene using the broad-range plasmid pMR11. The resultant strains were verified by PCR and sequencing.

To characterize the cell growth rate of ΔmucR strain, the bacterial strains were analyzed in TSB medium at 37°C with the same initial density of 1 ×10⁶ CFU/ml. The result showed that the growth rate of ΔmucR was almost the same as its parent strain RM6/66 and the complementation strain CmucR at different time points (Figure 1B).

To assess the virulence of ΔmucR strain, the ability of ΔmucR to multiply within the cultured macrophage RAW264.7 was tested. As shown in Figure 2, the intracellular bacterial load of ΔmucR strain was almost the same as that of RM6/66 and CmucR at 1 h post-infection, while the bacterial load of ΔmucR was significantly reduced at 24 and 48 h (both with p < 0.05) in murine macrophage compared to RM6/66 and CmucR strains.

To assess the virulence of ΔmucR strain, the behaviors of ΔmucR and RM6/66 strains in an infected mouse model at 1 and 4 weeks were also analyzed. The spleen weight of the mice infected by the ΔmucR mutant infection group was significantly lighter than that of the RM6/66 infection group at both 1 (p < 0.05) and 4 (p < 0.001) weeks post-infection (Figure 3A). In addition, the CFU of ΔmucR mutant recovered from mice spleens was almost the same as that of RM6/66 at 1-week post-infection. However, the CFU number of mucR mutant recovered from the mice spleens was significantly lower at 4 weeks post-infection (p < 0.01) (Figure 3B).

As shown in Figure 4A, the survival ratio of ΔmucR strain was not significantly affected in acidic TSB medium compared with RM6/66 strain and the complemented strain CmucR (Figure 4A). When exposed to a hypertonic environment, the survival ratio of ΔmucR was similar to that of the parental strain RM6/66 and CmucR (Figure 4B). However, when ΔmucR strain was cultured at 42°C for 72 h, the survival ratio of the mutant strain was significantly reduced than that of RM6/66 and CmucR (Figure 4B).

The growth patterns of ΔmucR, B. canis RM6/66, and the complemented strain in the low iron medium were also measured. In the concentrations evaluated, the presence of 2,2-dipyridyl restricted the growth of all three strains. As compared with B. canis RM6/66, the growth of ΔmucR was significantly reduced at 2.5 mM 2,2-dipyridyl (p < 0.05) and abolished at a higher concentration (Figure 4C).

The susceptibilities of RM6/66, ΔmucR, and CmucR to β-lactam and non-β-lactam drugs were examined using standard procedures. The sensitivity of mucR mutant to Ciprofloxacin, Gentamicin, and Tetracycline was similar to that of the parental strain RM6/66 or CmucR. However, this mutant showed a higher sensitivity to all the three types of β-lactam agents tested. Such enhancement in susceptibility, estimated by MICs, was two-fold compared with its parent strain (Table 1).

Previous studies on the role of MucR in B. melitensis 16M demonstrated that MucR protein could repress the expression of several flagellar genes (11). Thus, the expression of 15 flagellar genes in the mucR mutant strain was analyzed using qRT-PCR. Our results showed that the expression level of eight flagellar genes (ftcR, fliC, flgC, flhA, flgB, fliP, flgK, and fliF) was significantly increased in mucR mutant compared with its parent strain B. canis RM6/66 (Table 2).

To uncover the underlying mechanism of virulence attenuation of ΔmucR strain, changes of the transcriptional profile of gene expression of ΔmucR or RM6/66 were analyzed using transcriptomic analysis. The transcriptional data set was submitted to the Gene Expression Omnibus (GEO) repository (National Center for Biotechnology Information, NCBI) with access number GSE155748. To confirm the RNA-seq data, 12 genes were chosen for qRT-PCR. Transcriptional data of all selected genes gave identical tendencies by both methods RNA-seq and qRT-PCR (Table 3).

In total, 694 genes exhibited significant difference (log₂ FC > 1 or < -1 and FDR <0.05). Among these genes, 409 genes were significantly up-regulated and 285 genes were significantly down-regulated in ΔmucR compared to its parent strain RM6/66 (Figures 5A,B). Genes, such as DK60_1986, DK60_2665, DK60_1987, K60_1068, DK60_1531, DK60_1985, DK60_2016, DK60_2832, DK60_1984, and aqpZ2, were among the top 10 up-regulated genes, while DK60_2150, DK60_1727, DK60_895, bfr, DK60_3051, DK60_2926, ffh, DK60_1851, atpC, and DK60_1852 were among the top 10 down-regulated genes.

According to the result of clusters of orthologous genes (COG) analysis, DEGs were mainly involved in translation, ribosomal structure and biogenesis, signal transduction mechanisms, energy production and conversion, intracellular trafficking, secretion and vesicular transport, and extracellular structures (Figure 6A). The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis demonstrated that the genes which exhibited significant difference were primarily enriched in the ribosome, oxidative phosphorylation, aminoacyl-tRNA biosynthesis, and protein export (Figure 6B).