Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes were similar in both the RNA-seq and qRT-PCR results. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism.

Author summary: Iron response regulator (Irr) plays a key role in iron metabolism of Brucella. In this study, the Brucella melitensis M5-90 irr mutant appeared significant different of growth and iron utilization rate compared with that of the Brucella melitensis M5-90 strain. However, the specific binding sites of Irr in Brucella are not fully understood. Here, we used Dap-seq to identify the potential target genes of Irr in the genome of Brucella melitensis M5-90. A total of seven genes were identified as target genes for Irr and the expression of levels of these seven genes were identified using qRT-PCR. In addition, Six out of seven genes namely rirA, membrane protein, hypothetical protein, ftrA, zntA and 2Fe-2S binding protein interact with the Irr protein. Furthermore, the mutation of rirA markedly affected the iron utilization and growth rate of Brucella. Overall, these results provide valuable insights for the exploration of Brucella iron metabolism.

Brucella is a Gram-negative, facultative intracellular pathogen that causes brucellosis, a worldwide zoonotic disease. Annually, more than 500,000 cases of human brucellosis occur globally [[1]]. This disease is characterized by undulant fever and chronic debilitating symptoms, such as endocarditis, spondylitis, arthritis, and meningitis; in livestock, it causes abortion and infertility [[2]–[4]]. Several studies have shown that Brucella melitensis is the predominant pathogen responsible for human or animal brucellosis in many provinces in China, including Xinjiang, Inner Mongolia, and Shanxi [[4]–[12]]. However, the pathogenic mechanisms of Brucella are not well understood. During the invasion of the host tissue, the organism invades and multiplies within professional and nonprofessional phagocytes and eventually establishes persistent infection in the host [[13]]. Metal metabolism plays a role in the expression of virulence genes required by Brucella to replicate or regulate the immune response in a host [[14]–[19]].

Iron is an indispensable micronutrient required by nearly all organisms as it is involved in many cellular processes, including energy metabolism, electron transport, and nucleotide biogenesis [[20]–[24]]. Although iron is available in the environment, it is scarce inside host cells to prevent oxidative damage to itself or replication of pathogens, and the change in environmental iron concentration is also an important signal for the induction of expression of virulence genes [[22]]. Hence, many pathogens have evolved acquisition mechanisms to obtain iron in the iron-restricted environment of mammalian cells. For example, bacteria obtain iron through the assimilation of heme or from iron-binding proteins by producing siderophores (a small molecule with an extremely high affinity for iron) when the intracellular iron concentration drops below a threshold [[25]]. Currently, two Brucella siderophores (2,3-dihydroxybenzoic acid (2,3-DHBA) and brucebactin) have been identified [[26]].

Iron response regulator (Irr) is a member of Fur family of transcriptional regulators that was first identified as a regulator of heme biosynthesis in Bradyrhizobium japonicum [[28]]. Previous studies have revealed that B. abortus Irr is also involved in heme biosynthesis and siderophore production under conditions of iron limitation. In addition, the ability of B. abortus to secrete brucebactin was significantly reduced when irr was knocked out [[30]]. A study has shown that bhuA is a crucial heme transporter, the B. abortus bhuA mutant cannot utilize heme as an iron resource in vitro, and Irr can regulate the expression of bhuA by directly binding to the promoter region of bhuA [[32]]. In addition, Brucella heme utilization oxygenase Q (bhuQ) was identified as a heme oxygenase in B. abortus 2308; it is cotranscribed with the iron-responsive regulator rirA, and both genes are regulated by Irr [[33]]. In addition to Irr, rhizobial iron regulator A (RirA) was first identified as an iron-responsive regulator in Rhizobium leguminosarum [[34]]. A previous study reported that RirA protein is active under iron-sufficient conditions, repressing iron uptake and the expression of rirA was regulated by Irr in Agrobacterium tumefaciens [[36]]. It can be inferred from these results that numerous genes associated with iron metabolism may exist in the Brucella genome and they might also be regulated by Irr. Therefore, the aim of this study is to screen the other iron metabolism genes regulated by Irr in Brucella and to further understand the function of Irr in Brucella iron metabolism.

B. melitensis M5-90 was donated by the Center of Chinese Disease Prevention and Control (Beijing, China). The B. melitensis irr mutant and B. melitensis rirA mutant have been constructed in a previous study [[37]]. B. melitensis M5-90 (M5-90), B. melitensis M5-90 irr mutant (irr mutant) and B. melitensis rirA (rirA mutant) were respectively cultured in normal, iron-sufficient or iron-deficient tryptic soy broth (TSB) (Difco, BD, USA) at 37°C and 150 rpm. TSB with 0.45 mM of the iron chelator 2,2′-dipyridyl (DIP, Sigma-Aldrich) or 50 μM FeCl3 (MACKLIN, Shanghai, China) was used as an iron-deficient or iron-sufficient medium, respectively. These two strains were monitored over a 44 h period at 37°C and 150 rpm. The absorbance value of the cell suspension was adjusted to 0.001 in a 20 μL volume and inoculated into 20 mL of TSB. The absorbance value was measured at 600 nm wavelength every 4 h for 44 h with a Nanodrop 2000 spectrophotometer (Thermo, USA). Additionally, the quantification of colony-forming units/mL (CFU/mL) for these three strains was performed using McFarland turbidimeter (BEIJING HELI KECHUANG TECHNOLOGY DEVELOPMENT CO.LTD, Beijing, China), and bacterial CFU were monitored in triplicate at 4 h intervals throughout the incubation period. All experiments with Brucella strain were performed in a Biosafety level 3 facility according to the regulations of Center for Disease Prevention and Control (CDC) of China.

The iron utilization rates of M5-90, the irr mutant and the rirA mutant in normal TSB, iron-deficient TSB, or iron-sufficient TSB were measured via 1, 10-phenanthroline chelation with ferrous iron, yielding an orange-red color complex with maximum absorption at 512 nm [[38]]. Briefly, during the growth kinetics assay of these three strains, 2 mL cell suspension of M5-90, irr mutant and rirA mutant from the different iron concentrations of TSB was collected at each time point and centrifuged at 12,000 × g for 5 min. The supernatant was transferred into a new tube, and 600 μL of 1, 10-phenanthroline (5 nM, Sigma-Aldrich) and 400 μL of L-ascorbate (0.5 mM, Sigma-Aldrich) were added and incubated for 2 h at 25°C. Finally, the absorbance value was measured at 512 nm wavelength with a Nanodrop 2000 spectrophotometer (Thermo, USA).

Details of wheat cell-free reaction have been described previously [[39]]. Cell-free protein expression was performed using the TNT SP6 Wheat Germ Master Mix Kit (Promega, Madison, WI). The primers for the irr gene are as follows: irr forward, 5΄-CCATATGATGCATTCTTCACATACCCA-3΄; irr reverse, 5΄-CTCTAGATCAGCGGGCCTGACGGCG-3΄. The polymerase chain reactions (PCRs) were incubated at 95°C for 5 min. Then 35 cycles were performed as follows: 30 s at 95°C, 40 s at 57°C, and 7 min at 72°C. The amplified PCR product was subcloned into a pDAP-Halo-Kan vector (Zoobio, Nanjing, China) using the Seamless cloning kit (Beyotime, Shanghai, China), and the recombinant plasmid was used as a transcription template. The protein expression volume was 50 μL, comprising 30 μL Wheat Germ Master Mix, 1 μg pDAP-Halo-Kan-irr plasmid, and nuclease-free water to make 50 μL; this was incubated at 25°C for 2 h. The expressed protein was confirmed using western blot analysis and an anti-Halo tag antibody (Promega, Madison, WI).

The genomic DNA (gDNA) of M5-90 was extracted using a Genomic DNA Extraction Kit (Zoobio, Nanjing, China). Purified gDNA was fragmented to 100–500 bp via sonication for 17 min at 20% amplitude and keeping it for 30 s on and 30 s off on ice. The size of the fragmented gDNA was checked using 1.5% agarose gel. DNA end-repair and dA-tailing were performed on 1 μg of fragmented gDNA via the NEXTflex Rapid DNA-Seq Kit (Zoobio, Nanjing, China). Ligation of end-repaired and adenylated DNA to DAP-adaptor (Mich Scientific, Beijing, China) was performed using the NEXTflex TM Enzyme Mix (Zoobio, Nanjing, China) according to the manufacturer´s instructions.

Dap-seq experiments were performed in duplicate, and beads in negative control group were incubated without protein. Ten microliters of Halo-tag magnetic beads (Yeasen, Shanghai, China) were washed three times in a 1.5 mL tube with 600 μL of sterilized PBS containing 0.01% Tween 20 (binding buffer). Twenty-five microliters of wheat cell-free expressed protein was added to 25 μL of binding buffer (containing washed beads) and incubated for 1 h on a rotating wheel at 25°C. Next, 50 μL of binding buffer was added and used to wash the bead-protein complexes five times. The supernatant was discarded, 25 μL of binding buffer (containing 100 μM MnCl2) and an equal volume of the gDNA library were added, and then the mixture was placed on a rotating wheel for further incubation at 25°C for 1 h. A volume of 50 μL of binding buffer was added and used to wash the formed bead-protein-DNA complexes five times to remove unbound DNA. Finally, 30 μL of 50 mM Tris-HCl, with pH 8.5 was added to suspend the complexes, and incubated for 10 min at 98°C. After incubation, the samples were cooled at 4°C for 5 min, removing the beads, and the released DNA was collected and stored at -20°C for PCR amplification as described previously [[40]]. The sample was sequenced using a different indexed pair of primers.

The DNA sequencing was performed using the Illumina Hiseq 2500 sequencer, and 150-bp pair-end reads were generated; all the reads were aligned to the genome of M5-90 using Bowtie2 (version 2.3.4.2), and then peak calling was conducted using MACS2 (version 2.1.1.20160309). Peaks of two duplicate samples were merged using Irreproducible Discovery Rata (IDR, v2.0.2), and the reliability of the repeated peaks was scored. The conserved motif of the region of the peaks was analyzed using MEME (v5.3.0) and the annotation of the peaks was completed using HOMER (v4.11). The distribution frequency of the reads near the transcriptional start site was analyzed using deepTools (v3.3.1).

The expression levels of the genes identified in the Dap-seq experiment were further analyzed using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from M5-90 or M5-90 irr mutant cells with TRIzol (CWBIO, Beijing, China). Each group has three replicates. Briefly, the RNA concentration and quality were evaluated using a Nanodrop 2000 spectrophotometer (Thermo Fisher, USA). The extracted RNA was reverse-transcribed to cDNA using a First Strand cDNA Synthesis Kit (Beyotime, Shanghai, China) according to the manufacturer's protocol. qRT-PCR was performed using SYBR (CWBIO, Beijing, China) and a ThermoFisher QuantStudio 3 RT PCR-Well Q3 (Thermo Fisher, USA). The primers for the target genes are listed in S1 Table. The reaction conditions used are as follows: initial denaturation at 95°C for 5 min followed by 46 cycles of 95°C for 30 s and annealing at 56°C or 60°C for 30 s. The data were normalized according to the expression level of the 16S ribosomal RNA and the expression level of each gene was calculated using the 2-ΔΔCT method.

The DNA probes containing either wild type or mutated promoters of the candidate target genes were labeled using EMSA probe biotin labeling kit (Beyotime, Shanghai, China); in addition, unlabeled DNA probes were used for competition assays. Specific primers of the promoters of the target genes were listed in S2 Table. EMSAs were performed according to the manufacture´s instruction (Beyotime, Shanghai, China). Briefly, biotin-labeled DNA probes were incubated at 0.5 nM for 5 min at 37°C in an EMSA buffer (10 mM Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10% glycerol, 0.1 mg/mL BSA, and pH 8) containing 25 ng/μL poly(dI-dC). In addition, 100 nM unlabeled DNA probes (200-fold excess and containing 100 μM MnCl2) were incubated with the labeled probes for competition assays. A total of 0.5 μg (100 nmol/L) Irr protein was added and incubated for an additional 15 min at 37°C. The samples were separated on a 6% non-denaturing SDS-PAGE gel and run at 100 V and 4°C in cold 0.5 × Tris-borate buffer. The DNA bands were detected using BeyoECL Plus.

Graph plotting and all the data analyses were performed using R (v4.0.5) with the "ggplot2" package and the unpaired Student's t test. All performed experiments were were repeated at least three times and the results are presented as the mean ± standard deviation. Asterisks in the figures indicate significance (* P < 0.05, ** P < 0.01, *** P < 0.005, and ns = not significant).

Many studies have reported that Irr is pivotal for iron metabolism and is involved in heme biosynthesis under iron-limited conditions [[41]–[44]]. Hence, to investigate the effect of irr on Brucella growth in vitro, M5-90 and the irr mutant were grown in normal, iron-limited, and iron-sufficient TSB, and the absorbance values were measured at each time point. For the normal TSB, the growth rate of the irr mutant reduced significantly from 32–44 h, compared with that of the M5-90 strain (Fig 1A). However, the growth rate of the irr mutant was higher than that of the M5-90 strain from 16–28 h and 36–44 h under iron-limited conditions (Fig 1B). Under iron-normal or sufficient conditions, both strains exhibited a comparable growth pattern. However, it is noteworthy that the M5-90 strain demonstrated a higher growth rate in comparison to the irr mutant strain (Fig 1A and 1C). Additionally, the quantification of CFU/mL or CFUs for these two strains was performed under varying iron conditions at multiple time points. The results were obtained regarding CFU/mL or CFUs were found to be similar with those obtained from absorbance measurements (S1 and S2 Figs). These results suggest that irr can significantly affect Brucella growth, especially under iron-limited concentrations.

Graph: Fig 1 Growth curves of M5-90 and M5-90 irr mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The asterisk positioned atop each time point denotes the statistically significant contrast in growth between M5-90 and M5-90 irr mutant across various temporal intervals. The iron concentration in normal, iron-limited or sufficient TSB were 3.42 μg/mL, 3.07 μg/mL, 27.68 μg/mL. The standard deviation value in many time points cannot be presented, because the value is less than 1%.

To further understand whether the influence of irr on Brucella growth was completed via iron utilization, M5-90 and the irr mutant were grown in normal TSB, iron-limited, and iron-sufficient TSB. The residual iron in the medium was determined at each time point. For the normal TSB, the iron utilization of the M5-90 was lower than that of irr mutant from 20–44 h (Fig 2A), whereas the iron utilization of the M5-90 was significantly higher than that of irr mutant from 32–44 h under iron-limited conditions (Fig 2B). Under iron-sufficient conditions, the iron utilization of the M5-90 was higher than that of irr mutant from 12–44 h (Fig 2C). These results indicate that irr can affect Brucella growth via iron utilization, especially under iron-limited condition.

Graph: Fig 2 Iron utilization assays of M5-90 and M5-90 irr mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The iron concentration in normal, iron-limited or sufficient TSB were 7.11 μM/mL (3.42 μg/mL), 6.37 μM/mL (3.07 μg/mL), 57.4 μM/mL (27.68 μg/mL). The standard deviation value in many time points cannot be presented, because the value is less than 1%.

Given that Irr protein can affect growth and iron utilization of Brucella especially under iron-limited conditions, it is postulated that the numerous genes related to iron metabolism could be modulated by the Irr protein. Thus, to investigate the underlying mechanisms of Irr involvement in iron metabolism. The Daq-seq was used to identify the Irr-bound DNA sequences from the Brucella genome. The Irr protein was purified and confirmed using Western blotting (S3A Fig), and the fragmented Brucella genome was checked using agarose gel (S3B Fig). After DNA sequencing, aligning, and peak calling, seven peaks were generated on the Irr-bound region, and seven genes were identified in the Brucella genome (Fig 3A and S3 Table); of these, three genes were located in chromosome I (NC_003317.1) and four genes were located in chromosome II (NC_003318.1) (S4 Fig). In addition, three motifs of the Irr protein were identified in the Brucella genome (Fig 3B), which is consistent with the results obtained in previous studies [[33], [45]], and all of these sequences are A/T-rich. Overall, these results indicate that seven genes were identified as target genes of the Irr protein for regulating iron metabolism in Brucella genome.

Graph: Fig 3 Data generated by Dap-seq.(A) Peaks generated by Dap-seq. The regions of Irr-bound DNA produced seven peaks when mapped to the B. melitensis M5-90 genome. (B) Enriched DNA motifs obtained with MEME-Dap-seq on relevant Dap-seq peaks corresponding to near summit regions.

A total of seven Irr-bound genes have been identified previously. To further confirm the expression levels of these genes in M5-90 and the irr mutant, RNA was isolated from these two strains grown to log phase (32 h) in iron-limited TSB, and cDNA was synthesized. Of these seven genes, the expression levels of five genes (BME_RS09560, membrane protein, RirA, Iron transporter (ftrA), and BME_RS16825) were higher in the irr mutant than in the M5-90 strain (Fig 4). The expression levels of two genes (2Fe-2S binding protein and cation-transporting P-type ATPase (zntA)) were lower in the irr mutant than in the M5-90 strain (Fig 4). Therefore, these seven genes were regulated by irr and might interact directly with the Irr protein to regulate iron metabolism.

Graph: Fig 4 Identification of the expression levels of the seven genes using qRT-PCR.Total RNA was isolated from M5-90 or the M5-90 irr mutant and cDNA was synthesized. 2Fe-2S binding protein, Cation-transporting P-type ATPase (zntA), FtrA, Hypothetical protein (BME_RS09560), Hypothetical protein (BME_RS16825), Membrane protein, and RirA genes were analyzed using qRT-PCR. The expression levels of the targets gene were normalized by the expression of 16 S. The Y-axis represents the gene expression in the M5-90 irr mutant or M5-90.

To further verify the interactions of the Irr protein and the seven genes, EMSA was performed to determine whether the Irr protein can directly bind to these seven genes. As shown in Fig 5, the promoter regions of six out of the seven genes were able to form protein-DNA complexes with the Irr protein, and these genes are RirA (BME_RS13665, gene ID: 29595065), membrane protein (BME_RS01725, gene ID: 29593120), hypothetical protein (BME_RS09560, gene ID: 29594811), FtrA (BME_RS14525, gene ID: 29595411), cation-transporting P-type ATPase (zntA) (BME_RS10660, gene ID: 29595149), and 2Fe-2S binding protein (BME_RS13655, gene ID: 29595949). Collectively, these results indicate that the Irr protein can directly bind to rirA, membrane protein, hypothetical protein, ftrA, zntA, and 2Fe-2S binding protein to participate in iron metabolism in Brucella.

Graph: Fig 5 Genetic organization of the seven genes in M5-90 and the Irr protein interacts directly with the promoters of these genes in an EMSA.(A) Irr protein interacts with BME_RS13665 (rirA). The red and blue arrows indicate rirA and YbaK gene sequences. The red and underlined sequences represent the putative Irr binding sites (ICE-box); lane 1: positive probe, lane 2: positive probe + positive protein, lane 3: positive probe + positive protein + positive unlabeled probe, lane 4: Irr protein, lane 5: probe BME_RS13665-1, lane 6: probe BME_RS13665-1 + Irr protein, lane 7: probe BME_RS13665-1 + unlabeled probe BME_RS13665-1 + Irr protein, lane 8: probe BME_RS13665-1 mutation + Irr protein, lane 9: probe BME_RS13665-2, lane 10: probe BME_RS13665-2 + Irr protein, lane 11: probe BME_RS13665-2 + unlabeled probe BME_RS13665-2 + Irr protein, and lane 12: probe BME_RS13665-2 mutation + Irr protein. (B) Irr protein interacts with BME_RS01725 (membrane protein). The red and blue arrows indicate BME_RS01725 and MFS gene sequences. The red and underlined sequences represent the putative Irr binding site (ICE-box); lane 1: probe BME_RS01725, lane 2: probe BME_RS01725 + Irr protein, lane 3: probe BME_RS01725 + unlabeled BME_RS01725 + Irr protein, and lane 4: probe BME_RS01725 mutation + Irr protein; (C) Irr protein interacts with BME_RS09560 (hypothetical protein). The red and blue arrows indicate BME_RS09560, BME_RS09555, and BME_RS09565 gene sequences. The red and underlined sequences represent the putative Irr binding site (ICE-box); lane 1: probe BME_RS09560, lane 2: probe BME_RS09560 + Irr protein, lane 3: probe BME_RS09560 + unlabeled BME_RS09560 + Irr protein, and lane 4: probe BME_RS09560 mutation + Irr protein. (D) Irr protein interacts with BME_RS14525 (Iron transporter). The red and blue arrows indicate BME_RS14525 and GuaA gene sequences. The red and underlined sequences represent the putative Irr binding site (ICE-box); lane 1: probe BME_RS14525, lane 2: probe BME_RS14525 + Irr protein, lane 3: probe BME_RS14525 + unlabeled BME_RS14525 + Irr protein, and lane 4: probe BME_RS14525 mutation + Irr protein; (E) Irr protein interacts with BME_RS10660 (cation-transporting P-type ATPase). The red and blue arrows indicate BME_RS10660 and HemN gene sequences. The red and underlined sequences represent the putative Irr binding site (ICE-box); lane 1: probe BME_RS10660, lane 2: probe BME_RS10660 + Irr, lane 3: probe BME_RS10660 + unlabeled probe BME_RS10660 + Irr protein, and lane 4: probe BME_RS10660 mutation + Irr protein. (F) Irr protein interacts with BME_RS13655 (2Fe-2S binding protein). The red and blue arrows indicate BME_RS13655 and antibiotic biosynthesis monooxygenase gene sequences. The red and underlined sequences represent the putative Irr binding site (ICE-box); lane 1: probe BME_RS13655, lane 2: probe BME_RS13655 + Irr protein, lane 3: probe BME_RS13655 + unlabeled probe BME_RS13655 + Irr protein, and lane 4: probe BME_RS13655 mutation + Irr protein. (G) Irr protein interacts with BME_RS16825 (hypothetical protein). The red and blue arrows indicate BME_RS16825 and BME_RS09375 gene sequences. The red and underlined sequences represent the putative Irr binding site (ICE-box); lane 1: probe BME_RS16825, lane 2: probe BME_RS16825 + Irr protein, lane 3: probe BME_RS16825 + unlabeled probe BME_RS16825 + Irr protein, and lane 4: probe BME_RS16825 mutation + Irr protein.

The RirA is another iron response regulator and it also involved in iron uptake and energy metabolism [[35], [43]]. Hence, to further investigate the role of rirA in iron metabolism of Brucella, M5-90 and the rirA mutant were cultured in normal TSB, iron-limited, and iron-sufficient TSB. The residue iron in the medium was determined at each time point. For the normal TSB, the iron utilization of the M5-90 was lower than that of rirA from 16–24 h and 32–44 h (Fig 6A); under iron-limited conditions, the iron utilization of the M5-90 was lower than that of rirA from 16–32 h, whereas, the iron utilization of the M5-90 was higher than that of rirA from 40–44 h (Fig 6B). In order to elucidate the dissimilar phenotype observed in M5-90 under iron-depleted conditions, as compared to the rirA mutant strain, an investigation was conducted into the expression of irr and rirA genes during mid-exponential, late-exponential and stationary phases of growth. Results showed a progressive enhancement in gene expression of both irr and rirA from 16 h to 40 h. While the expression of rirA started to decline after 40 h and till 45 h, there was a rapid increase in the expression of irr during the same time frame. (S5 Fig). In addition, the iron utilization of the rirA was higher than that of M5-90 from 16–28 h and 36–44 h (Fig 6C). Collectively, these results suggested that rirA can affect iron metabolism of Brucella at different iron concentration conditions, especially under iron-sufficient and normal conditions, these two strains have similar iron metabolism pattern.

Graph: Fig 6 Iron utilization assays of M5-90 and M5-90 rirA mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The iron concentrations in normal, iron-limited or sufficient TSB were 7.12 μM/mL (3.43 μg/mL), 6.38 μM/mL (3.08 μg/mL), 57.4 μM/mL (27.68 μg/mL). The standard deviation value in many time points cannot be presented, because the value is less than 1%.

Previous study reported that iron is a fundamental micronutrient for Brucella, and plays a role in its growth [[46]]. Therefore, to investigate whether the rirA can affect Brucella growth via iron utilization, M5-90 and the rirA mutant were cultured in normal TSB, iron-limited, and iron-sufficient TSB. The absorbance values were measured at each time point. For the normal TSB, the growth rate of the rirA was lower than that of the M5-90 from 20–24 h and 40–44 h (Fig 7A). However, the growth rate of the rirA mutant was higher than that of the M5-90 from 16–24 h, and the growth rate of the rirA mutant was lower than that of the M5-90 from 28–44 h under iron-limited conditions (Fig 7B). For the iron-sufficient conditions, the growth rate of the rirA was lower than that of the M5-90 from 16–20 h and 36–44 h (Fig 7C). In addition, the quantification of CFU/mL or CFUs for these two strains was performed under varying iron conditions at multiple time points. The results were obtained regarding CFU/mL or CFUs were found to be similar with those obtained from absorbance measurements (S6 and S7 Figs). Overall, these results demonstrated that the rirA can affect Brucella growth at different iron concentration conditions via iron utilization.

Graph: Fig 7 Growth curves of M5-90 and M5-90 rirA mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The asterisk positioned atop each time point denotes the statistically significant contrast in growth between M5-90 and M5-90 irr mutant across various temporal intervals. The iron concentrations in normal, iron-limited or sufficient TSB were 3.43 μg/mL, 3.08 μg/mL, 27.68 μg/mL. The standard deviation value in many time points cannot be presented, because the value is less than 1%.

Iron is required by all life forms owing to its involvement in many fundamental biological processes, including photosynthesis, N2 fixation, methanogenesis, H2 production and consumption, respiration, the trichloroacetic acid (TCA) cycle, oxygen transport, gene regulation, and DNA biosynthesis [[47]]. B. melitensis is a Gram-negative intracellular pathogen that causes abortion and infertility in livestock. As with other bacteria, iron is an essential micronutrient for Brucella, and plays a role in its growth and virulence [[46], [48]]. However, maintaining iron homeostasis is also crucial for Brucella, because reactive oxygen can be produced in an iron-replete environment, which is not in favor of the survival of Brucella. Several studies have suggested that Irr is a major iron response regulator in many species and participates in heme biosynthesis [[28], [30], [49]]. The expression of the Irr protein is controlled by cellular iron levels, and is degraded under iron-sufficient conditions when heme is biosynthesized [[30], [32], [42]]. However, the Irr protein is activated under iron-limited conditions and represses heme biosynthesis [[30]]. Heme is a key iron source for Brucella growth, therefore, it might be the reason for the higher growth rate of the irr mutant than that of M5-90 under iron-limited conditions (Fig 1B). Under normal or iron-sufficient conditions, the Irr protein is less expressed or degraded. Remarkably, the growth rate of M5-90 surpasses that of the irr mutant under both normal and iron-sufficient circumstances (Fig 1A and 1C). This leads to the inference that the irr may exert an impact on the growth of Brucella in normal or iron-sufficient conditions through elusive mechanisms, despite being in iron-sufficient conditions, the mutation of irr was found to alter the expression of various genes associated with iron metabolism. Nonetheless, the underlying mechanisms responsible for this phenomenon warrant further investigation. In addition, the mutation of irr reduced the iron utilization of B. melitensis under iron-limited conditions (Fig 2B), and that phenotype may be produced by the Irr protein interacting with zntA or 2Fe-2S binding protein identified in this study (Fig 5E and 5F). The expression levels of these two genes were significantly reduced when irr was mutated (Fig 4). However, the M5-90 strain and irr mutant have different patterns of iron utilization under normal or iron-sufficient conditions (Fig 2A and 2C). The reason for that phenomenon may be attributed to the characteristics of the Irr protein, and it can serve as either a repressor or an activator of many genes based on the cellular iron level [[43]]. Despite the degradation of Irr protein under iron-sufficient conditions, the expression of other genes involved in iron metabolism can still be affected by irr, potentially influencing the growth rate and iron utilization of Brucella.

The RirA protein is another iron response regulator and was initially discovered in Rhizobium leguminosarum [[35]]. RirA participates in many physiological processes, such as iron uptake, energy metabolism, and heme biosynthesis [[43]]. RirA can be activated under iron-sufficient conditions to inhibit the iron uptake system. However, under iron-limited conditions, the expression of RirA was repressed by Irr to reduce iron consumption for maintaining essential processes in Agrobacterium tumefaciens [[36]]. In this study, the mutation of the rirA resulted in a significant enhancement of iron uptake of Brucella, especially under iron normal or -sufficient conditions, which further corroborated that the RirA can be activated under iron-sufficient conditions to inhibit iron uptake for maintaining iron homeostasis. In addition, the higher iron utilization of rirA mutant might be the reason for its lower growth rate under iron normal or -sufficient conditions, because the excessive iron uptake was not benefit for the growth of Brucella [[46]]. Whereas, the iron utilization of rirA mutant was not consistent under iron-limited conditions, the iron utilization of the rirA mutant was higher than that of the M5-90 from 16–32 h, but was lower than that of the M5-90 from 36–44 h. Additionally, the growth rate of the rirA mutant was also inconsistent under iron-limited conditions, whereas, the underlying mechanisms responsible for the observed phenotype may potentially be linked to the fluctuating expression patterns of irr or rirA, which in turn be influenced by the iron concentration within the medium. In this work, the expression level of rirA (BME_RS13665) was higher in the irr mutant than that in the M5-90 strain (Fig 4), and the promoter of the rirA can form a complex with the Irr protein (Fig 5A). Hence, Irr can directly inhibit rirA expression under iron-limited conditions, which is consistent with the results obtained from Agrobacterium tumefaciens [[36]]. Furthermore, the RirA contains the mixture of both [2Fe–2S] and [4Fe–4S] forms, and the [4Fe–4S] can inhibit the expression of many genes involved in iron uptake; however, under low iron conditions, the [4Fe–4S] was converted to the [2Fe–2S] to alleviate the repression effect [[50]]. Therefore, the Irr might regulate the functions of RirA via the conversion reaction from [4Fe–4S] to [2Fe–2S] for maintaining iron homeostasis under iron limited conditions (Fig 8). The FtrA have been identified a periplasmic iron-binding protein and act as a ferrous iron transporter in Brucella [[51]]. The non-haem iron utilization of Brucella was reduced when the ftrA gene was mutated and the iron-responsive expression of ftrA was dependent on the irr under iron-limited conditions. Interestingly, the ftrA (BME_RS14525) was also identified in our results, however, the expression of the ftrA in this work was contrary to the results obtained from a previous study [[51]]. The expression of ftrA was enhanced when irr was mutated in this work. This difference may be attributed to the iron concentration in TSB, and the iron concentration in the iron-deficient medium may not reach a threshold to trigger the irr to promote the expression of ftrA for transporting ferrous iron. Whereas, further studies are required to validate this hypothesis.

Graph: Fig 8 Model showing the proposed role of Irr and RirA in Brucella iron metabolism.Under iron-limited conditions, Irr remains stable and exerts a repressive effect on the expression of genes responsible for iron consumption or heme biosynthesis to ensure the maintenance of essential processes. On the other hand, Irr promotes the expression of genes attributed to iron transport. In iron-sufficient conditions, the degradation of Irr allows the expression of multiple genes associated with iron consumption and heme biosynthesis. When intracellular iron levels exceed a certain threshold, the 2Fe-2S clusters within RirA undergo conversion into 4Fe-4S clusters, resulting in the suppression of genes involved in iron uptake.

The MbfA is an inner membrane protein identified as a key iron exporter in B. japonicum [[52]]. The mbfA mutant displayed severely defective in iron export activity and contains more than two folds of intracellular iron level than the parent strain. Additionally, it has been described that the mbfA can be repressed by Irr under iron-limited conditions for iron homeostasis [[53]]. In the current study, we discovered that the sequence identity of the mbfA and the membrane protein (BME_RS01725) is 65%, hence, it can be inferred that these two genes may have some similar functions. Additionally, the expression of membrane protein was also inhibited by irr under iron-limited conditions and the Irr protein can directly bind to the promoter of membrane protein for iron regulation.

P-type ATPases make up a superfamily of transport proteins that are mediated by ATP hydrolysis [[55]]. Inorganic cations are mainly substrates for these proteins, including K+, Na+, Mg2+, Ca2+ and Zn2+[[56]]. Each P-type ATPase can import its substrates from the periplasm to the cytoplasm or export it from cytoplasm to the periplasm. Experimental evidence suggested that the zntA is a Zn-specific exporter that can export Zn2+ out of the cell, which prevents the toxicity of Zn2+ in Brucella [[18]]. In our results, the zntA was also identified as a Irr regulon in DAP-seq. Hence, the Irr protein might also involve in Zn2+ transport for maintaining zinc level in Brucella. In addition to rirA and ftrA(iron transporter), three other genes, namely, membrane protein, hypothetical protein and 2Fe-2S binding protein, could also bind to the Irr protein. However, the specific functions relate to these genes remain unknown in Brucella and it is also the limitation of this study. Additionally, the Irr protein can also bind to the promoter regions of dhbCEBA, bhuA, and bhuQ to regulate iron metabolism in Brucella [[31]–[33]]. However, these genes were not identified in our study, this discrepancy may be related to the different iron concentrations under the experimental conditions, because the expression of bhuA and bhuQ was determined by iron concentration or irr per se, specifically, the expression of Brucella abortus 2308 bhuA was enhanced under iron-limited conditions, but was reduced under iron-sufficient conditions or when irr was mutated [[32]].

In summary, seven genes related to iron metabolism were identified in this study, and six out of the seven genes can interact with the Irr protein and may participate in iron metabolism. It is also important to determine the role of membrane protein (BME_RS01725), hypothetical protein (BME_RS09560) and 2Fe-2S binding protein (BME_RS13655) in Brucella iron metabolism. In addition, the rirA was identified as a target gene for Irr and also participated in iron metabolism and Brucella growth. Overall, the results obtained in this study provide valuable information for the exploration of Brucella iron metabolism.

S1 Fig.

Growth curves of M5-90 and M5-90 irr mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The asterisk positioned atop each time point denotes the statistically significant contrast in growth between M5-90 and M5-90 irr mutant across various temporal intervals.

(TIF)

S2 Fig.

The number of CFUs of M5-90 and M5-90 irr mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The asterisk positioned atop each time point denotes the statistically significant contrast in growth between M5-90 and M5-90 irr mutant across various temporal intervals.

(TIF)

S3 Fig. Confirmation of cell-free expressed Irr protein using western blot analysis and identification of fragmented genome of B. melitensis M5-90.

(A) Western blot analysis of Irr protein; 20 μg of the Irr protein was separated using 4–12% SDS-page and immunoblotted with anti-Halo tag antibody. The anticipated molecule mass (51 kDa) of Irr is shown; lane M: Protein Marker (Sangon Biotech), lane 1: Irr protein; (B) Agarose gel analysis of fragmented Brucella genome; lane M: Marker B (Sangon Biotech), lane 1: fragmented Brucella genome.

(TIF)

S4 Fig. The distribution of peaks in the chromosome.

The height represents the quality of peaks.

(TIF)

S5 Fig. The gene expression of irr and rirA was determined at mid- exponential, late-exponential and stationary culture phases of M5-90 in iron-limited conditions.

Total RNA was isolated from M5-90 and cDNA was synthesized. The expression levels of the targets gene were normalized by the expression of 16 S.

(TIF)

S6 Fig.

Growth curves of M5-90 and M5-90 rirA mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The asterisk positioned atop each time point denotes the statistically significant contrast in growth between M5-90 and M5-90 irr mutant across various temporal intervals.

(TIF)

S7 Fig.

The number of CFUs of M5-90 and M5-90 rirA mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The asterisk positioned atop each time point denotes the statistically significant contrast in growth between M5-90 and M5-90 irr mutant across various temporal intervals.

(TIF)

S1 Table. The primers used for qRT-PCR.

(DOCX)

S2 Table. The primers used in EMSA experiments.

(DOCX)

S3 Table. The seven sequences identified in Dap-seq.

(XLSX)

S4 Table. The raw data of qRT-PCR for identification of the expression levels of the seven genes.

(CSV)

S1 Data. The raw data of the Dap-seq replica_1.

(CSV)

S2 Data. The raw data of the Dap-seq replica_2.

(CSV)

We thank LetPub (http://www.letpub.com/) for its linguistic assistance during the preparation of this manuscript, and Zoobio company for its assistance in DNA probe design in EMSA experiment.