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%.
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%.
Fig 3.
(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.
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.
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.
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%.
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%.
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.