Control of bacterial iron homeostasis by manganese

Abstract

Perception and response to nutritional iron availability by bacteria are essential to control cellular iron homeostasis. The Irr protein from Bradyrhizobium japonicum senses iron through the status of heme biosynthesis to globally regulate iron-dependent gene expression. Heme binds directly to Irr to trigger its degradation. Here, we show that severe manganese limitation created by growth of a Mn(2+) transport mutant in manganese-limited media resulted in a cellular iron deficiency. In wild-type cells, Irr levels were attenuated under manganese limitation, resulting in reduced promoter occupancy of target genes and altered iron-dependent gene expression. Irr levels were high regardless of manganese availability in a heme-deficient mutant, indicating that manganese normally affects heme-dependent degradation of Irr. Manganese altered the secondary structure of Irr in vitro and inhibited binding of heme to the protein. We propose that manganese limitation destabilizes Irr under low-iron conditions by lowering the threshold of heme that can trigger Irr degradation. The findings implicate a mechanism for the control of iron homeostasis by manganese in a bacterium.

Fig. 1.

Effect of iron and manganese on the growth and cellular iron content of B. japonicum parent and mntH strains. Growth media were inoculated with 5 × 10⁵ cells mL⁻¹ of parent strain (closed symbols) or the mntH mutant (open symbols) and grown in media using pyruvate as the carbon source containing 20 μM FeCl₃ (squares) or no exogenous iron (circles) and (A) no exogenous manganese or (B) 50 μM MnCl₂. Unsupplemented media contained 0.4 μM manganese and 0.3 μM iron. (C) Cellular iron content of the parent strain and mntH mutant grown in low-iron and low- or high-manganese media. Cells were grown in iron-limited media containing either no added manganese (−) or 50 μM MnCl₂ (+). Iron content of whole cells was determined by atomic absorption spectroscopy. The data are presented as the average of three replicates ± the standard deviation.

Fig. 2.

Effect of manganese on the expression of Irr-regulated genes and promoter occupancy by Irr. (A–C). mRNA transcripts of bll4920, blr3555, and blr6519 obtained from cells grown under different metal conditions were analyzed by qPCR. The data are expressed as the relative starting quantity (SQ) of the respective mRNAs normalized to the housekeeping gene gapA, and presented as the average of three replicates ± the standard deviation. (D–F) Cross-linking of parent strain cells grown under different conditions of iron and manganese, followed by co-IP using anti-Irr antiserum was carried out as described in Materials and Methods. The mock experiment was carried out without antibody. Immunoprecipitated DNA was analyzed by qPCR using primers delimiting the promoter regions of the respective genes. The data are expressed as the relative SQ of the respective pull-down DNA normalized to the mock pull-down samples and presented as the average of three replicates ± the standard deviation. The normalized SQ values obtained were considered to be directly proportional to the Irr promoter occupancy of the respective genes.

Fig. 3.

Effect of metals on Irr levels in B. japonicum. Cells were grown under different metal conditions and steady-state levels of Irr were detected by immunoblotting using anti-Irr antibodies. GroEL was used as a control for an unregulated protein, and was detected using anti-GroEL antibodies. Thirty micrograms of protein was loaded per lane.

Fig. 4.

Effect of different iron and manganese conditions on Irr levels in cells of the wild-type or a heme-deficient strain. Cells were grown in media supplemented (+) or unsupplemented (−) with FeCl₃ (Fe) or MnCl₂ (Mn). Media were also supplemented with 60 nM heme, which is necessary for growth of the mutant strain. Steady-state levels of Irr and GroEL were determined as by immunoblotting as described in Fig. 3.

Fig. 5.

Effect of manganese on the absorption spectra of heme bound to purified recombinant Irr. (A) Shows 8 μM Irr and 4 μM heme, in the presence of 100 μM dithionite as a reductant, and the absorption spectrum taken between 380 and 480 nm. The sample was then titrated with 2, 4, 6, 8, 10, 20 μM MnCl₂, and the spectrum was taken after each addition. The maximum peak was observed with no metal, and the minimum absorption at 20 μM MnCl₂. (B). The experiment was carried out as described in A, using either no metal (none), 20 μM ZnCl₂ (Zn), or 20 μM FeSO₄ (Fe).

Fig. 6.

Effect of manganese on the secondary structure of purified recombinant Irr. Far UV CD spectra of 8 μM Irr were recorded in the absence (black) and presence of increasing concentrations of manganese (brown, blue, red, and green, representing 8, 16, 40, and 80 μM MnCl₂, respectively).

Fig. 7.

Effect of manganese on Irr levels in the presence of exogenously added heme. Cells of the heme-deficient mutant strain ΔhemAH were grown under different concentrations of heme, in the presence (+) or absence (–) of manganese. Irr and GroEL proteins were measured in cells by immunoblotting as described in Fig. 3.