Iron response regulator protein IrrB in Magnetospirillum gryphiswaldense MSR-1 helps control the iron/oxygen balance, oxidative stress tolerance, and magnetosome formation

Abstract

Magnetotactic bacteria are capable of forming nanosized, membrane-enclosed magnetosomes under iron-rich and oxygen-limited conditions. The complete genomic sequence of Magnetospirillum gryphiswaldense strain MSR-1 has been analyzed and found to contain five fur homologue genes whose protein products are predicted to be involved in iron homeostasis and the response to oxidative stress. Of these, only the MGMSRv2_3149 gene (irrB) was significantly downregulated under high-iron and low-oxygen conditions, during the transition of cell growth from the logarithmic to the stationary phase. The encoded protein, IrrB, containing the conserved HHH motif, was identified as an iron response regulator (Irr) protein belonging to the Fur superfamily. To investigate the function of IrrB, we constructed an irrB deletion mutant (ΔirrB). The levels of cell growth and magnetosome formation were lower in the ΔirrB strain than in the wild type (WT) under both high-iron and low-iron conditions. The ΔirrB strain also showed lower levels of iron uptake and H2O2 tolerance than the WT. Quantitative real-time reverse transcription-PCR analysis indicated that the irrB mutation reduced the expression of numerous genes involved in iron transport, iron storage, heme biosynthesis, and Fe-S cluster assembly. Transcription studies of the other fur homologue genes in the ΔirrB strain indicated complementary functions of the Fur proteins in MSR-1. IrrB appears to be directly responsible for iron metabolism and homeostasis and to be indirectly involved in magnetosome formation. We propose two IrrB-regulated networks (under high- and low-iron conditions) in MSR-1 cells that control the balance of iron and oxygen metabolism and account for the coexistence of five Fur homologues.

FIG 1

Primary sequence alignment of Fur and Irr proteins by the ClustalW program. Four Fur homologues were from M. gryphiswaldense MSR-1: MGMSRv2_3137 (Fur; GenBank accession no. YP_008939008), MGMSRv2_1721 (IrrA; GenBank accession no. YP_008937596), MGMSRv2_3149 (IrrB; GenBank accession no. YP_008939020), and MGMSRv2_3660 (IrrC; GenBank accession no. YP_008939529). One related Fur sequence was from P. aeruginosa: Fur-Pa (GenBank accession no. NP_253452). Three related Irr sequences were from B. japonicum (Irr-Bj; accession no. YP_005605653), R. leguminosarum (Irr-Rl; accession no. WP_017962725), and A. tumefaciens (Irr-At; accession no. WP_003493135). Black, pink, or blue shading indicates that eight, six, or fewer than five proteins, respectively, share the same amino acids at a given site. The conserved histidine residue motif (HHH) is boxed in red. The heme regulatory motif is boxed in black. The second heme-binding site is indicated by red asterisks.

FIG 2

(A and B) Growth rates of the MSR-1 WT, ΔirrB (irrB-deficient mutant), and CF3149 strains in culture with 30 μM DIPy (low-iron conditions) (A) or 60 μM ferric citrate (high-iron conditions) (B). (C) Magnetic responses (Cmag) under high-iron conditions. The ΔirrB strain showed clear reductions in the levels of growth and magnetosome formation under low-iron conditions.

FIG 3

TEM images, intracellular iron contents, and H₂O₂ tolerances of the three MSR-1 strains. (A to C) WT, ΔirrB, and CF3149 cells, respectively, viewed by conventional TEM. Bars, 500 nm. (D to F) WT, ΔirrB, and CF3149 cells, respectively, at a higher magnification. Bars, 200 nm. Arrows indicate magnetosomes. The level of magnetosome formation was notably reduced in the ΔirrB strain. (G) Cells were grown in SLM supplemented with 20, 40, or 60 μM ferric citrate, and the intracellular iron content was determined by ICP-OES. (H) Cells were grown in SLM supplemented with four different concentrations of H₂O₂, as shown, and the OD₅₆₅ was measured by spectrophotometry. Growth in 200 μM H₂O₂ was normal for the WT, greatly reduced (13% of the WT value) for the ΔirrB strain, and partially restored for the complemented strain CF3149. In 300 μM H₂O₂, growth was close to normal for the WT (OD₅₆₅, 0.7) but greatly reduced for the ΔirrB strain and CF3149. The growth of all three strains was completely inhibited in 500 μM H₂O₂. The experiments were performed in triplicate.

FIG 4

Transcription levels of fur and two fur-like genes in the WT and ΔirrB strains under high-iron (A) and low-iron (B) conditions. Means were compared using Student's t test; differences with P values of <0.05 were considered significant. Under high-iron conditions, the irrA transcription level was 5.5-fold higher in the ΔirrB strain than in the WT (P < 0.05). Under low-iron conditions, the irrA level in the ΔirrB strain was 2-fold higher than that in the WT, but the irrC and fur levels were lower.

FIG 5

Transcription patterns and predicted interactions among IrrB and related proteins. (A) Expression of eight genes in the WT relative to that in the ΔirrB strain (fold change) under high-iron (filled bars) and low-iron (open bars) conditions, as evaluated by RT-qPCR. (B) Interactions among IrrB and related proteins, predicted using the online tool STRING 9.1. The network nodes represent proteins encoded by the MGMSRv2_3149 (irrB; red), MGMSRv2_2122, MGMSRv2_3112, MGMSRv2_3313, MGMSRv2_3702, and MGMSRv2_3703 genes. Lines between nodes indicate predicted associations between the corresponding proteins. (C) Expression of bfrA, bfrB, and a gene with unknown function in the WT relative to that in the ΔirrB strain (fold change) under high-iron and low-iron conditions, as determined by RT-qPCR.

FIG 6

Proposed IrrB-regulated networks in MSR-1 cells. Under high-iron conditions, IrrB indirectly regulates genes for heme biosynthesis (hemA, hemB, hemC), iron storage (bfrA, bfrB), and an unknown pathway. Under low-iron conditions, IrrB regulates genes for iron transport (bhuA), Fe-S cluster assembly (sufA), and nitrogenase metallocluster biosynthesis (nifS, nifU).