Fur in Magnetospirillum gryphiswaldense influences magnetosomes formation and directly regulates the genes involved in iron and oxygen metabolism

Abstract

Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe(3)O(4)). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense.

Figure 1. β-galactosidase activity of E. coli strain H1780 carrying fiu-lacZ fusion.

Bars represent fur mutant (H1780) reporter strain, H1780 containing E. coli (pEF) and M. gryphiswaldense (pRF) fur genes on pUC18 vector, and a vector control. Cells were grown in LB medium with 100 µM FeCl₃ (black bar), or supplemented with 200 µM DIPy (iron chelator) (white bar). Each assay was performed in three independent experiments, each in triplicate. Values shown are means with S.D., statistically significant (P<0.05) difference for strains grown under low-iron vs. high-iron condition.

Figure 2. Growth curves measured by OD₅₆₅ of WT, F4, and F4C strains in SLM added with 1 mM H₂O₂.

Experiments were performed in triplicate, and representative results are shown.

Figure 3. Comparative sensitivity of WT, F4, and F4C strains to streptonigrin (SNG).

Cells were treated without or with 1 µg/ml SNG for 5 days at 30°C. Cultures were diluted and spotted on agar plates with SLM. Numbers above each image indicate 10-fold serial dilutions.

Figure 4. TEM micrographs of WT (A), F4 (B), and F4C (C) strains.

Cells were grown in SLM added with 60 µM ferric citrate for 36 h. Bar, 500 nm.

Figure 5. Detection of ChIP DNAs from WT and F4 strains under low-iron and high-iron conditions.

a, d, g, j: positive controls (input). b, e, h, k: negative controls (no antibody). c: high-iron WT. f: low-iron WT. i: high-iron F4. l: low-iron F4. Primer of rpsJ gene (Table 3) is included as an additional negative control which codes for a conserved 30S ribosomal S10 protein and is not regulated by Fur .