Desulfovibrio magneticus RS-1 contains an iron- and phosphorus-rich organelle distinct from its bullet-shaped magnetosomes

Abstract

Intracellular magnetite crystal formation by magnetotactic bacteria has emerged as a powerful model for investigating the cellular and molecular mechanisms of biomineralization, a process common to all branches of life. Although magnetotactic bacteria are phylogenetically diverse and their crystals morphologically diverse, studies to date have focused on a few, closely related species with similar crystal habits. Here, we investigate the process of magnetite biomineralization in Desulfovibrio magneticus sp. RS-1, the only reported species of cultured magnetotactic bacteria that is outside of the alpha-Proteobacteria and that forms bullet-shaped crystals. Using a variety of high-resolution imaging and analytical tools, we show that RS-1 cells form amorphous, noncrystalline granules containing iron and phosphorus before forming magnetite crystals. Using NanoSIMS (dynamic secondary ion mass spectroscopy), we show that the iron-phosphorus granules and the magnetite crystals are likely formed through separate cellular processes. Analysis of the cellular ultrastructure of RS-1 using cryo-ultramicrotomy, cryo-electron tomography, and tomography of ultrathin sections reveals that the magnetite crystals are not surrounded by membranes but that the iron-phosphorus granules are surrounded by membranous compartments. The varied cellular paths for the formation of these two minerals lead us to suggest that the iron-phosphorus granules constitute a distinct bacterial organelle.

Fig. 1.

RS-1 forms round granules before magnetite crystals after the addition of iron to iron-starved cells. Transmission electron micrographs of RS-1 cells at various times after iron addition. At time 0, before iron addition, no magnetite crystals or granules are visible. Three hours after iron addition, round granules (enlarged in Inset) are present throughout the cytoplasm. Twenty-two hours after iron addition, granules (Upper Right Inset) are still present and magnetite crystals (Lower Left Inset) begin to appear. Fifty hours after iron addition, almost no granules remain and chains of magnetite crystals (enlarged in Inset) are present. [Scale bars = 500 nm and 100 nm (Insets).]

Fig. 2.

NanoSIMS analysis of biomineralization in RS-1. Iron-starved RS-1 cells were given 200 μM ferric (⁵⁶Fe) malate for 3 h, then transferred to medium containing 200 μM ferric (⁵⁷Fe) malate. At each time point, thin sections of cells were imaged by TEM (Far Left) and analyzed by NanoSIMS for ¹²C¹⁴N⁻, ³¹P⁻, ⁵⁶Fe¹⁶O⁻, and ⁵⁷Fe¹⁶O⁻. The CN and P signals indicate the presence of ubiquitous organic molecules and thus highlight the entire cellular space. When present, the Fe-P granules emit a strong ³¹P signal over the fainter background signal. The far-right panels show a dual merge of the sum of both FeO⁻ signals (green) and the ³¹P⁻ signal (red). (A) Fe-P granules in a cell grown with ⁵⁶Fe for 3 h. (B) A single magnetite crystal in a cell grown with ⁵⁶Fe for 3 h then ⁵⁷Fe for 18.5 h. (C) A cluster of Fe-P granules in a cell grown with ⁵⁶Fe for 3 h then ⁵⁷Fe for 18.5 h. (D) A chain of magnetite crystals in a cell grown with ⁵⁶Fe for 3 h then ⁵⁷Fe for 47 h. (Scale bars, 200 nm.)

Fig. 3.

RS-1 contains numerous and varied intracellular membranous compartments. (A and B) TEM images of cryo-ultrathin sections of RS-1 cells grown in iron-free medium. (B) Arrows highlight invaginations of the inner membrane. (C) Cryo-ultrathin sections of RS-1 cells, grown for 24 h in medium with iron. Arrows highlight magnetite crystals. (D) Enlargement of crystals from C. Arrow highlights halo seen around some crystals in underfocus conditions.

Fig. 4.

Cryo-electron microscopy of fully hydrated cells reveals intracellular membranous compartments and membranes around the Fe-P granules, but no membranes are visible around the magnetite crystals. Images are 19.2-nm thick sections taken from reconstructed cryo-electron tomograms. (A) Fe-P inclusions from a cell grown in medium with iron for 4.5 h. (B) Magnetite crystals from a cell grown in medium with iron for 56 h. (C) Thin, elongated compartments. (D) An invagination of the inner membrane and a round vesicle. (E) Multiple round vesicles. (Scale bars, 100 nm.)

Fig. 5.

Electron tomography analysis of a section of RS-1, grown for 96 h with iron, shows a chain of magnetite crystals with no membranes. (A) A tomographic x-y slice of ∼40 nm thickness of the bacteria. Highlighted are part of the magnetosome chain, outer membrane (long arrow), periplasmic space (large arrowhead), inner membrane (small arrowhead), and intracellular membrane compartments (short arrows). (B) A series of tomographic x-y slices of ∼7 nm thickness from bottom to top of the magnetosome chain. (C) The magnetosome chain superposed with a central section of the volume. The volume was manually segmented using IMOD, and each magnetosome was given a different color.