Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation

Abstract

Bacterial magnetosomes are intracellular compartments that house highly ordered magnetite crystals. By using Magnetospirillum sp. AMB-1 as a model system, we show that magnetosome vesicles exist in the absence of magnetite, biomineralization of magnetite proceeds simultaneously in multiple vesicles, and biomineralization proceeds from the same location in each vesicle. The magnetosome-associated protein, MamA, is required for the formation of functional magnetosome vesicles and displays a dynamic subcellular localization throughout the growth cycle of magnetotactic bacteria. Together, these results suggest that the magnetosome precisely coordinates magnetite biomineralization and can serve as a model system for the study of organelle biogenesis in noneukaryotic cells.

Fig. 1.

Characterization of magnetosome dynamics by electron microscopy. (A) TEM image of an ultrathin section of Magnetospirillum sp. AMB-1 grown under iron-rich conditions and embedded in epoxy resin. A chain of magnetite crystals can be observed in the cells. (B) AMB-1 cells grown without iron do not contain magnetite chains. (C) Higher magnification of the cell shown in B reveals potential empty magnetosome vesicles. (D) TEM image of cryo-ultrathin section of iron-starved AMB-1 showing a long chain of empty magnetosome vesicles. The three large white inclusions are polyhydroxybutyrate (PHB) granules. A magnetosome chain (arrows) is seen on top of these PHB granules. (E) Higher magnification of magnetosomes in D. Arrows point to magnetosome chains devoid of magnetite. (F) TEM image of a cryo-ultrathin section of AMB-1 cells 7 h after the addition of iron to iron-starved cultures. Small magnetite crystals show the early stages of crystal growth within vesicles having relatively uniform size and shape. It is evident that simultaneous nucleation and growth of magnetite crystals occurs within the full-sized vesicles. (G) TEM image of a cryo-ultrathin section of AMB-1 after 20 h of growth in iron-rich medium. Structures resembling empty magnetosomes are seen at the ends of chains of fully grown magnetite crystals. (H) Regular spacing of magnetite crystals 2 h after the addition of iron. Whole cells of AMB-1 were imaged in TEM without sectioning. The spacing between magnetite crystals is very even, with an average of 57 ± 7 nm. (Scale bars: 200 nm in D-G.)

Fig. 2.

Characterization of ΔmamA. (A) Schematic of the mamABE gene cluster (25). mamA, mamB, and mamE are designated as A, B, and E, respectively. Stars indicate the positions of transposon insertions in mnm1 and mnm2. mamA, deleted in subsequent experiments, is shaded gray. (B) Representative time-course experiment after the addition of iron to iron-starved WT (•) and ΔmamA (▪) strain. ΔmamA cells have taken up less iron than WT cells after 24 h of growth. Iron uptake is normalized to cell number by dividing the μM of iron depleted from the growth medium by the OD at 400 nm. (C) Measurement of response (turning) to a magnetic field for the experiment shown in B. OD₄₀₀ measurements were made with a magnet parallel or perpendicular to the light source. ΔmamA cells have a clear defect in turning in a magnetic field compared to WT cells.

Fig. 3.

ΔmamA cells have a defect in activating their magnetosome vesicles. (A-F) TEM images of whole cells of WT and ΔmamA after the addition of iron to iron-starved cells. At 2 h both WT (A) and ΔmamA (B) cells have started synthesizing magnetite. The crystals get progressively larger at 3.5 h (C and D) and are full-sized after 21 h (E and F). At all times, however, WT cells have more crystals per chain than ΔmamA cells. (G and H) TEM images of cryo-ultrathin sections of ΔmamA cells grown in the absence (G) or presence (H) of iron, revealing the presence of chains of empty vesicles similar to WT (Fig. 1 E and F). (Scale bars: 200 nm.)

Fig. 4.

MamA displays a dynamic localization during the growth of AMB-1. The top images are the GFP localization, the middle images are the cell membrane stain with FM4-64, and the bottom images are the overlay of the GFP and FM4-64 images. (A) MamA-GFP localizes to a thin line within the cell during the logarithmic growth phase. The localization is from one end of the cell to the other, and at times some membrane localization is also observed. (B) In stationary phase, MamA-GFP displays a punctate pattern with one to four spots within the cell. (Scale bars: 5 μm.)