Induction of <i>mamB</i> and <i>mamB</i>-GFP expression enables <i>de novo</i> magnetosome formation.

<p>(Ai): Progression of growth (OD₅₆₅ [circles]) and magnetic response (C_mag [triangles]) over time after induction of <i>mamB</i> expression with 2 mM IPTG in strain MamB_ind. IPTG was added at time point 0 (black triangle). TEM micrographs of formaldehyde-fixed cells were utilized to determine the number of magnetite particles per cell (box plots) and average magnetite particle diameter (white boxes) at certain time points. (Aii): Western blot with immune-detection against MamB after SDS-PAGE with whole cell samples from experiment (i) taken at certain time points. The cell density of the samples was normalized based on OD₅₆₅. (Aiii): Examples of TEM micrographs of formaldehyde-fixed cells obtained at different time points of experiment (i). Black arrows indicate the positions of single or multiple magnetite crystals (tiny particles after one hour of induction might stem from background). (B): Details of x-y slices from cryo-electron tomograms acquired with MamB_ind cells, plunge-frozen at a various time points after induction with 2 mM IPTG in a separate experiment. (i) and (ii): Details from two cells 2 hours post induction. (iii-v): Details from one cell 3 hours post induction. (v-viii): Details from two cells 4 hours post induction. Putative DMMs are indicated by black arrows, the magnetosome filament by white arrows. Scale bars: 100 nm. (C): 24 hours time-lapse live-cell fluorescent microscopy of induced MamB-EGFP_ind strain. Cells were grown at 30°C on sealed 1% agarose pads containing modified FSM medium and 3 mM IPTG. Fluorescence and corresponding bright field images from various indicated time points after induction are shown. White arrowheads indicate accumulation of fluorescent patches at midcell, while white arrows indicate linear fluorescence signals within cells. Scale bar: 2 μm.</p

Cryo-electron tomograms of different MSR-1 mutant strains.

<p>Segmented tomograms show representative phenotypes of mutants (compare with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006101#pgen.1006101.s025" target="_blank">S1 Table</a>). The inner and outer membrane of the cells are depicted in blue, wild type-like magnetosome membranes (MMs) in yellow [black arrow in x-y slices], iron-minerals in red and the magnetosome filament in green [white arrow in x-y slices]. Distinguishable dense magnetosome membrane-like structures (DMMs) are depicted in dark yellow [red arrows in x-y slices], emphasizing the differential appearance in contrast to wild type-like MMs. Full tomograms are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006101#pgen.1006101.s017" target="_blank">S3</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006101#pgen.1006101.s022" target="_blank">S8</a> Videos. (A): Δ<i>mamI</i> cell containing wild type-like MMs that partially enclose mineral particles. (B): Δ<i>mamN</i> cell containing a dense chain of empty and partially magnetite-filled wild type-like MMs. (C): Δ<i>mamL</i> cell with x-y slice detail (Box 1), showing small wild type-like MMs, partially containing crystals, and potential DMMs. (Di): Δ<i>mamB</i> cell displaying some putative isolated DMMs. (Dii): x-y slice detail of another tomogram shows putative DMMs (dashed red arrows) and a “mini-inclusion” structure (blue arrow) occasionally seen also in tomograms of the wild type and several other mutants. Asterisks marks polyhydroxyalkanoate inclusion that also occurred in all other analyzed strains. (Ei): Δ<i>mamQ</i> cell with two x-y slice details (Box 1 and Box 2). Box 1 shows filament-attached DMMs, Box 2 shows putative wild-type like MM. (Eii): x-y slice sections of another tomogram show four putative DMMs of which some appear continuous with the cytoplasmic membrane. Scale bars: 50 nm (F): Δ<i>mamM</i> cell with two x-y slice details (Box 1 and Box 2) showing filament attached DMMs.</p

Ultrastructural analysis of magnetosome membranes from wild type.

<p>A): Segmented cryo-electron tomogram of cell with selected details from x-y slices of tomographic reconstruction (Box 1–4) and a y-z slice, illustrating information loss by the missing wedge. The outer and cytoplasmic membrane (CM) are depicted in blue, magnetosome membranes (MMs) in yellow, magnetite crystals in red and the magnetosome filament in green. Scale bars in boxes: 100 nm. (B): Segmented cryo-electron tomogram of aerobically cultivated cell that contains MMs (some with small crystal). Full tomogram is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006101#pgen.1006101.s016" target="_blank">S2 Video</a>. (C): Panel with details from x-y slices of tomographic reconstructions of MSR-1 wild type cells, showing MMs that contain magnetite crystals of different sizes. The magnetosome filament is indicated by white arrows. The halo visible around magnetite crystals (red arrow shows examples) is caused by missing wedge effects and might obscure MM identification. (Cii): Section of x-y slice from tomogram showing MM that is continuous with CM and contains a small crystal. Numbers in image represent average value for all measured MM diameters (blue) (n = 289), approximate values for the annulus diameter to the periplasmic space of continuous MMs (green) and approximate values for the length of the protruding neck between the CM and MM (red). Scale bars: 100 nm.</p

Hypothetical model for magnetosome membrane formation.

<p>The model suggests that magnetosome membrane proteins (colorful shapes) are recruited to certain sites of the cytoplasmic membrane in a hierarchical manner, with the key proteins MamB, MamM, MamQ and MamL (labeled in shades of blue, yellow and red) acting as nucleating factors, which is followed by recruitment of MamI, MamE, MamO and other magnetosome proteins. Since MamB was found most important for magnetosome membrane (MM) formation, it might act as the initial landmark protein to prime complex formation at certain sited within the cytoplasmic membrane. After a critical size and composition of the multi-protein assembly is reached, the formed lipid-protein complex induces rapid invagination to form the magnetosome lumen. Diffusion from the periplasm into this lumen is blocked. Later, several further magnetosome proteins might become recruited into the MM, which eventually becomes detached to form magnetosome vesicles. The absence of MamB strongly inhibits MM formation, while the absence of either MamM, MamQ or MamL might cause a disturbed protein composition, which leads to the formation of aberrant dense magnetosome membrane-like structures (DMMs) that lack magnetite crystals or blocks magnetosome formation at an immature state.</p

Localization of MamI-GFP in several mutants and complementation/localization assay of mutated MamL-GFP.

<p>(A) Effects of exchange of basic amino acid residues in the C-terminus of MamL, fused to EGFP by an alpha-helical linker. Quantitative analysis of magnetite crystal number/cell (grey) and magnetite crystal sizes (white) of MSR Δ<i>mamL</i> complemented with transposon-integrated P_{<i>mamDC45</i>}-<i>mamL-egfp</i>, P_{<i>mamDC45</i>}-<i>mamL</i>_K77Q-R78Q<i>-egfp</i>, P_{<i>mamDC45</i>}-<i>mamL</i>_K72Q<i>-egfp</i>, P_{<i>mamDC45</i>}-<i>mamL</i>_{K63Q-K66Q-K68Q}<i>-egfp</i>, P_{<i>mamDC45</i>}-<i>mamL</i>_H67Y<i>-egfp</i>, P_{<i>mamDC45</i>}-<i>mamL</i>_R64Q-R65Q<i>-egfp</i> and P_{<i>mamDC45</i>}-<i>mamL</i>_{all neutral}<i>-egfp</i>. Box plots are indicating 10^th and 90^th percentiles (whiskers), 25^th and 75^th percentiles (box), median and outliers. Over 100 cells and 200 magnetosomes where analyzed for each strain. For quantitative analysis of MamL_(mutant)-EGFP localization (colorful bars in background), fluorescence patterns were grouped into three classes (examples are indicated; chain and aligned patches are visualized as one class). Scale bars: 2 μm. (B) Quantitative analysis of localization of plasmid expressed P_{<i>mamDC45</i>}-<i>mamI</i>-<i>egfp</i> in MSR-1, MSR 1B, Δ<i>mamB</i>, Δ<i>mamM</i>, Δ<i>mamQ</i> and Δ<i>mamL</i>. The localization patterns in individual cells were grouped into four different classes (examples are indicated; boundary of cells are outlined). More than 100 cells where analyzed for each strain. Scale bars: 2 μm.</p

Diameters of magnetosome membranes and similar structures from wild type and several mutant strains.

<p>Diameters of magnetosome membranes (MMs) or dense MM-like structures were measured from cryo-electron tomograms. Box plots are indicating 10^th and 90^th percentiles (whiskers), 25^th and 75^th percentiles (box), median and outliers. The number of measured membranes [n] and analyzed cells [n(cells)] are indicated. The mean value and the standard deviation (SD) of the diameters are given for each strain.</p

Bacterial Magnetosome Biomineralization - A Novel Platform to Study Molecular Mechanisms of Human CDF-Related Type-II Diabetes

<div><p>Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all organisms. CDFs were found to be involved in numerous human health conditions, such as Type-II diabetes and neurodegenerative diseases. In this work, we established the magnetite biomineralizing alphaproteobacterium <i>Magnetospirillum gryphiswaldense</i> as an effective model system to study CDF-related Type-II diabetes. Here, we introduced two ZnT-8 Type-II diabetes-related mutations into the <i>M. gryphiswaldense</i> MamM protein, a magnetosome-associated CDF transporter essential for magnetite biomineralization within magnetosome vesicles. The mutations' effects on magnetite biomineralization and iron transport within magnetosome vesicles were tested <i>in vivo</i>. Additionally, by combining several <i>in vitro</i> and <i>in silico</i> methodologies we provide new mechanistic insights for ZnT-8 polymorphism at position 325, located at a crucial dimerization site important for CDF regulation and activation. Overall, by following differentiated, easily measurable, magnetism-related phenotypes we can utilize magnetotactic bacteria for future research of CDF-related human diseases.</p></div

Thermodynamics of zinc binding to MamM-CTD dimer and mutants, as measured by Isothermal Titration Calorimetry.

<p>Thermodynamics of zinc binding to MamM-CTD dimer and mutants, as measured by Isothermal Titration Calorimetry.</p

Metal binding sites of MamM-CTD and V260R mutant.

<p>MamM-CTD presents a central binding site formed by symmetrical Asp249-His285 and two symmetrical peripheral binding sites formed by His264-Glu289. The twisted dimeric fold of V260R allows the formation of the central putative binding site and two alternative peripheral symmetrical binding sites between Glu268-His264, each from a different monomer.</p

Structural overlay of the wild type MamM-CTD in green and V260R mutant in purple.

<p>(A) Monomers overlay present a similar metallochaperone-like fold. In contrast to wild type MamM-CTD structure that presents a flexible C-terminal tail, one of V260R mutant monomers presents an additional beta-sheet (b4). A 2.05 Å 2F_o – F_c electron density omit map was calculated and is presented around b4. The map is countered at 1.0 σ (light brown). (B) Dimer packing overlay reveals altered and twisted dimer packing for V260R mutant.</p