Genetic and ultrastructural analysis reveals the key players and initial steps of bacterial magnetosome membrane biogenesis

Magnetosomes of magnetotactic bacteria contain well-ordered nanocrystals for magnetic navigation and have recently emerged as the most sophisticated model system to study the formation of membrane bounded organelles in prokaryotes. Magnetosome biosynthesis is thought to begin with the formation of a dedicated compartment, the magnetosome membrane (MM), in which the biosynthesis of a magnetic mineral is strictly controlled. While the biomineralization of magnetosomes and their subsequent assembly into linear chains recently have become increasingly well studied, the molecular mechanisms and early stages involved in MM formation remained poorly understood. In the Alphaproteobacterium Magnetospirillum gryphiswaldense, approximately 30 genes were found to control magnetosome biosynthesis. By cryo-electron tomography of several key mutant strains we identified the gene complement controlling MM formation in this model organism. Whereas the putative magnetosomal iron transporter MamB was most crucial for the process and caused the most severe MM phenotype upon elimination, MamM, MamQ and MamL were also required for the formation of wild-type-like MMs. A subset of seven genes (mamLQBIEMO) combined within a synthetic operon was sufficient to restore the formation of intracellular membranes in the absence of other genes from the key mamAB operon. Tracking of de novo magnetosome membrane formation by genetic induction revealed that magnetosomes originate from unspecific cytoplasmic membrane locations before alignment into coherent chains. Our results indicate that no single factor alone is essential for MM formation, which instead is orchestrated by the cumulative action of several magnetosome proteins

Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium.

Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacterium Magnetospirillum gryphiswaldense by chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of the mms6 operon resulted in the formation of increasingly larger crystals (increase of ∼35%), the duplication of all major magnetosome operons (mamGFDC, mamAB, mms6, and mamXY, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of the mam and mms clusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches

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

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

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

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

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

Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors

Redirecting T&nbsp;cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However, the effectiveness of therapeutic T&nbsp;cells is constrained by the endogenous T&nbsp;cell response: certain facets of natural response programs can be toxic, whereas other responses, such as the ability to overcome tumor immunosuppression, are absent. Thus, the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T&nbsp;cells: they can drive a la carte cytokine secretion profiles, biased T&nbsp;cell differentiation, and local delivery of non-native therapeutic payloads, such as antibodies, in response to antigen. SynNotch T&nbsp;cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases