Tethered Magnets Are the Key to Magnetotaxis: Direct Observations of Magnetospirillum magneticum AMB-1 Show that MamK Distributes Magnetosome Organelles Equally to Daughter Cells.

Magnetotactic bacteria are a unique group of bacteria that synthesize a magnetic organelle termed the magnetosome, which they use to assist with their magnetic navigation in a specific type of bacterial motility called magneto-aerotaxis. Cytoskeletal filaments consisting of the actin-like protein MamK are associated with the magnetosome chain. Previously, the function of MamK was thought to be in positioning magnetosome organelles; this was proposed based on observations via electron microscopy still images. Here, we conducted live-cell time-lapse fluorescence imaging analyses employing highly inclined and laminated optical sheet microscopy, and these methods enabled us to visualize detailed dynamic movement of magnetosomes in growing cells during the entire cell cycle with high-temporal resolution and a high signal/noise ratio. We found that the MamK cytoskeleton anchors magnetosomes through a mechanism that requires MamK-ATPase activity throughout the cell cycle to prevent simple diffusion of magnetosomes within the cell. We concluded that the static chain-like arrangement of the magnetosomes is required to precisely and consistently segregate the magnetosomes to daughter cells. Thus, the daughter cells inherit a functional magnetic sensor that mediates magneto-reception.IMPORTANCE Half a century ago, bacterial cells were considered a simple "bag of enzymes"; only recently have they been shown to comprise ordered complexes of macromolecular structures, such as bacterial organelles and cytoskeletons, similar to their eukaryotic counterparts. In eukaryotic cells, the positioning of organelles is regulated by cytoskeletal elements. However, the role of cytoskeletal elements in the positioning of bacterial organelles, such as magnetosomes, remains unclear. Magnetosomes are associated with cytoskeletal filaments that consist of the actin-like protein MamK. In this study, we focused on how the MamK cytoskeleton regulates the dynamic movement of magnetosome organelles in living magnetotactic bacterial cells. Here, we used fluorescence imaging to visualize the dynamics of magnetosomes throughout the cell cycle in living magnetotactic bacterial cells to understand how they use the actin-like cytoskeleton to maintain and to make functional their nano-sized magnetic organelles.

A protein-protein interaction in magnetosomes: TPR protein MamA interacts with an Mms6 protein.

Magnetosomes are membrane-enveloped bacterial organelles containing nano-sized magnetic particles, and function as a cellular magnetic sensor, which assist the cells to navigate and swim along the geomagnetic field. Localized with each magnetosome is a suite of proteins involved in the synthesis, maintenance and functionalization of the organelle, however the detailed molecular organization of the proteins in magnetosomes is unresolved. MamA is one of the most abundant magnetosome-associated proteins and is anchored to the magnetosome vesicles through protein-protein interactions, but the identity of the protein that interacts with MamA is undetermined. In this study, we found that MamA binds to a magnetosome membrane protein Mms6. Two different molecular masses of Mms6, 14.5-kDa and 6.0-kDa, were associated with the magnetosomes. Using affinity chromatography, we identified that the 14.5-kDa Mms6 interacts with MamA, and the interaction was further confirmed by pull-down, immunoprecipitation and size-exclusion chromatography assays. Prior to this, Mms6 was assumed to be strictly involved with biomineralizing magnetite; however, these results suggest that Mms6 has an additional responsibility, binding to MamA.

A comparison of the surface nanostructure from two different types of gram-negative cells: Escherichia coli and Rhodobacter sphaeroides.

Bacteria have been studied using different microscopy methods for many years. Recently, the developments of high-speed atomic force microscopy have opened the doors to study bacteria in new ways due to the fact that it uses much less force on the sample while imaging. This makes the high-speed atomic force microscope an indispensable technique for imaging the surface of living bacterial cells because it allows for the high-resolution visualization of surface proteins in their natural condition without disrupting the cell or the activity of the proteins. Previous work examining living cells of Magnetospirillum magneticum AMB-1 demonstrated that the surface of these bacteria was covered with a net-like structure that is mainly composed of porin molecules. However, it was unclear whether or not this feature was unique to other living bacteria. In this study we used the high-speed atomic force microscope to examine the surface of living cells of Escherichia coli and Rhodobacter sphaeroides to compare their structure with that of M. magneticum. Our research clearly demonstrated that both of these types of cells have an outer surface that is covered in a network of nanometer-sized holes similar to M. magneticum. The diameter of the holes was 8.0±1.5 nm for E. coli and 6.6±1.1 nm for R. sphaeroides. The results in this paper confirm that this type of outer surface structure exists in other types of bacteria and it is not unique to Magnetospirillum.

Characterization of uncultured giant rod-shaped magnetotactic Gammaproteobacteria from a freshwater pond in Kanazawa, Japan.

Magnetotactic bacteria (MTB) are widespread aquatic bacteria, and are a phylogenetically, physiologically and morphologically heterogeneous group, but they all have the ability to orientate and move along the geomagnetic field using intracellular magnetic organelles called magnetosomes. Isolation and cultivation of novel MTB are necessary for a comprehensive understanding of magnetosome formation and function in divergent MTB. In this study, we enriched a giant rod-shaped magnetotactic bacterium (strain GRS-1) from a freshwater pond in Kanazawa, Japan. Cells of strain GRS-1 were unusually large (~13×~8 µm). They swam in a helical trajectory towards the south pole of a bar magnet by means of a polar bundle of flagella. Another striking feature of GRS-1 was the presence of two distinct intracellular biomineralized structures: large electron-dense granules composed of calcium and long chains of magnetosomes that surround the large calcium granules. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that this strain belongs to the Gammaproteobacteria and represents a new genus of MTB.

A magnetosome-associated cytochrome MamP is critical for magnetite crystal growth during the exponential growth phase.

Magnetotactic bacteria use a specific set of conserved proteins to biomineralize crystals of magnetite or greigite within their cells in organelles called magnetosomes. Using Magnetospirillum magneticum AMB-1, we examined one of the magnetotactic bacteria-specific conserved proteins named MamP that was recently reported as a new type of cytochrome c that has iron oxidase activity. We found that MamP is a membrane-bound cytochrome, and the MamP content increases during the exponential growth phase compared to two other magnetosome-associated proteins on the same operon, MamA and MamK. To assess the function of MamP, we overproduced MamP from plasmids in wild-type (WT) AMB-1 and found that during the exponential phase of growth, these cells contained more magnetite crystals that were the same size as crystals in WT cells. Conversely, when the heme c-binding motifs within the mamP on the plasmid was mutated, the cells produced the same number of crystals, but smaller crystals than in WT cells during exponential growth. These results strongly suggest that during the exponential phase of growth, MamP is crucial to the normal growth of magnetite crystals during biomineralization.

Subcellular localization of the magnetosome protein MamC in the marine magnetotactic bacterium Magnetococcus marinus strain MC-1 using immunoelectron microscopy.

Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe3O4) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

Magnetotactic bacteria from Pavilion Lake, British Columbia.

Pavilion Lake is a slightly alkaline, freshwater lake located in British Columbia, Canada (50°51'N, 121°44'W). It is known for unusual organosedimentary structures, called microbialites that are found along the lake basin. These deposits are complex associations of fossilized microbial communities and detrital- or chemical-sedimentary rocks. During the summer, a sediment sample was collected from near the lake's shore, approximately 25-50 cm below the water surface. Magnetotactic bacteria (MTB) were isolated from this sample using a simple magnetic enrichment protocol. The MTB isolated from Pavilion Lake belonged to the Alphaproteobacteria class as determined by nucleotide sequences of 16S rRNA genes. Transmission electron microscopy (TEM) revealed that the bacteria were spirillum-shaped and contained a single chain of cuboctahedral-shaped magnetite (Fe3O4) crystals that were approximately 40 nm in diameter. This discovery of MTB in Pavilion Lake offers an opportunity to better understand the diversity of MTB habitats, the geobiological function of MTB in unique freshwater ecosystems, and search for magnetofossils contained within the lake's microbialites.

Collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 1975 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet. This property can be exploited when trying to isolate MTB from environmental samples. One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM). Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.

Atomic force microscopy (AFM) was used in concert with transmission electron microscopy (TEM) to image magnetotactic bacteria (Magnetospirillum gryphiswaldense MSR-1 and Magnetospirillum magneticum AMB-1), magnetosomes, and purified Mms6 proteins. Mms6 is a protein that is associated with magnetosomes in M. magneticum AMB-1 and is believed to control the synthesis of magnetite (Fe(3)O(4)) within the magnetosome. We demonstrated how AFM can be used to capture high-resolution images of live bacteria and achieved nanometer resolution when imaging Mms6 protein molecules on magnetite. We used AFM to acquire simultaneous topography and amplitude images of cells that were combined to provide a three-dimensional reconstructed image of M. gryphiswaldense MSR-1. TEM was used in combination with AFM to image M. gryphiswaldense MSR-1 and magnetite-containing magnetosomes that were isolated from the bacteria. AFM provided information, such as size, location and morphology, which was complementary to the TEM images.

In vitro evolution of a peptide with a hematite binding motif that may constitute a natural metal-oxide binding archetype.

Phage-display technology was used to evolve peptides that selectively bind to the metal-oxide hematite (Fe2O3) from a library of approximately 3 billion different polypeptides. The sequences of these peptides contained the highly conserved amino acid motif, Ser/Thr-hydrophobic/aromatic-Ser/Thr-Pro-Ser/Thr. To better understand the nature of the peptide-metal oxide binding demonstrated by these experiments, molecular dynamics simulations were carried out for Ser-Pro-Ser at a hematite surface. These simulations show that hydrogen bonding occurs between the two serine amino acids and the hydroxylated hematite surface and that the presence of proline between the hydroxide residues restricts the peptide flexibility, thereby inducing a structural-binding motif. A search of published sequence data revealed that the binding motif (Ser/Thr-Pro-Ser/Thr) is adjacent to the terminal heme-binding domain of both OmcA and MtrC, which are outer membrane cytochromes from the metal-reducing bacterium Shewanella oneidensis MR-1. The entire five amino acid consensus sequence (Ser/Thr-hydrophobic/ aromatic-Ser/Thr-Pro-Ser/Thr) was also found as multiple copies in the primary sequences of metal-oxide binding proteins Sil1 and Sil2 from Thalassiosira pseudonana. We suggest that this motif constitutes a natural metal-oxide binding archetype that could be exploited in enzyme-based biofuel cell design and approaches to synthesize tailored metal-oxide nanostructures.