Recent advances in studies on magnetosome-associated proteins composing the bacterial geomagnetic sensor organelle.

Magnetotactic bacteria (MTB) generate a membrane-enclosed subcellular compartment called magnetosome, which contains a biomineralized magnetite or greigite crystal, an inner membrane-derived lipid bilayer membrane, and a set of specifically targeted associated proteins. Magnetosomes are formed by a group of magnetosome-associated proteins encoded in a genomic region called magnetosome island. Magnetosomes are then arranged in a linear chain-like positioning, and the resulting magnetic dipole of the chain functions as a geomagnetic sensor for magneto-aerotaxis motility.

Structures of oxygen dissociation intermediates of 400 kDa V2 hemoglobin provide coarse snapshots of the protein allostery.

Ever since the historic discovery of the cooperative oxygenation of its multiple subunits, hemoglobin (Hb) has been among the most exhaustively studied allosteric proteins. However, the lack of structural information on the intermediates between oxygenated and deoxygenated forms prevents our detailed understanding of the molecular mechanism of its allostery. It has been difficult to prepare crystals of intact oxy-deoxy intermediates and to individually identify the oxygen saturation for each subunit.

Coarse snapshots of oxygen-dissociation intermediates of a giant hemoglobin elucidated by determining the oxygen saturation in individual subunits in the crystalline state.

Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now.

Diversity of physical properties of bacterial extracellular membrane vesicles revealed through atomic force microscopy phase imaging.

Bacteria release nanometer-scale extracellular membrane vesicles (MVs) to mediate a variety of biological processes. We analyzed individual MVs under physiological conditions by phase imaging of high-speed atomic force microscopy to assess the physiological heterogeneity of MVs isolated from bacterial cultures. Phase imaging makes it possible to map the physical properties of an individual, fragile MV in an isolated MV population containing a broad variety of vesicle diameters, from 20 to 150 nm.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms.

Measuring magnetosomal pH of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using pH-sensitive fluorescent proteins.

Magnetotactic bacteria synthesize uniform-sized and regularly shaped magnetic nanoparticles in their organelles termed magnetosomes. Homeostasis of the magnetosome lumen must be maintained for its role accomplishment. Here, we developed a method to estimate the pH of a single living cell of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using a pH-sensitive fluorescent protein EGFP.

Tethered Magnets Are the Key to Magnetotaxis: Direct Observations of 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.

High-Speed Atomic Force Microscopy Reveals Loss of Nuclear Pore Resilience as a Dying Code in Colorectal Cancer Cells.

Nuclear pore complexes (NPCs) are the sole turnstile implanted in the nuclear envelope (NE), acting as a central nanoregulator of transport between the cytosol and the nucleus. NPCs consist of ∼30 proteins, termed nucleoporins. About one-third of nucleoporins harbor natively unstructured, intrinsically disordered phenylalanine-glycine strings (FG-Nups), which engage in transport selectivity.

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.

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.

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.

The structure of a deoxygenated 400 kDa haemoglobin reveals ternary- and quaternary-structural changes of giant haemoglobins.

The quaternary structures of invertebrate haemoglobins (Hbs) are quite different from those of vertebrate Hbs. The extracellular giant Hbs of molecular masses of about 400 and 3600 kDa are composed of a dome-shaped dodecameric subassembly which consists of four individual globin subunits. Several crystal structures of 400 kDa Hbs from annelids have been reported, including structures in oxygenated and partially unliganded states, but the structure of the fully deoxygenated state has not been reported.

Purification, characterization, and crystallization of Crocodylus siamensis hemoglobin.

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form.

DnaJ-promoted binding of DnaK to multiple sites on σ32 in the presence of ATP.

The Escherichia coli DnaK chaperone system is a canonical heat shock protein 70 (Hsp70) chaperone system comprising Hsp70, Hsp40, and a nucleotide exchange factor. Although Hsp40 is known to facilitate the effective binding of Hsp70 to substrates, the role of Hsp40 in Hsp70-substrate interactions has not yet been fully elucidated. Using the E.

Structures of reduced and ligand-bound nitric oxide reductase provide insights into functional differences in respiratory enzymes.

Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N2O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non-heme Fe center. We report herein on the structures of the reduced and ligand-bound forms of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3-2.

Purification and characterization of coacervate-forming cuticular proteins from Papilio xuthus pupae.

The Papilio xuthus (Lepidoptera: Papilionidae) pupa expresses novel soluble proteins that undergo reversible temperature-dependent coacervate-formation. We purified two coacervate-forming proteins, PX-1 and PX-4, from the wings of pharate adults. PX-1 and PX-4 form coacervates upon warming.

Analysis of magnetotactic behavior by swimming assay.

Prokaryotic organelles called magnetosomes allow magnetotactic bacteria to navigate along geomagnetic field lines. In this study, we modified a swimming assay commonly used to assess bacterial motility to develop a new method of assessing magnetotactic motility. By this method, the swimming assay was performed in an artificial magnetic field.

Single-molecule imaging on living bacterial cell surface by high-speed AFM.

Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells.

Synergistic binding of DnaJ and DnaK chaperones to heat shock transcription factor σ32 ensures its characteristic high metabolic instability: implications for heat shock protein 70 (Hsp70)-Hsp40 mode of function.

Escherichia coli heat shock transcription factor σ(32) is rapidly degraded by ATP-dependent proteases, such as FtsH and ClpYQ. Although the DnaK chaperone system (DnaK, DnaJ, and GrpE) promotes σ(32) degradation in vivo, the precise mechanism that is involved remains unknown. Our previous results indicated that σ(32) mutants containing amino acid substitution in the N-terminal half of Region 2.

Structural basis of biological N2O generation by bacterial nitric oxide reductase.

Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N(2)O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution.

Visualization and structural analysis of the bacterial magnetic organelle magnetosome using atomic force microscopy.

The unique ability of magnetotactic bacteria to navigate along a geomagnetic field is accomplished with the help of prokaryotic organelles, magnetosomes. The magnetosomes have well-ordered chain-like structures, comprising membrane-enveloped, nano-sized magnetic crystals, and various types of specifically associated proteins. In this study, we applied atomic force microscopy (AFM) to investigate the spatial configuration of isolated magnetosomes from Magnetospirillum magneticum AMB-1 in near-native buffer conditions.

Direct evidence that extracellular giant hemoglobin is produced in chloragogen tissues in a beard worm, Oligobrachia mashikoi (Frenulata, Siboglinidae, Annelida).

In Oligobrachia mashikoi, a mouthless and gutless polychaete known as a beard worm, sites of production of extra-cellular giant hemoglobin were examined with whole-mount in-situ hybridization and semi-quantitative RT-PCR. An RNA probe was prepared from mRNA of the A2-globin subunit. Clear signals were obtained from a peritoneal membrane covering the trophosome in the posterior body in all seven individuals examined in this study.