Magnetic bacteria synthesize magnetite crystals with species-dependent morphologies. The molecular mechanisms that control nano-sized magnetite crystal formation and the generation of diverse morphologies are not well understood. From the analysis of magnetite crystalassociated proteins, several low molecular mass proteins tightly bound to bacterial magnetite were obtained from Magnetospirillum magneticum strain AMB-1. These proteins showed common features in their amino acid sequences, which contain hydrophobic Nterminal and hydrophilic C-terminal regions. The Cterminal regions in Mms5, Mms6, Mms7, and Mms13 contain dense carboxyl and hydroxyl groups that bind iron ions. Nano-sized magnetic particles similar to those in magnetic bacteria were prepared by chemical synthesis of magnetite in the presence of the acidic protein Mms6. These proteins may be directly involved in biological magnetite crystal formation in magnetic bacteria.Magnetic bacteria synthesize nano-sized magnetic particles of an iron oxide, magnetite (Fe 3 O 4 ); an iron sulfide, greigite (Fe 3 S 4 ); or a combination of greigite and iron pyrite (Fe 2 S) (1-3). These particles are individually covered with a stable lipid bilayer membrane that mainly consists of lipid and protein (4). The mineral size, type, and morphology of bacterial magnetic particles (BMPs) 1 are highly controlled within bacterial species or strains (5). The species-specific control of BMP formation has focused attention on the possible roles of the surrounding membrane structures.The molecular mechanism of BMP synthesis is a multistep process, including vesicle formation, iron transport, and magnetite crystallization (5, 6). Recent molecular studies have postulated the steps of BMP synthesis (7-9). Several proteins located on or in the BMP membrane have been isolated and analyzed in Magnetospirillum magneticum strain AMB-1. The first event of BMP synthesis is the formation of vesicles. Invagination of the cytoplasmic membrane is primed by a BMP membrane-specific GTPase (Mms16) to form the intracellular vesicle (7). MpsA, a homolog of an acetyltransferase containing a CoA-binding motif, is also considered to be involved in this process (8). The second process in BMP synthesis is iron transport into the BMP vesicles. It appears that ferric iron is reduced on the cell surface, taken into the cytoplasm, transported into the BMP vesicle, and finally oxidized to produce magnetite. The magA gene was isolated through transposon mutagenesis in strain AMB-1 (9). This gene encodes an integral membrane protein that is involved in the transport of iron into the BMP vesicles. The last process is crystallization of magnetite within the vesicle, but the process remains unclear.Other proteins associated with the BMP membrane have been partially characterized in magnetic bacteria to date. To understand the molecular mechanism of magnetite crystallization in M. magneticum AMB-1, several proteins tightly bound to the bacterial magnetite crystals were isolated and characterized. A new class of mi...

Circulating tumor cells (CTCs) are tumor cells circulating in the peripheral blood of patients with metastatic cancer. Detection of CTCs has clinical significance in cancer therapy because it would enable earlier diagnosis of metastasis. In this research, a microfluidic device equipped with a size-selective microcavity array for highly efficient and rapid detection of tumor cells from whole blood was developed. The microcavity array can specifically separate tumor cells from whole blood on the basis of differences in the size and deformability between tumor and hematologic cells. Furthermore, the cells recovered on the microcavity array were continuously processed for image-based immunophenotypic analysis using a fluorescence microscope. Our device successfully detected approximately 97% of lung carcinoma NCI-H358 cells in 1 mL whole blood spiked with 10-100 NCI-H358 cells. In addition, breast, gastric, and colon tumor cells lines that include EpCAM-negative tumor cells, which cannot be isolated by conventional immunomagnetic separation, were successfully recovered on the microcavity array with high efficiency (more than 80%). On an average, approximately 98% of recovered cells were viable. Our microfluidic device has high potential as a tool for the rapid detection of CTCs and can be used to study CTCs in detail.

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Biomineralization, the process by which minerals are deposited by organisms, has attracted considerable attention because this mechanism has shown great potential to inspire bottom-up material syntheses. To understand the mechanism for morphological regulation that occurs during biomineralization, many regulatory proteins have been isolated from various biominerals. However, the molecular mechanisms that regulate the morphology of biominerals remain unclear because there is a lack of in vivo evidence. Magnetotactic bacteria synthesize intracellular magnetosomes that comprise membraneenveloped single crystalline magnetite (Fe 3 O 4 ). These nanosized magnetite crystals (<100 nm) are bacterial species dependent in shape and size. Mms6 is a protein that is tightly associated with magnetite crystals. Based on in vitro experiments, this protein was first implicated in morphological regulation during nano-sized magnetite biomineralization. In this study, we analyzed the mms6 gene deletion mutant (⌬mms6) of Magnetospirillum magneticum (M. magneticum) AMB-1. Surprisingly, the ⌬mms6 strain was found to synthesize the smaller magnetite crystals with uncommon crystal faces, while the wild-type and complementation strains synthesized highly ordered cubo-octahedral crystals. Furthermore, deletion of mms6 gene led to drastic changes in the profiles of the proteins tightly bound to magnetite crystals. It was found that Mms6 plays a role in the in vivo regulation of the crystal structure to impart the cubo-octahedral morphology to the crystals during biomineralization in magnetotactic bacteria. Magnetotactic bacteria synthesize magnetite crystals under ambient conditions via a highly controlled morphological regulation system that uses biological molecules.

Magnetic particles offer high technological potential since they can be conveniently collected with an external magnetic field. Magnetotactic bacteria synthesize bacterial magnetic particles (BacMPs) with well-controlled size and morphology. BacMPs are individually covered with thin organic membrane, which confers high and even dispersion in aqueous solutions compared with artificial magnetites, making them ideal biotechnological materials. Recent molecular studies including genome sequence, mutagenesis, gene expression and proteome analyses indicated a number of genes and proteins which play important roles for BacMP biomineralization. Some of the genes and proteins identified from these studies have allowed us to express functional proteins efficiently onto BacMPs, through genetic engineering, permitting the preservation of the protein activity, leading to a simple preparation of functional protein-magnetic particle complexes. They were applicable to high-sensitivity immunoassay, drug screening and cell separation. Furthermore, fully automated single nucleotide polymorphism discrimination and DNA recovery systems have been developed to use these functionalized BacMPs. The nano-sized fine magnetic particles offer vast potential in new nano-techniques.

Prokaryotes are known to have evolved one or more unique organelles. Although several hypotheses have been proposed concerning the biogenesis of these intracellular components, the majority of these proposals remains unclear. Magnetotactic bacteria synthesize intracellular magnetosomes that are enclosed by lipid bilayer membranes. From the identification and characterization of several surface and transmembrane magnetosome proteins, we have postulated that magnetosomes are derived from the cytoplasmic membrane (CM). To confirm this hypothesis, a comparative proteomic analysis of the magnetosome membrane (MM) and CM of the magnetotactic bacterium, Magnetospirillum magneticum AMB-1, was undertaken. Based on the whole genome sequence of M. magneticum AMB-1, 78 identified MM proteins were also found to be prevalent in the CM, several of which are related to magnetosome biosynthesis, such as Mms13, which is tightly bound on the magnetite surface. Fatty acid analysis was also conducted, and showed a striking similarity between the CM and MM profiles. These results suggest that the MM is derived from the CM.

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Magnetotactic bacteria are ubiquitous microorganisms that synthesize intracellular magnetite particles (magnetosomes) by accumulating Fe ions from aquatic environments. Recent molecular studies, including comprehensive proteomic, transcriptomic, and genomic analyses, have considerably improved our hypotheses of the magnetosome-formation mechanism. However, most of these studies have been conducted using pure-cultured bacterial strains of a-proteobacteria. Here, we report the whole-genome sequence of Desulfovibrio magneticus strain RS-1, the only isolate of magnetotactic microorganisms classified under d-proteobacteria. Comparative genomics of the RS-1 and four a-proteobacterial strains revealed the presence of three separate gene regions (nuo and mamAB-like gene clusters, and gene region of a cryptic plasmid) conserved in all magnetotactic bacteria. The nuo gene cluster, encoding NADH dehydrogenase (complex I), was also common to the genomes of three iron-reducing bacteria exhibiting uncontrolled extracellular and/or intracellular magnetite synthesis. A cryptic plasmid, pDMC1, encodes three homologous genes that exhibit high similarities with those of other magnetotactic bacterial strains. In addition, the mamAB-like gene cluster, encoding the key components for magnetosome formation such as iron transport and magnetosome alignment, was conserved only in the genomes of magnetotactic bacteria as a similar genomic island-like structure. Our findings suggest the presence of core genetic components for magnetosome biosynthesis; these genes may have been acquired into the magnetotactic bacterial genomes by multiple gene-transfer events during proteobacterial evolution.