Biochemical and Proteomic Analysis of the Magnetosome Membrane in Magnetospirillum gryphiswaldense

We analyzed the biochemical composition of the magnetosome membrane (MM) in Magnetospirillum gryphiswaldense. Isolated magnetosomes were associated with phospholipids and fatty acids which were similar to phospholipids and fatty acids from other subcellular compartments (i.e., outer and cytoplasmic membranes) but were present in different proportions. The binding characteristics of MM-associated proteins were studied by selective solubilization and limited proteolysis. The MM-associated proteins were further analyzed by various proteomic approaches, including one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Edman and mass spectrometric (electrospray ionization-mass spectrometry-mass spectrometry) sequencing, as well as capillary liquid chromatography-mass spectrometry-mass spectrometry of total tryptic digests of the MM. At least 18 proteins were found to constitute the magnetosome subproteome, and most of these proteins are novel for M. gryphiswaldense. Except for MM22 and Mms16, all bona fide MM proteins (MMPs) were encoded by open reading frames in the mamAB, mamDC, and mms6 clusters in the previously identified putative magnetosome island. Eight of the MMPs display homology to known families, and some of them occur in the MM in multiple homologues. Ten of the MMPs have no known homologues in nonmagnetic organisms and thus represent novel, magnetotactic bacterium-specific protein families. Several MMPs display repetitive or highly acidic sequence patterns, which are known from other biomineralizing systems and thus may have relevance for magnetite formation.     

A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria

In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions. Except for mamA, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of M. magnetotacticum and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated. The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation.     

Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.     

Snapping magnetosome chains by asymmetric cell division in magnetotactic bacteria

The mechanism by which prokaryotic cells organize and segregate their intracellular organelles during cell division has recently been the subject of substantial interest. Unlike other microorganisms, magnetotactic bacteria (MTB) form internal magnets (known as magnetosome chain) for magnetic orientation, and thus face an additional challenge of dividing and equipartitioning this magnetic receptor to their daughter cells. Although MTB have been investigated more than four decades, it is only recently that the basic mechanism of how MTB divide and segregate their magnetic organelles has been addressed. In this issue of Molecular Microbiology, the cell cycle of the model magnetotactic bacterium, Magnetospirillum gryphiswaldense is characterized by Katzmann and co-workers. The authors have found that M. gryphiswaldense undergoes an asymmetric cell division along two planes. A novel wedge-like type of cellular constriction is observed before separation of daughter cells and magnetosome chains, which is assumed to help cell cope with the magnetic force within the magnetosome chain. The data shows that the magnetosome chain becomes actively recruited to the cellular division site, in agreement with the previous suggestions described by Staniland et al. (2010), and the actin-like protein MamK is likely involved in this fast polar-to-midcell translocalization. With the use of cryo-electron tomography, an arc-shaped Z ring is observed near the division site, which is assumed to trigger the asymmetric septation of cell and magnetosome chain.     

A Large Gene Cluster Encoding Several Magnetosome Proteins Is Conserved in Different Species of Magnetotactic Bacteria

In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions. Except for mamA, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of M. magnetotacticum and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated. The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation.     

Comparative genome analysis of four magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function

Magnetotactic bacteria (MTB) are a heterogeneous group of aquatic prokaryotes with a unique intracellular organelle, the magnetosome, which orients the cell along magnetic field lines. Magnetotaxis is a complex phenotype, which depends on the coordinate synthesis of magnetosomes and the ability to swim and orient along the direction caused by the interaction with the Earth's magnetic field. Although a number of putative magnetotaxis genes were recently identified within a conserved genomic magnetosome island (MAI) of several MTB, their functions have remained mostly unknown, and it was speculated that additional genes located outside the MAI might be involved in magnetosome formation and magnetotaxis. In order to identify genes specifically associated with the magnetotactic phenotype, we conducted comparisons between four sequenced magnetotactic Alphaproteobacteria including the nearly complete genome of Magnetospirillum gryphiswaldense strain MSR-1, the complete genome of Magnetospirillum magneticum strain AMB-1, the complete genome of the magnetic coccus MC-1, and the comparative-ready preliminary genome assembly of Magnetospirillum magnetotacticum strain MS-1 against an in-house database comprising 426 complete bacterial and archaeal genome sequences. A magnetobacterial core genome of about 891 genes was found shared by all four MTB. In addition to a set of approximately 152 genus-specific genes shared by the three Magnetospirillum strains, we identified 28 genes as group specific, i.e., which occur in all four analyzed MTB but exhibit no (MTB-specific genes) or only remote (MTB-related genes) similarity to any genes from nonmagnetotactic organisms and which besides various novel genes include nearly all mam and mms genes previously shown to control magnetosome formation. The MTB-specific and MTB-related genes to a large extent display synteny, partially encode previously unrecognized magnetosome membrane proteins, and are either located within (18 genes) or outside (10 genes) the MAI of M. gryphiswaldense. These genes, which represent less than 1% of the 4,268 open reading frames of the MSR-1 genome, as yet are mostly of unknown functions but are likely to be specifically involved in magnetotaxis and, thus, represent prime targets for future experimental analysis.     

The presumptive magnetosome protein Mms16 is a poly(3-hydroxybutyrate) granule-bound protein (phasin) in Magnetospirillum gryphiswaldense

The Mms16 protein has been previously found to be associated with isolated magnetosomes from two Magnetospirillum strains. A function of this protein as a magnetosome-specific GTPase involved in the formation of intracellular magnetosome membrane vesicles was suggested. Here we present a study of the Mms16 protein from Magnetospirillum gryphiswaldense to clarify its function. Insertion-duplication mutagenesis of the mms16 gene did not affect the formation of magnetosome particles but resulted in the loss of the ability of M. gryphiswaldense cell extracts to activate poly(3-hydroxybutyrate) (PHB) depolymerization in vitro, which was coincident with loss of the most abundant 16-kDa polypeptide from preparations of PHB granule-bound proteins. The mms16 mutation could be functionally complemented by enhanced yellow fluorescent protein (EYFP) fused to ApdA, which is a PHB granule-bound protein (phasin) in Rhodospirillum rubrum sharing 55% identity to Mms16. Fusions of Mms16 and ApdA to enhanced green fluorescent protein (EGFP) or EYFP were colocalized in vivo with the PHB granules but not with the magnetosome particles after conjugative transfer to M. gryphiswaldense. Although the Mms16-EGFP fusion protein became detectable by Western analysis in all cell fractions upon cell disruption, it was predominantly associated with isolated PHB granules. Contrary to previous suggestions, our results argue against an essential role of Mms16 in magnetosome formation, and the previously observed magnetosome localization is likely an artifact due to unspecific adsorption during preparation. Instead, we conclude that Mms16 in vivo is a PHB granule-bound protein (phasin) and acts in vitro as an activator of PHB hydrolysis by R. rubrum PHB depolymerase PhaZ1. Accordingly, we suggest renaming the Mms16 protein of Magnetospirillum species to ApdA, as in R. rubrum.     

Characterization of the bacterial magnetosome membrane.

Intact magnetosomes of Aquaspirillum magnetotacticum were purified from broken cells by a magnetic separation technique. Electron microscopic and chemical analyses revealed the magnetite to be enclosed by a lipid bilayer admixed with proteins. Lipids were recovered in fractions expected to contain (i) neutral lipids and free fatty acids, (ii) glycolipids and sulfolipids, and (iii) phospholipids (in a weight ratio of 1:4:6). Phospholipids included phosphatidylserine and phosphatidylethanolamine. Two of the numerous proteins detected in the magnetosome membrane were not found in other cell membranes or soluble fractions.     

Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor

Media and growth conditions were optimized for the microaerobic cultivation of Magnetospirillum gryphiswaldense in flasks and in a fermentor, resulting in significantly increased cell and magnetosome yields, compared with earlier studies. A reliable method was established for the automatic control of low dissolved oxygen tensions (pO(2)) in the fermentor (oxystat). Growth and magnetosome formation by M. gryphiswaldense, M. magnetotacticum and Magnetospirillum sp. AMB-1 were studied at various oxygen concentrations. Despite differences in their growth responses with respect to oxygen, we found a clear correlation between pO(2) and magnetosome formation in all three Magnetospirillum strains. Magnetite biomineralization was induced only below a threshold value of 20 mbar O(2) and optimum conditions for magnetosome formation were found at a pO(2) of 0.25 mbar (1 bar = 10(5) Pa). A maximum yield of 6.3 mg magnetite l(-1) day(-1) was obtained with M. gryphiswaldense grown under oxystat conditions, which is the highest magnetosome productivity reported so far for a magnetotactic bacterium. In conclusion, the presented results provide the basis for large-scale cultivation of magnetospirilla under defined conditions.     

Molecular analysis of a subcellular compartment: the magnetosome membrane in<Emphasis Type="Italic"> Magnetospirillum gryphiswaldense</Emphasis>

The ability of magnetotactic bacteria (MTB) to orient and migrate along magnetic field lines is based on magnetosomes, which are membrane-enclosed intracellular crystals of a magnetic iron mineral. Magnetosome biomineralization is achieved by a process involving control over the accumulation of iron and deposition of the magnetic particle, which has a specific morphology, within a vesicle provided by the magnetosome membrane. In Magnetospirillum gryphiswaldense, the magnetosome membrane has a distinct biochemical composition and comprises a complex and specific subset of magnetosome membrane proteins (MMPs). Classes of MMPs include those with presumed function in magnetosome-directed uptake and binding of iron, nucleation of crystal growth, and the assembly of magnetosome membrane multiprotein complexes. Other MMPs comprise protein families of so far unknown function, which apparently are conserved between all other MTB. The mam and mms genes encode most of the MMPs and are clustered within several operons, which are part of a large, unstable genomic region constituting a putative magnetosome island. Current research is directed towards the biochemical and genetic analysis of MMP functions in magnetite biomineralization as well as their expression and localization during growth.Abbreviations MM Magnetosome membrane - MMP Magnetosome membrane protein - MTB Magnetotactic bacteria     

Origin of magnetosome membrane: proteomic analysis of magnetosome membrane and comparison with cytoplasmic membrane

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.     

The cation diffusion facilitator proteins MamB and MamM of Magnetospirillum gryphiswaldense have distinct and complex functions, and are involved in magnetite biomineralization and magnetosome membrane assembly

Magnetotactic bacteria form chains of intracellular membrane-enclosed, nanometre-sized magnetite crystals for navigation along the earth's magnetic field. The assembly of these prokaryotic organelles requires several specific polypeptides. Among the most abundant proteins associated with the magnetosome membrane of Magnetospirillum gryphiswaldense are MamB and MamM, which were implicated in magnetosomal iron transport because of their similarity to the cation diffusion facilitator family. Here we demonstrate that MamB and MamM are multifunctional proteins involved in several steps of magnetosome formation. Whereas both proteins were essential for magnetite biomineralization, only deletion of mamB resulted in loss of magnetosome membrane vesicles. MamB stability depended on the presence of MamM by formation of a heterodimer complex. In addition, MamB was found to interact with several other proteins including the PDZ1 domain of MamE. Whereas any genetic modification of MamB resulted in loss of function, site-specific mutagenesis within MamM lead to increased formation of polycrystalline magnetite particles. A single amino acid substitution within MamM resulted in crystals consisting of haematite, which coexisted with magnetite crystals. Together our data indicate that MamM and MamB have complex functions, and are involved in the control of different key steps of magnetosome formation, which are linked by their direct interaction.     

Magnetosome chains are recruited to cellular division sites and split by asymmetric septation

Magnetotactic bacteria navigate along magnetic field lines using well-ordered chains of membrane-enclosed magnetic crystals, referred to as magnetosomes, which have emerged as model to investigate organelle biogenesis in prokaryotic systems. To become divided and segregated faithfully during cytokinesis, the magnetosome chain has to be properly positioned, cleaved and separated against intrachain magnetostatic forces. Here we demonstrate that magnetotactic bacteria use dedicated mechanisms to control the position and division of the magnetosome chain, thus maintaining magnetic orientation throughout divisional cycle. Using electron and time-lapse microscopy of synchronized cells of Magnetospirillum gryphiswaldense, we confirm that magnetosome chains undergo a dynamic pole-to-midcell translocation during cytokinesis. Nascent chains were recruited to division sites also in division-inhibited cells, but not in a mamK mutant, indicating an active mechanism depending upon the actin-like cytoskeletal magnetosome filament. Cryo-electron tomography revealed that both the magnetosome chain and the magnetosome filament are spilt into halves by asymmetric septation and unidirectional indentation, which we interpret in terms of a specific adaptation required to overcome the magnetostatic interactions between separating daughter chains. Our study demonstrates that magnetosome division and segregation is co-ordinated with cytokinesis and resembles partitioning mechanisms of other organelles and macromolecular complexes in bacteria.     

A hypervariable 130-kilobase genomic region of Magnetospirillum gryphiswaldense comprises a magnetosome island which undergoes frequent rearrangements during stationary growth

Genes involved in magnetite biomineralization are clustered in the genome of the magnetotactic bacterium Magnetospirillum gryphiswaldense. We analyzed a 482-kb genomic fragment, in which we identified an approximately 130-kb region representing a putative genomic "magnetosome island" (MAI). In addition to all known magnetosome genes, the MAI contains genes putatively involved in magnetosome biomineralization and numerous genes with unknown functions, as well as pseudogenes, and it is particularly rich in insertion elements. Substantial sequence polymorphism of clones from different subcultures indicated that this region undergoes frequent rearrangements during serial subcultivation in the laboratory. Spontaneous mutants affected in magnetosome formation arise at a frequency of up to 10(-2) after prolonged storage of cells at 4 degrees C or exposure to oxidative stress. All nonmagnetic mutants exhibited extended and multiple deletions in the MAI and had lost either parts of or the entire mms and mam gene clusters encoding magnetosome proteins. The mutations were polymorphic with respect to the sites and extents of deletions, but all mutations were found to be associated with the loss of various copies of insertion elements, as revealed by Southern hybridization and PCR analysis. Insertions and deletions in the MAI were also found in different magnetosome-producing clones, indicating that parts of this region are not essential for the magnetic phenotype. Our data suggest that the genomic MAI undergoes frequent transposition events, which lead to subsequent deletion by homologous recombination under physiological stress conditions. This can be interpreted in terms of adaptation to physiological stress and might contribute to the genetic plasticity and mobilization of the magnetosome island.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

Fatty acid binding proteins in multiple myeloma

Fatty acid binding proteins (FABPs) transport fatty acids (FA) into cells as an energy source, and their inhibition suppressed tumor proliferation in solid tumors. Multiple myeloma (MM) is a hematologic malignancy, known for disrupted protein metabolism including high proteasome activity, where proteasome inhibitors made a dramatic improvement in its treatment. Recent discovery found FABPs as a novel metabolic pathway in MM, which will have an impact on understanding the biology and on therapeutic application in MM.     

Fatty Acid Replacements in a Fatty Acid Auxotroph of Escherichia coli

Unsaturated fatty acids having structural features which are different from those of the monoenoic acids normally synthesized by Escherichia coli can serve as growth factors for an auxotroph requiring unsaturated fatty acids. These analogues were incorporated into the phospholipids, as shown by gas-liquid and thin-layer chromatographic analysis of the phospholipid fatty acid composition. Some of these fatty acids were cisDelta(5)- and cis-Delta(9)-tetradecenoic, cis-Delta(11)-eicosenoic, cis,cis-Delta(11,14)-eicosadienoic, cis,cis,cis-Delta(11,14,17)-eicosatrienoic, trans-Delta(9)- and trans-Delta(11)-octadecenoic acids. Although partial degradation of some of these analogues to shorter even-chain homologues occurred, chain elongation of the exogenous fatty acids was not detected. Trans-olefinic acids were utilized without stereochemical or positional isomerization. These studies provide a basis for exploring the properties of the fatty acids and phospholipids required for the formation, structure, and function of membranes.     

Adsorption of amino acids on the magnetite-(111)-surface: a force field study

Magnetite (Fe₃O₄) is an important biomineral, e.g., used by magnetotactic bacteria. The connection between the inorganic magnetite-(111)-surface and the organic parts of the bacteria is the magnetosome membrane. The membrane is built by different magnetosome membrane proteins (MMPs), which are dominated by the four amino acids glycine (Gly), aspartic acid (Asp), leucine (Leu) and glutamic acid (Glu). Force field simulations of the interaction of the magnetite-(111)-surface and the main amino acid compounds offer the possibility to investigate if and how the membrane proteins could interact with the mineral surface thus providing an atomistic view on the respective binding sites. In a force field simulation the four amino acids were docked on the Fe-terminated magnetite-(111)-surface. The results show that it is energetically favorable for the amino acids to adsorb on the surface with Fe-O-distances between 2.6 Å and 4.1 Å. The involved O-atoms belong to the carboxyl-group (Asp and Glu) or to the carboxylate-group (Gly, Leu and Glu). Electrostatic interactions dominate the physisorption of the amino acids. During the simulations, according to the frequency of the best results, the global minimum for the docking interaction could be attained for all amino acids analyzed.     

Somaclonal variation and chilling tolerance improvement in rice: Changes in fatty acid composition

The relationship between chilling tolerance of six rice cultivars – Facagro 57, Facagro 76, Fujisaka 5, Kirundo 3, Kirundo 9 and IR64 -and the fatty acid composition in total lipids, phospholipids, galactolipids and neutral lipids from leaves was studied. Higher double bond index and proportions of linolenic acid in the phospholipid and galactolipid classes were related to cultivar chilling tolerance, but this was not so for the total lipids nor the neutral lipid class. The somaclonal families derived from Facagro 76, Kirundo 3 and Kirundo 9 that showed enhanced chilling tolerance as compared to their original parental cultivar were analyzed for fatty acid composition in phospholipids and galactolipids from leaves. Altered proportions in fatty acid composition in phospholipids, galactolipids or both were found in the somaclonal families derived from Facagro 76 and Kirundo 9, but not from Kirundo 3. These changes most usually resulted in higher double bond index and higher proportions in linoleic and linolenic acids which were related either to lower ratio of C16 to C18 fatty acids or to higher unsaturation in the C18 fatty acid fraction. Different mechanisms thus seem to be implicated in the altered fatty acid composition of somaclones, which may be related to the chilling tolerance improvement of some somaclonal families.