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.     

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     

The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals

Magnetospirillum gryphiswaldense and related magnetotactic bacteria form magnetosomes, which are membrane-enclosed organelles containing crystals of magnetite (Fe₃O₄) that cause the cells to orient in magnetic fields. The characteristic sizes, morphologies, and patterns of alignment of magnetite crystals are controlled by vesicles formed of the magnetosome membrane (MM), which contains a number of specific proteins whose precise roles in magnetosome formation have remained largely elusive. Here, we report on a functional analysis of the small hydrophobic MamGFDC proteins, which altogether account for nearly 35% of all proteins associated with the MM. Although their high levels of abundance and conservation among magnetotactic bacteria had suggested a major role in magnetosome formation, we found that the MamGFDC proteins are not essential for biomineralization, as the deletion of neither mamC, encoding the most abundant magnetosome protein, nor the entire mamGFDC operon abolished the formation of magnetite crystals. However, cells lacking mamGFDC produced crystals that were only 75% of the wild-type size and were less regular than wild-type crystals with respect to morphology and chain-like organization. The inhibition of crystal formation could not be eliminated by increased iron concentrations. The growth of mutant crystals apparently was not spatially constrained by the sizes of MM vesicles, as cells lacking mamGFDC formed vesicles with sizes and shapes nearly identical to those formed by wild-type cells. However, the formation of wild-type-size magnetite crystals could be gradually restored by in-trans complementation with one, two, and three genes of the mamGFDC operon, regardless of the combination, whereas the expression of all four genes resulted in crystals exceeding the wild-type size. Our data suggest that the MamGFDC proteins have partially redundant functions and, in a cumulative manner, control the growth of magnetite crystals by an as-yet-unknown mechanism.     

New Vectors for Chromosomal Integration Enable High-Level Constitutive or Inducible Magnetosome Expression of Fusion Proteins in Magnetospirillum gryphiswaldense

The alphaproteobacterium Magnetospirillum gryphiswaldense biomineralizes magnetosomes, which consist of monocrystalline magnetite cores enveloped by a phospholipid bilayer containing specific proteins. Magnetosomes represent magnetic nanoparticles with unprecedented magnetic and physicochemical characteristics. These make them potentially useful in a number of biotechnological and biomedical applications. Further functionalization can be achieved by expression of foreign proteins via genetic fusion to magnetosome anchor peptides. However, the available genetic tool set for strong and controlled protein expression in magnetotactic bacteria is very limited. Here, we describe versatile vectors for either inducible or high-level constitutive expression of proteins in M. gryphiswaldense. The combination of an engineered native P_mamDC promoter with a codon-optimized egfp gene (Mag-egfp) resulted in an 8-fold increase in constitutive expression and in brighter fluorescence. We further demonstrate that the widely used P_tet promoter is functional and tunable in M. gryphiswaldense. Stable and uniform expression of the EGFP and β-glucuronidase (GusA) reporters was achieved by single-copy chromosomal insertion via Tn5-mediated transposition. In addition, gene duplication by Mag-EGFP–EGFP fusions to MamC resulted in further increased magnetosome expression and fluorescence. Between 80 and 210 (for single MamC–Mag-EGFP) and 200 and 520 (for MamC–Mag-EGFP–EGFP) GFP copies were estimated to be expressed per individual magnetosome particle.     

The biomineralization of magnetosomes in <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis>

Magnetotactic bacteria (MTB) are major constituents of natural microbial communities in sediments and chemically stratified water columns. The ability of MTB to migrate along magnetic field lines is based on specific intracellular structures, the magnetosomes, which, in most MTB, are nanometer-sized, membrane-bound magnetic particles consisting of the iron mineral magnetite (Fe₃O₄). A broad diversity of morphological forms has been found in various MTB. The unique characteristics of bacterial magnetosomes have attracted a broad interdisciplinary research interest. The magnetosome membrane (MM) in Magnetospirillum gryphiswaldense contains a number of specific Mam proteins. Several mam genes were analyzed and assigned to different genomic regions. Many of the Mam proteins are highly conserved in other MTB but display low sequence similarity to any proteins from nonmagnetic organisms. Electronic Publication     

Analysis of Magnetosome Chains in Magnetotactic Bacteria by Magnetic Measurements and Automated Image Analysis of Electron Micrographs

Magnetotactic bacteria (MTB) align along the Earth''s magnetic field by the activity of intracellular magnetosomes, which are membrane-enveloped magnetite or greigite particles that are assembled into well-ordered chains. Formation of magnetosome chains was found to be controlled by a set of specific proteins in Magnetospirillum gryphiswaldense and other MTB. However, the contribution of abiotic factors on magnetosome chain assembly has not been fully explored. Here, we first analyzed the effect of growth conditions on magnetosome chain formation in M. gryphiswaldense by electron microscopy. Whereas higher temperatures (30 to 35°C) and high oxygen concentrations caused increasingly disordered chains and smaller magnetite crystals, growth at 20°C and anoxic conditions resulted in long chains with mature cuboctahedron-shaped crystals. In order to analyze the magnetosome chain in electron microscopy data sets in a more quantitative and unbiased manner, we developed a computerized image analysis algorithm. The collected data comprised the cell dimensions and particle size and number as well as the intracellular position and extension of the magnetosome chain. The chain analysis program (CHAP) was used to evaluate the effects of the genetic and growth conditions on magnetosome chain formation. This was compared and correlated to data obtained from bulk magnetic measurements of wild-type (WT) and mutant cells displaying different chain configurations. These techniques were used to differentiate mutants due to magnetosome chain defects on a bulk scale.     

Proteomic analysis of irregular,bullet‐shaped magnetosomes in the sulphate‐reducing magnetotactic bacterium Desulfovibrio magneticus RS‐1

Recent molecular studies on magnetotactic bacteria have identified a number of proteins associated with bacterial magnetites (magnetosomes) and elucidated their importance in magnetite biomineralisation. However, these analyses were limited to magnetotactic bacterial strains belonging to the α‐subclass of Proteobacteria. We performed a proteomic analysis of magnetosome membrane proteins in Desulfovibrio magneticus strain RS‐1, which is phylogenetically classified as a member of the δ‐Proteobacteria. In the analysis, the identified proteins were classified based on their putative functions and compared with the proteins from the other magnetotactic bacteria, Magnetospirillum magneticum AMB‐1 and M. gryphiswaldense MSR‐1. Three magnetosome‐specific proteins, MamA (Mms24), MamK, and MamM, were identified in strains RS‐1, AMB‐1, and MSR‐1. Furthermore, genes encoding ten magnetosome membrane proteins, including novel proteins, were assigned to a putative magnetosome island that contains subsets of genes essential for magnetosome formation. The collagen‐like protein and putative iron‐binding proteins, which are considered to play key roles in magnetite crystal formation, were identified as specific proteins in strain RS‐1. Furthermore, genes encoding two homologous proteins of Magnetococcus MC‐1 were assigned to a cryptic plasmid of strain RS‐1. The newly identified magnetosome membrane proteins might contribute to the formation of the unique irregular, bullet‐shaped crystals in this microorganism.     

Inactivation of the Flagellin Gene flaA in Magnetospirillum gryphiswaldense Results in Nonmagnetotactic Mutants Lacking Flagellar Filaments

Magnetotactic bacteria synthesize magnetosomes, which cause them to orient and migrate along magnetic field lines. The analysis of magnetotaxis and magnetosome biomineralization at the molecular level has been hindered by the unavailability of genetic methods, namely the lack of a means to introduce directed gene-specific mutations. Here we report a method for knockout mutagenesis by homologous recombination in Magnetospirillum gryphiswaldense. Multiple flagellin genes, which are unlinked in the genome, were identified in M. gryphiswaldense. The targeted disruption of the flagellin gene flaA was shown to eliminate flagella formation, motility, and magnetotaxis. The techniques described in this paper will make it possible to take full advantage of the forthcoming genome sequences of M. gryphiswaldense and other magnetotactic bacteria.     

Bacterial Magnetosome Biomineralization - A Novel Platform to Study Molecular Mechanisms of Human CDF-Related Type-II Diabetes

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all organisms. CDFs were found to be involved in numerous human health conditions, such as Type-II diabetes and neurodegenerative diseases. In this work, we established the magnetite biomineralizing alphaproteobacterium Magnetospirillum gryphiswaldense as an effective model system to study CDF-related Type-II diabetes. Here, we introduced two ZnT-8 Type-II diabetes-related mutations into the M. gryphiswaldense MamM protein, a magnetosome-associated CDF transporter essential for magnetite biomineralization within magnetosome vesicles. The mutations'' effects on magnetite biomineralization and iron transport within magnetosome vesicles were tested in vivo. Additionally, by combining several in vitro and in silico methodologies we provide new mechanistic insights for ZnT-8 polymorphism at position 325, located at a crucial dimerization site important for CDF regulation and activation. Overall, by following differentiated, easily measurable, magnetism-related phenotypes we can utilize magnetotactic bacteria for future research of CDF-related human diseases.     

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.     

Magnetosome Expression of Functional Camelid Antibody Fragments (Nanobodies) in Magnetospirillum gryphiswaldense

Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surface of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific expression of a red fluorescent protein (RFP)-binding nanobody (RBP) in vivo was accomplished by genetic fusion of RBP to the magnetosome protein MamC in the magnetite-synthesizing bacterium Magnetospirillum gryphiswaldense. We demonstrate that isolated magnetosomes expressing MamC-RBP efficiently recognize and bind their antigen in vitro and can be used for immunoprecipitation of RFP-tagged proteins and their interaction partners from cell extracts. In addition, we show that coexpression of monomeric RFP (mRFP or its variant mCherry) and MamC-RBP results in intracellular recognition and magnetosome recruitment of RFP within living bacteria. The intracellular expression of a functional nanobody targeted to a specific bacterial compartment opens new possibilities for in vivo synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to manipulate bacterial structures in live cells.     

A Tailored galK Counterselection System for Efficient Markerless Gene Deletion and Chromosomal Tagging in Magnetospirillum gryphiswaldense

Magnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation of Magnetospirillum gryphiswaldense using a single antibiotic resistance cassette and galK-based counterselection for marker recycling. We demonstrate the potential of this technique by genomic excision of the phbCAB operon, encoding enzymes for polyhydroxyalkanoate (PHA) synthesis, followed by chromosomal fusion of magnetosome-associated proteins to fluorescent proteins. Because of the absence of interfering PHA particles, these engineered strains are particularly suitable for microscopic analyses of cell biology and magnetosome biosynthesis.     

Expression of Green Fluorescent Protein Fused to Magnetosome Proteins in Microaerophilic Magnetotactic Bacteria

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.     

Magnetosome membrane engineering to improve G protein-coupled receptor activities in the magnetosome display system

Magnetotactic bacterium, Magnetospirillum magneticum, produces biogenic magnetic nanoparticles termed magnetosomes, which are primarily composed of a magnetite core and a surrounding lipid bilayer membrane. We have fabricated human transmembrane protein-magnetosome complexes by genetic engineering with embedding the transmembrane proteins of interest, in particular G protein-coupled receptors (GPCRs), in the magnetosome membrane. The magnetosomes provide a promising platform for high throughput ligand screening towards drug discovery, and this is a critical advantage of the magnetosome display system beyond conventional membrane platforms such as liposomes and lipid nano-discs. However, the human GPCRs expressed on the magnetosomes were not fully functionalized in bacterial membranes the most probably due to the lack of essential phospholipids such as phosphatidylcholine (PC) for GPCR functionalization. To overcome this issue, we expressed two types of PC-producing enzymes, phosphatidylcholine synthase (PCS) and phosphatidylethanolamine N-methyltransferase (PMT) in M. magneticum. As a result, generation and incorporation of PC in cell- and magnetosome-membranes were demonstrated. To the best of our knowledge, M. magneticum is the second bacterial species which had the PC-incorporated lipid membrane by genetic engineering. Subsequently, a GPCR, thyroid-stimulating hormone receptor (TSHR) and PCS were simultaneously expressed. We found that PC in the magnetosome membrane assisted the binding of TSHR and its ligand, indicating that the genetic approach demonstrated in this study is useful to enhance the function of the GPCRs displayed on the magnetosomes.     

The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization

Magnetospirillum gryphiswaldense MSR‐1 synthesizes membrane‐enclosed magnetite (Fe₃O₄) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome‐associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome‐directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi‐disciplinary approach to define the role of MamB during magnetosome formation. Using site‐directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo‐electron tomography, we show that MamB is most likely an active magnetosome‐directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport‐independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C‐terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation.     

The magnetosome proteins MamX,MamZ and MamH are involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense

Magnetospirillum gryphiswaldense uses intracellular chains of membrane‐enveloped magnetite crystals, the magnetosomes, to navigate within magnetic fields. The biomineralization of magnetite nanocrystals requires several magnetosome‐associated proteins, whose precise functions so far have remained mostly unknown. Here, we analysed the functions of MamX and the Major Facilitator Superfamily (MFS) proteins MamZ and MamH. Deletion of either the entire mamX gene or elimination of its putative haem c‐binding magnetochrome domains, and deletion of either mamZ or its C‐terminal ferric reductase‐like component resulted in an identical phenotype. All mutants displayed WT‐like magnetite crystals, flanked within the magnetosome chains by poorly crystalline flake‐like particles partly consisting of haematite. Double deletions of both mamZ and its homologue mamH further impaired magnetite crystallization in an additive manner, indicating that the two MFS proteins have partially redundant functions. Deprivation of ΔmamX and ΔmamZ cells from nitrate, or additional loss of the respiratory nitrate reductase Nap from ΔmamX severely exacerbated the magnetosome defects and entirely inhibited the formation of regular crystals, suggesting that MamXZ and Nap have similar, but independent roles in redox control of biomineralization. We propose a model in which MamX, MamZ and MamH functionally interact to balance the redox state of iron within the magnetosome compartment.