René Uebe

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 earths 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.

Deletion of a fur-like gene affects iron homeostasis and magnetosome formation in Magnetospirillum gryphiswaldense.

Magnetotactic bacteria synthesize specific organelles, the magnetosomes, which are membrane-enveloped crystals of the magnetic mineral magnetite (Fe(3)O(4)). The biomineralization of magnetite involves the uptake and intracellular accumulation of large amounts of iron. However, it is not clear how iron uptake and biomineralization are regulated and balanced with the biochemical iron requirement and intracellular homeostasis. In this study, we identified and analyzed a homologue of the ferric uptake regulator Fur in Magnetospirillum gryphiswaldense, which was able to complement a fur mutant of Escherichia coli. A fur deletion mutant of M. gryphiswaldense biomineralized fewer and slightly smaller magnetite crystals than did the wild type. Although the total cellular iron accumulation of the mutant was decreased due to reduced magnetite biomineralization, it exhibited an increased level of free intracellular iron, which was bound mostly to a ferritin-like metabolite that was found significantly increased in Mössbauer spectra of the mutant. Compared to that of the wild type, growth of the fur mutant was impaired in the presence of paraquat and under aerobic conditions. Using a Fur titration assay and proteomic analysis, we identified constituents of the Fur regulon. Whereas the expression of most known magnetosome genes was unaffected in the fur mutant, we identified 14 proteins whose expression was altered between the mutant and the wild type, including five proteins whose genes constitute putative iron uptake systems. Our data demonstrate that Fur is a regulator involved in global iron homeostasis, which also affects magnetite biomineralization, probably by balancing the competing demands for biochemical iron supply and magnetite biomineralization.

Distinguishing magnetic particle size of iron oxide nanoparticles with first-order reversal curves

Magnetic nanoparticles encompass a wide range of scientific study and technological applications. The success of using the nanoparticles in various applications demands control over size, dispersibility, and magnetics. Hence, the nanoparticles are often characterized by transmission electron microscopy (TEM), X-ray diffraction, and magnetic hysteresis loops. TEM analysis requires a thin layer of dispersed particles on the grid, which may often lead to particle aggregation thus making size analysis difficult. Magnetic hysteresis loops on the other hand provide information on the bulk property of the material without discriminating size, composition, and interaction effects. First order reversal curves (FORCs), described as an assembly of partial hysteresis loops originating from the major loop are efficient in identifying the domain size, composition, and interaction in a magnetic system. This study presents FORC diagrams on a variety of well-characterized biogenic and synthetic magnetite nanoparticles. It also introduces deconvoluted reversible and irreversible components from FORC as an important method for obtaining a semi-quantitative measure of the effective magnetic particle size. This is particularly important in a system with aggregation and interaction among the particles that often leads to either the differences between physical size and effective magnetic size. We also emphasize the extraction of secondary components by masking dominant coercivity fraction on FORC diagram to explore more detailed characterization of nanoparticle systems.

The MagA protein of magnetospirilla is not involved in bacterial magnetite biomineralization

Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria.

Cation diffusion facilitators transport initiation and regulation is mediated by cation induced conformational changes of the cytoplasmic domain.

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all domains of life. CDFs were shown to be involved in several human diseases, such as Type-II diabetes and neurodegenerative diseases. In this work, we employed a multi-disciplinary approach to study the activation mechanism of the CDF protein family. For this we used MamM, one of the main ion transporters of magnetosomes – bacterial organelles that enable magnetotactic bacteria to orientate along geomagnetic fields. Our results reveal that the cytosolic domain of MamM forms a stable dimer that undergoes distinct conformational changes upon divalent cation binding. MamM conformational change is associated with three metal binding sites that were identified and characterized. Altogether, our results provide a novel auto-regulation mode of action model in which the cytosolic domains conformational changes upon ligand binding allows the priming of the CDF into its transport mode.

Experimental mixtures of superparamagnetic and single‐domain magnetite with respect to Day‐Dunlop plots

Day-Dunlop plots are widely used in paleomagnetic and environmental studies as a tool to determine the magnetic domain state of magnetite, i.e., superparamagnetic (SP), stable single-domain (SD), pseudosingle-domain (PSD), multidomain (MD), and their mixtures. The few experimental studies that have examined hysteresis properties of SD-SP mixtures of magnetite found that the ratios of saturation remanent magnetization to saturation magnetization and the coercivity of remanence to coercivity are low, when compared to expected theoretical mixing trends based on Langevin theory. This study reexamines Day-Dunlop plots using experimentally controlled mixtures of SD and SP magnetite grains. End-members include magnetotactic bacteria (MSR-1) as the SD source, and a commercial ferrofluid or magnetotactic bacteria (ΔA12) as the SP source. Each SP-component was added incrementally to a SD sample. Experimental results from these mixing series show that the magnetization and coercivity ratios are lower than the theoretical prediction for bulk SP magnetic size. Although steric repulsion was present between the particles, we cannot rule out interaction in the ferrofluid for higher concentrations. The SP bacteria are noninteracting as the magnetite was enclosed by an organic bilipid membrane. Our results demonstrate that the magnetization and coercivity ratios of SD-SP mixtures can lie in the PSD range, and that an unambiguous interpretation of particle size can only be made with information about the magnetic properties of the end-members.

Genetic and Ultrastructural Analysis Reveals the Key Players and Initial Steps of Bacterial Magnetosome Membrane Biogenesis

Magnetosomes of magnetotactic bacteria contain well-ordered nanocrystals for magnetic navigation and have recently emerged as the most sophisticated model system to study the formation of membrane bounded organelles in prokaryotes. Magnetosome biosynthesis is thought to begin with the formation of a dedicated compartment, the magnetosome membrane (MM), in which the biosynthesis of a magnetic mineral is strictly controlled. While the biomineralization of magnetosomes and their subsequent assembly into linear chains recently have become increasingly well studied, the molecular mechanisms and early stages involved in MM formation remained poorly understood. In the Alphaproteobacterium Magnetospirillum gryphiswaldense, approximately 30 genes were found to control magnetosome biosynthesis. By cryo-electron tomography of several key mutant strains we identified the gene complement controlling MM formation in this model organism. Whereas the putative magnetosomal iron transporter MamB was most crucial for the process and caused the most severe MM phenotype upon elimination, MamM, MamQ and MamL were also required for the formation of wild-type-like MMs. A subset of seven genes (mamLQBIEMO) combined within a synthetic operon was sufficient to restore the formation of intracellular membranes in the absence of other genes from the key mamAB operon. Tracking of de novo magnetosome membrane formation by genetic induction revealed that magnetosomes originate from unspecific cytoplasmic membrane locations before alignment into coherent chains. Our results indicate that no single factor alone is essential for MM formation, which instead is orchestrated by the cumulative action of several magnetosome proteins.

Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium

ABSTRACT Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earths weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacterium Magnetospirillum gryphiswaldense by chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of the mms6 operon resulted in the formation of increasingly larger crystals (increase of ∼35%), the duplication of all major magnetosome operons (mamGFDC, mamAB, mms6, and mamXY, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of the mam and mms clusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches. IMPORTANCE Before our study, it had remained unknown how the upper sizes and numbers of magnetosomes are genetically regulated, and overproduction of magnetosome biosynthesis had not been achieved, owing to the difficulties of large-scale genome engineering in the recalcitrant magnetotactic bacteria. In this study, we established and systematically explored a strategy for the overexpression of magnetosome biosynthesis genes by genomic amplification of single and multiple magnetosome gene clusters via sequential chromosomal insertion by transposition. Our findings also indicate that the expression levels of magnetosome proteins together limit the upper size and number of magnetosomes within the cell. We demonstrate that tuned overexpression of magnetosome gene clusters provides a powerful strategy for the precise control of magnetosome size and number.

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.

Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance.

Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins’ proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human.