Emanuel Katzmann

Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization.

Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches.

Loss of the actin-like protein MamK has pleiotropic effects on magnetosome formation and chain assembly in Magnetospirillum gryphiswaldense

Magnetotactic bacteria synthesize magnetosomes, which are unique organelles consisting of membrane‐enclosed magnetite crystals. For magnetic orientation individual magnetosome particles are assembled into well‐organized chains. The actin‐like MamK and the acidic MamJ proteins were previously implicated in chain assembly. While MamK was suggested to form magnetosome‐associated cytoskeletal filaments, MamJ is assumed to attach the magnetosome vesicles to these structures. Although the deletion of either mamK in Magnetospirillum magneticum, or mamJ in Magnetospirillum gryphiswaldense affected chain formation, the previously observed phenotypes were not fully consistent, suggesting different mechanisms of magnetosome chain assembly in both organisms. Here we show that in M. gryphiswaldense MamK is not absolutely required for chain formation. Straight chains, albeit shorter, fragmented and ectopic, were still formed in a mamK deletion mutant, although magnetosome filaments were absent as shown by cryo‐electron tomography. Loss of MamK also resulted in reduced numbers of magnetite crystals and magnetosome vesicles and led to the mislocalization of MamJ. In addition, extensive analysis of wild type and mutant cells revealed previously unidentified ultrastructural characteristics in M. gryphiswaldense. Our results suggest that, despite of their functional equivalence, loss of MamK proteins in different bacteria may result in distinct phenotypes, which might be due to a species‐specific genetic context.

Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments

ABSTRACT In this report, we describe the selective cloning of large DNA fragments from magnetotactic metagenomes from various aquatic habitats. This was achieved by a two-step magnetic enrichment which allowed the mass collection of environmental magnetotactic bacteria (MTB) virtually free of nonmagnetic contaminants. Four fosmid libraries were constructed and screened by end sequencing and hybridization analysis using heterologous magnetosome gene probes. A total of 14 fosmids were fully sequenced. We identified and characterized two fosmids, most likely originating from two different alphaproteobacterial strains of MTB that contain several putative operons with homology to the magnetosome island (MAI) of cultivated MTB. This is the first evidence that uncultivated MTB exhibit similar yet differing organizations of the MAI, which may account for the diversity in biomineralization and magnetotaxis observed in MTB from various environments.

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.

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.

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.

Clone libraries and single cell genome amplification reveal extended diversity of uncultivated magnetotactic bacteria from marine and freshwater environments.

Magnetotactic bacteria (MTB), which orient along the earths magnetic field using magnetosomes, are ubiquitous and abundant in marine and freshwater environments. Previous phylogenetic analysis of diverse MTB has been limited to few cultured species and the most abundant and conspicuous members of natural populations, which were assigned to various lineages of the Proteobacteria, the Nitrospirae phylum as well as the candidate division OP3. However, their known phylogenetic diversity still not matches the large morphological and ultrastructural variability of uncultured MTB found in environmental communities. Here, we used analysis of 16S rRNA gene clone libraries in combination with microsorting and whole-genome amplification to systematically address the entire diversity of uncultured MTB from two different habitats. This approach revealed extensive and novel diversity of MTB within the freshwater and marine sediment samples. In total, single-cell analysis identified eight different phylotypes, which were only partly represented in the clone libraries, and which could be unambiguously assigned to their respective morphotypes. Identified MTB belonged to the Alphaproteobacteria (seven species) and the Nitrospirae phylum (two species). End-sequencing of a small insert library created from WGA-derived DNA of a novel conspicuous magnetotactic vibrio identified genes with highest similarity to two cultivated MTB as well as to other phylogenetic groups. In conclusion, the combination of metagenomic cloning and single cell sorting represents a powerful approach to recover maximum bacterial diversity including low-abundant magnetotactic phylotypes from environmental samples and also provides access to genomic analysis of uncultivated MTB.

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

ABSTRACT Magnetotactic bacteria (MTB) align along the Earths 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.

The FtsZ-like protein FtsZm of Magnetospirillum gryphiswaldense likely interacts with its generic homolog and is required for biomineralization under nitrate deprivation.

Midcell selection, septum formation, and cytokinesis in most bacteria are orchestrated by the eukaryotic tubulin homolog FtsZ. The alphaproteobacterium Magnetospirillum gryphiswaldense (MSR-1) septates asymmetrically, and cytokinesis is linked to splitting and segregation of an intracellular chain of membrane-enveloped magnetite crystals (magnetosomes). In addition to a generic, full-length ftsZ gene, MSR-1 contains a truncated ftsZ homolog (ftsZm) which is located adjacent to genes controlling biomineralization and magnetosome chain formation. We analyzed the role of FtsZm in cell division and biomineralization together with the full-length MSR-1 FtsZ protein. Our results indicate that loss of FtsZm has a strong effect on microoxic magnetite biomineralization which, however, could be rescued by the presence of nitrate in the medium. Fluorescence microscopy revealed that FtsZm-mCherry does not colocalize with the magnetosome-related proteins MamC and MamK but is confined to asymmetric spots at midcell and at the cell pole, coinciding with the FtsZ protein position. In Escherichia coli, both FtsZ homologs form distinct structures but colocalize when coexpressed, suggesting an FtsZ-dependent recruitment of FtsZm. In vitro analyses indicate that FtsZm is able to interact with the FtsZ protein. Together, our data suggest that FtsZm shares key features with its full-length homolog but is involved in redox control for magnetite crystallization.

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.