RESUMO
Magnetospirillum gryphiswaldense MSR-1 synthesizes membrane-enclosed magnetite (Fe3 O4 ) 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.
Assuntos
Proteínas de Bactérias/metabolismo , Biomineralização , Óxido Ferroso-Férrico/metabolismo , Magnetossomos/metabolismo , Magnetospirillum/enzimologia , Alelos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Difusão Dinâmica da Luz , Óxido Ferroso-Férrico/química , Magnetossomos/química , Magnetospirillum/genética , Mutagênese Sítio-Dirigida , Domínios Proteicos , Difração de Raios XRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Magnetospirillum/metabolismo , Nitratos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Magnetospirillum/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The alphaproteobacterium Magnetospirillum gryphiswaldense synthesizes magnetosomes, which are membrane-enveloped crystals of magnetite. Here we show that nitrite reduction is involved in redox control during anaerobic biomineralization of the mixed-valence iron oxide magnetite. The cytochrome cd1-type nitrite reductase NirS shares conspicuous sequence similarity with NirN, which is also encoded within a larger nir cluster. Deletion of any one of these two nir genes resulted in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. However, whereas nitrite reduction was completely abolished in the ΔnirS mutant, attenuated but significant nitrite reduction occurred in the ΔnirN mutant, indicating that only NirS is a nitrite reductase in M. gryphiswaldense. However, the ΔnirN mutant produced a different form of periplasmic d(1) heme that was not noncovalently bound to NirS, indicating that NirN is required for full reductase activity by maintaining a proper form of d1 heme for holo-cytochrome cd(1) assembly. In conclusion, we assign for the first time a physiological function to NirN and demonstrate that effective nitrite reduction is required for biomineralization of wild-type crystals, probably by contributing to oxidation of ferrous iron under oxygen-limited conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Citocromos/metabolismo , Óxido Ferroso-Férrico/metabolismo , Heme/análogos & derivados , Magnetospirillum/enzimologia , Nitrito Redutases/metabolismo , Anaerobiose , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocromos/química , Citocromos/genética , Heme/metabolismo , Ferro/metabolismo , Magnetossomos , Magnetospirillum/classificação , Nitrito Redutases/química , Nitrito Redutases/genética , Nitritos/metabolismo , OxirreduçãoRESUMO
Magnetotactic bacteria (MTB), which orient along the earth's 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.
Assuntos
Bactérias/classificação , Bactérias/genética , Biodiversidade , Água Doce/microbiologia , Sedimentos Geológicos/microbiologia , Bactérias/ultraestrutura , DNA Bacteriano/genética , Ecossistema , Genoma Bacteriano/genética , Biblioteca Genômica , Magnetismo , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
The magnetosomes of many magnetotactic bacteria consist of membrane-enveloped magnetite crystals, whose synthesis is favored by a low redox potential. However, the cellular redox processes governing the biomineralization of the mixed-valence iron oxide have remained unknown. Here, we show that in the alphaproteobacterium Magnetospirillum gryphiswaldense, magnetite biomineralization is linked to dissimilatory nitrate reduction. A complete denitrification pathway, including gene functions for nitrate (nap), nitrite (nir), nitric oxide (nor), and nitrous oxide reduction (nos), was identified. Transcriptional gusA fusions as reporters revealed that except for nap, the highest expression of the denitrification genes coincided with conditions permitting maximum magnetite synthesis. Whereas microaerobic denitrification overlapped with oxygen respiration, nitrate was the only electron acceptor supporting growth in the entire absence of oxygen, and only the deletion of nap genes, encoding a periplasmic nitrate reductase, and not deletion of nor or nos genes, abolished anaerobic growth and also delayed aerobic growth in both nitrate and ammonium media. While loss of nosZ or norCB had no or relatively weak effects on magnetosome synthesis, deletion of nap severely impaired magnetite biomineralization and resulted in fewer, smaller, and irregular crystals during denitrification and also microaerobic respiration, probably by disturbing the proper redox balance required for magnetite synthesis. In contrast to the case for the wild type, biomineralization in Δnap cells was independent of the oxidation state of carbon substrates. Altogether, our data demonstrate that in addition to its essential role in anaerobic respiration, the periplasmic nitrate reductase Nap has a further key function by participating in redox reactions required for magnetite biomineralization.
Assuntos
Óxido Ferroso-Férrico/metabolismo , Magnetospirillum/enzimologia , Nitrato Redutase/metabolismo , Anaerobiose , Meios de Cultura/química , Deleção de Genes , Perfilação da Expressão Gênica , Magnetospirillum/genética , Magnetospirillum/crescimento & desenvolvimento , Magnetospirillum/metabolismo , Redes e Vias Metabólicas/genética , Nitrato Redutase/genética , Nitratos/metabolismo , Oxirredução , Compostos de Amônio Quaternário/metabolismoRESUMO
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.
Assuntos
Divisão Celular Assimétrica , Proteínas de Bactérias/metabolismo , Citocinese , Magnetossomos/metabolismo , Magnetospirillum/citologia , Magnetospirillum/metabolismo , Proteínas de Bactérias/genética , Magnetossomos/genética , Magnetospirillum/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Óxido Ferroso-Férrico/metabolismo , Membranas Intracelulares/metabolismo , Magnetossomos/metabolismo , Magnetospirillum/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Análise Mutacional de DNA , Deleção de Genes , Magnetospirillum/genética , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estabilidade Proteica , Homologia de Sequência de AminoácidosRESUMO
Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that orient along magnetic fields using membrane-coated magnetic nanocrystals of magnetite (Fe(3) O(4) ) or greigite (Fe(3) S(4) ), the magnetosomes. Previous phylogenetic analysis of MTB has been limited to few cultivated species and most abundant members of natural populations, which were assigned to Proteobacteria and the Nitrospirae phyla. Here, we describe a single cell-based approach that allowed the targeted phylogenetic and ultrastructural analysis of the magnetotactic bacterium SKK-01, which was low abundant in sediments of Lake Chiemsee. Morphologically conspicuous single cells of SKK-01 were micromanipulated from magnetically collected multi-species MTB populations, which was followed by whole genome amplification and ultrastructural analysis of sorted cells. Besides intracellular sulphur inclusions, the large ovoid cells of SKK-01 harbour â¼175 bullet-shaped magnetosomes arranged in multiple chains that consist of magnetite as revealed by TEM and EDX analysis. Sequence analysis of 16 and 23S rRNA genes from amplified genomic DNA as well as fluorescence in situ hybridization assigned SKK-01 to the candidate division OP3, which so far lacks any cultivated representatives. SKK-01 represents the first morphotype that can be assigned to the OP3 group as well as the first magnetotactic member of the PVC superphylum.
Assuntos
Bactérias/classificação , Bactérias/ultraestrutura , Magnetossomos/microbiologia , Filogenia , Bactérias/genética , Óxido Ferroso-Férrico/análise , Genes de RNAr , Genoma Bacteriano , Sedimentos Geológicos/microbiologia , Alemanha , Hibridização in Situ Fluorescente , Lagos/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Célula ÚnicaRESUMO
Genes for magnetosome formation in magnetotactic bacteria are clustered in large genomic magnetosome islands (MAI). Spontaneous deletions and rearrangements were frequently observed within these regions upon metabolic stress. This instability was speculated to be due to RecA-dependent homologous recombination between the numerous sequence repeats present within the MAI. Here we show that a RecA-deficient strain of Magnetospirillum gryphiswaldense (IK-1) no longer exhibits genetic instability of magnetosome formation. Strain IK-1 displayed higher sensitivity to oxygen and UV irradiation. Furthermore, the lack of RecA abolished allelic exchange in the mutant. Cells of strain IK-1 displayed a slightly altered (i.e., more elongated) morphology, whereas the absence of RecA did not affect the ability to synthesize wild-type-like magnetosomes. Our data provide evidence that the observed genetic instability of magnetosome formation in the wild type is due predominantly to RecA-mediated recombination. In addition, increased genetic stability could make strain IK-1 a useful tool for the expression of genes and further genetic engineering, as well as for biotechnological production of bacterial magnetosomes.
Assuntos
Proteínas de Bactérias/metabolismo , Ilhas Genômicas/genética , Magnetossomos/genética , Magnetospirillum/genética , Magnetospirillum/metabolismo , Proteínas de Bactérias/genética , Magnetospirillum/ultraestrutura , Microscopia Eletrônica de Transmissão , Mutação , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Magnetossomos/metabolismo , Magnetossomos/ultraestrutura , Magnetospirillum/citologia , Magnetospirillum/fisiologia , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Tomografia com Microscopia Eletrônica , Deleção de GenesRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Deleção de Genes , Ferro/metabolismo , Magnetossomos/metabolismo , Magnetospirillum/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Análise por Conglomerados , Citosol/química , Escherichia coli/genética , Teste de Complementação Genética , Homeostase , Magnetospirillum/química , Magnetospirillum/genética , Magnetospirillum/crescimento & desenvolvimento , Filogenia , Proteoma/análise , Regulon , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Análise Espectral/métodosRESUMO
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.
Assuntos
DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Genes Bacterianos , Ilhas Genômicas , Família Multigênica , Organelas/genética , Microbiologia da Água , Clonagem Molecular , DNA Bacteriano/química , Ordem dos Genes , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
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.