Genome-Wide Identification of Essential and Auxiliary Gene Sets for Magnetosome Biosynthesis in Magnetospirillum gryphiswaldense.

Magnetotactic bacteria (MTB) stand out by their ability to manufacture membrane-enclosed magnetic organelles, so-called magnetosomes. Previously, it has been assumed that a genomic region of approximately 100 kbp, the magnetosome island (MAI), harbors all genetic determinants required for this intricate biosynthesis process. Recent evidence, however, argues for the involvement of additional auxiliary genes that have not been identified yet. In the present study, we set out to delineate the full gene complement required for magnetosome production in the alphaproteobacterium Magnetospirillum gryphiswaldense using a systematic genome-wide transposon mutagenesis approach. By an optimized procedure, a Tn5 insertion library of 80,000 clones was generated and screened, yielding close to 200 insertants with mild to severe impairment of magnetosome biosynthesis. Approximately 50% of all Tn5 insertion sites mapped within the MAI, mostly leading to a nonmagnetic phenotype. In contrast, in the majority of weakly magnetic Tn5 insertion mutants, genes outside the MAI were affected, which typically caused lower numbers of magnetite crystals with partly aberrant morphology, occasionally combined with deviant intracellular localization. While some of the Tn5-struck genes outside the MAI belong to pathways that have been linked to magnetosome formation before (e.g., aerobic and anaerobic respiration), the majority of affected genes are involved in so far unsuspected cellular processes, such as sulfate assimilation, oxidative protein folding, and cytochrome c maturation, or are altogether of unknown function. We also found that signal transduction and redox functions are enriched in the set of Tn5 hits outside the MAI, suggesting that such processes are particularly important in support of magnetosome biosynthesis.IMPORTANCE Magnetospirillum gryphiswaldense is one of the few tractable model magnetotactic bacteria (MTB) for studying magnetosome biomineralization. So far, knowledge on the genetic determinants of this complex process has been mainly gathered using reverse genetics and candidate approaches. In contrast, nontargeted forward genetics studies are lacking, since application of such techniques in MTB has been complicated for a number of technical reasons. Here, we report on the first comprehensive transposon mutagenesis study in MTB, aiming at systematic identification of auxiliary genes necessary to support magnetosome formation in addition to key genes harbored in the magnetosome island (MAI). Our work considerably extends the candidate set of novel subsidiary determinants and shows that the full gene complement underlying magnetosome biosynthesis is larger than assumed. In particular, we were able to define certain cellular pathways as specifically important for magnetosome formation that have not been implicated in this process so far.

Uptake and persistence of bacterial magnetite magnetosomes in a mammalian cell line: Implications for medical and biotechnological applications.

Magnetotactic bacteria biomineralize intracellular magnetic nanocrystals surrounded by a lipid bilayer called magnetosomes. Due to their unique characteristics, magnetite magnetosomes are promising tools in Biomedicine. However, the uptake, persistence, and accumulation of magnetosomes within mammalian cells have not been well studied. Here, the endocytic pathway of magnetite magnetosomes and their effects on human cervix epithelial (HeLa) cells were studied by electron microscopy and high spatial resolution nano-analysis techniques. Transmission electron microscopy of HeLa cells after incubation with purified magnetosomes showed the presence of magnetic nanoparticles inside or outside endosomes within the cell, which suggests different modes of internalization, and that these structures persisted beyond 120 h after internalization. High-resolution transmission electron microscopy and electron energy loss spectra of internalized magnetosome crystals showed no structural or chemical changes in these structures. Although crystal morphology was preserved, iron oxide crystalline particles of approximately 5 nm near internalized magnetosomes suggests that minor degradation of the original mineral structures might occur. Cytotoxicity and microscopy analysis showed that magnetosomes did not result in any apparent effect on HeLa cells viability or morphology. Based on our results, magnetosomes have significant biocompatibility with mammalian cells and thus have great potential in medical, biotechnological applications.

The oxygen sensor MgFnr controls magnetite biomineralization by regulation of denitrification in Magnetospirillum gryphiswaldense.

BACKGROUND: Magnetotactic bacteria are capable of synthesizing magnetosomes only under oxygen-limited conditions. However, the mechanism of the aerobic repression on magnetite biomineralization has remained unknown. In Escherichia coli and other bacteria, Fnr (fumarate and nitrate reduction regulator) proteins are known to be involved in controlling the switch between microaerobic and aerobic metabolism. Here, we report on an Fnr-like protein (MgFnr) and its role in growth metabolism and magnetite biomineralization in the alphaproteobacterium Magnetospirillum gryphiswaldense. RESULTS: Deletion of Mgfnr not only resulted in decreased N2 production due to reduced N2O reductase activity, but also impaired magnetite biomineralization under microaerobic conditions in the presence of nitrate. Overexpression of MgFnr in the WT also caused the synthesis of smaller magnetite particles under anaerobic and microaerobic conditions in the presence of nitrate. These data suggest that proper expression of MgFnr is required for WT-like magnetosome synthesis, which is regulated by oxygen. Analyses of transcriptional gusA reporter fusions revealed that besides showing similar properties to Fnr proteins reported in other bacteria, MgFnr is involved in the repression of the expression of denitrification genes nor and nosZ under aerobic conditions, possibly owing to several unique amino acid residues specific to MTB-Fnr. CONCLUSIONS: We have identified and thoroughly characterized the first regulatory protein mediating denitrification growth and magnetite biomineralization in response to different oxygen conditions in a magnetotactic bacterium. Our findings reveal that the global oxygen regulator MgFnr is a genuine O2 sensor. It is involved in controlling expression of denitrification genes and thereby plays an indirect role in maintaining proper redox conditions required for magnetite biomineralization.

Ferromagnetic resonance of intact cells and isolated crystals from cultured and uncultured magnetite-producing magnetotactic bacteria.

Most magnetotactic bacteria (MB) produce stable, single-domain magnetite nanocrystals with species-specific size, shape and chain arrangement. In addition, most crystals are elongated along the [111] direction, which is the easy axis of magnetization in magnetite, chemically pure and structurally perfect. These special characteristics allow magnetite crystal chains from MB to be recognized in environmental samples including old sedimentary rocks. Ferromagnetic resonance (FMR) has been proposed as a powerful and practical tool for screening large numbers of samples possibly containing magnetofossils. Indeed, several studies were recently published on FMR of cultured MB, mainly Magnetospirillum gryphiswaldense. In this work, we examined both uncultured magnetotactic cocci and the cultured MB M. gryphiswaldense using transmission electron microscopy (TEM) and FMR from 10 K to room temperature (RT). The TEM data supported the FMR spectral characteristics of our samples. The FMR spectra of both bacteria showed the intrinsic characteristics of magnetite produced by MB, such as extended absorption at the low field region of the spectra and a Verwey transition around 100 K. As previously observed, the spectra of M. gryphiswaldense isolated crystals were more symmetrical than the spectra obtained from whole cells, reflecting the loss of chain arrangement due to the small size and symmetrical shape of the crystals. However, the FMR spectra of magnetic crystals isolated from magnetotactic cocci were very similar to the FMR spectra of whole cells, because the chain arrangement was maintained due to the large size and prismatic shape of the crystals. Our data support the use of FMR spectra to detect magnetotactic bacteria and magnetofossils in samples of present and past environments. Furthermore, the spectra suggest the use of the temperature transition of spectral peak-to-peak intensity to obtain the Verwey temperature for these systems.

The terminal oxidase cbb3 functions in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense.

The biomineralization of magnetosomes in Magnetospirillum gryphiswaldense and other magnetotactic bacteria occurs only under suboxic conditions. However, the mechanism of oxygen regulation and redox control of biosynthesis of the mixed-valence iron oxide magnetite [FeII(FeIII)2O4] is still unclear. Here, we set out to investigate the role of aerobic respiration in both energy metabolism and magnetite biomineralization of M. gryphiswaldense. Although three operons encoding putative terminal cbb3-type, aa3-type, and bd-type oxidases were identified in the genome assembly of M. gryphiswaldense, genetic and biochemical analyses revealed that only cbb3 and bd are required for oxygen respiration, whereas aa3 had no physiological significance under the tested conditions. While the loss of bd had no effects on growth and magnetosome synthesis, inactivation of cbb3 caused pleiotropic effects under microaerobic conditions in the presence of nitrate. In addition to their incapability of simultaneous nitrate and oxygen reduction, cbb3-deficient cells had complex magnetosome phenotypes and aberrant morphologies, probably by disturbing the redox balance required for proper growth and magnetite biomineralization. Altogether, besides being the primary terminal oxidase for aerobic respiration, cbb3 oxidase may serve as an oxygen sensor and have a further role in poising proper redox conditions required for magnetite biomineralization.

Optimization of magnetosome production and growth by the magnetotactic vibrio Magnetovibrio blakemorei strain MV-1 through a statistics-based experimental design.

The growth and magnetosome production of the marine magnetotactic vibrio Magnetovibrio blakemorei strain MV-1 were optimized through a statistics-based experimental factorial design. In the optimized growth medium, maximum magnetite yields of 64.3 mg/liter in batch cultures and 26 mg/liter in a bioreactor were obtained.

Salinity dependence of the distribution of multicellular magnetotactic prokaryotes in a hypersaline lagoon.

Candidatus Magnetoglobus multicellularis is an unusual magnetotactic multicellular microorganism composed of a highly organized assemblage of gram-negative bacterial cells. In this work, the salinity dependence of Ca. M. multicellularis and its abundance in the hypersaline Araruama Lagoon, Brazil were studied. Viability experiments showed that Ca. M. multicellularis died in salinities upper than 55 per thousand and lower than 40 per thousand. Low salinities were also observed to modify the cellular assemblage. In microcosms prepared with different salinities, the microorganism grew better at intermediate salinities whereas in high or low salinities, the size of the population did not increase over time. The concentrations of Ca. M. multicellularis in the lagoon were related to salinity; sites with lower and higher salinities than the lagoon average contained less Ca. M. multicellularis. These results demonstrate the influence of salinity on the survival and distribution of Ca. M. multicellularis in the environment. In sediments, the abundance of Ca. M. multicellularis ranged from 0 to 103 microorganisms/ml, which represented 0.001% of the counts of total bacteria. The ability of Ca. M. multicellularis to accumulate iron and sulfur in high numbers of magnetosomes (up to 905 per microorganism) suggests that its impact on the sequestration of these elements (0.1% for biogenic bacterial iron) is not proportional to its abundance in the lagoon.

Salinity dependence of the distribution of multicellular magnetotactic prokaryotes in a hypersaline lagoon

Candidatus Magnetoglobus multicellularis is an unusual magnetotactic multicellular microorganism composed of a highly organized assemblage of gram-negative bacterial cells. In this work, the salinity dependence of Ca. M. multicellularis and its abundance in the hypersaline Araruama Lagoon, Brazil were studied. Viability experiments showed that Ca. M. multicellularis died in salinities >55 per-mille and <40 per-mille. Low salinities were also observed to modify the cellular assemblage. In microcosms prepared with different salinities, the microorganism grew better at intermediate salinities whereas in high or low salinities, the size of the population did not increase over time. The concentrations of Ca. M. multicellularis in the lagoon were related to salinity; sites with lower and higher salinities than the lagoon average contained less Ca. M. multicellularis. These results demonstrate the influence of salinity on the survival and distribution of Ca. M. multicellularis in the environment. In sediments, the abundance of Ca. M. multicellularis ranged from 0 to 103 microorganisms/ml, which represented 0.001% of the counts of total bacteria. The ability of Ca. M. multicellularis to accumulate iron and sulfur in high numbers of magnetosomes (up to 905 per microorganism) suggests that its impact on the sequestration of these elements (0.1% for biogenic bacterial iron) is not proportional to its abundance in the lagoon (AU)

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Greigite magnetosome membrane ultrastructure in 'Candidatus Magnetoglobus multicellularis'.

The ultrastructure of the greigite magnetosome membrane in the multicellular magnetotactic bacteria 'Candidatus Magnetoglobus multicellularis' was studied. Each cell contains 80 membrane-enclosed iron-sulfide magnetosomes. Cytochemistry methods showed that the magnetosomes are enveloped by a structure whose staining pattern and dimensions are similar to those of the cytoplasmic membrane, indicating that the magnetosome membrane likely originates from the cytoplasmic membrane. Freeze-fracture showed intramembrane particles in the vesicles surrounding each magnetosome. Observations of cell membrane invaginations, the trilaminar membrane structure of immature magnetosomes, and empty vesicles together suggested that greigite magnetosome formation begins by invagination of the cell membrane, as has been proposed for magnetite magnetosomes.

Greigite magnetosome membrane ultrastructure in «Candidatus Magnetoglobus multicellularis»

The ultrastructure of the greigite magnetosome membrane in the multicellular magnetotactic bacteria «Candidatus Magnetoglobus multicellularis» was studied. Each cell contains 80 membrane-enclosed iron-sulfide magnetosomes. Cytochemistry methods showed that the magnetosomes are enveloped by a structure whose staining pattern and dimensions are similar to those of the cytoplasmic membrane, indicating that the magnetosome membrane likely originates from the cytoplasmic membrane. Freeze-fracture showed intramembrane particles in the vesicles surrounding each magnetosome. Observations of cell membrane invaginations, the trilaminar membrane structure of immature magnetosomes, and empty vesicles together suggested that greigite magnetosome formation begins by invagination of the cell membrane, as has been proposed for magnetite magnetosomes (AU)

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Grazing protozoa and magnetosome dissolution in magnetotactic bacteria.

Magnetotactic bacteria show an ability to navigate along magnetic field lines because of magnetic particles called magnetosomes. All magnetotactic bacteria are unicellular except for the multicellular prokaryote (recently named 'Candidatus Magnetoglobus multicellularis'), which is formed by an orderly assemblage of 17-40 prokaryotic cells that swim as a unit. A ciliate was used in grazing experiments with the M. multicellularis to study the fate of the magnetosomes after ingestion by the protozoa. Ciliates ingested M. multicellularis, which were located in acid vacuoles as demonstrated by confocal laser scanning microscopy. Transmission electron microscopy and X-ray microanalysis of thin-sectioned ciliates showed the presence of M. multicellularis and magnetosomes inside vacuoles in different degrees of degradation. The magnetosomes are dissolved within the acidic vacuoles of the ciliate. Depending on the rate of M. multicellularis consumption by the ciliates the iron from the magnetosomes may be recycled to the environment in a more soluble form.

Cell viability in magnetotactic multicellular prokaryotes / Viabilidad celular en procariotas multicelulares magnetotácticos

A magnetotactic multicellular prokaryote (MMP) is an assembly of bacterial cells organized side by side in a hollow sphere in which each cell faces both the external environment and an internal acellular compartment in the center of the multicellular organism. MMPs swim as a unit propelled by the coordinated beating of the many flagella on the external surface of each cell. At every stage of its life cycle, MMPs are multicellular. Initially, a spherical MMP grows by enlarging the size of each of its cells, which then divide. Later, the cells separate into two identical spheres. Swimming individual cells of MMPs have never been observed. Here we have used fluorescent dyes and electron microscopy to study the viability of individual MMP cells. When separated from the MMP, the cells cease to move and they no longer respond to magnetic fields. Viability tests indicated that, although several cells could separate from a MMP before completely losing their motility and viability, all of the separated cells were dead. Our data show that the high level of cellular organization in MMPs is essential for their motility, magnetotactic behavior, and viability (AU)

Un procariota multicelular magnetotáctico (MMP en inglés) es un conjunto de células bacterianas dispuestas una al lado de otra en una esfera hueca en la que cada célula se enfrenta tanto hacia el ambiente externo como hacia un compartimento acelular interno en el centro del organismo multicelular. Los MMPs pueden nadar como una unidad propulsadas por el batir coordinado de los numerosos flagelos de la superficie exterior de cada célula. Todas las fases del ciclo vital de los MMPs son multicelulares. Inicialmente, un MMP esférico crece debido al aumento del tamaño de cada célula, y entonces las células se dividen. Después, las células se separan, formando dos esferas idénticas. Nunca se han observado células individuales de MMPs nadando. Describimos el estudio de la viabilidad de las células individuales de MMP realizado con tinciones fluorescentes y el microscopio electrónico. Cuando se separan del MMP, las células dejan de moverse y ya no responden a campos magnéticos. Las pruebas de viabilidad indican que todas las células separadas están muertas. Varias células se pueden separar de un MMP antes de que ésta pierda completamente su movilidad y viabilidad. Nuestros datos indican que el alto nivel de organización celular de los MMPs es esencial para su movilidad, comportamiento magnetotáctico y viabilidad (AU)

Cell viability in magnetotactic multicellular prokaryotes.

A magnetotactic multicellular prokaryote (MMP) is an assembly of bacterial cells organized side by side in a hollow sphere in which each cell faces both the external environment and an internal acellular compartment in the center of the multicellular organism. MMPs swim as a unit propelled by the coordinated beating of the many flagella on the external surface of each cell. At every stage of its life cycle, MMPs are multicellular. Initially, a spherical MMP grows by enlarging the size of each of its cells, which then divide. Later, the cells separate into two identical spheres. Swimming individual cells of MMPs have never been observed. Here we have used fluorescent dyes and electron microscopy to study the viability of individual MMP cells. When separated from the MMP, the cells cease to move and they no longer respond to magnetic fields. Viability tests indicated that, although several cells could separate from a MMP before completely losing their motility and viability, all of the separated cells were dead. Our data show that the high level of cellular organization in MMPs is essential for their motility, magnetotactic behavior, and viability.