Magnetotactic bacteria are microorganisms that respond tomagnetic fields. We have studied the surface ultrastructure ofMagnetospirillum magnetotacticum and uncultured magne-totactic bacteria from a marine environment using trans-mission electron microscopy and freeze-etching. Numerous membrane vesicles were observed on the surface of Magneto-spirillum magnetotacticum bacteria. All uncultured magneto-tactic bacteria presented membrane vesicles on their surface inaddition to an extensive capsular material and an S-layer for-med by particles arranged in a hexagonal symmetry. We didnot observe any indication of electron-dense precipitation onthe surface of these microorganisms. Our results indicate thatmembrane vesicles are a common characteristic of magneto-tactic bacteria in natural sediments.

Key words: cell surface ultrastructure – freeze-etching –magnetotactic bacteria – Magnetospirillum – membrane ve-sicles

Introduction

Magnetotactic bacteria produce nano-sized, mostlymembrane-bound magnetic particles called magneto-somes that allow them to orient along magnetic fieldlines and find a suitable region in sediment where oxygen and nutrient requirements are met (Frankel et al., 1997). Magnetosome membranes have not beenfound in all MB species. It is not clear if all strains with

projectile-shaped magnetite crystals have magnetosomemembranes. For instance, in Magnetobacterium bavari-cum, the presence of a (protein) template at the base ofthe magnetite crystals has been suggested (Hanzlik et al., 2002). Natural enrichments of magnetotactic bac-teria contain a variety of different morphological types.Each morphotype is usually associated with a particularcrystalline habit of magnetic mineral (Schuler and Fran-kel, 1999). Most magnetotactic bacteria synthesizemagnetosomes that contain magnetite crystals (Fe3O4)whereas magnetotactic multicellular aggregates (Linsand Farina, 1999) and few other bacteria produce ironsulfides magnetosomes (Posfai et al., 1998).

Prokaryotes depend on diffusion to survive and havehigh surface area to volume ratio, which contributes totheir high capacity of interaction with the environment(Douglas and Beveridge, 1998). Bacterial cells, becauseof their small size and charge characteristics of theirsurface, can significantly contribute to the precipitationand accumulation of metal ions (Douglas and Beve-ridge, 1998). Thus, to study the surface characteristicsof magnetotactic bacteria is fundamental to understandthe interaction of microorganisms with the environmentand their role in the mobilization and metabolization of metals.

Few reports are available on the structural char-acteristics of the surface of magnetotactic bacteria (Valiand Kirschvink, 1990, Freitas et al., 2003), which is thefirst gateway for iron uptake in these microorganisms.Information, on the surface ultrastructure, may providea structural framework for the interpretation of physio-logical data and interrelations of these bacteria with theenvironment. We have recently studied the envelope

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Microbiol. Res. (2003) 158, 317–320http://www.urbanfischer.de/journals/microbiolres

Membrane vesicles in magnetotactic bacteria

Ulysses Lins1*, Marcos Farina2 and Bechara Kachar3

1 Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio deJaneiro, 21941-590, Rio de Janeiro, Brasil

2 Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, CCS, Bloco F, 21941-590, Rio de Janeiro, RJ,Brasil.

3 Section on Structural Cell Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, 20892, USA.

Accepted: July 22, 2003

Abstract

Corresponding author : Ulysses Linse-mail : ulins@micro.ufrj.br

ultrastructure of uncultured magnetotactic cocci (Frei-tas et al., 2003). Here, we report on the presence ofnumerous membrane vesicles on the surface of thecultivated species Magnetospirillum magnetotacticumbesides the uncultured magnetotactic bacteria that wehave studied by transmission electron microscopy andfreeze-etching techniques. Results indicate that mem-brane vesicles are commonplace in both cultivated mag-netotactic spirillum and uncultivated magnetotacticcocci.

Materials and methods

Magnetospirillum magnetotacticum (ATCC31632) cul-tivated in a chemically defined medium (Blakemore et al., 1979) was purchased from ATCC. Cultivated cellswere harvested at the stationary phase by centrifuga-tion at 10,000 g for 15 min. For harvesting uncultivated magnetotactic bacteria (Spring et al., 1998), samples(sediment and water) from Itaipu lagoon (43° 04′ W ×22° 57′ S), near Rio de Janeiro city, were collected and

stored in bottles that were left undisturbed at the labor-atory under dim light for several weeks. Magnetotacticbacteria were magnetically harvested with a speciallydesigned glass chamber (Lins and Farina, 1999) andexposed to the properly aligned magnetic field of ahomemade coil. After approximately 15 minutes ofexposition to the field, magnetotactic bacteria were harvested with a capillary tube and used in subsequentprocessing.

For freeze-etching experiments, magnetotactic bac-teria were fixed in a solution containing glutaraldehyde2.5% in cacodylate buffer 0.1 M for 30 minutes at roomtemperature, washed several times in distilled water andplaced on cushions of 12.5% gelatin attached to a metalsupport by a piece of filter paper. The support was then quick frozen by impact against a copper block, previously cooled by liquid nitrogen or by contact witha liquid nitrogen-cooled sapphire block of a Life CellCF0100 quick-freezing machine (Research and Manu-facturing Co, Tucson, AR). Frozen specimens were thentransferred to a Balzers freeze-fracture machine andfractured at –115°C. After fracturing, the exposed sur-face of some of the samples was etched for 10 minutes

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Fig. 1. Envelope structure of magnetotactic bacteria. (a) Freeze etching of uncultured magnetotactic bacteria where membranevesicles (arrowheads) can be seen. The magnetosome chains (M) remained attached to the cell surface after replication; (b) Highmagnification of the boxed region in (a) showing three membrane vesicles (arrowheads) as well as particles (arrow) from the S-layer; (c) Transmission electron microscopy image of a magnetotactic bacterium from the Itaipu lagoon showing the cell surface membrane with two membrane vesicles (arrowheads) filled with electron dense material; Scale bar indicates 0,5 µm in (a), 100 nm in (b), 50 nm in (c).

Microbiol. Res. 158 (2003) 4 319

approximately at –100°C cooled again to –115°C thenrotatory-shadowed with platinum at 15° and carbon at90°. Replicas were cleaned in sodium hypoclorite, col-lected on copper grids and examined with a ZeissEM902 transmission electron microscope.

Results and discussion

Freeze-etching experiments revealed the substructure of the cell surface of uncultured and cultured magneto-tactic bacteria. Freeze-etching images showed nume-rous membrane vesicles on the cell surface of cocci(Figures 1a–1c) and Magnetospirillum magnetotacti-cum cells (Figure 2). Magnetosomes (Figure 1a) re-mained attached to the replicas after freeze-etching and

consequently facilitated the identification of magneto-tactic bacteria in the replica. Replicas of the unculturedbacteria revealed the presence of para-crystalline sur-face structures similar to S-layers (Sleytr and Beve-ridge, 1999) distributed in domains of the cell surface(Figure 1b, arrow). The S-layer particles (Figure 1b,arrow) were periodically organized in a hexagonalsymmetry configuration. Numerous membrane vesiclescould be observed (Figure 1b, arrowheads) protrudingfrom the periodic cell surface. Observation of sectionedmaterial revealed that an electron dense material filledthese vesicles (Figure 1c, arrowheads). We could notdetermine the chemical nature of the vesicular interior.Figure 2b shows a cell of Magnetospirillum magneto-tacticum where the cytoplasm, magnetosome mem-brane profiles (Figure 2b, small arrows) and a flagellum(Figure 2b, arrow) could be observed. Numerous spher-

Fig. 2. (a) High magnification view of the cell surface of Magnetospirillum magnetotacticum showing numerous spherical(small arrowheads) and oval-shaped (large arrowheads) membrane vesicles; (b) Low magnification view of Magnetospirillummagnetotacticum where the granular cytoplasm (Cy), two magnetosome membrane profile (small arrows), a flagellum (largearrow) and membrane vesicles (arrowheads) can be seen; Scale bar indicates 200 nm in (a) and 0,5 µm in (b).

Acknowledgements

We thank Laboratório de Biologia Celular e Tecidual, UENFand Laboratório de Ultraestrutura Celular Hertha Meyer,UFRJ for facilities. This work was partially supported byCAPES-PROCAD, CNPq (PRONEX), FUJB, FAPERJ Brazi-lian programs.

References

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ical or oval-shaped membrane vesicles could be ob-served on the surface of different Magnetospirillummagnetotacticum cells (Figures 2a and 2b, arrowheads).Again, the vesicles seemed to be protruding from thesurface of the cell.

The physicochemical and structural qualities of thecell surface of magnetotactic bacteria provide the basisfor the interaction of these microorganisms with theenvironment and their relation to magnetotaxis. Theanalysis of their fine structure contributes with impor-tant information complementary to the biochemicalmeasurements and other physiological studies. Becauseof the procedures involved and the characteristics of thespecimen preparation required, the standard electronmicroscopy techniques may not provide a resolutionsufficient to visualize the structural relationships be-tween the elements of bacterial surface. Thus, interpre-tations about the ultrastructural morphology of biologi-cal samples are strongly limited by sample preparationtechniques, which may destroy the fine structure of theorganic phases. We believe that physical methods wehave applied for studying the membrane vesicles in thepresent work, such as fixation followed by fast-freezingwith mildly chemical manipulation (Heuser et al., 1979)contribute significantly to the minimization of artifactsduring the study of these soft biological structures. In the present study, we have used the quick-freezingdeep-etching technique to investigate the fine structureand micro-architecture of the cell surface of the uncul-tured magnetotactic cocci from the Itaipu lagoon andMagnetospirillum magnetotacticum. A noticeable fea-ture of the cell surface of both bacteria was the presenceof membrane vesicles. Membrane vesicles are common-place in gram-negative bacteria (Beveridge, 1999).They are commonly filled with components consideredto be secretion products. They seem to be commonlysecreted in natural communities of bacteria (Beveridge,1999), as is the case of magnetotactic bacteria fromsediments. Vesicles protruding from the cell surfacewere observed in ultrastructural studies of the cultivatedspecies Magnetospirillum magnetotacticum (Balkwill et al., 1980) and were here confirmed with the quick-freezing deep-etching technique (Heuser et al., 1979).Our results and previous reports suggest that membranevesicles are also a structural feature common to bothcultivated spirilla and uncultivated magnetotactic cocci.They role in magnetotaxis, if any exists, remains to bedetermined as we could no determine the chemical nature of their vesicular material. We intend to addressthis question in the future.

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