Bazylinski, Dennis A.

Frankel, Richard B.

Garratt-Reed, Anthony J.

Heywood, Brigid R.

Mann, Stephen

English

DigitalCommons@CalPoly

Controlled Biosynthesis of Greigite (Fe3S4) in Magnetotactic Bacteria B. R. Heywood School of Chemistry, University of Bath, Bath BA2 7AY, UK D. A. Bazylinski Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA A. Garratt-Reed Center for Materials Science and Engineering, Massachusetts Institute of Technol­ogy, Cambridge, MA 02139 USA S. Mann School of Chemistry, University of Bath, BATH BA 27 AY R.B. Frankel Department of Physics, California Polytechnic State University, San Luis Obispo, CA 93407, USA Several species of aquatic bacteria have been identified which employ the earth's geomagnetic field to direct their motion towards suitable habitats. A common feature of these magnetotactic bacteria is the presence of discrete in­tracellular magnetic inclusions, magne­tosomes, aligned in chains along the long axis of the organism. The size and orientation of these individual mag­netic particles impart a permanent mag­netic dipole moment which is re­sponsible for the magnetotactic re­sponse. Until recently only crystals of the mixed-valence iron oxide, mag­netite (Fe30 4), were identified in these bacteria. Now, however, magnetic and non magnetic iron sulfide minerals have been identified in multicellular magnetotactic bacteria collected from sulfidic environments [1, 2]. These studies showed the presence of discrete intracellular crystals but were in con­flict with regard to their mineralogical identification, viz. pyrrhotite (FeSI +J [2] or greigite (Fe3S4) and pyrite (FeS2) [1]. This discovery is of profound im­portance to the study of biomineral­ization, prokaryotic metabolism [3] and, possibly, the origin of life [4]. Here we report the presence of intra­cellular greigite crystals in two different types of rod-shaped single-celled mag­netotactic bacteria collected from sulfide-rich sites. The greigite particles are often organized in chains, have nar­row size distributions (50 - 90 nm), and adopt specific crystallographic habits (cubo-octahedral and rectangular prismatic) associated with particular cell types. The prismatic crystals are unusual since they do not conform to the symmetry relations of the greigite cubic lattice. Our results indicate that the biomineralization of greigite in magnetotactic bacteria is highly reg­ulated and there are close similarities to the biosynthesis of bacterial mag­netite (Fe304) [5, 6]. The formation of novel morphologies of biogenic greigite suggests that such crystals may provide a diagnostic marker in the characteriza­tion of sources of remanent magnetiza­tion in recent and ancient sediments [7]. Bacteria were collected from jars of sulfide-rich sediment and water sampled from salt-marsh pools at the Neponset River and Woods Hole, Mas­sachusetts, and at Morro Bay, Cali­fornia. Permanent magnets were placed on the sampling jars with the south magnetic pole positioned just above the water-sediment interface and bacteria that accumulated near the pole were drawn up with a Pasteur pipette and examined by light microscopy. The bacteria collected in this manner in­cluded the multicellular magnetotactic organism described previously [1, 2, 8, 9], as well as several morphological types of magnetotactic rod-shaped bac­teria. The bacteria were subsequently deposited unstained on amorphous car­bon films on nickel grids for electron microscopy. They were examined by scanning transmission electron micros­copy (STEM), high-resolution transmission electron microscopy (HRTEM), and energy-dispersive x­ray analysis (EDXA), and electron dif-Fig. 1. Transmission electron micrographs ot two types of magnetotactic rod-shaped bacteria collected from sulfidic environ­ments containing a) cubo-octahedral and b) rectangular prismatic iron sulfide inclu­sions. Bar 1 /lm (a), 500 nm (b) Fig. 2. Fe, S, and °elemental density maps for rectangular prismatic inclusions within a rod­shaped magnetotactic bacterium. The area analyzed is shown in the upper left-hand corner. Maps were produced by recording the respective x-ray intensities with the energy-dispersive x-ray detector at each position as the electron beam was slowly rastered across the area analyzed. Fe and S, but not 0, correlate with particle position. Similar results were obtained for the cubo-octahedral particles in the smaller rod. Bar 500 nm fraction. All crystallographic and ele­mental analyses were done on particles located within intact bacteria. Transmission electron micrographs of two of the predominant types of mag­netotactic rod are shown in Fig. 1. One type (Fig. Ib) was a large (ca. 3 x 2 (tm) organism with chains and clusters of predominantly rectangular electron­dense particles. Individual cells con­tained, on average, 57 crystals of mean dimensions 69 x 50 nm with a variable aspect ratio (1.0 to 2.0). The crystals exhibited well-defined end faces but the sides were often irregular. A second cell type (Fig. la) was a smaller (ca. 2.5 x 1.3 (tm) organism with a single chain of, on average, 26 well-defined cu­boidal electron-dense particles of mean dimension, 67 nm. Elemental mapping of the particles by EDXA (Fig. 2) showed that both types of particles con­sisted of iron and sulfur but not ox­ygen. Identification of the mineral phase in both cell types was made by indexing single-crystal electron diffraction pat­terns and lattice images of individual particles. This was necessary because the d-spacings of all the iron sulfides are similar, making identification from limited powder diffraction data alone unreliable. A single-crystal electron dif­fraction pattern from a rectangular particle in the larger rod-shaped bacte­rium is shown in Fig. 3a. The pattern corresponds to the < 100> zone of greigite, (Fe3S4). Superposition of the pattern and corresponding image (see inset) indicates that the crystals are elongated along one of the a axes and the well-defined end faces and less regular side faces are of [100J form. These observations were confirmed by crystallographic analysis of greigite particles aligned along different direc­tions e.g. [112], [215], and [110], and by lattice imaging (data not shown). Thus, the crystals have an idealized morphology based on a rectangular prism of six cubic [100J faces. Single-crystal electron diffraction pat­terns from particles in the smaller rod­shaped bacteria were also indexed to greigite (Fig. 3b). Diffraction patterns corresponding to the [112], [213], and [011] zones of greigite were obtained. Analysis of the diffraction patterns and associated images, as well as lattice images of the crystals, indicated that the particles were cubo-octahedral in morphology with well-defined [111J faces and smaller truncated [100J faces. These results in conjunction with pre­vious observations [1] suggest that the biomineralization of greigite may be a common occurrence in magnetotactic bacteria inhabiting sulfidic environ­ments. Interestingly, the rod-shaped bacteria appear to maintain greater control over mineralization compared with a previously described multicellu­lar organism. The latter produces crys­tals of both greigite and pyrite (FeS2) [1] and the morphologies are not well­defined [1,2], By contrast, the rod­shaped bacteria specifically mineralize greigite and regulate the crystal habit. While the deposition of cubo-octa­hedral crystals may reflect an equilib­rium form involving minimal biological intervention, the formation of elongated cubic crystals indicates that the biosynthetic mechanism involves disruption of the symmetry relation­ships in the isometric lattice of greigite. A similar effect has also been observed with bacterial magnetites [5]. One pos-Fig. 3. Single-crystal greigite (Fe3SJ electron diffraction patterns recorded from iron sulfide inclusions and associated images (in­sets). a) Prismatic rectangular crystal viewed down the [001] zone. Reflection A, (220) (3.50 A); reflection B, (400) (2.47 A); reflection C, (220) (3.50 A); angles: (220) 1\(400) = 45°; (220) 1\ (220) = 90°. b) Cubo­octahedral crystal viewed down the [213] zone. Reflection A, (222) (2.86 A); reflec­tion B, (422) (2.017 A); reflection C, (331) (2.28 A); angles: (222 1\422 = 61.5°; 222 1\331 = 82.3°). Scale bars (insets) 10 nm sibility is that the greigite crystals de-velop within vesicles that are extended along one direction, imparting a spatial constraint in crystal growth. But the as-sociation of a preferred crystallo-graphic axis « 100 » with the direc-tion of elongation suggests that the greigite crystals are oriented during nucleation, indicating that mineraliza-tion is also regulated at the molecular level. Assuming that the 67-nm particles in the smaller rod are single-magnetic-do-mains, it is possible to calculate the average permanent magnetic dipole per cell [10]. A greigite density of 4.1 g/cm 3 from the crystallographic data (space group, Fd3m, a = 9.88 A, 8 formula units per unit cell) and a measured sat-uration magnetization of 30 ernulg [11] yields a permanent magnetic dipole mo-ment per particle, m = 3.65 x 10-14 emu. For particles arranged in a chain, the total moment is the sum of the indi-vidual particle moments [10]. Thus, the total moment of a cell with a chain of 26 particles, M = 9.5 X 10-13 emu. In the geomagnetic field, Bo = 0.5 Gauss, the magnetic energy, M x Bo =:; 4 X 10-13 erg, which is more than ten times thermal energy at 300 K. Thus, the chain of greigite particles has a large enough permanent magnetic dipole mo-ment so that the migration speed of the cell along geomagnetic field lines would be more than 80 070 of its forward speed [10]. If some particles in the chain were nonmagnetic pyrite, as they are in the case of the multicellular organism [1], the migration efficiency would be correspondingly reduced. The average permanent magnetic dipole moment of the larger rod-shaped cells is not so easily calculated because the relative orientations of the individual particle moments in the clusters are not known. The fact that the larger rods contain more particles on average could reflect the magnetically less efficient arrange-ment of the elongated particles com-pared to the ordered chains in the smaller rod-shaped cells. The foregoing analysis suggests that the rod-shaped bacteria specifically miner-alize greigite, not pyrite, in connection with magnetotaxis. This specificity argues against the view that intracellu-lar greigite transforms to pyrite on a time scale comparable to the lifetime of the cell [l]. Thus, the multicellular bac-terium recently described [1], which contains greigite and pyrite, may be ca-pable of simultaneously and separately mineralizing the two iron sulfides. Since pyrite does not contribute to the magnetotactic response, its role may be related to the maintenance of homeo-stasis of iron andlor sulfide [3], or to other metabolic processes in the cells. We thank R.P. Blakemore for discus-sions. R.B.F. was supported by the US National Science Foundation and B.R.H. by BP International Ltd. I. Mann, S., Sparks, N.H.C., Frankel, R.B., Bazylinski, D.A., Jannasch, H.W.: Nature 343, 258(1990) 2. Farina, M., Motta de Esquivel, D., Lins de Barros, H.G.P.: ibid. 343, 256 (1990) 3. Williams, R.J .P.: ibid. 343, 213 (1990) 4. Wachterhauser, G.: Syst. Appl. Micro-bioI. 10, 207 (1988) 5. Mann, S., Frankel, R.B., in: Biomineral-ization: Chemical and Biochemical Per-spectives, p. 389 (eds. Mann, S., Webb, J., Williams, R.J.P.). Weinheim: VCR 1989 6. Blakemore, R.P.: Ann. Rev. Microbiol. 36,217 (1982) 7. Demitrack, A., in: Magnetite Biominer-alization and Magnetoreception in Organisms, p. 625 (eds. Kirchvink, J .L., Jones, D.S., Macfadden, B.F.). New York: Plenum 1985 8. Rodgers, F.G., Blakemore, R.P., Blake-more, N.A., Frankel, R.B., Bazylinski, D.A., Maratea, D., Rodgers, C.: Arch. Microbiol. 154, 18 (1990) 9. Farina, M., Lins de Barros, H.C.P., Es-quivel, D.M.S., Danon, J.: BioI. Cell 48, 85 (1983) 10. Frankel, R.B.: Ann. Rev. Biophys. Bioeng. 13, 85 (1984) II. Spender, R.M., Coey, J.M.D., Morrish, A.H.: Can. J. Phys. 50,2313 (1972) 

Controlled Biosynthesis of Greigite (Fe\u3csub\u3e3\u3c/sub\u3eS\u3csub\u3e4\u3c/sub\u3e) in Magnetotactic Bacteria

https://digitalcommons.calpoly.edu/cgi/viewcontent.cgi?article=1150&amp;context=phy_fac

Controlled Biosynthesis of Greigite (Fe\u3csub\u3e3\u3c/sub\u3eS\u3csub\u3e4\u3c/sub\u3e) in Magnetotactic Bacteria

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