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 Technol­ ogy, 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 cell types. The prismatic crystals are been identified which employ the unusual since they do not conform to the earth's geomagnetic field to direct their symmetry relations of the greigite motion towards suitable habitats. A cubic lattice. Our results indicate that common feature of these magnetotactic the biomineralization of greigite in bacteria is the presence of discrete in­ magnetotactic bacteria is highly reg­ tracellular magnetic inclusions, magne­ ulated and there are close similarities tosomes, aligned in chains along the to the biosynthesis of bacterial mag­ long axis of the organism. The size and netite (Fe304) [5, 6]. The formation of orientation of these individual mag­ novel morphologies of biogenic greigite netic particles impart a permanent mag­ suggests that such crystals may provide netic dipole moment which is re­ a diagnostic marker in the characteriza­ sponsible for the magnetotactic re­ tion of sources of remanent magnetiza­ sponse. Until recently only crystals of tion in recent and ancient sediments [7]. the mixed-valence iron oxide, mag­ Bacteria were collected from jars of netite (Fe30 4), were identified in these sulfide-rich sediment and water bacteria. Now, however, magnetic and sampled from salt-marsh pools at the non magnetic iron sulfide minerals Neponset River and Woods Hole, Mas­ have been identified in multicellular sachusetts, and at Morro Bay, Cali­ magnetotactic bacteria collected from fornia. Permanent magnets were placed sulfidic environments [1, 2]. These on the sampling jars with the south studies showed the presence of discrete magnetic pole positioned just above the intracellular crystals but were in con­ water-sediment interface and bacteria flict with regard to their mineralogical that accumulated near the pole were identification, viz. pyrrhotite (FeSI +J drawn up with a Pasteur pipette and [2] or greigite (Fe3S4) and pyrite (FeS2) examined by light microscopy. The [1]. This discovery is of profound im­ bacteria collected in this manner in­ portance to the study of biomineral­ cluded the multicellular magnetotactic ization, prokaryotic metabolism [3] organism described previously [1, 2, 8, and, possibly, the origin of life [4]. 9], as well as several morphological Here we report the presence of intra­ types of magnetotactic rod-shaped bac­ cellular greigite crystals in two different teria. The bacteria were subsequently types of rod-shaped single-celled mag­ deposited unstained on amorphous car­ netotactic bacteria collected from bon films on nickel grids for electron sulfide-rich sites. The greigite particles microscopy. They were examined by are often organized in chains, have nar­ scanning transmission electron micros­ Fig. 1. Transmission electron micrographs row size distributions (50 - 90 nm), and copy (STEM), high-resolution ot two types of magnetotactic rod-shaped bacteria collected from sulfidic environ­ adopt specific crystallographic habits transmission electron microscopy ments containing a) cubo-octahedral and (cubo-octahedral and rectangular (HRTEM), and energy-dispersive x­ b) rectangular prismatic iron sulfide inclu­ prismatic) associated with particular ray analysis (EDXA), and electron dif- sions. Bar 1 /lm (a), 500 nm (b) terns from particles in the smaller rod­ shaped 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 [111 J faces and smaller truncated [100J faces. These results in conjunction with pre­ vious observations [1] suggest that the biomineralization of greigite may be a common occurrence in magnetotactic bacteria inhabiting sulfidic environ­ ments. Interestingly, the rod-shaped bacteria appear to maintain greater control over mineralization compared with a previously described multicellu­ lar organism. The latter produces crys­ tals of both greigite and pyrite (FeS2) [1] and the morphologies are not well­ defined [1,2], By contrast, the rod­ shaped bacteria specifically mineralize greigite and regulate the crystal habit. While the deposition of cubo-octa­ Fig. 2. Fe, S, and °elemental density maps for rectangular prismatic inclusions within a rod­ hedral crystals may reflect an equilib­ shaped magnetotactic bacterium. The area analyzed is shown in the upper left-hand corner. rium form involving minimal biological Maps were produced by recording the respective x-ray intensities with the energy-dispersive intervention, the formation of x-ray detector at each position as the electron beam was slowly rastered across the area elongated cubic crystals indicates that analyzed. Fe and S, but not 0, correlate with particle position. Similar results were obtained the biosynthetic mechanism involves for the cubo-octahedral particles in the smaller rod. Bar 500 nm disruption of the symmetry relation­ ships in the isometric lattice of greigite. A similar effect has also been observed fraction. All crystallographic and ele­ single-crystal electron diffraction pat­ with bacterial magnetites [5]. One pos- mental analyses were done on particles terns and lattice images of individual located within intact bacteria. particles. This was necessary because Transmission electron micrographs of the d-spacings of all the iron sulfides two of the predominant types of mag­ are similar, making identification from netotactic rod are shown in Fig. 1. One limited powder diffraction data alone type (Fig. Ib) was a large (ca. 3 x 2 unreliable. A single-crystal electron dif­ (tm) organism with chains and clusters fraction pattern from a rectangular of predominantly rectangular electron­ particle in the larger rod-shaped bacte­ dense particles. Individual cells con­ rium is shown in Fig. 3a. The pattern tained, on average, 57 crystals of mean corresponds to the < 100> zone of dimensions 69 x 50 nm with a variable greigite, (Fe3S4). Superposition of the aspect ratio (1.0 to 2.0). The crystals pattern and corresponding image (see exhibited well-defined end faces but the inset) indicates that the crystals are sides were often irregular. A second cell elongated along one of the a axes and Fig. 3. Single-crystal greigite (Fe3SJ electron type (Fig. la) was a smaller (ca. 2.5 x the well-defined end faces and less diffraction patterns recorded from iron 1.3 (tm) organism with a single chain regular side faces are of [100J form. sulfide inclusions and associated images (in­ of, on average, 26 well-defined cu­ These observations were confirmed by sets). a) Prismatic rectangular crystal boidal electron-dense particles of mean crystallographic analysis of greigite viewed down the [001] zone. Reflection A, dimension, 67 nm. Elemental mapping particles aligned along different direc­ (220) (3.50 A); reflection B, (400) (2.47 A); of the particles by EDXA (Fig. 2) tions e.g. [112], [215], and [110], and reflection C, (220) (3.50 A); angles: (220) showed that both types of particles con­ by lattice imaging (data not shown). 1\(400) = 45°; (220) 1\ (220) = 90°. b) Cubo­ octahedral crystal viewed down the [213] sisted of iron and sulfur but not ox­ Thus, the crystals have an idealized zone. Reflection A, (222) (2.86 A); reflec­ ygen. morphology based on a rectangular tion B, (422) (2.017 A); reflection C, (331) Identification of the mineral phase in prism of six cubic [100J faces. (2.28 A); angles: (222 1\422 = 61.5°; 222 both cell types was made by indexing Single-crystal electron diffraction pat­ 1\331 = 82.3°). Scale bars (insets) 10 nm sibility is that the greigite crystals de­ with magnetotaxis. This specificity velop within vesicles that are extended argues against the view that intracellu­ along one direction, imparting a spatial lar greigite transforms to pyrite on a constraint in crystal growth. But the as­ time scale comparable to the lifetime of sociation of a preferred crystallo­ the cell [l]. Thus, the multicellular bac­ graphic axis « 100 » with the direc­ terium recently described [1], which tion of elongation suggests that the contains greigite and pyrite, may be ca­ greigite crystals are oriented during pable of simultaneously and separately nucleation, indicating that mineraliza­ mineralizing the two iron sulfides. tion is also regulated at the molecular Since pyrite does not contribute to the level. magnetotactic response, its role may be Assuming that the 67-nm particles in related to the maintenance of homeo­ the smaller rod are single-magnetic-do­ stasis of iron andlor sulfide [3], or to mains, it is possible to calculate the other metabolic processes in the cells. average permanent magnetic dipole per cell [10]. A greigite density of 4.1 g/cm3 from the crystallographic data (space We thank R.P. Blakemore for discus­ group, Fd3m, a = 9.88 A, 8 formula sions. R.B.F. was supported by the US units per unit cell) and a measured sat­ National Science Foundation and uration magnetization of 30 ernulg [11] B.R.H. by BP International Ltd. yields a permanent magnetic dipole mo­ ment per particle, m = 3.65 x 10-14 emu. For particles arranged in a chain, I. Mann, S., Sparks, N.H.C., Frankel, the total moment is the sum of the indi­ R.B., Bazylinski, D.A., Jannasch, vidual particle moments [10]. Thus, the H.W.: Nature 343, 258(1990) total moment of a cell with a chain of 2. Farina, M., Motta de Esquivel, D., Lins 26 particles, M = 9.5 X 10- 13 emu. In de Barros, H.G.P.: ibid. 343, 256 (1990) 3. Williams, R.J .P.: ibid. 343, 213 (1990) the geomagnetic field, B = 0.5 Gauss, o 4. Wachterhauser, G.: Syst. Appl. Micro­ =:; X the magnetic energy, M x Bo 4 bioI. 10, 207 (1988) 13 10- erg, which is more than ten times 5. Mann, S., Frankel, R.B., in: Biomineral­ thermal energy at 300 K. Thus, the ization: Chemical and Biochemical Per­ chain of greigite particles has a large spectives, p. 389 (eds. Mann, S., Webb, enough permanent magnetic dipole mo­ J., Williams, R.J.P.). Weinheim: VCR ment so that the migration speed of the 1989 cell along geomagnetic field lines would 6. Blakemore, R.P.: Ann. Rev. Microbiol. be more than 80 070 of its forward speed 36,217 (1982) [10]. If some particles in the chain were 7. Demitrack, A., in: Magnetite Biominer­ alization and Magnetoreception in nonmagnetic pyrite, as they are in the Organisms, p. 625 (eds. Kirchvink, J.L., case of the multicellular organism [1], Jones, D.S., Macfadden, B.F.). New the migration efficiency would be York: Plenum 1985 correspondingly reduced. The average 8. Rodgers, F.G., Blakemore, R.P., Blake­ permanent magnetic dipole moment of more, N.A., Frankel, R.B., Bazylinski, the larger rod-shaped cells is not so D.A., Maratea, D., Rodgers, C.: Arch. easily calculated because the relative Microbiol. 154, 18 (1990) orientations of the individual particle 9. Farina, M., Lins de Barros, H.C.P., Es­ quivel, D.M.S., Danon, J.: BioI. Cell moments in the clusters are not known. 48, 85 (1983) The fact that the larger rods contain 10. Frankel, R.B.: Ann. Rev. Biophys. more particles on average could reflect Bioeng. 13, 85 (1984) the magnetically less efficient arrange­ II. Spender, R.M., Coey, J.M.D., Morrish, ment of the elongated particles com­ A.H.: Can. J. Phys. 50,2313 (1972) 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