crystal growth of bacterial magnetite Structure, morphology and

Reprintedfrom Nature,Vol. 3 10. No. 5976. pp. 405-401. 2 AugustI 984 @ Macmillan JournalsLtd., 1984

Structure, morphology and crystal growth of bacterial magnetite Stephen Mann*, Richard B. Frankelt & Richard P. Blakemore* * Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OXI 3QR, UK t Francis Bitter National Magnet Laboratory, MassachusettsInstitute of Technology, Cambridge, Massachusetts02139, USA f Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824, USA

Recent high-resolution transmission electron microscopy (HRTEM) studies of the structure and morphology of bacterial magnetite (FerOa) crystals isolated from a magnetotactic coccust and fro- an unidentified bacterium extracted from sediment2have shown the crystals to be well ordered single-domain particles with a morphology basedon a hexagonalprism of {011i facestruncated by specific low index planes. We report here a HRTEM study of intact magnetite crystals (magnetosomes)in the microaerophilic bacterium Aquaspirillum magnetotacticuttr, grown in pure culture3'4. Our aim has been to investigate the structure, morphology and crystal growth of the magnetite particles in the lig_ht of a recent Mossbauer spectroscopy study of this organism' which indicated, in addition to magnetite, the presenceof hydrated iron(ur) oxide phasestogetherwith the magnetosomes.Our results show that the mature particles are well ordered single-domain crystals of magnetite with a morphology very diflerent from previously studied crystals and basedon an octahedral prism of il11) faces truncated by {100} faces. We also show the first direct evidencefor both crystalline and non-crystalline phaseswithin individual magnetosomes,The results are important in aiding elucidation of the crystal growth mechanismsof biogenic magnetite.

Fig. I Chain of magnetite particles imaged within an intact unstained A. magnetotacticumcell, showing a combination of large and small particles. Particle A has a characteristic morphology seen in many mature crystals (see Fig. 3). Particle B is irregular in form and appears to be at a different stagein development from pa(icle A. Lattice images of this particle showed crystalline and n o n - c r y s t a l l i n ez o n e s( F i g . 4 ) . T h e c l u s t e ro f f o u r p a ( i c l e s n e a r t o B w a s a l s o i m a g e d a s s i n g l e c r y s t a l sw i t h l o c a l i z e d a m o r p h o u s r e g i o n s . T h e y a r e s p a c i a l l y s e p a r a t ea n d t h e r e f o r e n o t a m u l t i d o m a i n a g g r e g a t eS . c a l eb a r , 1 0 0n m .

Fig, 2 Selected-area electrondiffractionpatternfrom a mature magnetitecrystalof characterilticmorphology.The singlecrystal patterncorresponds to the [0] l] zone.The (200)and (200)reflections arisefrom doubledifiraction.Cameralength, I l5 cm. Cells of A. magnetotacticumarc -3 pm long and contain, on average, 20 intracellular enveloped magnetite particles of diameter 40-50 nm which are organized in a single chain that traversesthe cell longitudinally". Cells of A. magnetotacticum synthesizemagnetite only in microaerobic conditions, accumulating Fe some 20,000-40,000-fold over the extracellular concentrationr. The magnetite particles are in the single magnetic domain size range and the chain of magnetosomesimparts a permanent magnetic dipole moment parallel to the cell's axis of mobility such that the bacteria orient in the geomagneticfield7. Intact, unfixed cells were dried down onto carbon-coated electron microscope grids and investigated in a JEOL 200CX high-resolution transmission electron microscope operating at 200 keV with a LaBu electron source and a point-to-point resolution of 2.46 A. Many cells showed chains of preferentially aligned magnetiteparticles of distinct morphology. Other chains were observedto contain a combination of large, well developed magnetosomesand smaller particles of irregular form, suggesting the presencewithin the chains of crystals at different stages of development(Fig. l). Crystals of distinct morphology (such as crystal A in Fig. l) showed lattice images consistent with the cubic (Fd3m) inverse spinel structure of magnetite. The lattice images were well defined and run continuously throughout the particles, thus indicating that they are single-crystal domains. Lattice fringes corresponding to the {1 1l}, {222}, {220}, {2OO}and {400} planes were imaged and by considering the relative orientation of these fringes, the zone of projection was identified as [0lI]. This was confirmed by selected-areaelectron diftraction patterns (Fig. 2). The morphology of these mature crystals was inferred from the relative directions of the fringes and crystal edges and is based on an octahedron of {1 l 1} faces truncate_dby {100} faces (Fig. 3a, b). Thus, crystals imaged in the [011] zone often showed edgesmeeting at characteristicanglesof 125'and 110o,corres p o n d i n g t o t h e i n t e r s e c t i o no f ( 1 0 0 ) a n d ( l 1 l ) a n d o f ( T l l ) and ( I I I ) faces. In other mature crystals, these angles were distorted from their theoretical value; this may be a consequence of slight crystal misalignment with respectto the electron beam or it may be a real growth eflect. On the basis of the above morphology, we determined the crystal alignment in bacteria containing chains ofwell developed crystals. The crystals were found to be preferentially aligned, with the Ill] direction parallel to the chain axis. This result is important with regard to magnetotaxis, as the easy axis of magnetizationin Fe3Oaalso lies along the [1] l] direction. Our results indicate that the cellular environment in which

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*+i#*u# Fig.4 HRTEM image of particle B of Fig. I, showing the coe x i s t e n c eo f c r y s t a l l i n ea n d n o n - c r y s t a l l i n ep h a s e s .T h e c r y s t a l l i n e z o n es h o w sw e l l o r d e r e d( 2 2 2 ) l a t t i c ef r i n g e sa n d i s a s i n g l ed o m a i n . The fringes extend into the amorphous phase in a preferential d i r e c t i o n .T h e s u p e r i m p o s e db l a c k d a s h e dl i n e i n d i c a l e st h e e x t e n t of the low contrast edge of the particle against the background c a r b o n n o i s e o f t h e g r i d . S c a l eb a r , 5 n m .

crystalimagedin the Fig.3 a, HRETEM imageof a magnetite crystaland a single-domain well ordered a zone, showing [Oit] prismof { I I I } basedon an octahedral morphology churacteristi. facestruncatedby { 100}faces.Thelatticefringesshowncorrespond to the (100)face'Note to the (022)planesand run perpendicular on the thatthe crysialedgesarenot smoothandshowoutgrowths well developed {l I l} faces(arrow).Scalebar, l0 nm' b, Idealized morphologyfor biogenicmagnetitecrystalsfrom A. magnetotacticrn. An octahedralprismof {lll} iacesis truncatedby {100} ofthe {lll} the stabilization faces.Sucha crystalform indicates planesoverotherlow indexfacessuchas {100} the crystals grow is often well defined and influencestheir morphology in a way which is different from that found for other magnetotactic bacteria''t. Many factors can influence the crystalmorphotogy of biogenicsolidsn,so it may not be surprising that the morphology ol the bacterialmagnetitesseemsto be speciesspecific.It is unclear why some mature crystalsin A. magnelotacticutrdo not attainthe characteristicoctahedralmorphology; perhapsthe crystalgrowth processesare very susceptible to chemical fluctuations in the intra- and extracellular environments. Latticeimagingof the irregularparticles(for example,particle B in Fig. I ) showed the presenceof contiguouscrystallineand non-crystallineregions within the magnetosome(Fig' 4)' The crystaltinezone was always single domain, with well ordered lattice planesof magnetite.No other crystallinephases,such as to 7-FeObH, were observed.The latticefringesoften appeared extend into the amorphous region in a preferential direction'

Thesemulti-phaseparticlesprobably representthe early stages of magnetite formation, with the non-crystalline material corresponding to the hydrated iron (rrt) oxide phasesidentified by Mossbauerspectroscopy3. On the basis of these results,a mechanism of crystal formation can be oflered. The crystallization of bacterial magnetite occurs within a localizedregionofthe cell and involvesa non-crystalline precursorof hydratediron(Itt) oxide. The lattice imagessuggest that a growing crystalfront ol magnetiteextendsinto the amorphous gel. The solid-state rearrangement could occur through a solution front at the interface of the two phases. The final crystalis singledomain and often has a characteristicoctahedral morphology, indicating that crystal growth is slow and that the { I 11} planes are stable. Becausemany crystals are preferentially aligned with the [111] direction parallel to the chain axis, both seem to be under biological nucleation and growttrrProcesses control. The origin of aqueous Fe2* ions in the crystal growth mechanism is unclear, although Fe'* ions have been detected in A. magnetotacticums.They could arise from partial reduction of the hydrated iron(tlt) oxide phaseat the solid-stateinterface due to the changes in the local redox potential or they could be transported directly from the cytoplasm or periplasmic space into the crystallizingzone. The adsorption of Fe'* ions at the hydrated iron(ttt) oxide surface is probably the trigger for magnetiteformation'. We thank N. Blakemorefor help in preparationof the samples. R.B.F. was in part supportedby the Office of Naval Research. The Francis Bitter Laboratory is supported by the NSF.

Received 7 March; accepted I June 1984. Mann. S.. Moench. T. T. & Williams, R. J. P. Proc. R. )^o.. B 221.31J5-l9l (1984). N4atsuda.T.. Ilndo, J.. Osakabe, N. & Tonomura, A. Nature 302,4l l-412 (1981) Blakemore. R. P., Maratea. D. & wolfe, R S i Bdcl. 140,720 729 (1919) Maratea. D. & Blakemore, R. P. /n/. I S,lrl. Bd.t. 31,452 455 (1981) \N Bio.hid bioqhF Frankel, R. B.. Papaefthymoeu, G. C., Blakemore, R. t'. & O'Brien, A . r o 7 6 3 . 1 4 7 - 1 5 9( 1 9 8 1 ) . 6 . B a l k w i l l , D . L . , M a r a t e a . D & B l a k e m o t c ,R P I B o ( t . l 4 l , 1 3 9 9 - 1 4 0 8( 1 9 8 0 ) . ?. Frankel, R. B. & Blakemore, R. P. J. Magn magn Muter. 15-18, 1562 1564 ( 1980). 8. Mann, S. Strutr. Bontling 54, 127-174 ( 1983).

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