American Mineralogist, Volume 83, pages 1469-1481, 1998

Iron sulfidesfrom magnetotacticbacteria: Structure, composition, and phasetransitions

Mrnfu,v PosrlIr''x Pprnn R. Busrcrrl DENNTsA. BlzyLrNSKrr2aNo RrcnlRD B. FnaNrrl3

lDepaftmentsof Geology and Chemistry/Biochemistry,Arizona State University, Tempe,Arizona 85287, U.S.A. '?Departmentof Microbiology, Immunology and PreventiveMedicine, Iowa State University, Ames, Iowa 50011, U.S.A. rDepartmentof Physics,Californa Polytechnic State University, San Luis Obispo, California 93407, U.S A.

AesrRAcr Using transmissionelectron microscopy, we studied the structuresand compositionsof Fe sulfides within cells of magnetotacticbacteria that were collected from natural habitats. Ferrimagnetic greigite (Fe.S.) occurred in all types of sulfide-producing magnetotactic bacteriaexamined. Mackinawite (tetragonalFeS) and, tentatively, sphalerite-typecubic FeS were also identified. In contrastto earlier reports,we did not find pyrite (FeSr)or pyrrhotite (Fe, ,S). Mackinawite converted to greigite over time within the bacteria that were de- posited on electron microscope grids and stored in air. Orientation relationshipsbetween the two minerals indicate that the cubic-close-packedS substructureremains unchanged during the transformation; only the Fe atoms rearrange. Neither mackinawite nor cubic FeS are magnetic, and yet they are aligned in chains such that when convertedto magnetic greigite, the probable easy axis of magnetization, [100], is parallel to the chain direction. The resulting chains of greigite are ultimately responsiblefor the magnetic dipole moment of the cell. Both greigite and mackinawite magnetosomescan contain Cu, depending on the sampling locality. Becausebacterial mackinawite and cubic FeS are unstableover time, only greigite crystals are potentially useful as geological biomarkers.

INrnooucrroN are no subsequentreports of pyrrhotite, and the presence Magnetotactic bacteria synthesizemembrane-bounded, and role of pyrite in magnetotacticbacteria remains un- intracellular, ferrimagnetic crystals called magnetosomes clear (Bazylinski and Moskowitz 1997). Although infor- that cause the bacteria to orient along geomagneticfield mation exists about magnetite formation (Frankel et al. lines (Blakemore 1975). Depending on the species, the 1983;Gorby et al. 1988),nothing has been reported about bacteria contain either magnetite, Fe.O4 (Frankel et al. the mode of formation of greigite in bacteria. In a brief 1979), greigite, Fe.So(Mann et al. 1990; Heywood et al. study, we reportedthat greigite forms by solid-statetrans- 1990), or both (Bazylinski et al. 1993a).The bacteria ap- formation from mackinawite (tetragonal FeS) (P6sfai et pear to exercise a high degree of control over the sizes, al. 1998); here we discussour findings in more detail. morphologies, and crystallographic orientations of these The discovery of nanometer-scalemagnetite and Fe minerals. Individual magnetosomesare usually arranged sulfides in the Martian meteorite ALH8400I and their in chains, with the easy magnetizationaxis of the crystals suggestedbiogenic origin (McKay et al. 1996) raises the aligned parallel to the chain, providing for the largestpos- question of whether inorganically formed minerals can be sible magnetic dipole moment of the cell (Frankel and distinguishedfrom those producedby magnetotacticbac- Blakemore 1989).A review of magnetotacticbacteria and teria. Our goals were to identify the Fe sulfide magneto- the magnetic properties of biogenic minerals is given by somes and study their defects and compositions to un- Bazylinski and Moskowitz (1997). derstandtheir mechanismof formation and to determine There is some uncertainty as to which Fe sulfidesoccur whether the crystals are any different from nonbiogeni- inside the magnetotacticbacteria. Intracellular Fe sulfides cally formed Fe sulfides. In this study, we present new are known to be produced by a many-celled, magneto- findings concerning intracellular, bacterially produced Fe tactic prokaryote (MMP) and severalmorphological types sulfides.We discussmicrosffuctural featuresof magnetite of single-celled, rod-shaped bacteria (Bazylinski et al. magnetosomesin a companion paper (Devouard et al. 1994). Only greigite was observedin the rod-shapedspe- 1998). cies (Heywood et al. 1991), whereasin MMP Mann et al. (1990) found greigite and pyrite (FeS,), and Farina et ExpnmrrnNTAL METHoDS al. (1990) identified monoclinic pyrrhotite (Fe,S,). There Magnetotactic bacteria that produce Fe sulfides only have been described from anaerobic,brackish-to-marine * Present address: Department of Earth and Environmental Sciences,University of Veszpr6m,Veszpr6m, Hungary. E-mail: aquatic habitats containing HrS and have not yet been [email protected] isolated and cultivated in axenic cultures. We therefore 0003-004x/98/1I 12-r 469S05.00 1469 1470 POSFAI ET AL,: IRON SULFIDES IN BACTERIA

collected cells from natural environments that include a The Fe sulfide crystals are enclosed within the cells coastal saltwater pond (Salt Pond, Woods Hole, MA, and could not be extracted by the same methods as the U.S.A.), a seriesof shallow (<0.5 m deep) salrmarsh magnetiteparticles (Devouard et al. 1998). Therefore,the pools in the Parker River Wildlife Refuge (Rowley, MA, electron beam must travel through amorphous organic U,S.A.), and the Sweet Springs Nature Preserve(Morro material that produces a strong background intensity in Bay, CA, U.S.A.). the SAED patterns against which the Bragg reflections Salt Pond is a well-characteized, small anaerobicbasin were commonly difficult to see. The cell contents were that stratifies chemically during the summer months. It is also frequently a causeof contamination under the elec- -5.5 m deep and has marine and freshwaterinput (Wak- tron beam, which hinders the use of convergent-beam eham et al. 1984,1987). The anaerobichypolimnion has electron diffraction (CBED) and makes high-resolution relatively high concenffationsof HrS (up to 5 mM), gen- imaging arduous. The cell may contain some S or Fe eratedby anaerobicsulfate-reducing bacteria. From April outside of the magnetosomes,introducing an unavoidable or May to about September,the anaerobic zone rises to error into the EDS analyses of magnetosomeparticles. within 3 m of the surface, with steep O, and HrS gradi- Despite theseproblems, TEM can still provide useful in- ents. The largest numbers of Fe-sulfide-producingmag- formation about the Fe sulfide masnetosomes. netotacticbacteria occur in the anaerobiczone just below the oxic-anoxic interface where HrS becomesdetectable. Rnsurrs Water samples from Salt Pond were collected from dis- Types of sulfide-producingorganisms and (Wakeham crete depthsby pumping et al. 1987). Samples magnetosomemorphologies were storedat room temperaturein glassor polycarbonate bottles that were completely filled to exclude air bubbles. Both the rod-shaped magnetotactic bacteria and the At the time our samples were collected (summer of MMP occurred in the samples from all collecting sites. 1997), the pH was -1.4 and the salinity -29 ppt at the The MMP consists of 15 to 20 cells that are attachedto depths from which the sampleswere drawn. The concen- one another forming a spherical a9gregateof cells (Fig. trations of HrS ranged from 0 to about 900 p.M, as de- 1a). This organism appearsto be a geographically wide- termined by the method of Cline (1969). Concentrations spread magnetotactic species that is motile as a single of parliculate Fe,* (likely including some magnetosomes) unit. Individual cells are only flagellated on their outer and soluble Fe2*were about 6 p,M and 5 pM, respective- sides, providing for the means of motility (Farina et al. ly, determined as described by Lovley and Phillips 1983;Rodgers et al. 1990).The averagenumber of mag- (1987). netosomechains within individual cells varies depending Samples were collected from the salt-marshpools by on where and when the sampleswere collected.However, simply filling jars with mud and overlying water, exclud- the chains are fairly uniformly distributed among the cells ing air bubbles as describedabove. The pH of these sam- of a given MMP Most magnetosomechains seemto have ples ranged from 7 .2 to 7.8, and the salinity varied from a common (E-W) orientation in the exceptionally well- 13 to 32 ppt. H,S was detected in all samples but its preserved MMP in Figure la. In an intact organism the concentrationwas not determined. chains are probably aligned parallel to one another.How- Magnetotactic bacteria were enriched within hours of ever, the cells commonly disaggregateon the TEM grid collection by placing a bar magnet with the south-end (Fig. 2a), and in this case the original chain orientations next to the side of the jar. Drops from Pasteurpipettes are not preserved.Most of the single-celled, rod-shaped were depositedonto carbon- and Formvar-coatedNi grids bacteria contain double or sometimesmultiple chains of for ffansmission electron microscopy (TEM). The speci- Fe sulfides(Fig. 3). mens were kept in a grid box in air before and between The Fe sulfides range from -30 to -120 nm in di- TEM studies that were performed between a few days amete! with most crystals in the 60 to 90 nm range. This and three months after sample collection. observation is consistent with earlier results (Bazylinski We used a JEOL 2000FX elecffon microscopeoperated et al. 1990) and means that most grains are in the mag- at a 2O0 kV accelerating voltage and equipped with a netic single-domainsize range (Bazylinski and Moskow- double-tilt (*30"; +45') goniometer stage.Particle mor- itz 1997). phologies and structural defects were observed in TEM In conffast to the well-defined crystal habits of mag- images.An ultra-thin-window KEVEX detectorwas used netite magnetosomes(Mann et al. 1984; Devouard et al. to study compositions by energy-dispersiveX-ray spec- 1998; see also Figure 3a), most sulfide crystals are sub- trometry (EDS). Pyrite and Cu sulfide standards were hedral. They are equidimensional or slightly elongated used for obtaining experimental k-factors for thin-film (Fig. 1b), or resemble "barrels" or "egg-timers" in two- analysis. For high-resolution imaging we used a JEOL dimensional projection. Elongated, arrowhead-shaped 4000EX TEM operated at 400-kV accelerating voltage greigite crystals occur in some MMPs (Fig. 2b) and rod- and a 200-kV, Akashi 0028 microscope.Fe sulfide sffuc- shapedbacteria (arrows in Fig. 3b). This morphology has tures were identified from single-crystal, selected-area not been previously reported for greigite crystals. Grains electron diffraction (SAED) patternsthat were taken from with similar morphologies from a variety of magnetotac- particles tilted into zone-axis orientations. tic microorganisms were all identified as magnetite in POSFAI ET AL.: IRON SULFIDES IN BACTERIA 14',7|

a

\

b

wffi

Frcunr 1. (a) A well-preserved,many-celled, magnetotacticprokaryote (MMP) from Salt Pond that consistsof about 20 indi- vidual cells. Fe sulfide magnetosomechains are marked by arrows. (b) Fe sulfide magnetosomesfrom an MMP from Salt Pond. The arrows point to contrast featuresthat indicate structuraldefects. 1112 ET AL.: IRON SULFIDES IN BACTERIA lu **' \

1pm

c

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FIGURE2. (a) A disaggregatedMMP next to a diatom skeleton(D) from Salt Pond. The arrows mark some of the magnetosome chains;S indicatessalt (NaCl) crystalsresulting from seawater. (b) A higher magnificationimage of the boxed areain a, showing grcigite crystals with rectangular(X) and arrowhead-shaped(Y) morphologies.(c) SAED pattern and (d) HRTEM image of crystal X in b (all in the correspondingorientations), indicating a well-ordered greigite structure. POSFAI ET AL.: IRON SULFIDES IN BACTERIA 1473

There is some ambiguity in distinguishing between mackinawite and the presumedcubic FeS magnetosomes on the basis of SAED patterns.Cubic FeS has the sphal- erite structure (De M6dicis 1970; Takeno et al. 1970); it has not been found in nature, likely becauseit converts to mackinawite (Murowchick and Barnes 1986; Murow- chick 1989; Lennie and Vaughan 1996). The geomeffies of some of our SAED pattems could be interpreted as different projections of either mackinawite or cubic FeS, and the observed d-spacingsare intermediateto those of the two structures(P6sfai et al. 1998). However, previous results support the interpretationthat some magnetosome crystals are cubic FeS; we believe that the pyrite that was identified by Mann et al. (1990) and Bazylinski et al. (1990) is more likely cubic FeS (P6sfaiet al. 1998). We found that mackinawite within bacterial cells con- verts to greigite over time (Fig. 4; see also P6sfai et al. 1998). The SAED pattern in Figure 4a was obtainedfrom a mackinawite crystal in a single-celled bacterium less than one week after the specimenwas collected.The pat- tern in Figure 4b was obtained ten days later from the samecrystal. The appearanceof new reflectionsalong the arrowed reciprocal lattice rows in Figure 4b and the changed d-spacingsof the remaining ones show that the original mackinawite transformedinto greigite. No traces 1ttm of the mackinawite reflections are observable in Figure J 4b, indicating an essentially complete conversion. Hori- Frcunn 3. Typical rod-shaped,sulfide-producing bacteria. uchi (1971) observed mackinawite that was coated with (a) A largebacterium from SweetSprings with a doublechain amorphous S converting to greigite as a result of pro- of Fe sulfidemagnetosomes (S) attachedto a smallercell that longed exposureto the electron beam, and Lennie et al. containsonly magnetitecrystals (M). The magnetitemagneto- (1997) transformedmackinawite to greigite by heating it somesproduce more uniform diffractioncontrast than the Fe in the TEM. Contrary to the results of those studies,we (b) sulfides. A multiplechain of sulfidemagnetosomes from a did not observe changesin this crystal (either as macki- rod-shapedbacterium from Salt Pond with many arrowhead- nawite or as greigite) while it was exposedto the electron shapedand elongated crystals (arrowed). The right partis a dig- occurred during those itally processedversion of theTEM image,showing the outlines beam; instead the transformation of thesulfide crystals. ten days while the sample was stored between the two studies and shows that mackinawite is a precursor to greigite in these bacteria. previousstudies (Bazylinski et al. L993a;1995).In gen- Greigite crystals typically show non-uniform, blotchy eral, the identities of the sulfides seem unrelated to mor- diffraction contrast in the electron microscope (Figs. lb phologies, and no relationship is obvious between cell and 5a), a characteristicappearance that can result from types (MMP or rod-shaped)and magnetosomeshapes. thicknessvariations, lattice strain associatedwith defects, or the combined effects of both. Greigite crystals com- Structures and phase transformations monly contain defects along their (222)-type planes, as We identified the structuresof about 100 magnetosome also observedby Heywood et al. (1991).Some resemble crystals from cells of the MMP and rod-shapedbacteria. stacking faults that change the stacking of the close- Our SAED results are summarizedand compared to cal- packed S+Fe layers (Fig. 5c), whereasothers seem to be culated data and published observationsin a brief report associatedwith disorder in the Fe distribution, without (P6sfai et al. 1998; Table l). We found that most mag- affecting the S arrangement. We therefore believe that all netosome crystals are greigite in both types of bacteria greigite crystals likely form from a precursorphase (such (see the SAED patterns in Figs. 2c, 4b, and 5b) and that as mackinawite) by solid-state transformation, and that mackinawite (Fig. 4a) can also occur in both types of the planar defects are rerru:Iantsof the parent sffucture. organisms. A third phase, cubic FeS with the sphalerite Some magnetosomecrystals seem to be in the process structure, was tentatively identified (P6sfai et al. 1998). of transformingfrom mackinawite to greigite. Such greig- Neither mackinawite nor cubic FeS had been reported ite crystals contain bandsparallelto (222) that are several previously from magnetotacticbacteria. None of the mag- atomic layers thick and have spacingsthat are consistent netosomesfrom either organism appearedto be pyrite or with the distancebetween close-packedlayers in macki- pyrrhotite. nawite (P6sfai et al. 1998). The two structures are ori- r474 POSFAI ET AL.: IRON SULFIDES IN BACTERIA

Taele 1. EDS analyses(at%) of individualsulfide magnetosome crystals

Sample Fe (Fe+Cu)/S Structure Ref. to figs. SP MMP 55 1 41.5 3.4 0.82 gr SP MMP 555 42.2 2.3 080 gr Jts MMP 55.0 +Jl 19 082 gr Jts MMP 54.8 432 20 082 gr SP MMP 57.4 42.6 bl 074 gr SP MMP 52.1 457 2.1 0.92 nl SP MMP CJ.Y 44.1 2.1 0.86 nl SP MMP 55.4 41 I 2.7 0.81 nl SP MMP 539 4.0 0.86 nl SP MMP 539 423 3.8 086 gr Jts MMP 52.4 424 48 089 nl SP MMP 54_0 43.1 29 0.85 nl SP MMP 53.9 40.6 5.5 086 gr SP MMP 51.5 44.1 4.4 094 nl SP 539 41.5 4b 0.86 gr l,lMP 500 41.9 8.1 1.00 SP l/MP 52.9 439 J.1 089 mkt BinFig7 51I 436 +o 0.93 SP YMP 52.O 42.3 5.7 093 MK AinFig 7 CU. I JY.O 97 097 SP MMP 54.6 40.0 54 083 gr SP MMP 45.6 44.3 101 1.20 grr CinFig 7 SP MMP 51.4 409 7.7 0.95 mk/grf D in Fig. 7 SP MMP 51 6 43.0 5.4 094 grlmkt SP MMP 51 7 44.1 4.2 093 gr Fig. 5 SP MMP cz,J 407 b4 089 gr SP MMP 53.8 41.4 4.8 086 gr SP MMP 54.0 31 085 gr SP MMP 50.6 39.2 102 0.98 gr PR MMP 600 40.0 bl 0.67 gr PR MMP 540 460 bl 0.8s gr PR MMP 55s 455 bl oa2 gr PR SC cbJ 437 bl 0.78 gr PR JU 555 44.5 bl 0.80 gr PR SC 51.5 48.5 bl 094 MK Fig.4a PR sc cJ. / +o.J bl 0.86 gr Fig 4b JD an 52.7 38.2 9-1 0.90 gr SS SC 57.4 394 3.2 0.74 gr SS SC co.4 412 2.4 0.77 gr SS SC 61.0 367 23 0.64 gr SS JU 552 44.8 bl 0.81 gr SS SC 53.9 43.9 086 gr SS SC 52.5 43.7 38 090 gr SS SC CJ.J 41.0 55 0.87 gr

NotesiSP : Salt Pond,MA; PR : ParkerRiver Wildlife Refuge, MA; SS : SweetSprings Nature Preserve, Morro Bay, CA; MMP : many-celled, magnetotacticprokaryote; SC : single-celledbacterium . Duplicateanalysis of the same (preceding)magnetosome; bl : belowdetection limit; gr : greigite;mk : mackinawite;ni : not identified t Stronglydisordered

ented with respectto one anotherwith (011).//(222)" and [110]/[00]9, such that the cubic close-packedS sub- b structure is continuous across the interfaces. The same \ t orientation relationship was observed by Lennie et al. \/ I (1997) for mackinawite thermally converted to greigite. Eight randomly chosen magnetosomesfrom a rod- shaped bacterium were likely cubic FeS (Fig. 6a), but 'i1o 'm4 \. may have been mackinawite (the problem of distinguish- , ing mackinawite from cubic FeS on the basis of SAED pattems is discussedabove). Regardlessof whether they are cubic FeS or mackinawite, thesecrystals are probable '11i \ .4& precursorsto greigite. The HRTEM image (Fig. 6c) of a crystal that seems typical of this magnetosome chain Frcunn 4. SAED patternsshowing the transformationof (a) (marked mackinawite into (b) greigite in a rod-shapedbacterium from shows a strongly disordered area X) and abun- Parker River. (b) Greigite pattern was obtained ten days later dant defects (marked by arrows) along both sets of cubic than mackinawite. The arrows in b mark reciprocal lattice rows close-packedplanes that are seen edge-on in this orien- containingreflections that are absent in a. tation. Although the quality of the image does not permit POSFAI ET AL.: IRON SULFIDES IN BACTERIA r4'75

b

Frcunn 5. (a) HRTEM image and (b) conesponding SAED pattern of a greigite magnetosomefrom an MMP from Salt Pond. The arrows in a mark planar defects atong (222) and (272) planes. Enlargements(c and d) of the respectiveboxed regions in a, showing two planar faults (arrowed) and an ordered area, respectively. t476 POSFAI ET AL.: IRON SULFIDES IN BACTERIA a c

Frcunn 6. (a) Low-magnification image of a double magnetosomechain from a rod-shapedbacterium from Salt Pond; the arrows mark the randomly chosen crystals from which SAED patterns were obtained (b) SAED pattern and (c) HRTEM image of the crystal marked A in a; the reflections are indexed according to a sphalerite-typecubic FeS cell The arrows in c mark defectsalong the close-packedlayers, and X marks a disorderedarea. (d) The boxed region, enlarged,shows two planar faults. the identification of the atomic structure at the defects, Positionsand orientations of magnetosomes we assumethat a greigite-like configuration of Fe atoms within chains is presentalong the defects.If correct, then the crystal in Mackinawite (Fig. 4a), whether orderedor strongly dis- Figure 6c can be regardedas being in the initial stage of ordered (Fig. 7a), was found only at the ends of magne- transformation (with few atomic layers of greigite in its tosome chains. Crystals A and B in Figure 7a produced structure), whereas the crystal in Figure 5 is the end- SAED patterns that could be interpreted as mackinawite product of the conversion and contains only a few layers [001] and [1ll] projections,respectively. The patternbe- of the precursor structure. longing to A shows additional spots as a result of scat- POSFAI ET AL.: IRON SULFIDES IN BACTERIA 147',|

FrcunE 7. (a) Fe sulfide magnetosomechain from an MMP from Salt Pond. Crystal A is mackinawite (its SAED pattern is in the upper right), B is poorly ordered mackinawite, C is dis- ordered greigite (its SAED pattern is in the lower left), and D is a heavily disorderedcrystal. (b) HRTEM image of greigite crys- tal C in a, showing defects along (222) and (222) planes (anowed).

tering from other crystals within the TEM selected-area aperture. Diffuse Bragg reflections in the SAED pattern from B (data not shown) indicate that this crystal is poor- ly ordered and probably a mixture of mackinawite and greigite. Crystal C produced a [10]*.",,." SAED pattern (lower-left corner in Fig. 7a); however, the faint reflec- tions along the arrowed reciprocal lattice rows suggest that the spinel-type ordering of Fe atoms is not complete (comparethis pattern with the one in Fig. 5b). The high- resolution image of the same crystal shows abundantde- fects along (222)-type planes (arrowed) and patchesthat are misoriented with respect to the rest of the crystal (marked on the right). Becausethe chains likely increase in length by the addition of new crystals at the ends (Frankel and Blakemore 1989), the disorderedmackina- wite (A and B) and greigite (C) crystals are presumably the youngestin the chain and did not completely convert to greigite. Contrary to the idea that chain formation in bacteriais a magnetism-relatedphysical process(Kirschvink 1990), our observations suggestthat the particles are arranged in chains whether or not they are magnetic. The studied crystals within the bacterium in Figure 6 were all mack- inawite or cubic FeS, or a mixture of the two, but not greigite. Becausemackinawite is diamagnetic (Bertaut et al. 1965) and at room temperaturecubic FeS is paramag- netic (Wintenberger et al. 1978), these crystals were aligned in a chain before they had acquired a permanent magnetic dipole moment. Elongated crystals typically have their long sides ap- proximately parallel to the chain direction. Greigite crys- tals are preferentially elongated along one of the cube axes and thus aligned with [100] parallel to the chain. This crystallographicorientation was recognizedby Hey- wood et al. (1991)and can alsobe observedfor all greig- ite images and SAED patterns in this paper (Figs. 2b,c; '7a 5a,b; crystal C in Figs. and b; all SAED patterns are presentedin their correct orientations with respectto the correspondingimages). Because [100] is likely the easy axis of magnetization in greigite (Bazylinski and Mos- kowitz 199"7),the maximum magnetic moment of the cell is obtained by this preferred orientation of greigite crystals. b The precursor FeS crystals, if elongated, are also aligned with their long sidesapproximately parallel to the chain (seeFig. 6a). Besides,the crystals are oriented such that when they convert to greigite as described above, then [100]*.",r,,"will be parallel to the chain. This orien- 1478 POSFAI ET AL.: IRON SULFIDES IN BACTERIA tational relationship is illustrated by the SAED patterns netotactic bacteria.The presenceof mackinawite was not in Figure 6b (in which 200..n,"r.. would become 400g*iri," previously reported, possibly because it converted to on conversion) and Figure 7a (by the upper-right pattern, greigite in samplesthat were stored for more than one or in which 110.".*,"".,,.would become 400r*,.,* on two weeks. The presenceof cubic FeS magnetosomesis conversion). also likely (P6sfai et al. 1998). The d-spacingsobtained from the crystals shown in Figure Compositions 6 are intermediatebe- tween those of cubic FeS and mackinawite and suggest In addition to Fe and S, most analyzedmagnetosomes that transitional structuresoccur, with the Fe atomspartly contain variable Cu up to -10 at7o. Such Cu-bearing in mackinawite and partly in sphalerite-type affange- magnetosomes were previously described exclusively ments. Future examination of fresh samplesmay provide from the MMP (Bazylinski et al. 1993b); however, we additional information regarding the identities of greigite found that both types of bacteria can contain either Cu- precursors. free or Cu-bearing sulfide crystals, depending on where Our data indicate that the bacteriaprobably do not syn- the bacteria were collected (Table 1). The sufide magne- thesize greigite crystals directly. Instead, they initially tosomes from the MMPs or the rod-shaped organisms produce either cubic FeS or mackinawite. Our evidence from Rowley, MA contain no detectableCu, whereasCu- indicates (Fig. a) that mackinawite magnetosomeparti- bearing Fe sulfide magnetosomesappear typical of bac- cles convert to greigite. Other possible ffansformations teria collected from Salt Pond and Morro Bay. Thus, the that may occtu in magnetotacticbacteria include the cu- Cu content likely depends on Cu availability and is in- bic FeS -+ mackinawite -+ greigite and the cubic FeS -+ dependentof the mineral phase.This observation is con- greigite series. Similar sequenceswere also observed in sistent with our assumption that greigite forms from experimental studies of the cubic FeS -+ mackinawite mackinawite or cubic FeS, becausethen the Cu content (Murowchick 1989; Lennie and Vaughan 1996) and of the precursor phasedetermines whether the end-prod- mackinawite -+ greigite conversions (Rickard 1969b; uct greigite will contain Cu. Mackinawite was reported Schoonenand Barnes 1991; Lennie et al. 1997). able to contain up to 8.8 wt%oCu in its structure (Clark All three structureshave a cubic close-packedS frame- 1970), and crystal-chemical considerationssuggest that work (Fig. 8). The S array can be preserved along the greigite could also contain Cu (Vaughanet al. 1971). Ba- cubic FeS --> mackinawite + greigite conversion series, zylinski et al. (1993b) speculatedthar bacteria might in- except it slightly contractsas indicated by the decreasing corporate Cu into sulfides as a means of detoxification, spacingsof adjacentclose-packed layers. "Mixed" mack- although the physiological function of Cu in the cell, if inawite/greigite planar (222)- one exists, remains unclear. crystals and defects along type planes of greigite indicate that the mackinawite --> The Fe(+Cu)/S ratio in most al,ralyzedmagnetosomes greigite conversion likely takes place is higher than 0.75, the value for stoichiometric greigite. along the close- packed planes through the movement of Fe atoms be- Even though our estimated metal/S etror can be as high tween neighboring layers (P6sfai et al. 1998). as +0.1 (for reasonsmentioned in the experimental sec- S Disor- dered structures tion), and the analysisof Cu seemsparticularly inaccurate can result when the transformation is incomplete. (differences exist in Cu contentsbetween duplicate anal- The occurrenceof yses, Table l), the excess metal in greigite seemsto be Cu in these sulfides is also notable. There significant. When mackinawite or cubic FeS convert to has been interest in whether magnetotacticbacteria greigite, one fourth of the Fe atoms should be lost. The are able to incorporate transition metals other than iron (Bazylinski crystal in Figures 4a and 4b was analyzed.before and into their magnetosomes and Moskowitz after the transformation; its composition changed from 1997). Cleafly, at least the sulfide producers we studied FeonoSto FeoroSas it converted from mackinawite to can. The Cu content may have an effect on the rate of greigite (Table 1). The direction of the change is consis- conversion to greigite. The crystals in Figure 6 and the tent with the loss of Fe, but the amount lost is less than disorderedmackinawite crystals in Figure 7 did not con- expected. vert to greigite one month and 2r/zmonths after sample A possible sink for the surplus Fe from the mackina- collection, respectively. All these crystals contained Cu wite --> greigite conversion could be amorphous nano- (see Table 1), whereas the completely converted crystal phaseFe-(OH) that may form when Fe reacts with O, or in Figure 4 had no detectableCu. H,O (Lennie et al. 1997). Fe- and O-rich regions sur- Biological significance rounding the Fe sulfide magnetosomeswere reported by Farina et al. (1990); if such amorphousenvelopes exist, Biomineralization of greigite in magnetotacticbacteria they would skew our analysesof greigite crystals toward resemblesthe processesof inorganic, sedimentaryFe sul- higher metaVSratios. fide formation; in sediments,the amorphousFe sulfide -+ (cubic FeS -+) mackinawite -+ greigite transition series DrscussroN is thought to be the main pathway for greigite formation Formation of sulfide magnetosomes (Morse et al. 1987;Schoonen and Barnes1991). Greigite Greigite and mackinawite were unequivocally identi- then convefts to pyrite over time when excessS is present fied as mineral phasesin the Fe sulfide-producingmag- (Berner 19671Rickard 1969b). Similar transformations POSFAI ET AL.: IRON SULFIDES IN BACTERIA 1479

-s (111) (011) -$ d=3.12A d=2.

A cubicFeS B mackinawite,FeS UC grelgite,greigite,F%lLF%S4 FIcunn E. The structuresof (a) cubic FeS, (b) mackinawite, "down" oriented) S-tetrahedrathat project as isoscelestriangles and (c) greigite in [T0], [100]. and [T0] projections,respec- in this projection; in mackinawite, they Rll half of both types of tively. The upper part of the image shows ball-and-stickmodels, tetrahedralsites, marked Tl and T2 (b). Tl and T2 are crystal- whereascoordination polyhedra are illustrated in the lower part. lographically equivalent positions; the distinction is made here The arrangementof S atoms is the same in all three structures, only to aid visualization. In the spinel-type structureof greigite with the cubic-close-packedlayers parallel to (111), (011), and (c), half of all octahedralsites (half of 01 and all 02 positions (222) in a, b, and c, respectively. In cubic FeS, the Fe atoms along the direction of projection) and one-eighthof both T1 and completely fill one (marked T1 in a) of the two ("up" and T2 positions are filled.

occur in sulfides that are produced by biologically in- that the formation of magnetosomesand chain assembly duced mineralization (BM); the extracellular conversion are separatelycontrolled by the bacteria (Bazylinski et al. of mackinawite to greigite and then to pyrite was ob- 1995). The time between two cell divisions is probably servedin batch culturesof Desulfuvibriodesulfuricans, a not enough for mackinawite to convert to greigite. How- dissimilatory sulfate-reducingbacterium (Rickard 1969a). ever, when magnetotacticbacteria divide, their magneto- It is likely that the MMP and other magnetotacticmi- some chains are presumably divided between the two croorganismsthat produce Fe sulfide also belong to the daughter cells (Blakemore 1982); therefore, these cells group of dissimilatory sulfate-reducingbacteria (Delong inherit crystals that are alreadyferrimagnetic greigite, im- et al. 1993); in addition, thesemicroorganisms exist under parting the cell with a magnetic dipole moment that is conditions where relatively high concenffations of HrS necessaryfor its magnetotacticresponse. occur (Bazylinski et al. 1990). Based on the similarities to nonbiogenic and BIM processesof sulfide formation, Geologicalsignifi cance the greigite particles in cells of magnetotactic bacteria Greigite is an important component of sedimentaryFe could be expectedto convert to pyrite (Bazylinski et al. sulfides (Morse et al. 1987) and the main carrier of mag- 1990). However, our results indicate that this final step netic remanencein many types of young sediments(Hoff- of the conversion sequenceis absent in magnetotactic mann 1992). Greigite was also reported in soil (Stanjek bacteria. The formation of pyrite from greigite is a slow et al. 1994). In locations where large numbers of mag- process(Berner 1970), thus its presencewithin the rela- netotactic bacteria produce Fe sulfides, magnetosome tively fresh bacteriathat we studied may not be apparent. crystals probably contribute to the deposition of greigite On the other hand, ffuncation of the reaction sequenceat in sedimentsand thus likely to the magnetic remanence. greigite appearsto make sensein that the further conver- However, given that greigite transforms to pyrite in en- sion to pyrite would result in the formation of a non- vironments where there is excess S, it is doubtful that magnetic phasethat is of no use for the bacterium, as far greigite would last long in the geologic record (Berner as magnetotaxisis concerned. 1967). Thus, the geological significance of bacterial Our results also indicate that magnetotactic bacteria greigiteremains unclear. synthesizenon-magnetic sulfides de novo, and align them In principle, the size distributions, morphologies, and in chains (as in Figs. 6 and 7) prior to the crystals be- microstructural features of greigite magnetosomesmight coming magnetic. These observations support the idea allow bacterial greigite to be distinguishedfrom its non- 1480 POSFAI ET AL.: IRON SULFIDES IN BACTERIA biogenic counterparts,although such a distinction is not Bazylinski,D.A, Heywood,B.R., Mann, S., and Frankel,RB (1993a) straightforward. Horiuchi et al. (1974) synthesizedgreig- Fe.Ooand Fe.Soin a bacterium.Nature, 366, 218 A J, Abedi,A, andFrankel, R.B (1993b) ite Bazylinski,D.A., GarraFReed, crystals with sizes that were approximately in the Copper associationwith iron sulfide magnetosomesin a magnetotactic samerange as those in bacteria.In addition, the synthetic bacterium Archives of Microbiology, 160, 35-42 crystals contain planar defects parallel to (111)-rype Bazylinski,D.A, Garatt-Reed,AJ, and Frankel,RB. (1994) Electron planes, as do the bacterially produced grains. The elon- microscopic studies of magnetosomesin magnetotacticbacteria. Mi- gated, arrowhead-shapedmorphologies of some magne- croscopy Researchand Technique,27,389-401. Bazylinski,D.A, Frankel,R B , Heywood,B.R., Mann, S , King, J.W., tosome greigite crystals may also not be uniquely char- Donaghay,PL, and Hanson,A K (1995) Controlled biomineralization acteristic of biogenic greigite. Horiuchi et al. (1974) of magnetite(Fe.O,) and greigite (Fe.Sn)in a magnetotacticbacterium. synthesizedneedle-shaped greigite from solution at pH 5. Applied and EnvironmentalMicrobiology, 61, 3232-3239 These grains were elongated along [110], whereas the Berner,R A ( 1967) Thermodynamicstability of sedimentaryiron sulfides long axis of magnetosomecrystals is parallel to American Journal of Science,265, 773-785 [00]. -(1970) Sedimentary pyrite formation. American Journal of Sci- Greigite synthesizedat pH 9 and 200 "C in the presence ence,268,l-23. of Mn ions typically contained spinel-type twins (Hori- Bertaut, E.F, Burlet, P, and Chappert,J (1965) On the absenceof mag- uchi et al. 1974). We did not observe such twins in bac- netic order in tetragonalFeS Solid StateCommunications, 3, 335-338. terial geigite, although twinning in magnetite magneto- Blakemore, R.P (1975) Magnetotacticbacteria. Science, 190, 377-379 -(1982) Magnetotacticbacteria Annual Reviews in Microbiology, somes appears to be relatively common (Buseck et al. 36,217-238 1997;Devouard et al. 1998). Buseck,PR, Devouard,B., Majhi, P, P6sfai,M., Bazylinski,D.A., and The occurrenceof small (tens of nanometers)pyrrho- Frankel, R.B (1997) Bacterial magnetiteand Fe sulfides Annual Meet- tite crystals associatedwith magnetitein the Martian me- ing of the Geological Society of America, Abstracts,A 129 Salt Lake teorite ALH84001 was cited as evidence of past life on City, Utah. (1970) from Vlakfontein, Transvaal: (McKay Clark, AH Nickelian mackinawite Mars et al. 1996). We did not identify pynhotite A discussionAmerican Mineralogist, 55, 1802-1807. in terrestrial magnetotacticbacteria, and so its presence Cline, J D (1969) Spectrophotometricdetermination of hydrogen sulfide in ALH8400I seemsirrelevant to the conffoversy about in natural waters Limnology and Oceanography,14,454-458 past biogenic activity on Mars. 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