the prokaryotes || magnetotactic bacteria

CHAPTER 1.26Magnetotactic Bacteria

Magnetotactic Bacteria

STEFAN SPRING AND DENNIS A. BAZYLINSKI

Introduction

Magnetotactic bacteria are Gram-negative,motile prokaryotes that synthesize intracellularcrystals of magnetic iron oxide or iron sulfideminerals. These apparently membrane-boundedcrystals are called magnetosomes (Balkwill et al.,1980) and cause the bacteria to orient andmigrate along geomagnetic field lines. Magneto-tactic bacteria are indigenous in sediments orstratified water columns where they occur pre-dominantly at the oxic-anoxic transition zone(OATZ) and the anoxic regions of the habitat orboth. They represent a diverse group of microor-ganisms with respect to morphology, physiologyand phylogeny. Despite the efforts of a numberof different research groups, only a few represen-tatives of this group of bacteria have been iso-lated in axenic culture since their discovery by(Richard P. Blakemore, 1975), and even fewerhave been adequately described in the literature.Therefore, little is known about their metabolicplasticity, whereas their diverse morphology andphylogeny has been analyzed to some extent byculture-independent methods. To date, the onlyvalidly described species of magnetotactic bacte-ria are members of the genus Magnetospirillum.Representatives of this genus have been isolatedreproducibly from various aquatic environmentsand can be grown relatively easily in mass cul-ture. Therefore, most of the knowledge about themetabolism and biochemistry of magnetotacticbacteria relies on results obtained with strains ofthis genus.

Ecology

Magnetotactic bacteria are ubiquitous andcommon in sediments of freshwater or marinehabitats, but also in stratified water columns(Bazylinski et al., 1995) and wet soils (Fassbinderet al., 1990). The occurrence of magnetotacticbacteria appears to be dependent on thepresence of opposing gradients of reduced andoxidized compounds, usually represented byreduced sulfur species and oxygen, in the sedi-

ments or water columns. It has been shown insome cases that the distribution and abundanceof these bacteria in the environment might alsobe dependent on the availability of soluble iron(Stolz et al., 1986). The highest numbers of mag-netotactic bacteria are observed at the OATZ ofsediments or stratified water columns. Magneto-tactic bacteria can therefore be considered astypical examples of gradient organisms. In onestudy (Fig. 1), the number of a morphologically-distinct magnetotactic bacterium, “Magnetobac-terium bavaricum,” at the OATZ in a freshwatersediment was determined to be up to 7

¥ 105 livecells per cm3 (Spring et al., 1993).

“Magnetobacterium bavaricum” is very large(average volume ca. 25.8

± 4.1

mm3) and couldaccount for approximately 30% of the microbialbiovolume in this layer of the sediment. In someenvironments, magnetotactic bacteria, particu-larly those that produce iron sulfide minerals, canalso be detected in the anoxic region of the hab-itat, but are only rarely found at sites or regionsof water columns or sediments exposed to highlevels of oxygen.

The detection of magnetotactic bacteria inenvironmental samples is relatively easy due totheir permanent magnetic dipole moment. Onesimple method is to put a drop of water or sedi-ment on a microscope slide and place a bar mag-net on the microscope stage in such a way thatall the magnetotactic bacteria are guided in onedirection until they reach and accumulate at theedge of a drop of water and/or sediment wherethey can be visualized. Alternatively, it is possi-ble to magnetically enrich for higher numbers ofcells by placing the south pole (in the NorthernHemisphere; the north pole of the magnet isused in the Southern Hemisphere) of a bar mag-net adjacent to the outer wall of a jar filled withsediment and water. If magnetotactic bacteriaare abundant in the sample, a brownish or some-times grayish to white (if the cells contain ele-mental sulfur globules) spot consisting mainly ofmagnetotactic bacteria will form next to theinside of the glass wall closest to the south poleof the bar magnet. This material can easily beremoved from the jar with a Pasteur pipette and

Prokaryotes (2006) 2:842–862DOI: 10.1007/0-387-30742-7_26

CHAPTER 1.26 Magnetotactic Bacteria 843

examined as described above. Magnetotacticbacteria commonly enrich (i.e., increase in num-bers) in sediment samples in jars or aquariastored in dim light at room temperature. Theprocess may take several weeks to months, how-ever. In several cases, successions of differentmagnetotactic bacterial morphotypes havebeen observed during the enrichment process.Astonishingly, magnetotactic bacteria sometimesremain active for several years in the aquariawithout addition of any nutrients.

When magnetotactic bacteria die and lyse,magnetosome crystals sometimes remain stablein sediments at sites where these bacteria wereabundant, resulting in a change of the remanentmagnetization of those sediments. Fossil magne-tosome crystals consisting of magnetite (“mag-netofossils”) have been retrieved from manysites including deep sea sediments up to 50 mil-lion years old (Petersen et al., 1986) and ancientconsolidated sediments up to 2 billion years old(Chang and Kirschvink, 1989a; Chang et al.,1989b). The bacterial origin of particles likethose shown in Fig. 2 is assumed based on theunique morphology and size distribution of thesemagnetite crystals (see Magnetite-type Magne-tosomes in this Chapter). Magnetite particles ofsimilar sizes and shapes to those of magnetotac-tic bacteria have also been discovered within theMartian meteorite ALH84001 where they are

thought to be the result of biological processesand therefore have been used as evidence for thepresence of life on ancient Mars (McKay et al.,1996).

Nanometer-sized magnetite particles have alsobeen found in soil samples. There the occurrenceof magnetite seems to be strongly dependent onenvironmental conditions favoring the growth ofmagnetotactic bacteria, a fact which is also ofsome archaeological interest. It has been specu-lated that changes in the magnetic susceptibilityof top soils, which frequently indicate locationsof buried remains of archaeological objects, arecaused by magnetotactic bacteria (Fassbinderand Stanjek, 1993). It is thought that the decayof wooden posts or palisades in wet soil leads tolocalized sites rich in organic material where thegrowth of magnetotactic bacteria is stimulated,thereby leading to the localized production andaccumulation of magnetite. However, it is alsopossible that the magnetite is produced by iron-reducing bacteria through a biologically-inducedmineralization process. In this case, Fe2

+, result-ing from the bacterial enzymatic reduction ofFe3

+, reacts chemically with ferrihydrite to formextracellular magnetite.

Magnetotaxis, Chemotaxis and Aerotaxis

Soon after the discovery of magnetotactic bacte-ria, a model was proposed to explain the functionof the bacterial magnetosome and the biologicaladvantage of magnetotaxis. The original theory,

Fig. 2. Brightfield TEM image of a magnetic separate fromsurface sediments collected from the Irish Sea. Note the pres-ence of parallelepipedal, cubo-octahedral, and tooth-shapedcrystals of magnetite, presumed “magnetofossils” left frommagnetotactic bacteria. Courtesy of Z. Gibbs.

150 µm

Fig. 1. Vertical distribution of “Magnetobacterium bavari-cum” in the stratified sediment of a freshwater lake inBavaria, Germany (Lake Chiemsee). The analyzed sedimentsample was stored in an aquarium for several weeks beforethe measurements. The crosshatched bars indicate counts forsuccessive depth fractions (3 mm intervals). Oxygen wasmeasured every 1 mm (solid circles). A color photograph ofthe corresponding section of the aquarium is shown on theleft. The upper brownish gray layer followed by a thin reddishbrown and then a gray layer is characteristic of Lake Chiem-see sediments. The water-sediment interface corresponds to0 mm.

– 5

0

0 50 100 150 200 250

0 1 2 3 4 5 6 7

5

10

15

Dep

th [m

m]

Oxygen [mmol/l]

Cells [105/cm3]

844 S. Spring and D.A. Bazylinski CHAPTER 1.26

proposed by Blakemore (1975), was based on theassumption that all magnetotactic bacteria aremicroaerophilic and indigenous in sediments.Richard B. Frankel and co-workers clearlyshowed that these bacteria passively align andactively swim along the inclined geomagneticfield lines as a result of their magnetic dipolemoment. Blakemore called this behavior magne-totaxis and proposed that magnetotaxis helps toguide the cells down to less oxygenated regionsof aquatic habitats at the surface of sediments.Once cells have reached their preferred micro-habitat they would presumably stop swimmingand adhere to sediment particles until conditionschanged, as for example, when additional oxygenwas introduced. By this mechanism, a conven-tional aerotactic response which is a threedimensional search problem could be reduced toan one dimensional search problem in whichcells only swim downward, thereby increasingthe efficiency of the organism in finding an opti-mal oxygen concentration in the sediments. Thistheory is supported by the predominant occur-rence of magnetotactic bacteria that are North-seeking (i.e., swim in the direction indicated bythe North-seeking pole of a magnetic compassneedle) under oxic conditions in the Northernhemisphere whereas bacteria are predominatelySouth-seeking in the Southern hemisphere. Dueto the negative and positive sign of the geomag-netic field inclination in the Northern and South-ern hemispheres, respectively, magnetotacticbacteria in both hemispheres therefore swimdownward toward the sediments (Blakemore,1982).

Recent findings, including the discovery oflarge populations of magnetotactic bacteria atthe OATZ of chemically stratified aquatic habi-tats and the isolation of obligately microaero-philic, coccoid magnetotactic bacterial strains,make it necessary to rethink this view of magne-totaxis. The traditional model does not com-pletely explain how bacteria in the anoxic zoneof a water column benefit from magnetotaxis,nor does it explain how the magnetotactic cocciform microaerophilic bands of cells in semi-solidoxygen gradient medium. Spormann and Wolfe(1984) showed earlier that magnetotaxis is some-how controlled by aerotaxis in some magnetotac-tic bacteria, but this alone does not help toexplain all observed effects of magnetotaxis.More recently, it was demonstrated (with purecultures of magnetite-producing magnetotacticbacteria) that magnetotaxis and aerotaxis worktogether in these bacteria (Frankel et al., 1997).The behavior observed in these strains has beenreferred to as “magneto-aerotaxis,” which is amore accurate description than magnetotaxisbecause these cells do not try to reach a distinctmagnetic pole or field as the term magnetotaxis

implies. Thus, the term magnetotaxis is amisnomer.

The traditional model also fails to explain thevarious types of magnetotactic behavior whichhad been observed by several authors butwithout recognizing the fundamental differ-ences between these behaviors (Moench andKonetzka, 1978; Blakemore et al., 1980;Spormann and Wolfe, 1984). Only when distinctmorphotypes of magnetotactic bacteria were iso-lated and grown in pure culture for detailed stud-ies using thin, flattened capillaries (Frankel et al.,1997), it became clear that two types of mecha-nisms have been observed, which apparentlyoccur in different bacteria, termed polar andaxial.

The distinction can be seen by examinationof cells in wet mounts under oxic conditionsusing a microscope and a magnet of a few gaussparallel to the plane of the slide (Fig. 3). Polarmagnetotactic bacteria, particularly themagnetotactic cocci, swim persistently along themagnetic field lines without reversing their direc-tion or turning. If the magnetic field is reversed,the bacteria reverse their swimming directionand continue swimming persistently in the samedirection relative to the magnetic field. Bacteriafrom Northern hemisphere habitats predomi-nately swim parallel to the magnetic field,corresponding to northward migration in thegeomagnetic field. Bacteria from the Southernhemisphere swim antiparallel to the magneticfield. It was this consistent swimming behaviorthat led to the discovery of magnetotacticbacteria by Blakemore (1975). On the otherhand, axial magnetotactic bacteria, especially thefreshwater spirilla, orient and swim in both direc-tions along the magnetic field lines with frequentreversals of swimming direction and some accu-mulating in approximately equal numbers onboth sides of the water drop (Fig. 3a).

The distinction between polar and axialmagneto-aerotaxis can also be seen in flattenedcapillary tubes containing suspensions of cells inreduced medium with one or both ends of thecapillary tube open. In the first situation, whereone end of the capillary is open (the right end ofthe capillaries in Fig. 3b) and the other sealed, asingle oxygen gradient forms beginning at theopen end of the capillary. Cells of strain MC-1 inthese capillaries rotate 180

∞ after a reversal of B,the magnetic field, and the band separates intogroups of cells swimming in opposite directionsalong B, away from the position of the bandbefore the reversal. A second reversal results inthe reformation of a single band. Cells of M.magnetotacticum also rotate 180

∞ in these capil-laries but the band of cells does not separate andremains intact (Fig. 3). In the second situation(not shown), where both ends of the capillary


tubes are open, diffusion of oxygen into the endsof the tubes creates an oxygen gradient at eachend of the tube, oriented in opposite directions.Polar magnetotactic bacteria incubated in a mag-netic field oriented along the long axis of thetube form an aerotactic band at only one end ofthe tube, whereas axial magnetotactic bacteriaform bands at both ends of the tube. Thus forpolar magnetotactic bacteria the magnetic fieldprovides an axis and direction for motility,whereas for axial magnetotactic bacteria themagnetic field only provides an axis of motility,pointing to different magneto-aerotactic mecha-nisms occurring in two types of bacteria.

Axial Magneto-Aerotaxis

Almost all magnetotactic spirilla available inaxenic culture and grown in liquid media, exhibitaxial magneto-aerotaxis (Figs. 3 and 4). Other

bacteria that show axial magnetotaxis aremicroaerophilic or anaerobic chemoheterotro-phs, or facultative chemolithoautotrophic bacte-ria that are either monopolarly or bipolarlyflagellated. In most habitats, axial magnetotacticbacteria appear to represent only a very smallfraction of the total count of magnetotactic bac-teria, although these organisms are harder todetect in wet mounts using a microscope. Cellsrepresenting this type of magnetotaxis werereferred to as two-way swimmers because in ahomogeneous medium they swim in either direc-tion along the magnetic field, B (Fig. 4). In thepresence of an oxygen gradient, cells swim par-allel or antiparallel to B with aerotaxis determin-ing the direction of migration. Therefore, anaerotactic band of cells forms at both ends of thetube in capillaries where both ends are open,whereas cells displaying a polar magnetotaxisform only one band at the end of the tube cor-responding to their magnetic polarity. The aero-tactic, axial magnetotactic spirilla appear to usea temporal sensory mechanism for oxygen detec-tion as do most microaerophilic bacteria studiedso far (Frankel et al., 1998). Changes in oxygenconcentration measured during swimming deter-mine the sense of flagellar rotation. Cells movingaway from the optimal oxygen concentrationconsequently reverse their swimming direction.In this model, changes in oxygen concentrationare measured within short intervals implying thatthese bacteria must be actively motile in order toquickly measure and respond to concentrationgradients in their habitat. The combination of apassive alignment along geomagnetic field lineswith an active, temporal, aerotactic responseprovides the organism with an efficient mecha-nism to find the microoxic or suboxic zone in itshabitat. Therefore the term magneto-aerotaxis isalso an appropriate descriptive term for thistactic behavior.

Fig. 4. Sequence showing magnetotactic spirilla displayingaxial magnetotaxis. For the video, see the online version ofThe Prokaryotes.

Polar Magneto-Aerotaxis

The large majority of naturally-occurring magne-totactic bacteria display polar magnetotaxis(Figs. 3 and 5). The following mechanism forpolar magnetotaxis was proposed based onexperimental data obtained with an axenicculture of a marine magnetotactic coccus. It wasdemonstrated that these cocci can swim in bothdirections along a static magnetic field, B, with-out the need of turning around by reversing thesense of flagellar rotation. It seems that a two-state sensory mechanism determines the sense offlagella rotation leading to parallel or antiparal-lel swimming along the geomagnetic field lines.

Fig. 3. Two types of magnetotaxis. (a) Depictions of the polarmagnetotactic behavior of strain MC-1 and axial magneto-tactic behavior of Magnetospirillum magnetotacticum inwater drops under oxic conditions on a microscope slide (B,magnetic field; arrow points northward). Cells of strain MC-1 swim persistently parallel to B (North-seeking motility) andaccumulate at the edge of the drop. When B is reversed, cellscontinue to swim parallel to B (North-seeking motility) andaccumulate at the other side of the drop. Cells of M. magne-totacticum swim in either direction relative to B and continueto do so when the field is reversed. (b) Illustrations of aero-tactic bands of strain MC-1 and M. magnetotacticum in flatcapillaries. The right ends of the capillaries are open to airand the left ends are sealed. After reversal of B, cells of strainMC-1 rotate 180

∞ and the band separates into groups of cellsswimming in opposite directions along B, away from theposition of the band before the reversal. A second reversalresults in the reformation of a single band. Cells of M. mag-netotacticum also rotate 180

∞ but the band of cells remainsintact. Figure adapted from Frankel et al. (1997).

Magnetospirillummagnetotacticum

Strain Mc-1

B

B

B

B

b

a


Under higher than optimal oxygen tensions, thecell is presumably in an “oxidized state” andswims persistently parallel to B (Fig. 5), i.e.,downward in the Northern hemisphere. Underreducing conditions or suboptimal oxygen con-centrations, the cell switches to a second state,the “reduced state”, which leads to a reversal ofthe flagellar rotation and to a swimming antipar-allel to B (upward in the Northern hemisphere).This two-state sensing mechanism results in anefficient aerotactic response, provided that theoxygen-gradient is oriented correctly relative toB, so that the cell is guided in the right directionto find either reducing or oxidizing conditions.This is especially important because adaptation,which would lead to a spontaneous reversal ofthe swimming direction, was never observed incontrolled experiments with the cocci. The redoxsensor, which controls this two-state response,might be similar to the FNR (fumarate andnitrate reduction) transcription factor found inEscherichia coli and other bacteria. The FNRfactor is sensitive to oxygen and activates geneexpression in the reduced state thereby promot-ing the switch between aerobiosis and anaerobi-osis in E. coli (De Graef et al., 1999). The sensorymechanism in the examined magnetotactic cocciis not only affected by oxygen. Cells exposed tolight of short wavelengths (

=500 nm) alsoshowed a response similar to a switch to the “oxi-dized state” (Frankel et al., 1997).

Fig. 5. Sequence showing magnetotactic cocci displayingpolar magnetotaxis. For the video, see the online version ofThe Prokaryotes.

Revised Model of Magnetotaxis

Based on these observations, we would like toextend the current model of a magnetically-guided aerotaxis (magneto-aerotaxis) to a morecomplex redoxtaxis. In this case, the unidirec-tional movement of magnetotactic bacteria in adrop of water would be only one aspect of asophisticated redox-controlled response. Onehint for the possible function of polar magneto-taxis could be that most of the representativemicroorganisms are characterized by possessingeither large sulfur inclusions or magnetosomesconsisting of iron-sulfides. Therefore, it may bespeculated that the metabolism of these bacte-ria, being either chemolithoautotrophic or mix-otrophic, is strongly dependent on the uptake ofreduced sulfur compounds which occurs in manyhabitats only in deeper regions at or below theOATZ due to the rapid chemical oxidation ofthese reduced chemical species by oxygen orother oxidants in the upper layers. To overcomethe problem of separated pools of electrondonor and acceptor, several strategies have been

developed by sulfide-oxidizing bacteria. Micro-organisms belonging to the genus Thioploca, forexample, use nitrate, which is stored intracellu-larly (most of the internal space of the cell isvacuolar) to oxidize sulfide and have developedvertical sheaths in which bundles of motile fila-ments are located. It is assumed that Thioplocauses these sheaths to efficiently move in a verti-cal direction in the sediment, thereby accumu-lating sulfide in deeper layers and nitrate inupper layers (Huettel et al., 1996). For somemagnetotactic bacteria, it might also be neces-sary to perform excursions to anoxic zones oftheir habitat in order to accumulate reduced sul-fur compounds. In our model, shown in Fig. 6,we propose that polar magnetotaxis helps toguide bacteria, depending on their internalredox-state, either downward to accumulatereduced sulfur species or upward to oxidizestored sulfur with oxygen. Thus, we hypothesizethat magnetotactic bacteria displaying polarmagnetotaxis alternate between two internalredox states. The “oxidized state” would resultfrom the almost complete consumption ofstored sulfur, the assumed electron donor. Inthis state, cells seek deeper anoxic layers wherethey could replenish the depleted stock ofelectron donor using nitrate or other com-pounds as alternative electron acceptor. Finally,they would reach a “reduced state.” Accordingto our model, cells in this redox state wouldhave accumulated a large amount of sulfurwhich cannot be efficiently oxidized underanaerobic conditions leading to a surplus ofreduction equivalents. Therefore, cells mustreturn to the microoxic zone where oxygen isavailable to them as an electron acceptor. Inaddition, oxygen may be necessary for the syn-thesis of magnetosomes in some bacteria(Blakemore et al., 1985). The advantage of polarmagnetotaxis is that an oxygen gradient is notnecessary for efficient orientation in the anoxiczone, thereby enabling a rapid return of the cellalong large distances to the preferred microoxicconditions. A further benefit would be that cellsavoid the waste of energy by constant move-ment along gradients, but instead can attach toparticles in preferred microniches until theyreach an unfavorable internal redox state thattriggers a magnetotactic response either parallelor antiparallel to the geomagnetic field lines. Inany case, greater than optimal concentrations ofoxygen would switch cells immediately to an“oxidized state” provoking the typical down-seeking response of magnetotactic bacteria visi-ble under the microscope. The observation ofsignificant numbers of microaerophilic, magne-totactic bacteria in the anoxic zone of somesediments and the attachment of “Magnetobac-terium bavaricum” to sediment particles in


microcolony-like aggregates, fits well with thismodel, which is summarized in the followingFig. 6.

Morphologic and Phylogenetic Diversity

Morphotypes

The diversity of magnetotactic bacteria isreflected by the high number of different mor-photypes found in environmental samples ofwater or sediment. Commonly observed mor-photypes include coccoid to ovoid cells, rods,vibrios and spirilla of various dimensions. One ofthe more unique morphotypes is an apparentlymulticellular bacterium referred to as the MMPmany-celled magnetotactic prokaryote.

Regardless of their morphology, all magneto-tactic bacteria studied so far are motile by meansof flagella and have a cell wall structure charac-teristic of Gram-negative bacteria. The arrange-ment of flagella differs and can be either polar,bipolar, or in tufts. Another trait which showsconsiderable diversity is the arrangement ofmagnetosomes inside the bacterial cell. In themajority of magnetotactic bacteria, the magneto-somes are aligned in chains of various lengthsand numbers along the cell’s long axis of the cell,which is magnetically the most efficient orienta-tion. However, dispersed aggregates or clustersof magnetosomes occur in some magnetotacticbacteria usually at one side of the cell, whichoften corresponds to the site of flagellar inser-tion. Besides magnetosomes, large inclusionbodies containing elemental sulfur, polyphos-

phate, or poly-

b-hydroxybutyrate are common inmagnetotactic bacteria collected from the natu-ral environment and in pure culture.

The most abundant type of magnetotactic bac-teria occurring in environmental samples, espe-cially sediments, are coccoid cells possessing twoflagellar bundles on one somewhat flattened side.This bilophotrichous type of flagellation gaverise to the tentative genus “Bilophococcus” forthese bacteria (Moench, 1988). One representa-tive strain of this morphotype is in axenic culture(see Other Magnetotactic Strains in Pure Cul-ture in this Chapter). In contrast, two of the mor-phologically more conspicuous magnetotacticbacteria, regularly observed in natural samplesbut never isolated in pure culture, are the MMPand a large rod containing large numbers ofhook-shaped magnetosomes (“Candidatus Mag-netobacterium bavaricum”).

The MMP, a Many-Celled MagnetotacticProkaryote A magnetotactic aggregation ofcells that swims as an entire unit and not as sep-arate cells was first reported and described byFarina et al. (1983). Similar morphotypes werelater found also in sulfide-rich marine and brack-ish waters and in sediments along the coasts ofNorth America and Europe (Mann et al., 1990a).The MMP (for many-celled magnetotacticprokaryote) consists of about 10 to 30 coccoid toovoid Gram-negative cells, roughly arranged ina sphere with a diameter ranging from approxi-mately 3 to 12

mm (Fig. 7).Cells are asymmetrically multiflagellated on

their outer surfaces exposed to the external sur-roundings. Magnetosomes consist of the mag-netic iron-sulfide greigite, Fe3S4, and severalnonmagnetic precursors to greigite (see “Iron-Sulfide Type Magnetosomes”). The magneto-some crystals are generally pleomorphicalthough cubo-octahedral, rectangular prismatic,and tooth-shaped particles have also beenobserved in cells. They are usually looselyarranged in short chains or clusters in individualcells. The total magnetic moment of the MMPwas determined and ranges from 5

¥ 10

-16 to 1

¥10

-15 Am2, which is sufficient for an effectivemagnetotactic response. The type of magneto-taxis displayed by the MMP appears to be polar,but aggregates have been observed to reversedirection. Under oxic conditions in a uniformmagnetic field, the swimming speed in the pre-ferred direction averages 105

mm/s. After reach-ing the edge of a water drop, aggregatessometimes spontaneously reverse their swim-ming direction and show short excursions of 100to 500

mm with twice the speed of the forwardmotion in the opposite direction of their polarity(Rodgers et al., 1990) as shown in Fig. 8. This so

Fig. 6. Hypothetical model of the function of polar magne-totaxis in bacteria (Northern hemisphere). Cells are guidedalong the geomagnetic field lines depending on their “redox-state” either downward to the sulfide-rich zone or upward tothe microoxic zone, thereby enabling a shuttling betweendifferent redox layers.

geomagnetic field lines

oxidixed state[ox]in> optimal

direction of motilityparallel to magnetic field

balanced redox state I

direction of motilityontiparalldl tomagnetic field

reduced state[Red]in > optimal

balanced redox state II [S2–]

[O2]

Fe3+

Fe2+

S2–

O2


called “ping pong” motion seems to be a pecu-liarity of this organism.

Fig. 8. Sequence showing the typical “ping-pong” motility ofthe MMP. For the video, see the online version of TheProkaryotes.

In one study, it was reported that individualcells within the aggregate are connected by inter-cellular membrane junctions (Rodgers et al.,1990a; 1990b). However, the cohesive forceamong individual cells seems to be relativelyweak because a lowering of the osmotic pressureleads to an immediate disruption of the aggre-gate into single nonmotile cells.

“Candidatus Magnetobacterium bavaricum” Thefirst phenotypic description of this morphotypeby Vali et al. (1987) was based on cells collectedfrom material retrieved from the littoral sedi-ments of a large freshwater lake in SouthernGermany (Lake Chiemsee). Later, similar bacte-ria were also found in sediments of other fresh-water habitats in Germany and Brazil. After thedetermination of its phylogenetic relationship(Spring et al., 1993), this organism was given thecandidatus status due to its unusual phenotypictraits which distinguish it from all othermagnetotactic bacteria. “Magnetobacteriumbavaricum” displays polar magnetotaxis and is

preferentially found in the microoxic zone ofsediments, although significant numbers are alsofound in anoxic regions of their habitat (Fig. 1).In situ hybridizations using a specificfluorescently-labeled oligonucleotide probe tar-geting the 16S rRNA of this organism enabledthe detection of microcolonies of this bacteriumon microscope slides immersed into sediment forseveral weeks (Fig. 9). Thus, there appears to bea tendency for “Magnetobacterium bavaricum”to adhere to particles located in microsites withpreferred environmental conditions.

Cells of “M. bavaricum” are large rods havingdimensions of 1–1.5 x 6–9

mm and are motile bya polar tuft of flagella. The most impressive traitof this bacterium is the extremely high numberof magnetosomes per cell. A single cell may con-tain up to a thousand hook-shaped magneto-somes usually arranged in 3–5 rope-shapedbundles oriented parallel to the long axis of thecell (Fig. 10).

The magnetosomes consist of magnetite(Fe3O4) and have a length of 110–150 nm. Theaverage total magnetic moment per cell wasexperimentally determined to be approximately3

¥ 10

-14 Am2, which is about an order of magni-tude higher than that of most other magnetotac-tic bacteria. The presence of large sulfurinclusions is typical for this bacterium and seems

Fig. 7. Brightfield TEM micrographsof the many-celled magnetotacticprokaryote (MMP). (a) An unstained,single MMP revealing the numerousgreigite-containing magnetosomeswithin the organism mostly arrangedin short chains. (b) Negatively-stainedpreparation (2.5% ammonium molyb-date, pH 7.0) of a single MMP that isdisrupted to reveal separated individ-ual cells. (c) Thin-section of an MMPagain showing its many-celled nature.(d) Negatively-stained individual cellof the MMP. Note the asymmetricdistribution of flagella which coverthe cell on one side, the pleomor-phism of the greigite-containing mag-netosomes, and the electron-lucentvacuoles resembling poly-b-hydroxy-butyrate (PHB) granules.

1 µm

0.5 µm

0.5 µm

5 µm

a b

dc


to be dependent on environmental conditions. Inan unidirectional magnetic field, cells swim for-ward (i.e., northward in the Northern Hemi-sphere) with an average speed of 40

mm/s withthe flagella wound around the rotating cell. Gra-dients of some chemical substances lead to areversal of the sense of flagellar rotation result-ing in a swimming in the opposite direction for ashort time.

Composition and Structure of Magnetosome Crystals

The magnetosome mineral phase in magnetotac-tic bacteria are tens-of-nanometer-sized crystalsof an iron oxide and/or an iron sulfide. The min-eral composition of the magnetosome is specificenough for it to be likely under genetic control,in that cells of several cultured magnetite-producing magnetotactic bacteria still synthesizean iron oxide and not an iron sulfide, even whenhydrogen sulfide is present in the growth medium

Fig. 9. In situ hybridization of a microscope slide grown overwith sediment bacteria using fluorescently labeled oligonu-cleotide probes. (a) Phase contrast micrograph. (b

+c) Samefield viewed with epifluorescence microscopy enabling thedetection of a specific probe binding to a signature region ofthe 16S rRNA of “M. bavaricum” (b), and of a probe withbroad specifity hybridizing with the 16S rRNA of most knownbacteria (c).

a 100 µm

b

c

Fig. 10. Brightfield transmission electron microscope (TEM)micrographs of “M. bavaricum.” (a) Whole cell displayingbundles of magnetosome chains and sulfur globules. (b)Hook-shaped magnetite-type magnetosomes. Courtesy of M.Hanzlik.

a

b

1 µm

100 nm


(Meldrum et al., 1993a; Meldrum et al., 1993b).The size of the magnetosome mineral crystals alsoappears to be under control of the organismbecause the large majority of magnetotactic bac-teria contain crystals displaying only a verynarrow size range, from about 35 to 120 nm(Frankel et al., 1998). Magnetite and greigite par-ticles in this range are stable single magneticdomains (Butler and Banerjee, 1975; Diaz-Rizziand Kirschvink, 1992). Smaller particles wouldbe superparamagnetic at ambient temperatureand would not have stable, remanent magnetiza-tion. Larger particles would tend to form multipledomains, reducing the remanent magnetization.However, in some uncultured bacteria from theSouthern Hemisphere exceptionally large mag-netite-magnetosomes have been observed insome uncultured bacteria from the Southernhemisphere (Fig. 11), having dimensions wellabove the theoretically determined size limits ofsingle domain magnetite (Spring et al., 1998). Itremains unclear if the crystals in these bacteriaare still of single-domain size or are multi-domainparticles and why such unusually large crystalsare formed by certain bacteria, but, interestingly,it seems that the crystal-size corresponds with thesize and/or the growth phase of these bacteria,i.e., large cells possess larger crystals than smallercells of the same type.

In contrast to chemically synthesized magne-tite and greigite crystals, biologically producedmagnetosome mineral particles display a rangeof well-defined morphologies which can be clas-sified as distinct idealized types (Fig. 12).

The consistent narrow size range (Devouardet al., 1998) and morphologies of the intracellu-lar magnetosome particles represent typical fea-tures of a biologically controlled mineralizationand are clear indications that the magnetotacticbacteria exert a high degree of control over thebiomineralization processes involved in magne-tosome synthesis.

Magnetite-Type Magnetosomes The iron oxide-type magnetosomes consist solely of magnetite,Fe3O4. The particle morphology of the magnetitecrystals in magnetotactic bacteria varies but isextraordinarily consistent within cells of a singlebacterial species or strain (Bazylinski et al.,1994). Three general morphologies of magnetiteparticles have been observed in magnetotacticbacteria using transmission electron microscopy(TEM; Blakemore et al., 1989; Mann et al.,

Fig. 11. Unusually large magnetite crystals identified in coc-coid magnetotactic bacteria retrieved from a lagoon near Riode Janeiro, Brazil. Small black dots represent gold-labeledantibodies detecting a specifically bound polynucleotideprobe complementary to a highly variable region of the 16SrRNA of these cells.

0.5 µm

Fig. 12. Idealized magnetite (a–d) and greigite (e–f) crystalmorphologies derived from high resolution TEM studies ofmagnetosome crystals from magnetotactic bacteria: (a) and(e) cubo-octahedrons; (b), (c), and (f) variations of pseudo-hexagonal prisms; (d) elongated cubo-octahedron. Numberswithin parentheses refer to the faces of the crystal latticeplanes on the surface of the crystal. Figure adapted fromHeywood et al. (1991) and Mann and Frankel (1989).

a b

(111)

(111)(111)

d

(011)

(111)

(001)

(111)

(110)

(111)

(100)

(111)

(010)

(111)

(100)

(010)(001)

(111)

(010)

(111)

(211)

(111)

(100)e f

(101)(011)

001

101 011 110

011110

100

(010)

c

(100)

(111)

(111)

111

101

(010)


1990a; Stolz, 1993; Bazylinski et al., 1994). Theyinclude: 1) roughly cuboidal (Balkwill et al.,1980; Mann et al., 1984); 2) parallelepipedal(rectangular in the horizontal plane of projec-tion; Moench and Konetzka, 1978; Towe andMoench, 1981; Moench, 1988; Bazylinski et al.,1988); and 3) tooth-, bullet-, or arrowhead-shaped (anisotropic; Mann et al., 1987a; Mann etal., 1987b; Thornhill et al., 1994).

High resolution TEM and selected area elec-tron diffraction studies have revealed that themagnetite particles within magnetotactic bacteriaare of relatively high structural perfection andhave been used to determine their idealized mor-phologies (Matsuda et al., 1983; Mann et al.,1984a; 1984b; 1987a; 1987b; Meldrum et al.,1993a; Meldrum et al., 1993b). These morpholo-gies are all derived from combinations of {111},{110} and {100} forms (a form refers to the equiv-alent symmetry related lattice planes of the crys-tal structure) with suitable distortions (Devouardet al., 1998). The roughly cuboidal particles arecubo-octahedra ([100]

+ [111]), and the parallel-epipedal particles are either pseudohexahedralor pseudooctahedral prisms. Examples are shownin Fig. 12a–d. The cubo-octahedral crystal mor-phology preserves the symmetry of the face-cen-tered cubic spinel structure, i.e., all equivalentcrystal faces develop equally. The pseudo-hexahedral and pseudo-octahedral prismatic par-ticles represent anisotropic growth in whichequivalent faces develop unequally (Mann andFrankel, 1989; Devouard et al., 1998). The syn-thesis of the tooth-, bullet- and arrowhead-shaped magnetite particles (Figs. 10b, 13c)appears to be more complex than that of the otherforms. They have been examined by high resolu-tion TEM in one uncultured organism (Mann etal., 1987a; Mann et al., 1987b) and their idealizedmorphology suggests that growth of these parti-cles occurs in two stages. The nascent crystals arecubo-octahedra which subsequently elongatealong the [111] axis parallel to the chain direction.

Whereas the cubo-octahedral form of magne-tite can occur in inorganically-formed magnetites(Palache, 1944), the prevalence of elongatedpseudohexahedral or pseudo-octahedral habitsin magnetosome crystals imply anisotropicgrowth conditions, e.g., a temperature gradient,a chemical concentration gradient, or an aniso-tropic ion flux (Mann and Frankel, 1989). Thisaspect of magnetosome particle morphology hasbeen used to distinguish magnetosome magne-tite from detrital or magnetite produced bybiologically induced mineralization (by theanaerobic iron-reducing bacteria), using electronmicroscopy of magnetic extracts from sediments(e.g., Petersen et al., 1986; Chang andKirschvink, 1989a; Chang et al., 1989b; Stolz etal., 1986; Stolz et al., 1990; Stolz, 1993).

Fig. 13. Morphologies of intracellular magnetite (Fe3O4)particles produced by magnetotactic bacteria. (a) Darkfieldscanning-transmission electron microscope (STEM) image ofa chain of cubo-octahedra in cells of an unidentified rod-shaped bacterium collected from the Pettaquamscutt Estu-ary, Rhode Island, USA, viewed along a [111] zone axis forwhich the particle projections appear hexagonal. (b) Bright-field TEM image of a chain of prismatic crystals within a cellof strain MV-2, a marine vibrio, with parallelepipedalprojections. (c) Brightfield TEM image of tooth-shaped(anisotropic) magnetosomes from an unidentified rod-shaped bacterium collected from the PettaquamscuttEstuary.

100 nm

100 nm

100 nma

b

c


Iron-Sulfide Type Magnetosomes Virtually allfreshwater magnetotactic bacteria have beenfound to synthesize magnetite as the mineralphase of their magnetosomes. In contrast, manymarine, estuarine, and salt marsh species pro-duce iron sulfide-type magnetosomes consistingprimarily of the magnetic iron sulfide, greigite,Fe3S4 (Heywood et al., 1990; Heywood et al.,1991; Mann et al., 1990b; Pósfai et al., 1998a;1998b). Reports of non-magnetic iron pyrite(FeS2; Mann et al., 1990b) and magnetic pyrrho-tite (Fe7S8; Farina et al., 1990) have not beenconfirmed and may represent misidentificationsof additional iron sulfide species occasionallyobserved with greigite in cells (Pósfai et al.,1998a; Pósfai et al., 1998b). Currently recognizedgreigite-producing magnetotactic bacteriaincludes the MMP (Farina et al., 1983; Rodgerset al., 1990a; 1990b; DeLong et al., 1993) and avariety of relatively large, rod-shaped bacteria(Bazylinski et al., 1990; Bazylinski et al., 1993a;Heywood et al., 1990; Heywood et al., 1991;Bazylinski and Frankel, 1992).

The iron sulfide-type magnetosomes containeither particles of greigite (Heywood et al., 1990;Heywood et al., 1991) or a mixture of greigiteand transient non-magnetic iron sulfide phasesthat appear to represent mineral precursors togreigite (Pósfai et al., 1998a; Pósfai et al., 1998b).These phases include mackinawite (tetragonalFeS) and possibly a sphalerite-type cubic FeS(Pósfai et al., 1998a; Pósfai et al., 1998b). Basedon TEM observations, electron diffraction, andknown iron sulfide chemistry (Berner, 1967;Berner, 1970; Berner, 1974), the reaction schemefor greigite formation in the magnetotactic bac-teria appears to be: cubic FeS

Æ mackinawite(tetragonal FeS)

Æ greigite (Fe3S4; Pósfai et al.,1998a; Pósfai et al., 1998b).

The de novo synthesis of non-magnetic crys-talline iron sulfide precursors to greigite alignedalong the magnetosome chain indicates thatchain formation within the cell does not involvemagnetic interactions. Interestingly, under thestrongly reducing, sulfidic conditions at neutralpH in which the greigite-producing magnetotac-tic bacteria are found (Bazylinski et al., 1990;Bazylinski and Frankel, 1992), greigite particleswould be expected to transform into pyrite(Berner, 1967; Berner, 1970) which has not beenunequivocally identified in magnetotactic bacte-ria. It is not known if and how cells prevent thistransformation.

As with magnetite, three particle morpholo-gies of greigite have been observed in magneto-tactic bacteria (Fig. 14): 1) cubo-octahedral (theequilibrium form of face-centered cubic greigite)(Heywood et al., 1990; Heywood et al., 1991); 2)pseudo-rectangular prismatic as shown in Fig. 14and 12e–f (Heywood et al., 1990; Heywood et al.,

Fig. 14. Morphologies of intracellular greigite (Fe3S4) parti-cles produced by magnetotactic bacteria. (a) BrightfieldSTEM image of cubo-octahedra in an unidentified rod-shaped bacterium collected from the Neponset River estuary,Massachusetts, USA. (b) Brightfield STEM image ofrectangular prismatic particles in an unidentified rod-shapedbacterium collected from the Neponset River estuary, Mas-sachusetts, USA. (c) Brightfield TEM image of tooth-shapedand rectangular prismatic particles from the many-celledmagnetotactic prokaryote (MMP), courtesy of M. Pósfai andP. R. Buseck.

100 nm

200 nm

50 nm

a

b

c


1991); and 3) tooth-shaped (Pósfai et al., 1998a;Pósfai et al., 1998b).

Like that of their magnetite counterparts, themorphology of the greigite particles alsoappears to be species- and/or strain-specific,although confirmation of this observation willrequire controlled studies of pure cultures ofgreigite-producing magnetotactic bacteria, noneof which is currently available. One clear excep-tion to this rule is the MMP (Farina et al., 1983;Bazylinski et al., 1990; Bazylinski et al., 1993a;Mann et al., 1990b; Rodgers et al., 1990a; 1990b;Bazylinski and Frankel, 1992). This unusualmicroorganism, found in salt marsh pools allover the world and some deep sea sediments,has been found to contain pleomorphic, pseu-dorectangular prismatic, tooth-shaped, andcubo-octahedral greigite particles. Some of theseparticle morphologies are shown in Fig. 7 and14c. Therefore the biomineralization process(es)appear(s) to be more complicated in this organ-ism than in the rods with greigite-containingmagnetosomes or in magnetite-producing,magnetotactic bacteria.

Magnetite and Greigite Crystals in a SingleBacterium One slow-swimming, rod-shapedbacterium, collected from the OATZ from thePettaquamscutt Estuary, was found to containarrowhead-shaped crystals of magnetite andrectangular prismatic crystals of greigite co-organized within the same chains of magneto-somes (this organism usually contains twoparallel chains of magnetosomes) (Bazylinskiet al., 1993b; Bazylinski et al., 1995). In cells ofthis uncultured organism, the magnetite andgreigite crystals occur with different, mineral-specific morphologies and sizes and are posi-tioned with their long axes oriented along the

chain direction. Both particle morphologies havebeen found in organisms with single mineralcomponent chains (Mann et al., 1987a; Mann etal., 1987b; Heywood et al., 1990; Heywood et al.,1991), which suggests that the magnetosomemembranes surrounding the magnetite andgreigite particles contain different nucleationtemplates and that there are differences inmagnetosome vesicle biosynthesis. Thus, it seemslikely that two separate sets of genes control thebiomineralization of magnetite and greigite inthis organism.

Phylogeny

The phylogeny of many morphotypes of magne-totactic bacteria, including both those in pureculture and those collected from natural environ-ments, has been determined by sequencing their16S rRNA genes. To date, representatives of themagnetotactic prokaryotes are phylogeneticallyassociated with three major lineages within theBacteria. Although most are located within theProteobacteria, “Magnetobacterium bavaricum”is affiliated with another phylum, the newly des-ignated Nitrospira group. Those within the Pro-teobacteria are distributed among the delta- andalpha-subclasses. The uncultured greigite-pro-ducing, MMP and the magnetite-producing, sul-fate-reducing magnetotactic bacterium RS-1,which is available in pure culture, are located inthe delta-subclass, whereas members of thegenus Magnetospirillum and various vibrios andcoccoid magnetotactic bacteria, all of whichproduce magnetite, belong to the alpha-subclass(Fig. 15). Although these results suggest that thetrait of magnetotaxis in bacteria has multipleevolutionary origins (DeLong et al., 1993), it isalso possible that the ability of magnetosome

Fig. 15. Phylogenetic tree based on16S rRNA sequences showing thepositions of cultured and unculturedmagnetotactic bacteria within thealpha-subclass of Proteobacteria.Sequences of uncultured magnetotac-tic bacteria retrieved from freshwaterhabitats are in blue, from marine hab-itats in red, and from a lagoon in pink.

Rhodopseudomonas palustris

Brevundimonas diminuta Paracoccus denitriticansAgrobacterium vitis

Magnetotactic coccus MC1

Unculturedmagnetotactic

bacteria

10%

mabrj58

macpa11mabrj12macpa19

macpa119macca81mabca92macth12

macca13macth24macmp17 macca38

Magnetospirillum magnetotacticumM. gryphiswaildense

Sphingomonas capsulata

Magnetotactic vibria MV1

Oceanospirillum pusillum

Rhodospirillum sallnarum

Rhodopila giobiformis


formation was spread among various phyloge-netic groups of bacteria and even eukaryotes bylateral gene transfer.

To date, most of the 16S rRNA sequences ofmagnetotactic bacteria retrieved from environ-mental samples form a deep-branching groupwithin the alpha-subclass (Fig. 15). This phyloge-netic assemblage consists (up to now) exclusivelyof bacteria displaying magnetotaxis. Similarityvalues of 16S rRNA sequences within this mono-phyletic group of magnetotactic bacteria rangefrom 88.0 to 99.3%. Using in situ hybridizationwith fluorescently-labeled oligonucleotideprobes, it was demonstrated that members of thiscoherent phylogenetic cluster represent thedominant fraction of magnetotactic bacteria inmany environments like lagoons, marine andfreshwater sediments (Spring et al., 1992; Springet al., 1994; Spring et al., 1998). Magnetotacticbacterial morphotypes in this group, asevidenced by in situ hybridization, are mainlyrepresented by coccoid to ovoid bacteria, butalso include one rod- to vibrio-shaped bacterium(mabcs92; Fig. 15). Despite continuous effort inseveral laboratories, most members of this grouphave resisted attempts (in several laboratories)at isolation to axenic culture. One major reasonmay be their adaptation to and requirement forgradient systems not easily replicated in syn-thetic growth media. The only exception is themarine magnetotactic coccus strain MC-1, whichcan be cultivated in a synthetic oxygen gradientmedium.

Cultivation and Physiology

The Genus Magnetospirillum

Taxonomy Magnetospirilla are found in fresh-water habitats where they usually occur in lownumbers as, for example, compared with themagnetotactic cocci. These clockwise spirillahave dimensions of 0.2–0.7 by 1–20 mm and dis-play an axial magneto-aerotaxis, at least whengrown in liquid culture. The genus Magnetospir-illum currently comprises the two validlydescribed species, M. magnetotacticum and M.gryphiswaldense, and several partially character-ized strains. M. magnetotacticum was the firstmagnetotactic bacterium isolated and grown inpure culture and was originally assigned to thegenus Aquaspirillum based on a number of phe-notypic characteristics (Maratea and Blakemore,1981). At that time, this genus contained a largenumber of phylogenetically diverse, nonpho-totrophic, freshwater spirilla with the type spe-cies A. serpens phylogenetically located amongthe beta-subclass of the Proteobacteria. Phyloge-netic analyses of Magnetospirillum strains laterrevealed that they all belong to a phylogenetic

branch within the alpha-subclass of the Proteo-bacteria and are closely related to phototrophicspirilla of the genus Phaeospirillum (Fig. 15; Bur-gess et al., 1993; Schüler et al., 1999). Therefore,it was justified to propose the new genus Mag-netospirillum for these strains (Schleifer et al.,1991). Members of this genus can be distin-guished from other freshwater spirilla by theirability to produce membrane-enveloped cubo-octahedral magnetite crystals, averaging about42 nm in diameter (Balkwill et al., 1980; Schleiferet al., 1991), arranged in a single chain within thecell. Other characteristic traits of members ofthis genus include bipolar monotrichous flagella-tion and a preference for microoxic growth con-ditions. Several strains of magnetospirilla cangrow also anaerobically with nitrate as terminalelectron acceptor or aerobically with atmo-spheric concentrations of oxygen. Magnetite syn-thesis appears to only occur under microaerobicconditions in most species while Magneto-spirillum strain AMB-1 appears to synthesizemagnetite under anaerobic conditions as well(Matsunaga and Tsujimura, 1993). Preferredsubstrates are intermediates of the tricarboxylicacid cycle and acetate. Carbohydrates are notutilized. Catalase and oxidase may be present ornot. The guanine-plus-cytosine content of DNAranges from 64 to 71 mol% (Burgess et al., 1993).

Biochemistry and Molecular Biology ofMagnetosome Formation There has been muchinterest in the elucidation of magnetosomeformation because the crystals synthesized bymagnetotactic bacteria are of great structuralperfection, have consistent particle morpholo-gies and narrow size distributions, possibleindications that the particles may have novelmagnetic, physical and/or electrical properties.Understanding the factors controlling the bio-mineralization of iron in magnetosome synthesiswithin bacteria could also be helpful for the elu-cidation of similar processes in animals and manor for the artificial synthesis of biominerals.Despite the dedicated and elaborate efforts instudying magnetosome synthesis in bacteria,published results are rather sparse. This is partlydue to the lack of a significant number of mag-netotactic bacteria strains and the difficulty inculturing them reproducibly in the laboratory,which would be a prerequisite for the establish-ing of biochemical or genetic model systems.Nevertheless, some interesting results have beenobtained using the few available, but fastidiousMagnetospirillum strains.

In general, the bacterial magnetite synthesiscan be divided into three steps. Initially, extracel-lular iron has to be transported across the cellwall to the inside of the cell. Once within the cell,iron must accumulate in specialized compart-


ments, the magnetosome vesicles. There, the ironpresumably precipitates and transforms or growsinto a single-magnetic-domain magnetite crystalwith a specific morphology. It is assumed that themembrane vesicle is synthesized prior to the pre-cipitation of iron but since there is currently littleevidence to support this idea, it is possible thatthe precipitation of iron and crystal nucleationoccurs first and the magnetosome membranethen forms around the growing crystal. Theuptake of iron from the surrounding environ-ment by cells of Magnetospirillum strains hasbeen analyzed by several groups (Paoletti andBlakemore, 1986; Nakamura et al., 1993b;Schüler and Baeuerlein, 1996; Schüler andBaeuerlein, 1998). Generally, the results suggestthat iron is taken up by the cell in the ferric formand transported across the membrane by anenergy-dependent reductive process. Iron-binding siderophores were thought to beinvolved in iron uptake by M. magnetotacticum(Paoletti and Blakemore, 1986), which appearedto produce a hydroxamate siderophore underhigh, but not low, iron conditions. However, thisfinding was never confirmed by other laborato-ries. Spent culture fluid stimulates the uptake offerric iron in M. gryphiswaldense although therewas no evidence for the production of a sidero-phore by this species. This stimulation may bedue to the production of unknown compounds,produced by cells during growth, which mediateiron uptake by an unrecognized novel mecha-nism (Schüler and Baeuerlein, 1996). In thisrespect, it is noteworthy that most magnetotacticbacteria are adapted to microenvironments, likethe oxic-anoxic transition zone of sediments,where soluble iron is available to the cell insufficient quantities for magnetite synthesis (gen-erally about 10–20 mM iron; Blakemore et al.,1979). Thus, magnetotactic bacteria probablyhave no need for high-affinity transport systemslike many other aerobic bacteria growing underiron deficient conditions. This is consistentwith experiments performed with cells ofM. gryphiswaldense. Under iron deficient condi-tions, cells of M. gryphiswaldense do not or can-not distinguish between the use of incorporatediron either as a cofactor for cellular proteins orfor magnetite synthesis and store this essentialelement as an inorganic mineral, magnetite, atthe expense of their own growth (Schüler andBaeuerlein, 1996). The marine magnetotacticvibrio, strain MV-1, behaves similarly.

The fate of iron taken up by cells was studiedby Frankel et al. (1983) in M. magnetotacticumusing Fe57 Moessbauer spectroscopy. It was pro-posed that the ferrous iron taken up by cells isimmediately reoxidized to form a low-densityhydrous Fe(III) oxide. It is not yet clear if thisstep takes place in the cytoplasm or in the mag-

netosome vesicles. How iron is transported fromthe cell membrane into the magnetosome vesicleis also not known. Iron is precipitated within themagnetosome vesicle presumably through adehydration step as ferrihydrite (a high-densityFe(III) hydroxide). Finally, magnetite (Fe3O4) isproduced by the reduction of one-third ofthe Fe(III) ions in ferrihydrite and furtherdehydration steps. The crystallization pro-cess(es) involved in magnetite formation isapparently linked closely to the magnetosomemembrane and may be controlled by specificproteins present in this membrane. The chemicaltransformation of amorphous Fe(III) precursorsto crystalline magnetite is sensitive to environ-mental conditions like ion concentration, pH andredox potential (Mann et al., 1990c) which haveto be therefore precisely regulated by the mag-netosome membrane or by conditions within themagnetosome membrane vesicle. Growth of themagnetite crystal, i.e., its orientation, shape andsize, must also be under strict control becausethese characteristics are specific for one strainand/or species of bacteria and to a great extentindependent from the growth conditions(Bazylinski et al., 1994).

Because the magnetosome membrane seemsto play a key role in the synthesis of magnetitecrystals, its structure and composition has beenanalyzed in several studies. By analyzing themagnetosome membrane in this way, some cluesrelating to how magnetite biomineralizationoccurs within the cell may be found. Gorby etal. (1988) showed that the magnetosomemembrane in M. magnetotacticum has anarchitecture similar to that of the cytoplasmicmembrane and consists of a lipid bilayer andnumerous proteins, some of which appear to beunique to the magnetosome membrane. Okudaet al. (1996) found three proteins with molecularweights of 12, 22 and 28 kDa, specifically asso-ciated with the magnetosome membrane inM. magnetotacticum. They successfully identi-fied and sequenced the gene encoding for the22 kDa protein, which was found to belong to afamily of protein import receptors common inmitochondria and peroxisomes. The role of thisprotein in magnetosome synthesis remainsunclear however. A gene likely involved in mag-netite synthesis was identified and characterizedby Matsunaga et al. (1992). They used a geneticapproach using the microorganism Magnetospir-illum strain AMB-1, which forms colonies ofmagnetite-forming cells on agar surfaces,thereby facilitating the screening for non-magnetic mutants. The gene, designated magA,encodes for a membrane protein showingsequence similarities to some cation efflux pro-teins. Based on experiments with the recombi-nant protein, it was proposed that the MagA


protein plays a role in the energy-dependenttransport of iron across membranes.

Enrichment and Isolation Magnetotactic spi-rilla have been repeatedly isolated from variousfreshwater habitats, so that it is possible to givesome guidelines for their succesful enrichmentand isolation.

Several morphotypes of magnetotactic bacte-ria can be enriched in the laboratory by puttingmud and overlying water from a sampling siteinto aquaria or jars, which are loosely coveredand stored in dim light. After several days toweeks, the number of magnetotactic bacterialcells generally increases significantly. Magneto-tactic spirilla, however, are in most cases notamong the dominating morphotypes and there-fore are only rarely detected using light micros-copy. Consequently, the usefulness of thismethod for the enrichment of representatives ofthe genus Magnetospirillum remains question-able. To date, no selective growth media areknown for the cultivation of magnetotactic spi-rilla, so that a successful isolation procedure willin most cases depend on the purity of the inocu-lum. Because of the magnetic dipole moment ofthese bacteria, physical separation fromnonmagnetotactic contaminants is possible. Acommonly used method for the separation ofmagnetotactic bacteria from sediment sampleswas described by Moench and Konetzka (1978).They concentrated magnetotactic bacterial cellsusing a bar magnet (e.g., stirring bar) fixed to theouter wall of a jar filled with sediment and water.Directed magnetotactic bacteria eventually accu-mulate at the side of the jar and become concen-trated enough to form (opposite to the magnet)a brownish spot from where they can be trans-ferred into a sterile cap using a Pasteur pipette.The sample containing the concentrated magne-totactic cells still cotains non-magnetotacticbacteria, so that a further purification step isadvisable before it is used as an inoculum forgrowth media. The “capillary racetrack” devisedby Spormann and Wolfe (Wolfe et al., 1987) hasbeen successfully used for this purpose (Schüleret al., 1999).

All isolated strains of magnetospirilla, with thepossible exception of Magnetospirillum strainAMB-1, appear to prefer low oxygen tensionsfor growth and magnetite synthesis. Thus the cre-ation and maintenance of microoxic conditionsin growth media is especially important for theisolation of these organisms starting from smallinocula. The growth medium should contain 10–20% of sterilized mud or water from the respec-tive habitat and low concentrations of agar toallow the establishment of a semisolid oxygengradient. Suitable carbon sources are intermedi-ates of the tricarboxylic acid cycle, e.g., malate or

succinate. An oxygen-sulfide gradient mediumwas successfully used by Schüler et al. (1999) forthe effective isolation of magnetotactic spirillafrom a freshwater pond. Screw-capped culturetubes are filled with 1 ml of solid sulfide agar(4 mM Na2S, 1.5% agar, pH 7.4) and overlaidwith 10 ml of slush-agar. The slush-agar consistsof (per 800 ml deionized water): 200 ml of fil-tered pond water, 1 ml of vitamin elixir, 2 ml ofmineral elixir (Wolin et al., 1963), 0.05 g, sodiumsuccinate, 0.05 g, yeast extract, 0.05 g, NH4Cl,0.05 g, MgSO4, 0.5 mM potassium phosphatebuffer (pH 7.0); 2 mg of resazurin and 2 g of agar.After adjusting the pH to 7.0 and autoclaving,sterile solutions of ferric citrate and neutralizedcysteine·HCl are added (final concentrations 10mM and 0.01%, respectively). The culture tubescan be inoculated after the establishment of sul-fide and oxygen gradients within the medium,which takes about 24 hours. Several days toweeks of incubation at room temperature in thedark may be required until growth becomesapparent, usually as fluffy pinpoint colonies.

Although the original inoculum always con-tains various types of magnetotactic bacteria, inmost cases only magnetotactic spirilla grow andare eventually isolated in pure culture. Modifica-tions of this medium may eventually prove usefulfor the isolation of hitherto uncultured types ofmagnetotactic bacteria.

Following isolation, most strains of magneto-spirilla can be cultured in liquid media withoutadded water or mud from the sampling site.However, the gas composition of the headspaceof the cultures is crucial for good growth andmagnetite synthesis by most species. The maxi-mum concentration of oxygen allowing growthand/or magnetite synthesis differs among thedescribed Magnetospirillum strains. M. magneto-tacticum grows optimally and produces the high-est number of magnetosomes at an oxygentension of 1% in the headspace and tolerateshigher initial oxygen concentrations only if alarge number of cells is inoculated into thegrowth medium. In contrast, Magnetospirillumstrain AMB-1 grows (but does not produce mag-netosomes) aerobically under atmospheric con-centrations of oxygen (approximately 21% O2;Matsunaga et al., 1991b). Magnetite synthesis isinhibited by all Magnetospirillum strains whencells are cultured under oxygen concentrationsabove 2 to 6% (Blakemore et al., 1985; Schülerand Baeuerlein, 1998).

Strain MV-1, a Facultatively Anaerobic Magnetotactic Vibrio

A marine magnetotactic vibrioid to helicoid bac-terium, strain MV-1, was isolated by Bazylinskiet al. (1988). Cells of strain MV-1 are small, rang-


ing from 1–5 mm by 0.2–0.5 mm, and possess asingle, unsheathed, polar flagellum (Fig. 16a).Cells grow and synthesize pseudohexahedralprismatic crystals of magnetite, averaging 53 by35 nm in size (Fig. 16b; Sparks et al., 1990), intheir magnetosomes microaerobically and anaer-obically, with nitrous oxide as the terminal elec-tron acceptor. Cells appear to produce moremagnetite under anaerobic conditions thanunder microaerobic conditions (Bazylinski et al.,1988) and, like M. magnetotacticum, synthesize anumber of magnetosome membrane proteinsthat are not present in other cellular fractions(Dubbels et al., 1998). A stable, spontaneousnonmagnetotactic mutant strain of MV-1 thatdoes not produce magnetosomes has recentlybeen isolated and partially characterized(Dubbels and Bazylinski, 1998).

Strain MV-1 is nutritionally versatile beingable to grow chemoorganoheterotrophicallywith organic and some amino acids as carbon and

energy sources, and chemolithoautotrophicallywith thiosulfate or sulfide as energy sources oxi-dizing them to sulfate, and carbon dioxide as thesole carbon source (Kimble and Bazylinski,1996). Cells produce intracellular sulfur depositswhen grown with sulfide (Kimble and Bazylinski,1996). As do virtually all aerobic chemolithoau-totrophic bacteria, strain MV-1 uses the Calvin-Benson cycle for autotrophic carbon dioxide fix-ation (McFadden and Shively, 1991). Cell-freeextracts from thiosulfate-grown cells of strainMV-1 show ribulose bisphosphate carboxylase/oxygenase (rubisCO) activity (Kimble andBazylinski, 1996), and recently (Dean and Bazy-linski, 1999a) the gene for a form II rubisCOenzyme (cbbM) was cloned and sequenced fromstrain MV-1. There was no evidence for a cbbLgene (encodes for form I rubisCO enzymes) inDNA hybridization analyses despite using cbbLgene probes from several different organisms.Because many uncultured magnetotactic bacte-ria collected from natural habitats thrive inoxygen-sulfide inverse gradients, as previouslymentioned, and contain internal sulfur deposits(Moench, 1988; Spring et al., 1993; Frankel andBazylinski, 1994; Iida and Akai, 1996; Kimbleand Bazylinski, 1996), it seems many species arelikely chemolithoautotrophs that obtain energyfrom the oxidation of sulfide and perhaps otherreduced sulfur sompounds. Using pulsed-fieldgel electrophoresis (PFGE), the genome ofstrain MV-1 was found to consist of a single,circular chromosome of approximately 3.7 Mb(Dean and Bazylinski, 1999b). There was no evi-dence of linear chromosomes or extrachromo-somal DNA such as plasmids. The guanine-plus-cytosine content of the DNA of this strain is52.9 mol% as determined by HPLC and53.5 mol% by Tm.

A virtually identical strain to strain MV-1, des-ignated MV-2, was isolated from the Pettaquam-scutt Estuary (DeLong et al., 1993; Meldrum etal., 1993b). Cells of this strain produce the samemorphological type of magnetite crystals asstrain MV-1 (Meldrum et al., 1993b) and displaymany of the same phenotypic traits as strainMV-1 (such as anaerobic growth with nitrousoxide as a terminal electron acceptor, het-erotrophic growth with organic and amino acids,and chemolithoautotrophic growth on reducedsulfur compounds). However, strain MV-2 showsslightly different restriction fragment patterns inpulsed-field gels than strain MV-1 using the samerestriction enzymes (Dean and Bazylinski,1999b). As with strain MV-1, the genome ofstrain MV-2 consists of a single, circular chromo-some of a similar size, about 3.6 Mb (Dean andBazylinski, 1999b). The guanine-plus-cytosinecontent of the DNA of this strain is 56.2 mol%as determined by HPLC and 56.6% by Tm.

Fig. 16. Brightfield TEM image of negatively stained cell andmagnetosomes of strain MV-1. (a) Cell stained with uranylacetate showing a single polar flagellum and a chain ofmagnetite-containing magnetosomes. (b) Preparation ofpurified magnetosomes from strain MV-1 stained with 2%aqueous sodium phosphotungstate, pH 7.0. The “magneto-some membrane” is visualized as an electron-lucent areasurrounding each individual crystal and is easily removedwith detergents such as sodium deodecyl sulfate.

100 nm

1 µm

a

b


Strain RS-1, a Sulfate-Reducing Magnetotactic Bacterium

It was thought for a long time that all magneto-tactic bacteria are obligate or facultativemicroaerophiles (Magnetospirillum strain AMB-1 and the marine vibrio, strain MV-1, growanaerobically with nitrate and nitrous oxide,respectively, as well as with oxygen) adapted tothe microoxic zone of their environment. Withthe isolation of an obligately anaerobic strainfrom a sulfidic freshwater habitat by Sakaguchiet al. (1993), this assumption is clearly incorrect.Cells of this organism, designated strain RS-1,are 0.9–1.5 by 3–5 mm with a helicoid to rod-shaped morphology and possess a single polarflagellum. They exhibit an axial magnetotaxiscoupled with a strong anaerotaxis reflecting theirobligate anaerobic metabolism. According to therevised model of magnetotaxis, they may havedeveloped an axial magnetotaxis because theydo not have to oscillate between microoxic andanoxic zones of their habitat, which would selectfor polar magnetotaxis.

Strain RS-1 is a dissimilatory sulfate-reducing,chemoorganoheterotrophic bacterium that uti-lizes a variety of organic substrates, e.g., pyru-vate, lactate, ethanol and fumarate). Cells canuse sulfate or fumarate as electron acceptor butnot oxygen. They are catalase positive and oxi-dase negative. The guanine-plus-cytosine contentof DNA was determined by HPLC to be66 mol%.

Sequencing of the 16S rRNA gene of strainRS-1 showed that it is phylogenetically affiliatedto the delta-subclass of the Proteobacteria(Kawaguchi et al., 1995). The nearest neighborsin a phylogenetic tree are members of the genusDesulfovibrio, typical representatives of the obli-gately anaerobic, dissimilatory sulfate-reducingbacteria. In contrast to Desulfovibrio sp., cells ofstrain RS-1 are able to produce intracellularbean-shaped crystals of magnetite, responsiblefor its magnetotactic response. Consequently, theproduction of magnetosomes consisting of mag-netite is found in bacteria belonging to threedifferent phylogenetic groups, viz. the a- and d-subclasses of Proteobacteria and the Nitrospiragroup (“Magnetobacterium bavaricum”), indi-cating multiple evolutionary origins of intracel-lular magnetite synthesis or lateral gene transferbetween different phylogenetic groups.

Other Magnetotactic Strains in Pure Culture

Several other pure cultures of magnetotacticbacteria exist, but they appear to be obligatemicroaerophiles and grow poorly (D. A. Bazylin-ski, unpublished results). Hence, very little isknown about them. Strain MC-1 (Fig. 17), a

marine biliophotrichous coccus, was isolatedfrom water collected from the PettaquamscuttEstuary, a chemically-stratified semi-anaerobicbasin in Rhode Island, USA. Cells of this strainproduce pseudohexahedral prisms of magnetite,averaging 72 by 70 nm in size (when grownautotrophically), and grow chemolithoau-totrophically with thiosulfate or sulfide as anelectron and energy source (Meldrum et al.,1993a; Frankel et al., 1997). Cells may also beable to grow chemoorganoheterotrophically.Like all magnetotactic cocci observed, cells ofstrain MC-1 show polar magneto-aerotaxisregardless of whether they are grown in liquid orsemi-solid oxygen gradient media. This strain hasa genome size of approximately 4.5 Mb asdetermined by pulsed-field gel electrophoresis(Dean and Bazylinski, 1999b). The guanine-plus-cytosine content of the DNA of strain MC-1, asdetermined by HPLC, is 55.8 mol%. This organ-ism has not been completely characterized anddescribed.

Strain MV-4 (Fig. 18), a small marine spiril-lum, was isolated from sulfide-rich mud andwater collected from School Street Marsh,Woods Hole, Massachusetts, USA. Cells of thisstrain produce elongated octahedrons of magne-tite, averaging 61 by 52 nm in size, and growchemolithoautotrophically with thiosulfate orchemoorganoheterotrophically with succinate(Meldrum et al., 1993b). Unlike most freshwatermagnetotactic spirilla, this strain shows polarmagneto-aerotaxis at least when grown in semi-solid oxygen gradient media. Like strain MC-1,

Fig. 17. Brightfield TEM image of a cell of the bilophotric-hously flagellated marine coccus strain MC-1 negativelystained with uranyl acetate. Note the two flagellar bundles(fb), the presence of pili (p), sulfur globules(s), and chain ofFe3O4-containing magnetosomes (m).

0.5 µm

fbp

ms


this strain has not been completely characterizedand described.

Biotechnological Applications

It was not long after the discovery of magneto-tactic bacteria that publications of physical stud-ies and of commercial and medical applicationsinvolving the magnetotactic cells, isolated mag-netosomes and/or magnetite crystals began toappear. It is clear that magnetotactic bacterialcells and their magnetic crystals have novel phys-ical, magnetic and possibly electrical properties.In addition, in certain types of applications,bacterial magnetite offers several advantagescompared to chemically synthesized magnetite.Bacterial magnetosome particles, unlike thoseproduced chemically, have a consistent shape, anarrow size distribution within the single mag-netic domain range, and a membrane coatingconsisting of lipids and proteins. The magneto-some envelope allows for easy couplings of bio-active substances to its surface, a characteristicimportant for many applications.

Magnetotactic bacterial cells have been usedto determine south magnetic poles in meteoritesand rocks containing fine-grained magnetic min-erals (Funaki et al., 1989; Funaki et al., 1992) andfor the separation of cells after the introductionof magnetotactic bacterial cells into granulocytesand monocytes by phagocytosis (Matsunaga etal., 1989). Magnetotactic bacterial magnetitecrystals have been used in studies of magneticdomain analysis (Futschik et al., 1989) and inmany commercial applications including: theimmobilization of enzymes (Matsunaga andKamiya, 1987); the formation of magnetic anti-bodies in various fluoroimmunoassays

(Matsunaga et al., 1990) involving the detectionof allergens (Nakamura and Matsunaga, 1993a)and squamous cell carcinoma cells (Matsunaga,1991a), and the quantification of IgG (Nakamuraet al., 1991); the detection and removal ofEscherichia coli cells with a fluorescein isothio-cyanate conjugated monoclonal antibody, immo-bilized on magnetotactic bacterial magnetiteparticles (Nakamura et al., 1993c); and the intro-duction of genes into cells, a technology in whichmagnetosomes are coated with DNA and “shot”using a particle gun into cells that are difficultto transform using more standard methods(Matsunaga, 1991a). Unfortunately, the prereq-uisite for any large scale commercial applicationis mass cultivation of magnetotactic bacteria orthe introduction and expression of the genesresponsible for magnetosome synthesis into abacterium, e.g., E. coli, that can be grown rela-tively cheaply to extremely large yields.Although some progress has been made, theformer has not been achieved with the availablepure cultures.

Acknowledgements. We thank F.C. Meldrum, M.Pósfai, P.R. Buseck, and M. Hanzlik for use offigures and L. Cox, D. Schüler and R.B. Frankelfor helpful discussions and suggestions concern-ing this chapter. S.S. is grateful to K.-H. Schleiferand the Deutsche Forschungsgemeinschaft(DFG) for continuous support. Research in thelaboratory of D.A.B. is supported by U.S.National Science Foundation grant CHE-9714101 and U.S. National Aeronautics andSpace Administration grant NAG 9-1115.

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the prokaryotes || magnetotactic bacteria

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