Genetic Dissection of the mamAB and mms6 Operons Reveals a GeneSet Essential for Magnetosome Biogenesis in Magnetospirillumgryphiswaldense

Anna Lohße,a Sarah Borg,a Oliver Raschdorf,a Isabel Kolinko,a Éva Tompa,b Mihály Pósfai,b Damien Faivre,c Jens Baumgartner,c

Dirk Schülera*

Ludwig-Maximilians-Universität München, Departement Biologie I, Mikrobiologie, LMU Biozentrum, Planegg Martinsried, Germanya; University of Pannonia, Departmentof Earth and Environmental Sciences, Veszprém, Hungaryb; Max-Planck-Institut für Kolloid- und Grenzflächenforschung, Potsdam, Germanyc

Biosynthesis of bacterial magnetosomes, which are intracellular membrane-enclosed, nanosized magnetic crystals, is controlledby a set of >30 specific genes. In Magnetospirillum gryphiswaldense, these are clustered mostly within a large conserved genomicmagnetosome island (MAI) comprising the mms6, mamGFDC, mamAB, and mamXY operons. Here, we demonstrate that thefive previously uncharacterized genes of the mms6 operon have crucial functions in the regulation of magnetosome biomineral-ization that partially overlap MamF and other proteins encoded by the adjacent mamGFDC operon. While all other deletionsresulted in size reduction, elimination of either mms36 or mms48 caused the synthesis of magnetite crystals larger than those inthe wild type (WT). Whereas the mms6 operon encodes accessory factors for crystal maturation, the large mamAB operon con-tains several essential and nonessential genes involved in various other steps of magnetosome biosynthesis, as shown by singledeletions of all mamAB genes. While single deletions of mamL, -P, -Q, -R, -B, -S, -T, and -U showed phenotypes similar to thoseof their orthologs in a previous study in the related M. magneticum, we found mamI and mamN to be not required for at leastrudimentary iron biomineralization in M. gryphiswaldense. Thus, only mamE, -L, -M, -O, -Q, and -B were essential for forma-tion of magnetite, whereas a mamI mutant still biomineralized tiny particles which, however, consisted of the nonmagnetic ironoxide hematite, as shown by high-resolution transmission electron microscopy (HRTEM) and the X-ray absorption near-edgestructure (XANES). Based on this and previous studies, we propose an extended model for magnetosome biosynthesis in M.gryphiswaldense.

Magnetotactic bacteria (MTB) orient along Earth’s magneticfield lines to navigate to their growth-favoring microoxic

habitats within stratified aquatic sediments (1). This behavior isenabled by the synthesis of ferrimagnetic intracellular organellestermed magnetosomes (2). In the alphaproteobacterium Magne-tospirillum gryphiswaldense and related MTB, magnetosomes con-sist of crystals of the magnetic iron oxide magnetite (Fe3O4) en-closed by the magnetosome membrane (MM), which contains aspecific set of about 30 proteins (3, 4). The biosynthesis of mag-netosomes is a complex process that comprises the (i) invagina-tion of vesicles from the inner membrane (5, 6), (ii) sorting ofmagnetosome proteins to the MM (7), (iii) iron transport andcrystallization of magnetite crystals (8), (iv) crystal maturation(7), and (v) assembly as well as positioning of mature crystals intoa linear chain along a filamentous cytoskeletal structure (6, 9).Each step is under strict genetic control, and responsible geneswere found to be located mostly within a genomic magnetosomeisland (MAI) (10, 11), comprising the mms6 operon (mms6op),mamGFDC operon (mamGFDCop), mamAB operon (mamABop),and mamXY operon (mamXYop) (10–12). These operons werefound to be highly conserved also in the closely related Magneto-spirillum magneticum (13–17). It has been shown that the regionsbetween and flanking the identified magnetosome operons haveno functional relevance for magnetosome biosynthesis in M. gry-phiswaldense and M. magneticum (7, 18). In M. gryphiswaldense,the mms6, mamGFDC, mamAB, and mamXY operons are tran-scribed as single polycistronic messengers under the control of thePmms6, PmamDC, PmamH, and PmamXY promoters, respectively (19,20). A mutant with a deletion of mamGFDCop, encoding the most

abundant magnetosome proteins, retained the ability to formmagnetic, although smaller and less regular, magnetosomes, whileplasmidal overexpression of the entire mamGFDC operon yieldedmagnetite particles even larger than those produced by the wildtype (WT) (21). Elimination of the corresponding region R3 in M.magneticum, comprising in addition parts of mms6op, caused asevere biomineralization defect, resulting in cells with reducedmagnetosome sizes and numbers (7). Deletion of the entiremamXY operon resulted in smaller and misshaped magnetosomeparticles in M. gryphiswaldense (18), whereas no obvious pheno-type was observed for mamXYop deletion in M. magneticum (7).

mms6op of M. gryphiswaldense comprises the genes mgr4074,mms6, mmsF, mgr4071 (renamed here mms36), and mgr4070 (re-named here mms48; see Fig. S1 in the supplemental material),which was previously predicted to encode a TPR (tetratricopep-tide repeat)-like protein (18). A mutant in which the entiremms6op was deleted (�A10) was also severely impaired in the

Received 3 April 2014 Accepted 6 May 2014

Published ahead of print 9 May 2014

Address correspondence to Dirk Schüler, dirk.schueler@uni-bayreuth.de.

* Present address: Dirk Schüler, University of Bayreuth, Department ofMicrobiology, Bayreuth, Germany.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01716-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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biomineralization of magnetite crystals, which exhibited defectsin crystal morphology, size, and organization. However, the indi-vidual functions of mgr4074, mms6, mmsF, mms36, and mms48 aswell as their contribution to the strong phenotype correspondingto �mms6op have remained unknown. In M. magneticum, themms6 cluster was described to comprise only amb0955 (mgr4074),amb0956 (mms6), and amb0967 (mmsF) and to lack homologs ofmms48 and mms36 (22). Single gene deletions of mms6 in M.magneticum by different groups revealed inconsistent phenotypes.Whereas Tanaka et al. (23) reported an important regulatoryfunction of Mms6 for magnetosome morphology, Murat et al.observed only minor effects on magnetosome biosynthesis afterdeletion of mms6 in vivo (22, 24). In vitro, the small (12.76 kDa inM. gryphiswaldense and 14.69 kDa in M. magneticum) Mms6 pro-tein was shown to be tightly bound to isolated bacterial magnetitecrystals as visualized by atomic force microscopy and transmis-sion electron microscopy (TEM) (25, 26). In vitro crystallizationexperiments suggested that Mms6 and peptides mimicking it haveiron-binding activity and affected the formation of cubo-octahe-dral crystal morphologies (27, 28).

In contrast to the smaller accessory operons, mamABop wasfound to contain genes absolutely essential for magnetosome bio-synthesis in M. gryphiswaldense and M. magneticum (18, 22).Whereas mamABop was found to be sufficient to support at leastsome rudimentary biomineralization of small magnetite crystalseven in the absence of all other magnetosome operons in bothstrains (18, 22), the mamXY, mamGFDC, mms6, and mamABoperons were required all together for magnetite biomineraliza-tion upon their transfer into the foreign host Rhodospirillumrubrum (29).

A recent comprehensive genetic dissection of mamABop in M.magneticum revealed that mamH, -P, -R, -S, and -T encode acces-sory functions for magnetosome synthesis, since mutants displayvarious biomineralization defects, whereas mamU and -V had noobvious magnetosome phenotype (7). As in M. gryphiswaldense(see below), mamK and mamJ were implicated in magnetosomechain assembly, but their loss did not affect biomineralization (30,31). However, deletions of mamI, -E, -L, -M, -N, -O, and -Q as wellas mamB (codeleted with their respective orthologs) fully abol-ished magnetosome synthesis in M. magneticum (7, 32). WhereasMamI, -L, -Q, and -B were suggested to be essential for vesiclegenesis, MamE, -O, -M, and -N were classified to be mainly re-quired only for magnetite crystallization (7). The discovery of asmall “magnetosome islet” in the genome of M. magneticum withfurther copies of mamE, -J, -K, -L, -M, and -F as well as mamDsuggested genetic redundancy that has to be clarified with respectto determination of the minimal essential gene set (33).

In M. gryphiswaldense, the 16.4-kb mamAB operon contains 17genes (mamH, -I, -E, -J, -K, -L, -M, -N, -O, -P, -A, -Q, -R, -B, -S,-T, and -U) (see Fig. 3). Only a few genes of mamABop so far havebeen analyzed individually in this organism. The actin-like pro-tein MamK forms a filamentous structure for magnetosome as-sembly and interacts with the acidic protein MamJ, which is in-volved in connecting magnetosomes to the filament. Bothproteins, however, have no or only minor effects on biomineral-ization (6). Deletion of mamH caused a moderate decrease ofmagnetosome number and size, whereas codeletion of mamH andits partial homolog mamZ had a considerably stronger effect, withonly very few or no regular crystals detectable in the cells (20).Deletion of mamE, -O, -M, and -B either resulted in a total inhi-

bition of crystal nucleation or prevented MM vesicle synthesis (18,34, 35). However, mamI, -L, -N, -P, -A, -Q, -R, -S, -T, and -U havenot yet been analyzed individually by mutagenesis, and it has re-mained unknown whether they have functions similar to or dis-tinct from those of their corresponding orthologs in M. magneti-cum. Finally, it is not clear which genes constitute the minimal setof essential determinants for magnetosome biomineralization inM. gryphiswaldense.

In this study, we analyzed the functional relevance of proteinsencoded by mms6op and mamABop for the biosynthesis of mag-netic minerals in M. gryphiswaldense. We demonstrate that be-sides Mms6 and MmsF, mms6op of M. gryphiswaldense encodestwo further important regulators (Mms36 and Mms48) for mag-netosome biomineralization. Whereas deletions of mamA, -R, -S,-T, and -U resulted in phenotypes similar to those observed fordeletion of homologous genes in M. magneticum, we show that incontrast to the case with M. magneticum, M. gryphiswaldense�mamN and �mamI mutants still synthesize particles, thus fur-ther shrinking the minimal gene set for iron biomineralization tomamE, -L, -M, -O, -Q, and -B. Finally, we propose an extendedmodel for magnetosome biosynthesis.

MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. Bacterial strains andplasmids used in this study are listed in Table S1 in the supplementalmaterial. WT and mutant strains of M. gryphiswaldense were grown inliquid modified flask standard medium (FSM) at 30°C under microaero-bic conditions if not otherwise specified (8, 36). Therefore, cells werecultivated in flasks, closed with butyl-rubber stoppers after incubationwith a gas mixture of 2% O2 and 98% N2, or in purged jars. For anaerobicrequirements, O2 was excluded from the gas mixture, while aerobic con-ditions were generated through free gas exchange with air. Escherichia colistrains were cultivated as previously described (37), and lysogeny brothmedium was supplemented with 1 mM DL-�,ε-diaminopimelic acid(DAP) for cultivation of E. coli strain BW29427 as well as WM3064. Forselection of antibiotic-resistant cells, media were supplemented with 25g/ml of kanamycin (Km), 12 g/ml of tetracycline (Tet), and 15 g/ml ofgentamicin (Gm) for E. coli strains and 5 g/ml of Km, 5 g/ml of Tet, and 20g/ml of Gm for M. gryphiswaldense strains.

Molecular and genetic techniques. Oligonucleotide sequences (seeTable S2 in the supplemental material) were deduced from the workingdraft genome sequence of M. gryphiswaldense (GenBank accession num-ber CU459003), and sequences were purchased from Sigma-Aldrich(Steinheim, Germany). Genetic fragments were amplified by standardPCR procedures with Phusion polymerase (NEB GmbH, Frankfurt amMain, Germany), and generated plasmids were sequenced with an ABI3700 capillary sequencer (Applied Biosystems, Darmstadt, Germany),utilizing BigDye Terminator v3.1. Data were analyzed with Vector NTIAdvance 11.5 (Invitrogen, Darmstadt, Germany) or MacVector 7.2.3(Oxford Molecular, Oxford, United Kingdom) software.

Generation of unmarked deletion mutants. Markerless single genedeletions within the mamAB, mms6, and mamGFDC operons were par-tially realized with the pORFM_galK plasmid (59). The vector was di-gested with BamHI and KpnI to insert the approximately 1-kb down-stream and upstream fragments of mamI, -L, -N, -P, -Q, -R, -S, -T, and -U,mms36, mms48, and mmsF_mms6. For integration of homologous regionsof mamA and mamL, the plasmid was digested with BamHI/NotI andNsi/SpeI, respectively. Oligonucleotides used to amplify the 5= and 3=flanking sequence from M. gryphiswaldense by PCR are listed in Table S3in the supplemental material. Both fragments were linked by an overlapPCR with the first and last listed corresponding oligonucleotide, sub-cloned into pJet1.2/blunt, sequenced, and ligated into the digestedpORFM_galK vectors. Generated plasmids were termed pAL_�mamI,

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pOR�mamL, pAL_�mamN, pAL_�mamP, pAL_�mamA, pAL_�mamQ,pAL_�mamR, pAL_�mamS, pAL_�mamT, pAL_�mamU, pAL_�mms36,pAL_�mms48, and pAL_�mmsF �mms6. Deletion of mms6, mmsF, andmamF and double deletion of mmsF and mamF was accomplished by double-crossover method. Oligonucleotides for amplification of flanking sections arelisted in Table S3. Regions were cloned into pJet1.2/blunt and sequenced.Plasmid pCM184 was digested with ApaI/SacI, and 3= end regions were in-serted for deletion of mms6, mmsF, and mamF. Generated plasmids weredigested with EcoRI/NdeI, and 5=flanking sequence was inserted, resulting inpCM184_mms6 3=5=, pCM184_mmsF 3=5=, and pCM184_mamF 3=5=. Thegenerated plasmids were examined by restriction analysis with a set of differ-ent enzymes or PCR and transferred into WT M. gryphiswaldense by conju-gation using E. coli BW29427 as a donor strain as described elsewhere (18).Genomic insertion mutants were selected on Km plates, cultivated in 100�l of FSM overnight at 30°C, and scaled up to 1 ml. Proper plasmidintegration was verified by PCR, and if necessary counterselection wasimplemented. Positive excision strains were verified by PCR, and mutantswere termed the �mamI, �mamL, �mamN, �mamP, �mamA, �mamQ,�mamR, �mamS, �mamT, �mamU, �mms48, �mms36, and �mmsF�mms6 mutants. Mutants generated by double crossover were cultivatedin 10 ml of FSM, and excision of the Km resistance gene was induced afterconjugation with the Cre expression plasmid pCM157. Generated strainswere termed the �mmsF and �mms6 mutants. For double deletion ofmmsF and mamF, the plasmid pCM184_mamF 3=5= was introduced into�mmsF, and deletion was verified as described above, resulting in the�mmsF �mamF strain.

Complementation of generated mutants and GFP localization. ForMamC-green fluorescent protein (GFP) localization experiments, plas-mid pFM236 was integrated into the genomes of the �mamI, �mamL,�mamN, �mamP, �mamA, �mamQ, �mamR, �mamS, and �mamTmutants. For construction of pAL_mamIg, mamI was amplified with oli-gonucleotides AL394/AL395 and inserted into pCL6 after digestion withNdeI/EcoRI. Plasmids for complementation of the other mutant strainswere derivatives of pBAM1 and labeled pBAM_mamL, -P, -S, -T, -R, -A,and -N, pBAM_mms48, pBAM_mms36, and pBAM_mgr4074. Oligonu-cleotides for amplification of the genes are listed in Table S3 in the sup-plemental material. Genes were cloned between the NdeI/EcoRI or NdeI/PacI sites (for mamN) of pBAM_GFDC under the control of the PmamDC

promoter. Plasmids pBam_mms36 and pBam_mms48 were also used foroverexpression studies in the WT. For complementation of the �mmsF,�mms6, �mmsF_mamF, �mmsF_mms6, and �A10 mutants, correspond-ing genes were amplified with oligonucleotides listed in Table S3. Geneswere inserted into pAP150 after digestion with BamHI/NdeI, resulting inpAL_mmsF, pAL_mms6, pAL_PmamDC_mms6op, and pAL_PmamDC_mms6,F,4074. Plasmid pBBRMCS2 was digested with NsiI/EcoRI for in-tegration of genes mms6, mmsF, and mgr4074 after amplification withAL125/AL136, generating pAL_Pmms6_mms6,F,4074. For transcomple-mentation assays, the plasmids were transferred to the respective deletionmutants by conjugation. �mmsF_mms6 and �mmsF_mamF were com-plemented with pAL_mms6 and pAL_mmsF, respectively. PlasmidspBam_mgr4074, pAL_PmamDC_mms6op, pAL_Pmms6_mms6,F,4074, andpAL_PmamDC_mms6,F,4074 were used for complementation studies inthe �A10 mutant.

Analytic methods. Optical density and magnetic response (Cmag)were analyzed photometrically at 565 nm (38). The applied magnetic fieldfor Cmag measurements was about 70 mT, which is able to magnetize verysmall or irregular magnetosomes within the superparamagnetic state. In-tracellular iron concentrations were measured after incubation under an-aerobic conditions as described previously (39).

Phase-contrast and fluorescence microscopy. M. gryphiswaldensestrains with genomic egfp were grown in 5 ml of FSM in six-well plates for16 h at 30°C and 2% O2 without agitation. Cells were immobilized onagarose pads (FSM salts in H2O, supplemented with 1% agarose) andimaged with an Olympus BX81 microscope equipped with a 100UPLSAPO100XO objective (numerical aperture of 1.40) and a Hamamatsu

Orca AG camera. Olympus cell software was used to capture and analyzeimages.

TEM and HRTEM. Magnetosome phenotypes of cells with respect tosize, shape, and number per cell were examined by transmission electronmicroscopy (40), for which cells were concentrated and adsorbed ontocarbon-coated copper grids. Cells were imaged with an FEI Morgagni 268(FEI, Eindhoven, Netherlands) electron microscope at an acceleratingvoltage of 80 kV. Bright-field TEM images and selected-area electron dif-fraction (SAED) patterns were recorded on image plates using a PhilipsCM20 microscope operated at 200 kV and fitted with a Noran Voyagerenergy-dispersive X-ray detector. High-resolution transmission electronmicroscopy (HRTEM) was performed using a JEOL 3010 microscopeoperated at 297 kV and equipped with a Gatan imaging filter (GIF) for theacquisition of electron energy loss spectra and energy-filtered composi-tional maps. For TEM data processing and interpretation, the softwarepackages DigitalMicrograph and SingleCrystal were used.

X-ray absorption spectroscopy. Bacterial cultures (90 to 135 ml) werepelleted by centrifugation (5 min at 9,000 � g and 4°C) and washed 3times by resuspension with 5 ml of Tris-buffered saline (TBS; pH 7.6) andcentrifugation. Pellets were then resuspended in 100 �l of TBS plus 25 �lof glycerol and frozen in liquid nitrogen on sample holders with Kaptonfilm support. Samples were shipped to the European Synchrotron Radia-tion Facility (ESRF) on dry ice, where they were stored at �80°C untilmeasurement. Fe K-edge X-ray absorption near-edge structure (XANES)spectra were recorded at the undulator beamline ID26 of the ESRF. Weused an Si (311) double-crystal monochromator and focusing mirrorsgiving a beam spot size of �200 by 400 �m2 on the samples. Data wererecorded in fluorescence detection mode using a Rowland-type spectrom-eter equipped with 4 Ge (440) analyzer crystals and an Si photodiode.During all measurements, samples were cooled to around 10 K using aliquid He cryostat. XANES spectra were recorded with 0.1-eV steps from7,100 to 7,200 eV. To improve data quality, 10 to 100 XANES scans wererecorded for each sample. Data were averaged using PyMca 4.6.2 afterevaluation for iron photoreduction. Averaged spectra were normalizedand fitted using Demeter 0.9.16. As reference materials, we used spinachferredoxin (Sigma-Aldrich), hematite (20- to 60-nm grain size; Alfa Ae-sar), magnetite, ferrihydrite, and phosphate-enriched ferric oxyhydrox-ides (described in reference 41).

RESULTSDeletion mutagenesis of the mms6 operon and mamF. After re-assessment of annotation and correction of the MmsF N terminus(see Fig. S1 in the supplemental material), we generated variousunmarked in-frame single and double deletions of all mms6op

genes as well as of mamF (localized in the adjacent mamGFDCoperon), which is highly similar (61% amino acid [aa] identitybetween the products) to mmsF (see Table 1 and Fig. 1 for over-view of deletions and resulting phenotypes). We found the hypo-thetical mgr4074 to be poorly conserved, and its chromosomalreintegration into the �mms6op strain (�A10 [18]) did not allevi-ate the severe biomineralization defects of the parent strain. Wetherefore consider mgr4074 a pseudogene with no role in biomin-eralization, although further studies are needed to address theexpression and putative localization of its gene product. The�mms6 strain had slightly smaller crystals (30 nm; wild type[WT], 36 nm) that were scattered throughout the cell, eitheraligned in irregularly spaced “pseudochains” (i.e., with �10 crys-tals per chain) or approximating WT-like chain configurations(Table 1 and Fig. 1). Crystals between 30 and 35 nm were predom-inant (WT, 40 to 45 nm), but particles larger than 60 nm wereabsent (WT, �70 nm; see Fig. S2 in the supplemental material).The average crystal number per cell was reduced to 30 (WT, 34particles per cell), and the magnetic response of a �mms6 culture

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was slightly weaker than that of the WT (Cmag(�mms6), 1.7 0.1;Cmag(WT), 2.0 0.1 [Table 1]).

The Cmag of �mmsF cells was similar to that of �mms6 cells.Magnetosomes displayed variable intracellular arrangements,such as one or more short chains, partially scattered crystals, or theabsence of any chain-like alignment (Fig. 1). Mean crystal sizeswere reduced to �30 nm, whereas the particle number was onlyslightly lower than in the WT (Table 1; see also Fig. S2 in thesupplemental material). Since the high similarity, 61%, betweenMmsF and MamF suggested possible functional redundancy,mamF was eliminated both alone and in combination with mmsF.In the �mamF mutant, MamC, encoded downstream of mamF inthe same operon, was found to be properly expressed by immu-nodetection, indicating that deletion of mamF had no polar effect(data not shown). The mean crystal sizes (34 nm) and numbers(34 per cell) were similar in the �mamF mutant to those in theWT. However, the combined excision of both genes within the�mmsF �mamF mutant resulted in a more drastic decrease in size(25 nm) and number (27 crystals per cell [Fig. 1 and Table 1; seealso Fig. S2]). Thus, loss of MmsF had a more pronounced effecton crystal size, number, and alignment than loss of MamF, and theadditive effect of their combined deletion suggested that both pro-teins are involved in size control.

Double deletion of mmsF and mms6 reduced the size to 24 nmand the number to 24 crystals per cell (Table 1). However, ironcontent, size, and number of magnetite particles as well as Cmag

(1.6 0.1) of the �mmsF �mms6 strain were still higher than inthe �A10 strain (in which the entire mms6op has been deleted)(Cmag, 1.0 0.1), with particles of 20 to 25 nm prevailing in bothstrains (Fig. 1 and Table 1; see also Fig. S2 in the supplementalmaterial). HRTEM images of particles from the �A12 strain, inwhich the entire mms6 and mamGFDC operons had been deletedtogether (18), revealed fringes spaced in correspondence to mag-netite. This indicates that the deleted genes alone do not have a

critical role in magnetite formation (Fig. 2). In contrast to thestrong size reduction observed in all other mutant strains, dele-tions of mgr4070 and mgr4071 (renamed into mms48 und mms36according to their predicted protein masses of 48 and 36 kDa,respectively) unexpectedly caused a substantial increase in meancrystal size. Particles synthesized by both strains resembled WTcrystals in shape but were significantly larger in the �mms36 (39nm) and �mms48 (46 nm [Fig. 1 and Table 1; see also Fig. S2])mutants. This is equivalent to a mean size increase of about 30%compared to that of WT for the latter strain, in which crystalsbetween 50 and 60 nm were most abundant, with a maximum sizeof up to 85 nm (see Fig. S2). However, both strains synthesizedfewer particles than the WT (�mms36 mutant, 22 per cell;�mms48 mutant, 16 per cell), and whereas in the WT magneto-some chains of larger particles at midchain are usually flanked bynumerous smaller crystals, those characteristic small crystals (15to 25 nm) were less frequent at the chain ends in the �mms36 and�mms48 mutants (Fig. 1 and Table 1; see also Fig. S2). Thus, thepredominance of larger (30 nm) particles partly accounted forthe substantially increased mean crystal size. However, despite thereduced particle numbers per cell, overall magnetite biomineral-ization was increased, as evident by the increased iron content ofboth deletion strains (21% more iron than for the WT). Genomicexpression of additional copies of mms48 and mms36 did not sig-nificantly change mean particle size (WT::mms36 strain, 35 nm;WT::mms48 strain, 33 nm [Table 1]), but the size distribution wasshifted toward smaller crystals for both strains. Crystals between30 and 45 nm were predominant in the WT::mms36 and WT::mms48 strains, whereas particles larger than 60 nm were not ob-served, unlike for WT crystals, which were most frequently be-tween 40 and 45 nm, with a maximum size up to 70 nm (data notshown). Whereas particle number was WT-like for overexpres-sion of Mms36 (32 per cell), crystal number was increased for theWT::mms48 strain (40 per cell [Table 1]). Interestingly, cells con-

TABLE 1 Characterization of the generated mutants

Relevantgenotype ofstrain

Deletedgene(s)

Magneticresponse(Cmag)a

Avg magnetosomesize (nm)a

Magnetosome size(% of WT)

No. ofmagnetosomesper cella

Maximumsize (nm)

WT 2.0 0.1 35.6 13.0 100 34.3 8.4 69.2�A10 mms6 operon Intermediate 19.7 6.9 55.3 16.8 6.2 39.6�mms6 mms6 WT 30.4 9.0 85.4 29.7 6.2 57.3�mmsF mmsF Intermediate 28.6 8.0 80.3 30.4 7.4 60.9�mms48 mms48 WT 46.4 14.8 130.3 16.0 5.6 82.8�mms36 mms36 WT 39.1 12.9 109.8 22.2 7.3 67.6�mamF mamF WT 33.6 10.4 94.4 33.6 10.4 64.9�mmsF �mamF mmsF, mamF WT 25.0 7.4 70.2 26.6 9.3 47.4�mmsF �mms6 mmsF, mms6 Intermediate 24.1 6.4 67.7 24.1 6.8 44.8WT::mms36 WT 34.6 9.2 97.2 31.6 10.1 57.2WT::mms48 WT 33.2 8.2 93.3 40.2 10.5 58.0�mamI mamI None 14.8 7.1 41.6 9.9 4.3 34.4�mamL mamL None�mamN mamN Weak 17.7 7.1 49.7 11.0 7.4 41.3�mamP mamP Intermediate 21.9 15.3 61.5 19.3 10.0 72.8�mamA mamA WT 35.2 13.1 98.9 10.2 4.7 73.4�mamQ mamQ None�mamR mamR WT 29.0 11.0 81.5 33.5 10.5 70.2�mamS mamS WT 22.0 7.5 61.8 34.8 12.0 44.2�mamT mamT WT 28.9 10.3 81.2 32.0 8.2 60.1�mamU mamU WT 37.3 13.0 104.8 31.8 8.4 65.3a Values are means standard deviations.

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taining double chains were more abundant for the WT::mms48strain (67%, compared to 28% for the WT::mms36 strain and 32%for the WT; see Fig. S3 in the supplemental material).

In summary, all proteins encoded by mms6op are involved incontrol of magnetosome size and/or number. The previously ob-served severe biomineralization defects in the �mms6op strain arethus due to the loss not of a single gene but of several genes, whichpoints toward a cumulative effect on magnetosome synthesis byvarious proteins encoded by mms6op.

Deletion analysis of the mamAB operon: mamE, -L, -M, -O,-Q, and -B are essential for iron biomineralization. First, anno-tations of all 17 mamABop genes were reassessed. Sequences cor-responding to the N termini that were conserved between all threeclosely related magnetospirilla M. gryphiswaldense, M. magneti-cum, and M. magnetotacticum were considered the most likelytranslation starts. Annotations were corrected accordingly formamI and mamL (see Fig. S4 in the supplemental material) andexperimentally confirmed by the ability of genes to complementtheir respective gene deletions. In addition to the previous dele-tions of the mamABop genes, we constructed 10 single in-framedeletions comprising mamI, -L, -N, -P, -A, -Q, -R, -S, -T, and -U.As expected, all resulting deletion strains displayed WT-likegrowth and morphologies. However, deletion mutants were im-paired in magnetosome biomineralization to variable extents.Based on their magnetic responses, mutants were considered ei-ther (i) magnetically responsive, with variable but significant Cmag

(�mamN, -P, -A, -R, -S, -T, and -U mutants), or (ii) entirelynonmagnetic, without any detectable Cmag (�mamI, -Q, and -L

mutants) (Table 1). TEM analysis confirmed that the first group ofstrains were still able to synthesize magnetosome-like particles butdisplayed various distinct phenotypes with respect to crystal mor-phology, size, and number per cell (Fig. 3). �mamU cells werehardly distinguishable from WT cells and produced 32 cubo-oc-tahedral crystals per cell with a size of 37 nm. All other mutantsshowed a drastically decreased magnetosome size, number,and/or alignment. Magnetosomes of the �mamA strain had aWT-like size, 35 nm, but their number was substantially de-creased, to 10 per cell. �mamS particles exhibited a widely spacedlinear chain-like arrangement within the cell. Whereas the crystalsize was strongly decreased (22 nm), the crystals were present inabout same numbers as in the WT (35 particles per cell). The�mamT strain also synthesized irregularly spaced magnetosomechains, whereas in some cells larger magnetosomes appeared atthe chain center and formed condensed “pseudochains.” Due tothe prevalence of smaller crystals, the mean particle size was de-creased to 29 nm, whereas their number was WT-like (32 particlesper cell). Several �mamR cells showed scattered magnetosomeslacking any chain-like alignment or showed short, densely spacedchains flanked by smaller particles with irregular morphologies orWT chains (average size, 29 nm; number per cell, 34). �mamPcells at first glance seemed to contain only a few (i.e., not morethan six magnetosomes; mean, three) particles larger than for theWT (59 nm on average). However, at closer inspection, numerousvery small and irregularly shaped crystals flanking the larger crys-tals became apparent, with an average size of 16 nm (see arrows inFig. 3). In total, �mamP cells synthesized, on average, only 19

FIG 1 Molecular organization of the mms6 and mamGFDC operons in M. gryphiswaldense and TEM micrographs of generated deletion mutants. Dark blue barsindicate extent of gene deletions generated in this study. Light blue bars indicate gene deletion mutants generated previously (18, 21). Scale bars, 1 �m.

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crystals, with a mean size of 22 nm. HRTEM of the two distinctparticle types revealed that the lattice fringes for the larger crystalscorresponded clearly to magnetite, whereas in contrast, thesmaller and poorly crystalline particles produced lattice fringescharacteristic for hematite (Fig. 2). The �mamN mutant showed avery weak but detectable magnetic response (Cmag, 0.1). TEM con-firmed the presence of a few (11 per cell) tiny, widely spaced crys-tals with a size of only 18 nm. HRTEM images of these particlesand their Fourier transforms indicated that these crystals have thestructure of magnetite.

The �mamI, �mamL, and �mamQ mutants represent the sec-ond class of mutants with no detectable magnetic response (Cmag,0). The �mamQ and �mamL mutants were entirely devoid of anyclearly recognizable crystalline electron-dense structures (Fig. 2).In the �mamL mutant, occasionally a few tiny (around 10 nm)structures were observed (not shown); however, these were diffi-cult to discern unambiguously from the cellular body and thebackground. The relevance and identity of these structures remainto be verified in future studies with higher resolution. Carefulanalysis of �mamI cells, however, revealed the presence of a few(10 per cell) electron-dense particles with highly irregular or elon-gated morphologies and a size of 15 nm (Fig. 3). As shown byHRTEM, the nuclei within �mamI cells were composed of severalsmall grains that formed thin aggregates (Fig. 2). Lattice fringeswere observed in only two particles, and according to the Fourier

transforms of the HRTEM images, the spacing between the fringesis very close to the d (012) and d (014) spacing in hematite. XANES(X-ray absorption near-edge structure) spectra obtained fromwhole �mamI and �mamN cells were clearly distinct from thoseof pure magnetite as in the WT and suggest that the ferrous com-pounds are predominantly Fe-S clusters (proteins) that accountfor around 40% of the total iron content in the cells (see Fig. S5 inthe supplemental material). Magnetite was clearly present in�mamN cells (around 50% of total iron), whereas the low fit qual-ity for �mamI did not allow us to reliably determine the structureof the Fe present in the bacteria apart from Fe-S (see supplementalmaterial for more detailed information). However, the overall lineshape appears most consistent with an amorphous or only poorlyordered Fe compound as suggested by HRTEM.

Intracellular localization of the magnetosome chain markerMamC-GFP. All mutant strains could be complemented by eithergenomic reintegration or plasmidal transfer (see the supplementalmaterial). We studied the ability of all mutants to properly localizethe abundant MamC magnetosome protein, which served as amarker for magnetosome chain localization in previous studies (6,35, 42). To this end, MamC was tagged by a chromosomal in-frame enhanced GFP (EGFP) insertion on pFM236 that shows inWT cells a continuous straight-line fluorescence signal (see Fig. S6in the supplemental material). Within �mamN, -P, -S, and -Tstrains, MamC-GFP localized as shorter structures but still

FIG 2 TEM and HRTEM micrographs and their corresponding Fourier transforms of electron-dense particles in various generated deletion strains of M.gryphiswaldense. The Fourier transforms were obtained from the images of entire particles or from the boxed areas in each corresponding HRTEM image. Thelattice fringe spacings correspond to the structures of either magnetite (A, D, E, F, and G) or hematite (B and C) in the �A12, �mamI, �mamN, and �mamPmutants. H, hematite; M, magnetite.

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showed a linear localization running along the inner curvature ofthe cell. Within �mamI cells, a short but still elongated fluores-cence signal at midcell was observed. Thus, MamI, -N, -P, -R, -S,and -T are not required for proper MamC localization. On thecontrary, in �mamA, -Q, and -L cells, no defined position of theMamC-GFP signal was detectable, but instead a diffuse spot likeaccumulation within the cell was predominant (see Fig. S6). In the�mamA mutant, the magnetosome formation was not inhibited,even though MamC is misplaced within this strain, which suggeststhat MamA may interact with MamC.

DISCUSSIONThe Mms6 operon encodes nonessential magnetosome proteinscrucial for proper crystal growth. As in M. magneticum (reduc-tion of size of crystals by 19% [22]), we observed only minorbiomineralization defects upon deletion of mms6 in M. gryphis-waldense (15% size reduction). Only a 20% size reduction was alsoseen after mmsF deletion in M. gryphiswaldense, which, however,is weaker than its deletion phenotype in M. magneticum (52% sizereduction [22]). However, double deletion of mms6 and mmsFresulted in an almost 32% size reduction, which suggests a certainfunctional overlap between Mms6 and MmsF. We found func-tional redundancy between mmsF and mamF (carried on the ad-jacent mamGFDC operon), since their double deletion exacer-bated defects in crystal maturation (30% size reduction). Hence,the loss of several genes together contributed to the strong mag-netosome defect (45% size reduction) observed after deletion ofthe entire mms6 operon (�A10), which indicates a cumulativeregulation of magnetosome biomineralization.

mms6op of M. gryphiswaldense contains two additional genes,named mms36 (mgr4071) and mms48 (mgr4070), that are ex-pressed under magnetosome-forming conditions (18) but had notyet been studied by deletion analysis in either M. gryphiswaldenseor M. magneticum. Surprisingly, their deletion caused the synthe-sis of larger magnetite crystals instead of size reduction. Since noconserved domains or motifs are present in Mms36 and -48, apartfrom weak similarity to proteins involved in porphyrin synthesis(Mms36, 29% to uroporphyrinogen III synthase of Rhodospiril-lum rubrum; Mms48, 28% to HemY-like proteins, possibly in-volved in porphyrin biosynthesis [43, 44]), their precise in vivofunctions are difficult to infer. They might either be inhibitors ofcrystal growth (32) or recruit other inhibitory proteins to the MMin order to prevent excessive crystal growth.

Genetic analyses of the mamAB operon: MamU, -T, -S, -R, -A,-N, and -I are not essential for iron biomineralization in M. gry-phiswaldense. We found that deletion of several genes (mamU,-T, -S, -R, -A, -P, -Q, and -L) from mamABop of M. gryphis-waldense essentially phenocopied the deletions of their orthologsin M. magneticum (7). Loss of mamU, -T, -S, -R, -A, -P, and -N didnot entirely abolish biomineralization of magnetite crystals. As inM. magneticum, deletion of MamU did not have any detectablemagnetosome phenotype in M. gryphiswaldense. MamT of M.

magneticum as well as of M. gryphiswaldense contains a doublecytochrome c motif (CXXCH), referred to as the magnetochromedomain, necessary for heme binding (45). It was speculated thatMamT therefore transfers electrons to balance the ferric-to-fer-rous iron ratio form required for magnetite formation (45). De-letion of mamT in M. gryphiswaldense resulted in smaller magne-tosome particles as in M. magneticum, supporting its previouslypredicted function in crystal maturation. As in the �mamT mu-tant, deletion of mamR in M. gryphiswaldense resulted in smallercrystals and partially modified chain formation, similar to thephenotype observed for �mamR AMB. Thus, MamR is involvedin controlling particle number and size, as also suggested forMamR of M. magneticum (7). We also confirmed a key role forMamS in M. gryphiswaldense, which has similarity to the putativeserine proteases and magnetochrome domain-containing pro-teins MamE and -X (20, 32). However, MamS itself lacks a mag-netochrome domain, which argues against its direct participationin redox control. The TPR domain-containing protein MamA wasspeculated to play a role in activation of biomineralization (5). Itwas suggested that MamA self-assembles through its putative TPRdomain and concave site to form a large homooligomeric scaffoldsurrounding the magnetosomes (46, 47), whereas its convex siteinteracts with other magnetosome-associated proteins, likeMamC, and several unidentified proteins (46, 47). However, as inM. magneticum, the deletion of mamA in M. gryphiswaldense hadonly a weak effect (5), suggesting that these interactions are notessential or can be partly compensated by other proteins. In�mamP M. gryphiswaldense, particles larger than those synthe-sized by the WT were flanked by smaller and poorly crystallineparticles, similar to the case with M. magneticum (48). MamPcontains two closely spaced magnetochrome domains and wasspeculated to interact with MamE, -X, and -T through its PDZdomain and to somehow regulate the electron transport requiredfor biomineralization of the mixed-valence iron oxide magnetite(20, 32, 48). Magnetosomes in �mamP M. gryphiswaldense show acrystallization defect (magnetite crystals flanked by flakes) similarto that of the mamX mutant of M. gryphiswaldense (20), whichmight indicate involvement of this gene in the same step of mag-netosome biosynthesis. In contrast, phenotypes of �mamT(smaller particles) and �mamE (total loss of electron-dense par-ticles) of M. gryphiswaldense are distinct from the phenotype ofthe �mamP strain, suggesting that some or all of the magneto-chrome proteins have different or additional functions. However,MamE also contains, besides the magnetochrome domains, a pro-tease and double PDZ domains, which might cause the differencesbetween the generated mutants upon their deletion (32). Thus,further analyses are needed to explain the different observed mu-tant phenotypes, such as the specific deletion of the different mag-netochrome domains. MamP from M. magneticum catalyzed theformation of ferrihydrite and magnetite from iron solutions invitro, indicating that MamP binds and oxidizes iron (48). How-ever, this ability of MamP is not essential in vivo, as the �mamP

FIG 3 Molecular organization and deletion analysis of the mamAB operon of M. gryphiswaldense as well as representative TEM micrographs of cells andmagnetosome morphologies observed within the generated excision mutants. The highly conserved mamAB operon encodes 17 magnetosome proteins (MamH,-I, -E, -J, -K, -L, -M, -N, -O, -P, -A, -Q, -R, -B, -S, -T, and -U) in M. gryphiswaldense and was found to be essential and sufficient to maintain magnetite biogenesis(18, 22). Red arrows, genes essential for magnetosome crystal formation; blue arrows, genes nonessential for particle formation; gray bars, nonessential genes forwhich deletion strains were previously generated (6, 9, 20); blue bars, nonessential genes for which deletions were generated in this study; dark red bars, essentialgenes for which deletions were generated previously (34, 35); light red bars, essential genes for which deletions were generated in this study; white arrows,magnetosome chains or particles; black arrows, PHB granules.

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mutant of M. gryphiswaldense continued to biomineralize parti-cles of magnetite. Potentially this might be due to unchecked min-eral growth after deletion of a major redox regulator (49) and/orthe ability of other magnetochrome proteins, like MamX, -E, and-T, to partially compensate the loss of MamP.

In addition to the strong similarities between several AMB andM. gryphiswaldense mutants, we also found several striking differ-ences between the two species. MamN was described to be essen-tial for magnetosome biosynthesis, as indicated by the absence ofelectron-dense crystals in M. magneticum (7). However, our TEM,HRTEM, and XANES analyses revealed the presence of magnetiteparticles within �mamN M. gryphiswaldense. Because of its simi-larity to the human permease P, which is predicted to regulate theintraorganelle pH of melanosomes together with an ATP-drivenproton pump (50), MamN was speculated to regulate pH condi-tions within the magnetosome vesicles by export of protons re-leased by the precipitation of magnetite (2Fe3�� Fe2�� 4H2O¡Fe3O4 � 8H� [8, 51]), and thus the observed phenotype of the�mamN strain might be due to alteration of the intramagneto-somal pH.

Another gene which exhibited a strikingly distinct deletionphenotype between M. magneticum and M. gryphiswaldense ismamI, which encodes a small magnetospirillum-specific magne-tosome protein (70 aa) with no significant homology to already

characterized proteins. In M. magneticum, MamI was found to beessential for the biosynthesis of magnetosome membrane vesiclesand, consequently, iron biomineralization (7). In contrast, exci-sion of mamI in M. gryphiswaldense did not entirely abolish thebiomineralization of electron-dense iron-rich particles, but themutant still synthesized tiny and poorly crystalline nonmagneticparticles, which in some cases were shown to consist of hematite.Recent findings for M. gryphiswaldense and M. magneticum indi-cate that the observed poorly ordered iron (oxyhydr)oxide phasesare precursors to the magnetite phase in bacteria (41, 52). In ad-dition, the ability of MamP to precipitate ferrihydrite and magne-tite in vitro suggests that magnetite may be formed through astepwise phase transformation process (48). Such a biosyntheticphase transformation requires a precise control of iron supersat-uration, pH, and redox potential levels (53), suggesting that MamImay be involved at an early stage of magnetite nucleation by reg-ulation of proper conditions within the vesicles.

Only MamE, -L, -M, -O, -B, and -Q are essential for ironbiomineralization in M. gryphiswaldense. In addition to the pre-viously identified mamE (34), mamM (35), mamO (34), andmamB (35) genes, we also demonstrated mamQ and most proba-bly mamL to be essential genes for magnetosome synthesis in M.gryphiswaldense, since their deletions abolished the biomineraliza-tion of clearly distinguishable electron-dense particles. MamQ

FIG 4 Hypothetical model for magnetosome biosynthesis in M. gryphiswaldense. Magnetosome biosynthesis depends on various steps including variousmagnetosome proteins. Magnetosome vesicle formation (I) is induced by the proteins MamQ, MamB, and MamL. MamY was found to regulate vesicle shape(23). MamA forms a multiprotein complex surrounding the magnetosome membrane (47), and MamE is involved in localization of magnetosome proteins bya protease-independent process (32). The heterodimer of the CDF transporters MamB and MamM transports ferrous iron into the magnetosome vesicles (35),and ferric iron is taken up by MamH and MamZ (20) or formed by oxidation of ferrous iron within the vesicles. MamI is involved in magnetite nucleation. MamOwas speculated to be directly involved in precipitation of iron oxide particles (32). The crystal growth is affected by several magnetosome proteins, includingMamE, which proteolytically remove a growth inhibitor(s) or activate growth-promoting proteins (32). Based on the conserved CXXCH heme-binding motifswithin MamE, MamT, MamP, and MamX, it has been speculated that the proteins form a complex for electron transport to regulate electron flow (20, 45). MamSand MamR control crystal size by an unknown mechanism. MamN exhibits similarity to H� translocation proteins and might be involved in crystal growth byregulating intramagnetosomal pH (51). Mms6 is tightly bound to the magnetosome crystals (26, 28) and assembles into coherent micelles for templating crystalgrowth (57). mms48 (Mms48) and mms36 (Mms36) act as inhibitors of crystal growth or recruit inhibiting proteins of particle growth by an unknownmechanism. The small, hydrophobic proteins MamG, MamF, MamD, and MamC control in a cumulative manner the growth of magnetite crystals (21).Magnetosomes were assembled into chains by the interaction of MamJ with the actin-like MamK filament that is also involved in chain positioning (6, 9, 58). OM,outer membrane; IM, inner membrane; MP, magnetosome protein. Underlined proteins are proteins analyzed in this study by single gene deletion of encodinggenes. Proteins in brackets are nonessential proteins encoded by mamXYop.

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shares homology with the LemA protein, which is conserved inseveral bacteria but whose function is uncertain (12, 55).MamQ has a high content of �-helices that are somewhat rem-iniscent of the EFC/BAR domain of formin binding protein 17(7). BAR domains have the ability to sense and stabilize mem-brane curvatures (56), and their weak similarity to MamQmight hint toward related functions in MM vesicle genesis ofthe protein. The small protein MamL has no predicted functionbut was shown to be essential for magnetosome membranegenesis in M. magneticum (7).

Despite the metabolic and genetic similarities between M.magneticum and M. gryphiswaldense, previous studies already sug-gested that the functions of orthologous genes might be somewhatdistinct in these organisms (6, 14, 18). Apart from the possibilitythat the tiny magnetite and hematite particles in �mamN and�mamI of M. gryphiswaldense simply had escaped detection in thecorresponding mutants of AMB (7), this might possibly due to thedifferent genetic context, with only about 50% of all genes sharedby the genomes of these two strains (14). In fact, it cannot beexcluded that further genes outside the MAI partially compensatethe loss of deleted genes, as observed in a recent study, in which amagnetosome islet outside the MAI compensated the deletion ofmamK in M. magneticum (6, 33).

In summary, whereas in M. magneticum eight proteins (MamI,-E, -L, -M, -N, -O, -B, and -Q) were found to be essential formagnetosome biomineralization, in M. gryphiswaldense only sixproteins (MamE, -M, -O, -B, -Q, and most probably MamL) areessential for at least some rudimentary iron biomineralizationand, if including MamI, seven proteins for the biosynthesis ofmagnetite-containing magnetosomes. This leads to an expandedmodel of magnetosome biosynthesis in M. gryphiswaldense (Fig.4). However, it remains to be shown whether these essential mag-netosome proteins are also sufficient for vesicle formation andcrystallization even in the absence of the other factors encoded bythe mamAB and other magnetosome operons.

ACKNOWLEDGMENTS

This work was funded by the Deutsche Forschungsgemeinschaft (grantsDFG Schu1080/13-1 and 15-1) and the European Union (projectBio2MaN4MRI no. 245542). This research was further supported by theMax Planck Society and the European Research Council through a start-ing grant to D.F. (project MB2 no. 256915). A.L. was supported by theKonrad-Adenauer-Stiftung.

We acknowledge the European Synchrotron Radiation Facility forprovision of synchrotron radiation facilities. We thank J.-D. Cafun, P.Glatzel, and C. Lapras for assistance in using beamline ID26 at ESRF. Weare thankful to R. Susen, A. Hähle, A. Singer, T. Perez Gonzalez, and G.Morin for experimental support.

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