magnetosomes: how do they stay in shape?

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Magnetosomes in Magneto-Tactic Bacteria

J Mol Microbiol Biotechnol 2013;23:81–94 DOI: 10.1159/000346655

Magnetosomes: How Do They Stay in Shape?

Dorothee Murat

Laboratoire de Chimie Bactérienne, Unité Mixte de Recherche 7283, Aix-Marseille Université, Centre National de la Recherche Scientifique, Marseille , France

address specific proteins to these compartments. Then, if eu-karyotes did not invent organelles or the nucleus, who did? Are bacteria with a complex cell plan providing us with an unexpected opportunity to investigate how organelles came to exist? Is it possible that the mechanisms leading to cell compartmentalization in eukaryotes were invented by bacteria? Or, by studying how bacterial organelles are formed, will we discover new ways to control membrane cur-vature, target proteins, organize and segregate organelles?

Copyright © 2013 S. Karger AG, Basel

The Magnetosome Chain: A Magnetic Bacterial

Organelle

Magnetosomes are magnetic nanocompartments found in the so-called magnetotactic bacteria (MTB). This phylogenetically and morphologically diverse group of aquatic bacteria was first described in a report pub-lished in 1975 describing bacterial cells that consistently swam toward the magnetic north and could align with and swim parallel to the Earth’s magnetic field lines [Blakemore, 1975]. Years later, Richard Frankel found out that these unusual bacteria had also been serendipi-tously discovered by an Italian graduate student 12 years

Key Words

Magnetosomes · Magnetotactic bacteria · Magnetosome island

Abstract

Biology textbooks taught us that eukaryotes could be easily distinguished from the far less complex bacteria. One crite-rion is that eukaryotes can segregate their DNA into a lipid-bounded compartment called a nucleus which isolates DNA replication and transcription from the rest of the cytoplasmic content. The second criterion is that eukaryotes can com-partmentalize their cytoplasm so as to isolate specific path-ways, enzymes and chemical reactions in membrane-bound-ed subcellular compartments called organelles. Time and high resolution imaging taught us that the story is a little more complicated. In fact, bacteria too can isolate cell com-ponents in subcellular compartments, including, in rare cas-es, their DNA. Clearly, some bacteria also have the capacity to isolate reactions that require a specific chemistry or that generate toxic byproducts within specialized organelles. De-spite the significant advances made in the field of bacterial cell biology in the past 15 years, little is known about the mechanisms employed by bacteria to shape, position and segregate organelles, or how the cells can discriminate and

Published online: April 18, 2013

Dorothee Murat Laboratoire de Chimie Bactérienne, Unité Mixte de Recherche 7283 Aix-Marseille Université, Centre National de la Recherche Scientifique FR–13402 Marseille Cedex 20 (France) E-Mail dmurat @ imm.cnrs.fr

© 2013 S. Karger AG, Basel1464–1801/13/0232–0081$38.00/0

www.karger.com/mmb

Murat

J Mol Microbiol Biotechnol 2013;23:81–94DOI: 10.1159/000346655

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earlier, but his work has only recently been edited and published [Bellini, 2009]. The restriction of the cell move-ment to one dimension along the magnetic field lines is believed to make the bacteria search for their niche more efficient [Bazylinski and Frankel, 2004]. This ability to orient in the Earth’s magnetic field is due to the presence in the cells of an intracellular compass needle made of magnetic nanocrystals organized in a chain. MTB are found in a great variety of freshwater and marine envi-ronments such as lakes, ponds, marshes and oceans, as well as extremely harsh places such as Badwater Basin in Death Valley, California [Lefevre et al., 2011].

The first images of the magnetosome chain of Magne-tospirillum magnetotacticum MS-1, formerly Aquaspiril-lum strain MS-1, revealed that each electron-dense nano-crystal is in fact surrounded by what appeared to be one or several layers of organic material [Balkwill et al., 1980] ( fig. 1 ). At the time, Blakemore and colleagues hypothe-sized that this layer could be a lipid membrane, which was confirmed a few years later when the lipid analysis of pu-rified magnetosomes was undertaken. Interestingly, the composition of the magnetosome membrane did not sig-nificantly differ from that of the inner membrane [Gorby et al., 1988; Grunberg et al., 2004; Tanaka et al., 2006] indicating that the so-called magnetosome membrane is formed by invagination of the inner membrane. The pres-ence of lipid-bounded nanocompartments in the mag-netic cells indicated that this bacterium was capable of forming, organizing and segregating up to 30 membrane-bounded subcellular compartments dedicated to the syn-thesis of a single magnetic mineral. These criteria define magnetosomes as bacterial organelles.

For the purpose of this review, after giving an overview of the multiple strategies that were taken to identify fac-tors implicated in magnetosome formation, I will focus on four aspects of magnetosome biology which each highlights magnetosome properties as a bacterial organ-elle: magnetosome membrane formation, targeting of magnetosome proteins, organelle positioning and at last, biomineralization.

Magnetosome Gene and Protein Identification

Many strategies were employed to identify the con-stituents of magnetosome organelles. All the data ob-tained converged toward one particular region of the MTB chromosome that is essential for magnetosome for-mation and was eventually named the Magnetosome Is-land (MAI) [Schubbe et al., 2003].

Proteomics Thanks to the nanocrystal magnetic properties, mag-

netosomes can easily be purified from cell extracts, along with the membrane that surrounds them [Grunberg et al., 2004; Okuda et al., 1996; Tanaka et al., 2006]. After cells are lysed, and the membranes are properly sheared, mag-netosomes can be immobilized on magnetic beads that can be successively magnetized and demagnetized. The protein content of purified magnetosomes from three species of Magnetospirilla was analyzed and led to the identification of many proteins that were specifically en-riched in the magnetosome fraction [Grunberg et al., 2004; Okuda et al., 1996; Tanaka et al., 2006]. By subject-ing the magnetosome fraction to different treatments, from the mildest to the harshest, putative magnetosome proteins could be grouped into different classes: periph-erally associated, membrane-embedded and crystal-asso-ciated magnetosome proteins [Arakaki et al., 2003]. The proteomic approach indicated that in addition to canon-ical inner membrane proteins, the membrane surround-ing each magnetic nanoparticle contains a set of proteins that are exclusively found in the magnetosome fraction. The first magnetosome protein that was identified is one of the most abundant and was named MamA [Okuda et al., 1996]. A large body of work done in MSR-1 identified the genes coding for most of the magnetosome proteins (named Mam and Mms proteins) and showed that they were in close proximity on the chromosome [Schubbe et al., 2003; Ullrich et al., 2005]. This was an important mile-stone in the study of these microorganisms as this work revealed that the previously identified magnetosome pro-teins were encoded by neighboring gene clusters grouped within a conspicuous region of the chromosome of ap-proximately 130 kb in MSR-1.

It is important to note that this approach presents lim-itations. First, in addition to the proteins enriched in the magnetosome fraction, a large amount of inner mem-brane proteins, outer membrane and cytoplasmic pro-teins were found in the magnetosome fraction, which suggests cross contamination of the fractions [Grunberg et al., 2004; Tanaka et al., 2006]. Second, this approach was used to identify magnetosome-associated proteins in Desulfovibrio magneticus RS-1 before its genome was se-quenced [Matsunaga et al., 2009]. This work identified a few conserved known magnetosome proteins associated with the purified magnetosomes, including six predicted integral membrane proteins [Matsunaga et al., 2009], yet the presence of membranes around RS-1 magnetite crys-tals could not be confirmed yet [Byrne et al., 2010]. It is important to keep in mind that nonspecific cell material



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probably copurifies with the magnetic nanoparticles, and this caveat is particularly relevant in the case of AMB-1 for which the magnetosome membrane and the inner membrane are contiguous, making the clear separation between these two membrane networks difficult (see ‘One Organelle, Different Architectures’ below). As a consequence, the presence of a protein in the organelle should be verified in vivo.

Random Mutagenesis Random transposon mutagenesis was adapted to M.

magneticum AMB-1 in order to screen for mutants pre-senting a magnetosome formation defect, that is, cells that could no longer be pulled by a bar magnet [Komeili et al., 2004]. One study allowed for the identification of mutants for which the polar transposon insertions affect-ed or completely prevented magnetosome formation. These insertions all occurred within an 18-gene cluster, the mamAB cluster, which encodes most of the Mam pro-teins [Grunberg et al., 2001].

Genome Analysis of Spontaneous Nonmagnetic Mutants Spontaneous nonmagnetic variants occur rather fre-

quently (estimated around 10 –5 for MSR-1) [Ullrich et al., 2005] in MTB as they are cultivated in the laboratory. The frequency with which these spontaneous mutants occur were reported to increase with oxygen and temperature related stresses (up to 10 –2 ) [Ullrich et al., 2005]. When comparing the genome of the spontaneous nonmagnetic mutants in MSR-1 and AMB-1 to that of wild-type strains, it appeared that the mutants had lost a large genomic re-gion containing most of the mam and mms genes [Fu-kuda et al., 2006; Grunberg et al., 2001; Ullrich et al., 2005]. As this unstable region was also enriched for mo-bile genetic elements such as insertion sequences and transposons, as its GC content was different from that of the rest of the chromosome and as magnetosome forma-tion was directly dependent upon its presence, this ge-nomic region was named the magnetosome island (MAI). In 2006, sequencing of the M. magneticum sp. AMB-1 genome also revealed the existence of a 98 kb MAI flanked by 1,100 direct repeats that is essential for magnetosome formation and which contains all the mam and mms genes [Fukuda et al., 2006]. Since then, MAI-like genom-ic regions have been found in most sequenced MTB.

Altogether, these complementary approaches identi-fied a conspicuous and unstable region in MTB chromo-some essential for magnetosome organelle formation and encoding most if not all predicted magnetosome proteins.

Since then, the MAI became the target of directed genet-ic approaches which helped identify the proteins essential for magnetosome formation.

The Magnetosome Membrane

Magnetosomes are nanocompartments made of a mineral and a membrane component. A single magnetic nanocrystal of either the iron oxide magnetite (Fe 3 O 4 ) or the iron sulfide greigite (Fe 3 S 4 ) is biomineralized within the boundaries of a lipid bilayer which constitutes a com-plete magnetosome. This definition of what makes a mag-netosome can be considered the general consensus as a lipid membrane was clearly identified in all but one MTB. This exception will be discussed in the following section [Byrne et al., 2010]. The membrane that surrounds each crystal is called the magnetosome membrane. Its lipid composition is quantitatively and qualitatively similar to that of the inner membrane (neutral lipids, glycolipids, phospholipids …) [Gorby et al., 1988; Grunberg et al., 2004; Tanaka et al., 2006].

Compartments Can Form in the Absence of a Crystal While the presence of a mineral is essential for the cells

to align in the magnetic field, the absence of a crystal does not necessarily indicate that the membrane component of the organelle is absent. In fact, while the electron-dense magnetic particles can easily be made out by transmission electron microscopy (TEM), magnetosome membrane observation requires cryosectioning and staining prior to observation by TEM, or electron cryotomography (ECT), which are both time consuming and technically challeng-ing. When M. magneticum sp. AMB-1 cells were grown in the absence of iron it was shown that a chain of empty membrane compartments is assembled in the cells, and the size and shape of the compartments are the same as those of magnetite-filled magnetosomes [Komeili et al., 2004]. This demonstrates that in AMB-1 not only can the membrane compartments exist in the absence of crystals, but they also can be assembled into a chain while empty. The fact that the empty compartments were present in the absence of crystals demonstrated that the membranes do not grow as the crystals do. After the addition of iron to the culture, crystals at different stages of maturation could be seen in the preformed magnetosome chain where they could grow until they completely filled the volume of the magnetosome [Komeili et al., 2004] ( fig. 1 ). Thus, the final size of the crystal produced by the bacte-rium is limited by the size of the magnetosome mem-

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brane. Practically, without high resolution imaging, the absence of a magnetic particle can suggest that the cells are affected in some aspect of biomineralization. There are no reports of small tooth- or bullet-shaped crystals growing within the boundaries of a larger magnetosome membrane.

One Organelle, Different Architectures High resolution imaging of intact magnetosome chains

was first performed in M. magneticum sp. AMB-1 and M. gryphiswaldense sp. MSR-1 [Komeili et al., 2006; Scheffel et al., 2006], two closely related freshwater species which are to this day the best characterized MTB ( fig. 1 , 2 ). Both are spirillum-shaped magnetite-producing Gram nega-tive α-proteobacteria. They produce 20–30 magneto-somes organized in one chain in AMB-1, and up to two in MSR-1, running parallel to the long axis of the cell from pole to pole. ECT on these two species provided us with important information about magnetosome chain archi-tecture and pinpointed unexpected differences that exist between the magnetosomes of these phylogenetically close species.

In AMB-1, where the chain of empty compartments is established prior to magnetite synthesis, ECT revealed the presence of permanent membrane connections be-tween the magnetosomes and the inner membrane [Ko-meili et al., 2006]. These connections suggest that there is: (1) a way to segregate and maintain the magnetosome proteins at the organelle even after the magnetosome compartments are formed, and (2) some kind of physical barrier preventing the magnetosome content from mix-ing with that of the periplasm as it is thought that magne-tite nucleation requires a particular physico-chemical en-vironment. Magnetite crystals have never been reported to form in the periplasm.

Scheffel et al. [2006] showed that when MSR-1 is cul-tivated in the absence of iron, empty membrane nano-compartments are present in the cell. However, the emp-ty magnetosomes are dispersed in the cytoplasm and it is only upon addition of iron, when magnetite biomineral-ization is initiated, that the magnetite-containing magne-tosomes assemble into a chain [Scheffel et al., 2006]. This strongly suggests that the magnetosome organelles are present in the cell as vesicles that are separated from the inner membrane so as to freely move in the cytoplasm upon magnetite synthesis ( fig. 2 ). No connections be-tween the magnetosomes and the inner membrane could be made out in MSR-1. In this bacterium, magnetosome chain formation is driven by both magnetic interactions between neighboring crystals and a dedicated cytoskele-

ton that would act as a track to guide individual magne-tosomes. These differences are particularly striking con-sidering that these two bacteria are both phylogenetically and morphologically close and that they possess a very similar magnetosome gene pool.

More recently, several more distantly related MTB species were isolated and imaged, and parts of their ge-nome were sequenced. One species, the greigite-produc-ing δ-proteobacterium Candidatus Magnetoglobus mul-ticellularis, has a unique multicellular life cycle and as-sembles into a roughly spherical magnetic structure containing 10–30 cells [Keim et al., 2004; Simmons and Edwards, 2007; Zhou et al., 2012]. This bacterium was im-aged and part of its magnetosome gene content was re-vealed [Abreu et al., 2008, 2011]. In this bacterium, greigite nanocrystals are also enclosed within a mem-brane, a characteristic that seems to be conserved regard-less of the nature of the magnetic mineral produced. In some cases, membrane connections between the magne-tosomes and the inner membrane could be observed ( fig. 3 ). Using culture-independent techniques, the au-thors identified two magnetosome gene clusters similar to those identified in Magnetospirilla which strongly sug-gests that magnetosome formation probably occurs via similar mechanisms in this morphologically unique MTB. Similar observations were made in the sulfate-reducing δ-proteobacterium BW-1 which can produce magnetite and/or greigite-containing magnetosomes. In these cells, an electron dense layer observed around the greigite crys-tals also suggested the presence of a lipid membrane around them ( fig. 4 ) [Lefevre et al., 2011].

Candidatus M. bavaricum, an uncultivated MTB from the deep branching phylum Nitrospira, was the first non-proteobacterial MTB to be isolated and imaged with high resolution techniques [Jogler et al., 2011]. This bacterium is unique in that its large cells (up to 10 μm in length) can accommodate up to a thousand bullet-shaped magnetite crystals organized in multiple bundles, parallel to the long axis of the cell [Jogler et al., 2010]. Each bullet-shaped magnetite crystal of M. bavaricum is also surrounded by a lipid membrane ( fig. 5 ) [Jogler et al., 2011]. Altogether, the results obtained for a diverse sample of MTB strongly suggests that magnetite formation depends on the pres-ence of a membrane across all MTB. Yet the analysis of the δ-proteobacterium D. magneticus sp. RS-1 led to an unexpected finding. This sulfate-reducing bacterium produces twelve to fifteen bullet-shaped magnetite crys-tals [Byrne et al., 2010] around which three high-resolu-tion imaging techniques failed to demonstrate the pres-ence of a lipid membrane. Even though it is possible that



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the absence of a visible membrane around the crystals could be the consequence of a lack of contrast in close proximity to the magnetite crystals, it is also possible that in this bacterium the presence of a membrane is only transient during magnetosome formation and that once matured, the crystals could be released in the cytoplasm as proposed by the authors [Byrne et al., 2010]. The pres-

ence of a permanent connection between the magneto-somes and the inner membrane remains to be investi-gated in the non- Magnetospirilla .

How Do Magnetosomes Stay in Shape? How do bacteria control membrane curvature and

build organelles with a distinct and reproducible size and

Col

or v

ersi

on a

vaila

ble

onl

ine

Fig. 1. Magnetosome architecture in AMB-1. ECT of M. magneti-cum sp. AMB-1 reveals that magnetosomes are invaginations of the inner membrane. a General features of AMB-1 cells highlight-ed in a 12-nm-thick section of an ECT reconstruction. OM = Out-er membrane; IM = inner membrane; PG = peptidoglycan layer;R = ribosomes; B = outer membrane bleb; CR = chemoreceptor bundle; PHB = polyb-hydroxybutyrate granule; G = gold fiduciary marker; MG = magnetosome chain. Scale bar = 500 nm. Represen-tative magnetosomes containing no magnetite ( b ), small ( c ), me-dium-sized ( d ) and fully-grown ( e ) crystals are invaginations of the inner membrane. Scale bar = 50 nm [Komeili et al., 2006]. Fig. 2. Cryoelectron tomography of wild-type MSR-1. a Tomo-graphic slice showing magnetosome vesicles which are either emp-ty or contain a growing crystal (arrow) connected to a filamentous structure. b A surface-rendered representation of the segment

shown in a (vesicles are in yellow, magnetite crystals are in red and filamentous structure are in green) [Scheffel and Schuler, 2007]. Fig. 3. Transmission electron micrograph of ‘ Candidatus M. mul-ticellularis’ magnetosomes. Detail of the border of a cell showing a membrane similar to the cell membrane around the magnetic crys-tal which seem connected (arrow) [Abreu et al., 2008]. Scale bar = 100 nm. Fig. 4. TEM images of a stained thin section of a BW-1 cell show-ing that an electron-dense layer surrounds the greigite crystals as indicated by the white arrows [Lefevre et al., 2011]. Fig. 5. TEM ultrathin sections of high-pressure frozen and freeze-substituted M. bavaricum cells showing strands of magnetosomes aligned parallel to a tubular filamentous structure indicated by an asterisk. Black arrowheads point at the magnetosome membrane [Jogler et al., 2011]. Scale bar = 20 nm.

*

1 2

3 4 5

a

b c d e

a b

100 nm

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shape? How, when and where are they formed from the inner cell membrane? How organelle-producing bacteria generate and maintain membrane curvature remains largely unknown. Several families of proteins involved in membrane curvature control are known in eukaryotes where they are essential for membrane remodeling dur-ing endocytosis, filopodia protrusion and organelle bio-genesis [McMahon and Gallop, 2005]. Considering that no obvious homologs of the eukaryote curvature-gener-ating proteins have been identified in bacterial genomes thus far, including MTB genomes, it suggests that these microorganisms may have evolved different strategies to generate and maintain membrane curvature.

Four Conserved Genes Are Essential but Not Sufficient for Magnetosome Membrane Formation in AMB-1 Within a species, magnetosomes are amazingly consis-

tent in size and shape. The diameter, spacing and distance from the inner membrane are constant suggesting that the bacterium must be able to control these parameters by adjusting the nature and quantity of the proteins ad-dressed to these organelles. In M. magneticum AMB-1, magnetosomes are bulb-shaped compartments with an outside diameter of about 60 nm that stands on a 5–10 nm pedestal of inner membrane ( fig. 1 ). Adjacent magneto-somes are not in direct contact. A matrix surrounds the magnetosome membrane which is thought to be mostly made of the peripherally associated magnetosome pro-tein MamA [Yamamoto et al., 2010]. This is supported by the crystallographic data showing how MamA self-as-sembles into larger polymeric structures [Zeytuni et al., 2011]. This protein matrix may have a direct role in con-trolling the spacing of magnetosomes, it may serve as an anchoring platform for other peripherally associated magnetosome proteins [Okuda and Fukumori, 2001; Zeytuni et al., 2011] and it may potentially help prevent two consecutive magnetosome membranes from merg-ing with one another.

A lot of what we know about the factors controlling magnetosome membrane formation comes from the ge-netic analysis of the MAI in M. magneticum sp. AMB-1 for which both genetic tools and a complete genome se-quence were made available a few years ago [Matsunaga et al., 2005]. A conserved genetic trait of MTB is the pres-ence on their chromosome of a conspicuous region called MAI which is essential for magnetosome formation. In AMB-1, this region is unstable and its spontaneous loss upon cultivation of the bacteria leads to the appearance in the population of nonmagnetic variants [Fukuda et al.,

2006]. These bacteria, which no longer display the char-acteristic magnetic behavior, are not only impaired in magnetite biomineralization, they are in fact incapable of forming the magnetosome compartments that are a pre-requisite for magnetite synthesis [Komeili et al., 2004]. In AMB-1, this 100-gene region which comprises most of the magnetosome genes was therefore proposed to code for proteins controlling inner membrane invagination. Yet no protein predicted to play such a role could be iden-tified on the basis of its sequence. In a comprehensive genetic analysis where all the genes in the MAI were sys-tematically deleted, it was shown that one conserved 18-gene cluster called the mamAB cluster was the only region of the MAI that was essential for magnetosome mem-brane formation. Four single-gene deletion mutants were impaired in producing magnetosome compartments [Murat et al., 2010]. The products of these four genes (MamI, MamL, MamQ and MamB) are well conserved in MTB (summarized in table 1 ) which reinforces the idea that these proteins may play an essential role in magneto-some formation. MamI and MamL are unique to MTB [Jogler et al., 2009] and no proteins with similar features could be identified in bacteria synthesizing other kinds of membrane-bounded organelle. These two proteins are predicted to encode small transmembrane proteins (around 6 and 8 kD, respectively) each containing two transmembrane segments and leaving less than 40 amino acids accessible from either the periplasm/magnetosome lumen or the cytoplasm. Therefore, the transmembrane helices themselves or the few residues that are not embed-ded in the inner membrane must be essential for the func-tion of these proteins, by influencing how they are insert-ed in the membrane or by mediating homo or hetero-meric interactions.

The two other genes essential for magnetosome invag-ination code for proteins that are conserved in MTB as well as in non-MTB. MamQ belongs to the LemA family of proteins which is a surface-exposed protein of un-known function first identified in Listeria [Lenz et al., 1996]. The 3-dimensional structure of its Thermotoga maritima homolog was solved and revealed that the larg-est part of the protein is made of four alpha helices. MamB, which was also shown to be essential for magne-tosome membrane formation in MSR-1 [Uebe et al., 2011], belongs to the highly conserved family of cation diffusion facilitators (CDF) responsible for zinc, cadmi-um and cobalt transport. One CDF transporter was in-volved in ferrous iron transport [Grass et al., 2005]. Both mamB and mamQ are present in two copies in the MAI in AMB-1 (100% identity at the nucleotide level) [Murat



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et al., 2010] and the phenotype of mamB and mamQ dele-tions could only be observed in a strain of AMB-1 lacking both paralogs. In general, CDF transporters seem to be important for magnetosome formation as several pro-teins of this family are present in the conserved magneto-some gene clusters. The mamAB cluster in AMB-1 en-codes two more transporters of this family, MamM and MamV. MamM is particularly well conserved in MTB but MamV is not. While in AMB-1, the deletion of mamM completely abolishes magnetite nucleation, in MSR-1, MamM plays a role in controlling the number of crystal nucleation sites [Uebe et al., 2011]. In addition, MamM can form heterodimers with MamB in MSR-1, an interac-tion that is important for MamB stability.

In the recently isolated greigite and magnetite-pro-ducing BW-1 strain, two independent mamAB -like clus-ters were found [Lefevre et al., 2011]. mamI and mamL are only present in one cluster while mamB and mamQ are present in both. One MamB is most closely related to MamB proteins from α-proteobacteria and therefore thought to participate in magnetite biomineralization, the second copy (MamB * ) is most closely related to its homolog in the greigite-producing δ-proteobacterium Candidatus M. multicellularis and was therefore pro-posed to be implicated in the formation of greigite mag-netosomes. The same holds true for MamQ and MamQ * ( table 1 ). From these observations, one could propose that while MamI and MamL play a structural role in mag-

netosome assembly and do not provide specificity for magnetite or greigite membranes, MamQ and MamB may help determine mineral specificity in addition to their role in establishing the membrane. Since MamB is predicted to be a transporter, it is likely that this protein allows for the coupling of membrane invagination with the accumulation of cations in the right amount inside the magnetosomes accordingly to the mineral that will be produced in it.

A fifth protein, MamY, was proposed to play a direct role in magnetosome membrane remodeling in AMB-1 [Tanaka et al., 2010]. It is also conserved in the MAI of many MTB and was reported to share some distant ho-mology with BAR-related domains, coiled-coil proteins which participate in membrane curvature control in eu-karyotes [McMahon and Gallop, 2005]. Considering the low level of similarity, one cannot exclude the possibility that the highly helical nature of MamY is responsible for this similarity. While the deletion of a region including mamY did not have any observable effect on magneto-some formation in a previous study [Murat et al., 2010], a mamY deletion was reported to produce smaller crystals in larger magnetosome compartments [Tanaka et al., 2010]. Interestingly, the purified protein can tubulate li-posomes in vitro which led the authors to propose a role for MamY in disconnecting the magnetosomes from the inner membrane. While this model does not take into consideration the fact that in AMB-1 the magnetosome

Table 1. Conservation of magnetosome genes important for membrane invagination and biomineralization

Phylum Pureculture

Genomesequenced

MamI MamL MamQ MamB Mineral Crystal symmetry MamC MamD Mms6 MmsF

AMB-1 α-prot. yes complete 1 1 2 2 magnetite cubic + + + +MSR-1 α-prot. yes contigs 1 1 1 1 magnetite cubic + + + +MS-1 α-prot. yes contigs 1 1 2 2 magnetite cubic + + + +MV-1 α-prot. yes partial

(MAI)1 1 1 1 magnetite elongated truncated

hexaoctahedrons+ + + +

BW-1 δ-prot. yes partial 1 1 2 2 magnetiteand greigite

bullet-shapedpleomorphic

ND ND ND ND

MC-1 α-prot. yes complete 1 0 1 1 magnetite cubic + – + +M. bav nitrospira no partial 1 0 2 1 magnetite bullet-shaped ND ND ND NDCandidatus M.multicellularis

δ-prot. no partial 0 0 1 1 greigite pleomorphic ND ND ND ND

RS-1 δ-prot. yes complete 0 0 1 1 magnetite bullet-shaped – – – –QH-2 α-prot. yes complete1 1 1 1 1 magnetite cubic + + + +MO-1 ND yes complete1 1 1 1 1 magnetite cubic + + + 2

1 Genome sequence is not publically available yet. ND = Not determined.

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do not pinch off the membrane, it is possible that MamY participates in establishing magnetosome architecture.

Models for Curvature Control in Magnetosome Formation In eukaryotes, three main mechanisms can cause

membrane curvature: membrane scaffolding by curved proteins or complexes, insertion of wedge-like amphipa-thic helices into the membrane and protein-protein crowding [Kirchhausen, 2012; Stachowiak et al., 2012]. Considering how little information we can extract from the primary sequence of the proteins implicated in mag-netosome membrane formation, each model can be envi-sioned as well as others that would be specific to MTB to trigger inner membrane invagination.

First and foremost, the results obtained so far do not allow us to draw a full picture of magnetosome mem-brane formation as the expression of MamI, MamL, MamQ and MamB in a mutant lacking the 18-gene mamAB operon is not sufficient to trigger magnetosome formation [Murat et al., 2010]. There are two main hy-potheses that could explain this result: at least two essen-tial factors remain to be identified in the mamAB cluster, or, magnetosome membrane formation or stability is de-pendent upon the presence of a minimum amount of magnetosome proteins ready to be packed in the organ-elle.

Magnetosome Membranes Are Formed thanks to the Coordinated Action of Membrane-Remodeling Proteins According to this first hypothesis, some proteins es-

sential for magnetosome membrane formation remain to be identified. While mamI , mamL , mamQ and mamB do not appear to be sufficient for magnetosome formation, two studies recently reported that in the absence of the MAI, the mamAB cluster is sufficient for the production of magnetosome organelles in MSR-1 and in AMB-1 [Lohsse et al., 2011; Murat et al., 2012]. This result clearly demonstrates that the missing proteins, if any, are present within the mamAB cluster, or that they must be encoded on the chromosome, outside the MAI. The fact that the single gene deletion strategy did not identify other pro-teins essential for magnetosome membrane formation in the MAI [Murat et al., 2010] suggests that the deletion of two other mamAB genes and not just one would be es-sential for the formation of the membranes in AMB-1.

Biochemical and structural characterization of the candidate proteins will be necessary to propose a model for membrane curvature control in MTB. It was previ-

ously proposed that MamL could participate to mem-brane curvature generation thanks to a particularly charged carboxy-terminal tail. In light of the wedge-like insertion model, even though it does not seem to form a clear amphipathic helix, this tail could insert itself in the lipid bilayer where it would locally generate curvature [Komeili, 2012].

A Set of Proteins Controls Membrane Invagination while Another Set Is Needed to Stabilize the Invagination The strain in which the sufficiency of MamI, MamL,

MamQ and MamB was tested lacks all other MamAB pro-teins, which represents the majority of magnetosome-as-sociated proteins. It is possible that in a context where more than 50% of the magnetosome content is missing, the compartments could form but not be stable and that the lack of magnetosome proteins would cause the mag-netosomes to collapse. According to this model, cells need to control the amount of magnetosome proteins produced to keep the size and shape of the magnetosome compartments consistent.

Is Protein Crowding Sufficient? According to the protein-protein crowding model that

was recently proposed and tested by C. Hayden and col-laborators, the coverage of a membrane above ∼ 20% of its surface is sufficient to generate membrane bending [Stachowiak et al., 2012]. In their work, the authors showed that even proteins that are unrelated to mem-brane curvature control, such as GFP, can bend mem-branes when sufficiently concentrated [Stachowiak et al., 2012]. Therefore, one cannot exclude the possibility that the four proteins identified in AMB-1 for magnetosome membrane formation are as essential because they can ac-cumulate and locally form primal supramolecular com-plexes on which other magnetosome proteins can assem-ble. This model could explain why the four proteins are essential but not sufficient in the absence of the rest of the magnetosome proteins.

Let’s Get Organized

Magnetosomes present a quite large range of architec-tures in MTB where the number, size and morphology of the crystals vary between species. From one chain con-taining 12–15 crystals in RS-1 to several bundles of chains grouping hundreds of crystals in M. bavaricum, what is common to all these organelles is the fact that they are



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organized into chains. This organization is crucial for or-ganelle function as clumped magnetosomes would not al-low for the cells to align in the magnetic field and would make magnetic organelle transmission to the progeny more complex. In eukaryotes, organelles and the compo-nents of the cytoplasm are organized by a cytoskeleton made of three main components (tubulin, actin and in-termediate filaments) for which many bacteria have ho-mologs (MreB and FtsZ for example) [Thanbichler and Shapiro, 2008]. In addition to chromosomal bacterial ac-tin and tubulin homologs, MTB have a unique actin-based cytoskeleton which helps organize the magneto-somes into chains [Komeili et al., 2006; Pradel et al., 2006; Scheffel and Schuler, 2007]. MamK, a unique actin-like protein which forms a specific bacterial actin clade, was shown to be responsible for magnetosome chain organi-zation in AMB-1 [Komeili et al., 2006]. In a Δ mamK AMB-1 mutant, magnetosomes are scattered and found around the cell membrane instead of being aligned into a chain. This protein forms a network of parallel short fila-ments flanking the magnetosome chain and is particu-larly well conserved in MTB. MamK is even present in two copies on the MAI of the magnetic vibrio MV-1 [Jo-gler et al., 2009]. The genome of AMB-1 encodes for a MamK-like actin (54.5% identity with MamK) in a locus comprising several other magnetosome gene paralogs named the magnetosome islet [Rioux et al., 2010]. Both MamK and a MamK-like protein were shown to form polymers in vitro [Ozyamak et al., 2012; Rioux et al., 2010; Taoka et al., 2007] but a direct role of the MamK-like pro-tein in magnetosome chain assembly remains to be shown in vivo. It was proposed that the MamK-like protein could help MamK organize the magnetosome chain and that the simultaneous deletion of mamK-like and mamK would lead to a more severe effect on magnetosome orga-nization [Rioux et al., 2010]. In MSR-1, the deletion of mamK has a pleotropic effect of magnetosome organiza-tion as it leads to shorter, interrupted chains of magneto-somes producing smaller magnetite crystals [Katzmann et al., 2010]. Therefore, deletion of mamK affects magne-tosome organization in both AMB-1 and MSR-1, but in different ways. Similarly, the deletion of another con-served gene located directly upstream of mamK , mamJ , also differentially affects magnetosome organization in AMB-1 and MSR-1. Its deletion causes the magnetosome chain to collapse into aggregated magnetosomes [Scheffel et al., 2006] in MSR-1 while it does not affect chain orga-nization in AMB-1 [Draper et al., 2011]. In the latter, however, another protein named LimJ (60–70% similar-ity to MamJ) works with MamJ to control MamK fila-

ment dynamics. In a double Δ mamJ Δ limJ mutant, the MamK filaments are static and have a tendency to assem-ble in thick bundles in between magnetosomes [Draper et al., 2011].

Magnetosomes clearly have in common a dedicated cytoskeleton that organizes the organelles even though some differences exist in how its components work. The results obtained in Magnetospirilla so far suggest that each species, according to its specific gene catalog and the particularities of its chain or chains, has evolved specific ways to organize the magnetosome organelles.

Reaching the Magnetosomes

The fact that a particular set of proteins is associated with the magnetosomes demonstrated that the bacterium is capable of sorting its proteins, in particular integral membrane proteins, to the magnetosome membrane. The proposed role for these proteins is to help build and shape the compartments and make them chemically suit-able for magnetite synthesis. The cell has to bring the cor-rect set of proteins, and probably the right amount of these proteins, so as to build a magnetosome.

The search for a signal sequence common to all identi-fied integral magnetosome membrane proteins failed to identify a conserved motif that would suggest a specific sorting mechanism of magnetosome proteins. While in-vestigating the function of the MAI genes in AMB-1, a class of mutants which did not produce magnetite but still formed membrane invaginations was identified [Murat et al., 2010]. The presence of empty magnetosomes in these mutants could be explained by their incapacity to take up iron, to establish the proper chemical environment for magnetite nucleation in the magnetosomes, or to prop-erly localize magnetosome proteins essential for biomin-eralization. By following the localization of several GFP-tagged magnetosome proteins in these mutants, it was shown that in one of them, lacking the putative mem-brane-bound protease MamE, some magnetosome pro-teins were no longer correctly localized to the magneto-somes [Murat et al., 2010; Quinlan et al., 2011]. The fact that the magnetosome membranes can still be formed and aligned in the cell while a subset of magnetosome proteins are not properly localized to the organelles indi-cated that magnetosome invaginations can form prior to and independently from magnetosome protein targeting. The proteins for which localization was followed partici-pate at different stages of magnetosome formation: from the initial step of membrane invagination (MamI) to the

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final step of magnetite biomineralization (MamC). The fact that the magnetosome membranes are present in the Δ mamE mutant while the localization of MamI is affected suggests that this protein may participate in magneto-some membrane formation by transiently localizing at the organelle, or that some MamI is still localized in the Δ mamE mutant. By generating point mutations in MamE’s protease catalytic site, it was shown that the pro-tease function of the protein was not essential for magne-tosome protein targeting [Quinlan et al., 2011]. However, a protease-deficient mutant of this protein had lost the ability to restore complete magnetite crystal formation in a Δ mamE mutant. The fact that the protease activity of MamE is not essential for protein localization to the mag-netosomes suggests that MamE would act as an anchor-ing platform and recruit magnetosome proteins through specific protein-protein interactions. Like other DegQ homologs, MamE possesses two PDZ domains located in the periplasm/magnetosome lumen, which could be held responsible for such interactions. In MSR-1, the MamE PDZ1 domain was shown to interact with MamB [Uebe et al., 2011]. In MSR-1, it was also found that MamC is mislocalized in the absence of MamB or MamM, which were also shown to play multiple roles during magneto-some formation [Uebe et al., 2011]. This result also sug-gests that some magnetosome proteins might be recruited to the organelles by interacting with preassembled pro-tein complexes.

MTB: Biomineralization Experts

The unique properties of magnetosomes have inspired a lot of research in fields as varied as material sciences, chemistry and synthetic biology. Synthetically produced magnetite nanocrystals are used in medical sciences and nanotechnologies for which the magnetic properties of the nanoparticles, which are directly correlated with their size, must be tightly controlled [Lang et al., 2007]. Unfor-tunately, de novo magnetite synthesis techniques mostly allow for the production of crystals which are either too small (superparamagnetic) or too large (multiple domain crystals) for these applications. Magnetite crystals pro-duced by MTB are strikingly consistent in size and shape and always fall within the single magnetic domain range (30–120 nm in length) and confer a permanent magnetic dipole moment to the cell. In addition, in MTB, magnetite is conveniently produced at ambient temperature and under atmospheric pressure. While crystal size and ge-ometry vary across different species, they are highly con-

sistent within one species, which suggested that magne-tite crystal properties are genetically controlled. The search for proteins that can control crystal properties has motivated a lot of work in our field, aiming at using these proteins for bioinspired synthesis of magnetite nanopar-ticles in vitro. While a dozen putative biomineralization mutants were reported in the literature, a direct role for these proteins in magnetite synthesis in vitro was only confirmed for one of them [Amemiya et al., 2007; Ara-kaki et al., 2010; Wang et al., 2012]. A lot of work remains to be done to evaluate if and how these biomineralization candidates participate in magnetite synthesis.

Magnetosome proteins can be classified according to the kind of interaction they make with the magneto-somes. Four magnetosome proteins were found to direct-ly associate with the crystals and in consequence they were proposed to play a role in biomineralization: MamC, MamD, Mms5 and Mms6 [Arakaki et al., 2003]. A role of MamC, MamD and Mms6 in controlling magnetite crys-tal growth was confirmed in vivo in MSR-1 and/or in AMB-1 [Scheffel et al., 2008b; Tanaka et al., 2011; Murat et al., 2012] but the effect of their deletion was surpris-ingly mild. While MamC represents the most abundant magnetosome protein in MSR-1, its deletion did not af-fect magnetite synthesis significantly unless three other genes cotranscribed with it (mamD , mamF and mamG) are also deleted [Scheffel et al., 2008b]. Even then, the four-gene deletion only leads to a reduction of 25% of crystal size. Similarly, the deletion of Mms6, which is the only magnetosome protein which once purified was shown to facilitate magnetite synthesis in vitro, only has little effect on magnetite crystal synthesis in AMB-1 [Ara-kaki et al., 2010; Murat et al., 2012]. These results were surprising in that these four proteins were the most likely biomineralization candidates in MTB. They are encoded by conserved neighboring gene clusters in most MTB (AMB-1, MS-1, MSR-1, MV-1). In α-proteobacteria, mms6 is located directly upstream of the conserved mmsF gene, which product was found to copurify with magne-tosomes in MSR-1. In AMB-1, it was found that the dele-tion of mmsF had a much stronger effect on magnetite synthesis than the deletions of either the mamCDF cluster or mms6 . Moreover, in an AMB-1 strain where these five biomineralization genes are deleted, the expression of MmsF alone is sufficient to restore wild-type magnetite biomineralization [Murat et al., 2012]. Therefore, magne-tite synthesis can fully proceed in the absence of these four genes as long as MmsF is expressed. This suggests that many factors can contribute to magnetite biominer-alization but can have redundant functions in this pro-



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cess. It is likely that these proteins can regulate each oth-er’s activity in vivo [Murat, 2012].

The current model for crystal formation in MTB is that small proteins are able to bind to specific faces of the growing crystal where they would locally inhibit crystal growth so as to determine its elongation axis or axes. The capacity to bind to the crystals was verified in vitro for the Mms6 protein and remains to be tested for all other biomineralization candidates.

To date, the deletion of about a dozen MTB proteins was shown to affect magnetite crystal size or geometry to different extents in AMB-1 and MSR-1. For example, four mutants producing aberrant magnetite crystals were iso-lated in AMB-1 (Δ mamP , Δ mamR , Δ mamS and Δ mamT ) [Murat et al., 2010]. This was particularly informative as each mutant was affected in magnetite synthesis in a dif-ferent manner. For example, crystal size was reduced to the same extent in two mutants (Δ mamR and Δ mamS ) but while the crystals had a typically wild-type geometry in one mutant, they were aberrantly shaped in the other mutant. This demonstrated that crystal size and geometry could be independently controlled.

Biomineralization mutants include mutants in iron-related proteins like Fur (ferric uptake regulator) or the FeoB system [Qi et al., 2012; Rong et al., 2008] for which a defect in magnetite synthesis could be expected. How-ever, a biomineralization defect is harder to explain when cells are mutated for cytoskeletal elements such as the ac-tin-like protein MamK or the bacterial tubuline FtsZ - like protein [Ding et al., 2010; Katzmann et al., 2010]. The rather high number as well as the nature of the putative biomineralization mutants described in the literature suggests that some of the biomineralization phenotypes reported are most likely secondary consequences of an altered chemistry in the magnetosomes or to a different protein composition of the organelle.

This data was obtained from Magnetospirilla species which produce crystals of cubic symmetry. Until very re-cently, it was still unclear whether the biomineralization genes were common to MTB species producing bullet-shaped magnetite crystals or greigite crystals. Surprising-ly, the mamCDF and mms genes are not conserved in the δ-proteobacterium RS-1 which produces bullet-shaped magnetite crystals ( table 1 ). Recent reports identified mamAB -related gene clusters in two greigite-producing MTB Candidatus M. multicellularis and BW-1. This in-dicates that a similar pool of magnetosome genes could control magnetosome formation regardless of the min-eral produced within it [Abreu et al., 2011; Lefevre et al., 2011]. However, the presence of the biomineralization

mamCDF and mms genes has not been confirmed yet in these organisms. If they are indeed absent, this would in-dicate that biomineralization of greigite is handled by a different set of biomineralization proteins.

Conclusions

The MAI: Is That All It Takes? The exponential improvements in the fields of DNA

synthesis and DNA sequencing have revolutionized the way we think about molecular biology. Complete meta-bolic pathways and whole genomes can be synthesized de novo and manipulated for the purpose of applications [Gibson et al., 2010; Moon et al., 2012]. As in MTBthe genes responsible for magnetosome formation are grouped within a dense locus on the chromosome, an ob-vious question has been: is that all it takes? This question was addressed in two independent studies performed in MSR-1 and AMB-1. In both cases, it was shown that in the absence of the rest of the MAI, the mamAB gene clus-ter was in fact sufficient for the formation of aligned elec-tron-dense particles resembling magnetosomes in the cell cytoplasm [Lohsse et al., 2011; Murat et al., 2012]. The particles were identified as being magnetite in AMB-1 and ECT, confirming that the crystals were formed with-in a membrane compartment the dimensions of which were similar to those of wild-type magnetosomes [Murat et al., 2012]. In AMB-1, the expression of MmsF in addi-tion to the mamAB cluster significantly improves magne-tite crystal growth, although overall crystals remain smaller than wild-type ones [Murat et al., 2012]. Even though these results are encouraging as they indicate that magnetosome formation can be assumed by as few as 19 MAI genes, it is still not clear whether other non-MAI genes are essential for magnetosome formation. This small number of genes could be used for the development of an autonomous genetic cassette dedicated to heterog-enous magnetosome expression in technologically or in-dustrially relevant microorganisms. One of the main ca-veats of heterogeneous magnetosome expression resides in the fact that the receiving organism would have to be able to take up close to 4% of its dry weight in iron while E. coli uptakes the equivalent of 0.022%. As our under-standing of magnetosome and magnetite chemistry re-main limited, it is difficult to predict whether MTB, in addition to the MAI, encode specific metabolic pathways that are too essential for the compartmentalized produc-tion of magnetic minerals. Moreover, the dosage and tim-ing of magnetosome gene expression might be essential

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Got Genetic Tools? The complete genomes of five freshwater MTB are

currently available (AMB-1, MS-1, MSR-1, MC-1 and RS-1) and the sequence of M. blakemori MV-1 MAI is known. All but one genome (RS-1 is a δ-proteobacterium) belong to α-proteobacteria, and three belong to the genus Magnetospirilla , which limits the general conclusions that can be made about magnetosome gene evolution. The ge-nomes of two novel species of marine MTB have been recently sequenced and should be made available soon (MO-1 from the Mediterranean Sea, France [Lefevre et al., 2009], and QH-2 from the Yellow Sea, China [Zhu et al., 2010]). At last, the sequences of some magnetosome genes were made available for two greigite-producing MTBs [Abreu et al., 2011; Lefevre et al., 2011] which re-vealed that a similar magnetosome gene pool could be central to the formation of greigite-containing magneto-somes. The ultrastructural analysis of magnetosomes in different bacteria can only highlight the fact that magne-tosomes come in many different flavors, however, some general features such as the organization in a chain and the requirement for a membrane for crystal biomineral-ization seem to be conserved. The presence in the genome of at least a subset of conserved magnetosome genes is also clearly common to all MTB, yet the numbers and types of genes vary a lot from species to species.

Even if the accumulation of genomic and structural data is essential for a better understanding of magneto-some organelle evolution, across time and phyla, the ca-pacity to grow and genetically manipulate MTB is cruelly lacking. The difficulties to isolate and grow MTB axeni-cally and most importantly the lack of genetic tools in all but two species considerably limits the conclusions one can make from genomic data. From where the magneto-some field stands, it is essential that genetic tools be de-veloped for more MTB species, in particular non-α-proteobacteria, so that morphological differences and biomineralization specifics can be correlated with a par-ticular gene set. Lastly, in addition to genomics, phyloge-netics and genetics, a solid investment in magnetosome protein biochemistry including protein-protein interac-tions will be the next logical path to take to understand how magnetosomes are assembled at the molecular level and allow for a better understanding of bacterial organelle biogenesis.

Aknowledgments

I would like to thank Dr. L.-F. Wu, Dr. C.-L. Santini and Dr. E. Mauriello for their critical reading of the manuscript and sugges-tions. I also thank Claudine Médique, Valérie Barbe (Genoscope, Evry, France) and Dr. L.-F. Wu (Laboratoire de Chimie Bactéri-enne, Marseille, France) for giving me access to the genomes of QH-2 and MO-1. Dr. D. Murat is supported by a grant from the Fondation pour la Recherche Médicale SPF20110421349.



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magnetosomes: how do they stay in shape?

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