A Look into the Biochemistry of Magnetosome Biosynthesis in

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A look into the biochemistry of magnetosome biosynthesis in magnetotactic bacteria Shiran Barber-Zucker, and Raz Zarivach ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01000 • Publication Date (Web): 08 Dec 2016 Downloaded from http://pubs.acs.org on December 9, 2016

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ACS Chemical Biology

A look into the biochemistry of magnetosome biosynthesis in magnetotactic

1

bacteria

2 3

Authors: Shiran Barber-Zucker and Raz Zarivach*

4 5

Authors' affiliation:

6

Department of Life Sciences, the National Institute for Biotechnology in the Negev and Ilse

7

Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev,

8

Beer Sheva, 8410501, Israel

9 10

* Correspondence should be addressed to Raz Zarivach, Department of Life Sciences, Ben-

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Gurion University of the Negev, P.O.B. 653, Beer Sheva 8410501, Israel. Tel: +972-8-

12

6461999, Fax: +972-8-6472970, Email: [email protected]

13 14 15 16

ACS Paragon Plus Environment


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Abstract

17

Magnetosomes are protein-rich membrane organelles that encapsulate magnetite or greigite

18

and whose chain-alignment enables magnetotactic bacteria (MTB) to sense the geomagnetic

19

field. As these bacteria synthesize uniform magnetic particles, their biomineralization

20

mechanism is of great interest among researchers from different fields, from material

21

engineering to medicine. Both magnetosome formation and magnetic particle synthesis are

22

highly controlled processes that can be divided into several crucial steps: membrane

23

invagination from the inner-cell membrane, protein sorting, the magnetosomes' arrangement

24

into chains, iron transport and chemical environment regulation of the magnetosome lumen,

25

magnetic particle nucleation and finally crystal growth, size and morphology control. This

26

complex system involves an ensemble of unique proteins that participate in different stages

27

during magnetosome formation, some of which were extensively studied in recent years. Here

28

we present the current knowledge on magnetosome biosynthesis with a focus on the different

29

proteins and the main biochemical pathways along this process.

30 31


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Keywords

32

Magnetotactic bacteria: Gram negative bacteria that can align themselves according to the

33

geomagnetic field and by that reach a suitable environment for their survival.

34

Magnetosome: a subcellular organelle in magnetotactic bacteria, comprising a magnetic iron

35

mineral enclosed in a protein-rich membrane.

36

Biomineralization: the formation of minerals by living organisms.

37

Magnetosome island: a genomic region in magnetotactic bacteria that encodes for proteins

38

that participate in magnetosome formation.

39

Cation diffusion facilitator: a conserved family of divalent transition metal cation

40

transporters that usually utilize the proton motive force to excrete the metals from the

41

cytoplasm.

42

Major facilitator superfamily: a conserved family of uniporters and cotransporters that

43

facilitates the movement of small molecules through the membrane.

44

Magnetochrome: a small, c-type cytochrome domain that is found uniquely in magnetotactic

45

2+

bacteria and participates in Fe oxidation inside the magnetosome.

46

Protein raft: a defined area in the membrane that is condensed with transmembrane proteins

47

and, similarly to a lipid raft, has specific properties that allow a certain function.

48 49 50 51 52



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Introduction

53

Many organisms are thought to have the ability to sense the geomagnetic field, including 1

54

birds, fish, insects and even humans . In 1963 and 1975 Salvatore Bellini and Richard

55

Blakemore, respectively, independently discovered magnetotactic bacteria (MTB), a group of

56

Gram-negative bacteria that can be found in sediments and aquatic environments and navigate

57

2–4

passively according to the geomagnetic field lines . Since their discovery, MTB have

58

become the most characterized group of organisms that can orient according to a magnetic

59

field. MTB are highly divergent and affiliated to Alpha-, Gamma- and Deltaproteobacteria

60

classes of the Proteobacteria phylum, as well as to the Nitrospirae, Latescibacteria and

61

5–7

Omnitrophica phyla . Today, there are cultivated strains from all the Proteobacteria

62

classes8, but mostly the study of Alphaproteobacteria Magnetospirillum magneticum AMB-1

63

(AMB-1) and Magnetospirillum gryphiswaldense MSR-1 (MSR-1) strains in recent years

64

9

contributed to the understanding of the molecular mechanism of MTB .

65

In all MTB strains, the ability to navigate according to the geomagnetic field lines is due to

66

the formation of a chain-like arrangement of magnetosomes, MTB-exclusive organelles in

67

which each is composed of a magnetic particle (magnetite, Fe3O4, or greigite, Fe3S4)

68

encapsulated in a protein-rich membrane10,11. The magnetic particle size and shape varies

69

between different MTB strains but are conserved within each strain, which makes their

70

biomineralization mechanism of great interest to the biotechnology community. The magnetic

71

particle size range is usually ~30–120 nm, the size of a single magnetic domain, and only the

72

alignment of several magnetosomes into a chain or chains (strain-dependent) creates a dipole

73

12

moment . The magnetosome chain's dipole enables the rotation of the bacterium to the

74

direction of the geomagnetic field and allows for its movement according to the field lines

75

using its flagella. This behavior, named magnetotaxis, enables the bacterium to reach a

76

13

suitable, usually oxic-anoxic zone in aquatic ecosystems .

77

The magnetosome membrane and lumen differ from the cytoplasm and its membrane in their

78

compositions, creating together a distinctive environment that enables the biomineralization

79

14

of the magnetic particle . The formation of the magnetosomes themselves and their

80

alignment are highly controlled processes and require a unique set of proteins, most of which

81

are encoded in a conserved genomic segment named the magnetosome island (MAI)

14–16

. The

82

MAI is found in all MTB species and contains a few operons: the most conserved and

83

essential mamAB operon is found in all characterized MTB strains, and other operons such as

84

5,17

mamCDFG, mamXY and mms6 are specific to Alphaproteobacteria

. The proteins encoded

85

by these operons are named magnetosome membrane-associated (Mam) and magnetic particle

86

membrane-specific (Mms) proteins and were widely studied in the past decade. Other sets of

87

magnetosome-related genes are the magnetosome-associated Deltaproteobacteria (mad)

88


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genes in Deltaproteobacteria, Nitrospirae and Omnitrophica strains and the magnetosome

89

genes in Nitrospirae (man), which started to be characterized recently and whose encoded

90

proteins are thought to have a parallel role to some of the Mam and Mms proteins, but in

91

18,19

different strains

.

92

Mam, Mms, Mad and other related proteins participate in concerted processes that result in

93

the formation of an adult, functioning magnetosome chain or chains. Magnetosome formation

94

begins with membrane invagination to create a separated environment from the cytoplasm,

95

combined with the sorting of specific proteins that are needed for the biomineralization, some

96

of which are exclusively found in the magnetosome. Next, magnetosome alignment into

97

chains and magnetic particle nucleation starts. The final step is the magnetic particle growth

98

into a specific size and morphology (Figure 1)14,20,21. Recent reviews in the field of MTB have

99

9

focused on different scientific aspects of magnetosome biogenesis, such as genetic studies , 22

20

chemical pathways that lead to magnetite formation , protein function

100

and single-protein

101

studies . A comprehensive structural study of magnetosome proteins presented structural

102

modeling with an overview of these proteins studies to gain better understanding of their

103

23

21

function . Here, we review all of the pathways in the magnetosome formation scheme with

104

an emphasis on protein structural studies and the most studied and defined biochemical

105

processes that occur during magnetite biosynthesis: the transport of iron to, into and inside the

106

2+

magnetosome, the oxidation of Fe

and pH regulation to enable magnetite nucleation, and

magnetite growth and morphology control.

107 108 109

Constructing the organic envelope: membrane invagination and protein sorting and

110

activation

111

Nucleation of magnetite or greigite particles requires a specific chemical environment, with a

112

basic pH and a local high iron concentration, for example, and can result in the formation of

113

toxic byproducts24,25. In MTB, the formation of the lipid bilayer magnetosome membrane

114

(MM) creates a distinct environment that allows magnetic particle biomineralization and

115

protects the cell from unnecessary, harmful byproducts26. The MM contains a mass of

116

proteins that facilitate the formation of the required environment and hence creates the

117

21,27,28

suitable space for the particle nucleation and growth

. Many studies have showed that the

118

MM invaginates from the inner cell membrane (CM), a process that is utilized by a few

119

11,29

magnetosome-associated proteins

. The invagination of the MM does not depend on

120

magnetic particle formation, as empty magnetosome vesicles can be found prior to the

121

26

biomineralization process and in non-magnetic MTB strains . It was recently shown that in

122

AMB-1 the membrane grows to a certain size, and only if and when biomineralization starts

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does the membrane expand to a larger size that can contain the mature magnetite30. In some

124

MTB species, at least some of the magnetosomes stay attached to the CM, which can enable

125

better orientation of the whole bacterium in response to the magnetic field

9,29

. It is also

126

speculated that the CM-attached magnetosomes often have an open channel to the periplasmic

127

space that can facilitate the exchange of compounds such as iron with the periplasmic space

128

29,31

. However, it is believed that

129

they are not fully open but at least partially closed, maybe by a plug of proteins, since in an

130

open state the magnetosome lumen is exposed to unnecessary components that can be harmful

131

and affect normal magnetite formation. Yet, the magnetosomes do not remain attached to the

132

CM in all species32, suggesting a variation in membrane biogenesis between different species.

133

A few studies have shown the relationship between MM invagination and the magnetosome-

134

and provide an advantage in nucleation and magnetite growth

associated proteins MamB, Q, L, and Y (and in AMB-1, also MamI)

33,34,28,35

. Recently it was

135

shown by Raschdorf et al. that in the absence of the mamAB operon in MSR-1, only the

136

complementation with seven mam genes – mamL, Q, B, I, E, M, O – resulted in proper

137

magnetosome vesicle synthesis, suggesting that all of these proteins have a critical role in

138

33

membrane biogenesis . This study also found that the most crucial protein for MM formation

139

is MamB, which belongs to the cation diffusion facilitator protein family and hence may also

140

33,36

have a role in ferrous transport into the magnetosome

. All of these proteins are thought to

141

be involved in the physical bending of the membrane or in protein recruitment near the MM

142

nucleation points in the CM, and by that to facilitate its invagination20,21,37.

143

Although invaginated from the CM, the MM protein composition is different from that of the

144

CM, hence protein sorting and activation are needed to enable the formation of the MM itself

145

10,27,38,39

and of the magnetic particle

. Over the years, many studies have associated protein

146

sorting with MamA and MamE, and therefore they will be discussed here in detail. Yet,

147

recently it was suggested that MamL also has a role in the recruitment of magnetite

148

33

maturation-related proteins to the magnetosome . MamA is a conserved cytoplasmic 40

149

protein whose structures from different MTB phyla were solved. All structures contain five

150

(and putatively six) tetratricopeptide repeat (TPR) motifs that create three protein-protein

151

41–43

interaction sites: a concave site, a convex site and a putative TPR

(Figure 2a). Self-

152

assembly of MamA creates homo-oligomers that coat the MM from its cytoplasmic side and

153

44

enables interaction with other magnetosome-associated proteins (such as Mms6 , as will be

154

discussed later), presumably with its convex site41,45 (Figure 2a). MamE is an HtrA/DegP

155

serine protease that was shown, in a few studies, to affect magnetosome-associated proteins’

156

localizations and therefore to have a role in protein sorting28,46. MamE contains a trypsin-like

157

domain, two cytochrome domains and two PDZ domains. In the absence of MamE, no

158

magnetic particles are formed and a few magnetosome proteins changed their localization,

159


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and in MamE protease's catalytic domain mutated form, smaller magnetic particles were 46,47

160

. These particles are smaller than are required to form a magnetic dipole, which

161

raised the hypothesis that the cells produce small magnetic particles and only when some

162

obtained

46

signal activates MamE does the maturation of the particles to their paramagnetic form start .

163

MamE’s proteolytic activity includes a self-cleaving activity and cleavage of the

164

magnetosome-associated proteins MamO and MamP, processes that require MamO's ion-

165

47–50

transporter TauE domain

. Stimulated by substrates, MamE was shown to sequentially

166

auto-cleave small fragments from its C-terminal in vivo and in vitro. Furthermore, ligand-

167

47

binding to both PDZ domains activates its auto-processing . Although MamE contains two

168

cytochrome domains, there is no experimental evidence that these domains or their redux-

169

47

related activity impacts MamE’s proteolytic regulation . Altogether, a switch-like mode of

170

regulation was suggested for MamE, in which the protease domain requires a specific

171

environment to be activated and the balance between its active and inactive states is needed

172

for a proper biomineralization process47.

173

A recent study suggested that, firstly, a membrane of a certain size is formed with the same

174

interior conditions as the periplasm, and once proper conditions for magnetite formation are

175

achieved, magnetite nucleation starts followed by membrane and magnetite growth (Figure

176

1). The size restriction prior to nucleation allows supersaturation of iron to facilitate

177

30

nucleation . Together with the switch-like mode that was proposed for MamE, it was

178

suggested

179

that

proteolysis by MamE is

the

switch that

controls the

crystal

47

nucleation/membrane growth .

180 181

Lining up: alignment of magnetosomes into chains

182

The magnetic particles inside the magnetosomes are in the size of a single magnetic domain,

183

therefore only the establishment of a magnetosome chain or chains (strain-dependent) can

184

result in a dipole moment and the weak-geomagnetic field sensing. Although it seems logical

185

that the chains will be formed mostly due to the magnetic attraction between the

186

magnetosomes, only a few studies showed this relationship and most of the studies associated

187

29,51–53

chain formation with the protein MamK

. With the cytoplasmic, acidic and putatively

188

unstructured protein MamJ serving as an anchor, the magnetosomes are attached to a long,

189

filamentous structure made of the actin-like protein MamK that stretches from one end of the

190

cell to the other

54,55,52

. MamK contains an ATP binding site (as other actin-like proteins do)

191

and its assembly depends on the hydrolysis of ATP in all studied species, on salt

192

56–58

concentrations and on the presence of magnesium in some strains

. As MamK’s surface is 57

largely hydrophobic, the salt concentration is thought to control and limit filament assembly .


193 194


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A 6.5 Å resolution EM structure of MamK confirmed its previously suggested double56,58

195

. This was supported by a recent study

196

that achieved an EM structure of AMB-1 MamK filaments at 3.6 Å resolution combined with

197

stranded, non-staggered organization (Figure 2b)

59

a monomeric 1.8 Å resolution crystal structure . MamK monomers are made of four

198

canonical domains (two domains that are each divided into two subdomains: IA, IIA, IB and

199

56,59

. The

200

transition from monomer to filament assembly involves the binding of subdomain IIA to both

201

subdomains IB and IIB from the previous protomer, which results in them getting closer to

202

each other. The tighter cleft that is formed in the polymerization and the movement of key

203

residues are thought to facilitate the ATP hydrolysis into ADP59. The longitudinal contacts in

204

each strand are based on hydrophobic interactions, electrostatic attraction, salt bridges and

205

hydrogen bonds between the positively charged top edges and negatively charged bottom

206

edges of the monomers (Figure 2b). Big gaps separate MamK's strands and there is a small

207

number of interactions between the strands and a small interaction area compared to other

208

IIB), with an ADP molecule found in a cavity between two domains (Figure 2b)

56,59

actin-like proteins (Figure 2b)

. Overall, the most current study on AMB-1 MamK

209

suggested that after invagination, magnetosomes are aligned discontinuously to an axis in a

210

MamK-independent manner and that MamK filaments have a role in closing the gaps to

211

create a long, continuous magnetosome chain, either by recruiting misaligned magnetosomes

212

into these empty locations or by pulling the existing magnetosomes together to create a more

213

30

compact packing .

214 215

Starting to biomineralize: nucleation of magnetic particle

216

To initiate biomineralization – that is, to enable iron condensation into magnetite nuclei – a

217

high concentration of iron and a proper chemical environment are required. As one of the key

218

aspects in biomineralization, deciphering the proteins' nucleation-related mechanisms has

219

been of great interest to researchers in the MTB field. The main steps that were characterized

220

in recent years regarding magnetite nucleation and that will be discussed here are: iron

221

transport from the cytoplasm, through the MM and within the magnetosome; pH regulation in

222

the magnetosome; the oxidation of Fe2+ to Fe3+; and the formation of ferrihydrite as a

223

precursor and its transformation to magnetite (Figure 3a).

224

In order to form magnetite, an effective mechanism for iron transport into the magnetosomes

225

is needed. In the Deltaproteobacteria strain RS-1, which lives in anoxic environments where

226

iron is mostly in ferrous form, it was recently shown that ferrous ions accumulates in the cell

227

as FeP granules and is then converted to other forms of iron: ferritin, magnetite and mostly

228

60

ferrous FeS

(although there are multiple intramembrane organelles in RS-1, there is


229

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contradicting evidence as to whether the magnetite itself is enclosed in a lipid membrane61,62,

230

therefore the compartmentalization of the iron forms is not clear). In AMB-1, the model

231

bacterial strain that lives under microoxic conditions, it was shown that Fe is stored in a

232

phosphate-rich ferric hydroxide phase in the cytoplasm, which is consistent with prokaryotic

233

ferritins and the expected iron state in the microoxic environment. Then, Fe and P are

234

22,63

. The current literature regarding

235

magnetosome proteins leads to the assumption that the iron is transported into the

236

magnetosomes in the form of ferrous rather than ferric ions. In that case, the existence of a

237

phosphate-rich ferric hydroxide phase before the Fe-P separation requires redox factors in the

238

cytoplasm that should be found very close to the magnetosome membrane. These factors are

239

presently unknown but we speculate that the membrane-associated ferritins, which can store

240

ferric ions and cause the nucleation of the FeP phase, have at least a partial role in the

241

oxidation-reduction process. Nevertheless, the difference in the environment redox conditions

242

and the iron forms suggests a link between these factors in MTB60. The magnetosomal cation

243

diffusion facilitator (CDF) proteins MamB and MamM are associated with iron transport into

244

separated and the Fe is transported into the magnetosome

36

the magnetosome . CDF proteins transport divalent transition metal cations from the

245

cytoplasm to the extracellular environment or into intercellular compartments, usually by

246

exploiting the proton motive force. These proteins are conserved in all domains of life and are

247

crucial for normal function of the cell and, as such, they are of great interest and were widely

248

64

studied in the past two decades . CDF proteins’ structures consist of a six-helix

249

transmembrane domain (TMD) and typically a cytoplasmic C-terminal domain (CTD) with a

250

36,64

metallocheparone-like fold, and they usually form homodimers

. The MamM CTD

251

structure from MSR-1 was solved and well characterized (Figure 3b); it binds iron and zinc in

252

vitro, which facilitates a CTD conformational change to a tighter packing and this, in turn,

253

was suggested to promote a conformational change of the TMD to allow cation transport

254

65

through the membrane . Deletion of full mamM or only its CTD and specific point mutations

255

within its CTD result in no magnetic particle formation36,66. Furthermore, specific loss-of-

256

structure of its CTD and mutations in its TM and CTD putative metal binding sites caused

257

36,65–67

defects in magnetite formation

. MamM stabilizes MamB and both were shown to

258

interact, presumably via their CTDs, suggesting that they are forming not only homodimers

259

36

but also heterodimers . In contrast to MamM, deletion of mamB results in the lack of the MM

260

and mutations within its TMD metal binding-site abolished magnetite formation28,36. MamB

261

and MamM are mainly found in the MM rather than the CM and were shown to have a role in

262

the accumulation of iron in the magnetosome specifically, meaning that a special mechanism

263

is required for their location-related function, maybe by interaction with other magnetosome-

264

related proteins36. The difference between their phenotypes and the fact that they cannot

265



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compensate for each other suggests that MamB and MamM have distinct roles in

266

magnetosome formation in general and in magnetite nucleation in particular.

267

By exploiting the proton motive force for metal transfer, the CDF proteins are also assumed

268

36

to affect the pH in the magnetosome lumen . MamB and MamM most likely cause to the

269

increasing of the magnetosome lumen pH and by that optimize the conditions for magnetite

270

growth that requires basic pH (the formation of magnetite releases eight protons per

271

magnetite: 2Fe3+ + Fe2+ + 4H2O Fe3O4 + 8H+)31. Since the number of released protons in

272

magnetite formation is much larger than the consumed iron, the CDF proteins are not

273

sufficient and other systems are required for proton extermination. Another protein that was

274

+

+

suggested to have a role in pH regulation is MamN, a Na /H antiporter homolog in whose

275

absence no crystals (AMB-1) or smaller crystals (MSR-1) were produced21,28,35. Additionally,

276

50

another protein, MamP, also helps in exporting protons, as will be discussed below .

277

Containing the major facilitator superfamily (MFS) domain, MamH and MamZ are suspected

278

68

to take part in the regulation of the chemical environment in the magnetosome . Concerning

279

MamH, its predicted structure contains a negative cavity and its deletion results in a decrease

280

in magnetic response – both suggest a role in iron transport. Besides the MFS domain, MamZ

281

contains also a ferric reductase TM component; the combination of these domains suggests

282

that MamZ has an active part in iron transport or as a mediator

21,68

.

283

In the presence of ferric reductases and the CDF proteins, magnetosomes accumulate Fe2+

284

rather than Fe3+. Since magnetite formation requires Fe3+ as well, oxidation of the ferrous

285

form into the ferric is required. The ferric reductase domain of MamZ is not the only

286

component that is thought to take part in this process; each of MamP, MamX, MamT and

287

MamE contain CXXCH heme-binding c-type cytochrome motifs – magnetochromes – which

288

68–70

are specific to MTB and represent a new type of cytochrome 70

conserved in all MTB

. MamE and MamP are 46–48,50

and their deletion causes defects in magnetite formation

289

. The

290

crystal structure of the soluble part of MamP from MO-1 strain sheds light on the

291

magnetochrome domain and redox control in MTB50. The MamP monomers contain a flexible

292

arm, a PDZ domain and two small c-type cytochrome domains (magnetochrome domain 1 or

293

2, also known as MCR 1 or 2), and they form dimers or tetramers in a pH-dependent manner

294

(Figure 3c). The 23-residue magnetochrome domain contains a single-heme binding site and

295

is one of the smallest heme-binding domains known in nature. Two iron cations can be bound

296

50

2+

within an in-dimer cavity that is surrounded by the MCRs . MamP was shown to oxidize Fe

297

in alkaline pH in vitro, with maximum activity at a pH that fits in vitro magnetite synthesis –

298

2+

four Fe are oxidized per MamP dimer

48,50

. The reduction potential of AMB-1 MamP is in a 2+

different range than other c-type cytochromes, with lower overpotential to oxidize Fe 3+

can prevent the re-reduction of Fe

299

that

300

to Fe . This suggests a mechanism for MamP in

301

2+


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controlling the Fe2+-Fe3+ ratio within the magnetosome so that the magnetite can grow 48

302

without defects . The dimension and nature of the MamP cavity, the presence of a conserved

303

proton exit channel at its bottom and the four hemes within the dimer (Figure 3c) are well-

304

2+

2+

+ 7H2O 

305

(2Fe2O3*H2O) + 12H + 4e . Overall, MamP is thought to oxidize ferrous to ferrihydrite as a

306

suited for the chemistry of ferrihydrite formation from Fe : 4Fe +

50

2+

precursor of magnetite, and once MamP is fully reduced, the addition of Fe

causes the

50

307

transformation of ferrihydrite to magnetite, similar to magnetite formation in vitro .

308

Nonetheless, although MamP and also MamT heme-binding sites were shown to be required

309

for proper magnetite crystallization in AMB-1, their deletion and mutants retain the ability to

310

form magnetite. Therefore, it is assumed that they both probably have a more important role

311

in crystal growth than in nucleation48,71.

312

As mentioned before, MamO's TauE transporter domain is required for activation of MamE

313

protease activity. As TauE proteins were suggested to act as transporters of sulfur-containing

314

47,49

organic molecules, and with the evidence regarding MamE’s ligand-dependent activity

, it

315

is possible that the activation of MamE depends on a specific chemical composition inside the

316

magnetosome that is controlled by MamO. Due to the similarity between sulfate and

317

phosphate compounds, we are tempted to suggest that MamO transports phosphate-containing

318

organic molecules that originate in the periplasmic space. As phosphate inhibits magnetite

319

formation72, it can explain why it should be excreted before the magnetite nucleates and

320

19

grows. In contrast, as MamO is also found in greigite-forming MTB it is possible that it has

321

a role in controlling the sulfur concentration for proper magnetite and greigite nucleation.

322

Since both scenarios seem sensible, further studies on MamO's TauE domain are required to

323

elucidate its role. Biochemical and structural analysis of MamO's serine-protease domain

324

indicates that MamO also has a role in iron transport for magnetite nucleation inside the

325

49

magnetosome . Although sharing high similarity to serine proteases, MamO protease activity

326

is degenerated and its crystal structure lacks features that are essential for protease activity.

327

Nonetheless, the crystal structure of this domain revealed a di-histidine transition metal

328

binding site that is required for magnetite formation in vivo (Figure 3d), which suggests that

329

the protease domain of MamO binds iron and by that leads the ions into the crystal lattice,

330

facilitating its nucleation49.

331

The protein MamI presumably contains two TM helices with an inter-magnetosome loop that

332

was thought to take part in the MM banding during invagination, but was recently shown to

333

21,73

bind magnetite in vitro

. No MM biogenesis was observed in AMB-1 cells that lack

334

mamI , while in MSR-1 not only was the MM formed but also small iron oxide particles

335

28

33,35

were observed

. Altogether, this suggests that MamI has a role in the transition from 73

unordered condensed iron oxides to magnetite .


336 337


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Page 12 of 31

338 The final touchup: control of size and morphology

339

One of the main reasons that MTB interest researchers in the biotechnology field is that each

340

MTB strain biomineralizes magnetic particles in uniform size and morphology yet in a unique

341

strain-dependent form. Magnetite growth involves a set of proteins that controls the final size

342

and shape of the particles, some of which have been intensively studied during recent years

343

20,21

by the MTB research community

. There are six, small, membrane-integrated proteins in

344

the Alphaproteobacteria strains encoded by the mamCDFG and mms6 operons that were well

345

characterized and their function will be discussed here in detail: MamC (or Mms13), MamD

346

(or Mms7), MamF, MamG (or Mms5), MmsF and Mms6. As these proteins are not encoded

347

by Deltaproteobacteria, Omnitrophica and Nitrospirae stains – which form bullet-shaped

348

magnetite particles or greigite particles – it is thought that some of the mad and man genes

349

18,19

encode proteins with similar roles, but thus far there is no evidence for that

.

350

MamC, D and G and Mms6 were shown to be tightly bound to magnetite and, as their

351

encoding genes are absent from all bullet-shape synthesizing strains that were studied so far,

352

they are thought to be involved specifically in cubo-octahedral shaped magnetite particles’

353

74,75

. They all contain a conserved hydrophobic N-terminal domain (NTD)

354

with 1–2 TM helices, which in all of them but MamC contain a leucine-glycine (LG) repeat

355

morphology control

21,27,74

that is also found in other known biomineralization-related proteins

. This domain was

356

hypothesized to create a self-assembly of these proteins – together, in some combination of

357

them or separately – which forms a wide inner-lumen domain that might interact with the

358

23,75

magnetite more steadily

.

359

An early study on the deletion of AMB-1 mms6 phenotypes showed a decrease in magnetite

360

size, a different length-to-width ratio (elongated crystals) compared to WT, and a high energy

361

76

crystal face, which suggest that in its absence magnetite crystallization is not finished . A

362

later study showed similar-but-milder phenotypic effects when mms6 was deleted in the same

363

strain and suggested that the differences in the earlier study were due to lower expression

364

levels of MmsF – another important protein for magnetite size regulation77. When comparing

365

in vitro magnetite synthesis without and with Mms6, the latter showed a larger fraction of

366

magnetite rather than other iron oxides’ forms, a narrower size distribution of magnetite

367

74,78,79

particles, and particles with a similar shape to that of magnetosomes' magnetite

. Mms6

368

contains a cytoplasmic, random-coiled NTD, a TM helix and an acidic CTD that is found

369

21,23

inside the magnetosome lumen

. It has been known for years that Mms6 undergoes 27,74

proteolytic cleavage to create a functional 6 kDa C-terminal truncated protein

370

, but it was

371

recently shown that both the full and truncated versions of Mms6 are found in the MM and

372


Page 13 of 31

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that the full length protein interacts with MamA44. It is not known what the function is of 23,80

373

, but we

374

speculate that Mms6 NTD, when it appears, binds to MamA in order to create a specific

375

distribution of Mms6 in the MM to enable the cubo-octahedral shape of the magnetite

376

particles. In contrast to the NTD, the Mms6 acidic CTD function was well studied: the CTD

377

Mms6 NTD, as it is cleaved in some strains and does not appear in others

74,78,81

by itself can form homogenous magnetite crystals in vitro and was shown to bind iron

. It

378

was recently shown by NMR studies that the 20 C-terminal residues of Mms6 bind Fe more

379

2+

3+

specifically than Fe , which suggests that it facilitates the formation of magnetite from the 82

380

ferrous-rich environment inside the magnetosome . It was also shown that the pH impacts the

381

function of Mms6 (both in iron binding and magnetite precipitation) and its micelle

382

23,82,83

morphology

. In vivo studies of Mms6 have just recently indicated that the LG repeat and

383

the C-terminal acidic domain are required for its localization within the MM and onto the

384

magnetic particle surface, and that three specific acidic residues within the C-terminus are

385

crucial for its function in crystal growth and morphology control84. As Mms6 was shown to

386

self-assemble in vitro, since it has a similar function in magnetite formation in vitro and in

387

vivo and based on experimental evidences, it is hypothesized that specific hydrophobic

388

interactions cause its self-assembly in the MM as well (presumably via the LG repeat and

389

maybe together with MamD and MamG, which also contain this repeat). This assembly will

390

create a protein raft in the membrane with a large acidic surface inside the presumably-basic

391

magnetosome lumen and bind both iron forms in a favorable energetic geometry that

392

promotes magnetite formation specifically and by that affects its growth23 (Figure 4a).

393

MmsF and MamF are homologous proteins whose co-deletion causes a decrease in crystal

394

35,77

. Each protein contains three TM helices, in which the loops between the

395

two N-terminal helices in both proteins and the C-terminal tail of MmsF are rich with acidic

396

size and number

residues and are found in the magnetosome lumen

21,85

. Hence, they are assumed to bind

397

magnetite and by that to impact the maturation phase of magnetite formation. MamF in MSR-

398

1 was shown to form stable oligomers in vivo whereas MmsF was shown to self-assemble in

399

vitro

27,85

. In a co-precipitation assay with iron, the latter assemblies were shown to form

400

similar magnetite particles to AMB-1's, which further signifies its contribution to proper

401

85

magnetite formation .

402

MamC is the most abundant protein in the MM whose encoding gene deletion results in

403

slightly smaller magnetite particles and was suggested to have a role in magnetite size

404

75,86,87

control

. MamC contains two integral TM helices with an acidic inter-lumen helical loop

405

whose structure was recently solved by X-ray crystallography21,88 (Figure 4b). The helical

406

loop contains two charge-separated regions and it can bind magnetite particles. As with the

407

88

full-MamC, the loop improves magnetite formation in vitro . It contains two ~8 Å distant


408


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Page 14 of 31

acidic residues that impact the ability of the loop to bind magnetite; as the iron atoms are

409

separated by ~6 Å in the cubo-octahedral magnetite and considering the conformational

410

freedom of these residues, it was suggested that the two residues interact directly with the iron

411

88

ions in the magnetite surface and serve as a template for magnetite nucleation and growth

412

(Figure 4b). Since in in vitro precipitation of iron with MamC forms magnetite with missing

413

corners, it was suggested that MamC might also impact crystal morphology by a face-specific

414

89

interaction .

415

Deletion of the mms7 gene in AMB-1 (also known as mamD) results in a decrease in

416

magnetite particle minor axis size and a different crystal face compared to WT bacteria

75,90

.

417

Recently, it was shown that mms7 expression induction in AMB-1 changes the morphology of

418

the crystal from elongated dumbbell-shaped (no Mms7) to a spherical particle (high

419

90

expression levels of mms7) . Mms7 contains the LG repeat in its MM-integral hydrophobic

420

NTD and a hydrophilic, inter-lumen CTD that is thought to interact with the magnetic

421

21

particle . When the latter domain was introduced into an in vitro mineralization assay 2+

3+

422

together with MamP, it did not influence green rust (a mixed valence Fe -Fe form of iron

423

oxide) formation as a precursor to magnetite. However, whereas in the lack of Mms7 the

424

green rust was fully oxidized with exposure to air, the Mms7 C-terminal peptide somehow

425

48

protected the iron oxide form and maintained the green rust . Altogether, Mms7 is suggested 2+

3+

426

to have a role not only in size and morphology control but also in regulation of the Fe -Fe

427

ratio in the crystal surface and templating of the crystal lattice.

428

MamG is a MamD and Mms6 homolog and is found exclusively in the MM27,87,91. It contains

429

two TM helices and the loop that connects them is suspected to interact with magnetite,

430

21

similar to the MamC loop . When deleting each protein, similar phenotypes were observed

431

with smaller magnetite size in all dimensions, which suggests a role of MamG in size

432

75

control .

433

Together, all of these proteins constitute the vast majority of the MM proteins and some of

434

them have a similar impact on the bacterium’s function. The fact that the bacteria put so much

435

effort into producing such a complicated system shows how important the specific

436

morphology and size of the magnetic particles are in these bacteria, for a reason that is not yet

437

clear. Nonetheless, nature gave us a wonderful living model that we can utilize for the

438

synthesis of uniform magnetic particles in vitro for different bio/nanotechnology applications

439

– a field of a broad interest that is now being studied by many groups around the world.

440 441

Acknowledgments

442


Page 15 of 31

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The authors are supported by the Israel Ministry of Science, Technology and Space, the Israel

443

Science Foundation (grant n° 761676), the European Molecular Biology Organization and

444

CMST COST Action CM1306. We would like to thank S. Cronin for his help with the

445

manuscript.

446 447

** The authors declare no competing financial interest.

448 449



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Page 16 of 31

Figure legends (1)–(2)

451

Magnetosome membrane invaginates from the inner cell membrane, (3) The lumen

452

composition is modified, (4) Magnetic particle nucleation, (5) Magnetosome membrane and

453

magnetic particle growth, and (6)–(8) Alignment of magnetosomes into chains.

454

Figure 2. Proteins that participate in magnetosome organization. a) MamA participates in

455

protein sorting. Up: MamA self-assembly coats the magnetosome membrane and interacts

456

with magnetosome-associated proteins. Down: AMB-1 MamA structure (PDB code: 3AS541)

457

exhibits five TPR motifs that create concave and convex protein-protein interaction sites. b)

458

MamK assembly into filaments aligns the magnetosomes into a chain or chains (PDB code:

459

Figure

1.

Magnetosome

450 formation

scheme.

Left-to-right

magnetosomes:

56

5jyg ). Up: Surface representation of MamK monomers assembled into double-stranded

460

(green and blue) non-staggered filaments. Down: Four monomers in the filaments’ assembly,

461

with AMP and residues that participate in the subunits' interactions presented as sticks.

462

Intermolecular interactions are enlarged: in the longitudinal (i-1, i) interface hydrophobic

463

interactions between domains IB (i-1) and IA (i) are presented whereas in the longitudinal (i,

464

i+1) interface the electrostatic interactions between domains IIB (i) and IIA (i+1) are

465

presented. The inter-strand assembly (top right, green and blue) relies on a small number of

466

92

interactions. All images were produced using PyMol .

467

Figure 3. Structure and function of proteins that participate in magnetosome lumen chemical

468

composition modification and magnetic particle nucleation. a) General scheme of

469

magnetosome lumen chemical environment modification: iron is stored in the cytoplasm in

470

2+

the form of FeP, which breaks down to Fe and P (Magnetospirillum); MamB (pink), MamM

471

(purple), MamH and MamZ (dark blue and green) participate in iron transport into the

472

magnetosome; MamE (yellow), MamP (light green), MamT (brown) and MamX (red) in the

473

2+

oxidation of Fe ; MamB, MamM, MamN (light blue) and MamP in the extraction of protons;

474

MamO (orange) in iron transport to the magnetic particle surface and in S/P extraction. b)

475

MamM CTD dimer structure in the apo form (PDB code: 3w5y65). Putative binding site

476

residues are presented as sticks. c) MamP dimer structure in the iron-bound state (PDB code:

477

4jj350) with the heme ligands bound in each of the four magnetochrome domains. Close-up

478

views, left-to-right: the acidic residues in the iron binding pocket and the histidine residues in

479

the proton channel are shown as sticks and magnetochrome domains 1 and 2 with their

480

CXXCH motifs presented as sticks. In all close up views, the heme ligands are presented as

481

49

yellow sticks. d) MamO protease domain structure in Ni-bound state (PDB code: 5hma ).

482

The histidine pair is presented as sticks with the bound nickel in yellow.

483


Page 17 of 31

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Figure 4. Magnetite size and morphology control. a) The presumed assembly of the

484

Alphaproteobacteria proteins MamC (yellow), MamD (purple), MamF (orange), MamG

485

(red), Mms6 (blue) and MmsF (green) in the membrane forms an inter-lumen negative

486

surface that can interact with the magnetic particle’s surface and affect its size and shape. The

487

greater the abundance of the protein in the magnetosome, the more it appears in the figure

488

(ratio is not exact; MamC and MamF are the most and second-most abundant proteins in the

489

membrane, all others are highly abundant but as their ratios are unknown they appear the

490

same number of times in the figure). b) Crystal structure of MamC inter-lumen loop (PDB

491

88

code: 5e7u ) shows two acidic residues that face the same direction and are thought to

492

interact with the magnetic particle’s iron atoms, and a positively charged arginine that faces

493

the negatively charged membrane surface.

494 495



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Page 18 of 31

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Abstract figure 65x27mm (300 x 300 DPI)



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Figure 1 Figure 1 140x31mm (300 x 300 DPI)


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