Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
1
Probing the nanostructure and arrangement of bacterial 1
magnetosomes by small-angle x-ray scattering 2
3
Sabine Rosenfeldt# 1,2, Cornelius N. Riese# 3, Frank Mickoleit3, Dirk Schüler3, Anna S. 4
Schenk*1,4 5
6
# These authors contributed equally to this work 7
8
1Bavarian Polymer Institute (BPI), 2Physical Chemistry 1, 3Dept. Microbiology, 9
4Physical Chemistry - Colloidal Systems; University of Bayreuth, D-95447 Bayreuth 10
11
* corresponding author: Jun.-Prof. Dr. Anna S. Schenk, Physical Chemistry - 12
Colloidal Systems, University of Bayreuth, D-95447 Bayreuth; E-Mail: 13
[email protected]; Tel.: +49-921-55-3915 14
15
16
Abstract 17
Magnetosomes are membrane-enveloped single-domain ferromagnetic nanoparticles 18
enabling the navigation of magnetotactic bacteria along magnetic field lines. Strict 19
control over each step of biomineralization generates particles of high crystallinity, 20
strong magnetization and remarkable uniformity in size and shape, particularly 21
interesting for many biomedical and biotechnological applications. However, to 22
understand the physicochemical processes involved in magnetite biomineralization, 23
precise and permanent monitoring of particle production is required. Common 24
techniques such as transmission electron microscopy (TEM) or Fe measurements 25
AEM Accepted Manuscript Posted Online 11 October 2019Appl. Environ. Microbiol. doi:10.1128/AEM.01513-19Copyright © 2019 Rosenfeldt et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
2
only allow for semi-quantitative assessment of the magnetosome formation without 26
routinely revealing quantitative structural information. In this study, lab-based small-27
angle x-ray scattering (SAXS) is explored as a means to monitor the different stages 28
of magnetosome biogenesis in the model organism Magnetospirillum 29
gryphiswaldense. SAXS is evaluated as a quantitative stand-alone technique to 30
analyse the size, shape and arrangement of magnetosomes in cells cultivated under 31
different growth conditions. By applying a simple and robust fitting procedure based 32
on linearly aligned spheres, it is demonstrated that the SAXS datasets contain 33
information on both the diameter of the inorganic crystal and the protein-rich 34
magnetosome membrane. The analyses reveal a narrow particle size distribution with 35
an overall magnetosome radius of 19 nm in Magnetospirillum gryphiswaldense. 36
Furthermore, the averaged distance between individual magnetosomes is 37
determined, revealing a chain-like particle arrangement with a center-to-center 38
distance of 53 nm. Overall, these data demonstrate that SAXS can be used as a 39
novel stand-alone technique allowing for the at-line monitoring of magnetosome 40
biosynthesis, thereby providing accurate information on the particle nanostructure. 41
42
43
Importance 44
This study explores lab-based small-angle x-ray scattering (SAXS) as a novel 45
quantitative stand-alone technique to monitor the size, shape and arrangement of 46
magnetosomes during different stages of particle biogenesis in the model organism 47
Magnetospirillum gryphiswaldense. The SAXS datasets contain volume-averaged, 48
statistically accurate information on both the diameter of the inorganic nanocrystal 49
and the enveloping protein-rich magnetosome membrane. As a robust and non-50
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
3
destructive in-situ technique, SAXS can provide new insights into the 51
physicochemical steps involved in the biosynthesis of magnetosome nanoparticles as 52
well as their assembly into well-ordered chains. The proposed fit model can be easily 53
adapted to account for different particle shapes and arrangements produced by other 54
strains of magnetotactic bacteria, thus rendering SAXS a highly versatile method. 55
56
Keywords: Small-Angle X-Ray Scattering; SAXS; Magnetospirillum 57
gryphiswaldense; Magnetosomes; Magnetotactic bacteria, Magnetic nanostructure 58
59
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
4
Introduction 60
61
In order to facilitate their orientation and migration along the Earth’s magnetic field, 62
magnetotactic bacteria (MTB) biomineralize magnetosomes enabling them to 63
navigate towards favorable habitats in the oxic-anoxic transition zone. In the 64
alphaproteobacterium Magnetospirillum gryphiswaldense (Fig. 1a) these magnetic 65
nanoparticles consist of a cuboctahedral, monocrystalline core of chemically pure 66
magnetite (Fe3O4), enveloped by a protein-rich biological membrane (magnetosome 67
vesicle) aligned in a linear chain of about 40 particles (Fig. 1b) (1–4). Due to strict 68
control of each step of magnetosome biogenesis, particles with unprecedented 69
characteristics (with respect to crystallinity, particle size, shape and magnetization; 5, 70
6) are synthesized. However, despite of considerable efforts, the physicochemical 71
processes and steps involved in the biomineralization of well-ordered chains of 72
magnetite nanocrystals are still not understood. This is partially due to a lack of 73
practical lab-scale methods to assess bulk magnetosome formation in growing cells. 74
In addition, magnetosomes are of increasing interest as biogenic magnetic 75
nanoparticles as they have been already successfully explored in a wide range of 76
potential biotechnical and biomedical applications including magnetic hyperthermia 77
(7, 8), as contrast agents (9, 10) or particle based immune-assays (11–13). Yet, 78
bioproduction of magnetosome particles depends on highly controlled growth 79
conditions (14, 15) and requires precise and permanent monitoring of bulk 80
magnetosomes biomineralization during bacterial cultivation. 81
Commonly used proxies to estimate magnetosome formation, such as the magnetic 82
orientation of cells (cmag [16, 17]) and their iron content, lack sufficient specificity 83
and do not provide information about the size, structure and arrangement of particles. 84
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
5
In contrast, transmission electron microscopy (TEM) allows the precise determination 85
of particle numbers, sizes, and shapes as well as their chain assemblies. 86
Nonetheless, gathering statistically significant datasets from time-resolved 87
experiments on cultures is very laborious and time consuming. Furthermore, the 88
analysis is often hindered by preparation or drying artefacts. X-ray radiation based 89
techniques such as extended X-ray absorption fine structure (EXAFS) and X-ray 90
absorption near edge structure (XANES) were already applied for characterization of 91
magnetite biomineralization, however, quantitative TEM measurements were still 92
necessary to address magnetosome size distribution (18, 19). 93
As an alternative, small-angle X-ray scattering (SAXS) has been used to assess the 94
structural properties of bacterial magnetosomes (20–22). This technique represents a 95
volume-averaging method to study nanoscale interfaces based on electron density 96
heterogeneities within a sample. SAXS experiments performed on a laboratory based 97
setup provide access to size, shape and orientation of nanoscale objects spanning a 98
size range of 1–300 nm, and thus are ideally suited to study structural parameters 99
and aggregation behaviour of intermediates and final products formed during 100
magnetosome biomineralization, as the mature nanoparticles exhibit an average size 101
of about 40 nm (4, 12) and a substantial electron density contrast. During 102
magnetosome biogenesis, the forming particles characteristically undergo a 103
morphological transition leading to changes in the electron density distribution at 104
different growth stages. Such variations in contrast, e.g. evoked by vesicles 105
successively filling up with iron-rich magnetite mineral, are accessible by SAXS and 106
contribute to the scattering pattern in a characteristic way. 107
Krueger et al. (20) first combined small-angle neutron scattering (SANS) and SAXS 108
to investigate structural parameters of magnetite nanocrystals in magnetotactic 109
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
6
bacteria. Due to the restricted data quality of the individual scattering patterns, the 110
results were analysed by comparing the scattering profiles of samples with low and 111
high iron loading in the presence and absence of external magnetic fields. The 112
diameters of the magnetosomes biosynthesized during cultivation were estimated to 113
be 40 nm (20). Hoell et al. (21) later investigated M. gryphiswaldense by specialized 114
techniques based on both SANS and SAXS to access the size of the magnetosome 115
membrane and the magnetite crystals as well as their magnetization. More recently, 116
Molcan and co-workers (23) performed a comprehensive structural characterization 117
of magnetosome suspensions from M. magnetotacticum for potential hyperthermia 118
applications by applying a wide range of contrasting analytical techniques including 119
SAXS. The scattering data in the regime of low values of the scattering vector q were 120
fitted by a q-x power law, where the increase in the exponent x was interpreted as a 121
progressing fractal-like aggregation of magnetosomes. Orue et al. (22) focused on 122
the magnetic nanoarchitecture of magnetosomes from M. gryphiswaldense, but also 123
performed SAXS measurements on highly concentrated bacterial colloids. The 124
authors analysed the data sets by an indirect Fourier transformation method, which 125
usually relies on additional information regarding the geometry and size of possible 126
aggregate structures. Applying this strategy, Orue and coworkers (22) correlated the 127
first maximum of the pair distance distribution function to the size of one 128
magnetosome, and the subsequent maxima to the inter-particle distances of adjacent 129
particles. Although comparison with the results from direct visualization by cryo-130
electron tomography revealed a good agreement of the estimated magnetosome 131
size, a 15% lower value was estimated for the correlation distance. However, in all 132
SAXS studies on bacterial magnetosome, the method was used only on selected 133
specimens as a complementary tool to validate the results obtained from other 134
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
7
techniques such as TEM, electron tomography or SANS rather than as a stand-alone 135
analytical method. 136
In this study, SAXS is explored as a quantitative stand-alone bulk measurement 137
technique allowing time–efficient particle size analysis (in contrast to well-138
established, but laborious methods such as TEM). Despite the detailed structural 139
resolution of magnetosomes, which can be achieved by highly brilliant X-ray beams 140
produced at synchrotron sources (21), limited access as well as usually long 141
distances between production and measurement site render synchrotron SAXS 142
analysis unpractical for fast at-line magnetosome characterization. Accordingly, the 143
present study investigated concentrated cell suspensions of M. gryphiswaldense at 144
different growth stages using a lab-scale SAXS system with a rotating copper anode 145
as X-ray source. This in-house setup offers the advantage of easier access (in 146
contrast to synchrotron measurements) and close proximity to the fermentation 147
facility, thus enabling sampling and process control during magnetosome 148
biosynthesis. 149
150
151
Materials and Methods 152
153
Cultivation of M. gryphiswaldense 154
M. gryphiswaldense wild type was grown either aerobically or anaerobically at 28°C 155
in a BioflowTM 320 Eppendorf Bioprocess fermentor system (Jülich, Germany) using 156
a modified large-scale medium (14). Cells were harvested by low-spin centrifugation 157
(7,000 rpm, 10 min, 4°C; Beckmann Coulter Avanti J-26SXP, USA). Cell pellets were 158
washed and resuspended in 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-159
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
8
yl]ethanesulfonic acid (HEPES), pH 7.0, before transferring the suspension into glass 160
capillaries ( = 1 mm, Hilgenberg, Germany) for further analysis. 161
162
Structural characterization of magnetosome nanoparticles 163
Transmission electron microscopy (TEM). 164
For TEM analysis of whole cells, specimens were directly deposited onto carbon-165
coated copper grids. TEM imaging was performed on a Jeol Jem 1400+ (Freising, 166
Germany) operated at an acceleration voltage of 80 kV. Images were recorded with a 167
Gatan Erlangshen ES500W CCD camera. Average particle sizes were measured by 168
data processing using the ImageJ software package v1.52i. 169
170
Small-Angle-X-ray Scattering (SAXS). 171
Nanostructure analysis of bacterial magnetososmes was performed on highly 172
concentrated bacterial suspensions at ambient conditions using a Double Ganesha 173
AIR system (SAXSLAB, Denmark). Monochromatic radiation with a wavelength of = 174
1.54 Å was generated by a rotating anode source (Cu, MicroMax 007HF, Rigaku 175
Corporation, Japan). All data sets were recorded with a position-sensitive detector 176
(PILATUS 300K, Dectris) placed at different distances from the sample to cover a 177
wide range of scattering vectors q with 0.002 Å-1 < q < 0.5 Å-1, where q is given as 178
2sin
4
with representing the wavelength of the incident beam and the 179
scattering angle. 180
The 2-dimensional scattering patterns were converted into 1-dimensional intensity 181
profiles of I(q) vs. q by radial averaging and subsequently normalized to the intensity 182
of the incident beam, the sample thickness and the accumulation time. Background 183
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
9
correction was performed by subtracting the signal of a glass capillary ( = 1 mm, 184
Hilgenberg, Germany) filled with HEPES buffer. The software SasView 4.2 was used 185
for data analysis. 186
187
188
Results and Discussion 189
190
A simplified model to describe the magnetosome chain 191
As SAXS represents an indirect technique acquiring information in reciprocal space, 192
appropriate models have to be established to derive structural parameters of the 193
sample in real space. Therefore, at first a model description of the magnetosomes 194
based on both, the architecture of individual particles and their arrangement within 195
the cell had to be developed in order to allow for a detailed evaluation of the x-ray 196
scattering data. 197
One magnetosome consists of a magnetite crystal, surrounded by a proteinaceous 198
membrane (Fig. 2a), which exerts strict control over the growth of the crystals, and 199
their arrangement into well-ordered chains (4, 12). Based on previous TEM imaging 200
of magnetosome chains within M. gryphiswaldense (24, 25), magnetosome 201
biogenesis is here considered as the formation of a single linear chain composed of 202
individual core-shell particles, which exhibit sharp phase boundaries and are closely 203
packed (Fig. 2c). In the case of a strictly monodisperse system, such an initial 204
intuitive approach would result in an x-ray scattering profile (Intensity I(q) vs. q) 205
exhibiting pronounced extrema and a strong decay in scattering intensity in the 206
regime of low q values, but fails to explain the additional scattering contribution seen 207
in the high q range. 208
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
10
Therefore, an alternative model is proposed, which contains all structural parameters 209
of interest to the here presented study: the magnetosome particle radius (R), the 210
degree of polydispersity and the distance between neighbouring particles (l). For 211
simplification, no indirect Fourier transformation was included in the evaluation. 212
Based on the model presented in Fig. 2b, it can be assumed that the x-ray contrast of 213
a magnetosome is mainly determined by the electron densities of magnetite (~1500 214
electrons/nm3) and the attached proteins, for which an electron density in the order of 215
650 electrons/nm3 is estimated by grey scale analysis of TEM micrographs. 216
In this model, the magnetite crystal was regarded as a sphere (rather than a facetted 217
object) decorated densely, but randomly with smaller spheres simulating the protein-218
crowded particle coverage (Fig. 2b). Furthermore, in order to represent the biological 219
heterogeneity more accurately, a certain polydispersity with respect to the 220
magnetosome radius had to be incorporated into the theoretical description. Under 221
these restrictions, in a multi-particle model the sharp phase boundary smears out and 222
the magnetosome effectively appears as a simple homogeneous sphere with radius 223
R. It has to be considered, though, that this simplified approach does not account for 224
contributions originating from empty vesicles, invaginations and the cellular matrix, as 225
these do not feature a significant x-ray contrast at the interior. A linear chain of such 226
spheres can then be assumed to describe the geometric arrangement of 227
magnetosomes within the cell (Fig. 2c and d). However, since in a helix-type folding 228
pattern the helical pitch size, i.e. the length of one single turn, would most probably 229
exceed a dimension of 200 nm (24, 25), distinguishing between a helical and a linear 230
chain-like arrangement is beyond the resolution limit of most conventional SAXS 231
setups. 232
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
11
Summarizing the here presented model, it is suggested that a magnetosome can be 233
simplified as a homogeneous large sphere with a typical size range of 10-70 nm 234
surrounded by 3-6 nm thick proteinaceous phospholipid bilayer that is invaginated 235
from several non-specific cytoplasmic membrane locations (4, 26). Analysis of the 236
magnetosome subproteom (i.e. the complement of proteins specifically associated 237
with the magnetosome membrane) suggested an unusually crowded protein 238
composition on the membrane, and a tight packing with transmembrane domains of 239
integral proteins (27). Depending on the size and folding (number of transmembrane 240
helices and extra-magnetosomal domains) the magnetosome proteins can be 241
modelled as small objects (spheres) typically in the size range of 4-10 nm (28). For 242
simplicity, a coiled (spherical) supramolecular arrangement/folding pattern is 243
assumed. 244
In the regime of small q (corresponding to large dimensions in real space), the x-ray 245
scattering pattern is insensitive to the fine details of the protein shell. Hence, while 246
the proteins significantly increase the contrast of the shell, they are not 247
distinguishable as separate small spherical objects. Consequently, the SAXS profile 248
is dominated by the overall size of the particles, and data analysis in the lower q 249
range yields the magnetosome radius R, i.e. the outer radius determined by the 250
thickness of the magnetosome membrane (shell). However, in the regime of high q 251
(corresponding to smaller dimensions in real space), x-ray scattering detects 252
fluctuations in the electron density distribution on a smaller length scale and therefore 253
is sensitive to finer structural motives such as the proteins themselves. 254
255
256
257
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
12
Calculation of the scattering function 258
The intensity I(q) is determined by the square of the amplitude of the scattered wave, 259
which, in turn, is given by equation 1 in case of a particle with the volume Vp and an 260
excess electron density distribution Δ(r). 261
Vp
drrqr
qrrqA 2sin
4 (eq. 1). 262
For a system of monodisperse spheres with radius R, eq. 1 can be expressed as a 263
function of the particle radius according to equation 2. 264
33 cossin3
3
4
qR
qRqRqRRqA
(eq. 2). 265
In order to calculate the intensity for polydisperse systems composed of different 266
particles species with size i, the squared amplitudes qAi
2 have to be added up and 267
weighted by their respective number densitypV
N. 268
269
X-ray scattering analysis of magnetosome particles 270
The scattering intensity I(q) originating from non-interacting small and large spheres 271
is given by the corresponding amplitudes A(q) according to equation 3 (29): 272
i i ji ij
ij
jsmallismallsmallbigsmallbigqd
qdqAqA
qd
qdqAqAAqAqI
sin2
sin,,
22 (eq. 3) 273
where d denotes the distance between the centres of gravity of the large and the 274
small spheres whereas dij represents the mutual distances between small spheres. 275
The first two terms are equal to the intensities of non-interacting spheres of the 276
corresponding size (cf. eq. 2). In the case of large spheres, the first term decays 277
rapidly with I(q) ~q-4, and therefore does not contribute significantly to the scattering 278
intensity at high values of q (cf. Fig. S1), whereas the second term associated with 279
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
13
the small spheres (high amount) plays the dominant role. Due to polydispersity in 280
real-world systems, the factor
qd
qdsincancels out the third term at high q. 281
Concomitantly, the average over the fourth term vanishes as the distance dij varies 282
randomly. 283
As discussed above, SAXS represents a low-resolution method at low scattering 284
angles. As a consequence, term 2, i.e. the sum of the contribution of the small 285
spheres, can be alternatively described as an additional contribution to the electron 286
density of the entire large sphere. The protein decoration may then be regarded as 287
independent small density fluctuations and thus according to scattering theory, the 288
intensity of these fluctuations will add up to describe the scattering of a much larger 289
object with high contrast (here corresponding to the magnetosome). 290
Therefore, particularly in view of potential applications in on-line analysis of 291
magnetosome biosynthesis, the SAXS data analysis of these biogenic particles can 292
be simplified by assuming that the total intensity is modelled by a term corresponding 293
to the large spheres and an additional contribution originating from the small objects, 294
e.g. I(q)=Isphere(q) + Ismall(q) (eq. 4). Irrespective of the particle morphology, the 295
scattering intensity I(q) for a dilute solution of small objects is given by Guinier’s law 296
2
2
2
3exp q
RcMqI
g
wsmall (eq. 5), where Δ denotes the electron density 297
contrast of the small spheres, c the weight concentration, Mw the molecular weight 298
and Rg the radius of gyration. For the subsequent analysis it is important to note that 299
Ismall(q) mainly contributes to the scattering intensity for q > 0.05 Å-1 (Fig. 3) and can 300
be approximated by Guinier’s law, which allows us to perform profile fitting based on 301
a minimum of adjustable parameters. 302
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
14
With the terms for the evaluation of magnetosome and protein radius at hand, 303
anaerobically grown, magnetosome-producing cells were analysed by TEM and 304
SAXS (Fig. 3). In this experiment, the radius of the magnetosome is derived from the 305
position of the first minimum (q ≈ 0.02 Å-1) as R = 19 nm ± 17% (Gaussian size 306
distribution). The radius of gyration of the small objects (proteins) is determined as 307
1.7 nm and correlates to the radius R of a solid sphere via equation 6, 308
RRg5
3 (eq. 6), yielding R = 2.2 nm. This value is comparable to the general 309
dimensions expected for the coiled proteins based on TEM imaging (28, 30). 310
Hence, it can be concluded that within the framework of the afore-mentioned 311
assumptions, the scattering pattern is well described by the proposed model for 312
q > 0.015 Å-1. 313
To further test this concept, magnetosome-free cells of M. gryphiswaldense 314
(aerobically grown), which are characterized by the lack of magnetite crystals, but still 315
contain in some cases (empty) membrane vesicles (26) were analysed. For this 316
purpose, SAXS data obtained from magnetite-free and magnetite-producing cells 317
(Fig. 3c-e) were compared. Intriguingly, both magnetite-free systems (cf. blue and 318
green curves in Fig. 3e) do not exhibit the characteristic oscillations observed for 319
producing cells in the mid q range (q [0.08 Å-1, 0.03 Å-1]), but instead show a 320
similar linear decay at low q (q < 0.01 Å-1). In contrast, at high values of q, where 321
small nanostructural features dominate the curve, the scattering profiles of 322
magnetosome-deficient cells and magnetosome-producing bacteria (i.e. cells that 323
synthesize magnetite cores enveloped by the magnetosome membrane) are almost 324
identical. In some cases, here exemplified by the green curve, even a clear kink is 325
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
15
observed (q ≈ 0.06 Å-1), indicating the presence of a huge amount of very well-326
defined small objects such as proteins, which are highly abundant in the cells. 327
These observations strongly support the assumption that the total scattering intensity 328
of one magnetosome (form factor scattering) can be deconvoluted into partial 329
intensities assigned to large and small spherical objects, Isphere(q) and Ismall(q). Based 330
on these results one can in principle even consider the magnetosome-deficient 331
phenotype as background when investigating the scattering observed for 332
anaerobically grown M. gryphiswaldense cells which biosynthesize magnetosomes. 333
However, while there were no phenotypical differences between the two specimens 334
of magnetite-free bacteria identified by TEM (Fig. 3c,d), their corresponding SAXS 335
profiles clearly showed a deviation, particularly in the regime of high q (Fig. 3e, blue 336
and green curves), which indicates a substantial biological variability in these 337
systems, and thus complicates the identification of a suitable background curve. 338
The assembly of the magnetosomes into ordered chains is the major reason for the 339
deviation between the experimentally observed scattering intensity at low q (Fig. 3e, 340
black curve) and the here applied fit model. Due to the high degree of order (aligned 341
magnetosomes), Bragg’s law is suited to determine inter-particles separation 342
distances, such that a correlation length of approximately 47 nm can be determined 343
based on the position of the first maximum (q ≈ 0.0134 Å-1). 344
345
Determination of structural magnetosome assembly into chains 346
Chain assembly results from the coordinated interplay of collaborative magnetic 347
interactions, which occur naturally between magnetite nanocrystals, in combination 348
with active protein-mediated bio-assembly mechanisms (31, 32). Cellular structures 349
stabilize the string of magnetic dipoles, thereby avoiding a magnetic moment induced 350
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
16
collapse into rings or irregular clusters (33). In this geometric arrangement, the actin-351
like MamK filament anchors the magnetosome chain and generates an intracellular 352
compass needle to maximize the cell response to even low magnetic fields 353
experienced under environmental conditions. The pole-to-midcell treadmilling growth 354
of MamK filaments provides the key driving force for magnetosome movement (34). 355
Typically, wild type cells of M. gryphiswaldense synthesize 1-2 magnetosome chains 356
that extend across approximately two-third of the length of the cell and are located at 357
mid-cell. An average chain is composed of 20-40 particles with a mean center-to-358
center distance of about 50–60 nm (22, 24, 25). 359
According to the decoupling approximation, the total scattering intensity is 360
proportional to the product of the scattering originating from one particle (form factor) 361
and the scattering due to inter-particle interactions (structure factor). The structure 362
factor S(q) is related to the probability distribution function of inter-particle distances 363
g(r) and can be expressed according to equation 7 for isotropic systems. 364
0
2 sin141 dr
qr
qrrgrqS (eq. 7) 365
In an ideal solution featuring no position or orientation correlations, the equation 366
S(q) = 1 can be assumed, whereas ordered crystal-like particle arrangements exhibit 367
S(q) > 2.85 according to the Hansen Verlet criterion (35). The here presented 368
approach to consider the data set obtained from the magnetite-deficient bacteria as 369
background signal for the scattering of magnetosome-containing cells affords the 370
possibility to experimentally estimate the particle-particle interaction via the 371
experimental structure factor S(q), where the latter is obtained by dividing the 372
scattering intensity of the magnetite-containing sample by the intensity of the 373
respective magnetite-deficient sample. 374
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
17
Next, magnetosome biomineralization and chain formation during cell growth were 375
investigated. Therefore, samples were drawn at different production time points and 376
subsequently analysed by TEM and SAXS (Fig. 4). Using the scattering data 377
obtained at time zero as background (Fig. 4a, black line), the experimental structure 378
factors were determined as S(q) = 1 at start, S(q) ≈ 1.8 after 8 h and S(q) ≈ 3.2 after 379
23 h of biomineralization, where this corresponds to the degree of particle interaction 380
and is accompanied by the evolution from a non-ordered to a highly ordered spatial 381
correlation of the magnetite cores into a single chain. In support of the here proposed 382
theoretical model, the extracted structural parameters are in very good agreement 383
with TEM results (Fig. 4b), which clearly demonstrate the 1D crystalline order of 384
magnetosomes after 23 h. 385
Such an ordered chain-like nanoparticle array resembles a chain as described by a 386
linear pearl model (36), which is based on the form factor for NS spheres with radius 387
R linearly joined by short strings of length l (cf. Fig. 2; the thickness of the strings is 388
not taken into account). The scattering intensity I(q) for such an assembly is given by 389
equation 8. 390
2
31
2 cossin3
sin2
qR
qRqRqR
nq
nqnNNV
V
NqI
S
S
N
n
SSpearlsN
(eq. 8). 391
This consideration accounts for an increase in the scattering intensity on length 392
scales of the order of q(2R+l) ≈ 2πk, where k is an integer. 393
The scattering data in Fig. 4c were obtained from fully mature M. gryphiswaldense 394
cells after subtraction of the scattering contribution Ismall(q). For comparison, the 395
theoretical scattering curve of spheres with a radius of 19 nm (± 17%, Gaussian size 396
distribution) and the intensity profile of 5 monodisperse spheres arranged in a line 397
with a center-to-center distance of 2R+l = 50 nm are depicted. The modelled 398
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
18
interaction distance of 50 nm is in very good agreement with the oscillation pattern in 399
the scattering data arising from inter-particle interactions (first maximum observed at 400
q ≈ 0.013 Å-1), and most importantly is confirmed by TEM analysis, which reveals a 401
separation distance of 2R+l = 53 ± 6.0 nm. 402
403
404
Conclusions 405
Routinely used methods to monitor magnetosome biosynthesis such as cmag (16, 406
17), iron content measurement and EXAFS (18) as well as TEM, can often only 407
provide a semi-quantitative assessment of magnetosome structure and content within 408
the investigated cells, thus rendering them comparably unpractical for on-line process 409
control and generating a strong demand for efficient quantitative bulk methods. 410
Therefore, the present study introduces SAXS as a non-destructive (i.e. leaving 411
membranes and proteins intact) stand-alone tool for process monitoring during 412
magnetosome production. 413
In order to model the here obtained scattering profiles, magnetosomes were 414
considered as polydisperse spheres arranged into a single linear chain, which is 415
reflected in the scattering signal at low q, whereas the cell’s densely packed protein 416
background dominates the signal at high q. Based on these simplifications, a linear 417
pearl model without indirect Fourier transformation was applied to describe 418
concentrated cell suspensions grown under anaerobic (magnetosome producing) 419
and aerobic (magnetosome-free) conditions. The linear pearl model based on five 420
polydisperse spheres turned out to be best suited for describing the experimental 421
data, yielding magnetosome sizes that could be essentially confirmed by TEM 422
analysis. In conclusion, this study demonstrates that SAXS measurements performed 423
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
19
on a laboratory instrument can be used as a convenient, readily accessible, and 424
potentially even automated method to monitor bulk magnetosome biosynthesis in 425
growing cells and cultures. Most importantly, SAXS is a volume-averaging, 426
quantitative technique providing statistically accurate information on size, shape and 427
arrangement of nanostructural motives in a sample. Additionally, the lab-scale in-428
house equipment employed in this study enables at-line analysis of magnetosome 429
biomineralization and perspectively even on-line process control during fermentation. 430
Therefore, SAXS has the potential to complement and partially replace laborious and 431
time-consuming quantitative TEM analyses, thereby allowing for routine 432
magnetosome characterization. Applying the here proposed model, structural 433
parameters such as the dimensions of the inorganic crystal core and the protein-rich 434
membrane as well as the arrangement and assembly of the magnetosomes can be 435
extracted with high statistical accuracy. In view of a more general applicability to 436
different species and their magnetosome mutants grown under different conditions, 437
the model can be easily adapted and modified to account for different particle shapes 438
and arrangements within the cell e.g. by implementing form factors for non-spherical 439
scattering objects. The general contrast situation with respect to the electron density 440
heterogeneities within the sample (i.e. Fe-containing mineral vs. organics) should not 441
be affected by the specific strain of bacteria used, thus rendering the proposed 442
method a highly versatile tool for elucidating further details in magnetosome 443
formation. 444
445
446
447
448
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
20
Acknowledgments 449
We thank the Bavarian Polymer Institute (BPI) for providing access to small-angle x-450
ray scattering facilities within the Keylab Mesoscale Characterization: Scattering 451
Techniques. The authors are grateful to M. Schlotter (Dept. Microbiology, University 452
of Bayreuth) for expert technical assistance and M. Völkl for preparing some of the 453
samples as part of his Master thesis at the Dept. Microbiology and at the Chair for 454
Process Biotechnology (Prof. R. Freitag). This work benefited from the use of the 455
SasView application, originally developed under NSF award DMR-0520547. SasView 456
contains code developed with funding from the European Union's Horizon 2020 457
research and innovation programme under the SINE2020 project, grant agreement 458
No 654000. 459
460
461
Funding 462
The authors gratefully acknowledge financial support by the DFG via the 463
Collaborative Research Center SFB 840. In addition, this project has received 464
funding from the European Research Council (ERC) under the European Union’s 465
Horizon 2020 research and innovation programme (grant agreement No 692637 to 466
D.S.). 467
468
469
470
471
472
473
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
21
References 474
475
1. Lefevre C.T , Bazylinski D.A.. 2016. Ecology, Diversity, and Evolution of 476
Magnetotactic Bacteria. Microbiology and Molecular Biology Reviews 77:497-477
526. 478
479
2. Lohße A, Borg S, Raschdorf O, Kolinko I, Tompa E, Pósfai M, Faivre D, 480
Baumgartner J, Schüler D. 2014. Genetic dissection of the mamAB and mms6 481
operons reveals a gene set essential for magnetosome biogenesis in 482
Magnetospirillum gryphiswaldense. J. Bacteriol. 196:2658-2669. 483
484
3. Barber-Zucker S, Keren-Khadmy N., Zarivach R. 2016. From invagination to 485
navigation: The story of magnetosome-associated proteins in magnetotactic 486
bacteria. Protein Science. 25:338-351. 487
488
4. Uebe R, Schüler D. 2016. Magnetosome biogenesis in magnetotactic bacteria. 489
Nat. Rev. Microbiol. 14:621-637. 490
491
5. Staniland SS, Ward B, Harrison A, van der Laan G, Telling N. 2007. Rapid 492
magnetosome formation shown by real-time X-ray magnetic circular dichroism. 493
Proc. Natl. Acad. Sci. U.S.A. 104:19524-19528. 494
495
6. Staniland SS, Rawlings AE. 2016 Crystallizing the function of the 496
magnetosome membrane mineralization protein Mms6. Biochem. Soc. Trans. 497
44:883-890. 498
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
22
499
7. Hergt R, Dutz S, Röder M. 2008. Effects of size distribution on hysteresis 500
losses of magnetic nanoparticles for hyperthermia. J. Phys. Condens. Matter 501
20:385214. 502
8. Alphandéry E, Guyot F, Chebbi I. 2012. Preparation of chains of 503
magnetosomes, isolated from Magnetospirillum magneticum strain AMB-1 504
magnetotactic bacteria, yielding efficient treatment of tumors using magnetic 505
hyperthermia. Int. J. Pharm. 434:444-452. 506
507
9. Heinke D, Kraupner A, Eberbeck D, Schmidt D, Radon P, Uebe R, Schüler D, 508
Briel A. 2017. MPS and MRI efficacy of magnetosomes from wild-type and 509
mutant bacterial strains. IJMPI 3:1706004. 510
511
10. Kraupner A, Eberbeck D, Heinke D, Uebe R, Schüler D, Briel A. 2017. 512
Bacterial magnetosomes - nature's powerful contribution to MPI tracer 513
research. Nanoscale 9:5788-5793. 514
515
11. Tanaka T, Takeda H, Ueki F, Obata K, Tajima H, Takeyama H, Goda Y, 516
Fujimoto S, Matsunaga T. 2004. Rapid and sensitive detection of 17β-estradiol 517
in environmental water using automated immunoassay system with bacterial 518
magnetic particles. J. Biotechnol. 108:153-159. 519
520
12. Mickoleit F, Schüler D. 2019. Generation of nanomagnetic biocomposites by 521
genetic engineering of bacterial magnetosomes. Bioinspired Biomim. 522
Nanobiomat. 8:86-98. 523
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
23
524
13. Vargas G, Cypriano J, Correa T, Leão P, Bazylinski D, Abreu F. 2018. 525
Applications of magnetotactic bacteria, magnetosomes and magnetosome 526
crystals in biotechnology and nanotechnology: mini-review. Molecules 527
23:2438. 528
529
14. Heyen U, Schüler D. 2003. Growth and magnetosome formation by 530
microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor. 531
Appl. Microbiol. Biotechnol. 61:536-544. 532
533
15. Fernández-Castané A, Li H, Thomas OR, Overton TW. 2017. Flow cytometry 534
as a rapid analytical tool to determine physiological responses to changing O2 535
and iron concentration by Magnetospirillum gryphiswaldense strain MSR-1. 536
Sci. Rep. 7:13118. 537
538
16. Schüler D, Uhl R, Bäuerlein E. 1995. A simple light scattering method to assay 539
magnetism in Magnetospirillum gryphiswaldense. FEMS Microbiol. Lett. 540
132:139-145. 541
542
17. Schüler D. 1999. Formation of magnetosomes in magnetotactic bacteria. J. 543
Mol. Microbiol. Biotechnol. 1:79-86. 544
545
18. Baumgartner J, Morin G, Menguy N, Gonzalez T P, Widdrat M, Cosmidis J, & 546
Faivre D. 2013. Magnetotactic bacteria form magnetite from a phosphate-rich 547
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
24
ferric hydroxide via nanometric ferric (oxyhydr) oxide intermediates. PNAS, 548
110:14883-14888. 549
550
19. Fdez-Gubieda ML, Muela A, Alonso J, García-Prieto A, Olivi L, Fernández-551
Pacheco R, Barandiarán JM. 2013. Magnetite biomineralization in 552
Magnetospirillum gryphiswaldense: time-resolved magnetic and structural 553
studies. ACS nano 7:3297-3305. 554
555
20. Krueger S, Olson GJ, Rhyne JJ, Blakemore RP, Gorby YA, Blakemore N. 556
1989. Small angle neutron and X-ray scattering from magnetite crystals in 557
magnetotactic bacteria. J. Magn. Magn. Mater. 82:17-28. 558
559
21. Hoell A, Wiedenmann A, Heyen U, Schüler D. 2004. Nanostructure and field-560
induced arrangement of magnetosomes studied by SANSPOL. PHYSICA B 561
350:E309-E313. 562
563
22. Orue I, Marcano L, Bender P, García-Prieto A, Valencia S, Mawass MA, Gil-564
Cartón D, Alba Venero D, Honecker D, García-Arribas A, Fernández Barquín 565
L, Muela A, Fdez-Gubieda ML. 2018. Configuration of the magnetosome 566
chain: a natural magnetic nanoarchitecture. Nanoscale 10:7407-7419. 567
568
23. Molcan M, Petrenko VI, Avdeev MV, Ivankov OI, Garamus VM, Skumiel A, 569
Jozefczak A, Kubovcikova M, Kopcansky P, Timko M. 2017. Structure 570
characterization of the magnetosome solutions for hyperthermia study. J. Mol. 571
Liq. 235:11-16. 572
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
25
573
24. Katzmann E, Müller FD, Lang C, Messerer M, Winklhofer M, Plitzko JM, 574
Schüler D. 2011. Magnetosome chains are recruited to cellular division sites 575
and split by asymmetric septation. Mol. Microbiol. 82:1316-1329. 576
577
25. Katzmann E, Eibauer M, Lin W, Pan Y, Plitzko JM, Schüler D. 2013. Analysis 578
of magnetosome chains in magnetotactic bacteria by magnetic measurements 579
and automated image analysis of electron micrographs. Appl. Environ. 580
Microbiol. 79:7755-7762. 581
582
26. Raschdorf O, Forstner Y, Kolinko I, Uebe R, Plitzko JM, Schüler D. 2016. 583
Genetic and ultrastructural analysis reveals the key players and initial steps of 584
bacterial magnetosome membrane biogenesis. PLoS Genet. 12:e1006101. 585
586
27. Raschdorf O, Bonn F, Zeytuni N, Zarivach R, Becher D, Schüler D. 2018. A 587
quantitative assessment of the membrane-integral sub-proteome of a bacterial 588
magnetic organelle. J. Proteomics 172:89-99. 589
590
28. Nudelman H, Zarivach R. 2014. Structure prediction of magnetosome-591
associated proteins. Front. Microbiol. 5:9. 592
593
29. Glatter O, Kratky O. 1982. Small angle X-ray scattering. Academic press, 594
London 595
596
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
26
30. Erickson HP. 2009. Size and shape of protein molecules at the nanometer 597
level determined by sedimentation, gel filtration, and electron microscopy. Biol. 598
Proced. Online, 11:32. 599
600
31. Klumpp S, Faivre D. 2012. Interplay of magnetic interactions and active 601
movements in the formation of magnetosome chains. PLoS ONE 7:e33562. 602
603
32. Ghaisari S, Winklhofer M, Strauch P, Klumpp S, Faivre D. 2017. 604
Magnetosome organization in magnetotactic bacteria unraveled by 605
ferromagnetic resonance spectroscopy. Biophys J. 113:637-644. 606
607
33. Bennet M, Bertinetti L, Neely RK, Schertel A, Körnig A, Flors C, Müller FD, 608
Schüler D, Klumpp S, Faivre D. 2015. Biologically controlled synthesis and 609
assembly of magnetite nanoparticles. Faraday discuss. 181:71-83. 610
611
34. Toro-Nahuelpan M, Müller FD, Klumpp S, Plitzko JM, Bramkamp M, Schüler 612
D. 2016. Segregation of prokaryotic magnetosomes organelles is driven by 613
treadmilling of a dynamic actin-like MamK filament. BMC Biol. 14:88. 614
615
35. Hansen JP, Verlet L. 1969. Phase transitions of the Lennard-Jones system. 616
Phys. Rev. 184:151-161. 617
618
36. Dobrynin AV, Rubinstein M, Obukhov SP. 1996. Cascade of transitions of 619
polyelectrolytes in poor solvents. Macromol. 29:2974-2979. 620
621
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
27
Figure Captions 622
623
Fig. 1 Magnetosome arrangement in M. gryphiswaldense. TEM micrograph of wild type cells 624
illustrating the chain-like particle arrangement (a), as well as a sketch of a single cell with a schematic 625
of a magnetosome consisting of one magnetite core and its surrounding magnetosome membrane (b). 626
The latter contains phospholipids and about 30 different Mam proteins with various functions in control 627
of magnetosome biomineralisation. (4) 628
629
Fig. 2 Form factor model of magnetosomes. (a) TEM micrograph of isolated magnetosomes from 630
M. gryphiswaldense (Mickoleit, unpublished data), exhibiting the characteristic core-shell structure. (b) 631
Sketch of a single magnetosome (as shown in Fig. 1) and the corresponding x-ray contrast model. 632
SAXS is sensitive to the different electron density distributions of the core and the surrounding shell, 633
which mainly consist of magnetite and proteins, respectively. Both contributions are modelled as 634
spheres of different sizes. The resulting contrasts contribute in a characteristic way to specific regions 635
in the scattering data, which consequently contain information on both the overall particle radius R as 636
well as the radius of gyration Rg of the individual proteins. (c) Chain-like arrangement of 637
magnetosomes within one cell as seen by TEM. Separation distances between single particles are 638
indicated by arrows, while the membranes surrounding the magnetite cores are highlighted by dashed 639
lines. (d) Model of the particle chain shown in (c). SAXS measurements detect the center-to-center 640
distances of two magnetosomes given by 2R + l. 641
642
Fig. 3 Comparison of magnetosome-free and producing cells of M. gryphiswaldense. (a) TEM 643
micrograph of representative magnetosome-producing cells grown under anaerobic conditions. (b) 644
Scattering patterns obtained from cell suspensions (blue circles) shown in (a). Additionally, the 645
calculated scattering patterns of small objects according to eq. 5 (solid black line) and polydisperse 646
spheres according to eq. 2 are shown (red line). The spheres exhibit a radius of 19 nm with a 647
Gaussian distribution with standard deviation of 17%. (c/d) TEM micrographs of representative, 648
magnetite-free cells of M. gryphiswaldense. (e) Scattering patterns of the corresponding 649
M. gryphiswaldense cell suspensions. The curves are presented with a vertical shift for better visibility: 650
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from
28
Open symbols, magnetite-producing M. gryphiswaldense cells as shown in (a); filled symbols, 651
magnetite-free cells (green or blue symbols correspond to cells shown in (c) or (d), respectively). 652
Additionally, the calculated scattering profile of the small particles is given for comparison (grey line). 653
654
655
Fig. 4 Scattering patterns and TEMs of M. gryphiswaldense at different sampling times during 656
cultivation. (a) Patterns at production start (t = 0 h, black line, no magnetite due to growth conditions), 657
after 8 h (red squares) and 23 h (blue circles), and (b) corresponding TEM micrographs. (c) The 658
scattering of fully matured cells (symbols, after subtraction of the scattering contribution Ismall(q)) are 659
compared to the form factor scattering of polydisperse, non-interacting spheres (thin blue line, R = 19 660
± 3 nm, Gaussian distribution) and the scattering of 5 monodisperse spheres arranged in a linear 661
chain (thin red line, R = 19 nm, l = 12 nm). A model allowing for polydispersity in the system is 662
represented by the green line. 663
on October 31, 2020 by guest
http://aem.asm
.org/D
ownloaded from