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Ultrastructure of the denitrifying methanotroph Candidatus Methylomirabilis1
oxyfera: a novel polygonal-shaped bacterium2
3
Ming L. Wua, Muriel C.F. van Teeseling
a, Marieke J.R. Willems
a, Elly G. van Donselaar
b,4
Andreas Klinglc,#
, Reinhard Rachelc, Willie J.C. Geerts
b, Mike S.M. Jetten
a, Marc Strous
d,e&5
Laura van Niftrika,*
6
7
aDepartment of Microbiology, Institute for Water and Wetland Research, Radboud University8
Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands9
bCellular Architecture & Dynamics, Utrecht University, Padualaan 8, 3584 CH Utrecht, The10
Netherlands11
cDepartment of Microbiology and Centre for Electron Microscopy, University of Regensburg,12
Universittsstrae 31, 93053 Regensburg, Germany13
dMPI for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany14
eCentre for Biotechnology, University of Bielefeld, Germany15
#Present address: Cell Biology, University of Marburg, D-35043 Marburg, Germany16
17
*Corresponding author: Laura van Niftrik, Department of Microbiology, Institute for Water and18
Wetland Research, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen,19
The Netherlands, e-mail: L.vanNiftrik@science.ru.nl, phone: 0031-24-3652563, fax: 0031-24-20
365283021
22
Section: Microbial Cell Biology23
Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.05816-11JB Accepts, published online ahead of print on 21 October 2011
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Running title: Ultrastructure of Candidatus Methylomirabilis oxyfera25
26
Keywords: cell shape, ultrastructure, electron microscopy, anaerobic methane oxidation,27
denitrification, methanotroph, Candidatus Methylomirabilis oxyfera28
29
Abbreviations: AMO, anaerobic methane oxidation; ET, electron tomography; ICM,30
intracytoplasmic membranes; FISH, fluorescence in situ hybridization; pMMO, particulate31
methane monooxygenase; SEM, scanning electron microscopy; S-layer, protein surface layer;32
TEM, transmission electron microscopy33
34
ABSTRACT35
Candidatus Methylomirabilis oxyfera is a newly discovered denitrifying methanotroph that is36
unrelated to previously known methanotrophs. This bacterium is a member of the NC1037
phylum and couples methane oxidation to denitrification through a newly discovered intra-38
aerobic pathway. In the present study, we report the first ultrastructural study of Ca. M. oxyfera39
using scanning electron microscopy, transmission electron microscopy and electron tomography,40
in combination with different sample preparation methods. We observed that Ca. M. oxyfera41
cells possess an atypical polygonal shape, distinct from other bacterial shapes described so far.42
Also, an additional layer was observed as outmost sheath which might represent a (glyco)protein43
surface layer. Further, intracytoplasmic membranes, which are a common feature among44
proteobacterial methanotrophs, were never observed under the current growth conditions. Our45
results indicate that Ca. M. oxyfera is ultrastructurally distinct from other bacteria by its46
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atypical cell shape, and from the classical proteobacterial methanotrophs by its apparent lack of47
intracytoplasmic membranes.48
49
INTRODUCTION50
Methanotrophic bacteria (methanotrophs) are included in the subset of bacteria known as51
methylotrophs. These organisms play a critical role in the global carbon cycle and are defined by52
their ability to utilize methane (CH4) as their sole carbon and energy source [reviewed in (6, 13,53
24, 34)]. Since their discovery over a century ago (33), methanotrophs were found in a variety of54
ecosystems, including soils, sediments, wetlands, fresh water and marine habitats.55
Until recently, methanotrophs were assigned to a rather limited group of bacteria within56
the - and -subclasses ofProteobacteria (13, 34). A major extension was made by the isolation57
of three Verrucomicrobia species, which were able to oxidize methane in extremely acidophilic58
environments (8, 16, 25), and the identification of the denitrifying methanotroph Candidatus59
Methylomirabilis oxyfera, in fresh water enrichment cultures (12, 15, 19, 20, 26). For Ca. M.60
oxyfera, various enrichment cultures using different inocula have been described; they61
show >97.5% 16S rRNA gene sequence similarity (40).62
So far, at least two properties set Ca. M. oxyfera apart from the other known63
methanotrophs. First, its phylogenetic association with the deep branching NC10 phylum (26),64
a phylum without any cultivated representatives in pure culture (27), opened a new phylogenetic65
branch within the otherwise well-defined methanotrophs. Second, Ca. M. oxyferais neither an66
aerobic methane oxidizer like all other known methanotrophs , nor is it sensu stricto an67
anaerobic methane oxidizerwith the only known case of anaerobic methane oxidation (AMO)68
represented by the consortium of methanotrophic archaea and sulphate reducing bacteria, through69
reverse methanogenesis (17). It seems that Ca. M. oxyferahas developed a new way of living70
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on methane, by combining AMO coupled to denitrification with normal respiration, through a71
newly discovered intra-aerobic pathway for the production of oxygen (Fig. 1). The oxygen is72
produced through an atypical denitrification pathway, which proceeds by the dismutation of nitric73
oxide into dinitrogen and oxygen. Part of the produced oxygen is then used for the activation and74
oxidation of methane (10); the remaining oxygen is proposed to be used in normal respiration, by75
terminal respiratory oxidases (39) (Fig. 1).76
With respect to cell shape, methanotrophs harbor a variety of types: rods, cocci, and77
occasionally crescent- and pear-shaped forms are described (13). There is, however, one78
ultrastructural feature of methanotrophs that is shared by most: the intracytoplasmic membranes79
(ICMs). These ICMs harbor the key enzyme for the methane oxidation, the particulate form of80
methane monooxygenase (pMMO). Some methanotrophs also posses the soluble form of this81
enzyme (sMMO), which resides in the cytoplasm (13, 34). The physical arrangement of pMMO82
in ICMs results in an increase of the amount of this enzyme, which can reach up to 80% of total83
ICM content, and might be reflected in an enhancement of metabolic speed (23, 29). With the84
exception ofMethylocella sp. (7), which contains only the soluble form of the methane85
monooxygenase enzyme, and the three currently known verrucomicrobial species (24), all other86
methanotrophs possess the pMMO enzyme as well as ICM structures. The ICMs occur in two87
main types of arrangements: as bundles of vesicular disks in -proteobacteria (type I88
methanotrophs), and as paired peripheral layers in -proteobacteria (type II methanotrophs) (13).89
Since its discovery in 2006 (26), some of the key features of Ca. M. oxyferahave been90
unraveled, including the genome, transcriptome and proteome as well as the major catabolic91
pathways (10, 40). However, unlike for many proteobacterial methanotrophs, knowledge on the92
ultrastructure of Ca. M. oxyferais so far non-existent. Being evolutionary completely unrelated93
to previously known methanotrophs, Ca. M. oxyfera is very interesting from an ultrastructural94
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point of view. Here, we investigated the ultrastructure of Ca. M. oxyfera using an array of95
electron microscopy techniques in combination with various sample preparation methods. We96
observed that Ca. M. oxyfera cells possess an atypical polygonal shape. Also, the outermost97
layer of the cell consisted of a putative protein surface layer (S-layer). Further, this study revealed98
that, at least under the growth conditions used in this study, Ca. M. oxyferadoes not develop99
ICMs.100
101
MATERIALS AND METHODS102
Ca. M. oxyferaenrichment culture. Samples were taken from a 15 L sequencing batch reactor103
containing the Ca. M. oxyferaenrichment culture [modified from (26)].104
105
Fluorescence in situ hybridization (FISH). Cells from the Ca. M. oxyferaenrichment culture106
were harvested and hybridizations with a fluorescent probe were performed as described107
previously (11), using a stringency of 50% formamide in the hybridization buffer. The probe was108
purchased as a Cy3-labeled derivative from Thermo Electron Corporation (Ulm, Germany). The109
following probe was used: S-*-DBACT-0193-a-A-18 for NC10 bacteria (26). The preparation110
was counterstained with DAPI and mounted with Vectashield (Vector Laboratories, Inc., CA).111
The percentage of Ca. M. oxyfera cells was estimated by counting the number of cells that112
hybridized with the S-*-DBACT-0193-a-A-18 probe and the number of cells that showed only a113
4',6'-diamidino-2-phenylindole(DAPI) signal, from a total of 600 counted cells.114
115
Sample preparation for cryo-scanning electron microscopy (Cryo-SEM). Cells from the Ca.116
M. oxyferaenrichment culture were frozen by both plunge freezing and high-pressure freezing117
methods. For plunge freezing, the cells were placed between two cryo stubs, forming a118
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sandwich, and plunge frozen in liquid nitrogen slush. For high-pressure freezing, cells were119
transferred into a 100-m cavity of a planchette (3-mm diameter, 0.1-0.2-mm depth; Engineering120
Office M. Wohlwend GmbH, CH-9466 Sennwald, Switzerland) and closed with the flat side of a121
lecithin-coated planchette (3 mm, 0.3-mm depth). The cells were then cryofixed by high-pressure122
freezing (Leica EMHPF, Leica Microsystems, Vienna, Austria) and transferred to cryo stubs.123
Next, samples were placed into a cryo transfer system (Gatan Alto 2500, Oxford, UK). The top124
cryo stub from plunge frozen samples was fractured by a razor. Subsequently, both samples were125
processed in the similar manner. The water layer was sublimated for 10 min at -80 C, and sputter126
coated with a thin layer of Au/Pd (60/40) for 45 s using a Cressington 208HR sputter coater fitted127
with a MTM-20 thickness Controller (Cressington Scientific Instruments Ltd, UK) and analyzed128
by cryo-SEM.129
For cryo-SEM, cells were taken from the 'Ca. M. oxyfera' enrichment culture at six130
different time points. In total 195 typical images were obtained containing different amounts of131
cells (ranging from one to ca. 30 cells).132
133
Sample preparation for transmission electron microscopy (TEM)134
135
Chemical fixation (tannic acid-mediated osmium impregnation), Epon-embedding and136
sectioning. Cells from the Ca. M. oxyfera enrichment culture were immersed for 30 min at137
room temperature in aldehyde-based fixative (1.5% glutaraldehyde and 2% paraformaldehyde, in138
0.08 M sodium cacodylate trihydrate buffer, pH 7.4), post-fixed with 1% osmium tetroxide139
(OsO4) and 1.5% K4[Fe(CN)6] for 90 min at 4C in darkness, incubated with 1% tannic acid in140
0.1 M sodium cacodylate trihydrate buffer pH 7.4 for 30 min at RT and treated with 1% OsO4 in141
distilled water for 30 min on ice in darkness. Finally, cells were dehydrated in a graded ethanol142
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series (70%, 80%, 90%, 96%, and 100% ethanol), gradually infiltrated with Epon resin and143
sectioned (70 nm sections) using a Reichert Ultracut E Microtome (Leica Microsystems, Vienna,144
Austria) and collected on carbon-Formvar-coated 100-mesh hexagonal copper grids.145
For chemical fixation, cells were taken from the 'Ca. M. oxyfera' enrichment culture at146
one time point. This sample was chemically fixed in triple using the described protocol both with147
and without the tannic acid-mediated osmium impregnation (6 samples). For each fixation, three148
Epon blocks were produced, used for thin sectioning and investigated by TEM (6 blocks). Based149
on contrast and ultrastructural preservation, the fixation with tannic acid-mediated osmium150
impregnation was used for further investigation. These blocks were extensively examined by151
TEM and in total 50 typical images were obtained containing different amounts of cells (ranging152
from one to ca. 50 cells). In instances where cells were counted, cells were chosen from the153
images at random at the best of our ability.154
155
Cryofixation, freeze-substitution, Epon-embedding and sectioning. Cells from the Ca. M.156
oxyfera enrichment culture were cryofixed by high-pressure freezing as described above.157
Freeze-substitution was performed in acetonecontaining 2% OsO4, 0.2% Uranyl acetate, and 1%
158
H2O (37). Subsequently, samples were kept at -90C for 47 h; broughtto -60C at 2C per hour;159
kept at -60Cfor 8 h; brought to -30C at 2C per hour; and
kept at -30C for 8 h in a freeze-160
substitution unit(AFS, Leica Microsystems, Vienna, Austria). Uranyl acetate was
removed by161
washing the samples four times for 30 min in theAFS device at -30C with acetone containing162
2% OsO4 and 1% H2O. Fixation was then continued for 1 h onice. OsO4 and H2O were removed163
by washing two times for 30 min on ice with anhydrous acetone. Samples were gradually164
infiltrated with Epon resin and polymerized for 72 h at 60C (22). Ultrathin sections (for TEM,165
70 nm) and semithin (for ET,400 nm) were cut using a Reichert Ultracut E Microtome (Leica166
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Microsystems, Vienna, Austria), and collected on carbon-Formvar-coated 100-mesh hexagonal167
and 50-mesh square copper grids, respectively. The ultrathin sections were post-stained with 20%168
Uranyl acetate in 70% methanol for 4 min and Reynolds lead citrate for 2 min (28).169
For cryofixation, cells were taken from 'Ca. M. oxyfera' enrichment cultures at four170
different time points. All four samples were freeze-substituted in duplo in both acetone171
containing 2% OsO4 and acetone containing 2% OsO4, 0.2% Uranyl acetate and 1% water (16172
samples in total). For each fixation, two Epon blocks were produced, used for thin sectioning and173
investigated by TEM (16 blocks). Based on contrast and ultrastructural preservation, the174
substitution in acetone containing 2% OsO4, 0.2% Uranyl acetate and 1% water was used for175
further investigation. These blocks were extensively examined by TEM and in total 131 typical176
images were obtained containing different amounts of cells (ranging from one to ca. 50 cells). In177
instances where cells were counted, cells were chosen from the images at random at the best of178
our ability.179
180
Electron tomography (ET). Electron tomography was performed as described previously (35).181
Ten-nanometer colloidal gold particles were applied to one surface of grids bearing 200-400-nm182
semi-thin sections of high-pressure frozen, freeze-substituted, and Epon-embedded Ca. M.183
oxyfera cells to serve as fiducial markers in the alignment of the tilt series. The sections were184
post-stained with 2% Uranyl acetate in water for 10 min. Specimens were placed in a high-tilt185
specimen holder, and dual axis datasets were automatically recorded at 200 kV using a Tecnai-20186
microscope (FEI Company, Eindhoven, The Netherlands) by rotating the grid 90 inside the187
microscope (Fischione rotation holder; Fischione Instruments, Pittsburgh, PA). The angular tilt188
range was from -65 to 65, with an increment of 1. Binned (2x2) images (1,024x1,024 pixels)189
were recorded using a charge-coupled device camera (TemCam F214; TVIPS GmbH, Gauting,190
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Germany). Automated data acquisition of the tilt series was carried out using Xplore 3D (FEI191
Company, Eindhoven, The Netherlands). Tomograms from each tilt axis were computed with the192
R-weighted back-projection algorithm and combined into one double-tilt tomogram using the193
IMOD software package (18). In total, 30 Ca. M. oxyfera cells were imaged in 6 double-tilt194
tomograms.195
196
Freeze-etching. Freeze-etching was performed on concentrated (by a 4 min centrifugation step at197
10000 or 12000 g) Ca. M. oxyferacells from the enrichment culture, of which 1.7 l per gold198
carrier was plunge frozen in liquid nitrogen by hand. The samples were then introduced into a199
Cressington Freeze-Etch machine at T < -170C and a pressure of below 10-6
bar. The samples200
were kept at -97C for 7 min before being fractured. Next, the water was left to sublimate from201
the samples for 4 min (freeze-etching) before the samples were shadowed with 1 nm Pt/C (angle202
45) and 10 nm C (angle 90). The biological material was removed from the replicas by203
overnight incubation in 70% sulfuric acid; the replicas were washed twice on bidistilled water204
and picked up with 700-mesh hexagonal copper grids before investigation by TEM.205
For freeze-etching, cells were taken from the 'Ca. M. oxyfera' enrichment culture at two206
different time points. The replicas were extensively examined by TEM and in total 180 typical207
images were obtained containing different amounts of cells (ranging from one to three cells).208
209
Transmission electron microscopy (TEM). Cells, ultrathin sections and replicas were210
investigated with a TEM at 60, 80 or 120 kV (CM12, Tecnai10 or Tecnai12, FEI Company,211
Eindhoven, The Netherlands) and images were recorded using a CCD camera (MegaView II,212
OSIS; 0124, TVIPS, Gauting).213
214
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Cryo-scanning electron microscopy (Cryo-SEM). The coated samples were analyzed with a215
field-emission SEM (JEOL JSM-6330F; Tokyo, Japan) at a sample temperature of216
-170C using an accelerating voltage of 3 kV.217
218
RESULTS219
Quantification ofCa. M. oxyferacells in the enrichment culture220
Until now, it has not been possible to grow Ca. M. oxyferain pure culture. In the culture used221
for this study, the level of enrichment was about 71% when assessed by fluorescence in situ222
hybridization (FISH) using a previously described oligonucleotide probe (26). Ca. M. oxyfera223
cells appeared as small rods with an intense DAPI signal in the center. They occurred as single224
cells or as multicellular aggregates, which occasionally included cells from other species that225
made up 29% of the community.226
227
Cryo-scanning electron microscopy (Cryo-SEM)228
The cell shape of Ca. M. oxyfera was investigated using cryo-SEM (Fig. 2). The general229
appearance of Ca. M. oxyferacells was similar when prepared with plunge freezing or high-230
pressure freezing methods. The rod-shaped cells were on average 1158 323 nm long and 259 231
64 nm wide (measured from a total of 50 cells). The cell surface had a relatively smooth232
appearance except for the presence of several distinct longitudinal ridges that ran along the entire233
cell length and joined at the cell poles in a circular, cap-like structure (Fig. 2B, inset). Different234
cells had different amounts of these longitudinal ridges resulting in a polygonal cell shape for Ca.235
M. oxyfera. Further, Ca. M. oxyferacells were observed to divide by binary fission in growing236
cultures (Fig. 2A).237
238
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Freeze-etching239
Freeze-etching also revealed the polygonal cell shape of Ca. M. oxyfera(Fig. 3), especially in240
cross section (Fig. 3C). The ridges had a granular appearance compared to the rest of the cell241
surface (Fig. 3A and B). Further, Ca. M. oxyferacells were observed to contain an additional242
layer, outside the cell wall, as outmost sheath (Fig. 3A and B). This layer could be a243
(glyco)protein surface layer (S-layer) with an oblique or square lattice symmetry (p2 or p4). The244
power spectrum (Fig. 3D) indicated a repetitive pattern with frequencies at (7 nm)-1
and in some245
cases at (5 nm)-1
. Those values are likely to correspond to a center-to-center spacing of the S-246
layer units. In comparison with S-layer center-to-center spacings found in literature, which are in247
the range of 3-35 nm (30), these are rather small values. However, small values have been248
previously found for p2 symmetry S-layers (32).249
250
Ultrathin sections of cryofixed, freeze-substituted and Epon-embedded cells251
Transmission electron microscopy of ultrathin sections of cryofixed, freeze-substituted and Epon-252
embedded Ca. M. oxyferacells showed a cell envelope typical of Gram-negative bacteria (Fig.253
4). The cell envelope had a total width of about 40 nm and consisted, from in- to outside, of a254
cytoplasmic membrane, peptidoglycan and an outer membrane (Fig. 4D). The peptidoglycan255
comprised an electron dense layer within the periplasmic space in close vicinity of the outer256
membrane (Fig. 4D). Ca. M. oxyfera cells differed from prototypical bacterial cell shapes.257
Transversely sectioned cells had a polygonal cell shape with variant numbers of sides for258
different cells and longitudinally sectioned cells showed cornered cell poles (Fig. 4). Inside the259
cytoplasm, electron light granules were observed which possibly contain reserve material (Fig.260
4B, white arrows). The nucleoid, which is visible as a densely stained area in the middle of the261
cell, occupied much of the cell content and appeared to be quite condensed (Fig. 4B, black arrow).262
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Surprisingly, intracytoplasmic membranes (ICM), commonly found in proteobacterial263
methanotrophs (13, 35), were never observed.264
265
Ultrathin sections of chemically fixed and Epon-embedded cells266
There was a significant variation in the appearance of the cell envelope in the chemically fixed267
cells compared to the cryofixed cells. The chemically fixed cells appeared more shrunk (Fig. 5),268
possibly due to dehydration, a phenomenon that is commonly reported for chemically fixed cells,269
particularly when glutaraldehyde is used as initial chemical fixative, in combination with270
dehydration at room temperature (14). In the chemically fixed cells, the cell wall was often271
collapsed while the ridges stayed in position resulting in a more pronounced star-like appearance272
of the cells (Fig. 5). The polygonal cell shape was the dominant morphotype in the sample from273
the enrichment culture; from 980 cells counted, cells with a polygonal shape made up 69%.274
In general, when comparing the chemically fixed cells with cryofixed cells, the bilayer of275
the outer membrane appeared thicker and denser, and the membranes appeared more distorted276
and wavy. In some instances, an additional layer with a thickness of about 8 nm was observed on277
top of the outer membrane (Fig. 5C, inset and D). This could correspond to the putative S-layer278
observed in the freeze-etching preparations.279
280
Electron tomography281
Three-dimensional imaging using electron tomography (ET) confirmed the polygonal shape of282
Ca. M. oxyferacells after high-pressure freezing and freeze-substitution (Fig. 6, Movies S1-S3283
in supplemental material). Occasionally, an electron dense, vesicular body with a diameter of284
about 55 nm was observed within the cytoplasm (Fig. 6C, arrow). These structures contained a285
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rather rough and irregular boundary and the absence of any membrane connections suggest that286
they are separate entities and possibly storage vesicles.287
288
DISCUSSION289
In the present paper, we performed a detailed ultrastructural study of the newly discovered290
denitrifying methanotroph Ca. M. oxyfera. This bacterium is a member of the deep-branching291
NC10 phylum, and thus evolutionary unrelated to the previously known methanotrophs (10, 26).292
To avoid misinterpretation due to artifacts inherent to one single technique or sample preparation293
method, we used scanning electron microscopy (SEM), transmission electron microscopy (TEM)294
and electron tomography (ET), in combination with various sample preparation methods,295
including plunge freezing, high-pressure freezing, chemical fixation, cryofixation, and freeze-296
etching, to investigate the ultrastructure of this bacterium.297
At a first glance, Ca. M. oxyferacells appeared as typical rod-shaped Gram-negative298
bacteria. However, a careful inspection revealed that Ca. M. oxyferapossesses a unique and not299
yet described ultrastructure. The cell wall contained multiple longitudinal ridges, which conferred300
a distinctive polygonal shape to the cells (Figs. 2-6). This atypical cell shape was observed in all301
the independent methods and sample preparations employed. The percentage of cells that302
depicted the polygonal shape (69%) was in the same range as assessed by FISH using specific303
probes for Ca. M. oxyfera (71%). Taken together, this strongly suggests that this polygonal304
shape is a real feature of Ca. M. oxyferaand not artificial.305
The mechanism by which a certain cell shape is acquired and maintained is not always306
clearly understood but it often involves exo- or endoskeletal elements. The exoskeleton-like307
(glyco)protein surface layer (S-layer) and peptidoglycan components are known to play a role in308
osmotic and mechanical cell stabilization, as well as shape maintenance (4, 9, 31). The309
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endoskeletal-like elements, such as the actin-like protein MreB and the tubulin-like protein FtsZ,310
are known to act as internal scaffolds that influence the cell shape (21, 41). Another endoskeletal311
protein is the intermediate filament crescentin (CreS). The presence of crescentin is essential for312
the formation of the vibrioid and helical cell shape ofCaulobacter crescentus and the absence of313
it leads to cells with a straight, rod cell shape (2). The genome of Ca. M. oxyferacontains both314
mreB (ORF identifier DAMO_3131) and ftsZ (ORF identifier DAMO_2292) genes, but no315
homologue of creS, the gene encoding crescentin, is found. However, these known shape-316
determining elements alone cannot explain how complex cell shapes, like the polygonal cell317
shape of Ca. M. oxyfera, are formed and maintained. The square shape ofHaloquadratum318
walsbyi (36) is an example of another complex and unusual cell shape. In this archaeon it is319
hypothesized that the square shape is derived from the presence of a cross-linked matrix of poly-320
gamma-glutamate that forms a capsule outside the cell (3). It is possible that the polygonal cell321
shape in Ca. M. oxyferais also derived from the presence of a capsular matrix, like suggested322
forH. walsbyi. If so, the composition of this matrix is most likely different from the one in H.323
walsbyi since Ca. M. oxyferalacks the genes encoding the poly-gamma-glutamate biosynthesis324
protein complex capBCA (1). However, in contrast to the prototypical Gram-negative bacteria325
where the outer membrane gives the bacteria a rough appearance when examined by SEM, the326
cell surface of Ca. M. oxyferawas relatively smooth (Fig. 2). This smoothness was consistent327
with the presence of an additional layer as outmost sheath, a putative S-layer (Fig. 3 and 5C, inset328
and D). This layer might play a role in cell shape maintenance and/or determination. Nevertheless,329
this hypothesis requires further investigation and the possibility that the polygonal cell shape is330
derived from an endoskeleton-like element cannot be ruled out.331
To our knowledge, the presence of a star-like cell shape was only reported once in332
literature (38). It was found in a branching, filamentous bacterium from a deep surface mine-333
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slime. The authors named the bacteria star-shaped bacteria due to their appearance as stars in334
transverse sections of chemically fixed cells. Unfortunately, there is no genetic information335
available about these star-shaped bacteria; otherwise, it would be interesting to investigate336
whether it is phylogenetically related to Ca. M. oxyfera, and whether both organisms share337
unique genes involved in cell shape determination.338
Strikingly, one ultrastructural feature was not observed in Ca. M. oxyferacells under the339
present growth conditions, namely ICMs. With the exception of verrucomicrobial species, ICMs340
are common to all known pMMO-containing methanotrophs (13, 34). The extension and341
arrangement of the ICM might, however, differ from species to species and with growth342
conditions (5). In this study, we did not observe ICMs of any kind in Ca. M. oxyferacells. Such343
negative results, however, must be interpreted cautiously because they do not necessarily imply a344
genetic incapability to produce such structures. Ca. M. oxyfera is an extremely slow-growing345
organism; the estimated doubling time is 1-2 weeks under laboratory conditions (11) with a346
metabolic rate of 1.7 nmol methane oxidized min-1
mg protein-1
(12). It was suggested that the347
slow metabolism might be due to sub-optimal growth conditions (i.e. lack of an essential growth348
cofactor) (40); but this still needs further investigation. One possible explanation for the lack of349
ICMs is that it is energetically disadvantageous for Ca. M. oxyferato produce ICMs since to do350
so, requires a considerable energy investment that does not comply with its slow metabolism.351
Hence, the question remains whether optimal growth conditions would trigger the development352
of ICMs, or whether the lack of these structures is an intrinsic property of Ca. M. oxyfera.353
In conclusion, we found that Ca. M. oxyfera cells possess an unusual polygonal cell354
shape. The mechanism of polygonal cell shape determination, however, remains a puzzle to be355
solved. The unique cell shape of Ca. M. oxyferaprovides clear differentiation from other356
morphotypes and might be valuable as a morphology-based tool for identification. Other357
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observations, such as the putative S-layer and the apparent absence of ICMs, are interesting and a358
challenge for future research on the formation of the atypical polygonal cell shape and the actual359
subcellular localization of the pMMO enzyme.360
361
ACKNOWLEDGEMENTS362
We would like to thank Katinka van de Pas-Schoonen for support maintaining the enrichment363
cultures, Bruno Humbel and Rob Mesman for operation of the high-pressure freezer, Geert-Jan364
Janssen for support operating the cryo-scanning electron microscope and Katharina F. Ettwig,365
Francisca Luesken, Bas Dutilh, Huub Op den Camp and Jan T. Keltjens for stimulating366
discussions. LvN is supported by the Netherlands Organization for Scientific Research (VENI367
grant 863.09.009), MLW by a Horizon grant (050-71-058) and MSMJ by ERC 232937.368
369
REFERENCES370
1. Ashiuchi, and Misono. 2002. Biochemistry and molecular genetics of poly-gamma-371
glutamate synthesis. Appl. Microbiol. Biotechnol. 59:9-14.372
2. Ausmees, N., J. R. Kuhn, and C. Jacobs-Wagner. 2003. The bacterial cytoskeleton: an373
intermediate filament-like function in cell shape. Cell 115:705-713.374
3. Bolhuis, H., P. Palm, A. Wende, M. Falb, M. Rampp, F. Rodriguez-Valera, F.375
Pfeiffer, and D. Oesterhelt. 2006. The genome of the square archaeon Haloquadratum376
walsbyi: life at the limits of water activity. BMC Genomics 7:169.377
4. Cabeen, M. T., and C. Jacobs-Wagner. 2005. Bacterial cell shape. Nat. Rev. Micro.378
3:601-610.379
5. de Boer, W., and W. Hazeu. 1972. Observations on the fine structure of a methane-380
oxidizing bacterium. Antonie van Leeuwenhoek38:33-47.381
7/31/2019 Ultra Structure of the Denitrifying Methanotroph Candiddatus Methylomirabilis Oxyfera_a Novel Polygonal Shaped
17/28
17
6. Dedysh, S. N. 2009. Exploring methanotroph diversity in acidic northern wetlands:382
Molecular and cultivation-based studies. Microbiology 78:655-669.383
7. Dedysh, S. N., Y. Y. Berestovskaya, L. V. Vasylieva, S. E. Belova, V. N. Khmelenina,384
N. E. Suzina, Y. A. Trotsenko, W. Liesack, and G. A. Zavarzin. 2004.Methylocella385
tundrae sp. nov., a novel methanotrophic bacterium from acidic tundra peatlands. Int. J.386
Syst. Evol. Microbiol. 54:151-156.387
8. Dunfield, P. F., A. Yuryev, P. Senin, A. V. Smirnova, M. B. Stott, S. Hou, B. Ly, J. H.388
Saw, Z. Zhou, Y. Ren, J. Wang, B. W. Mountain, M. A. Crowe, T. M. Weatherby, P.389
L. E. Bodelier, W. Liesack, L. Feng, L. Wang, and M. Alam. 2007. Methane oxidation390
by an extremely acidophilic bacterium of the phylum Verrucomicrobia. Nature 450:879-391
882.392
9. Engelhardt, H. 2007. Are S-layers exoskeletons? The basic function of protein surface393
layers revisited. J. Struct. Biol. 160:115-124.394
10. Ettwig, K. F., M. K. Butler, D. Le Paslier, E. Pelletier, S. Mangenot, M. M. M.395
Kuypers, F. Schreiber, B. E. Dutilh, J. Zedelius, D. de Beer, J. Gloerich, H. J. C. T.396
Wessels, T. van Alen, F. Luesken, M. L. Wu, K. T. van de Pas-Schoonen, H. J. M.397
Op den Camp, E. M. Janssen-Megens, K.-J. Francoijs, H. Stunnenberg, J.398
Weissenbach, M. S. M. Jetten, and M. Strous. 2010. Nitrite-driven anaerobic methane399
oxidation by oxygenic bacteria. Nature 464:543-548.400
11. Ettwig, K. F., S. Shima, K. T. v. d. Pas-Schoonen, J. Kahnt, M. H. Medema, H. J. M.401
o. d. Camp, M. S. M. Jetten, and M. Strous. 2008. Denitrifying bacteria anaerobically402
oxidize methane in the absence ofArchaea. Environ. Microbiol. 10:3164-3173.403
7/31/2019 Ultra Structure of the Denitrifying Methanotroph Candiddatus Methylomirabilis Oxyfera_a Novel Polygonal Shaped
18/28
18
12. Ettwig, K. F., T. van Alen, K. T. van de Pas-Schoonen, M. S. M. Jetten, and M.404
Strous. 2009. Enrichment and molecular detection of denitrifying methanotrophic405
bacteria of the NC10 phylum. Appl. Environ. Microbiol. 75:3656-3662.406
13. Hanson, R., and T. Hanson. 1996. Methanotrophic bacteria. Microbiol. Rev. 60:439-471.407
14. Hayat, M. A. 1986. Glutaraldehyde: role in electron microscopy. Micron Microsc. Acta408
17:115-135.409
15. Hu, S., R. J. Zeng, L. C. Burow, P. Lant, J. Keller, and Z. Yuan. 2009. Enrichment of410
denitrifying anaerobic methane oxidizing microorganisms. Environ. Microbiol. Rep.411
1:377-384.412
16. Islam, T., S. Jensen, L. J. Reigstad, . Larsen, and N.-K. Birkeland. 2008. Methane413
oxidation at 55 C and pH 2 by a thermoacidophilic bacterium belonging to the414
Verrucomicrobia phylum. Proc. Natl. Acad. Sci. USA 105:300-304.415
17. Knittel, K., and A. Boetius. 2009. Anaerobic oxidation of methane: progress with an416
unknown process. Ann. Rev. Microbiol. 63:311-334.417
18. Kremer, J.R., D.N. Mastronarde, J.R. McIntosh, 1996. Computer visualization of418
three-dimensional image data using IMOD. J. Struct. Biol. 116, 71-76.419
19. Luesken, F. A., T. A. van Alen, E. van der Biezen, C. Frijters, G. Toonen, C.420
Kampman, T. L. G. Hendrickx, G. Zeeman, H. Temmink, M. Strous, H. J. M. Op421
den Camp, and M. S. M. Jetten. 2011. Diversity and enrichment of nitrite-dependent422
anaerobic methane oxidizing bacteria from wastewater sludge. Appl. Microbiol.423
Biotechnol. DOI 10.1007/s00253-011-3361-9.424
20. Luesken, F. A., B. Zhu, T. A. van Alen, M. K. Butler, M. Rodriguez Diaz, B. Song, H.425
J. Op den Camp, M. S. Jetten, and K. F. Ettwig. 2011.pmoA primers for detection of426
anaerobic methanotrophs. Appl. Environ. Microbiol. 77: 3877-3880.427
7/31/2019 Ultra Structure of the Denitrifying Methanotroph Candiddatus Methylomirabilis Oxyfera_a Novel Polygonal Shaped
19/28
19
21. Margolin, W. 2009. Sculpting the bacterial cell. Curr. Biol. 19(17):R812-22.428
22. Mollenhauer, H. H. 1964. Plastic embedding mixtures for use in electron microscopy429
Stain Technol. 39:111-114.430
23. Nguyen, H.-H. T., S. J. Elliott, J. H.-K. Yip, and S. I. Chan. 1998. The particulate431
methane monooxygenase from Methylococcus capsulatus (Bath) is a novel copper-432
containing three-subunit enzyme. J. Biol. Chem. 273:7957-7966.433
24. Op den Camp, H. J. M., T. Islam, M. B. Stott, H. R. Harhangi, A. Hynes, S.434
Schouten, M. S. M. Jetten, N. K. Birkeland, A. Pol, and P. F. Dunfield. 2009.435
Environmental, genomic and taxonomic perspectives on methanotrophic Verrucomicrobia.436
Environ. Microbiol. Rep. 1:293-306.437
25. Pol, A., K. Heijmans, H. R. Harhangi, D. Tedesco, M. S. M. Jetten, and H. den Camp.438
2007. Methanotrophy below pH 1 by a new Verrucomicrobia species. Nature 450:874-439
878.440
26. Raghoebarsing, A. A., A. Pol, K. T. van de Pas-Schoonen, A. J. P. Smolders, K. F.441
Ettwig, W. I. C. Rijpstra, S. Schouten, J. S. S. Damst, H. J. M. Op den Camp, M. S.442
M. Jetten, and M. Strous. 2006. A microbial consortium couples anaerobic methane443
oxidation to denitrification. Nature 440:918-921.444
27. Rapp, M. S., and S. J. Giovannoni. 2003. The uncultured microbial majority. Ann. Rev.445
Microbiol. 57:369-394.446
28. Reynolds, E. S. 1963. Use of lead citrate at high pH as an electron-opaque stain in447
electron microscopy. J. Cell Biol. 17:208-212.448
29. Ribbons, D. W., and J. L. Michalover. 1970. Methane oxidation by cell-free extracts of449
Methylococcus capsulatus FEBS Letters 11:41-44.450
7/31/2019 Ultra Structure of the Denitrifying Methanotroph Candiddatus Methylomirabilis Oxyfera_a Novel Polygonal Shaped
20/28
20
30. Sleytr, U. B. 1997. I. Basic and applied S-layer research: an overview. FEMS Microbiol.451
Rev. 20:5-12.452
31. Sleytr, U. B., and T. J. Beveridge. 1999. Bacterial S-layers. Trends Microbiol. 7:253-453
260.454
32. Smarda, J., D. Smajs, J. Komrska, and V. Krzyznek. 2002. S-layers on cell walls of455
cyanobacteria. Micron 33:257-277.456
33. Sohngen, N. L. 1906. Uber bakterien, welche methan ab knhlenstoffnahrung and457
energioquelle gebrauchen. Parasitendk Infectionskr Abt. 2:513-517.458
34. Trotsenko, Y. A., J. C. Murrell, S. S. Allen I. Laskin, and M. G. Geoffrey. 2008.459
Metabolic aspects of aerobic obligate methanotrophy p. 183-229, Adv. Appl. Microbiol.,460
vol. Volume 63. Academic Press.461
35. van Niftrik, L., W. J. C. Geerts, E. G. van Donselaar, B. M. Humbel, R. I. Webb, J.462
A. Fuerst, A. J. Verkleij, M. S. M. Jetten, and M. Strous. 2008. Linking ultrastructure463
and function in four genera of anaerobic ammonium-oxidizing bacteria: Cell plan,464
glycogen storage, and localization of cytochrome c proteins. J. Bacteriol. 190:708-717.465
36. Walsby, A. E. 1980. Square bacterium. Nature 283:69-71.466
37. Walther, P., and A. Ziegler. 2002. Freeze substitution of high-pressure frozen samples:467
the visibility of biological membranes is improved when the substitution medium contains468
water. J. Microsc-Oxford 208:3-10.469
38. Wanger, G., T. C. Onstott, and G. Southam. 2008. Stars of the terrestrial deep470
subsurface: A novel star-shaped bacterial morphotype from a South African platinum471
mine. Geobiology 6:325-330.472
39. Wu, M. L., S. de Vries, T. A. van Alen, M. K. Butler, H. J. M. Op den Camp, J. T.473
Keltjens, M. S. M. Jetten, and M. Strous. 2011. Physiological role of the respiratory474
7/31/2019 Ultra Structure of the Denitrifying Methanotroph Candiddatus Methylomirabilis Oxyfera_a Novel Polygonal Shaped
21/28
21
quinol oxidase in the anaerobic nitrite-reducing methanotroph 'Candidatus475
Methylomirabilis oxyfera'. Microbiology 157:890-898.476
40. Wu, M. L., K. E. Ettwig, M. S. M. Jettenm, M. Strous, J. T. Keltjens, and L. van477
Niftrik. 2011. A new intra-aerobic metabolism in the nitrite-dependent anaerobic478
methane-oxidizing bacterium 'Candidatus Methylomirabilis oxyfera'. Biochem. Soc.479
Trans. 39: 243-248.480
41. Young, K. D. 2003. Bacterial shape. Mol. Microbiol. 49:571-580.481
482
FIGURE LEGENDS483
484
Figure 1. Postulated model for central catabolism and energy conservation in Ca. M. oxyfera.485
White diamonds, direction of proton flow. Abbreviations; bc1, cytochrome bc1 complex; mdh,486
methanol dehydrogenase; ndh, NAD(P)H dehydrogenase complex; nir, nitrate reductase; nod,487
nitric oxide dismutase; pmmo, particulate methane monooxygenase; Q, co-enzyme Q. Adapted488
from (40).489
490
Figure 2. Cryo-scanning electron micrographs of Ca. M. oxyferacells showing the longitudinal491
ridges along the cell length. (A) Plunge frozen Ca. M. oxyferacells undergoing cell division. (B)492
Plunge frozen Ca. M. oxyferacells showing the cap-like structure (inset) at the cell poles. Scale493
bars, 500 nm.494
495
Figure 3. Transmission electron micrographs of freeze-etched Ca. M. oxyferacells. (A and B)496
Ca. M. oxyferacells fractured longitudinally over the cell wall and displaying a putative S-layer.497
(C) Ca. M. oxyferacell fractured transversely through the cell. (D) FFT power spectrum of the498
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putative S-layer cut-out shown in B. Repetitive patterns occur after ca. 7 nm. Scale bars, 200 nm.499
500
Figure 4. Transmission electron micrographs of cryofixed, freeze-substituted, and Epon-501
embedded Ca. M. oxyferacells. (A) Overview showing the dominant polygonal cell shape in502
the sample. (B) Longitudinally sectioned Ca. M. oxyferacells showing electron light granules503
(white arrows) and condensed nucleoid (black arrow). (C) Longitudinally sectioned cells showing504
a Gram-negative cell envelope. (D) cut-out and density profile of part of the cell wall shown in C.505
The density profile is measured across the white line. The numbered white arrows in the density506
profile correspond to the numbered black arrows in the cell wall. om, outer membrane; pe,507
peptidoglycan; p, periplasm; cm, cytoplasmic membrane; c, cytoplasm; Scale bars, 500 nm.508
509
Figure 5. Transmission electron micrographs of chemically fixed and Epon-embedded Ca. M.510
oxyfera cells. (A) Overview showing the star-like cell shape caused by dehydration and cell wall511
collapse. Longitudinal (B-C) and transverse (D) sections showing the Gram-negative cell512
envelope and the presence of a putative S-layer on the top of the outer membrane; om, outer513
membrane; p, periplasm; cm, cytoplasmic membrane; c, cytoplasm; s, putative S-layer. Scale bars,514
200 nm.515
516
Figure 6. (A, B and C) Tomographic slices of Ca. M. oxyfera cells showing the polygonal cell517
shape. See also Movie S1, S2 and S3 in supplemental material, respectively. (D) Model of518
tomogram shown in A. Arrow, electron dense vesicular body; Scale bars, 500 nm.519
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