Published Ahead of Print 28 June 2013.
2013, 195(17):3940. DOI: 10.1128/JB.00712-13.
J. Bacteriol.
Morris, Victoria J. Orphan and Grant J. Jensen
Elitza I. Tocheva, Anne E. Dekas, Shawn E. McGlynn, Dylan
longum
Acetonema
in the Gram-Negative Bacterium
Polyphosphate Storage during Sporulation
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Polyphosphate Storage during Sporulation in the Gram-Negative
Bacterium
Acetonema longum
Elitza I. Tocheva
,
a
Anne E. Dekas
,
c
*
Shawn E. McGlynn
,
c
Dylan Morris
,
a
Victoria J. Orphan
,
c
Grant J. Jensen
a,b
Division of Biology,
a
Howard Hughes Medical Institute,
b
and Geological and Planetary Sciences,
c
California Institute of Technology, Pasadena, California, USA
Using electron cryotomography, we show that the Gram-negative sporulating bacterium
Acetonema longum
synthesizes high-
density storage granules at the leading edges of engulfing membranes. The granules appear in the prespore and increase in size
and number as engulfment proceeds. Typically, a cluster of 8 to 12 storage granules closely associates with the inner spore mem-
brane and ultimately accounts for
7% of the total volume in mature spores. Energy-dispersive X-ray spectroscopy (EDX)
analyses show that the granules contain high levels of phosphorus, oxygen, and magnesium and therefore are likely composed of
polyphosphate (poly-P). Unlike the Gram-positive
Bacilli
and
Clostridia
,
A. longum
spores retain their outer spore membrane
upon germination. To explore the possibility that the granules in
A. longum
may be involved in this unique process, we imaged
purified
Bacillus cereus
,
Bacillus thuringiensis
,
Bacillus subtilis
, and
Clostridium sporogenes
spores. Even though
B. cereus
and
B. thuringiensis
contain the
ppk
and
ppx
genes, none of the spores from Gram-positive bacteria had granules. We speculate that
poly-P in
A. longum
may provide either the energy or phosphate metabolites needed for outgrowth while retaining an outer
membrane.
B
acteria have the ability to store energy and nutrients such as
carbon, phosphate, and nitrogen in the form of granules
(
1
). Inorganic phosphorus (P
i
) is stored in the form of
polyphosphate (poly-P), chains of tens to hundreds of P
i
resi-
dues, linked by high-energy phosphoanhydride bonds (
2
). A
variety of roles for poly-P granules have been suggested in cell
membrane formation, transcriptional and enzymatic regula-
tion, stress and stationary-phase responses, and cation seques-
tration (
3
). Even though the mechanism underlying poly-P
accumulation is not clearly understood, the principal enzymes
involved in the metabolism of poly-P in bacteria have been
identified: two classes of poly-P kinases (PPK1 and PPK2) po-
lymerize the terminal phosphate of ATP onto a poly-P chain
and can also work in reverse to generate ATP from poly-P, and
exopolyphosphatase (PPX) hydrolyzes the terminal phosphate
from linear poly-P (
4
). Genes encoding PPK are present in
many bacteria, including various human pathogens (
5
). Dele-
tion of
ppk
affects growth, motility, quorum sensing, biofilm
formation, and virulence (
4
,
6
,
7
). In the opportunistic patho-
gen
Bacillus cereus
, the
ppx
mutant was also impaired in sporu-
lation (
8
).
Sporulation is a complex morphological process performed by
some members of the phylum
Firmicutes
when nutrients are lim-
ited (
9
). The process begins with an asymmetric cell division, fol-
lowed by the engulfment of the smaller compartment by the big-
ger, mother cell (
10
). At the end of sporulation, two membranes
and numerous protective layers surround the mature spore.
When the conditions are favorable again, the spore germinates
and a new cell is released via outgrowth (
10
). Our previous studies
on sporulation revealed that, unlike
Bacilli
and
Clostridia
, the
noncanonical Gram-negative organism
A. longum
retains both
spore membranes during outgrowth (
11
). Here, we describe how
during sporulation,
Acetonema longum
also forms small dense
bodies at the leading edges of engulfing membranes. The number
and size of these bodies increase as engulfment proceeds, reaching
a final number of 8 to 12 per mature spore. Using bioinformatics,
nanoscale secondary ion mass spectrometry (NanoSIMS), elec-
tron cryotomography (ECT), and energy-dispersive X-ray spec-
troscopy (EDX), we identify these bodies as poly-P storage gran-
ules (SGs) and discuss their possible roles in sporulation.
MATERIALS AND METHODS
Sample preparation.
Acetonema longum
strain APO-1 cells were grown as
described previously (
12
). Sporulating cells were harvested from cultures
entering stationary phase.
Bacillus subtilis
,
B. cereus
, and
Bacillus thurin-
giensis
cells were grown in one-fourth Luria-Bertani medium (Life Tech-
nologies), and sporulation was induced by suspending exponential-phase
cells in sporulation medium (
13
). One liter of sporulation medium con-
tains 3
M FeCl
3
·6H
2
O, 40 mM MgCl
2
·6H
2
O, 38 mM MnCl
2
·4H
2
O,
0.01 M NH
4
Cl, 75 mM Na
2
SO
4
,0.12MNH
4
NO
3
,0.05MKH
2
PO
4
, 0.25
mM morpholinepropanesulfonic acid (MOPS), pH 7.5, 0.02% glutamic
acid, 0.1 mM CaCl
2
, 4 mM MgSO
4
. Pure spores from
A. longum
,
B. sub-
tilis
,
B. cereus
, and
B. thuringiensis
were harvested by centrifugation and
purified from mother cells as described previously (
14
).
Clostridium
sporogenes
spores were a kind gift from Adrian Ponce. Cells and spores
were grown and purified as described previously (
15
).
Electron cryotomography.
Pure spores and cells were prepared for
ECT by plunge freezing in nitrogen-cooled liquid ethane. Images and
tilt-series were collected on an FEI Polara (FEI Company, Hillsboro, OR)
300-kV field emission gun (FEG) transmission electron microscope
equipped with a Gatan energy filter and a lens-coupled 4k-by-4k Ultra-
Cam camera (Gatan, Pleasanton, CA) or a Titan Krios (FEI Company,
Hillsboro, OR) microscope equipped with a Gatan energy filter and a K2
Summit direct detector (Gatan, Pleasanton, CA). Samples were imaged
Received
16 June 2013
Accepted
20 June 2013
Published ahead of print
28 June 2013
Address correspondence to Grant J. Jensen, Jensen@caltech.edu.
* Present address: Anne E. Dekas, Chemical Sciences Division, Lawrence Livermore
National Laboratory, Livermore, California, USA.
Supplemental material for this article may be found at
http://dx.doi.org/10.1128
/JB.00712-13
.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JB.00712-13
3940
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with 200 e
/Å
2
, a defocus of
10
m, and a tilt range from
60 to
60°.
Three-dimensional reconstructions and segmentations were produced
with IMOD (
16
).
Traditional electron microscopy (EM) of pure
A. longum
spores.
Pure spores were chemically fixed based on protocols developed by Saba-
tini et al. (
17
). Briefly, primary fixation was done in 2.5% glutaraldehyde
in buffer A (0.1 M sodium cacodylate buffer, pH 7.2). After three consec-
utive rinses in buffer A, the spores were fixed with 1% osmium tetroxide in
buffer A. Epon epoxy resin was sequentially dissolved in 50%, 70%, and
100% and infiltrated into the spores. Once fully infiltrated, the resin was
cured at 60°C for 2 days and then sectioned with an ultramicrotome. The
thickness of the sections was 300 nm. Projection images were collected on
a Tecnai T12 electron microscope.
NanoSIMS of
A. longum
spores.
Sections were prepared as described
above for traditional EM analysis. NanoSIMS analyses were performed
using a Cameca nanoscale secondary ion mass spectrometry (NanoSIMS)
50L instrument (Gennevilliers, France). A primary Cs
ion beam was
focused to an
100-nm spot size and scanned over the sample in 256 by
256 pixel rasters to generate secondary ions. Dwell time was 1 to 5 ms per
pixel, and raster size was 3 by 3
m. Five secondary ions (
12
C
,
16
O
2
,
31
P
,
32
S
, and
14
N
12
C
) were collected simultaneously using electron
multipliers.
EDX analysis of whole
A. longum
spores.
EDX analysis was per-
formed on an FEI Titan 80- to 300-kV scanning transmission electron
microscope equipped with an Oxford Instruments System Detector 7773
(FEI Company, Hillsboro, OR). Mature hydrated spores from
A. longum
FIG 1
Storage granule formation during sporulation in
A. longum
. (A) Tomographic slices through vegetative cell; (B) sporulating cell during early stages of
engulfment; (C and D) sporulating cell during later stages of engulfment; (E) mature spore; (F) germinating cell. Abbreviations: S, spore; M, mother cell; SG,
storage granule; IM, inner membrane, OM, outer membrane. Bar, 200 nm.
Poly-P Storage during Sporulation in
A. longum
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were placed on carbon-coated copper grids and air dried. The FEI trans-
mission EM (TEM) imaging and analysis (TIA) software package was used
to acquire data from a point measurement over a storage granule and
from an area over a whole spore. EDX spectra of different areas were
collected at 80 kV and a dosage of
50 e
/Å
2
.
Homology searches.
Several kinds of storage granules (SGs) have been
described in bacteria, including glycogen, poly-
-hydroxybutyrate (PHB),
polyphosphate, “sulfur-rich” granules, and “nitrogen-rich” granules (
18
–
22
).
We searched for genes encoding enzymes associated with the production of
storage granules in the genomes of
A. longum
and a number of “control”
species, including
Ralstonia eutropha
(known to form PHB granules) (
23
),
Caulobacter crescentus
(known to form polyphosphate granules) (
24
),
Esche-
richia coli
(known to store glycogen) (
25
), and
Allochromatium vinosum
(known to form sulfur globules). The well-known endospore-forming spe-
cies
B. subtilis
and
C. sporogenes
were also included as controls. To analyze the
distribution of enzymes responsible for storage granule formation, we con-
ducted BLAST searches of the several genomes using the blastp program with
low-complexity filtering disabled and a strict E value threshold of 1e
10 (
26
).
The query proteins used for these searches for glycogen storage were GlgBI
(NP_629578.1) and GlgBII (NP_631386.1); those for PHB granules were
PhaC (YP_726471.1), PhaP (YP_001171240.1), PhaZ1 (YP_725659.1), and
PhaZ2 (YP_727307.1); those for polyphosphate storage were PPK1
(NP_416996.1) and PPX (NP_416997.1); and those for sulfur globules were
SgpA (YP_003443861.1) and SgpB (YP_003442351.1).
Homology searches for just the poly-P enzymes in all sequenced spo-
rulating bacteria were performed using the Pfam domains PF02503,
PF03976, and PF02541 for PPK1, PPK2, and PPX, respectively.
RESULTS
ECT of sporulating
A. longum
cells reveals storage granules.
As
described in the work of Tocheva et al., cryotomograms of
250
A. longum
cells were recorded at different stages of sporulation
(
11
). Dense storage granules (SGs) were rarely observed (
1%) in
vegetative cells of
A. longum
(
Fig. 1A
) but were consistently found
in all prespores at the leading edges of engulfing membranes dur-
ing early stages of engulfment (
Fig. 1B
). The number and size of
the SGs increased as sporulation proceeded (
Fig. 1C
and
D
). Mea-
surements of the distance of the SGs to the closest leading edge of
engulfing membranes show a range of distances (from 67 nm to
272 nm), with the closest SG located 71
7 nm from a leading
edge. At the end of engulfment, all mature
A. longum
spores typ-
ically contained 8 to 12 storage granules with diameters of 40 to
120 nm, accounting for
7% of the spore volume (
Fig. 1E
). In
mature spores, the SGs remained clustered but were no longer
proximal to the inner spore membrane. The SGs persisted
throughout germination and outgrowth (
Fig. 1F
), though no spe-
cific localization with respect to the newly emerging cell was ap-
parent.
Appearance of SGs.
The SGs in
A. longum
appeared dense and
grossly spherical, surrounded by an even denser shell (
Fig. 2
).
Compared to other organisms, they closely resembled the size,
density, and shape of the poly-P storage granules observed in
Cau-
lobacter crescentus
and other organisms (see Fig. S4B in the sup-
plemental material) (
24
). The shell around the SGs was discontin-
uous, only partially covering the granule (
Fig. 2C
; white arrows
indicate the presence and black arrows indicate the absence of the
protein layer). No patterns in the positions of the shell patches
were recognized. In contrast to reports of an apparent membra-
nous shell surrounding poly-P storage granules in
Agrobacterium
FIG 2
Structural features of the storage granules in
A. longum
. (A) Segmentation of the sporulating
A. longum
cell from
Fig. 1D
shows that 9 storage granules
(represented as colored spheres) are clustered together and pressed against the inner spore membrane (green). Bar, 200 nm. (B) The storage granules are located
close to the leading edge of the engulfing membrane. Colored stars correspond to the colors of the storage granules in panel A. (C) The granules exhibit a variety
of roughly spherical shapes and are surrounded by a patchy surface layer. White arrows indicate areas of the presence of a proteinaceous layer; black arrows
indicate the absence of a layer. Bar, 50 nm.
Tocheva et al.
3942
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tumefaciens
and
Rhodospirillum rubrum
(
27
), the surrounding
shell in
A. longum
was both discontinuous and variable in thick-
ness and was therefore likely proteinaceous. The cores of the SGs
appeared granular and void of internal organization. Fourier
transforms of the images also failed to reveal internal order (data
not shown).
Traditional EM of mature
A. longum
spores.
Traditional EM
methods failed to preserve the SG consistently (see Fig. S1 in the
supplemental material). Using the same preparation method and
sections, sometimes the SGs were retained, and other times they
were lost, leaving “holes” in the section that are a well-known
artifact of chemical fixation and alcohol dehydration (
28
). The
inconsistency of SG preservation with traditional EM methods
complicated elemental data acquisition, and precautions were
therefore taken to analyze only dense (preserved) granules.
Mature spores from
Bacilli
and
Clostridia
lack SGs.
In order
to explore the role of poly-P SGs in sporulation in general, mature
spores of other sporulating bacteria were also imaged with ECT. In
contrast to
A. longum
, mature
B. subtilis
,
B. cereus
,
B. thuringiensis
,
and
C. sporogenes
lacked dense SGs (see Fig. S2 in the supplemen-
tal material).
Elemental mapping of
A. longum
spores using NanoSIMS.
To investigate the intracellular elemental distribution in a spore,
thin sections of three
A. longum
spores were analyzed with nano-
scale secondary ion mass spectrometry (NanoSIMS). Areas of in-
creased phosphorus concentration were observed within the
spores (see Fig. S3 in the supplemental material). Peaks were also
visible in the
31
P
/
14
N
12
C
ratio image, in patterns different from
those seen for the other elements (data not shown), demonstrat-
ing that they were not an artifact of sample topology or uneven
generation of secondary ions (see Fig. S3C). Due to the lower
sensitivity of NanoSIMS for phosphorus, the
31
P
signal for DNA
and RNA from the core of mature spores was not detected.
EDX.
To further explore the elemental composition of the SGs,
EDX was employed.
A. longum
spores and SGs were identified
using scanning-transmission electron microscopy (
Fig. 3
). EDX
spectra were then collected and showed elevated counts for O, P,
and Mg but not Na, S, Cl, Ca, Mn, and Cu within SGs. The counts
for phosphorus in the granules were
3-fold greater than those
for magnesium but half those for oxygen (
Fig. 3
). The copper and
some of the carbon detected likely came from the EM grid. The
distribution and overall shape of the storage granule cluster cor-
related well with elevated signals for P, O, and Mg ions in areal
analyses (
Fig. 4
).
Bioinformatics.
A. longum
possesses the genes known to me-
diate storage of polyphosphate and glycogen but not sulfur or
PHB (see Fig. S4A in the supplemental material). While PPK
and PPX are present in some
Bacilli
(
B. cereus
and
B. thurin-
giensis
, also imaged with tomography), most
Clostridia
(for
example,
C. sporogenes
) and some
Bacilli
(for example,
B. sub-
tilis
) lacked the genes associated with poly-P formation.
A.
longum
and
Pelosinus fermentans
were the only Gram-negative
endospore-forming
Firmicutes
that had been sequenced, and
both had
ppk
and
ppx
genes.
Volume calculations of SGs.
To examine the fate of the SGs
during germination and outgrowth, we performed volume calcu-
lations of the SGs in mature spores and germinating and outgrow-
ing
A. longum
cells (
n
25) (
Fig. 5
). Our results show that while
mature spores had clusters of SGs occupying
5
10
6
nm
3
, the
volume and number of the SGs gradually decreased in germinat-
ing cells (cells with hydrolyzed cortex,
Fig. 5C
and
D
)to
3.5
10
6
nm
3
. The volume and number of SGs continued to decrease
during initial stages of outgrowth (
Fig. 5E
and
F
) and were ulti-
mately the lowest in cells at later stages of outgrowth (total volume
of
1.2
10
6
nm
3
).
FIG 3
High-dose EDX point analysis of a storage granule. The elemental compositions within a storage granule and a random location outside the granule
but within the spore core are shown in red and blue, respectively. Major peaks are assigned. Data show elevated levels of P, O, and Mg in the storage granule
compared to the spore core. The inset shows a scanning-transmission EM image of the air-dried spore used for imaging, with crosses marking the
positions analyzed.
Poly-P Storage during Sporulation in
A. longum
September 2013 Volume 195 Number 17
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DISCUSSION
Here, we have called attention to our observation that
A. longum
spores contain highly dense storage granules. Following tomo-
graphic studies of sporulating cells, visual inspection and compar-
ison to characterized SGs in other organisms suggested that the
SGs observed in
A. longum
were enriched in phosphorus. EDX
analyses of the SGs in mature spores showed high concentrations
of phosphorus, oxygen, and Mg
2
indicative of concentrated
phosphate. Traditional EM images, bioinformatics analyses, and
NanoSIMS analysis support the conclusion that the granules are
likely composed of poly-P. Further analysis needs to be performed
to characterize the ratio and lengths of the phosphate chains in the
SGs, and we do not exclude the possibility that carbon may be
present in the granules as well. Previous studies have shown that
phosphate concentrates into granules by polymerizing into long
chains of polyanionic phosphate (
29
). Divalent cations have been
previously shown to form ionic bonds between separate phos-
phate groups, allowing for denser packing (
30
). In the case of
A.
longum
, the cation is likely Mg
2
, since Mg
2
levels in the granules
were also elevated. The high concentration of poly-P excludes
proteins and other organic carbon, thus explaining the slight dip
observed in the carbon EDX counts within granules (
Fig. 4
).
At this point, we can only speculate about the role of poly-P
SGs in
A. longum
sporulation. Boutte et al. showed that poly-P
appears to inhibit the swarmer-to-stalk transition in
Caulobacter
sp. under glucose exhaustion (
31
). Poly-P may play a similar reg-
ulatory role during sporulation in
A. longum
. Interestingly, previ-
ous work suggests that in the opportunistic pathogen
B. cereus
,
poly-P depolymerization may induce or at least promote efficient
sporulation in that organism (
8
). Alternatively, poly-P has been
shown to be associated with the nucleoid and important for chro-
mosomal packing or DNA segregation (
32
–
34
). Poly-P may there-
FIG 4
Lower-dose EDX area scan of a mature
A. longum
spore. Top left,
scanning-transmission EM image of the spore. Other panels, individual ele-
ment distributions within the scanned area. P, O, and Mg but not the other
elements are seen to be higher inside the storage granules than outside. (The
“K” in the panel titles corresponds to the atomic shell assessed for the X-ray
dispersion.)
FIG 5
Volume calculations of the storage granules in mature spores and ger-
minating and outgrowing
A. longum
cells. (A and B) Tomographic slices
through mature spores. The storage granules are segmented and represented as
spheres in different colors. (C and D) Spores that had hydrolyzed their cortex
but maintained an intact spore coat. (E and F) Early stages of outgrowth. (G
and H) Late stages of outgrowth. The total volume of the SGs is shown in each
panel.
Tocheva et al.
3944
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fore help pack and dehydrate the DNA in
A. longum
spores, but
again, such a role would not be general, since spores of other
species do not contain poly-P storage granules. The position of the
storage granules at the leading edge of engulfing membranes may
be a clue about their formation or use, but the significance re-
mains unclear.
One obvious difference between
A. longum
and other endo-
spore-forming bacteria, which may be relevant to its unique pro-
duction of poly-P SGs, is the presence of an outer membrane. The
basic topology of sporulation in all species produces two mem-
branes, both of which originate from the cytoplasmic membrane
of the mother cell and then later surround the mature spore (
11
,
35
). When Gram-positive cells germinate, the second membrane
dissociates together with the spore coat and is lost. In the case of
Gram-negative sporulating bacteria like
A. longum
, the new out-
growing vegetative cell retains the second membrane (
11
). In ad-
dition, this second membrane is at some point transformed from
an “inner” to an “outer” membrane.
A. longum
may therefore
store poly-P in SGs as a source of energy or building blocks for its
unique germination challenges. Toso et al. estimated that a 150-
nm-diameter poly-P granule contains about 6.5
10
5
pmol of
phosphate (
36
). Since mature
A. longum
spores contain approxi-
mately 10 100-nm-diameter poly-P granules, this is enough to
produce an ATP concentration within the small spore nearly
100
that of a normal exponentially growing cell (
10 mM [
37
]).
Considered another way, this is enough energy to approximately
double the number of proteins in the spore (
38
) or synthesize
enough fatty acids to cover 5
the area of both the inner and outer
spore membranes. Volume calculations of SGs in mature
A.
longum
spores and cells at different stages of germination and
outgrowth support this hypothesis. Our observations show a con-
sistent decrease in the size and number of the SGs from mature
spores to later stages of outgrowth (
Fig. 5
), suggestive of the SGs
being consumed during outgrowth. Furthermore, none of the
Gram-positive spore-forming bacteria that we characterized here
possess SGs in their mature spores.
Once
A. longum
becomes genetically tractable, mutation stud-
ies may help identify the role of poly-P. If sporulation efficiency is
impaired in a
ppk
mutant, for example, then accumulation of
poly-P may be necessary for engulfment. If a
ppx
mutant fails to
germinate, however, then a role of poly-P in outgrowth could be
considered.
ACKNOWLEDGMENTS
We acknowledge the use of electron microscopy facilities at the UCLA
Electron Imaging Center for NanoMachines at the California NanoSys-
tems Institute (CNSI) and thank Ivo Atanasov and Dan Taso for technical
assistance with EDX data collection and analysis. We thank Yunbin Guan
and John Eiler for assistance with the NanoSIMS measurements. We
thank Adrian Ponce for providing the
C. sporogenes
spores.
The NanoSIMS apparatus is housed within the Caltech Microanalysis
Center and is partially funded by the Gordon and Betty Moore Founda-
tion. This work was funded in part by the Howard Hughes Medical Insti-
tute, the Caltech Center for Environmental Microbial Interactions, and
gifts to Caltech from the Gordon and Betty Moore Foundation.
This work was partially performed under the auspices of the U.S. De-
partment of Energy by Lawrence Livermore National Laboratory under
contract DE-AC52-07NA27344.
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