Figure S1
. 300 nm-thick plastic section of
A. longum
spores. Traditional EM preparation methods result in
variability of poly-P storage granule preservation. Left: storage granules (black arrows) appear dense and
are well preserved; Right: the dense poly-P material is lost leaving an empty hole. A thin layer of dense
material (white arrows) surrounds in some empty storage granules. The spores represented here were picked
from the same plastic section.
A
B
core
core
Cortex
Cortex
Protein
coat
C
D
core
core
Protein
coat
Protein
coat
Protein
coat
Cortex
Cortex
Figure S2.
Spores of other species lack storage granules. Tomographic slices through mature spores
from A)
Clostridium sporogenes
and B)
Bacillus subtilis
that lack the genes associated with poly-P
formation. C)
B. cereus
and D)
B. thuringiensis
have the genes for poly-P formation. All imaged
Gram-positive organisms lack dense SGs in their mature spores. Scale bar 200 nm.
C
A
14N 12C
0
19
39
59
79
99
119
139
Cts
1
μ
m
14
N
12
C
-‐
31P
0
0
1
1
2
2
3
4
Cts
31
P
-‐
Alongum_thin_July2011_pt1.im
500nm
31P/12C
Smooth Width: 9
Masked by ROI:ROI 3
Raster size=3.006 Microns
Sigma Rel. to Image Average
Ratio
0.002
0.177
0.351
0.526
0.701
0.875
1.050
1.224
x10
-2
Abs #
σ
0.0
14.5
29.0
43.4
57.9
72.4
86.9
101.4
31P/14N 12C
Smooth Width: 9
Masked by ROI:ROI 3
Raster size=3.006 Microns
Sigma Rel. to Image Average
Ratio
0.052
0.603
1.154
1.706
2.257
2.808
3.359
3.910
x10
-2
Abs #
σ
0.0
3.0
6.0
9.0
12.0
15.0
18.1
21.1
32S/12C
Smooth Width: 9
Masked by ROI:ROI 3
Raster size=3.006 Microns
Sigma Rel. to Image Average
Ratio
0.054
1.238
2.422
3.606
4.790
5.974
7.158
8.342
x10
-2
Abs #
σ
0.0
31.0
62.0
93.1
124.1
155.1
186.1
217.2
32S/14N 12C
Smooth Width: 9
Masked by ROI:ROI 3
Raster size=3.006 Microns
Sigma Rel. to Image Average
Ratio
0.0599
0.1104
0.1610
0.2115
0.2621
0.3126
0.3631
0.4137
Abs #
σ
0.0
2.8
5.7
8.5
11.4
14.2
17.1
19.9
31
P
-‐
/
14
N
12
C
-‐
B
D
Figure S3
. Elemental composition of pure spores using NanoSIMS. A and B) Elemental analysis of three
A.
longum
spores. Distribution of
14
N
12
C
−
and
31
P
−
ions are shown in A and B
respectively. Cts indicates counts
of ions detected. The region of interest applied for the generation of ratio images in panel C is contoured in
white. C) Ratio image of
31
P
−
/
14
N
12
C
−
showing areas of elevated phosphorus ions in the spores. A smoothing
factor of 9 was applied to images A-C. Due to the lower sensitivity of NanoSIMS for phosphorus, the
31
P
−
signal for DNA and RNA from the core of mature spores is not readily detected.
D) Representative even
distribution of
12
C
−
,
19
F
−
,
31
P
−
and
35
Cl
−
in a single
B. thuringiensis
spore. Lighter colors represent higher counts.
Panel D reprinted from Ghosal et al 2010.
Figure S4
. Storage granule identification. A) Presence (black
squares) or absence (empty squares) of genes encoding granule
formation in
A. longum
and six other control organisms.
Abbreviations:
glgBI
and
glgBII
– glycogen-branching enzyme 1
and 2;
ppk
– polyphosphate kinase 1 or 2;
ppx
–
exopolyphosphatase;
sgpA
and
sgpB
– sulfur globule protein A
and B;
phaP
– phasin;
phaC
– poly(3-hydroxybutyrate)
polymerase;
phaZ1
and
phaZ2
– poly(3-hydroxybutyrate)
depolymerase.
A. longum
has the genes required for glycogen
and polyphosphate granule synthesis. B) Appearance of storage
granules in representative organisms. From top to bottom:
Negative-stained glycogen storage granules in
E. coli
appear as
large numbers of small granules. Reprinted from Leduc et al
1989. A tomographic slice through a
C. crescentus
cell showing
phosphorus-rich bodies with diameters ranging from 30 to 180
nm. Reprinted from Comolli et al 2006. Negative-stained
A.
vinosum
cells showing empty sulfur globules enclosed in a
protein envelope. Reprinted from Prange et al 2004. A
tomographic slice though PHB granules in
R. eutropha
. The
granules appear dense, uniform in texture and circular. Reprinted
from Beeby et al 2012.
Glycogen storage granules in
E. coli
PHB granules in
R. eutropha
Phosphorus rich storage granule
(black arrow) and carbon-‐rich body
(white arrow) in
C. crescentus
Glycogen storage
Polyphosphate storage
Sulfur storage
PHB
storage
glgBI
glgBII
ppk
ppx
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
C. crescentus
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
R. eutropha
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
A. vinosum
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
E. coli
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
C. sporogenes
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
B. sub@lis
C. sporogenes
C. crescentus
R. eutropha
A. vinosum
S. coelicolor
A. longum
E. coli
glgBI
glgBII
ppx
ppk
sgpA
sgpB
phaP
phaC
phaZ1
phaZ2
A. longum
A
B
Sulfur globules in
A. vinosum