97
FEMS Microbiology Letters 7 (1980) 97-101
0 Copyright Federation of European Microbiological Societies
Published by Elsevier/North-Holland Biomedical Press
LOCATION OF METAL IONS IN BACILLUS A EG, TERIl f SPORES BY
HIGH-RESOLUTION ELECTRON PROBE X-RAY MICROANALYSIS
KEITH JONHSTONE, DAVID J. ELLAR and TIMOTHY C. APPLETON *
Department of Biochemistry, University of Cambridge, Cambridge, CB2 IQW and *Physiological Laboratory,
Cambridge CB2 3EG, U.K.
Received 15 November 1979
Accepted 16 November 1979
1. Introduction
Dormant bacterial spores have no detectable metabolism, are extremely resistant and yet can germinate rapidly within minutes of exposure to germinants
and outgrow to form vegetative cells [I]. Spores also
contain much higher levels of calcium, manganese
and zinc than vegetative cells as a result of an
increased uptake of these cations during sporulation
[2]. These metal ions, which comprise up to 2.5% of
the spore dry weight are rapidly released on germination [3,4] and are believed to play an important role
in spore dormancy and resistance. Thus spores
formed with a reduced calcium content showed
decreased heat resistance [5]. A reduction in spore
manganese yielded spores with. a decreased radiation
resistance and a reduced requirement for heat shock
prior to germination [6,7]. Before any of these ions
can be implicated in models to explain spore dormancy, their precise cellular location must be determined. Previous attempts to locate these metal ions
by spodography [8,9], microincineration [ 101 and
low resolution X-ray microanalysis [11] did not conclusively identify these sites.
New methods of high resolution electron probe Xray microanalysis can analyse elemental distribution
at cellular and subcellular levels, with a limit of
detection of c. 1 attomole and a potential resolving
power of 10 nm [ 121. By use of electron probe
X-ray microanalysis Hutchinson et al. [ 131 were able
to show that calcium is a prominent constituent of
the y-particle of Blastocladiella emersonii. These
methods have also been used to study the develop-
ment of oospores of Saprolegnia [I41 and trichocysts
ofparamecium [ 151.With the advent of rapid freezing
techniques and cryo-ultramicrotomy, analysis of diffusable elements has been possible [ 161. We have
used these methods to determine the location of
metal ions in Bacillus megaterium spores.
2. Materials and Methods
Spores of Bacillus megaterium KM suspended in
deionised water at 100 mg dry weight per ml were
prepared as previously described [ 171, frozen at
-210°C and frozen sections 120-140 nm thick cut
at -70°C with glass knives in a cryostat as described
by Appleton [ 181. Sections were collected on formvarcoated nickel grids, freeze-dried at -7OoC, coated
with a 20-30 nm layer of carbon and examined in a
Jeol 100 CX electron microscope with an accelerating
voltage of 100 kV. Linescans and density area maps
for the calcium &line were produced by scanning
sections with an electron beam 10 nm in diameter
and the counts in the energy window of the calcium
&line analysed without smoothing by a Kevex X-ray
detector and a Link energy dispersive analysis system.
Point analyses were collected in 100 s under the same
conditions but using a static 10 nm diameter probe.
3. Results
Fig. 1 shows the scanning transmission electron
microscope (STEM) image of a freeze-dried frozen
Fig. 1. STEM (scanning transmission electron microscope) image of a freezedried section of Bacillus megaterium KM spores. The
linescan along the marked line is for the calcium K, line. A , background; B, coat;C, cortex;D, core. Bar represents 300 nm.
Fig. 2. Calcium density area map of the field shown in Fig. 1 .
99
is shown in Fig. 2. This also indicates a core location
for the majority of spore calcium. in order to locate
other elements in the spore, spot analyses were performed at the points, A, B, C and D marked on Fig. 1.
The energy dispersive X-ray spectra for these analyses
are shown in Fig. 3 and quantitated in Table 1 allowing for the volumes of the spore compartments. Due
to the difficulty of construction of suitable standards
section of Bacillus megaterium KM spores. Spore
coats, cortex and core are visible and similar in
appearance to the structures observed by conventional
electron microscopy [ 191. Superimposed on Fig. 1 is
a linescan along the marked line for the & line of
calcium. This analysis shows that the majority of
calcium is located in the spore core. The X-ray map
for the &line of calcium in the same fields as Fig. 1
n
A
A
I 1 I
I I
1
K CaOpg
2
0
1
1
1
1
1
1
1
1
1
1
4
1
I
I
I 1
Mn
6
Ni,
NiRZn
I I I
NaMgSi P S
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
8
1
1
1
1
1
1
1
1
1
10
1
1
1
1
I
1
1
1
1
J
Energy keV
Fig. 3. Energy dispersive X-ray spectra for spot analyses at each of the points marked on Fig. 1 . A, background; B, coat; C, cortex;
D, core.
100
TABLE 1
Percentage distribution of elements in B. rnegateriurn KM
spore compartments
Percentage in each location a
Elcment
Coats
Cortex
Core
1
9
4
0
26
4
41
71
98
6
12
18
0
47
5
33
26
1
93
79
78
99
26
91
21
3
1
~~
Ca
K
Mg
Mn
Na
P
S
Si
Zn
a Values based o n integral counts from Fig. 3 and coat, cortex a n d core volumes of 0.362 pm3,0.586 pm3 and 0.688
pm3, respectively.
no account has been taken of X-ray quenching. These
data demonstrate that the core contains most of the
calcium, potassium, magnesium, manganese and phosphorus present in the spore. Detectable amounts of
zinc and silicon are located in the coat, and coat plus
core respectively. The phosphorus and sulphur peaks
in t h e coat region may be correlated with its chemical
composition [20]. The phosphorus signal may also
arise from the outer spore membrane which is considered to be associated with the coats [21]..
Linescans for silicon (unpublished results) confirmed the high levels of silicon in the coats and also
the resolution of the method. We suggest that this
distribution of elements reflects that of the intact
spore in vivo since spores are perfectly viable after
freezing and in the conditions used to prepare freezedried frozen sections there is no opportunity for ions
to diffuse [ 181.
that to preserve electrical neutrality a major fraction
of spore divalent metal ions must be associated with
this DPA. Thus our results confirm that DPA must be
primarily located in the core and they support theories
of heat resistance that require a low concentration of
divalent metal ions in the cortex. The results are also
consistent with the concept of an osmoregulatory
cortex proposed by Gould and Dring [24]. The level
of metal ions observed in the cortex may be somewhat higher than actually exists in vivo, since electron
scatter may cause secondary excitation in the adjacent
coats and core. Examination of thin sections reduces
this scatter to a minimum. The biological significance
of the silicon observed in the coats and cortex is in
doubt since it may be derived from glass culture vessels. The divalent metal ions present in these outer
layers may be associated with this silicon rather than
cortex peptidoglycan.
A recent study has indicated that changes in the
concentration of free manganese in the developing
spore may regulate the activity of the manganese
dependent enzyme phosphoglycerate mutase [25].
One possible explanation for the low level of phosphoglycerate mutase activity in dormant spores was
suggested to be the reduction in free manganese concentration brought about by tight binding of manganese t o some spore component [25]. It is therefore
important to note that all manganese in the dormant
spore is located in the core. Since manganese is paramagnetic, its electron spin resonance spectrum [26]
could be used as a non-destructive probe to examine
the physical state of the spore core.
Acknowledgements
This work was supported by the Medical Research
Council and Science Research Council. We thank Jeol
U.K. Ltd. for generous provision of electron microscope facilities.
4. Discussion
0-Attenuation analysis [ 2 2 ] and UV irradiation
[23] suggested that pyridine-2,6dicarboxylic acid
(DPA) is primarily located in the spore core. DPA is a
strong divalent metal ion chelator which comprises up
to 10%of spore dry weight [20]. Consideration of
the total ionic composition of spores [20] indicates
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