Kinetics of Germination of Individual Spores of
Geobacillus stearothermophilus as Measured by Raman
Spectroscopy and Differential Interference Contrast
Microscopy
Tingting Zhou1,3, Zhiyang Dong1, Peter Setlow2, Yong-qing Li3*
1 Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, 2Department of Molecular, Microbial and Structural Biology, University of Connecticut Health
Center, Farmington, Connecticut, United States of America, 3Department of Physics, East Carolina University, Greenville, North Carolina, United States of America
Abstract
Geobacillus stearothermophilus is a gram-positive, thermophilic bacterium, spores of which are very heat resistant. Raman
spectroscopy and differential interference contrast microscopy were used to monitor the kinetics of germination of
individual spores of G. stearothermophilus at different temperatures, and major conclusions from this work were as follows.
1) The CaDPA level of individual G. stearothermophilus spores was similar to that of Bacillus spores. However, the Raman
spectra of protein amide bands suggested there are differences in protein structure in spores of G. stearothermophilus and
Bacillus species. 2) During nutrient germination of G. stearothermophilus spores, CaDPA was released beginning after a lag
time (Tlag) between addition of nutrient germinants and initiation of CaDPA release. CaDPA release was complete at Trelease,
and DTrelease (Trelease – Tlag) was 1–2 min. 3) Activation by heat or sodium nitrite was essential for efficient nutrient
germination of G. stearothermophilus spores, primarily by decreasing Tlag values. 4) Values of Tlag and Trelease were
heterogeneous among individual spores, but DTrelease values were relatively constant. 5) Temperature had major effects on
nutrient germination of G. stearothermophilus spores, as at temperatures below 65uC, average Tlag values increased
significantly. 6) G. stearothermophilus spore germination with exogenous CaDPA or dodecylamine was fastest at 65uC, with
longer Tlag values at lower temperatures. 7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly
and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the
dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of
Bacillus species.
Citation: Zhou T, Dong Z, Setlow P, Li Y-q (2013) Kinetics of Germination of Individual Spores of Geobacillus stearothermophilus as Measured by Raman
Spectroscopy and Differential Interference Contrast Microscopy. PLoS ONE 8(9): e74987. doi:10.1371/journal.pone.0074987
Editor: Adam Driks, Loyola University Medical Center, United States of America
Received June 21, 2013; Accepted August 7, 2013; Published September 13, 2013
Copyright: 
 2013 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Authors PS and YL acknowledge support by a Department of Defense Multi-disciplinary University Research Initiative through the U.S. Army Research
Laboratory and the U.S. Army Research Office under contract number W911F-09-1-0286. Authors TZ and ZD also acknowledge support by a grant from the
National Natural Science Foundation of China (No. 31228001) and a grant from the National High Technology Research and Development Program of China
(No. 2012AA092103). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: liy@ecu.edu
Introduction Concomitant with cortex hydrolysis, the core’s full rehydration
ultimately leads to resumption of enzyme activity, and initiation of
Many components of the spore germination machinery are metabolism and macromolecular synthesis in the core, and thus
conserved between spore forming members of the Bacillales [1]. spore outgrowth [2,5].
Bacillus subtilis spore germination can be initiated by a variety of B. subtilis spores contain three major GRs, termed GerA, GerB
chemicals, including nutrients, cationic surfactants, and enzymes, and GerK, each of which contains A, B and C subunits all of
as well as by hydrostatic pressure [2]. Nutrient germinants for which are required for GR function [2]. These GRs are encoded
spore germination generally include amino acids, purine deriva- by three tricistronic operons, each of which appears to encode a
tives, and sugars, and are species and strain specific. These single GR [1,2]. The GerD protein is also essential for proper GR
nutrient germinants interact with germination receptors (GRs) function, and the proteins encoded by the spoVA operon are
located in the inner spore membrane [2], stimulating the release of essential for DPA uptake in sporulation and probably CaDPA
the spore core’s large (,10% of spore dry wt) depot of pyridine- release during germination as well [1,6,7]. Geobacillus stearothermo-
2,6-dicarboxylic acid (dipicolinic acid [DPA]) and divalent cations,
+ philus is a Gram-positive spore-forming thermophile. Genomicpredominantly Ca2 , which are likely present as a 1:1 chelate analysis suggests that G. stearothermophilus has clear homologs of the
(CaDPA) [3]. CaDPA in the core is released and replaced by water B. subtilis GR genes as well as gerD and spoVAB, C, D genes, and
in stage I of spore germination, and CaDPA release then triggers genes encoding the cortex lytic enzymes CwlJ and SleB [1,8].
stage II of germination, a major event which is the hydrolysis of G. stearothermophilus spores are the most wet heat-resistant among
spores’ peptidoglycan cortex by cortex lytic enzymes (CLEs) [2,4]. spores of aerobic spore-forming bacteria, and can spoil a variety of
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Geobacillus stearothermophilus Spore Germination
types of foodstuffs [9–12]. These spores are also commonly used as Table 1. CaDPA level in individual G. stearothermophilus and
a biological indicator to evaluate the effectiveness of sterilization Bacillus spores*.
processes, in particular wet heat. However, the germination of
spores of G. stearothermophilus species is much less well studied than
that of spores of Bacillus species. Limited studies have shown that Spores CaDPA level (mM)
G. stearothermophilus spores germinate in response to low mol wt
G. stearothermophilus 382679
nutrient germinants including amino acids, purine and pyrimidine
nucleosides, and sugars. However, the kinetics of the germination B. cereus 3506105
of individual G. stearothermophilus spores and the heterogeneity B. subtilis 335642
among individual spores in a population has not been studied.
*CaDPA levels in 30 individual spores of various Bacillales species were
determined, and mean values and standard deviations were calculated as
described in Materials and Methods.
doi:10.1371/journal.pone.0074987.t001
In this study, we investigated the nutrient and non-nutrient
germination of multiple individual intact and decoated G.
stearothermophilus spores at various temperatures. We also measured
the CaDPA level and Raman spectra of individual G. stearothermo-
philus spores and compared these with those of spores of several
Bacillus species, as well as effects of different activation methods on
kinetics of germination of individual G. stearothermophilus spores.
This work has provided new information on the dynamics of and
the heterogeneity in the germination of G. stearothermophilus spores.
Materials and Methods
Bacterial Species Used and Spore Preparation
Spores of G. stearothermophilus NGB101 were prepared and
purified as described previously [13]. The Bacillus species used in
this work were Bacillus subtilis PS533 [14] and Bacillus cereus T
(originally obtained from H.O. Halvorson). Spores of these species
were prepared and stored as described [15,16]. All spores used in
this work were free (.98%) of growing or sporulating cells, as
determined by phase contrast microscopy.
Measurement of CaDPA Level and Raman Spectra of
Individual Spores by Laser Tweezers Raman
Spectroscopy
The CaDPA levels of individual spores of various species were
determined by laser tweezers Raman spectroscopy at 25uC [17].
Briefly, an individual spore was captured with laser tweezers, and
its Raman spectrum was acquired with an integration of 20 s and
a laser power of 20 mW at 780 nm. Spectra of 30 individual
spores were measured and averaged. The CaDPA level in an
individual spore was determined from the peak intensity at
1,017 cm21 in its Raman spectrum relative to the peak intensity of
the same Raman band from a CaDPA solution of known
concentration (50 mM) and by multiplying this concentration
value by the excitation volume of 1 fl to obtain attomoles of
CaDPA/spore [17]. Raman spectra of 30 individual spores of G.
stearothermophilus, B. subtilis and B. cereus at 25, 65, and 95uC were
also averaged for analysis of heat-induced changes in spores’
molecular components.
Activation of G. stearothermophilus Spores
Figure 1. Raman spectra of individual spores. Raman spectra of Unless noted otherwise, prior to germination experiments, G.
individual G. stearothermophilus (A), B. subtilis (B), and B. cereus stearothermophilus spores were activated by one of three methods: 1)
spores (C), measured at 25uC (curve a), 65uC (curve b) and 95uC
incubation in water at 100uC for 30 min followed by cooling in ice
(curve c), respectively. Curve d in Fig. 1(C) is the Raman spectrum of
single B. cereus spores that had lost their CaDPA at 95uC. All the spectra water for 15 min; 2) incubation in water at 30uC for 120 h; or 3)
were averages from 30 individual spores determined as described in incubation in 0.2 M sodium nitrite (pH 8.0) at 30uC for 17 h.
Methods. The dotted lines are the protein bands of amide I (1653/ Germination of unactivated G. stearothermophilus spores was also
1667 cm21) and amide III (1253 cm21), respectively. carried out in a few experiments.
doi:10.1371/journal.pone.0074987.g001
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Geobacillus stearothermophilus Spore Germination
Figure 2. Dynamics of nutrient germination of an optically trapped individual G. stearothermophilus spore. A heat activated (30 min,
100uC) spore was germinated at 65uC with 0.1 mM L-valine in 10 mM sodium phosphate buffer (pH 8.0), and the spore was monitored by Raman
spectroscopy and DIC microscopy as described in Methods. Time-lapse Raman spectra of the trapped spores after the addition of L-valine were
shown in (A). The indicated peaks at 661, 825, 1,017, 1,395 and 1,575 cm21 are the CaDPA bands. Normalized intensities of the CaDPA band at 101721
(#) and DIC images (%) as the function of incubation time were shown in (B). The CaDPA band intensities and DIC image intensities were normalized
to their initial values right after the addition of L-valine, and the DIC image intensity at 25 min was normalized to 0. The interval between Raman
spectrum acquisitions was 30 s, and the interval between DIC image acquisitions was 15 s. The inserts in Fig. 2(B) are the time-lapse DIC images of
the trapped spore with a scale bar of 2 mm. The DIC image of a single spore appears as two bright spots in DIC microscopy.
doi:10.1371/journal.pone.0074987.g002
Monitoring Germination of Single Spores by Raman Monitoring Germination of Multiple Individual Spores by
Spectroscopy and Differential Interference Contrast (DIC) DIC Microscopy
Microscopy The germination of a number of individual spores was
The germination of an individual G. stearothermophilus spore with simultaneously monitored with DIC microscopy [18]. Prior to
0.1 mM L-valine in 10 mM sodium phosphate buffer (pH 8.0) at germination, the spores were routinely activated at 100uC for
65uC was monitored simultaneously by Raman spectroscopy and 30 min unless noted otherwise. Briefly, 1 ml of heat-activated
8
DIC microscopy, as described [18,19]. Briefly, a single G. spores (10 spores/ml in water) was spread on the surface of a glass
stearothermophilus spore was optically captured immediately after coverslip glued to a clean and sterile sample container. The spores
the addition of 65uC 0.1 mM L-valine/10 mM sodium phosphate on the container were quickly dried in a vacuum chamber at room
buffer. Both the Raman spectra and DIC microscopy images of temperature so that they adhered to the coverslip. The spore
the trapped spore were recorded simultaneously for a period of container was then mounted on a microscope heat stage kept at
45 min with intervals of 30 s per spectrum and 15 s per image the appropriate temperature. Preheated germinant / buffer
frame, respectively. Note that the low concentration of L-valine solution was then added to the container, and a digital CCD
used in this experiment was to slow spore germination sufficiently camera (12 bits; 1600 by 1200 pixels) was used to record the DIC
to allow its measurement by Raman spectroscopy. images at a rate of 1 frame per 15 s for 60–120 min. These DIC
images were analyzed with a computation program in Matlab to
Table 2. Effect of activation methods on G. stearothermophilus spore germination*.
No. of spores examined
Activation method (% spore germination) Tlag (min) Trelease(min) DTrelease(min) Tlys DTlys(min)
No activation 458 (53.7) 12.666.2 14.166.2 1.460.8 19.566.2 5.462.8
100uC, 30 min 264 (98.1) 5.063.9 6.464.0 1.460.8 13.165.6 6.763.4
30uC, 0.2 M NaNO2, 17 h 248 (97.2) 4.463.2 5.563.3 1.160.6 11.565.6 6.062.9
30uC, 5 d 523 (81.1) 6.264.3 7.464.3 1.260.6 13.665.8 6.263.5
*Activated or unactivated spores were germinated at 65uC with 1 mM L-valine in 10 mM sodium phosphate buffer (pH 8.0) for 30 min, and kinetic parameters for all
germinations were determined by analysis of $248 spores that germinated as described in Methods.
doi:10.1371/journal.pone.0074987.t002
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Geobacillus stearothermophilus Spore Germination
Figure 3. Effects of different activation methods on germination of multiple individual G. stearothermophilus spores. Spores were
activated by various methods, and germinated at 65uC with 1.0 mM L-valine and 10 mM sodium phosphate buffer (pH 8.0), and germination of
individual spores was monitored by DIC microscopy as described in Methods. Germination of $248 individual spores (Table 2) that were activated in
0.2 M sodium nitrite (pH 8.0) at 30uC for 17 h (N), in water at 30uC for 120 h (m), in water at 100uC for 30 min (&), or without activation (*) was
shown in (a). Kinetics of germination of ten individual spores without activation (b); activated at 30uC for 120 h (c), and activated in 0.2 M sodium
nitrite (pH 8.0) for 17 h (d) was given in (b-d).
doi:10.1371/journal.pone.0074987.g003
locate each spore’s position and to calculate the summed pixel 30 min at 65uC [20]. This procedure removes much of the
intensity. The DIC image intensity of each spore was plotted as a spore’s coat protein as well as the spore’s outer membrane [20].
function of the incubation time (with a resolution of 15 s). The decoated spores were washed at least 10 times with 0.1 M
Unless noted otherwise, G. stearothermophilus spores were germi- NaCl by centrifugation to remove all traces of the decoating
nated at various temperatures in: (i) 1 mM L-valine in 10 mM solution and suspended in water. The decoated spores were then
sodium phosphate buffer (pH 8.0); (ii) 1 mM AGFK (a mixture of germinated with various agents with or without activation
1 mM each of L-asparagine, D-glucose, D-fructose, and potassium treatment as described above.
ions) in 10 mM sodium phosphate buffer (pH 8.0); (iii) 60 mM
CaDPA made to pH 7.4 with Tris base; and (iv) 1 mM Data Analysis
dodecylamine in 10 mM sodium phosphate buffer (pH 8.0). The DIC microscope that monitored individual spores was set
Except for dodecylamine and CaDPA germination, spores were such that the polarizer and analyzer were crossed, and thus the
routinely activated for 30 min at 100uC prior to germination DIC bias phase was zero. After adding pre-heated germinant/
experiments unless noted otherwise. buffer solution to spores on the coverslips, a digital CCD camera
was used to record the DIC images. These images were analyzed
Chemical Decoating of G. stearothermophilus Spores and with a Matlab program to locate each spore’s position and to
Germination of Decoated Spores calculate the averaged pixel intensity of an area of 20620 pixels
Spores of G. stearothermophilus at an optical density at 600 nm of that covered the whole individual spore on the DIC image. The
,10 were decoated by treatment with 1% sodium dodecylsulfate DIC image intensity of each individual spore was plotted as a
(SDS)–0.1 M NaOH–0.1 M NaCl–0.1 M dithiothreitol for function of the incubation time and the initial intensity (the first
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Geobacillus stearothermophilus Spore Germination
Table 3. Mean values and standard deviations of Tlag, Trelease, DTrelease, Tlys, and DTlys values for individual germinating G.
stearothermophilus spores*.
No. of spores
examined (% spore DTrelease
Strains and germination conditions germination) Tlag (min) Trelease (min) (min) Tlys DTlys (min)
1 mM L-valine 65uC, 30 min 264 (98.1) 5.063.9 6.464.0 1.460.8 13.165.6 6.763.4
55uC, 30 min 351 (63.8) 9.566.6 11.266.6 1.760.8 16.766.7 5.463.1
45uC, 30 min 275 (6.9) 10.666.7 12.866.1 2.261.0 16.866.9 4.061.8
Decoated, 65uC, 224 (89.7) 8.365.0 10.165.6 2.861.9 20.866.5 9.766.8
30 min
1 mM AGFK 65uC, 30 min 302 (94.7) 3.061.7 4.362.0 1.360.8 10.665.2 6.364.3
55uC, 30 min 407 (26.5) 6.864.5 8.564.6 1.760.6 13.865.1 5.362.0
45uC, 30 min 220 (16.4) 7.868.2 11.369.0 3.662.5 17.467.6 6.1610.7
Decoated, 65uC, 396 (76.0) 5.565.0 7.965.4 2.361.4 20.367.9 12.466.8
30 min
60 mM CaDPA (no 65uC, 120 min 380 (99.0) 4.463.9 5.363.8 2.561.2 7.863.7 0.960.5
activation)
25uC, 120 min 510 (68.8) 56.9626.5 60.4626.8 3.561.1 77.2626.4 23.469.1
Decoated, 65uC, 310 (66.1) 29.4625.8 31.2625.9 1.861.7 44.2629.8 13.0612.0
120 min
1 mM Dodecylamine 65uC, 120 min 557 (54.0) 21.7617.7 23.4617.9 1.761.1 36.3622.9 12.9613.1
(no activation)
55uC, 120 min 470 (11.5) 14.9619.8 16.7620.0 1.961.3 39.8623.0 23.0614.5
45uC, 120 min 515 (3.9) 67.8630.1 68.7630.2 0.960.8 78.5632.1 9.965.4
Decoated, 65uC, 432 (94.9) 5.163.1 8.363.4 3.261.5 – –
30 min
*Heat-activated G. stearothermophilus spores were germinated for 30 min with 1 mM L-valine or 1 mM AGFK at 65uC in 10 mM sodium phosphate buffer (pH 8.0),
unactivated G. stearothermophilus spores were germinated at 65uC for 120 min with 60 mM CaDPA or with 1 mM dodecylamine in 10 mM sodium phosphate buffer
(pH 8.0), and decoated G. stearothermophilus spores (heat-activated or unactivated) were germinated with different germinants at 65uC for 30 or 120 min. Kinetic
parameters for individual germinations were determined by analysis of $100 spores that germinated as described in Methods.
doi:10.1371/journal.pone.0074987.t003
DIC image recorded after the addition of the germinant) was Fig. 1 also shows the average Raman spectra of individual G.
normalized to 1 and the intensity at the end of measurements was stearothermophilus spores measured at 25, 65, and 95uC, in
normalized to zero. Invariably, the latter value had been constant comparison to spectra of B. subtilis and B. cereus spores. The peaks
for $10 min at the end of measurements. at 661, 824, 1,017, 1,395 and 1,575 cm21 are the bands due to
From the time-lapse DIC image intensity, we can determine the CaDPA, while the dotted lines denote the protein bands of amide I
21 21
time of completion of the rapid fall of ,75% in spore DIC image (1653/1667 cm ) and amide III (1253 cm ), respectively. The
21
intensity, which is concomitant with the time of completion of bands at 1,653 and 1,667 cm are assigned to the a-helical and
spore CaDPA release (Trelease). CaDPA release kinetics during nonregular structures of the amide I (peptide bond C = O stretch)
germinationof individual spores were described by the parameters of proteins, respectively [22–24]. Raman spectra of B. subtilis and
Tlag, Trelease and DTrelease [7,18]. We also defined the additional B. cereus spores (Fig. 1(B, C)) show that as the temperature was
21
germination parameters, Tlys and DTlys where Tlys is the time increased from 25 to 95uC, the intensity of the 1653 cm band
21
when spore cortex hydrolysis is completed as determined by the was slightly decreased and the intensity at 1667 cm slightly
completion of the fall in the spore’s DIC image intensity, and increased. Similarly, the peak of the 1253 cm
21 band (protein
DTlys = (Tlys-Trelease). amide III) was slightly shifted to the left at the higher
temperatures. This suggests that the structure of proteins in B.
Results subtilis and B. cereus spores had changed significantly from an a-
helical structure to a nonregular structure at high temperature,
Raman Spectra and Average CaDPA Level of Individual G. indicative of significant denaturation of proteins in these spores as
Stearothermophilus Spores found previously [24–26]. Indeed, when incubated at 95uC, some
CaDPA dominates the Raman spectra of individual spores of B. cereus spores lost their CaDPA, the 1653c m
21 band shifted to
1667 cm21, and the 1253 cm21 band shifted to 1235 cm21Bacillus species [21], and this was also the case for spores of G. (curve
stearothermophilus (Fig. 1). The intensity of the CaDPA-specific d in Fig. 1(C)), suggesting that significant protein denaturation
1,017 cm21 Raman band in the average spectrum from 30 took place after CaDPA release at 95uC. In contrast to results with
individual spores indicated that the CaDPA level in the core of G. B. subtilis and B. cereus spores, the Raman bands of protein amide I
stearothermophilus spores was , 21382 mM, and this value was only (1653/1667 cm ) were unchanged for G. stearothermophilus spores
slightly higher than the values for B. subtilis and B. cereus spores at 95uC (Fig. 1(A)), indicating that these spores’ proteins are stable
(Table 1). even at 95uC, consistent with these spores’ extremely high wet heat
resistance. The amide III band (1230–1300 cm21) region centered
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Geobacillus stearothermophilus Spore Germination
Figure 4. L-Valine germination of multiple individual G. stearothermophilus spores. Heat activated spores (30 min, 100uC) were germinated
at various temperatures with 1 mM L-valine in 10 mM sodium phosphate buffer (pH 8.0), and germination of individual spores was monitored by DIC
microscopy as described in Methods. Germination of $264 individual spores at 65uC(&), 55uC (N), or 45uC (m) was shown in (a). Kinetics of
germination of ten individual spores at 65uC (b), 55uC (c), or 45uC (d) was given in (b–d).
doi:10.1371/journal.pone.0074987.g004
at 1253 cm21 shifted to a lower wavenumber at 65uC and at 95uC spore usually continued to fall (but see below) until Tlys at ,
for B. cereus spores, but for G. stearothermophilus and B. subtilis spores, 9.6 min, corresponding to the completion of spore cortex
this change was less prominent. The Raman band at 783 cm21 hydrolysis, and then remained constant. As seen with the
seen at 25uC is attributed to ring breathing of cytosine/thymine/ germination of Bacillus spores [19], the termination point of the
uracil and the O–P–O symmetric stretch of the phosphodiester rapid fall in DIC image intensity precisely corresponded to the
bond in DNA and RNA [27,28]. At 95uC, the Raman band at completion of CaDPA release for G. stearothermophilus spores.
783 cm21 was nearly unchanged, suggesting that the double
helical structure of nucleic acids in G. stearothermophilus spores is Effect of Different Activation Methods on G.
stable at elevated temperature. stearothermophilus Spore Germination
Previous studies [29,30] have shown that germination of G.
Dynamics of Germination of Single G. stearothermophilus stearothermophilus spores becomes much more rapid if the spores are
Spores first given an activation treatment such as incubation in water for
Fig. 2 shows dynamics of an optically trapped individual G. short times at a high temperature, long times in water at a
stearothermophilus spore during L-valine germination at 65?C, as moderate temperature, or incubation in sodium nitrite at a
monitored by Raman spectroscopy and DIC microscopy. After the moderate temperature for intermediate times. The current work
addition of the germinant the CaDPA level as measured by the demonstrated that these different activation regimens led to
1017 cm21 band [17] and the DIC image intensity were nearly different kinetics of L-valine germination of individual G.
unchanged before Tlag at , 2.2 min. The intensity of the stearothermophilus spores at 65uC (Table 2; Fig. 3). All three
1017 cm21 band then quickly dropped to zero and the spore’s activation regimens increased the overall rates of spore germina-
DIC image intensity decreased ,70% by Trelease at , 3.2 min. In tion, almost completely by decreasing average Tlag values with
this experiment, the DIC image intensity of the G. stearothermophilus minimal if any effects on values for DTrelease and DTlys. Note also
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Geobacillus stearothermophilus Spore Germination
Figure 5. Germination of multiple individual G. stearothermophilus spores with AGFK at different temperatures. Heat activated (30 min,
100uC) spores were germinated at various temperatures with AGFK and 10 mM sodium phosphate buffer (pH 8.0), and germination of individual
spores was monitored by DIC microscopy as described in Methods. Germination of $220 individual spores at 65uC (m), 55uC (N), or 45uC (&) was
shown in (a). Kinetics of germination of ten individual spores at 65uC (b), 55uC (c), or 45uC (d) was given in (b–d).
doi:10.1371/journal.pone.0074987.g005
that for a number of the individual spores activated by various temperatures was due primarily to longer Tlag values as: i) many
regimens, following the initial rapid fall in DIC image intensity of spores did not even germinate in the observation times at the lower
, 60%, there was an lag of 5–20 min following Trelease and before temperatures, and thus have very long Tlag values; ii) DTrelease
the further fall in DIC image intensity. This was also seen in many values increased only slightly at lower temperatures; and iii) DTlys
other germination experiments (see below), although the reason for values were essentially unchanged at low and high temperatures.
this lag period is not clear.
Kinetics of Non-nutrient Germination of Individual G.
Kinetics of Germination of Multiple Individual G. stearothermophilus Spores
stearothermophilus Spores with L-valine or AGFK In addition to nutrients, spores can germinate with a variety of
Previous work [29] has shown that G. stearothermophilus spores are non-nutrients [2,3], including lysozyme, CaDPA, cationic surfac-
able to germinate in the presence of L-valine or AGFK. tants, high pressures and some salts. Unlike the case with nutrient
Consequently we used DIC microscopy to analyze the germina- germination, exogenous CaDPA induced germination of G.
tion of multiple individual G. stearothermophilus spores with L-valine stearothermophilus spores at 25uC (Fig. 6; Table 3). However,
or AGFK at multiple temperatures (Fig. 4 and 5; Table 3; and CaDPA germination of G. stearothermophilus spores was faster at
data not shown). G. stearothermophilus spores germinated faster with 65uC due largely to a much shorter average Tlag value than at
AGFK than L-valine at 65uC, and as expected, germination with 25uC, although the DTrelease values were almost identical at these
these nutrients was faster at 65uC than at 55uC or 45uC, while no two temperatures. The average DTlys value for CaDPA germina-
germination was observed when G. stearothermophilus spores were tion at 25uC was also much longer than for CaDPA germination at
incubated with 1 mM L-valine at 37uC or 25uC (Table 3; and data 65uC.
not shown). The slower germination of these spores at lower
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Geobacillus stearothermophilus Spore Germination
Figure 6. Germination of multiple individual G. stearothermo-
philus spores with CaDPA. Unactivated spores were germinated with
CaDPA at various temperatures, and germination of individual spores
was followed by DIC mNicroscopy as described in Methods. Germinationat 65uC (&) or 25uC ( ), with germination of $380 individual spores
examined was shown in (a). Kinetics of germination of ten individual
spores at 65uC (b) or25uC (c) was given in (b, c). Figure 7. Germination of multiple individual G. stearothermo-
doi:10.1371/journal.pone.0074987.g006 philus spores with dodecylamine. Unactivated spores were
germinated at various temperatures with 1 mM dodecylamine in
Another group of non-nutrient germinants is cationic surfac- 10 mM sodium phosphate buffer (pH 8.0), and germination of $470
individual spores was monitored by DIC microscopy. Germination at
tants, with dodecylamine being the one that has been best studied 65uC (&), 55uC ( ) and 45uC (m) was shown in (a). Kinetics of
[31].With 1 mM dodecylamine at 65uC, only , 50% of G. Ngermination of ten individual spores at 65uC (b) or 55uC (c) was given in
stearothermophilus spores germinated in 120 min, a slow germination (b, c).
compared to those with other germinants, and dodecylamine doi:10.1371/journal.pone.0074987.g007
germination was minimal at 45uC (Fig. 7; Table 3). As seen with
CaDPA germination at low and high temperatures, most of the decoated spores were markedly lower than with intact spores, as
decrease in the rate of germination with dodecylamine at the lower the average Tlag value increased .6-fold while the average Tlys
temperature was due to much longer average Tlag values. value increased,13-fold, although the average DTrelease value was
essentially unchanged from that for intact spores. Decoating also
Kinetics of Germination of Individual Decoated G. greatly increased the rate of dodecylamine germination of G.
stearothermophilus Spores stearothermophilus spores markedly, largely by decreasing the average
Tlag value (Table 3).Since at least some proteins involved in spore germination in
Bacillus species are located in the spore coats, in particular the CLE
CwlJ [2], we also examined the effect of chemical decoating on G. Discussion
stearothermophilus spores’ germination with nutrient and non- The work in this communication has revealed a number of
nutrient germinants, all at 65uC (Fig. 8; Table 3). With L-valine similarities in the properties of spores of G. stearothemophilus and
and AGFK, the rate of germination of decoated G. stearothermophilus Bacillus species, in particular the nearly identical DPA concentra-
spores decreased by , 15%, while Tlag values increased ,1.5 fold. tions in these spores’ core. However, there were some differences.
However, the amount and rate of CaDPA germination of the One was the lack of change in the Raman spectrum of proteins in
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Geobacillus stearothermophilus Spore Germination
Figure 8. Germination of multiple individual decoated G. stearothermophilus spores. Heat activated (30 min, 100uC (a,b), or unactivated
spores (c,d) were germinated at 65uC with 1 mM L-valine (a); 1 mM AGFK (b); 60 mM CaDPA (c); and 1 mM dodecylamine (d), in 10 mM sodium
phosphate buffer (pH 8.0), and germination of individual spores was monitored by DIC microscopy as described in Methods. The insets in the various
panels show the percentages of spore germination when $224 individual spores (Table 2) were monitored.
doi:10.1371/journal.pone.0074987.g008
G. stearothermophilus spores upon incubation at 95uC. This behavior, Bacillus species. Thus CaDPA release for all G. stearothermophilus
as well as the high temperature needed for heat activation of G. spore germinations examined began only after a highly variable
stearothermophilus spores, is undoubtedly a reflection of G. stear- Tlag period but DTrelease took only a few min, with Trelease followed
othermophilus being a thermophile, and is consistent with both the by cortex hydrolysis that was completed at Tlys. Almost always,
high temperature optimum for germination of spores of this DTlys was longer than DTrelease, and most of the heterogeneity in
species and their extremely high wet heat resistance compared to the germination between individual spores was in DTlag values, as
spores of B. cereus and B. subtilis [10]. The second difference, and a seen previously with spores of Bacillus species [18,32,33]. The
more intriguing one was the highly variable lag period between effects of activation treatments on the germination G. stearothermo-
Trelease and the initiation of the second fall in G. stearothermophilus philus were also largely, if not completely on Tlag values, as average
spores’ DIC image intensity during spore germination with all DTrelease and DTlys values in nutrient germination of unactivated
germinants tested, as this has been seen only rarely in germination and maximally activated spores were essentially identical. Optimal
of spores of Bacillus species [19,32]. While we have no good heat activation also decreases average Tlag values for nutrient
explanation for this difference, it is as if there is a much higher germination of spores of Bacillus species [34]. Since a major factor
threshold for the signal event that begins G. stearothermophilus spore determining the Tlag period for nutrient germination of spores of
cortex degradation by CLEs following CaDPA release than with Bacillus species is spores’ levels of functional GRs [33], this further
spores of Bacillus species. However, these signaling mechanisms are suggests that heat activation of G. stearothermophilus spores for
not well understood, so we have no good mechanistic explanation 30 min at 100uC makes these spores’ GRs optimally functional,
for this difference between spores of these two genera. perhaps by some conformational protein changes as has been
While there were the notable differences between G. stearother- suggested for spores of Bacillus species [35]. The mechanism of
mophilus and Bacillus spore properties noted above, the overall nitrite activation of spores has never been analyzed in detail, but
features of the nutrient and non-nutrient germination kinetics of could be due to covalent modification of the spore cortex by
individual spores of this species were very much like those of
PLOS ONE | www.plosone.org 9 September 2013 | Volume 8 | Issue 9 | e74987
Geobacillus stearothermophilus Spore Germination
nitrous acid [36]. However, this could equally well be due to average Tlag value for CaDPA germination increased , 7-fold in
nitrous acid modification of GRs. decoated G. stearothermophilus spores. Thus it seems most likely that
It was also notable that germination at suboptimal temperatures CwlJ is also the primary target of CaDPA in triggering
greatly increased Tlag values for nutrient germination of G. germination of G. stearothermophilus spores.
stearothermophilus spores, especially given that lower percentages of Along with nutrient germination, G. stearothermophilus spore
these spores germinated at lower temperatures in the observation germination with the non-nutrients CaDPA and dodecylamine
periods used. In contrast, there was essentially no effect on DTlys also decreased markedly at suboptimal temperatures, again largely
values as the germination temperature was lowered, indicating due to effects on Tlag. However, the latter effect is almost certainly
that the temperature sensitive step in nutrient germination of G. not on GRs, which are not involved in CaDPA and dodecylamine
stearothermophilus spores is in Tlag, and probably is on the GRs germination of spores of Bacillus species [31,37]. Indeed, as noted
themselves, although there was also a small increase in DTrelease above, CaDPA probably triggers G. stearothermophilus spore
times as germination temperature was lowered. The effect of germination by activating the CLE CwlJ, while in Bacillus spores
temperature on kinetics of the germination of individual spores has dodecylamine likely triggers germination by triggering the opening
not been studied with spores of Bacillus species. of the CaDPA channel in the spores’ inner membrane that is
Decoating of G. stearothermophilus spores had only a minimal composed at least in part of SpoVA proteins [41]. Interestingly,
effect on their nutrient germination, with the biggest effect being decoating of G. stearothermophilus spores significantly increased these
1.5 to 2-fold increases in DTlys values. The G. stearothermophilus spores’ germination with dodecylamine primarily by decreasing
genome has the genes for the two redundant CLEs, CwlJ and Tlag values, just as with spores of Bacillus species [31]. Why this
SleB, involved in cortex hydrolysis during spore germination in should be is not completely clear, but decoating may allow easier
Bacillus species. With Bacillus spores, decoating largely removes or
access of dodecylamine to the SpoVA CaDPA channel than in an
inactivates CwlJ [37], and presumably a decrease in CwlJ level is
intact spore.
the reason for the increased DTlys values in decoated G. In summary, the analysis of the dynamics of the germination of
stearothermophilus spores. However, we do not know if all G.
multiple individual G. stearothermophilus spores with a variety of
stearothermophilus CwlJ is inactivated by the decoating regimen we
germinants indicates that the general features of the germination
used. Indeed, decoating or loss of CwlJ by mutation increases
of these spores appear to be quite similar to those of spores of
values of DTrelease in nutrient germination of spores of several Bacillus species.
Bacillus species 6- to 10-fold [38,39], while the increase in decoated
G. stearothemophilus spores was at most 2-fold. Thus with G.
stearothermophilus spores either CwlJ is not essential for rapid Author Contributions
CaDPA release in spore germination, or some active CwlJ survives Conceived and designed the experiments: PS YL. Performed the
the decoating regimen used. We favor the latter possibility, since experiments: TZ YL. Analyzed the data: TZ PS YL. Contributed
CwlJ is essential for CaDPA germination of spores of Bacillus reagents/materials/analysis tools: PS YL. Wrote the paper: TZ ZD PS
species [37,40], while significant CaDPA germination still took YL. Designed the experiments, analysed the results, and revised the
place with decoated G. stearothermophilus spores. However, the manuscript: ZD.
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