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Applied and Environmental Microbiology, January 2000, p. 257-261, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Comparative Study of Pressure- and Nutrient-Induced
Germination of Bacillus subtilis Spores
Elke Y.
Wuytack,
Johan
Soons,
Filip
Poschet, and
Chris W.
Michiels*
Laboratory of Food Microbiology, Katholieke
Universiteit Leuven, Leuven, Belgium
Received 6 July 1999/Accepted 22 October 1999
 |
ABSTRACT |
Germination experiments with specific germination mutants of
Bacillus subtilis, including a newly isolated mutant
affected in pressure-induced germination, suggest that a pressure of
100 MPa triggers the germination cascades that are induced by the nutrient germinant alanine (Ala) and by a mixture of asparagine, glucose, fructose, and potassium ions (AGFK), by activating the receptors for alanine and asparagine, GerA and GerB, respectively. As
opposed to germination at 100 MPa, germination at 600 MPa apparently shortcuts at least part of the Ala- and AGFK-induced germination pathways. Inhibitors of nutrient-induced germination (HgCl2
and N
-P-tosyl-L-arginine methyl ester) also
inhibit pressure-induced germination at 600 MPa, suggesting that
germination at 600 MPa involves activation of a true physiological
germination pathway and is therefore not merely a physico-chemical
process in which water is forced into the spore protoplast.
| |
INTRODUCTION |
The capacity to form endospores
endows Bacillus subtilis with the ability to survive in
adverse environmental conditions. However, the success of this survival
strategy depends on the presence of an efficient mechanism for
returning to the vegetative state under favorable conditions
(14). A wide range of chemical and physical effectors can
trigger germination of spores (5). In B. subtilis, the two best-known germination pathways are those induced by the chemical effectors alanine (Ala) and the combination of
asparagine (Asn), glucose (Glu), fructose (Fru), and potassium ions
(AGFK) (13, 14). While the first steps of the Ala- and AGFK-induced germination cascades are different, both cascades converge
in a later stage. This has been demonstrated by the isolation of
specific mutants defective in the Ala response (gerA), in
the AGFK response (gerB and gerK), and in both
responses (gerD). It was subsequently hypothesized that the
gerA, gerB, and gerK gene products are
receptors of the germinants Ala, Asn, and Glu, respectively (4,
12, 14, 18).
The gerA-encoded Ala receptor of B. subtilis is
sufficient to trigger spore germination, while the
gerB-encoded Asn receptor cannot trigger spore germination
in absence of the Glu receptor (gerK) and Fru receptor
(encoding gene unknown) (4, 12, 14, 18). Finally, Ala may
also interact with the Asn receptor and induce germination, albeit with
lower efficiency (12, 18).
The function of the gerD gene product is unclear but it must
have an essential role in AGFK-induced germination since mutation of
the gerD gene blocks the AGFK-induced germination. Although not essential, the gerD gene product is also involved in
Ala-induced germination, since gerD mutants germinate very
slowly in the presence of Ala (9, 13, 14, 19).
Little is known about the biochemical reactions involved in triggering
spore germination, but activity of the major catabolic and anabolic
pathways, and metabolic conversion of the germinant in particular,
seems not to be required (10). Binding of the germinant to
its receptor is believed to generate some sort of allosteric
conformational change in this receptor (14, 20). The
receptor itself or another protein could in turn act as an ion
transporter or as an ion channel (14) to promote the efflux of Ca2+ and other ions and the influx of water in the
spore, ultimately resulting in the activation of preformed,
spore-specific degradative enzymes involved in cortex degradation
(11, 14). Alternatively, the conformational change of the
germinant receptor could activate these hydrolytic enzymes first, e.g.
by proteolytic cleavage. The redistribution of ions and water in the
spore protoplast would then be the consequence of cortex degradation.
As opposed to the mechanisms of nutrient-induced germination, the
mechanisms of pressure-induced germination remain largely unknown.
Based on the observation that some inhibitors of nutrient-induced germination also inhibited pressure-induced germination of
Bacillus cereus and Bacillus megaterium, Gould
and Sale (6) hypothesized that pressure-induced germination
proceeds by activation of enzymes in the nutrient-induced germination
cascade. Furthermore, it was observed that spores from which cations,
including those in the core, have been exchanged with protons by
exposure of the spores to a very acidic environment (so-called
H-spores) are defective in pressure- and nutrient-induced germination
(1, 15). In an earlier study, we demonstrated that spores
germinated at a pressure of 100 MPa undergo a number of events similar
to those during nutrient-induced germination and lose their resistance to various stresses to a similar degree as the latter. In contrast, spores germinated at 600 MPa retain their resistance to some stresses and do not initiate two key enzymatic reactions of nutrient-induced germination: the degradation of small, acid-soluble proteins and the
rapid generation of ATP (21).
The objective of the present work was to investigate in greater detail
the pathways of pressure-induced germination at 100 and 600 MPa and
their relationship to the pathways of nutrient-induced germination in
B. subtilis.
| |
MATERIALS AND METHODS |
Production of B. subtilis spore suspensions.
Strains of B. subtilis used in this study are listed in
Table 1. To induce sporulation, cells
from a 
80°C glycerol stock culture were grown at 37°C in a humid
atmosphere on the surface of nutrient agar CM3 (Oxoid, Basingstoke,
United Kingdom) supplemented with 0.06 g of MgSO4 per
liter and 0.25 g of KH2PO4 per liter. After 7 days, spores were harvested from the plates in sterile deionized water, washed twice, and were finally resuspended in sterile
deionized water to a concentration of 107 to
108 spores ml
1, and the spores were kept at
4°C for up to 1 month.
Germination treatment.
For pressure-induced germination,
aqueous spore suspensions were diluted twofold with 100 mM potassium
phosphate buffer (pH 7) and were pressurized (100 or 600 MPa at 40°C)
in heat-sealed sterile polyethylene bags in an 8-ml pressure vessel
thermostatted with an external water jacket (Resato, Roden, The
Netherlands). The compression rate was approximately 100 MPa/min, while
decompression was immediate.
For nutrient-induced germination, aqueous spore suspensions were
diluted twofold with germinant solution and were incubated
for 5 h
at 37°C. The germinant solutions used were 50 mM potassium
phosphate
buffer (pH 7) containing 10 mM
L-Ala (Ala-induced
germination)
or 50 mM potassium phosphate buffer (pH 7) containing 10 mM
L-Asn,
10 mM
D-Glu, and 10 mM
D-Fru (AGFK-induced
germination).
To determine the degree of germination, treated and control spore
suspensions were subjected to a heat treatment (80°C for
15 min) and
were subsequently serially diluted, plated on the
surface of tryptic
soy agar (Oxoid), and were incubated for 24
h at 37°C. The heat
treatment of 15 min at 80°C was found not
to kill ungerminated native
spores. Because the heat sensitivity
of H-spores is higher than that of
native spores (
2), the heat
treatment to determine the
degree of germination of H-spores was
10 min at 60°C instead of 15 min at 80°C. The percentage of ungerminated
spores was expressed as
the ratio of the colony counts of the
treated sample to the colony
counts of the control sample multiplied
by
100.
Selection of mutants affected in pressure-induced
germination.
A selection procedure consisting of consecutive
enrichment cycles was developed to isolate B. subtilis
mutants defective in pressure-induced germination. Each enrichment
cycle consisted of three steps. In the first step, spores were
subjected to a pressure treatment (100 or 600 MPa at 40°C) to induce
germination. In the second step, the pressure-germinated spores were
killed by a heat treatment (80°C for 15 min). Only ungerminated
spores survived this heat treatment. In the third step, the surviving spores were plated on nutrient agar and were allowed to germinate, grow, and sporulate again. These spores were then harvested and were
subjected to a new enrichment cycle. In principle, this procedure should result in mutants with reduced pressure germination but which
are still capable of germination on nutrient agar. A similar approach
has allowed the isolation of highly pressure-resistant mutants of
Escherichia coli MG1655 (7).
Preparation of H-spores.
Aqueous spore suspensions of
B. subtilis PY79 were diluted fourfold with 1 M HCl. After
3 h of incubation at room temperature, the spore suspensions were
centrifuged (2,650 × g for 15 min) and were
resuspended in 50 mM phosphate buffer (pH 7). The successful formation
of H-spores was confirmed by demonstrating their sensitivity at 80°C
and resistance at 60°C (2). PY79 as a wild-type strain was
used for the preparation of H-spore suspensions, because we have not
succeeded in reproducibly isolating H-spores from strains LMG7135 and 1604.
Chemical inhibition of pressure-induced germination.
Aqueous
spore suspensions, diluted fivefold with 50 mM phosphate buffer (pH 7)
containing an inhibitor of nutrient-induced germination, were pressure
treated (100 or 600 MPa at 40°C for 20 min). Inhibitors used were
N-P-tosyl-L-arginine methyl ester (50 mM)
(TAME) (3, 18), HgCl2 (3 mM) (8, 18),
and phenol (0.2% [wt/vol]) (16).
To estimate the degree of germination inhibition exerted by an
inhibitor, germination in the presence and absence of inhibitor
was not
measured by differential plating of heat-treated germinated
and control
spore suspensions as described above, but by measuring
the decrease in
optical density at 600 nm (OD
600) that accompanies
spore
germination. This was necessary because the toxicity of
HgCl
2 precludes the formation of colonies. The
OD
600 of the spore
suspension with and without an inhibitor
before and after germination
were measured, and the percentage of
inhibition exerted by the
inhibitor was then calculated as follows:
Reproducibility of results.
The data presented are either
means of three replicate experiments or from a single representative experiment.
| |
RESULTS |
Selection of B. subtilis mutants affected in
pressure-induced germination.
We attempted to select B. subtilis mutants showing reduced germination under pressure by
using a specific enrichment procedure. After each enrichment cycle at
100 MPa, the resulting spores were found to be less susceptible to
pressure-induced germination at 100 MPa. Therefore, the duration of the
pressurization in the first step of the enrichment cycle was gradually
increased in the consecutive enrichment steps in order to maintain a
sufficient level of germination. Four selection rounds at 100 MPa
resulted in mutant LMM2021, which germinated much slower at 100 MPa
than the wild-type LMG7135 from which it was derived (Fig.
1A). The germination deficiency of this
strain at 100 MPa was stable upon repeated subcultivation. Similar
attempts were undertaken to produce a B. subtilis mutant
that is unable to germinate at 600 MPa by using a pressure treatment of
600 MPa during the selection procedure. However, even after 10 enrichment cycles, these attempts remained unsuccessful.
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FIG. 1.
Germination of spores of B. subtilis LMG7135
(wild type) ( ) and LM2021 (pressure-selected mutant) ( ) induced
by 100 MPa (A), 600 MPa (B), Ala (C), or AGFK (D).
|
|
Effect of different germination stimuli on specific germination
mutants.
To investigate the relationship between the pathways of
pressure-induced and nutrient-induced spore germination, the
germination responses of the pressure-selected mutant LMM2021 (Fig. 1)
and the nutrient-induced germination mutants gerA,
gerB, gerD, and gerA gerB (Fig.
2) to both pressure and nutrient stimuli
were compared.
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|
FIG. 2.
Germination of spores of B. subtilis.
Symbols: wild-type strain (), gerA strain ( ),
gerB strain ( ), gerD strain ( ), and
gerA gerB strain () induced by 100 MPa (A) or 600 MPa
(B).
|
|
Figure
1 shows that while spores of the selected mutant LMM2101 could
not be induced to germinate at 100 MPa, they germinated
at wild-type
levels at 600 MPa. Interestingly, LMM2101 spores
also did not germinate
in the presence of Ala nor in the presence
of
AGFK.
All four nutrient-induced germination mutants germinated normally at
600 MPa or, in the case of the
gerA gerB mutant, even
better
than the wild type (Fig.
2B). At 100 MPa, the
gerA and
gerB single mutants germinated normally, but the mutants
that
are affected in both Ala- and AGFK-induced germination (
gerA
gerB double mutant and
gerD mutant) were also affected
in pressure-induced
germination at 100 MPa. Germination induced at 100 MPa was consistently
more inhibited in the
gerA gerB mutant
than in the
gerD mutant
(Fig.
2A).
Germination response of H-spores.
The germination response of
native and H-spores has been compared (Table
2). From the results, it was confirmed
that the exchange of spore minerals by protons inhibits Ala-induced and AGFK-induced germination. Pressure-induced germination of H-spores was
also completely inhibited at 100 MPa, but only slightly at 600 MPa.
Effect of inhibitors of nutrient-induced spore germination.
The effects of different inhibitors (TAME, HgCl2, and
phenol) on nutrient- and pressure-induced germination at 100 and 600 MPa were compared (Table 3). Two
inhibitors (HgCl2 and phenol) completely blocked both Ala-
and AGFK-induced germination, and these were found to also completely
block 100-MPa-induced germination. TAME inhibited only one of the two
nutrient-dependent germination pathways completely, and it was found to
exert only partial inhibition of 100-MPa-induced germination.
Inhibition of germination at 600 MPa was either nearly complete
(HgCl2), partial (TAME), or marginal (phenol).
| |
DISCUSSION |
Although it is well known that pressure may induce germination of
bacterial spores, the factors affecting pressure-induced germination
and its cellular and biochemical basis are poorly understood. In this
study, it was investigated if the B. subtilis germination
pathways triggered by nutrients (Ala and AGFK) and pressure share
common steps. Two different pressure levels (100 and 600 MPa) were used
throughout this study, because we have previously demonstrated some
remarkable differences between germination at 100 and 600 MPa
(21).
In a first step, we attempted to select mutants affected in
pressure-induced germination. Mutants unable to germinate at 100 MPa
could be readily obtained after sequential enrichment of the spores
remaining ungerminated after treatment at 100 MPa. In contrast, up to
10 consecutive rounds of enrichment by the same procedure but at 600 MPa did not result in mutants affected in germination at 600 MPa.
Interestingly, the mutant unable to germinate at 100 MPa germinated
normally at 600 MPa, but was completely blocked in Ala- and
AGFK-induced germination (Fig. 1). These observations suggest that the
pathway of germination at 100 MPa shares one or more steps with Ala-
and AGFK-induced germination. Previously, based on the observation that
degradation of small, acid-soluble proteins and rapid ATP formation do
not occur in spores germinated at 600 MPa, we proposed that germination
at 600 MPa, as opposed to germination at 100 MPa (21), would
be arrested at an early stage. These results additionally suggest that
germination at 600 MPa involves a pathway or mechanism that is at least
partly different from that followed at 100 MPa.
To further investigate the relationship between the pathways of
pressure-induced and nutrient-induced germination, we analyzed the
response of existing mutants affected in nutrient-induced germination
to pressure treatments at 100 and 600 MPa. The observation that the
nutrient-induced germination mutants gerA gerB and
gerD are affected in pressure-induced germination at 100 MPa
but not at 600 MPa (Fig. 2) supports the idea of a different pathway
for germination at 100 and 600 MPa. Further, since it is widely
accepted that gerA and gerB encode the receptors
for Ala and Asn, respectively, it can be concluded from these results
that treatment at 100 MPa can activate the germination cascade at the
level of these receptors. The fact that the gerA and
gerB mutants, in contrast to the gerA gerB
mutant, germinate normally at 100 MPa indicates that both the Ala
(gerA) and Asn (gerB) receptors are susceptible
to activation at 100 MPa. In this way, a mutant that is deficient in
only one of the nutrient-induced germination pathways will still
germinate at 100 MPa. Possibly, low pressure activates the nutrient
receptors by inducing conformational changes in their active sites. The fact that this activation occurs both in the Ala receptor and the Asn
receptor can probably be explained by the high degree of homology
between the sequences of the gerA and gerB
operons, which are believed to have evolved by gene duplication and
subsequent divergence from a common ancestral operon (4,
14). Germination at 600 MPa apparently shortcuts the Ala and AGFK
germination pathways at least partly, since neither gerA or
gerB, nor gerD, which is situated further
downstream in the germination cascade, are required for germination at
600 MPa. B. subtilis spores contain a high concentration of
cations (2, 17). It has been previously shown that the
exchange of spore cations with protons not only reduces the heat
resistance (2) but also the germination response of B. megaterium spores to pressure and nutrient germinants (1, 15). In the present study, we demonstrated that H-spores of B. subtilis are unable to germinate in Ala, in AGFK, or at
100 MPa (Table 2). Germination occurred at 600 MPa, although to a slightly lower extent than for the native spores. Therefore, the presence of spore cations appears to be important in the Ala-, AGFK-,
and 100-MPa-induced germination cascades, while they have a minor role
in the 600-MPa-induced germination cascade. Again, these observations
support the existence of a different pathway for germination at 600 MPa.
A final experiment to characterize the germination pathways followed
under different conditions examined the effect of specific inhibitors
of germination. Gould and Sale (6) already observed that
inhibitors of nutrient-induced germination also inhibited pressure-induced germination of B. cereus and Bacillus
coagulans spores, but their experiments were done only at low
pressure (at 40 and 60 MPa, respectively). In our work, we compared the
effect of some known inhibitors of nutrient-induced germination (TAME, HgCl2, and phenol) on germination by Ala, AGFK, and 100 and
600 MPa of pressure (Table 3). The results from this experiment confirm and complement the conclusions from the experiments with the
germination mutants. Inhibitors that block both the Ala and AGFK
pathways (HgCl2 and phenol) also block the 100-MPa pathway.
On the other hand, if the Ala and AGFK pathways are not completely
blocked (TAME), then the 100-MPa pathway is not either. Of particular interest is the finding that two of the three inhibitors also inhibit
germination at 600 MPa. This is the case for TAME (41% inhibition) and
for HgCl2 (90% inhibition). Inhibition by these compounds
is consistent with a role for specific enzymes in germination at 600 MPa. Germination at 600 MPa is, therefore, not merely a physico-chemical process in which water is forced into the spore protoplast, but involves activation of a true physiological germination pathway. At this time, our experiments do not allow us to conclude whether this 600-MPa pathway is really distinct from the 100-MPa pathway, or whether it corresponds to or overlaps with some lower part
of the 100-MPa germination pathway downstream of gerD.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Belgian Fund for
Scientific Research (N.F.W.O. G.0395.98) and K.U. Leuven
Onderzoeksfonds (OT/97/31). E.W. is a grant holder from the Flemish
Institute for the promotion of Scientific-Technological Research
(I.W.T.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Food Microbiology, Kard. Mercierlaan 92, B-3001 Heverlee, Belgium.
Phone: 32-16-32.15.78. Fax: 32-16-32.19.60. E-mail:
chris.michiels{at}agr.kuleuven.ac.be.
| |
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Applied and Environmental Microbiology, January 2000, p. 257-261, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
