Amino acid- and inosine-induced germination of Bacillus cereus ATCC 14579 spores was reversibly inhibited in the presence of 3 mM undissociated sorbic acid. Exposure to high hydrostatic pressure, Ca-dipicolinic acid (DPA), and bryostatin, an activator of PrkC kinase, negated this inhibition, pointing to specific blockage of signal transduction in germinant receptor-mediated germination

Abee, T.

Melis, C.C.J., van

Nierop Groot, M.N.

English

Wageningen Yield 

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2011, p. 2552–2554 Vol. 77, No. 7
0099-2240/11/$12.00 doi:10.1128/AEM.02520-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Impact of Sorbic Acid on Germinant Receptor-Dependent and
-Independent Germination Pathways in Bacillus cereus
C. C. J. van Melis,1,2 M. N. Nierop Groot,1,3* and T. Abee1,2
Top Institute Food and Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, Netherlands1; Food Microbiology Laboratory,
Wageningen University, Bomenweg 2, 6703 HD Wageningen, Netherlands2; and Food and Biobased Research,
Wageningen University & Research Centre, Bornse Weilanden 9, 6708 WG Wageningen, Netherlands3
Received 25 October 2010/Accepted 22 January 2011
Amino acid- and inosine-induced germination of Bacillus cereus ATCC 14579 spores was reversibly inhibited
in the presence of 3 mM undissociated sorbic acid. Exposure to high hydrostatic pressure, Ca-dipicolinic acid
(DPA), and bryostatin, an activator of PrkC kinase, negated this inhibition, pointing to specific blockage of
signal transduction in germinant receptor-mediated germination.
A wide range of food preservatives, including weak acids
such as lactates and sorbic acid (SA), are effective in mainte-
nance of food quality and safety (5, 14) and are therefore
widely used in industry. We previously assessed the impact of
SA on germination and outgrowth of Bacillus cereus spores
(16) and reported that at concentrations of 3 mM undissoci-
ated sorbic acid (HSA) and higher, amino acid- and inosine-
induced spore germination was blocked, with spores remaining
fully refractile. An earlier study on Clostridium botulinum and
B. cereus spores suggested that the mode of action of sorbic
acid was by competitive inhibition on the L-alanine/inosine
receptor (13). In later work performed by Blocher and Busta
(3), competitive inhibition was disputed, and evidence was
provided that led the authors to propose that inhibition oc-
curred after germinant binding and could involve alteration of
the spore’s inner membrane permeability or inhibition of
cortex lytic enzymes. Based on current knowledge, the B.
cereus ATCC 14579 genome encodes seven putative germi-
nant receptors, and spore germination in this organism can
be triggered not only by alanine, via the germinant receptor
GerR, but also by a range of other germinants, including
inosine, adenosine, glutamine, cysteine, threonine, and phe-
nylalanine (7).
This triggered us to assess the impact of sorbic acid on the
different Ger receptors by using inosine and selected amino
acids as germinants with both B. cereus ATCC 14579 wild-type
spores and gerR deletion mutant spores, which are insensitive
to alanine-induced germination (6). Spore preparations of the
B. cereus ATCC 14579 wild type and its gerR deletion mutant
were prepared for germination assays as described previously
(6, 16). In short, heat-activated spores (70°C for 15 min) of
both the wild type and gerR mutant were resuspended in mor-
pholineethanesulfonic acid (MES) buffer corresponding to the
test conditions prior to addition to a microtiter plate. Both
wild-type and gerR mutant spores were exposed to a 1 mM
concentration of one of the germinants L-alanine, L-glutamine,
or inosine, thereby triggering GerR, GerG and GerQ, and
GerI, respectively, as demonstrated by Hornstra et al. (7). The
spores were incubated at pH 5.5 with or without 3 mM HSA
and germinant (Fig. 1). To test the reversibility of HSA-ar-
rested germination, the spores were incubated for 2 h in the
presence of selected germinants before the pH was raised to
7.1 by the addition of NaOH in the HSA-exposed samples.
This instantaneously shifted the equilibrium between SA and
HSA, resulting in a decrease from 3 mM HSA to less than 0.2
mM, with a concomitant relief of inhibition of germination
(Fig. 1).
The data show that not only germination via L-alanine but
also that via the germinants inosine and glutamine is inhibited
by the presence of 3 mM HSA. Comparable results were ob-
tained for germination induced with cysteine, threonine, and
phenylalanine (data not shown). Thus, HSA blocks germina-
tion in B. cereus spores via the receptors GerR, GerG, GerQ,
and GerI and does not display a receptor-specific effect, as was
previously reported by Cortezzo for Bacillus subtilis spores (4).
In the latter study, HSA arrested germination only when trig-
gered by L-alanine in B. subtilis, but not when triggered by a
mixture of L-asparagine, D-glucose, D-fructose, and potassium
ions (AGFK). Notably, the concentration of sorbic acid used in
the latter study (5 mg/liter) was far below the range (250 to
2,000 mg/liter) that is applied by industry (14), and given that
experiments were performed at pH 6, the amount of undisso-
ciated acid was in the micromolar range.
Inhibition of germination by HSA was for all tested nutrients
a reversible process; when the pH was raised, spore germina-
tion is activated, as reflected in the drop in optical density
(OD) indicative of the transition from phase bright to phase
dark. Thus, in an extension of the previously reported data (3),
inhibition of nutrient-induced germination of B. cereus by HSA
does not involve competitive inhibition of (specific) germinant
receptors.
Although the exact mechanisms following nutrient binding
are not fully understood, several subsequent events are trig-
gered upon binding of the nutrient to the receptor. After being
committed to germinate, cations are released from the spores,
followed by the release of Ca2 and dipicolinic acid (DPA),
which subsequently activates the cortex lytic system (8), allow-
ing for expansion of the spore core by the uptake of water.
* Corresponding author. Mailing address: Top Institute Food and
Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, Netherlands.
Phone: (31) 317 480159. Fax: (31) 317 483011. E-mail: masja
.nieropgroot@wur.nl.
 Published ahead of print on 28 January 2011.
2552
Ca-DPA release involves the presence of SpoVA proteins that
have been suggested to act as functional components of a
specific channel (17). In this scenario, sorbic acid may display
its inhibiting effect either by (i) directly blocking the Ca-DPA
channel, (ii) inactivating the cortex lytic system, or (iii) pre-
venting the signal transduction between nutrient-activated re-
ceptor(s) and the Ca-DPA channel.
To assess whether sorbic acid inhibits germination by di-
rectly blocking the Ca-DPA channel or by inactivating the
cortex lytic system, dormant spores were exposed to either
hydrostatic pressure (HP) or to exogenous Ca-DPA in the
presence or absence of HSA. Ca-DPA is known to trigger
spore cortex degradation by directly activating cortex lytic en-
zyme Cw1J (9, 10). Exposure of dormant spores to relatively
high pressure (500 to 600 MPa) can induce germination either
by the activation of channels in the inner membrane or by
creating pores, with both possibilities resulting in the release of
Ca-DPA (2). The triggering of germination by 100 to 200 MPa
is assumed to involve activation of the nutrient receptors, even
in the absence of nutrients, followed by activation of Ca-DPA
channels (18, 19).
Heat-activated spores in MES buffer (pH 5.5) with or with-
out 3 mM HSA were exposed to temperature-controlled pres-
sure as previously described by Hornstra et al. (6) at 150 or 500
MPa or Ca-DPA at a final concentration of 50 mM. After
exposure to HP (30 min) or to Ca-DPA (60 min), part of the
samples was plated directly on brain heart infusion (BHI)
plates (total number of spores), while a second part was heat
treated for 10 min at 80°C before plating (only nongerminated
spores survive) to determine the extent of germination. CFU
were counted after overnight incubation at 30°C. Figure 2A
and B show that spores were able to germinate when exposed
to 50 mM Ca-DPA or 500 MPa, regardless of whether HSA
was present or not, thereby excluding an effect of sorbic acid on
the cortex lytic system or Ca-DPA release. Only germination of
B. cereus spores exposed to 150 MPa is affected by HSA,
indicating that receptor-mediated signaling is inhibited. The
fact that pressure-induced germination at 150 MPa without
FIG. 1. Impact of pH and sorbic acid on amino acid- and inosine-induced germination of spores derived from B. cereus ATCC 14579 and its
gerR mutant. Heat-activated spores of B. cereus ATCC 14579 (squares) (A and B) and its gerR mutant (circles) (A and C) were incubated at
pH 5.5 (open symbols) or pH 5.5 plus 3 mM HSA (closed symbols). At time zero, the following germinants were used to induce germination:
L-alanine (1 mM) (A), L-glutamine (1 mM) (B), and inosine (1 mM) (C). The vertical axis expresses the percentage of OD600 at the indicated time
points, relative to that determined at time zero. The drop in optical density signifies germination. After 2 h of incubation, the pH in sorbic
acid-stressed spore cultures was raised to pH 7.1 by addition of 2 M NaOH (indicated with the black arrow). Graphs show the results from triplicate
experiments.
FIG. 2. Impact of sorbic acid on B. cereus ATCC 14579 spore
germination induced by chemical and physical triggers. Heat-activated
spores of B. cereus ATCC 14579 were incubated in MES buffer at pH
5.5 with 3 mM HSA (gray bars) or without HSA (white bars). The
following germination triggers were used: a 1:1 chelate of 50 mM
calcium and dipicolonic acid (Ca-DPA) (A), high hydrostatic pressure
at either 150 or 500 MPa (B), and 10 M bryostatin (C). The germi-
nation percentages were determined by comparing the number of
germinated spores with the total number of spores present in each
sample. The percentage bars show the results from duplicate experi-
ments for high hydrostatic pressure and triplicate experiments for
Ca-DPA and bryostatin, respectively.
VOL. 77, 2011 IMPACT OF SORBIC ACID ON B. CEREUS GERMINATION PATHWAYS 2553
nutrients was still inhibited in the presence of sorbic acid sup-
ports the earlier assumption by Blocher and Busta (3) that a
role for sorbic acid in competitive inhibition at the nutrient
receptors is unlikely.
Recently, a novel mechanism for initiating spore germina-
tion was reported, in which germination is triggered by break-
down products of peptidoglycan (12). This route depends on
the presence of the eukaryote-like Ser/Thr protein kinase
PrkC, which is activated upon binding of cell wall peptidogly-
can fragments or muropeptides. This newly identified germi-
nation pathway appears to act independently from the germi-
nant receptor pathway. To test whether the PrkC pathway is
sensitive to sorbic acid, both HSA- and non-HSA-exposed
spores were induced to germinate by the addition of the cyclic
macrolide bryostatin, a potent activator of the kinase-induced
germination pathway (12). Heat-activated spores were incu-
bated in MES buffer at pH 5.5 without or with HSA while
being exposed to 10 M bryostatin. After incubation for 120
min, an aliquot of the sample was plated directly on BHI
plates, while a second aliquot was heat treated for 10 min at
80°C prior to plating (30°C overnight) to determine the pro-
portion of (germinated) spores.
Both control and HSA-exposed spores displayed a high per-
centage of germination when triggered with 10 M bryostatin
(Fig. 2C). The observed efficiency in the control (non-HSA-
exposed spores) is comparable to that of bryostatin-induced
germination of B. subtilis spores (12). B. cereus spore germi-
nation in the presence of 3 mM HSA appeared to be only
slightly reduced compared to that in non-HSA-exposed spores,
which shows that activation and signaling of germination via
the membrane-bound PrkC kinase is not prevented by HSA.
The results of this study allow us to draw several important
conclusions about the action of HSA on spore germination.
We demonstrated that HSA does not target specific germinant
receptors, and in agreement with that finding, a role for sorbic
acid in competitive inhibition of nutrients can thus be ex-
cluded. This is also supported by the fact that inhibition is
instantaneously released when pH is upshifted. Additionally,
the results show that inhibition of germination by sorbic acid
can be bypassed by triggering later events of the germination
pathway or by activating Ger protein receptor-independent
germination pathways, suggesting that sorbic acid specifically
interferes in the signaling between Ger receptors and putative
ion/Ca-DPA channels. Insights provided by the present study
may contribute to more efficient application of sorbic acid in
food preservation, especially in those cases that involve mild
thermal treatments. Here, it may be advisable to aim at inter-
mediate HSA concentrations that prevent outgrowth, but do
not completely inhibit germination, thereby maximizing spore
inactivation at the thermal treatment step. Additionally, the
data illustrate the importance of pH control in food preserva-
tion since small pH upshifts strongly affect HSA concentra-
tions, which could lead to outgrowth opportunities for spores.
The mode of action of sorbic acid remains to be elucidated;
however, two possible effects can be envisioned: (i) undissoci-
ated sorbic acid enters the spore and dissociates because of the
higher core pH (11), acidifying the spore’s core and interfering
with signaling; or (ii) the highly lipophilic undissociated sorbic
acid accumulates in the core membrane and in this way inter-
feres with signaling. The low mobility and reduced availability
of water in the spore’s core (15), the effects described for other
nonacid lipophilic compounds (4), and the fact that a high
concentration (15 mM) of undissociated acetic acid (pKa of
4.76, similar to that of sorbic acid, i.e., pKa 4.75) does not
inhibit germination (although outgrowth is blocked) (1), com-
bined with the immediate germination after pH upshift, sug-
gest that the second mode of action is the most likely.
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2554 VAN MELIS ET AL. APPL. ENVIRON. MICROBIOL.


A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments.

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure.

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species.

Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: phenotype and transcriptome analysis. Food Microbiol.

Characterization of germination receptors of Bacillus cereus ATCC 14579.

Characterization of spores of Bacillus subtilis which lack dipicolinic acid.

Comparative study of pressure- and nutrient-induced germination of Bacillus subtilis spores.

Comparative study of pressure-induced germination of Bacillus subtilis spores at low and high pressures.

Germination and outgrowth of spores of Bacillus cereus group members: diversity and role of germinant receptors. Food Microbiol.

gerR, a novel ger operon involved in L-alanine- and inosineinitiated germination of Bacillus cereus ATCC 14579.

How do spores germinate?

Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores.

Measurements of the pH within dormant and germinated bacterial spores.

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate.

Organic acids,

Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme.

Sorbic acid and sorbates,

The physical state of water in bacterial spores.

Impact of sorbic acid on germinant receptor-dependent and -independent germination pathways in Bacillus cereus

Name not available

Impact of sorbic acid on germinant receptor-dependent and -independent germination pathways in Bacillus cereus

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