ORIGINAL ARTICLE
Spore germination and germinant receptor genes in wildstrains of Bacillus subtilisO.M. Alzahrani1,2 and A. Moir1
1 Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK
2 Department of Biotechnology, Taif University, Taif, Saudi Arabia
Keywords
germination, receptor, spizizenii, spore,
subtilis.
Correspondence
Anne Moir, Department of Molecular Biology
and Biotechnology, University of Sheffield,
Firth Court, Western Bank, Sheffield S10 2TN,
UK.
E-mail: [email protected]
2014/0853: received 25 April 2014, revised 3
June 2014 and accepted 4 June 2014
doi:10.1111/jam.12566
Abstract
Aims: To compare the germination of laboratory and wild strains of Bacillus
subtilis.
Methods and Results: The spore germination of B. subtilis 168
(subsp. subtilis) was compared with that of the laboratory strain W23
(subsp. spizizenii) and desert-sourced isolates, including one member of
subsp. subtilis (RO-NN-1), strains TU-B-10, RO-E-2, N10 and DV1-B-1, (all
subsp. spizizenii), the B. mojavensis strain RO-H-1 and a B. subtilis natto
strain. All germinated in L-alanine, although some were slower, and some 10-
fold less sensitive to germinant. All germinated in calcium dipicolinate
(CaDPA). Germination in asparagine, glucose, fructose + KCl was slow and
incomplete in many of the strains, and decoating RO-NN-1 and W23 spores
did not restore germination rates. Comparing the sequences of B. subtilis
strains 168, RO-NN-1, W23, TU-B-10 and DV1-B-1, the operons encoding
GerA, B and K germinant receptors were intact, although the two additional
operons yndDEF and yfkQRST had suffered deletions or were absent in several
spizizenii strains.
Conclusions: Wild strains possess an efficient germination machinery for
L-alanine germination. AGFK germination is often less efficient, the gerB genes
more diverged, and the two germinant receptor operons of unknown function
have been lost from the genome in many subsp. spizizenii strains.
Significance and Impact of the Study: The two major subspecies of B. subtilis
have conserved GerA receptor function, confirming its importance, at least in
the natural environments of these strains.
Introduction
Spore germination and its prediction are of major applied
importance. Bacillus subtilis 168 has been extensively used
in identifying germination proteins, developing models
for germination and measuring germination kinetics, but
little has so far been published on the behaviour of close
relatives within the two major subspecies, subtilis and
spizizenii. This work describes the spore germination
behaviour of seven isolates from nature (six B. subtilis
and one B. mojavensis) and the laboratory strains W23
and 168. These strains were earlier used to sample gen-
ome diversity within B. subtilis, using a microarray-based
comparative genome hybridization technique (M-CGH;
Earl et al. 2007, 2008), where it was noted that many of
the 168 germination receptor genes exhibited divergence.
Our experiments aimed to test this prediction in physio-
logical terms. More recently, the genome sequence has
been partly or completely determined for several of these
(Earl et al. 2012), allowing comparison of germination
receptor operons at the sequence level.
Bacillus subtilis is the paradigm for the study of the
mechanism of bacterial endospore germination (Paredes-
Sabja et al. 2011; Christie 2012; Setlow 2013). The
response of spores to nutrient germinants is mediated by
germinant receptors, a likely complex of three proteins,
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology 741
Journal of Applied Microbiology ISSN 1364-5072
located in the inner membrane (Hudson et al. 2001). In
B. subtilis, such membrane-associated receptor complexes,
including those of different specificity, appear to be orga-
nized together in one or two foci per spore, called ‘ger-
minosomes’ (Griffiths et al. 2011), in association with,
and dependent upon, the GerD lipoprotein.
Germinant receptor genes are most often organized as
tricistronic operons. The germinant specificity of various
receptors has been elucidated in several species; some are
effective individually and others in combination (Christie
and Lowe 2007; Abee et al. 2011; Paredes-Sabja et al.
2011; Setlow 2013). In B. subtilis, five receptor operons
have been identified, three of which are clearly active
(Paidhungat and Setlow 2000). Of these, the best studied
is the gerA operon (Cooper and Moir 2011; Mong-
kolthanaruk et al. 2011). The three encoded proteins,
GerAA, GerAB and GerAC, form a membrane-associated
receptor that initiates germination in response to L-ala-
nine concentrations of >10 lmol l�1, in the presence of
K+ ions. The gerB and gerK operons encode receptors
that together mediate the germination response to the
cogerminant mixture AGFK (L-asparagine, glucose, fruc-
tose and K+ ions). The GerK receptor has a prominent
role in the interaction with sugars, while GerB responds
to L-amino acids, but not normally sufficiently to induce
germination without adjuncts (McCann et al. 1996; Paid-
hungat and Setlow 1999; Atluri et al. 2006). Two other
homologous operons, yndDEF and yfkQRT, are present in
the B. subtilis genome, but there is no evidence of their
function; they do not contribute to germination on rich
laboratory media (Paidhungat and Setlow 2000). As the
paradigm for the study of spore germination, the labora-
tory strain of B. subtilis 168 is the only member of this
species whose germination behaviour has been reported
in detail.
On the basis of DNA sequences of the polC, rpoB and
gyrA genes, DNA re-association and 16S rRNA sequence
(Nakamura et al. 1999), the Bacillus subtilis group has been
divided into two subspecies: subsp. subtilis and subsp.
spizizenii, including strains 168 and W23, respectively. The
likely history of these two laboratory strains has been con-
sidered (Zeigler et al. 2008; Zeigler 2011). The laboratory
168 strain was derived from B. subtilis Marburg by X-ray
mutagenesis (Burkholder and Giles 1947). The W23 strain
differs significantly from the 168 strain, for example having
ribitol rather than glycerol teichoic acids in the cell wall; it
is likely to be a direct descendant of B. subtilis strain
ATCC6633 (Zeigler 2011).
A more recent phylogeny (Rooney et al. 2009) con-
firmed the relationship between the two different subspe-
cies and suggested a third, subsp.inaquosorum. Their
analysis also included, amongst others, B. mojavensis, a
species closely related to, but distinct from, B. subtilis
(Roberts et al. 1994), which has been included in our
study.
Methods
Bacterial strains and culture conditions
Bacillus strains used are listed in Table 1. Spores were
prepared in DSM liquid medium (Schaeffer et al. 1965)
at 37°C, and harvested and washed as previously
described (Moir et al., 1979). Spores of all strains that
were directly compared were prepared at the same time,
so that washing and storage conditions were equivalent.
At least two independent spore preparations were exam-
ined for each strain.
Germination assays in nutrient germinants
Spores were heat-activated in distilled water at 70°C for
30 min and then cooled on ice before addition of
germinants. This standard activation treatment was not
optimized for individual strains. For measurement of ger-
mination rates under saturating conditions, spores were
diluted in microwell plates (to a spore density that would
be equivalent to an OD600 of 0�6 in a 1 cm light path) by
addition of prewarmed germinant to give a final concentra-
tion of 20 mmol l�1 L-alanine, 10 mmol l�1 Tris-HCl,
Table 1 List of strains used to compare germination physiology
Strain (subspecies) Origin
Bacillus subtilis 168 trpC2 (subtilis) Laboratory strain (Sammons et al., 1981)
RO-NN-1 (subtilis) Wild strain Mojave Desert, USA (Earl et al. 2007)
natto (subtilis) Wild strain Japan (Tanaka and Koshikawa 1977); (Earl et al. 2007)
TU-B-10 (spizizenii) Wild strain Sahara Desert, Nefta, Egypt; USDA (Earl et al. 2007)
DV1-B-1 (spizizenii) Wild strain Death Valley National Monument, California; USDA (Earl et al. 2007)
W23 (spizizenii) Laboratory strain USDA (Earl et al. 2007)
RO-E-2 (spizizenii) Wild strain Mojave Desert; USDA (Earl et al. 2007)
N10 (spizizenii) Wild strain Mediterranean Sea, Egypt (Earl et al. 2007)
RO-H-1 (B. mojavensis) Wild strain Mojave Desert, (Earl et al. 2007)
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology742
Germination in wild B. subtilis O.M. Alzahrani and A. Moir
20 mmol l�1 KCl, pH 7�4. Germination was carried out at
37°C, and the OD of the suspension was measured every
2 min for 120 min using a Victor2 1420 multilabel counter
Wallac Victor2 1420 multilabel counter (PerkinElmer,
Waltham, MA) with a 480 nm filter. Germination in
L-asparagine (30 mmol l�1), D-glucose (5�6 mmol l�1),
fructose (5�6 mmol l�1) and KCl (20 mmol l�1) was in
50 mmol l�1 Tris-HCl, pH 8�4, again at 37°C. For each
mutant, data were obtained for at least two independent
spore preparations, and routine experiments at saturating
germinant concentrations were carried out in duplicate for
each preparation. Spore suspensions were checked by phase
contrast microscopy to confirm the extent of germination
at 120 min. For all strains, results were very similar for
both spore preparations, and typical graphs in L-alanine
and AGFK are shown.
For calculation of germination kinetic data, spores
were diluted to an OD600 of 0�6 in varying concentrations
of L-ala (10 lmol l�1 to 100 mmol l�1), in germination
buffer (10 mmol l�1 Tris-HCl, 20 mmol l�1 KCl, pH
7�4) and measured as above. Data were obtained for three
experiments for each of two independent spore prepara-
tions. As in Mongkolthanaruk et al.(2011), the maximum
slope of each curve (germination rate) was determined.
The germination rate was then plotted against the con-
centration of germinant on a logarithmic scale, and the
maximum germination rate (Gmax, expressed as change in
OD min�1) and C50, the concentration for half-maximal
germination rate, were calculated.
CaDPA- mediated germination
Germination was measured on the basis of loss of heat
resistance (Jaye and Ordal 1965). Spores, without heat
activation, were resuspended to OD600 of 1 in
120 mmol l�1 DPA, adjusted to pH 8 with NaOH, and an
equal volume of CaCl2 (120 mmol l�1 adjusted to pH 8
with NaOH) was added to initiate germination in
60 mmol l�1 CaDPA. Germination was at room tempera-
ture (20°C). Samples were diluted in water, and the dilu-
tion was plated in triplicate on Nutrient Agar, both with
and without heating at 70°C for 30 min, to allow calcula-
tion of heat-resistant CFU. It was first confirmed that dor-
mant spores of all strains were fully resistant to this heat
challenge. Experiments were carried out on two indepen-
dent spore preparations, which gave similar results.
Germination of spores after spore-coat permeabilization
Spore coats were permeabilized chemically (Behravan
et al. 2000), by incubation at 37°C for 90 min in CHES
buffer (pH 8�6) containing 8 mol l�1 urea, 70 mmol l�1
dithiothreitol and 1% (w/v) SDS. They were then washed
five times with water and confirmed as phase-bright by
phase contrast microscopy. Increased spore-coat perme-
ability was confirmed by testing the sensitivity of spore
cortex to lysozyme, measuring the loss of OD480 in
30 lg ml�1 lysozyme, 50 mmol l�1 NaCl. Spores were
not heat-activated before germination, which was then
measured by loss of heat resistance as above. Experiments
were carried out on two independent spore preparations,
which gave similar results.
Results
Germination behaviour of the wild-type strains in
nutrient germinants
Germination was measured as the loss of OD of a spore
suspension over time after addition of germinant. Loss of
OD reflects late events in spore germination; as a result
of the loss of refractility of spores in the germination
process, the suspension will lose approximately 50% of
initial OD when all spores germinate. In this assay, the
behaviour of the population as a whole is being consid-
ered. By study of single spores, two distinct stages of ger-
mination were defined, microlag and microgermination
(Hashimoto et al. 1969). Microlag can be defined as the
time interval between exposure of a spore to the germi-
nant and the start of a detectable germination-associated
change, such as loss of refractility or DPA release. The
length of the microlag is often very variable between indi-
vidual spores in a spore suspension, as reported by
microscopic analysis (Coote et al. 1995) and Raman spec-
troscopy (Kong et al. 2010, 2011). In contrast, the micro-
germination time is very short (Zhang et al. 2010) so
that the overall time to germinate for a single spore is
dependent on the length of the microlag. Different factors
affect the microlag, including, for example, heat activa-
tion, pH, germinant concentration and germinant recep-
tor levels; superdormant spores, with a very long average
microlag, contain low levels of the relevant germinant
receptor (Ghosh et al. 2012).
The germination kinetics for spore suspensions of all
strains in 20 mmol l�1 L-alanine, well above the mini-
mum concentration needed to achieve maximum germi-
nation rates (Gmax), are shown in Fig. 1, and data
summarizing the overall germination behaviour of strains
in L-alanine are summarized in Table 2. All strains tested
germinated efficiently, suspensions losing >40% of initial
OD, in high L-alanine concentrations.
The three strains in subsp. subtilis (Fig. 1a) all germi-
nated well in excess L-alanine, but spores of strain RO-
NN-1 required a 10-fold higher concentration, and at sat-
urating concentrations of germinant still showed a much
longer microlag. The B. subtilis natto strain germinated
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology 743
O.M. Alzahrani and A. Moir Germination in wild B. subtilis
rapidly and sensitively, but unusually, spores germinated
less well at 100 mmol l�1 compared to 20 mmol l�1
(data not shown); a plausible explanation of this would
be that higher substrate concentrations might allow for-
mation of sufficient D-ala to cause competitive inhibi-
tion, by spore-associated alanine racemase activity,
although this was not tested. B. mojavensis strain R-OH-1
also germinated well, though showing a somewhat lower
sensitivity to germinant, and a slightly longer microlag,
than the fastest B. subtilis strains.
The strains in subsp. spizizenii all germinated in excess
L-alanine (Fig. 1b), although spores of every strain dem-
onstrated a long microlag compared to B. subtilis 168,
with TU-B-10 the slowest. Strains DV1-B-1 and RO-E-2
were approximately 10-fold less sensitive to L-alanine, as
judged by C50 (Table 2), whereas W23, TU-B-10 and
N10 had Gmax values close to those of the rapid
germinating B. subtilis 168 and natto strains, reflecting a
reasonable degree of synchrony in the germination
response, despite the longer microlag.
Germination in the combination of amino acids and
sugars (AGFK) is dependent on the joint function of
GerB and GerK receptors. Apart from the B. subtilis 168
strain, only B. mojavensis and the subsp. spizizenii strain
RO-E-2 germinated well and to completion (Fig. 2).
Most of the wild isolates germinated poorly, and only a
fraction of spores germinated within the 100 min of the
assay. Strain N10 showed no response at all.
Germination in CaDPA
Ca-DPA, a 1-1 chelate of Ca2+ with pyridine-2,6-dicar-
boxylic acid (dipicolinic acid, DPA), is classified as a
non-nutrient germinant and induces germination by
stimulation of CwlJ, a spore cortex lytic enzyme (Paid-
hungat et al. 2001), by a germinant receptor-independent
mechanism. All strains of the subsp. subtilis and spizize-
nii, as well as B. mojavensis, germinated in CaDPA
(Fig. 3), although there was a wide variation in the
response time between different wild strains. The overall
pattern of response did not correlate particularly with the
response to nutrient germinants; for example, RO-E-2,
slowest to germinate in CaDPA, was one of the faster ger-
minating strains in the spizizenii group in both L-alanine
and AGFK.
Overall, the germination behaviour could not be pre-
dicted on the basis of phylogenetic group. There was no
consistent pattern; different strains could show a long lag
or fractional germination, where only some spores
respond, in one germinant, but this did not predict
Table 2 Germination of Bacillus subtilis 168 and wild strains in
L-alanine
Strain Subspecies
Gmax in
L-ala*
C50 in L-ala
(910�4 mol l�1)
1604
(168)
subtilis 1�7 � 0�2 0�4 � 0�1
natto subtilis 3�4 � 0�3 0�2 � 0�04RO-NN-1 subtilis 0�7 � 0�1 4�4 � 1�4TU-B-10 spizizenii 0�95 � 0�1 0�55 � 0�1DV1-B-1 spizizenii 1�1 � 0�1 4�9 � 1
RO-E-2 spizizenii 1�1 � 0�2 3�3 � 0�6N10 spizizenii 1�0 � 0�2 0�5 � 0�3W23 spizizenii 1�2 � 0�2 0�5 � 0�1RO-H-1 B. mojavensis 1�8 � 0�3 2�4 � 0�4
C50 is the concentration (mol l�1) of germinant (L-alanine) required to
achieve 50% of maximum germination rate (Gmax) whose units are%
of initial OD lost per min.
*Data from germination in 20 mmol l�1 L-ala, a saturating concentra-
tion for all strains.
110
100
90
80
70
60
50
40
30
110
100
90
80
70
60
50
40
30
0 20 40 60 80 100
0 20 40 60 80 100
% In
itial
OD
% In
itial
OD
Time (min)
Time (min)
(a)
(b)
Figure 1 Germination in 20 mmol l�1 L-alanine measured as% OD
fall of spore suspensions. (a) Symbols are ●, 1604; ■, RO-NN-1; ▲,
natto; □, Bacillus mojavensis R-OH-1. (b) Symbols are M, TU-B-10; ▽,
N10; ◇, DV-1-B1; ○, W23; □, R-OE-2.
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology744
Germination in wild B. subtilis O.M. Alzahrani and A. Moir
whether they would be slow to respond to another germi-
nant.
Coat removal does not restore efficient germination of
slow-germinators
In a gerP mutant of B. subtilis or B. cereus, increasing the
permeability of the spore coat can increase germination,
apparently by enabling germinants to penetrate the spore
structure more rapidly (Behravan et al. 2000). Strains
chosen for chemical decoating included the B. subtilis 168
strain 1604 and strains RO-NN-1 and W23, both of
which showed a long lag in L-alanine and slow germina-
tion in AGFK. For B. subtilis 1604, germination in
100 mmol l�1 L-alanine of decoated spores was slightly
faster than germination of the intact spores (Fig. 4),
reducing the average lag before loss of heat resistance by
ca. 7 min. The rate and extent of germination of RO-
NN-1 spores were not improved by decoating. In the case
of W23, the germination response was poorer after deco-
ating. Germination of each strain was improved slightly,
relative to the equivalent nonpermeabilized spores, in the
AGFK combination germinant (Fig. 4), but the improve-
ment in germination rate was not higher for wild strains
than was seen for 168. Therefore, overall, the reluctance
to germinate seen in these wild strains relative to the 168
strain did not appear to be related to decreased coat
permeability.
Germinant receptor genes in the different subspecies
The germinant receptor operons in the completed
genome sequences of five strains, including RO-NN-1,
TU-B-10 and DV1-B-1, strain W23 and B. mojavensis
RO-H-1, were compared to those of B. subtilis 168. No
additional germinant receptors were encoded beyond
those already reported in B. subtilis 168.
These B. subtilis genome sequences are now available
for comparison in a public database (www.ncbi.nlm.nih.
100
90
80
70
60
50
110
100
90
80
70
60
50
40
0 20 40 60 80 100 120
0 20 40 60 80 100 120
Time (min)
Time (min)
% In
itial
OD
% In
itial
OD
(a)
(b)
Figure 2 Germination in a combination of asparagine, glucose, fruc-
tose and KCl (AGFK). (a) Symbols are ●, 1604; ■, RO-NN-1; ▲,
natto; □, Bacillus mojavensis R-OH-1. (b) Symbols are M, TU-B-10; ▽,
N10; ◇, DV-1-B1; ○, W23; □, R-OE-2.
Time (min)
% h
eat r
esis
tant
0
20
40
60
80
100
Time (min)
0 10 20 30 40 50 60
0 10 20 30 40 50 60
% h
eat r
esis
tant
0
20
40
60
80
100
(a)
(b)
Figure 3 Germination in CaDPA. (a) Symbols are ●, 1604; ■, RO-
NN-1; ▲, natto; □, Bacillus mojavensis R-OH-1. (b) Symbols are M,TU-B-10; ▽, N10; ◇, DV-1-B1; ○, W23; □, R-OE-2.
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology 745
O.M. Alzahrani and A. Moir Germination in wild B. subtilis
gov/genomes/geblast.cgi?taxid=1423). Table 3 compares
the proteins encoded by the three major operons of the
above strains with finished genome sequences, compared
to those of the laboratory strains B. subtilis 168 and W23.
The predicted GerA receptor proteins are the most
conserved in all strains, followed by the GerK receptor
subunits, followed by GerB protein subunits. The degree
of similarity correlates with the subspecies, except for
proteins encoded in the gerB operon, for which the labo-
ratory strain 168 is the least similar of the strains tested.
To explore this further, the corresponding gerBA gene
sequences were aligned with those of several more
recently sequenced B. subtilis strains, and an unrooted
phylogenetic tree generated (Fig. 5). Sequences cluster in
three distinct groups, with the cluster of subsp. subtilis
strains 168 and AUS198 as the most distant. Strain RO-
NN-1, unusually for a subsp. subtilis strain, is close to the
W23 (spizizenii) group.
The apparently silent germinant receptor operons of
B. subtilis 168 have not been conserved in all strains. The
yndDEF operon (Figure S1) is complete in strain RO-
NN-1, but has a frameshift in yndD. The operon is intact
in TU-B-10, but has been partially deleted in both W23
and DV1-B-1. The yfkQRST operon (Figure S2) has
suffered deletions in strains TU-B-10 and DV1-B-1. In
B. mojavensis RO-H-1, both yndDEF and yfkQRST ope-
rons are absent.
Discussion
Functionality of L-alanine germination, mediated by the
GerA receptor, was conserved in all the wild strains
tested, whereas GerB- and GerK-dependent AGFK germi-
nation was generally less efficient.
A long microlag is often correlated with slower maxi-
mum germination rate, reflecting increased asynchrony of
the population, and often fractional germination, as a rela-
tively superdormant fraction of spores fail to germinate
within the time frame of the experiment (Ghosh et al.
2009; Wei et al. 2010). This may result from a reduction in
the number of germinant receptor molecules, or a differ-
ence in effectiveness of the germination receptor apparatus.
In the experiments described here, some wild strains were
slow to germinate in L-alanine, with a long average micro-
lag, but germination of the bulk of spores in such popula-
tions was relatively synchronous, considering the longer
delay, and most or all of the spores germinated.
A longer microlag and inefficient germination might
reflect a slower passage of germinant through the spore
coats in some strains, as in a gerP mutant (Behravan
et al. 2000). However, in the case of the wild strains
tested here, decoating did not result in a rapid germina-
tion response—the longer lag remained. Testing the
expression of the gerA or gerB operons, or the levels of
the encoded proteins, in different strains was beyond the
scope of this study.
The three germinant receptors of B. subtilis 168 shown
to be functional in germination in rich laboratory media
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
0 10 20 30 40 50 60
0 10 20 30 40 50 60
0 10 20 30 40 50 60
% h
eat r
esis
tant
% h
eat r
esis
tant
% h
eat r
esis
tant
Time (min)
Time (min)
Time (min)
(a)
(b)
(c)
Figure 4 Effect of decoating on spore germination measured as loss
of heat resistance. (a), Strain 1604, (b), RO-NN-1, (c), W23. Circles
represent alanine germination, and squares germination in AGFK.
Closed symbols represent intact spores, and open circles represent
decoated spores.
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology746
Germination in wild B. subtilis O.M. Alzahrani and A. Moir
are the GerA, B and K receptors. These receptors had a
complete complement of intact ORFs in all the strains
examined. Sequence similarity within the gerA and gerK
operons reflects the phylogenetic relationship of the
B. subtilis subspecies, but this is not entirely the case for
genes of the gerB operon. Data are presented here for ger-
BA, but the same is the case for gerBB and gerBC genes
also (data not shown). Many wild strains had lost some
or all of the other two germinant receptor operons, ynd-
DEF and yfkQRST, for which no function has been
detected in the laboratory. The contrast with the conser-
vation of the other three germinant receptor operons
suggests that there has not been selective pressure to
maintain the receptors of unknown function.
No additional receptor operons were detected, in con-
trast to B. cereus, where different strains often have
additional receptor operons in addition to the five core
receptors (van der Voort et al. 2010).
The data presented here also demonstrate that the ger-
mination efficiency cannot be predicted from the molecu-
lar phylogeny of a particular receptor. Germination in
AGFK of the RO-NN-1 subsp. subtilis strain was slow,
like that of the subsp. spizizenii strains with which it clus-
ters in Fig. 5. However, germination in AGFK was very
efficient in the B. mojavensis strain RO-H-1 and DV1-B-1
strains, although their gerBA receptor genes are relatively
well diverged from the 168 strain. It is notable that gerBA
genome sequences of many of the subsp. subtilis strains,
including 168 and AUS198, shown in Fig. 5, but also
PS216, MB73/2, 6051-HGW and BSP1 (data not shown)
show very close conservation. The position of RO-NN-1,
in contrast, suggests a history of genetic exchange, intro-
ducing a spizizenii-like operon in this subtilis subspecies.
The observation by microarray-based comparative gen-
ome hybridization (Earl et al. 2007) that many of the
germinant receptor genes of B. subtilis strains exhibited
divergence has been confirmed by examination of gen-
ome sequences, but this analysis demonstrates that it pri-
marily reflects absence or loss of the two receptor
operons of unknown function, rather than the earlier
suggestion (Earl et al. 2008) that it might reflect evolu-
tion of germinant receptors to use alternative environ-
mental cues. The GerA and GerB/K receptor genes are
retained, although there is a tendency to slower germina-
tion in some strains, at least under standard laboratory
conditions used to test the function of these receptors.
Acknowledgements
We thank Ashlee Earl and Jacques Revel for early access
to genome sequence data, and for providing strains, and
the Government of Saudi Arabia for their award of a
PhD scholarship to OMA.
Conflict of interest
No conflict of interest declared.
Table 3 Percentage amino acid identity between germinant receptor proteins in different Bacillus subtilis strains. 168 and RO-NN-1 are subsp.
subtilis, whereas W23 and TU-B-10 are subsp. spizizenii. RO-H-1 is the more distant B. mojavensis
AA, AB,
AC vs 168
AA, AB,
AC vs W23
BA, BB,
BC vs 168
BA, BB,
BC vs W23
KA, KB,
KC vs 168
KA, KB,
KC vs W23
168 100 94,94,90 100 86,86,87 100 93,92,89
RO-NN-1 98, 96, 91 95, 93, 92 86, 86, 88 96, 99, 96 98, 96, 95 93, 92, 91
W23 94, 94, 90 100 86, 86, 87 100 93, 92, 89 100
TU-B-10 93, 92, 90 99, 97, 99 87, 86, 87 97, 94, 99 93, 93, 89 97, 98, 98
RO-H-1 93, 92, 84 95, 96, 88 86, 85, 86 95, 99, 97 88, 91, 84 89, 92, 86
W23 TU-B-10RO-NN-1
RO-H-1
AUS198, 168
gtP20KCTC 13429
DV1-B-1
BSn5
Figure 5 Unrooted tree showing the divergence between gerBA
gene sequences of Bacillus subtilis 168 and other closely related
strains. In addition to sequenced strains 168, RO-NN-1, W23, TU-B-
10 and DV1-B-1, and B. mojavensis RO-H-1, sequences included were
B. subtilis BSn5 (Deng et al. 2011), gtP20b (Fan et al. 2011) AUS198
(Earl et al. 2012) and B. subtilis subsp. inaquosorum strain KCTC
13429 (Yi et al. 2014). Sequences were compiled using Clustal
Omega (http://www.ebi.ac.uk) by the distance-joining method, and
the tree represented using the WUR Phylogenetic TreePlot server
(http://www.bioinformatics.nl/tools/plottree.html).
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology 747
O.M. Alzahrani and A. Moir Germination in wild B. subtilis
References
Abee, T., Groot, M.N., Tempelaars, M., Zwietering, M.,
Moezelaar, R. and van der Voort, M. (2011) Germination
and outgrowth of spores of Bacillus cereus group
members: diversity and role of germinant receptors. Food
Microbiol 28, 199–208.
Atluri, S., Ragkousi, K., Cortezzo, D.E. and Setlow, P. (2006)
Cooperativity between different nutrient receptors in
germination of spores of Bacillus subtilis and reduction of
this cooperativity by alterations in the GerB receptor. J
Bacteriol 188, 28–36.
Behravan, J., Chirakkal, H., Masson, A. and Moir, A. (2000)
Mutations in the gerP locus of Bacillus subtilis and Bacillus
cereus affect access of germinants to their targets in spores.
J Bacteriol 182, 1987–1994.
Burkholder, P.R. and Giles, N.H. Jr (1947) Induced
biochemical mutations in Bacillus subtilis. Am J Bot 34,
345–348.
Christie, G. (2012) Initiation of germination in Bacillus and
Clostridium spores. In Bacterial Spores: Current Research
and Applications ed. Abel-Santos, E. pp. 89–106. Norfolk,
UK: Caister Academic Press.
Christie, G. and Lowe, C.R. (2007) Role of chromosomal and
plasmid-borne receptor homologues in the response of
Bacillus megaterium QM B1551 spores to germinants. J
Bacteriol 189, 4375–4383.
Cooper, G.R. and Moir, A. (2011) Amino acid residues in the
GerAB protein important in the function and assembly of
the alanine spore germination receptor of Bacillus subtilis
168. J Bacteriol 193, 2261–2267.
Coote, P.J., Billon, C.M.P., Pennell, S., McClure, P.J.,
Ferdinando, D.P. and Cole, M.B. (1995) The use of
confocal scanning laser microscopy (CSLM) to study the
germination of individual spores of Bacillus cereus. J
Microbiol Methods 21, 193–208.
Deng, Y., Zhu, Y., Wang, P., Zhu, L., Zheng, J., Li, R., Ruan, L.,
Peng, D. et al. (2011) Complete sequence of Bacillus subtilis
BSn5, an endophytic bacterium of Amorphophallus konjac
with antimicrobial activity for the plant pathogen Erwinia
carotovora subsp. carotovora. J Bacteriol 193, 2070–2071.
Earl, A.M., Losick, R. and Kolter, R. (2007) Bacillus subtilis
genome diversity. J Bacteriol 189, 1163–1170.
Earl, A.M., Losick, R. and Kolter, R. (2008) Ecology and
genomics of Bacillus subtilis. Trends Microbiol 16, 269–
275.
Earl, A.M., Eppinger, M., Fricke, W.F., Rosovitz, M.J., Rasko,
D.A., Daugherty, S., Losick, R., Kolter, R. et al. (2012)
Whole-genome sequences of Bacillus subtilis and close
relatives. J Bacteriol 194, 2378–2379.
Fan, L., Bo, S., Chen, H., Ye, W., Kleinschmidt, K., Baumann,
H.I., Imhoff, J.F., Kleine, M. et al. (2011) Genome
sequence of Bacillus subtilis subsp. spizizenii
gtP20b, isolated from the Indian Ocean. J Bacteriol 193,
1276–1277.
Ghosh, S., Zhang, P.F., Li, Y.Q. and Setlow, P. (2009)
Superdormant spores of Bacillus species have elevated wet-
heat resistance and temperature requirements for heat
activation. J Bacteriol 191, 5584–5591.
Ghosh, S., Scotland, M. and Setlow, P. (2012) Levels of
germination proteins in dormant and
superdormant spores of Bacillus subtilis. J Bacteriol 194,
2221–2227.
Griffiths, K.K., Zhang, J.Q., Cowan, A.E., Yu, J. and Setlow, P.
(2011) Germination proteins in the inner membrane of
dormant Bacillus subtilis spores colocalize in a discrete
cluster. Mol Microbiol 81, 1061–1077.
Hashimoto, T., Frieben, W.R. and Conti, S.F. (1969)
Germination of single bacterial spores. J Bacteriol 98,
1011–1020.
Hudson, K.D., Corfe, B.M., Kemp, E.H., Feavers, I.M., Coote,
P.J. and Moir, A. (2001) Localization of GerAA and
GerAC germination proteins in the Bacillus subtilis spore.
J Bacteriol 183, 4317–4322.
Jaye, M. and Ordal, Z.J. (1965) Germination of spores of
Bacillus megaterium with divalent metal-dipicolinate
chelates. J Bacteriol 89, 1617–1618.
Kong, L.B., Zhang, P.F., Setlow, P. and Li, Y.Q. (2010)
Characterization of bacterial spore germination using
integrated phase contrast microscopy, Raman spectroscopy,
and optical tweezers. Anal Chem 82, 3840–3847.
Kong, L.B., Zhang, P.F., Wang, G.W., Yu, J., Setlow, P. and Li,
Y.Q. (2011) Characterization of bacterial spore
germination using phase-contrast and fluorescence
microscopy, Raman spectroscopy and optical tweezers.
Nat Protoc 6, 625–639.
McCann, K.P., Robinson, C., Sammons, R.L., Smith, D.A. and
Corfe, B.M. (1996) Alanine germination receptors of
Bacillus subtilis. Lett Appl Microbiol 23, 290–294.
Moir, A., Lafferty, E. and Smith, D.A. (1979) Genetic analysis
of spore germination mutants of Bacillus subtilis:
correlation of map position with phenotype. J Gen
Microbiol 111, 165–180.
Mongkolthanaruk, W., Cooper, G.R., Mawer, J.S.P., Allan,
R.N. and Moir, A. (2011) Effect of amino acid
substitutions in the GerAA protein on the function of the
alanine responsive germinant receptor of Bacillus subtilis
spores. J Bacteriol 193, 2268–2275.
Nakamura, L.K., Roberts, M.S. and Cohan, F.M. (1999)
Relationship of Bacillus subtilis clades associated with
strains 168 and W23: a proposal for Bacillus subtilis subsp
subtilis subsp nov and Bacillus subtilis subsp spizizenii
subsp nov. Int J Syst Bacteriol 49, 1211–1215.
Paidhungat, M. and Setlow, P. (1999) Isolation and
characterization of mutations in Bacillus subtilis that allow
spore germination in the novel germinant D- alanine. J
Bacteriol 181, 3341–3350.
Paidhungat, M. and Setlow, P. (2000) Role of Ger proteins in
nutrient and nonnutrient triggering of spore germination
in Bacillus subtilis. J Bacteriol 182, 2513–2519.
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology748
Germination in wild B. subtilis O.M. Alzahrani and A. Moir
Paidhungat, M., Ragkousi, K. and Setlow, P. (2001) Genetic
requirements for induction of germination of spores of
Bacillus subtilis by Ca2 + -dipicolinate. J Bacteriol 183,
4886–4893.
Paredes-Sabja, D., Setlow, P. and Sarker, M.R. (2011)
Germination of spores of Bacillales and Clostridiales
species: mechanisms and proteins involved. Trends
Microbiol 19, 85–94.
Roberts, M.S., Nakamura, L.K. and Cohan, F.M. (1994)
Bacillus mojavensis sp. nov., distinguishable from Bacillus
subtilis by sexual isolation, divergence in DNA sequence,
and differences in fatty acid composition. Int J Syst
Bacteriol 44, 256–264.
Rooney, A.P., Price, N.P.J., Ehrhardt, C., Swezey, J.L. and
Bannan, J.D. (2009) Phylogeny and molecular taxonomy
of the Bacillus subtilis species complex and description of
Bacillus subtilis subsp inaquosorum subsp nov. Int J Syst
Evol Microbiol 59, 2429–2436.
Sammons, R.L., Moir, A. and Smith, D.A. (1981) Isolation and
properties of spore germination mutants of Bacillus subtilis
168 deficient in the initiation of germination. J Gen
Microbiol 124, 229–241.
Schaeffer, P., Millet, J. and Aubert, J.P. (1965) Catabolic
repression of bacterial sporulation. Proc Natl Acad Sci U S
A 54, 704–711.
Setlow, P. (2013) Summer meeting 2013 – when the sleepers
wake: the germination of spores of Bacillus species. J Appl
Microbiol 115, 1251–1268.
Tanaka, T. and Koshikawa, T. (1977) Isolation and
characterization of four types of plasmids from Bacillus
subtilis (natto). J Bacteriol 131, 699–701.
van der Voort, M., Garc�ıa, D., Moezelaar, R. and Abee, T.
(2010) Germinant receptor diversity and germination
responses of four strains of the Bacillus cereus group. Int J
Food Microbiol 139, 108–115.
Wei, J., Shah, I.M., Ghosh, S., Dworkin, J., Hoover, D.G. and
Setlow, P. (2010) Superdormant spores of Bacillus species
germinate normally with high pressure, peptidoglycan
fragments, and bryostatin. J Bacteriol 192, 1455–1458.
Yi, H., Chun, J. and Cha, C.-J. (2014) Genomic insights into
the taxonomic status of the three subspecies of Bacillus
subtilis. Syst Appl Microbiol 37, 95–99.
Zeigler, D.R. (2011) The genome sequence of Bacillus subtilis
subsp. spizizenii W23: insights into speciation within the
B. subtilis complex and into the history of B. subtilis
genetics. Microbiology 157, 2033–2041.
Zeigler, D.R., Pragai, Z., Rodriguez, S., Chevreux, B., Muffler,
A., Albert, T., Bai, R., Wyss, M. et al. (2008) The origins
of 168, W23, and other Bacillus subtilis legacy strains. J
Bacteriol 190, 6983–6995.
Zhang, P.F., Garner, W., Yi, X.A., Yu, J., Li, Y.Q. and Setlow,
P. (2010) Factors affecting variability in time between
addition of nutrient germinants and rapid dipicolinic acid
release during germination of spores of Bacillus species. J
Bacteriol 192, 3608–3619.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 Representation of the yndDEF germinant
receptor operon and flanking genes.
Figure S2 Representation of the yfkQRST germinant
receptor operon and flanking genes.
Journal of Applied Microbiology 117, 741--749 © 2014 The Society for Applied Microbiology 749
O.M. Alzahrani and A. Moir Germination in wild B. subtilis