spore germination and germinant receptor genes in wild strains of bacillus subtilis

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


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

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0 20 40 60 80 100

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% 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.



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

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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

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tant

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(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.



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

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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.



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).



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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.



spore germination and germinant receptor genes in wild strains of bacillus subtilis

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