University of Groningen
Heat resistance of Bacillus spores
Berendsen, Erwin Mathijs
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Berendsen, E. M. (2016). Heat resistance of Bacillus spores: Natural variation and genomic adaptation
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Chapter 4
High-level heat resistance of spores
of Bacillus amyloliquefaciens and
Bacillus licheniformis results from
the presence of a spoVA operon in a
Tn1546 transposon
Erwin M. Berendsen
Rosella A. Koning
Jos Boekhorst
Anne de Jong
Oscar P. Kuipers
Marjon H.J. Wells-Bennik
Chapter 4
Abstract
Bacterial endospore formers can produce spores that are resistant against many food
processing conditions, including heat. Some spores may survive heating processes
aimed at production of commercially sterile foods. Recently, it was shown that a spoVA
operon present on a Tn1546 transposon in Bacillus subtilis profoundly increases the
wet heat resistance of spores. In this study, the presence of the Tn1546 transposon
was assessed in nine strains of Bacillus amyloliquefaciens and nine strains of Bacillus
licheniformis, and was found to be naturally present in several strains. Its effect on the
wet heat resistance of spores was investigated. The presence of the Tn1546 transposon
homologues and spoVA operon was confirmed by whole genome sequencing and
PCR detection. Strains of B. amyloliquefaciens and B. licheniformis carrying a Tn1546
transposon produced spores with significantly higher resistance to wet heat than their
counterparts that lacked this transposon. The spoVA operons encoded on the Tn1546
transposons of B. licheniformis and B. amyloquefaciens were cloned into B. subtilis 168,
and resulted in strains that produced high-level heat resistant spores. The finding that
spoVA genes on a transposon determine heat resistance properties of spores of strains
belonging to the B. subtilis group can contribute to improved control of these spores in
the food chain.
82
High-level heat resistance of spores of B. amyloliquefaciens and B. licheniformis
Introduction
Bacterial spore formers can survive harsh environmental conditions due to the
formation of endospores (spores) and they are ubiquitously found in nature (27, 40).
Since bacterial spores are widely present in nature, they can enter the food chain from
many different sources, for example via soil (13). The intrinsic resistance properties
of spores potentially result in survival during food processing, in which heating is one
of the most commonly applied treatments to reduce bacterial loads. Such treatments
put selective pressure on the microflora that is present, allowing for survival of those
strains that produce spores with high heat resistance (32). Surviving spores may
germinate upon exposure to certain environmental triggers, and can subsequently
resume vegetative growth, potentially resulting in food pathogenicity or food spoilage,
depending on the species (39).
Spores of mesophilic species belonging to the B. subtilis group are commonly found
in various food ingredients and food products. The B. subtilis group encompasses the
species B. subtilis, B. amyloliquefaciens, B. licheniformis, B. vallismortis, B. mojavensis, B.
atropheus and B. sonorensis, which are phylogenetically close, yet distinguishable (22).
These species can generally grow between temperatures of 30°C to 50°C, with reported
growth temperatures of B. licheniformis up to 58°C (51). The spores of B. subtilis, B.
amyloliquefaciens and B. licheniformis, are commonly found in various food ingredients
and food products including cocoa, herbs, spices, bread, soups, milk and milk powders
(20, 23, 26, 29, 42). These species are for instance well-known contaminants of raw
materials used in bread making (36, 41), and the spores can potentially even survive the
bread baking process (44). After spore survival, germination, and outgrowth, vegetative
cells of B. amyloliquefaciens, B. subtilis or B. licheniformis can result in spoiled bread, by
degradation of starch and the formation of extracellular polysaccharide, resulting in
ropy bread (41, 44, 45). Certain strains of B. licheniformis can produce a toxin, lichenisyn
A, that can lead to foodborne illness (21, 28, 38). Lichenisyn is a non-ribosomally
synthesized lipo-peptide that is heat-stable (14). Due to the pathogenic potential of
strains of B. licheniformis, it is critical to control these spores in the food chain (24).
4
The resistances of spores toward wet heat treatments that are applied in food processing
can vary significantly between strains within the B. subtilis group (4, 15, 20, 29). A
detailed analysis of the heat resistance of spores for fourteen strains of the B. subtilis
group revealed the presence of two distinct groups of strains that produce spores with
significantly different heat resistances of spores (4). For B. subtilis strains, we recently
demonstrated that the high-level heat resistance of spores resulted from the presence
of a spoVA operon (designated spoVA2mob, where mob indicates the presence on a mobile
genetic element) that is encoded on a Tn1546 transposon (3). In another study, we
83
Chapter 4
observed high-level heat resistance of spores of two B. amyloliquefaciens strains (B4140
and B425), with spores of strain B425 showing heat resistance levels similar to those of
spores of B. subtilis strains with a Tn1546 transposon (4). Given the fact that B. subtilis,
B. amyloliquefaciens and B. licheniformis are closely related, variation in resistance
of spores to wet heat in the latter two species may also result from the presence or
absence of a transposon.
In this study, we evaluated presence or absence of the Tn1546 transposon homologue
that mediates heat resistance of spores in B. subtilis in nine strains of B. amyloliquefaciens
and nine strains of B. licheniformis. This was performed by either genome analysis or by
PCR detection. In addition, the detailed spore heat inactivation kinetics were determined
for all eighteen strains, and phenotypic data on spore heat resistance correlated with
the presence or absence of the Tn1546 transposon. The spoVA2mob operons found in B.
amyloliquefaciens and B. licheniformis were introduced into B. subtilis to assess their
role in spore heat resistance.
Materials and methods
Bacterial strains used in this study
The strains used in this study for genomic and phenotypic analyses are listed in Table
1. In this study, nine strains of B. amyloliquefaciens and nine strains of B. licheniformis
were analyzed. For B. amyloliquefaciens strains B425 and B4140, the heat inactivation
kinetics of their spores were described previously (4). Strains of B. subtillis 168 and B.
subtilis B4146 were included in the genome analysis, and were previously analysed for
heat resistance of spores (4).
Genome analysis
84
Multiple sequence alignments were made for protein sequences of conserved
genes that were present in single copy in all genomes using MUSCLE (10). The core
genome phylogenetic tree was constructed using PHYML (12). To verify the presence
of the Tn1546 transposon and the encoded spoVA (designated spoVA2mob) operon, an
orthology matrix was constructed using Ortho-MCL (18) with the genomes of the four
B. amyloliquefaciens strains, the nine B. licheniformis strains, B. subtilis B4146 as a strain
that produces spores with high heat resistance and B. subtilis 168 as a reference strain,
producing spores of normal heat resistance. The genomic organization of the Tn1546
transposon was visualized using Artemis, the Artemis comparison tool (ACT), and
using microbial genomic context viewer (MGcV) (5, 6, 31). For the identified Tn1546
transposons, operon predictions were performed using FGENESB (www.softberry.
com). Additionally, manual sequence comparisons and searches for pseudogenes were
performed for all genes in the transposon.
High-level heat resistance of spores of B. amyloliquefaciens and B. licheniformis
Table 1. Strains used in this study.
Strain No
B425
B4140
10A5
FZB4210A18
DSM7-
DSM1060
101
SB42
B4089
B4090
B4091
B4092
B4094
B4121
B4123
B4124
B4125
B4062
B4146
168spoVA2mob
(B4090)
168spoVA2mob
(DSM7)
Species
Description
B. amyloliquefaciens Isolated from sterilized
milk
B. amyloliquefaciens Isolated from pizza
B. amyloliquefaciens Known as 10A5; NRRL
B-14393, isolated from
soil
B. amyloliquefaciens Known as 10A6; FZB42,
isolated from plant soil
B. amyloliquefaciens Known as 10A18;
CU8004
B. amyloliquefaciens Known as DSM7, isolated from soil
B. amyloliquefaciens Known as DSM1060
B. amyloliquefaciens Received as 101
B. amyloliquefaciens Received as SB42
B. licheniformis
Known as E5/T12,isolated from pea soup
B. licheniformis
Known as T1, isolated
from pea soup
B. licheniformis
Known as T29, isolated
from mushroom soup
B. licheniformis
Isolated from buttermilk powder
B. licheniformis
Isolated from camomile
tea
B. licheniformis
Isolated from sateh
pastry
B. licheniformis
Isolated from sateh
pastry
B. licheniformis
Isolated from pancakes
B. licheniformis
Isolated from pancakes
B. subtilis
Type strain 168
B. subtilis
Isolated from curry
sauce
B. subtilis
168 amyE::spoVA2mob
(B4090) specR
B. subtilis
168 amyE::spoVA2mob
(DSM7) specR
Tn1546
Yes
Genome sequence Reference
LQYP00000000
(4)
No
LQYO00000000
No (PCR) No
(4)
(34)
No
NC_009725
(9)
Yes
FN597644
(37)
Yes
LQYL00000000
No (PCR) No
No (PCR)
No (PCR)
No (PCR)
No
No
Yes
Yes
No
No
No
No
No
Yes
-
No
No
No
LKPM00000000
LQYM00000000
LQYK00000000
LKPN00000000
LKPO00000000
LKPP00000000
LKPQ00000000
LKPR00000000
NC_000964
NZ_JXHR01000000
-
(52)
(33)
This study
This study
(29)
(29)
(29)
This study
This study
This study
This study
4
This study
This study
(17)
(4)
This study
This study
Generic PCR primers were designed for the detection of the Tn1546 encoded genes
tnpA, spoVAC2mob and cls in the other strains of B. amyloliquefaciens. The primers were
designed based on aligned nucleotide sequences of the corresponding genes found in
B. subtilis, B. licheniformis and B. amyloliquefaciens using MUSCLE (10). These primers
(Table 2) were used for verification of the presence of these genes in the five strains of B.
amyloliquefaciens (10A5, FZB42, 10A18, 101 and SB42) for which no genome sequence
was available.
85
Chapter 4
Table 2 . Primers used in this study. Primers were designed for detection of tree genes of the Tn1546 transposon:
tnpA, spoVAC2mob and cardiolipin synthase (cls). The expected PCR fragment sizes for tnpA, spoVAC and cls, are 181
bp, 192 bp, and 687 bp, respectively. For the B. amyloliquefaciens strains 101, SB42, DSM1060, 10A5, and 10A18
a PCR was performed on genomic DNA for the detection of Tn1546 encoded tnpA, spoVAC and cls genes. The
spoVA2mob operon was cloned from B4090 and DSM7 into pDG1730 (11) using the primers EMB-1-F and EMB23-R.
Primer used for
Universal spoVAC2mob
Universal spoVAC2mob
Universal cls
Universal cls
Universal tnpA
Universal tnpA
EMB23
EMB23
Name primer
Uni-spoVAC-F
Uni-spoVAC-R
Uni-cls-F
Uni-cls-R
Uni-tnpA-F
Uni-tnpA-R
EMB1-F
EMB23-R
Heat inactivation kinetics of spores
DNA sequence 5’ to 3’
TGAGCAAACGGCCGGAAATC
ACCTACGCCCAGTACAAATC
TGAGACATTCGGTGCTTCAG
ATCAAGGGTGCTCAACTCTG
CAGCTTGGCTACAGCCTTAC
GGTTCGTTCCCTAAGCTCTC
CGCCGGATCCTGGAAAAGGGGTTATTATCG
GCGCCGGTCTCCAGCTTAAAAAATAGACACTTCTAAC
To verify whether the presence of the Tn1546 transposon influences the heat
resistance of spores, spores were prepared as described previously and subjected to
heat treatments. For the B. amyloliquefaciens strains B4140 and B425, the inactivation
kinetics were previously determined, and those data were included in the current
analysis (4). For all other B. amyloliquefaciens and B. licheniformis strains, spore crops
were prepared. The initial spore concentrations were determined by heating at 80°C for
10 minutes, followed by pour plating in nutrient agar, and incubation for 5 days at 37°C.
Spores were suspended at a concentration of 1 x 108 CFU/mL in peptone physiological
salt. The heat inactivation kinetics of spores were determined for one spore crop per
strain, at three different temperatures, using at least five time points. The inactivation
temperatures were selected on the basis of the resistance of the spores of a strain
(4). The inactivation data obtained were fitted with a log linear model, and decimal
reduction times (D-values) were calculated using equation 1.
t
Equation 1: log N(t ) = log N(0) − D
The D-values at 110°C were plotted versus temperature to visualize the strain variation
in heat resistance of spores in relation to the presence or absence of the Tn1546
transposon. Additionally, for all strains and groups of strains the z-value (i.e. the increase
in temperature required to achieve an additional log unit reduction), and the reference
D-value (Dref) at reference temperature (Tref) 110°C were calculated (46).
Cloning of the spoVA2mob operon
The spoVA2mob operon, including predicted promotor region, was cloned from B.
licheniformis B4090 and B. amyloliquefaciens DSM7 into plasmid pDG1730, as
86
High-level heat resistance of spores of B. amyloliquefaciens and B. licheniformis
previously described (3). The obtained constructs were transformed to B. subtilis 168
and integrated in the amyE locus, to verify the role of this operon in increased heat
resistance of spores. Spores were prepared for strains 168 amyE::spoVA2mob (DSM7),
168 amyE::spoVA2mob (B4090) and 168 as described above, and the heat resistance of
spores of these strains was assessed by heating at 100°C for 1 hour, followed by plating,
incubation and enumeration of survivors.
Results and discussion
Genome mining for the Tn1546 transposon
For B. subtilis strains, it has been demonstrated that the presence of a Tn1546 transposon
leads to a profound increase in heat resistance of the spores (3). Here, the presence of
the Tn1546 transposon was assessed in nine strains of B. amyloliquefaciens and nine
strains of B. licheniformis. The genome sequences of all nine strains of B. licheniformis
were available and in three of these strains the Tn1546 transposon was found (namely,
strains B4090, B4092 and B4094) (Figure 1). The genome sequences of four strains
of B. amyloliquefaciens were available, and the Tn1546 transposon was found in two
of these strains of B. amyloliquefaciens (namely in B425 and DSM7) (Figure 1). The
predicted protein for the transposase TnpA, which is part of the Tn1546 transposon,
was found in orthologous group OG3133 for all of the strains that carry the Tn1546
transposon. Genome sequences were not available for the other five strains of B.
amyloliquefaciens. PCR-based detection of the genes tnpA, spoVAC and cls (that are
present on the transposon and very well conserved) did not reveal the transposon in
these five strains, whereas the PCRs using primers for these three target genes were
positive for B. amyloliquefaciens strains B425 and DSM7. In short, two out of nine
strains of B. amyloliquefaciens and three out of nine strains of B. licheniformis contained
the Tn1546 transposon.
4
Heat resistance of spores is related to the presence of the Tn1546 transposon
The heat resistances of spores of B. amyloliquefaciens and B. licheniformis were
assessed in relation to the presence or absence of the Tn1546 transposon. Detailed heat
inactivation kinetics of spores were obtained for seven strains of B. amyloquefaciens
(including the two strains carrying the Tn1546 transposon) and nine strains of B.
licheniformis (Table 3). The inactivation kinetics of spores of B. amyloliquefaciens
strains B4140 and B425 have been reported previously (4) and are included in the
current analysis (Figure 2B). The heat resistance of spores was visualized by plotting
the reference decimal reduction time of spores per strain at the reference temperature
of 110°C (D110°C-values) in relation to the presence or absence of the Tn1546 transposon
(Table 3). The heat resistance of spores was plotted for nine strains of B. licheniformis
(Figure 2A) and nine strains of B. amyloliquefaciens (Figure 2B).
87
Cardiolipin synthase
yetF N-terminal
yetF C-terminal
Unknonw fuction
spoVAEb2mob
Unknonw fuction
spoVAD2mob
spoVAC2mob
Unknonw fuction
Unknonw fuction
Unknonw fuction
Unknonw fuction
Mn Catalase
ger(x)C
ger(x)A
tnpR
tnpA
tnpA
A
N-acetylmuramoyl L-alanine amidase
Chapter 4
(B. subtilis B4146)
(Site specific recombination)
(B. amyloliquefaciens B425)
(B. amyloliquefaciens DSM7)
(B. licheniformis B4090)
(Gene loss)
(B. licheniformis B4094)
(B. licheniformis B4092)
B
Core genome
Phylogentic tree
0.1
Strain name
Tn1546 transposon
Present / absent
Bacillus subtilis 168
Bacillus subtilis B4146
Bacillus amyloliquefaciens B425
Bacillus amyloliquefaciens DSM7
Bacillus amyloliquefaciens B4140
Bacillus amyloliquefaciens FZB42
Bacillus licheniformis B4123
Bacillus licheniformis B4121
Bacillus licheniformis B4125
Bacillus licheniformis B4090
Bacillus licheniformis B4089
Bacillus licheniformis B4094
Bacillus licheniformis B4092
Bacillus licheniformis B4124
Bacillus licheniformis B4091
+ (1x)
+
+ (3x)
+ (1x)
+ (1x)
+ (1x)
-
Figure 1. A) Overview of the Tn1546 transposons found in B. subtilis B4146, B. amyloliquefaciens B425 and DSM7,
and B. licheniformis B4090, B4092 and B4090. The predicted gene functions are indicated for the transposon of
B. subtilis B4146: a transposase gene (tnpA), a resolvase gene (res), an operon of N-acetylmuramoyl-L-alanine
amidase, gerKA and ger(X)C (Operon 1), an operon of a gene with unknown function and a manganese catalase
(Operon2), an operon of two genes with unknown functions, spoVAC, spoVAD, spoVAEb and two genes with
unknown functions (Operon 3, spoVA2mob), a fragmented yetF gene (Gene 4), and a cardiolipin synthase gene
(Gene 5). The transposons in B. amyloliquefaciens are smaller, probably due to site specific recombination
events, whereby operon 1 and operon 2 were lost. In B. licheniformis B4092 and B4094 the resolvase gene was
not present, which is most likely a result of gene loss. B) Maximum likelihood phylogenetic tree based on core
genome of single genes of fifteen strains of the B. subtilis group. The presence and copy number of the Tn1546
transposon was indicated behind the corresponding strains. B. subtilis B4146, B. licheniformis B4090, B4092, and
B4094 carry a single transposon. The B. amyloliquefaciens strain DSM7 carries three copies of the transposon,
while for strain B425 the copy number could not be determined.
Two strains of B. amyloliquefaciens, namely B425 and DSM7, contained the Tn1546
transposon. These strains produced spores that required significantly longer heating
times (p<0.001) at 110°C, i.e. approximately 15 times, to achieve one decimal reduction,
than spores of the other seven strains without this transposon. To illustrate the difference
in heat resistance of spores: heating at 110°C for 5 minutes results in 0.6 log reduction for
the strains with Tn1546 transposon, whereas the group without the Tn1546 transposon
would show more than 10.5 log reduction. Strains B. licheniformis B4090, B4092 and
B4094 contained the Tn1546 transposon, and the spores of these strains all required
88
High-level heat resistance of spores of B. amyloliquefaciens and B. licheniformis
longer heating times (p<0.001) (2.5 times) to reach a decimal reduction than the spores
of the six B. licheniformis strains that did not possess the Tn1546 transposon. Heating at
110°C for 5 minutes results in a calculated 3.4 log reduction for the strains carrying the
Tn1546 transposon, whereas the other group without the Tn1546 transposon would
show 8.5 log reduction.
The number of spoVA2mob operons in B. subtilis was found to correlate with the level
of heat resistance of spores; strains carrying three copies produced spores with the
highest level of heat resistance (3). B. licheniformis strains B4090, B4092 and B4094
contained a single copy of the Tn1546 transposon with a single spoVA2mob operon.
The heat resistances of spores of B. licheniformis with or without this operon were
significantly different, but relatively modest. For B. amyloliquefaciens, spores of strains
B425 and DSM7 showed comparable high-levels of heat resistance, which were
significantly higher than those of the spores of other B. amyloliquefaciens strains. Strain
DSM7 contains three Tn1546 transposable elements, and it is likely that strain B425
also contains multiple copies, however this remains to be established.
A
B
B. licheniformis
B. amyloliquefaciens
*** p<0.001
Decimal reduction time (min) at 110 ° C
*** p<0.001
10
10
1
1
0.1
4
0.1
No Tn1546
Tn1546
No Tn1546
Tn1546
Figure 2. Spore heat inactivation kinetics at 110 °C of A) Nine strains of B. licheniformis and B) Nine strains of B.
amyloliquefaciens. The closed circles and squares indicate the absence of a Tn1546 transposon in the strains of B.
licheniformis and B. amyloliquefaciens, respectively. The open circles and squares indicate the presence of at least
one Tn1546 transposon in the strains of B. licheniformis and B. amyloliquefaciens, respectively.
The spoVA2mob operon is responsible for increased heat resistance of spores
The introduction of the spoVA2mob operon originating from B. subtilis strain B4067 in
laboratory strain B. subtilis 168, has been shown to result in high-level heat resistance
of spores (3). To establish whether the spoVA2mob operons present in the Tn1546
transposons of B. licheniformis B4090 and B. amyloliquefaciens DSM7 have a functional
role, these were introduced into B. subtilis 168. The B. subtilis 168 mutants carrying the
spoVA2mob genes from B. licheniformis B4090 and B. amyloliquefaciens produced spores
with significantly higher heat resistance than the parent strains: after heating at 100°C
89
Chapter 4
Table 3. Detailed spore heat inactivation kinetics and calculated reference D-value (Dref ) at a reference
temperature (Tref ) of 110 °C of spores of nine strains of B. amyloliquefaciens and spores of nine strains of B.
licheniformis.
Species
Strain
Tn1546
Dref 110 °C
95%
(min)
upper PI
10A5
No
1.02
103.26
10A6
No
0.54
2.06
10A18
No
0.61
8.74
101
No
0.55
0.98
No
0.59
1.36
B. amyloliquefaciens SB42
B4140a
No
0.13
2.54
DSM1060 No
0.41
1.78
DSM7
Yes
5.71
99.87
B425a
Yes
10.80
25.62
B4089
No
0.50
4.21
B4091
No
0.58
3.33
B4121
No
0.65
3.64
B4123
No
0.50
1.95
B4124
No
0.72
6.40
B. licheniformis
B4125
No
0.69
12.01
B4090
Yes
1.48
5.17
B4092
Yes
1.64
3.48
B4094
Yes
1.24
22.24
a : The D110 °C was calculated based on previously published data (4).
95%
lower PI
0.01
0.14
0.04
0.31
0.26
0.01
0.09
0.33
4.55
0.06
0.10
0.12
0.13
0.08
0.04
0.42
0.77
0.07
z-value
(°C)
11.13
13.88
10.32
11.66
11.44
7.21
12.00
8.48
6.23
9.99
9.53
9.33
9.97
10.01
16.32
22.10
16.07
12.51
S.E.
1.09
1.54
2.53
0.88
0.68
0.17
0.33
2.12
0.41
2.31
1.74
1.45
1.30
2.18
2.72
3.01
0.85
1.65
for 60 minutes, the spores of B. subtilis 168 amyE::spoVA2mob (containing the spoVA2mob
operon of B. licheniformis B4090) showed survival of 4.0 log10 unit (± 0.4), and B. subtilis
168 amyE::spoVA2mob (containing the spoVA2mob operon of B. amyloliquefaciens DSM7)
showed survival of 1.7 log10 unit (± 0.5), whereas the spores of B. subtilis 168 were
inactivated below the detection limit. The control strain B. subtilis 168 amyE::spoVA2mob
(containing the spoVA2mob operon of B. subtilis B4067) (3), showed survival of 2.8 log10
unit (± 0.05).
The spoVA2mob operon differs from the spoVA operon (designated spoVA1) that is encoded
on the chromosome of B. subtilis (43), B. amyloliquefaciens and B. licheniformis. The
SpoVA proteins encoded in the spoVA1 operon are required for uptake of DPA during
the sporulation process and upon deletion or disruption of these genes in B. subtilis
168, sporulation is not completed (43). In addition, the SpoVA proteins are involved in
the release of Ca-DPA during the germination process (49, 50), with the SpoVAC protein
functioning as a mechano sensitive channel during germination (48). The SpoVAD
protein has a binding pocket whereby it can directly bind DPA (19). Both the spoVA1 and
the spoVA2mob operons contain genes encoding for SpoVAC, SpoVAD and SpoVAEb, while
the other genes in the operons are different.
90
High-level heat resistance of spores of B. amyloliquefaciens and B. licheniformis
It is known that environmental conditions during sporulation, such as temperature,
matrix and medium composition, can influence the heat resistance of spores of the B.
subtilis group (8, 25, 35). To allow for a direct comparison of the resistance properties
of spores of the different strains, the sporulation conditions were kept constant in this
study. Factors that are known to influence spore heat resistance include the composition
of the sporulation medium, and it is known that the addition of salts (Ca2+, Mn2+, Mg2+
and K+) results in higher resistances of spores to heat (8). Furthermore, the temperature
during sporulation is an important factor that influences heat resistance of B. subtilis
spores (25). In line with these findings, the heat resistance of spores of a B. licheniformis
strain was higher upon sporulation at 45°C, with a modeled optimum at 49°C, than at
lower temperatures such as 20°C (2). Overall, the environmental sporulation conditions,
the presence or absence of genetic elements such as the spoVA2mob operon, and the
storage conditions will ultimately determine the heat resistance properties of spores.
A
P-sigG
DUF
1657
Yhcn / YlaJ
spoVAC2mob
spoVAD2mob
spoVAEb2mob DUF
1657
DUF 421
DUF1657
spoVA2mob
operon
Viable spore count (log10 CFU mL-1)
B
12
N(0) 80 °C 10 min
N(t)100 °C 60 min
10
4
8
6
4
2
0
Bacillus subtilis
168
Bacillus subtilis
168
amyE::spoVA2mob
(B4067)
2mob
Bacillus subtilis
168
amyE::spoVA2mob
(B4090)
Bacillus subtilis
168
amyE::spoVA2mob
(DSM7)
Figure 3. A) Overview of the spoVA
operon, as initially found in B. subtilis strain B4146. The spoVA2mob operon
has a predicted sigma G binding site upstream of the first gene. The operon consists of seven genes: a gene of
unknown function with a predicted DUF1657 domain, a gene of unknown function with a predicted YhcN/YlaJ
domain, spoVAC, spoVAD, spoVAEb, a gene of unknown function with a predicted DUF1657 domain, and a gene
of unknown function with a predicted DUF421 and a DUF1657 domain. B) Spore counts after heating at 80 °C
for 10 minutes and at 100°C for 60 minutes for strains of B. subtilis 168, B. subtilis 168 amyE::spoVA2mob (B4067, data
from (3)), B. subtilis 168 amyE::spoVA2mob (B4090), and B. subtilis 168 amyE::spoVA2mob (DSM7). The downward arrow
indicates that the spores were inactivated below detection limit.
91
Chapter 4
It is therefore conceivable that spores produced under laboratory conditions do not
necessarily reach the heat resistance levels of spores that are present in food products
(20, 47).
Detailed analysis of the Tn1546 transposon
The composition of the Tn1546 transposon in B. licheniformis strains B4090, B4092 and
B4094 is shown in Figure 1A. In these strains, the Tn1546 transposon is highly similar to
the one found in B. subtilis B4146 (Figure 1A). The transposons found in B. subtilis and
B. licheniformis consist of genes that are required for transposition, and furthermore
contain three operons and two single genes. The Tn1546 transposon found in the B.
amyloliquefaciens strains DSM7 and B425 was smaller than the transposon found in B.
subtilis and B. licheniformis. In both B. amyloliquefaciens strains, the first two operons
were absent, possibly due to a site-specific recombination event, as a recombinase gene
and a hypothetical gene were present at that genomic location.
The evolutionary relatedness of the different strains and species was visualized in a
maximum likelihood core genome phylogenetic tree, based on concatenated protein
sequences of conserved genes present in single copy in all genomes (Figure 1B).
The species B. amyloliquefaciens, B. licheniformis and B. subtilis clustered in separate
branches of the phylogenetic tree. For B. amyloliquefaciens, the strains with the Tn1546
transposon clustered together, whereas this was not the case for B. licheniformis strains
carrying the Tn1546 transposon.
The genomic locations of the Tn1546 transposons were different for B. subtilis, B.
amyloliquefaciens and B. licheniformis. In B. subtilis, the transposon was found at two
genomic locations, namely in yitF and between yxjA and yxjB (3). In B. amyloliquefaciens,
three Tn1546 transposons were found in strain DSM7 at three different genomic
locations, namely between a gene encoding for a fructose-1,6-bisphophatase and a
hypothetical gene, between two hypothetical genes, and between a hypothetical gene
and rapK. For B. amyloliquefaciens strain B425 it was not possible to determine the
genomic locations and copy number of the Tn1546 transposon, since contig breaks
were present on both sides of the Tn1546 transposon. In B. licheniformis strains B4090,
B4092 and B4094, a single Tn1546 transposon was found integrated in a gene that
encodes a D-alanyl-D-alanine carboxypeptidase. For B. subtilis, it has been shown that
different insertion locations of the Tn1546 transposon led to high-level heat resistance
of B. subtilis spores (3). It remains to be established whether the location of the insertion
of the transposon in the genome of B. amyloliquefaciens and B. licheniformis plays a role
in the level of heat resistance of spores.
92
High-level heat resistance of spores of B. amyloliquefaciens and B. licheniformis
Detailed analysis revealed that some genes in the Tn1546 transposon were mutated and
present as pseudogenes in the transposon of some strains. The genes tnpA and tnpR in
the Tn1546 transposon (which are required for active transposition) were intact and
present in B. licheniformis strain B4090, and in B. amyloliquefaciens strains B425 and
DSM7. This suggests that active transposition of the element may be possible for these
strains, although active transposition of the Tn1546 transposon is believed to require a
plasmid intermediate, as has been described in Enterococcus faecium (2). Interestingly,
B. amyloliquefaciens DSM7, containing the intact tnpA and tnpR genes, contained three
Tn1546 transposons. The encoded proteins required for transposition potentially
allowed for internal transposition within the chromosome of strain DSM7. In B. subtilis
strain B4146 and in B. licheniformis strains B4092 and B4094, the transposition genes
are absent or not intact, suggesting that active transposition of the Tn1546 transposon
is not likely to occur in these strains. This does not mean that the transposons cannot
be transferred; transfer of genetic material including the Tn1546 transposon can be
mediated by other transfer mechanisms, such as phage transduction, as described
previously for B. subtilis (3), or via the uptake of external DNA via natural competence
(16).
Conclusions
Variation in heat resistance of spores exists between strains of different spore forming
species (4, 20, 29, 30). In this study, a genomic analysis revealed the presence of Tn1546
transposons in three strains of B. licheniformis and in two strains of B. amyloliquefaciens.
The presence of this transposon, containing the spoVA2mob operon, correlated with highlevel heat resistance of spores. A functional role of the spoVA2mob operon in increasing
the heat resistance of spores was demonstrated by cloning these operons in B. subtilis
168, resulting in spores with high-level heat resistance. Clearly, mere identification
of the species of spores in food products does not provide information on the heat
resistance levels of these spores. The knowledge obtained in this study on the spoVA2mob
operon can be used for specific detection of strains of the B. subtilis group that produce
spores with high-level heat resistance. Multiple DNA based methods can be used for
the detection of such genetic elements, such as whole genome sequencing and specific
PCR detection, among others (1, 7). The data presented in this study can be used for
improved control of bacterial spores in the food chain.
4
Acknowledgements
The authors have declared that no competing interests exist. The research was funded
by TI Food and Nutrition, a public-private partnership on pre-competitive research in
food and nutrition. The funding organization had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
93
Chapter 4
Supplementary Dataset 1
Orthology matrix constructed using Ortho-MCL with the genomes of the four B.
amyloliquefaciens strains, the nine B. licheniformis strains, B. subtilis B4146 as a strain
that produces spores with high heat resistance and B. subtilis 168 as a reference strain
that produces spores with normal heat resistance.
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