The invention relates to a kit and methods and means for the determination of the presence of heat resistant organisms. The invention further relates to the provision organisms wherein the heat resistance is modulated.

BACKGROUND

In the food industry, mesophilic, aerobic spore-forming bacteria are ubiquitously present (Anonymous, 2005; Gould, G. W. 2006). Their dormant endospores are highly resistant to environmental insults, and are able to survive various preservation regimes commonly used in the food industry. Heat treatment is commonly applied in food processing to inactivate bacteria and their spores. Insufficient heat treatment of bacterial spores may allow for survival of spores, potentially leading to food spoilage upon germination and outgrowth, and, in the case of food borne pathogens, to food poisoning (De Jonghe et al, 2010; Scheldeman et al, 2006). Depending on the spore heat resistance, heating regimes may exceed the required heat load, often negatively affecting product quality. Hence, knowledge on required heat load to inactivate spores in relation to product characteristics are important.

The heat resistance and germination properties of bacterial spores and their phenotypic variation are a major concern of the food industry (Eijlander et al, 2011; Hornstra et al, 2009). Different Bacillus species including Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Bacillus thermoamylovorans, Geobaccili and Bacillus sporothermodurans are able to form highly heat resistant spores that can survive heating regimes that are commonly used in food preservation (Scheldeman et al, 2005). Various spores belonging to the genera of Bacillus, Aneurinibacillus, and Paenibacillus are able to survive heat treatments of temperatures higher than 120°C (te Giffel et al, 2002).

The heat resistance of spores can vary between species and even between strains of one species. Variation in spore heat resistance between different strains of Bacillus sp. has been reported, but not extensively studied. Van Asselt and Zwietering, 2006, indicated that strain variation in B. cereus significantly influences spore heat resistance. Another example is B. sporothermodurans, which produces spores that are highly heat resistant and can survive UHT treatments. For this bacterium, clear differences were observed in decimal reduction times (JJ-value) at 100 °C for spores of strains of various isolation sources (Scheldeman et al, 2005). Variation in spore heat resistances of strains of B. subtilis isolated from different soups has also been reported by Oomes et al, 2007. Kort et al, 2005, compared the spore heat resistances of a laboratory strain of B. subtilis 168 with B. subtilis A163 which was isolated from peanut chicken soup, and found significant differences in spore heat resistances, namely a /J-value of 1.4 minutes at 105 °C for strain 168 and 0.7 minutes at 120 °C for strain A163. In a study performed by Lima et al, 2011, spores with high thermal resistance were isolated from cocoa powder. The spores with the highest thermal resistance mainly belonged to the B. subtilis group and displayed large variation in spore heat resistance after sporulation under laboratory conditions (Lima et al, 201 1). The observed variations in spore heat resistance within a species can complicate predictive modeling and design of e.g. food processes. Therefore, better insight into spore heat resistance is required including the effect of strain variation on spore heat resistance. The present invention illustrates the presence of two distinct groups of spore heat resistance within the species of B. subtilis and depicts genes involved in spore heat resistance.

DESCRIPTION OF THE INVENTION

In a study that focused on variation in spore heat resistance within the species B. subtilis, for eighteen strains of B. subtilis from which the spore heat resistance phenotype was determined, it was surprisingly found that these could be clustered into two groups. Using extensive analysis, multiple genes and a mobile genetic element were identified that correlate to the group of high spore heat resistance. In addition, the present invention provides for means of specific detection heat resistant spores in a sample. This is the first time that means for exclusive and specific detection of heat resistant spores are provided. Previous research (e.g. Oomes et al, 2007) enabled the classification of heat resistant and non-heat resistant spores, once isolated, into specific phylogenetic groups, but could not exclusively and specifically detect heat resistant spores in a sample. Accordingly, the invention provides a method for the determination of a heat resistant organism in a sample, preferably in a microorganism, more preferably in a spore of a microorganism.

In a first aspect, there is provided a method for the determination of the presence of a heat resistant organism in a sample from a product comprising, assessing whether in the sample at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotides are present encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%), at least 99% or most preferably 100% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 15-28, or selected from the group consisting of SEQ ID NO: 15-28 and 41-43, or selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 or 43, 25, 41 and 42, wherein the presence of at least one, at least two, at least three, at least four, at least five of such polynucleotide, preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, is a measure for the presence of a heat resistant organism in the sample. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 21 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100%) sequence identity with SEQ ID NO: 22 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%), at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 23 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%), at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 24 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 43 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 25 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 41 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%), at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 42 is determined. Said method is herein referred to as a method according to the invention; said polynucleotide is herein referred to as a polynucleotide according to the invention. In a method according to the invention and in any embodiment according to the invention wherein the presence of any polynucleotide according to the invention is determined, the presence of the polynucleotide may be detected by detecting the polynucleotide itself and/or a product of the polynucleotide may be determined to assess the presence of the polynucleotide; such product may be a transcript such as an RNA or a part thereof, or may be a polypeptide, or a part thereof, encoded by the polynucleotide. A polynucleotide according to the invention and/or an indigenous counterpart or paralog of a polynucleotide according to the invention may be present in the genome or may be present episomal such as on a plasmid. In such case, the determination of a polynucleotide according to the invention can or will be determined by assessing whether an additional copy of the polynucleotide is present.

As used herein, a heat resistant organism is an organism that is able to resist great heat or high temperature without being damaged; heat resistance is preferably assessed by determination of the germination properties of the organism. Multiple time-temperature combinations can be applied to assess heat resistance, such as an assay based on batch heating in capillary tubes, as used in the experimental section in "Spore enumeration" to distinguish two groups of spore heat resistance within the B. subtilis. Heating for one hour at 100°C, resulted in a 10.2 log reduction in viability for the low spore heat resistance group and a 0.1 log reduction for the high spore heat resistance group, using the \ogD _re f from the batch heating experiment. Using a similar approach, heating for 5 minutes at 110 °C resulted in a 18.6 log reduction for the low spore heat resistance group, and a 0.1 log reduction for the high spore heat resistance group. It should be noted that time-temperature combinations listed herein are based on spores prepared under laboratory conditions wherein thus excluding variations in spore heat resistance based on the history of the spores and a potential effect of the food matrix; the person skilled in the art however knows how to compensate for these differences.

Accordingly, heat resistance is herein preferably defined as at most 5 log, more preferably 4 log, more preferably 3 log, more preferably 2 log, more preferably 1 log, more preferably 0.5 log, more preferably 0.2 log, more preferably 0.1 log and most preferably 0.05 log reduction in viability of spores when heating in capillary tubes for one hour at 100°C or when heating in capillary tubes for 5 minutes at 110°C, preferably when heating in capillary tubes for 5 minutes at 1 10°C. Viability is preferably assessed using the assay as described in the experimental section in "Spore enumeration" . Modulation of heat resistant may have further implications than modulation of the heat resistance itself; it may e.g. result in alteration of sensitivity to certain compounds such as antibiotics, bactericidals etc such as e.g. vancomycin, nysin etc.

An organism is defined herein as any contiguous living system (such as animal, fungus, micro-organism, or plant). An organism may be either unicellular or multicellular wherein the cells are grouped into specialized tissues and organs. In a method or any other embodiment according to the invention, a heat resistant organism preferably is a microorganism or a spore of a microorganism, wherein the microorganism preferably is a bacterium, more preferably a Gram positive bacterium, more preferably a Bacillus strain, more preferably a Bacillus subtilis, even more preferably a B. subtilis selected from the group consisting of B. subtilis 4067, 4068, 4069, 4070, 4071, 4072, 4073, 4145, and 4146. B. subtilis 4067 has been deposited under the regulations of the Budapest Treaty at the "Centraal Bureau voor Schimmel cultures" (CBS), Uppsalalaan 8, 3584CT Utrecht, The Netherlands; the deposit number is CBS 137174. In a method or any other embodiment according to the invention, a sample relates to a portion, piece, or segment that is representative of a whole.

A product in a method or any other embodiment according to the invention may be any known product such as a food product or an ingredient, a waste stream and/or recycling stream. Examples of products are, but are not limited to herbs, spices, flours, dairy products such as milk and milk powder, cocoa, cocoa powder, rice, wheat bakery ingredients, bakery products, meat, meat products, pasta products, plant material, feed, soil, silage and compounds or products derived from these. The food product is preferably, but not limited to, a product comprising a plant material, more preferably a cocoa or cocoa powder comprising food product. A food product in a method or any other embodiment according to the invention may also comprise a compound of plant and/or animal origin. The food product may be intended for ingestion by an organism and subsequent assimilation by the organism's cells to produce energy, maintain life, and/or stimulate growth.

The term polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or isochemically modified, non-natural, or derivatized nucleotide bases. A polynucleotide in a method or any other embodiment according to the invention is preferably present as an insert in or close to a gene associated with sporulation, preferably a gene associated with cortex build-up, more preferably a gene having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with yitF (SEQ ID NO: 29) or yitG (SEQ ID NO: 30), even more preferably yitF (SEQ ID NO: 29). The at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotide(s) according to the invention and preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, are preferably present in a mobile genetic element, e.g. a transposon. Such mobile genetic element is preferably present as an insert in or close to a gene associated with sporulation, preferably a gene associated with cortex build-up, more preferably a gene having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 96%, at least 97%, at least 98%>, at least 99% or most preferably 100%> sequence identity with yitF (SEQ ID NO: 29) or yitG (SEQ ID NO: 30), even more preferably yitF (SEQ ID NO: 29). When the at least one polynucleotide is present in a mobile genetic element, the presence of the at least one polynucleotide may be assessed by determining the presence of the at least one polynucleotide itself and/or by determining the presence of a sequence associated with the mobile genetic element, such as a sequence flanking the mobile genetic element or an integrase, transposase etc. The size of the mobile genetic element comprising the at least one polynucleotide may be used to determine whether the at least one polynucleotide is present.

Herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably. Sequence identity is herein preferably defined as a percentage of identity and is determined by calculating the ratio of the number of identical nucleotides/amino acids in the sequence divided by the length of the total nucleotides/amino acids of said sequence, preferably minus the lengths of any gaps and is further defined elsewhere herein.

The method according to the invention depicted here above can conveniently be used for the selection of a batch of a product.

Accordingly, the invention further relates to a method for selecting a batch of a product comprising:

a. providing a sample of a batch of a product,

b. assessing whether in the sample at least one, at least two, at least three, at least four, at least five polynucleotide(s) is/are present encoding a polypeptide having at least

60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100%) sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 15-28, or selected from the group consisting of SEQ ID NO: 15-28 and 41-43, or selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 or 43, 25, 41 and 42, wherein the presence of at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen of such polynucleotide(s) preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, is a measure for the presence of a heat resistant organism in the sample, and

c. separating a batch wherein a heat resistant organism is present or separating a batch wherein a heat resistant organism is not present. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%), at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 21 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 22 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 23 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%), at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 24 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%), at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 43 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 25 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 41 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 42 is determined.

Said method is herein further referred to as a method according to the invention.

The methods depicted here above can conveniently be used for the production of a batch of an organism.

Accordingly, the invention relates to a method for the production of a batch of a microorganism, comprising:

a. culturing a batch of a microorganism,

b. providing a sample of the culture, before during or after step (a),

c. assessing whether in the sample at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotides are present encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%), at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 15-28, or selected from the group consisting of SEQ ID NO: 15-28 and 41-43, or selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 or 43, 25, 41 and 42, wherein the presence of at least one at least two, at least three, at least four, at least five of such polynucleotide(s) preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, is a measure for the presence of a heat resistant organism in the sample, and

d. separating a batch wherein a heat resistant organism is present or separating a batch wherein a heat resistant organism is not present.

Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100%) sequence identity with SEQ ID NO: 21 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 22 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%), at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 23 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 24 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 43 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%), at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 25 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%), at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 41 is determined. Preferably, the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 42 is determined.

Said method is herein further referred to as a method according to the invention.

In the methods according to the invention, assessment of the presence of the at least one polynucleotide according to the invention may be performed using any method known to the person skilled in the art. Such methods include detection of the polynucleotide itself, such as polymerase chain reaction (PCR) or Ligation Detection Reaction (LDR), liquid and solid hybridization assays such as Spot blot, Northern blot, Southern blot, and sequencing including whole genome sequencing. Also included are methods to detect a product of the polynucleotide, such as a transcript such as an RNA or a part thereof, or a polypeptide, or a part thereof, encoded by the polynucleotide. Protein detection assays such as Enzyme Linked Immune Sorbent Assay (ELISA), immune precipitation and Western blot are thus included. The assessment is preferably performed using the polymerase chain reaction (PCR). A Ligation Detection Reaction (LDR) is preferably performed essentially as described in WO2010/049489.

In a second aspect, the invention relates to means for assessing whether in a sample at least one polynucleotide is present encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 96%, at least 97%, at least 98%), at least 99% or most preferably 100% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 15-28, or selected from the group consisting of SEQ ID NO: 15-28 and 41-43, or selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 or 43, 25, 41 and 42, wherein the at least one polynucleotide preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, is preferably present as an insert in or close to a gene associated with sporulation, preferably a gene associated with cortex build-up, more preferably a gene having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 96%, at least 97%, at least 98%), at least 99% or most preferably 100% sequence identity with yitF (SEQ ID NO: 29) ovyitG (SEQ ID NO: 30), even more preferably yitF (SEQ ID NO: 29). The at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotide(s) preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, are preferably present in a mobile genetic element, e.g. a transposon. When the at least one polynucleotide is present in a mobile genetic element, the presence of the at least one polynucleotide may be assessed by determining the presence of the at least one polynucleotide itself and/or by determining the presence of a sequence associated with the mobile genetic element, such as a sequence flanking the mobile genetic element or an integrase, transposase etc. The size of the mobile genetic element comprising the at least one polynucleotide may be used to determine whether the at least one polynucleotide is present.

Said means are herein further referred to as a means according to the invention. Any means known to the person skilled in the art can be used. Preferably, a means according to the invention is selected from the group consisting of an oligonucleotide and a solid carrier comprising such oligonucleotide such as a biochip or an array. More preferably, a means according to the invention comprises or consists of an oligonucleotide that is suitable for use in the polymerase chain reaction (PCR). An oligonucleotide generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double- stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A preferred means according to the invention comprises an oligonucleotide selected from Table 1 or a part thereof: a more preferred means according to the invention is an oligonucleotide selected from Table 1. The invention is not limited to the oligonucleotides in Table 1; these are depicted as examples.

Table 1: Examples of PCR primers to verify presence/absence and location of a polynucleotide according to the invention.

Primer 5'-3' SEQ ID NO: l.yitF-F TGGGCTTCAACATTGGAGAC 31

l .tnpA-R TGGCTACAGCCTTACGTGAG 32

2.gerXC-F TTGGGTGGGCAAAGGCGAAG 33

2 spoVAC-R CCCACCAAATTGGGCCATTC 34

3.spoVAC-F GAAATCCGACGGTGGGTACG 35

3.hyp-R GGTCTGAGGCTCCTTGATTG 36

4.hyp-F TCAATCAAGGAGCCTCAGAC 37

Hyp IF GTAGGATCAGATGTTAAACAGTG 61

HyplR TAGAAACCTTTGTACTGAAGTTCC 62

Hyp2F TGCCTGAATGGTTAGATGTAGC 63

Hyp2R CCGTTCCAAGAGCAAATAGTTC 64

4.Cls-R AATAGTCTTGGTCGCCTGTG 38

5.Cls-F GGTTCCTCGCCACAATAATC 39

5 yitG-R CGCCCTGCTTGTCTGGTATG 40

Preferably, the means according to the invention are capable of detecting the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 21. Preferably, the means according to the invention are capable of detecting the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%>, at least 70%, at least 75%, at least 80%>, at least 85%>, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100%> sequence identity with SEQ ID NO: 22. Preferably, the means according to the invention are capable of detecting the presence of a polynucleotide encoding a polypeptide having at least 60%>, at least 65%>, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 23. Preferably, the means according to the invention are capable of detecting the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 24. Preferably, the means according to the invention are capable of detecting the presence of a polynucleotide encoding a polypeptide having at least 60%), at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 43. Preferably, the means according to the invention are capable of detecting the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 25. Preferably, the means according to the invention are capable of detecting the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 41. Preferably, the means according to the invention are capable of detecting the presence of a polynucleotide encoding a polypeptide having at least 60%), at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 42.

In a third aspect, the invention relates to a kit for the determination of the presence of a heat resistant organism in a sample comprising means for assessing whether in a sample at least one, at least two, at least three, at least four, at least five polynucleotide(s) is/are present encoding a polypeptide having at least at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100%) sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 15-28, or selected from the group consisting of SEQ ID NO: 15-28 and 41-43, or selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 or 43, 25, 41 and 42, wherein the at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotide(s) preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, is preferably present as an insert in or close to a gene associated with sporulation, preferably a gene associated with cortex build-up, more preferably a gene having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with yitF (SEQ ID NO: 29) or yitG (SEQ ID NO: 30), even more preferably yitF (SEQ ID NO: 29).

Preferably, the kit is capable of determining the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 21. Preferably, the kit is capable of determining the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 22. Preferably, the kit is capable of determining the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 23. Preferably, the kit is capable of determining the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 24. Preferably, the kit is capable of determining the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 43. Preferably, the kit is capable of determining the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 25. Preferably, the kit is capable of determining the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 41. Preferably, the kit is capable of determining the presence of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 42. Said kit is herein further referred to as a kit according to the invention.

Preferably, the means are means as defined in the second aspect of the invention, more preferably they are selected from the group consisting of an oligonucleotide and a solid carrier comprising such oligonucleotide such as a biochip or an array. More preferably, said means comprise or consist of an oligonucleotide that is suitable for use in the polymerase chain reaction (PCR) or Ligation Detection Reaction (LDR). More preferably, said means comprise an oligonucleotide or part thereof selected from the oligonucleotides in Table 1. In a fourth aspect, the invention relates to an organism derived from a parent organism wherein the expression of at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotide(s) encoding a polypeptide(s) having at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 15-28, or selected from the group consisting of SEQ ID NO: 15-28 and 41-43, or selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 or 43, 25, 41 and 42, is modulated as compared to the parent organism where the organism is derived from, when assayed under substantially identical conditions. Such organism is herein referred to as an organism according to the invention.

Preferably, in an organism according to the invention the expression of a polynucleotide encoding a polypeptide having at least 60%, at least 65%>, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%), at least 98%>, at least 99% or most preferably 100%> sequence identity with SEQ ID NO: 21 is modulated. Preferably, in an organism according to the invention the expression of a polynucleotide encoding a polypeptide having at least 60%>, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 22 is modulated. Preferably, in an organism according to the invention the expression of a polynucleotide encoding a polypeptide having at least 60%>, at least 65%>, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 23 is modulated. Preferably, in an organism according to the invention the expression of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 24 is modulated. Preferably, in an organism according to the invention the expression of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%), at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 43 is modulated. Preferably, in an organism according to the invention the expression of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 25 is modulated. Preferably, in an organism according to the invention the expression of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 41 is modulated. Preferably, in an organism according to the invention the expression of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 42 is modulated.

Preferably, the modulated expression of at the least one, at least two, at least three, at least four, at least five polynucleotide(s) according to the invention, preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, is enhanced expression or reduced expression. Preferably, an organism according to the invention is a recombinant organism. A recombinant organism is an organism that contains a different combination of alleles from either of its parents. More preferably an organism according to the invention is a microorganism or a spore of a microorganism, wherein the microorganism preferably is a bacterium, more preferably a Gram positive bacterium, more preferably a Bacillus strain, more preferably Bacillus subtilis, even more preferably a B. subtilis selected from the group consisting of B. subtilis 4067, 4068, 4069, 4070, 4071, 4072, 4073, 4145, and 4146. The organism according to the invention and an organism in any embodiment according to the invention may be a probiotic or spore thereof; it preferably has increased or decreased resistance to passage through the gut by its increased or decreased heat resistance.

In an organism according to the invention as described here above and in an organism in any embodiment according to the invention, at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotide(s) according to the present invention, preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, are preferably present.

Preferably, a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%), at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 21 is present. Preferably, a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 22 is present. Preferably, a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 23 is present. Preferably, a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 24 is present. Preferably, a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%), at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 43 is present. Preferably, a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or most preferably 100% sequence identity with SEQ ID NO: 25 is present. Preferably, a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%), at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 41 is present. Preferably, a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or most preferably 100% sequence identity with SEQ ID NO: 42 is present. More preferably, two, or three or four or five or more copies of at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotide(s) according to the present invention, preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, are present.

Preferably, two, or three or four or five or more copies of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or most preferably 100% sequence identity with SEQ ID NO: 21 are present. Preferably, two, or three or four or five or more copies of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 22 are present. Preferably, two, or three or four or five or more copies of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with SEQ ID NO: 23 are present. Preferably, two, or three or four or five or more copies of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or most preferably 100% sequence identity with SEQ ID NO: 24 are present. Preferably, two, or three or four or five or more copies of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or most preferably 100% sequence identity with SEQ ID NO: 43 are present. Preferably, two, or three or four or five or more copies of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100%) sequence identity with SEQ ID NO: 25 are present. Preferably, two, or three or four or five or more copies of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or most preferably 100% sequence identity with SEQ ID NO: 41 are present. Preferably, two, or three or four or five or more copies of a polynucleotide encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or most preferably 100% sequence identity with SEQ ID NO: 42 are present. In an organism according to the invention as described here above and in an organism in any embodiment according to the invention, the at least one, at least two, at least three, at least four, at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen polynucleotide(s) according to the invention, preferably selected from the group consisting of sequences: SEQ ID NO: 1-14, or from the group consisting of SEQ ID NO: 1-14 and 44-46, or from the group consisting of SEQ ID NO: 7, 8, 9, 10 or 46, 25, 44 and 45, are preferably present in a mobile genetic element, e.g. a transposon. Such mobile genetic element is preferably present as an insert in or close to a gene associated with sporulation, preferably a gene associated with cortex build-up, more preferably a gene having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or most preferably 100% sequence identity with yitF (SEQ ID NO: 29) or yitG (SEQ ID NO: 30), even more preferably yitF (SEQ ID NO: 29). In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that an inhibitor, a product or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

"Sequence identity" or "identity" in the context of amino acid- or nucleic acid-sequence is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Within the present invention, sequence identity with a particular sequence preferably means sequence identity over the entire length of said particular polypeptide or polynucleotide sequence. The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

"Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al, Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al, J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps). Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

A polynucleotide is represented by a nucleotide sequence. A polypeptide is represented by an amino acid sequence.

The word "about" or "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value.

All literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

FIGURE LEGENDS Figure 1: Plot of the estimated logD values, plotted against the temperature of eighteen strains of B. subtilis and corresponding 95% prediction intervals, determined in capillary tubes. The symbol · represents the data points from the lower spore heat resistance group and■ represents the data points of the higher spore heat resistance group, determined using batch heating.

Figure 2: (A) Overview of the transposon that leads to increased spore heat resitance of strain Bacillus subtilis 168 upon insertion, as demonstrated in 168-HR3 (top). Deletions constructed in Tnl546 of strain 168-HR3 are represented below. The deletion strains indicated with an arrow on the right hand side of the figure lost the high spore heat resistance phenotype.

(B) Overview of the native spoVA operon (lower) and the newly identified spoVA operon that is responsible for the increased spore heat resistance (upper).

In total seven genes are encoded in the newly identified operon that is responsible for increased spore heat resistance (Hypothetical protein comprising DUF1657, polypeptide SEQ ID NO: 21, polynucleotide SEQ ID NO: 7; Hypothetical protein comprising Yhcn/YijA, polypeptide SEQ ID NO: 22, polynucleotide SEQ ID NO: 8; spoVAC, polypeptide SEQ ID NO: 23, polynucleotide SEQ ID NO: 9; spoVAD, polypeptide SEQ ID NO: 24/43, polynucleotide SEQ ID NO: 10/46; spoVAEb, polypeptide SEQ ID NO: 25, polynucleotide SEQ ID NO: 11; Hypothetical protein SPVA1H comprising DUF1657, polypeptide SEQ ID NO: 41, polynucleotide SEQ ID NO: 44; Hypothetical protein SPVA2H comprising DUF421 and DUF 1657, polypeptide SEQ ID NO: 42, polynucleotide SEQ ID NO: 45).

In total seven genes are encoded in the native spoVA operon from Bacillus subtilis 168 (spoVAA, polypeptide SEQ ID NO: 47, polynucleotide SEQ ID NO: 54; spoVAB, polypeptide SEQ ID NO: 48, polynucleotide SEQ ID NO: 55; spoVAC, polypeptide SEQ ID NO: 49, polynucleotide SEQ ID NO: 56; spoVAD, polypeptide SEQ ID NO: 50, polynucleotide SEQ ID NO: 57; spoVAEb, polypeptide SEQ ID NO: 51, polynucleotide SEQ ID NO: 58; spoVAEa, polypeptide SEQ ID NO: 52, polynucleotide SEQ ID NO: 59; spoVAF, polypeptide SEQ ID NO: 53, polynucleotide SEQ ID NO: 60). It should be noted that the spoVAC, spoVAD and spoVAEb polynucleotide sequences and polypeptide sequences of the native operon and the newly identified operon are phylogenetically different. (C) Topology of the final gene (SpoVA2H) of the spoVA operon, which was found required for the increased spore heat resistance phenotype. The protein is membrane bound by three transmembrane segments, and possesses two domains of unknown function (DUF); DUF421 and DUF1657.

Figure 3: Spore survival of different strains, after heating spores for 60 minutes at 100°C. Strains indicated with an asterisk (*) were inactivated and not recovered (numbers below the detection limit) after heat treatment of 60 minutes at 100 °C. Figure 4: Spore heat inactivation data measured at 115°C for different strains of B. subtilis carrying either 0, 1, 2 or 3 additional spoVA operons that are responsible for increased heat resistance. The strains lacking the spoVA operon that confers heat resistance are least heat resistant (closed circles). Increasing number of the spoVA operon that mediates heat resistance results in increasing heat resistance: strains containing three copies of the spoVA operon that mediates heat resistance (open squares) have spores that are more heat resistance than when two (grey squares) copies or one (black squares) copy of the operon is/are present.

EXAMPLES

Example 1: Investigation of Bacillus strains with spores of high thermal resistance Materials, Methods and Results

Bacterial strains

The strains investigated in this study were ten food isolates, supplied by food manufacturers, and eight publically available strains (Bacillus genetics stock center, BGSC). All strains belong to the species B. subtilis and are listed in Table 2. Strain A163 is known to form spores with high thermal resistance properties (Cazemier et al, 2001; Kort et al, 2005; Oomes et al, 2004; Oomes et al, 2007). Strains 4068, 4069, 4071, 4072, and 4073 correspond with strains CC2, IIC14, CC16, RL45, and MC85 have been previously described (Omes et al, 2007). The strains 4143, 4145 and 4146 were not previously described (Berendsen et al. 2014, submitted for publication). Table 2: Strain used in this study, with corresponding strain numbers and isolation sources.

Species Strain - NIZO nr Isolation Spore heat Genome Reference received as source resistance seq

B. subtilis 1A700 4062 Not relevant Low Publically BGSC available

B. subtilis 1A96 4055 Not relevant Low Publically BGSC available

B. subtilis 1A747 4056 Not relevant Low Publically BGSC available

B. subtilis 2A9 4057 Type strain Low Publically BGSC spizizenii available

B. subtilis 2A11 4058 Sahara desert Low Publically BGSC available

B. subtilis 2A12 4059 Death valley Low Publically BGSC national available

monument

B. subtilis 3A1 4060 Not relevant Low Publically BGSC available

B. subtilis 3A27 4061 Mojave desert Low Publically BGSC available

B. subtilis A163 4067 Peanut High This study (Cazemier chicken soup et al,

2001; Kort et al, 2005; Oomes et al, 2004; Oomes et al, 2009)

B. subtilis CC2 4068 Curry cream High This study Oomes et soup al, 2007

B. subtilis IIC14 4069 Binding flour High This study Oomes et ingredient al, 2007

B. subtilis A162 4070 Peanut High This study Caspers et chicken soup al, 2011

B. subtilis CC16 4071 Curry cream High This study Oomes et soup al, 2007

B. subtilis RL45 4072 Red lasagna High This study Oomes et sauce al, 2007

B. subtilis MC85 4073 Curry soup This study Oomes et al, 2007

B. subtilis 50 4143 Surimi Low This study Berendsen et al. 2015

B. subtilis 1173 4145 Pasta High This study Berendsen et al. 2015

B. subtilis 1208 4146 Curry sauce High This study Berendsen et al. 2015

Spore preparation

Spore crops were prepared as described by Schaeffer et al. with slight modifications (Schaeffer et al., 1965). In short, the sporulation medium consisted of Nutrient Broth 8 g/L (NB, Difco), supplemented with ImM MgS0 ₄ , 13mM KC1, 0.13mM MnS0 ₄ , lmM CaCi ₂ , and a final pH of 7.0. For cultivation on plates the medium consisted of Nutrient Agar (NA, Difco) 23 g/L, supplemented with ImM MgS0 ₄ , 13mM KC1, 0.13mM MnS0 ₄ , ImM CaCl ₂ , with a final pH of 7.0 (NA). The Luria-Broth (LB) medium was inoculated from the -80°C stocks, and was incubated for 16 hours at 37 °C, with shaking at 200 rpm. The overnight cultures were diluted 100 times in sporulation medium and allowed to grow until an OD ₆ oonm of 0.6, subsequently 200 μΐ of a culture was spread on three agar plates per strain. The plates were incubated at 37 °C for seven days and spore formation was followed microscopically. The spores were harvested by swabbing the entire bacterial layer of three plates, combined in one tube, and washed by three successive steps in sterile water (5000 x g, 10 min, 4°C). The spore suspensions were stored in sterile water at 4°C for at least one month to allow for spore maturation, before being used in heat-inactivation experiments. Two independent spore crops were prepared for most strains.

Spore enumeration

To determine the initial spore count, spore suspensions were heated at 80 °C for 10 minutes to inactivate germinated spores and vegetative cells and to allow for activation of germination. Subsequently, samples were serially diluted in peptone water, and appropriate dilutions were pour-plated in Nutrient Agar (NA, Difco) in duplicate. Based on the initial spore yields, the spore suspensions were further diluted prior to batch inactivation experiments, as described below. The number of surviving spores was determined following different heat treatments by serially diluting the samples in peptone water and pour plating appropriate dilutions in Nutrient Agar (NA, Difco). All counts were performed after incubation for five days at 37 °C.

Batch inactivation

For each strain, the spore heat inactivation kinetics were determined in a batch heating system using capillary tubes. The experiments were performed twice per strain, using two independent spore preparations. For each spore preparation, the recoveries were determined using three different temperatures, each with at least five different time points. The inactivation kinetics were determined as previously described by Xu et al. (2006). In brief, the spore suspensions were diluted to an initial count of approximately 1 x 10 ⁸ colony forming units per milliliter (CFU/ml), in phosphate buffered saline (PBS), with a pH of 7.4. A capillary tube (0 _ext 1.0 mm, 0.8mm, length 150 mm, catalog no 612-2806, VWR, Amsterdam, The Netherlands) was filled with a spore suspension of 50 μΐ, which was subsequently heat sealed. Each tube was completely submerged in an oil bath at a selected temperatures for a given time and subsequently transferred to an ice-water bath for 10 minutes. The sealed capillary tubes were then incubated in a hypochlorite solution (525 ppm) for 10 minutes and washed with sterile peptone water. The capillary tubes were then transferred to 5 ml sterile peptone water and crushed with a magnetic stirrer. The spores were subsequently enumerated as described before. Using the same method, the initial spore count was determined for each spore suspension following a heat treatment of 80 °C for 10 minutes, 100 °C for 10 minutes, and in the absence of a heat activation, to establish optimal germination, which might require heat activation.

Data analysis

For eighteen strains, extensive spore inactivation data were obtained from two independent spore crops using batch heating. The inactivation data were fitted with the log-linear model in equation 1, to determine the JJ-value, the decimal reduction time, at the corresponding temperature, using Excel. The effective heating times for the capillary tubes were used (calculated based on a z-value of 10), thus omitting the heating and the cooling profiles. t

(1): logN(t) = logN(O)

D

To determine the temperature dependency of the JJ-value, the z- value was determined. The z-value was calculated per strain based on the D- values of two independent spore crops, as the negative reciprocal of the slope of the plot of logD against the

temperature, as displayed in equation 2.

(2): z = - 1 / slope(logD,r)

The logD values of all strains were plotted against the temperature to visualize strain variability. Based on this visualization, two groups of spore heat resistance were identified with logD values that cluster together. To compare the two groups with different spore heat resistances, the overall z-value, using equation 2, and subsequently the \ogD _re f at a reference temperature of 120 °C, were determined per group using equation 3. Based on the \ogD _re f at the reference temperature, JJ-values can be estimated at each desired temperature using equation 4.

(3): = intercept (log/), T)— T _re f/z

(4): \ogD _T = \ogD _ref - T - T _ref )/z

The 95% prediction interval (PI) of the logD _ref was calculated using the following equation:

Where t _DF is the student t-value with degrees of freedom (DF), a is the confidence level (a=0.05), and the residual sum of squares (RSS) is calculated from the data points deviating from the regression line.

Statistical analysis

An F-test was used to test significant differences, based on the plotting of the logD values against the temperature. The F-test was performed to test if the slope and the intercept of the logD of were significantly different. A confidence level of a = 0.05 was used. The F-test was applied to test whether the two groups of with presumed different spore heat resistances were indeed significantly different. Spore heat resistance For eighteen strains of B. subtilis spore heat resistance was determined using capillary tubes. The spore heat resistance, in the form of D- and z-value, was calculated per strain, and is presented in Table 3. Table 3 The calculated JJ-values per strain for the independent spore crops, at three different temperatures. The calculated z-value for each strain as determined.

Strain Temperature Spore crop 1 Spore crop 2 z value

(°C) D (min) S.E r ² D (min) S.E. r ² (°C) S.E r ²

4055 100 3.5 0.22 0.95 N.D. ^* 7.5 0.5 1.0

105 0.61 0.08 0.88 N.D.

110 0.16 0.01 0.97 N.D.

4056 100 3.9 0.51 0.88 N.D. 7.6 0.0 1.0

105 0.86 0.08 0.91 N.D.

110 0.19 0.02 0.94 N.D.

4057 100 1.7 0.16 0.92 N.D. 7.3 1.0 1.0

105 0.25 0.01 0.98 N.D.

110 0.08 0.00 0.96 N.D.

4058 100 2.4 0.17 0.96 N.D. 7.9 0.6 1.0

105 0.46 0.05 0.85 N.D.

110 0.13 0.01 0.94 N.D.

4059 100 1.5 0.22 0.87 N.D. 9.3 3.2 0.9

105 0.21 0.02 0.96 N.D.

110 0.13 0.03 0.38 N.D.

4060 100 4.4 0.4 0.90 2.9 0.4 0.91 7.5 0.6 1.0

105 0.83 0.09 0.90 0.65 0.06 0.91

110 0.21 0.02 0.90 0.13 0.02 0.82

4061 100 4.0 0.6 0.86 N.D. 6.4 0.2 1.0

105 0.60 0.06 0.90 N.D.

110 0.11 0.02 0.90 N.D.

4062 100 3.5 0.2 0.96 3 0.2 0.94 6.9 0.4 0.99

105 0.65 0.08 0.91 0.84 0.06 0.9

110 0.1 0.02 0.67 0.13 0.01 0.88

4067 115 18.1 1.9 0.89 10.2 0.6 0.94 6.3 0.7 0.96

120 1.8 0.1 0.99 1.58 0.05 0.99

125 0.24 0.01 0.99 0.53 0.05 0.94

4068 115 4.6 0.4 0.96 5.8 0.5 0.94 6.6 0.3 0.99

120 0.66 0.02 0.98 0.79 0.04 0.98

125 0.15 0 0.99 0.16 0.02 0.92

4069 115 3.7 0.2 0.95 2.6 0.2 0.96 7.3 0.3 0.99

120 0.57 0.02 0.98 0.58 0.06 0.96

125 0.12 0.01 0.92 0.14 0.01 0.93

4071 110 18.6 2.5 0.83 10.4 0.5 0.98 6.7 1 0.91

115 1.18 0.08 0.88 1.31 0.08 0.92

120 0.65 0.03 0.97 0.3 0.04 0.77 4072 115 3.1 0.3 0.91 2.7 0.1 0.93 7.8 0.7 0.97

120 0.99 0.05 0.96 0.6 0.02 0.96

125 0.12 0.01 0.96 0.18 0.02 0.9

4073 115 9.6 0.9 0.92 4.8 0.6 0.9 5.8 0.4 0.98

120 0.92 0.03 0.98 0.79 0.04 0.97

125 0.13 0.01 0.93 0.13 0.01 0.96

4140 100 3.3 0.4 0.94 3.3 0.6 0.85 7.2 0.2 1.0

105 0.6 0.06 0.96 0.58 0.09 0.95

110 0.13 0.02 0.98 0.14 0.02 0.94

4143 100 3.5 0.1 0.99 1.6 0.1 0.93 7.5 1.2 0.9

105 0.85 0.07 0.99 0.8 0.1 0.75

110 0.17 0.02 0.91 0.07 0.01 0.87

4145 115 17.6 2 0.91 9.1 0.3 0.97 6.1 0.6 0.97

120 3 0.4 0.92 1.5 0.1 0.99

125 0.26 0.02 0.97 0.33 0.01 0.99

4146 105 22.8 1.9 0.92 9.3 0.5 0.95 6.9 0.7 0.96

110 3 0.1 0.99 2.3 0.1 0.89

115 0.56 0.03 0.95 0.48 0.02 0.97

*N.D. = Not determined.

Based on the spore heat resistance (log -values), two distinct groups were depicted (Figure 1). The two groups were not significantly different in the z-value, however the two groups were significantly different in the intercept, signifying the spore heat resistance. The low spore heat resistance group consisted of strains 4055, 4056, 4057, 4058, 4059, 4060, 4061, 4062, and 4143. The high spore heat resistance group consisted of strains 4067, 4068, 4069, 4070, 4071 , 4072, 4073, 4145, and 4146. For the two groups of spore heat resistance the z-value, the \ogD _re f and the D ₁₂ o°c were calculated, which are presented in Table 4.

Table 4 The calculated z-values and logDref for the two groups of spore heat resistance.

z- Upper D 120 °c

Spore heat resistance value S.E. r ² (min) 95% PI (sec) n

Low heat resistance - group 7.4 0.0 0.78 -2.24 -2.04 0.34 36

High heat resistance - group 9.5 0.0 0.90 -0.09 0.14 48.48 48 Genome sequencing Genomes of ten Bacillus food isolates of B. subtilis were sequenced via next generation whole genome sequencing on the Illumina HiSeq 2000 platform. The 101 bases paired- end sequencing on a 500bp fragment library-prep was performed. The generated 101 base-long reads were mapped on a reference genome of B. subtilis 168 (NCBI Reference Sequence: NC 000964) with use of the Contiguator tool (Galardini et al, 2011). The Velvet software (Zerbini and Birney, 2008) was used for de novo assembly of the genomes and the RAST (Rapid Annotations using Subsystems Technology) version 4.0 server (Aziz et al, 2008) was used for the genome annotation. Features of the sequenced genomes of the individual strains are listed in Table 5.

Table 5: Details of sequencing of B. subtilis strains.

Genome size

Strain ID Strain (Mb) Coverage

4067 Bacillus subtilis A163 B4067 4.31 168

4068 Bacillus subtilis CC2 B4068 3.98 227

4069 Bacillus subtilis IIC14 B4069 4.09 434

4070 Bacillus subtilis A 162 B4070 4.28 237

4071 Bacillus subtilis CC16 B4071 4.20 323

4072 Bacillus subtilis RL45 B4072 4.09 197

4073 Bacillus subtilis MC85 B4073 4.13 285

4143 Bacillus subtilis B4143 4.15 214

4145 Bacillus subtilis B4145 4.40 258

4146 Bacillus subtilis B4146 4.26 313

Gene-trait matching

Gene- trait matching was performed based on the spore heat resistance phenotype, low or high, and the gene orthology (Li et al, 2003), using phenolink (Bayjanov et al, 2012). Target genes were identified, which were 100% present in high spore heat resistant strains, and 100% absent in low spore heat resistant strains. The identified target genes were located in one mobile genetic element, a transposon. Using the target genes present in the transposon in strain 4067, the presence/absence pattern of these genes was verified based on an orthology prediction as described before. The genes that are always present in transposon in the various high spore heat resistant strains are listed in Table 6. The nucleotide sequences of the target genes are listed in Table 7, and the amino acid sequences of the translated target genes are listed in Table 8.

Table 6: Genes present in the transposon of B. subtilis strain 4067, which are always found in correlation to the increased spore heat resistance.

Locus tag Predicted function

B4067 4748 TnpA transposase

B4067 4749 TnpA transposase

B4067 4855 N-acetylmuramoyl-L-alanine amidase (EC

3.5.1.28)

B4067 4717 Spore germination protein xc. bacillus

B4067 4718 hypothetical protein

B4067 4719 Manganese catalase (EC 1.11.1.6)

B4067 4767 hypothetical protein comprising DUF1657

B4067 4768 hypothetical protein comprising Yhcn/Yij A

B4067 4769 Stage V sporulation protein AC (SpoVAC)

B4067 4766 Stage V sporulation protein AD (SpoVAD)

B4067 4765 Stage V sporulation protein AE (SpoVAEb)

SpVAlH SpoVAlH hypothetical protein comprising

DUF1657

SpVA2H SpoVA2H hypothetical protein comprising

DUF421 and DUF 1657

B4067 4745 probable membrane protein yetF

B4067 4746 probable membrane protein yetF

B4067 4747 Cardiolipin synthetase (EC 2.7.8.-)

Table 7: Sequence ID of the genes correlated to increased spore heat resistance associated with increased spore heat resistance, with their nucleotide sequences.

SE Gene Nucleotide sequence

Q- encoding

ID for protein

1 TnpA ATGGGGAAATTAGGTTCTTATTCAAGACAAAACAGCTTGGCTA

CAGCCTTACGTGAGATGGGCCGAATAGAAAAAACGATCTTTA

transposase

TTTTGAATTATATTTCGGATGAATCATGA TnpA ATGAATGGACTGGCAAGAGCTATTTTCTTCGGAAAACAAGGA GAGCTTAGGGAACGAACCATACAGCATCAATTGCAAAGAGCC

transposase AGTGCCTTAAACATCATTATCAATGCCATCAGTATCTGGAATA

CTTTACATCTAACAAAGGCAGTTGAATATCAAAAACGGTCAG GTAGTTTTAATGAAGAATTATTGCACCATATGTCACCTCTAGG TTGGGAACATATTAATTTACTTGGAGAATACCATTTTAATTCG GAAAAAATGGTCTCGTTAGATTCTTTAAGACCCTTGAAACTTT CTTAA

N- ATGCAGGAAAAAGAAATTACCTTAAATATTTCCCATAGCATCC

GAAATCTCTTGGAAAACCATTATGAAGGCCTGCAAATTAAAA

acetylmuramo JQAGTAGAACAGCAGATATTACGCGCAGCTTGAAAGAACGTA yl-L-alanine CAGATGATGCGAATGCTTTCGGTACGGGGGCCAAAGGATGGA

TTTGTGGAAGAAGTGGGAGTCAATGTGGCGTTAATTCGGAAA

am ⁱ dase CGAATCAAATCAACCAGCATGGTTTATGA

GTGCAATTGCTTGATTTCTCGACAGTGGCCAAGCTGGAGGGAC AGCAAAAAGCGGAGCAGCCCCCGGTTTGGGTGGGCAAAGGCG

Spore AAGGTTCCTCGTTTACGGAGGCGGCAAATGATTTGTACAGTAC germination ATCACAACAACGCTTAAACTTGGGACAAATCTCTGCCATTTTG

TTCAGCGAGAGATTGATGAAGGAAAACAAAGTCGGAGAAGTG

protein xc. CTGGAATTGATCAACCGTTATCGTGAAATAAGGTACTTGGCCT

GGCCGTTTAGCACAAGAGAGCCTCCTGAAGAAATCTTGCTGG

bacillus hypothetical ATGAGTGAAAAATGGAAACTTAGTGATCCGTATGTTTATCAAG

CTTTAATGGGACTTCACAATCAATCCCTTGCTGTTCAAACGAC

prote ⁱ n CCACGGCTCTGTACGAGGTGTATTACGAGAGGTCATGCCTGAT

CATATCGTCATCATGATGGGCGGGACACCATTCTATGTACGTA CAGCACAAATAATTTGGTTTCATCGAGAATAA Manganese ATGATGAAACGCGTGAATAAAATTGCCATTGAGTTACCATATC

CAGAACATGGCGATATGAATGCTGCTGCAGCTGTACAAGAAT

catalase TAATGGGTGGAAAATTCGGTGAGATGTCCACCTTAAACAATTA

CATGTTTCAATCCTTTAATTTCCGCGGTAAAAAAAAGCTAAAG CCCTTTTATGATTTAATTGCAAGTATTACAGCGGAGGAATTTG GCCATGTTGAATTAGTAGCGAATGCAATTAATCTGCTCTCGAT TGGCAATACCATTCCTGGCAATCCTGATACCGCCCCACTGCAA AATGGAAAGGATGCCCGTTATTCTACACATTTTACAACCACTG CTCAAACAGCTTTTCCAGGTGACGGGATGGGAAGACCGTGGA ATGGGTCGTATGTAAATAACAGTGGAACGCTCGTTGAAGATCT ACTCGCCAATTATTTATTGGAAATCGGTGCTAGAAACCACAAA ATGCGTGTGTATGAAATGACGACCCATCCGACTGCCTTAGAGA TGACCGGATACTTGCTTGTCAGAGGTGGGACTCATATTATTGC CTATGCAAAAGCACTAGAAGTAGCTACAGGTGTGGATGTGGG TAAAATGCTTCCAGTTCCAAGTTTGGATAATAATAAGTTTGAT TATGCCAAAAAGTTTATGGATCAAGGATTGTACAATGTGTTGT ATACATGGGGAGAAGCGGATTATCGTGATATCAACCAAATTT GGAAAGGTGCAAACCCAGAAACAGGTGAAAGGCTGCATGTAA TTGACGGAATGCCTGAAGGTGCGCCTGTACCCGATTTTCCAGA

ACTTCCAGAGCAATTCGCTCCAGGGATTGATTTGGATGATTAC TATCGTATTTTAAAACGTTTAAAAAGCAATATGTAA

ATGACAGTAATAAATGACGTTAAAACAGCTCTAGCAGGATTG

AAAAGTGCTCAAGCAAGTTTTGAAACATTTGCTCTTGGTACAG

ATAATCAACAAGCTAAGCAACTTTACCAAGATGCAGCTAAGC

AAACCCAATCCGTTGTAGACAGCATTGAACCACGTGTTCAACA

AATCGAACAAGAAGAACCTCAATACAAACAACAGTAA

TAATGGTCATTGGTTTCGCATCAGGATGTAATGGTAATCAAAA

TGAATTTAGTCAAGGTAATAGTACTTTTGGTATTTCGCAAGTA

CATACAAGTAAGCCAATTGATCAATCCGTTGCCAATCATGCGA

AAGAAAAGATAATTGCCAAGGAAGATATTACAGACGTAAAAG

CTGTGAATACTGATAAAGAGCTTATGGCAGCGATTAAAGTTG

AGAATTTTGATCGATTTCGATTAAAGAGTATCGAGAAGTCAGT

GTTTCGACTGATAAAAAAATATTCTGGGAACTTGAAAAGGTC GAGCAAAGACTGAAAAAAAACGATATGAATAAGAAGAACTT AAAAAATGATTTGAACAAACTGAAAAGTCTTATGAAGGAACA AACCTAA

ATGTCTAACAATCAAAAGAAACAACTTACTCCTGTGCAACAG

GAATATCAAAAATTGCAAAAACAACGAGAGATCAAAAGACCG

GTTGTTAAAAATTGTATTAAGGCCTTTTTAATCGGTGGACTTA

TTTGTTTGATTGGTCAGCTTATTTCAGACTTTTACATTTATTAT

TTCGATTTTACTGAGCAAACGGCCGGAAATCCGACGGTGGGT

ACGTTAATTTTTATTACAATGCTTCTTACTGGTTTTGGCGTATA

TGATCGAATGGCCCAATTTGGTGGGGCTGGAACAGCTGTACCT

GTAACAGGTTTTGGCAATGCGGTTATATCGCCTGCCATTGAGC

ATCGAAGCGAAGGATTTGTACTGGGCGTAGGTGGCAACATAT

TTTGTCATTGCCTTAATTAAAACCACTTTAATCCAGTGGGGTG GTTTATAA

ATGATTTATCGGGAAGGACAACCCGTTCTTGCAGGGGCAAGC

GGTGCAGGCTGTTCAGCAACGGTAGTTTATGGGCATTTATTAA

ACCGCATGAAAAATGGTGAATTTAAACGCATGTTAGTTGTGGC

TACAGGTGCTTTGCTTTCACCGTTGTCCTTTCAGCAAAATGAA

ACAATTCCTTGTATCGCCCATGCAGTGTCAATTGAATACGGAG

GTGAACAATTAACATGA

CATTGGGCAAATTATGTTTGATGTATTTAAATTGACACCAGGT

CATACCTTAAGTGCTCTTGTAGTGATTGGGGCGCTTTTAGATG

GATTTGGACTATACGAGCCTTTGATTGATTTTGCTGGGGCAGG

GGCTACCGTTCCTATTACAAATTTTGGGAATTCACTTGTTCATG

GTGCGATGCAGGAAGCAGAAAAACATGGGTTAGTTGGTGTAC

TGACAGGAATGTTTGAAGTAACTAGTTCTGGTATTTCAGCTGC GGATAA TAGAAAAGCCATACGGAGATGCCAATGGGTTTAAACGATATC

GGACTTGTTATCTCATCAGGATTATCACCGAAAGCGGGATAGA

CGGCTGGGGAGAATGCGTTGATTGGCTCCCTGCTCTCCATGTC

GGTTTTACGAAGCGGATCATCCCATTTCTATTAGGAAAACAGG

CTGGCAGCCGCCTGTCATTAGTGCGCACGATTCAAAAATGGCA

CCAGCGGGCGGCCTCCGCTGTAAGCATGGCGCTGACAGAGAT

TGCAGCCAAAGCTGCGGATTGTTCGGTTTGCGAATTATGGGGC

GGGCGATACAGAGAGGAGATTCCTGTGTACGCGTCTTTTCAAT

CGTACTCAGACTCCCCGCAATGGATCAGCCGCTCAGTCTCCAA

TGTTGAAGCCCAGTTGAAAAAAGGTTTTGAGCAAATCAAAGT

CAAAATTGGCGGGACTTCATTCAAGGAGGATGTCCGGCACAT

CAATGCGCTGCAGCATACAGCAGGCAGCTCCATTACGATGATT

CTGGACGCAAACCAAAGCTACGATGCCGCTGCGGCTTTTAAAT

GGGAGCGCTATTTCTCCGAATGGACGAACATTGGCTGGCTTGA

GGAGCCTTTGCCCTTTGATCAGCCGCAGGATTACGCTATGCTT

CGAAGCCGTTTGTCTGTTCCTGTTGCCGGCGGAGAAAACATGA

AAGGCCCGGCGCAATATGTGCCTCTCCTTTCACAGCGCTGTTT

GGATATCATTCAGCCCGATGTCATGCATGTCAACGGAATTGAT

GAGTTTCGGGACTGCCTCCAGCTCGCGCGTTATTTCGGCGTCA

GAGCATCCGCCCATGCATATGACGGATCGCTCTCACGGCTCTA

TGCTTTATTTGCACAAGCCTGTCTGCCCCCATGGTCTAAAATG

AAGAACGATCACATTGAACCGATCGAGTGGGACGTCATGGAG

AATCCTTTTACAGATCTCGTCAGCCTGCAGCCTTCAAAAGGAA

TGGTCCACATCCCGAAAGGAAAAGGCATTGGCACAGAGATCA

ATATGGAGATCGTCAATCGCTACAAGTGGGATGGCTCAGCCT

ATTAA

yitG ATGGAATCATCAAAACAAAATAACGGAATGACGATTGTGGCA

ATCGGTTCAATCCCTTTAATATTAACCCTAGGAAACTCGATGC TTATTCCGATTTTGCCAAAAATGAAATCTGAACTTCATTTATC ACAATTTCAAGTCAGTCTCGTGATCACAGTATTTTCCTTGATTG CAGCATTTGCGATTCCGATTGTCGGTTATCTCGCAGACCGATT CTCAAGGAAAGTCATCATTATTCCCTGCTTAATTTTGTATGGT

ATGCCTATCCTTGGGTCATGGCAGGACGAGCGTTACAGGGAA

TCGGGGCAGCCGGAACCGGCCCAATTGCCATGGCACTGACGG

GAGATCTGTTTAAAGGCGCCCAGGAGAGCAAGGTGCTCGGCC

TTGTTGAAGCTTCAAACGGCATGGGGAAAGTGCTATCGCCGAT

CATTGGCTCGCTGATCGCCCTGCTTGTCTGGTATGGCGCGTTTT

TTGCCTTTCCTGTGTTTTGTATCATTTCGATCGTACTGACTTGG

ATCGGACAATATGCAAAAGGGCTGTTAAGTGTCTTTAAACATG AAGGCAGATGGCTGTTTACCGCTTATTTAGCCGGTGCGACCTG TTTGTTTACTTTATTCGGCATCCTGTTTTATCTCTCAGATGTTCT TGAAAAAACATACGACACCGACGGCGTGAAAAAAGGCTTAAT TTTAGCGATCCCGCTTTTGGTTATGTGTGTGACGTCCTATACAA CAGGAAGCAAAATCGGGCAAAAGCAATCGTTAATGAAAAAGC TGATCGTACTTGGATTGGCGTTCATGACCGTATCCTACGCGGC

TACTGAGCAGTATAGGCTCAGGTCTTGTCCTTCCTTGTGTGAA CAGTTTCATTACCGGGGCTGTCGGCAAAGAAAGACGGGGATT TGTCACCTCGCTGTACGGCTCTGTACGTTTCTTAGGGGTAGCG ATCGGTCCTCCGATTTTCGGCCGCCTGATGCAATGGTCCAGAC GATTCTTGTGATGATGCTCATCCATGTGAAGCAAAACAATGAA

GAAACAAAAGAAAAAGAAGACCCTAAAATGGCTGGCAATCG

GCTCCAGCCCGCTGAGGAAAGGTAG

ATGACAGTAGGATCAGATGTTAAACAGTGTTTTGCCAGTCTAA

AAGGGGTAGAAGCAAGTCTCTCAAGTTTAGCATTACGGACTCT

TGATGATGAATCGAAGCGAACTTTGCATGAGGCTATGATGGT

AGTACACGAAGTAACAAAAGATTTAAAAAAAAGAGTAGGAG

AACTAGAAGGAGAGGAACTTCAGTACAAAGGTTTCTAG

GTGCCTGAATGGTTAGATGTAGCTGTACGCTCAGTATTATTTC

AATTTCCGAACTAAGTTTCTTTGAATATGTCACCGGTATTTCTA TTGGGAATATTGGTGCAGAAGTGGCTATAGGACTTGAACGGA d CAATTTTATAGAAGGAAAAGCAACTGTATTTATCAAAGATGG

GAAGATTATGGAAGATAATTTAAAAAAGGAAAGATATACGAC

AGATGAACTGTTAGATGTCCTTCGCAAGAAGGATGTCTTTCAA

GTCTCTGATGTAGAATTTGCTGTTTTAGAGGCAACAGGTGATT

TATCTGTGATGCTAAAGAAAGAAAATCAACCTTTAACAGCAA

AGGATATAAACTTGAAGGTTGCTTCAATCAAGGAGCCTCAGA

CCATTATTATGGATGGTACAATAATGGATGAACCACTAGCCAC

GATTGGACGAAGTCGGGCTTGGCTACAAACTGAATTAGAAAA

ACTAGGGGTAACGATTGAAAATGTATTTCTGGGACAGGTTAAT

TCTTACGGAGAGTTAACGATTGATCTGTTTGACGATAAATTAC

AAGTCGCACCCCCACAAGAAAGACCCTTAATTCTTTCAACCTT

GAAAAAATGTCACGCAGACTTAGAACTATTTGCTCTTGGAACG

GAATCAAAAGACGCCAAACAAATGTATAGGTTAAACAGTGAA

AAATTACAAGAAGCTATTGATAAAGTGACCCCTATTTTAAAAG

GATAA

ATGCTAGCAGGTCATCGTACATGGATCTTCGAACAAAAGCCTG

TCATTATCTCCACTGGAACCGTTGGTGGACCATTTGAAGCCAA

TGGTGCTATCCCAGATGATTTCGATACTCTTCATGCTGATTTAT

GGCTTGGGCAGGATTCCTATGAGAAAGCACATAAAATCCTTTT

TGAAGAGGCTTGCCAAAAAGCCATGGAAAAAGGGGGCATACA

AAAGGATCAAGTTCAATTTATCCTAGCTGGGGACTTAATCAAT

CAAATCACCCCAACAAGTTTCGCTAGCAGAACGATTGGAGCG

CCTTATTTTGGCTTATTCGGTGCTTGCTCTACCTCGATGGAAGG

ACTGGCTCTAGGTGCTTATATTGTAAATACAAAAGGAGCGAA

ATATTTGTTAACAGGAGCTTCCAGTCATGATACAGCTGTTGAA

AAACAATTTCGGTATCCAACTGAGTATGGTGGACAAAAGCCA

CCTACGGCACAATGGACGGTTACTGGTGCAGGTGCAGCCCTAT

TAAGTGATTCTGGGGAAGGACCTCATGTCACATCTGCCACAAT

TGGTCGTGTAATCGATATGGGATTGACAGATCCATTTAACATG

GGAGGAGCTATGGCGCCAGCTGCGGTTGATACCATTGAAGCC

CATTTAAAGGAACGAAATGTTGAGCCATCTTATTACGATTTAA

TTGTAACCGGTGACCTTGGCCAAATCGGACAGGAAGTGTCCAT

GGATCTATTTAAAAAGCATGGAACTCCTATTAGTGAAGAACA

ATACCAAGATTGTGGCCTTATGATTTATCGGGAAGGACAACCC GTTCTTGCAGGGGCAAGCGGTGCAGGCTGTTCAGCAACGGTA

GTTTATGGGCATTTATTAAACCGCATGAAAAATGGTGAATTTA

AACGCATGTTAGTTGTGGCTACAGGTGCTTTGCTTTCACCGTT

GTCCTTTCAGCAAAATGAAACAATTCCTTGTATCGCCCATGCA

GTGTCAATTGAATACGGAGGTGAACAATTAACATGA

Native ATGGAACGACGAATATTTATCCGGCTTCGCCACCGAGTGCTGG

CACATCCAGGGGATATTATTACCGTTGGAGATGCCGCGCAAAT

spoVAA

AGAAGGGCAGCTTCAGCTGAAAAAGAAACTTTCGGCTATGCC

GCTTTATCAGGTGAGCGAAAAAGATAAAAATATCGTAATTCT

GGATATCATACAAGTCCTCAGAGCCATTCATTTACAAGACCCG

ACAATTGATGTTCAAACCGTAGGCGGAGCAGAAACCATTGTT

GAAATTCAGTATCGAAAGCGAAATTTATCAACGGTTCTATTTA

TCGGTGTCTGGCTGCTTCTGTTTATTGGATCGTGTCTTGCCATC

ATGAACTTTCATGAGGATGTAAGCATGAGAGATGTTCATATCG

CACTATATGAAATCATAACCGGAGAGAGGAATGACTATCCAT

ATTTGCTTCAAATCCCATACAGCATCGGTTTGGGACTGGGGAT

GAGCCCAGCCCGCTGGAGGTTGAGATGTTTAACTATCAGCTTG

ATCTCGATCAATATGTGGCCATGCATGAGAATCAAGAAACCA

TAAAGGATCTGCATGATCGTTAG

Native

ATGGGAATCATTCCGCGGCTGATGCAGCTCACCAAAACAATG

AGATTTGTTCAGGCTTATGAAGCGGCTGTTATTCTTGGCGCGG

TTTGCGGGGGATGGGAGACGCTTCATATGAATCATCTTTATCT

AACAAAATGGATAGCTGTGCCGGTCGGGCTTCTGGCAGGTTTG

TTCGTCGGGATGCTAGCGGCTGCTCTTACAGAGGTTTTAAATG

TCCTGCCGATTTTGGCGAAACGAATCGGGCTCAGAAGTAAAA

TCATTATTCTGTTAATGGCCATCGTGATTGGGAAAATCGCGGG

ATCTTTATTTCACTGGCTGTACTTTATTGACCATTCATAA

Native ATGACAAACATAAAAGAAAATTACAAATCAAAAGTGAAAACA

TATCAGCCTAAGCCGCCTTACGTCTGGAACTGTGTAAAAGCCT

spoVAC

TTTTAGTGGGCGGACTGATTTGTGCAATCGGGCAAGGTCTGCA

GGAATCCAACAGCTGCAACGCTGATATTAATTTCTGCCCTGCT

TACAGGGTTTGGAATCTATGACAGAATCGGACAATTCGCAGG

CGCAGGTTCAGCCGTACCTGTCACGGGTTTTGCCAACAGTATG

GCAAGTGCGGCTCTTGAATATAAAAGCGAAGGATTAGTGCTT

GGAGTAGCGACAAATATGTTTAAACTGGCAGGAAATGTTATT

GTTTTCGGAGTTGTAGCTGCATACATCGTTGGAATGATTCGGT

TTGCTTTTGAGAAACTGATGTCATAG

Native ATGAAATTAACAGGAAAGCAAACCTGGGTATTTGAACATCCC

ATATTTGTCAACTCAGCGGGAACGGCAGCAGGACCAAAGGAA

spoVAD

AAAGACGGACCGCTTGGCTCTTTATTTGATAAAACCTATGACG

AAATGCATTGTAATCAAAAAAGCTGGGAAATGGCGGAACGGC

AGCTGATGGAAGACGCTGTTAATGTTGCACTTCAAAAAAACA

ATCTGACAAAAGATGATATTGACCTATTGCTGGCAGGCGACTT

GCTGAACCAAAACGTGACGGCTAACTATGTGGCAAGACACTT

GAAAATCCCATTTCTTTGTATGTTTGGCGCCTGTTCAACATCA

ATGGAAACAGTGGCTGTCGCATCAGCCTTAGTTGATGGAGGG TTTGCCAAAAGGGCACTCGCTGCCACCAGCAGTCACAATGCA

ACTGCGGAAAGACAGTTTCGCTATCCGACAGAATACGGAGGC

CAAAAACCGGACACTGCTACCTCCACTGTAACCGGAAGCGGT

GCAGTTGTCATCAGTCAGACACCGGGTGATATCCAAATCACA

AGTGCGACTGTCGGGAAGGTTTCTGATTTAGGAATTACAGATC

CTTTTGATATGGGATCGGCTATGGCTCCGGCTGCAGCTGATAC

GATTAAGCAGCACTTTAAAGATCTCAACCGGACAGCGGATGA

CTATGATCTAATCCTGACAGGTGACCTGTCAGGCGTCGGTTCT

CCAATCGTAAAAGACATTTTAAAAGAAGATGGATATCCAGTC

GGCACAAAGCATGATGATTGCGGGCTTCTGATTTACACTCCGG

ATCAGCAGGTCTTTGCCGGGGGGAGCGGCTGTGCTTGTTCAGC

AAACTAAACCGTGTATTTGTCGTGGCGACCGGGGCGCTATTAA

GCCCGACGATGATCCAGCAAAAAGAAACCATCCCGACTATCG

CCCACGGGGTTGTATTTGAGCGTGCAGGAGGTGCATCTTAA

Native ATGGACTACCTTTTGGCTTTTGTCTGCGGCGGGGCCATCTGTA spoVAEb

TCATGTCATGACATCATTTGTTGTAATCGGTGCAATATTAGAT GGTTTCGGCATTTACGATAAATTTATTGAATTTGCCGGAGGAG GCGCCACCGTTCCGATCGTCAGCTTCGGTCACAGTCTTCTGCA CGGCGCCATGCATCAGGCGCATATACACGGATTTATCGGAATC

CTATTCTTTTCGCCTTTATCGTAGCTGTTATTTTCAAACCGAAA GGATAA

Native ATGACGACAAAACGCAAGGTCATACTGGTAACAGACGGGGAT

GTTTATGCCGCTAAAACAATTGAATACGCCGCAAGCAAAGTG

spoVAEa

GGAGGCCGATGTATTTCACAATCAAAAGGGAATCCTTCCACCC

GCAGCGGCGCCGAGCTTGTCAAGCTGATCGCCTCAGCACCGT

ATGATCCTGTATTTGTCATGTTTGACGACTCCGGCCTTCAAGG

AGAGGGCCCGGGAGAAGCCGCTCTAACCTATGTCGCAACCCA

TCCGTCTATTGAAGTGCTCGGTGTGATTGCGGTCGCGTCAAAA

ACACATCAGGCTGAATGGACCAGGGTCGATGTAAGCATTGAC

CGCAATGGAGAAATAACTGAATACGGCGTCGATAAAGTCGGG

GAAAGGGAATTTGATGACCACAGAATGAGTGGAGATACAGTC

TATTGCCTTGATAAACTTGATCTCCCGCTTATTGTCGGTATCGG

GGATATCGGCAAAATGGGCAGAAAAGACGATATCTCAAAAGG

GTCGCCAATCACCATGAAAGCGGTCGAGTTTATTTTAGAAAGG

AGCGGGTATCATGCCGGACCACAAGGAAGAGAAAATTCGGGT

TTATCGGAATCCAGCTAA

Native ATGCCGGACCACAAGGAAGAGAAAATTCGGGTTTATCGGAAT

CCAGCTAAAAATGAAGAGTATTTCAAAAACCGTGTTGGGATG

spoVAF

GGGACGAGCTATGATGTAGGTGTTCGCAAGCTCACGATTTTAG

ATAAAGAGATCCAGCTTTACTATCTAAATGGATTATGCGATAC

AGCCTATATTATTCATTTAATGAGAGAGCTTGTTGCCATTAAC

AATCGGAAAGAAGACCCTGATGAGCTGGTCGACATCGTCGAA

AACAGGCTGCTTAACGCCCAGGTCGAAAAAGTAAAAACCTTG

GATGAAACCACCGACCAAGTGCTGTCCGGGCTCGTCGCTGTCA

TTGTTGAAGGTGCAGGCTTCGCATTTATAATTGATGTCAGAAG

CTATCCGGGCAGAAACCCGGAAGAACCTGATACGGAAAAGGT

GGTCAGGGGAGCCAGAGACGGATTTGTTGAAAATATCGTCGT

CAATACGGCCCTTCTCAGAAGGCGGATCAGAGACGAACGCCT TCGAGTCAAGATGACTAAAGTTGGTGAGCGGTCAAAGACGGA

TTTAAGCATTTGTTATATAGAAGATATAGCTGATCCGGATCTG

GTTGAGATCGTAGAAAAAGAAATTGCATCCATTGATGTCGAT

GGATTAACAATGGCAGACAAAACTGTCGAGGAATTCATTGTT

AATCAAAGCTATAATCCTTTTCCGCTCGTGCGCTATACAGAAA

GACCGGATGTGGCCGCAAATCATGTTCTAGAAGGCCATGTTAT

CATTATAGTGGACACATCGCCGAGTGTGATCATTACCCCGACG

ACTTTATTTCACCATGTGCAGCATGCTGAAGAATATAGGCAGG

TATTCTTGCTTCTACTTTATTTCTTCCGATCTGGTTTCTCTTTGT GCTTCAGCCTGATCTGCTCCCGGACAACATGAAATTTATCGGT TTAAATAAAGATACGCATATCCCGATCATCCTGCAGATATTTT TAGCTGACTTAGGAATCGAGTTTCTCAGAATGGCTGCCATTCA CACACCGACGGCTTTATCGACAGCAATGGGTTTAATTGCCGCT

CTGAGGTGATTCTGTACGTCTCACTTGCGGCGATTGGAACCTT

TACGACGCCTAGTTATGAATTAAGCTTGGCAAACAAAATGAG

CCGTCTTGTCCTCATGATACTCGTTGCTTTATTTCATATAAAAG

GGCTCGTCATCGGATTTACAGTGCTAATTATTGCCATGGCAAG

CATCAAATCCCTTCAGACACCGTACTTATGGCCGCTGATTCCC

TTTAACGGAAAGGCGCTGTGGCAGGTTCTTGTCCGCACAGCGA

AACCTGGCGCAAAAGTAAGACCCAGCATCGTTCACCCCAAAA

ATCGCTTAAGGCAGCCTACCAATTCATAA

Table 8: Sequence ID of the polypeptides correlated to the increased spore heat resistance, with corresponding amino acid sequences.

SE Gene Amino acid sequence

Q- encoding

ID for protein

15 TnpA transposase MGKLGSYSRQNSLATALREMGRIEKTIFILNYISDES

16 TnpA transposase MNGLARAIFFGKQGELRERTIQHQLQRASALNIIINAISIWNT

LHLTKAVEYQKRSGSFNEELLHHMSPLGWEHINLLGEYHF

NSEKMVSLDSLRPLKLS

17 N-acetylmuramoyl- MQEKEITLNISHSIRNLLENHYEGLQIKMSRTADITRSLKERT

DDANAFGTGAKGWICGRSGSQCGVNSETNQINQHGL

L-alanine amidase

18 Spore germination MQLLDFSTVAKLEGQQKAEQPPVWVGKGEGSSFTEAANDL

YSTSQQRLNLGQISAILFSERLMKENKVGEVLELINRYREIR

protein xc. bacillus

YL AWPF STREPPEEILLATPFFRF SPNA

19 hypothetical protein MSEKWKLSDPYVYQALMGLHNQSLAVQTTHGSVRGVLRE

VMPDHIVIMMGGTPFYVRTAQIIWFHRE

20 Manganese catalase MMKRVNKIAIELPYPEHGDMNAAAAVQELMGGKFGEMST LN YMFQSFNFRGKKKLKPFYDLIASITAEEFGHVELVANAI

NLLSIGNTIPGNPDTAPLQNGKDARYSTHFTTTAQTAFPGDG

MGRPWNGSYVN SGTLVEDLLANYLLEIGARNHKMRVYE

MTTHPTALEMTGYLLVRGGTHIIAYAKALEVATGVDVGKM

LPVPSLDNNKFDYAKKFMDQGLYNVLYTWGEADYRDINQI

WKGANPETGERLHVIDGMPEGAPVPDFPELPEQFAPGIDLD

DYYRILKRLKSNM MTVINDVKTALAGLKSAQASFETFALGTDNQQAKQLYQDA

AKQTQSVVDSIEPRVQQIEQEEPQYKQQ

MKDKITTLKNLLFIIMVIGFASGCNGNQNEFSQGNSTFGISQ VHTSKPIDQSVANHAKEKIIAKEDITDVKAVNTDKELMAAI KVENFDRFRLKSIEKSVKSDLEKKYPDYKVFVSTDKKIFWE LEKVEQRLKKNDMNKKNLKNDLNKLKSLMKEQT

MSN QKKQLTPVQQEYQKLQKQREIKRPVVKNCIKAFLIG GLICLIGQLISDFYIYYFDFTEQTAGNPTVGTLIFITMLLTGFG VYDRMAQFGGAGTAVPVTGFGNAVISPAIEHRSEGFVLGV GGNIFKLAGAVILFGVFSAFVIALIKTTLIQWGGL

MIYREGQPVLAGASGAGCSATVVYGHLLNRMKNGEFKRM LVVATGALLSPLSFQQNETIPCIAHAVSIEYGGEQLT

MIYFWAFVVGGLICIIGQIMFDVFKLTPGHTLSALVVIGALL

DGFGLYEPLIDFAGAGATVPITNFGNSLVHGAMQEAEKHGL

VGVLTGMFEVTSSGISAAIVFGMIGALIFKPKG

MSIFLVKVIFIYIVFIVAVNILGKSALAQLTPHDFGAILFLS

MSNLRTTGYPDIHDNEYAILEATGEISIFPRKELVTITSKYLH MKVEYRGLPIAVVIEGKVQKRKLKFINKNEKWLKEELKAK GYLQIKDFFΎAAVRDTDHSLΉNKKDVND

MEWIFYLYLLNTFFILFIAIWEVRRPAKALNWIIIVLFLPVIGF

WLYLSISNPKFIHRKRLTCSKNESNKLPETFGASASVIANAL

RHFTVNGLRMGQVQVFN GIAKYDQLLVSLQKAQETIDLE

YFIYRNDQIGNRITELLIEKALNGVQVRFMRDSLGSYKFPRE

KIRQMVEAGIECRTIFPLRFPWILSNWNYRDHCKIVTIDRKE

SFTGGMNVGYEYTGLKPDVGFWRDTHLQIIGEATGDLQTV

FDVHWEIAVPERIKSKTNLKTKSEVTKINSIGRADHSKWSAE

LGSELSTLDDKEMDISGKTRTLHKAYIHTLEGNPGIPTPVIR

QAYFICITQATKTIDLTTPYFVPETDIIMALKTAVTRGVRVRL

LVPRHNNQKIVGLASRTYYGELIEAGVQIYQYDKGMIHAK

VLTIDEEIAAVGSANYDMRSFRLNYEVCQVVYSADVAREL TEQFERDLTDSVPLRIEDLVQRSLTERIVEQGARVLSPLL

MTVGSDVKQCFASLKGVEASLSSLALRTLDDESKRTLHEA MMVVHEVTKDLKKRVGELEGEELQYKGF

MPEWLDVAVRSVLFLALLFFITKWLGKKQISELSFFEYVTGI

SIGNIGAEVAIGLERNIFQGIIGIVIFAVIPFFAGLISLKSKRFR

NFIEGKATVFIKDGKIMEDNLKKERYTTDELLDVLRKKDVF

QVSDVEFAVLEATGDLSVMLKKENQPLTAKDINLKVASIKE

PQΉIMDGTIMDEPLATIGRSRAWLQTELEKLGVTIENVFLG

QVNSYGELTIDLFDDKLQVAPPQERPLILSTLKKCHADLELF

ALGTESKDAKQMYRLNSEKLQEAIDKVTPILKG

MLAGHRTWIFEQKPVIISTGTVGGPFEANGAIPDDFDTLHAD

LWLGQDSYEKAHKILFEEACQKAMEKGGIQKDQVQFILAG

DLINQITPTSFASRTIGAPYFGLFGACSTSMEGLALGAYIVNT

KGAKYLLTGASSHDTAVEKQFRYPTEYGGQKPPTAQWTVT

GAGAALLSDSGEGPHVTSATIGRVIDMGLTDPFNMGGAMA

PAAVDTIEAHLKERNVEPSYYDLIVTGDLGQIGQEVSMDLF

KKHGTPISEEQYQDCGLMIYREGQPVLAGASGAGCSATVV

YGHLLNRMKNGEFKRMLVVATGALLSPLSFQQNETIPCIAH

AVSIEYGGEQLT MERRIFIRLRHRVLAHPGDIITVGDAAQIEGQLQLKKKLSAM

PLYQVSEKDKNIVILDIIQVLRAIHLQDPTIDVQTVGGAETIV EIQYRKRNLSTVLFIGVWLLLFIGSCLAIMNFHEDVSMRDV HIALYEIITGERNDYPYLLQIPYSIGLGLGMIVFFNHIFKKRL NEEPSPLEVEMFNYQLDLDQYVAMHENQETIKDLHDR MIVSVLFIIFVGLGGGITVGAGFVAFLTVMGIIPRLMQLTKT

MRFVQAYEAAVILGAVCGGWETLHMNHLYLTKWIAVPVG LLAGLFVGMLAAALTEVLNVLPILAKRIGLRSKIIILLMAIVI GKIAGSLFHWLYFIDHS MTNIKENYKSKVKTYQPKPPYVWNCVKAFLVGGLICAIGQ

GLQNFYIHFFDFNEKTAGNPTAATLILISALLTGFGIYDRIGQ FAGAGSAVPVTGFANSMASAALEYKSEGLVLGVATNMFKL AGNVIVFGVVAAYIVGMIRFAFEKLMS MKLTGKQTWVFEHPIFVNSAGTAAGPKEKDGPLGSLFDKT

YDEMHCNQKSWEMAERQLMEDAVNVALQKNNLTKDDID LLLAGDLLNQNVTANYVARHLKIPFLCMFGACSTSMETVA VASAL VDGGFAKRALAATSSHNATAERQFRYPTEYGGQKP DTATSTVTGSGAVVISQTPGDIQITSATVGKVSDLGITDPFD MGSAMAPAAADTIKQHFKDLNRTADDYDLILTGDLSGVGS PIVKDILKEDGYPVGTKHDDCGLLIYTPDQQVFAGGSGCAC SAVVTYSHIFKQLREGKLNRVFVVATGALLSPTMIQQKETIP TIAHGVVFERAGGAS

51 Native spoVAEb MDYLLAFVCGGAICIVGQLLLDIFKLTPAHVMTSFVVIGAIL DGFGIYDKFIEFAGGGATVPIVSFGHSLLHGAMHQAHIHGFI GIGMGIFELTSAGISAAILFAFIVAVIFKPKG

52 Native spoVAEa MTTKRKVILVTDGDVYAAKTIEYAASKVGGRCISQSKGNPS

TRSGAELVKLIASAPYDPVFVMFDDSGLQGEGPGEAALTYV

ATHPSIEVLGVIAVASKTHQAEWTRVDVSIDRNGEITEYGV

DKVGEREFDDHRMSGDTVYCLDKLDLPLIVGIGDIGKMGR

KDDISKGSPITMKAVEFILERSGYHAGPQGRENSGLSESS

53 Native spoVAF MPDHKEEKIRVYRNPAKNEEYFKNRVGMGTSYDVGVRKL

TILDKEIQLYYLNGLCDTAYIIHLMRELVAINNRKEDPDELV

DIVENRLLNAQVEKVKTLDETTDQVLSGLVAVIVEGAGFAF

IIDVRSYPGRNPEEPDTEKVVRGARDGFVENIVVNTALLRRR

IRDERLRVKMTKVGERSKTDLSICYIEDIADPDLVEIVEKEIA

SIDVDGLTMADKTVEEFIVNQSYNPFPLVRYTERPDVAANH

VLEGHVIIIVDTSPSVIITPTTLFHHVQHAEEYRQAPSVGTFL

RWVRFFGILASTLFLPIWFLFVLQPDLLPDNMKFIGLNKDTH

IPIILQIFLADLGIEFLRMAAIHTPTALSTAMGLIAAVLIGQIAI

EVGLFSPEVILYVSLAAIGTFTTPSYELSLANKMSRLVLMIL

VALFHIKGLVIGFTVLIIAMASIKSLQTPYLWPLIPFNGKALW

QVLVRTAKPGAKVRPSIVHPKNRLRQPTNS

Determining the exact location of the insert

Primers for PCR were developed based on the strains 4067, 4068, 4069, 4070, 4071, 4072, 4073, 4145, and 4146, to determine the insertion site in the chromosome. PCR primers were designed based on conserved regions (based on sequence alignment with other strains) within the genes of interest. Additionally primers were designed in yitF and yitG to determine the insertion site of the transposon yitF. These primers are depicted in Table 1. In all strains with the high spore heat resistance phenotype a PCR product was formed using primers yitF-F and tnpA-R.

Deletion of the location insertion yitF

A disruption mutant of yitF, was obtained from the BGSC (BKE10970). Spores were prepared as described above, followed by a heat inactivation experiment at 100°C for 60 minutes. No survivors appeared after this heat treatment, indicating complete inactivation.

A disruption of the gene yitF in B. subtilis 168 did not result in an increase of spore heat resistance. Therefore the location yitF, where the transposon is integrated, is not solely responsible for the increased spore heat resistance phenotype. Identification of the transposon in other species

Based on comparative genomics, the transposon was additionally identified in certain strains of B. amyloliquefaciens and B. licheniformis that belong to the B. subtilis group. The transposon in B. amyloliquefaciens consisted of the genes encoding for Transposase, Resolvase, Site specific recombinase, hypothetical protein, lipoprotein, SpoVAC, SpoVAD, SpoVAE, hypothetical protein, hypothetical protein, Cardiolipin Synthetase. The B. amyloliquefaciens strains carrying the transposon do not carry the gene yitF, and thus the transposon has a different site of integration in the genome, which is still to be verified experimentally. The transposon found in B. licheniformis has the same composition as was found in B. subtilis. The location of the transposon on the chromosome in B. licheniformis,was not in yitF, but in a gene encoding for D- Alanyl-D- Alanine Carboxypeptidase. The role of the transposon and the correlation to the increased spore heat resistance in B. amyloliquefaciens and B. licheniformis is depicted in Example 2).

Discussion

In this study we establish that spores of different B. subtilis isolates display highly significant differences in heat resistance. Two distinct groups could be identified based on a thorough analysis of the spore heat resistances of eighteen strains, using a wide range of time-temperature combinations for heat exposure. This study thus provides a detailed description of variation in spore heat resistance of B. subtilis species, and renders a modeling approach using two spore inactivation kinetics for highly heat resistant strains versus lower heat resistant strains. The spore heat resistance varied within the B. subtilis species from a D ₁₂ o _° c of 0.34 seconds for the low spore heat resistance group to a D ₁₂ o _° c of 48.5 seconds for the high spore heat resistance group, thus a factor 142 different (Berendsen et al. 2014, submitted for publication).

Based on spore heat resistance, strains of the B. subtilis could be grouped into two clusters when plotting the logD values against temperature. Variation in spore heat resistance of strains within the B. subtilis species and B. subtilis group has been reported before (Lima et al, 2011 ; Oomes et al, 2007). For Clostridium perfringens, strain variation in spore heat resistance was observed with varying D _{90 °} c values ranging from 5.5 minutes to 120.6 minutes (Orsburn et al, 2008). In addition for B. cereus, where spore inactivation kinetics were globally assessed, strain variation was identified as significant factor (van Asselt et al, 2006). The high number of strains used in the current study allowed for a statistical analysis that rendered two groups with respect to spore heat resistance. In our analysis, strain B. subtilis A163 was incorporated (corresponding with strain nr. 4067) and showed Duo _° c values of 1.79 and 1.53 minutes, which is higher than the previously reported Duo _° c of 0.7 minutes by Kort et al, 2005. However, the reported z-value of 6.1 °C for this strain (Kort et al, 2005) was similar to the z-values found in this study, i.e. 6.3 °C (± 0.7 °C). A possible explanation for the difference in Duo _° c-value is the different preparation method of spores, on plates in this study, and in liquid medium for the other experiment (Kort et al, 2005). The phenomenon that spores produced on surfaces are more heat resistant than spores produced from planktonic cells in liquid media has previously been reported by Rose et al 2007, who observed higher spore heat resistances for B. subtilis when spores were prepared following growth on agar plates compared with liquid medium. There are multiple other factors known to contribute to the final spore heat resistance. Generally the higher the sporulation temperature, the higher the final spore heat resistance properties (Nicholson et al, 2000). The sporulation of B. subtilis in a natural or a processing environment might occur in biofilms, and complex colony growth allows the formation of more heat resistant spores (Lindsay et al, 2006; Veening et al, 2006). The composition of the sporulation medium is also important, including different salts added to the medium, such as magnesium, manganese, potassium, and in particular calcium, are known to increase the final heat resistance of spores of B. subtilis (Cazemier et al, 2001; Oomes et al, 2004; Oomes et al, 2009). Calcium is also required for a spore to reach full heat resistance, after release from the mother cell, in the maturation process (Sanchez-Salas et al 2011). In this study, the spores of all strains were allowed to form, and mature under the same conditions, to rule out the effect of variation in sporulation conditions on spore heat resistance. No variations in sporulation conditions were applied; the observed differences in spore heat resistance between strains are thus a specific property of the strain. A generally observed phenomenon is that the spore heat resistance decreases after re-sporulation under laboratory conditions (Lima et al, 2011; van Zuijlen et al, 2010). It is important to consider that the exact history of spores encountered in food matrices, such as the sporulation and maturation conditions, is not known. Multiple time-temperature combinations can be proposed, based on batch heating in capillary tubes, to distinguish the two groups of spore heat resistance within the B. subtilis group. Heating for one hour at 100°C, will result in a 10.2 log reduction for the low spore heat resistance group and a 0.1 log reduction for the high spore heat resistance group, using the logZ ⁾ _re from the batch heating experiment. Using a similar approach, heating for 5 minutes at 110 °C will result in a 18.6 log reduction for the low spore heat resistance group, and a 0.1 log reduction for the high spore heat resistance group. It should be noted that the proposed time-temperature combinations are based on spores prepared under laboratory conditions, and do not include variations in spore heat resistance based on the history of the spores and a potential effect of the food matrix.

A mobile genetic element was identified, that correlated to the increased spore heat resistance, using a gene-trait matching approach. The integration site of the mobile genetic element was verified by PCR, and the insertion was found in the same gene iyitF) for all strains producing high heat resistant spores. The disruption of the gene yitF, did not result in the increased spore heat resistance.

Conclusions

In this study the spore heat inactivation kinetics were determined in detail for eighteen stains of B. subtilis. Two distinct groups of spore heat resistance were identified, with batch heating using capillary tubes. The spore heat resistance within B. subtilis can be separated in two groups, indicating that spore heat resistance is not a species specific, but rather a strains specific property. Using a gene-trait matching approach, a mobile genetic element was identified, correlated to the increased spore heat resistance phenotype.

Example 2: Further investigation of Bacillus strains with spores of high thermal resistance

Materials and Methods

Strains, sporulation, and establishing spore heat resistance

For 18 strains of B. subtilis the spore heat resistance was characterized, and for all strains the genome sequence was either publically available, or recently sequenced (Table 2). Detailed spore heat inactivation kinetics were determined for 11 strains in example 1 and Berendesen et al, 2015. For the other 7 strains, spores were prepared and detailed inactivation kinetics were determined, as described in example 1. The variation in spore heat resistance of 18 strains was visualized by plotting the decimal reduction time (D) over temperature, and allowed for separation in two groups of spore heat resistance as was described in example 1 and Figure 1. Additionally, 9 strains of B. amyloliquefaciens and 9 strains of B. licheniformis were included for determination of spore inactivation kinetics. For ?, amyloliquefaciens strains B425 and B4140, the spore inactivation kinetics were determined previously, and for the other strains newly determined as described in example 1. The B. amyloliquefaciens strains SB42 and 101, and the B. licheniformis strains were isolated from food. For the B. amyloliquefaciens strains B425, B4140, FZB42 and DSM7, and for all B. licheniformis strains, the genome sequence was determined previously (Ruckert et al, 2011; Chen et al, 2007). Carry-over of transposon

A filter mating experiment was performed as described previously, to carry over the transposon, thereby establishing its role in increased spore heat resistance (Auchtung et al, 2005). High spore heat resistant strain B4067 was used as a donor, and strain 168 amyE: :spec as a recipient. After the mating procedure spores were prepared, and subjected to a heat treatment of 100 °C for 60 minutes which allows for selection of strains with increased spore heat resistance. Survivors were additionally selected on the basis of antibiotic resistance marker, tryptophan deficiency, and colony morphology. The presence of the transposon in the survivors was verified by PCR, and by re- sequencing of the genome (Chen et al, 2007) of the selected survivor, which was designated as strain 168-HR. During the mating experiment, clear lysis of strain 4067, after induction with mitomycinC (MMC), was observed. Strain 4067 was then induced to produce phages as described previously (Moineau et al, 1994) and DNA was isolated from the phages, and subjected to next generation sequencing. The sequence of the phage DNA were mapped against the DNA of the 4067 genome and visualized using Artemis (Carver et al, 2012).

Gene deletion and cloning Specific deletion mutants were constructed in strain 168-HR3 using the cre/lox system, as previously described with slight alterations (Lambert et al, 2007; Yan et al, 2008). The genetic make-up of the deletion mutants is depicted in Figure 2A. The PCR fragment lox71-cat-lox66 cassette from pNZ5319 was fused by overhang PCR with the flanking regions corresponding to flanking regions of the genes to be deleted. The fused fragment was cloned into a SwaI/Ecll36II restricted pNZ5319. Deletion mutants were constructed for the entire transposon, yitF, and for the predicted operons in the transposon. Correct deletion mutants were verified by PCR, and spore heat resistance was assessed as described here above. The Tn\546-spoVA operons from B. subtilis 4067, B. amyloliquefaciens DSM7, and B. licheniformis B4090 were cloned into pDG1730 (Guerout-Fleury, 2008). Constructs were integrated into the amyE locus of B. subtilis 168, followed by sporulation and assessing of the spore heat resistance as described above. Spore characterization

Total protein was extracted from spores. Bead beating (4 rounds, 40 seconds, 5m/s) was applied to 0.5 mL of spore suspension, followed by addition of 1 mL Urea (8M) Tris (lOmM), at pH 8 and incubation at room temperature for 1 hour. From the total protein extract 10 μg was digested in silutiuon, after reduction and alkylation, in- solution digested. The resulting purified and concentrated peptide mixture was analyzed by nanoflow CI 8 reversed phase liquid chromatography (Bruker Daltonics). The DP A content of spores were determined for strain 168 and 168-FIR3, as described previously (Kort et al, 2005). The core water content of the spores of 168 and 168-HR3 was determined as described previously (Lindsay et al, 1985).

Results and Discussion

Bacterial endospores are intrinsically resistant towards environmental insults, and are considered as one of sturdiest forms of life on earth (Nicholson et al, 2000; Sunde et al, 2009; Gould et al, 2006). The enigma of spore heat resistance has puzzled scientist for long periods of time (Sunde et al, 2009; Gerhardt et al, 2003). A spore-specific buildup ensures dormancy and resistance and a highly organized gene regulatory network is involved in the process of sporulation which is well studied in Bacillus subtilis (Sunde et al, 2009; Errington et al, 2003; Eijlander et al, 2014; Setlow et al, 2006). Major variation in heat resistance of spores within the species of B. subtilis has been observed in previous studies (Berendsen et al, 2015; Oomes et al, 2007; Lindsay et al, 1988). However, the underlying molecular mechanisms of the observed increased spore heat resistance remained unknown. Here we provide conclusive evidence that increased spore heat resistance of in specific strains of B. subtilis has a genomic determinant, i.e. a transposon-based spoVA operon, revealing a novel mechanism of adaptation, which was found in a wide variety of spore forming Bacilli. We found a crucial role for this spoVA operon, that was not known before (Nicholson et al, 2000; Sunde et al, 2009). In depth genomic analysis provided new insights in the abundance and spread of this spoVA operon, indicating a central role in the ecology and evolution of bacterial spore formers including pathogenic species.

Heat treatments during food processing are applied to ensure food safety but such treatments put continuous selective pressure on heat resistance of bacterial spore formers, and selects for the survival of the most resistant spores (Postollec et al, 2012). Horizontal gene transfer plays an important role in the acquisition of increased resistance of bacteria towards their respective selective pressure (Ochman et al, 2000). Understanding the genetic and molecular basis of the adaptations is essential for control of resistant bacteria.

Upon determination of the spore heat resistance of 18 B. subtilis strains, a clear separation into two groups of spore heat resistance was observed, as demonstrated in example 1 (Figure 1) and in Berendsen et al, 2015. By correlating the genome content on basis of orthology to the spore heat resistance phenotype, a Tnl546 transposon was identified, which was solely present in the strains which displayed an increased heat resistance of spores. The size and exact DNA sequences of the genes on the transposon varied slightly between the different strains.

Transfer of a genomic fragment was achieved from donor strain 4067 to the lab strain B. subtilis 168, which included the transposon integrated at genomic location yitF (BSU10970). In the filter-mating experiment active transposition did not occur, as anticipated based on the truncated tnpA gene and absence of resolvase gene in the donor strain 4067. However, 4067 showed lysis upon treatment with mitomycinC and phage particles could be isolated. The sequences of the DNA isolated from the phages contained the complete chromosome of 4067 at low levels. The selected strain was designated 168-HR3, and its spores displayed a significantly increased heat resistance (Figure 3). Subsequent deletion of Tnl546 from 168-HR3 resulted in the loss of the high spore heat resistance phenotype (Figure 3).

To identify the Tnl546 encoded genes responsible for the increased spore heat resistance, specific deletion mutants were constructed. Deletion of the spoVA operon from Tnl546 of 168-FIR3 resulted in the loss the increased heat resistance of spores (Figures 2 and 3). The Tnl546 spoVA operon is different from the native spoVA operon, although both contain genes encoding for SpoVAC, SpoVAD and SpoVAEb (Figure 2A, 2B). It is known that deletion of the native spoVA genes from B. subtilis 168 results in spores that do not complete sporulation, and they were proven to be essential for uptake and release of dipicolinic acid (DP A) (Tovar-Rojo et al, 2002; Ghosh et al, 2009; Ghosh* et al, 2009; Perez- Valdespino et al, 2013). Interestingly, the levels of DP A did not differ between 168 and 168-HR3 suggesting that there is no difference in the uptake of DP A.

More specifically, deletion of the final gene SpoVA2H (polynucleotide sequence SEQ ID NO: 45, polypeptide sequence SEQ ID NO: 42) from the spoVA operon in 168-HR resulted in the loss of spore heat resistance indicating that the encoded protein is required for increased spore heat resistance (Figure 3). This SpoVA2H gene encodes for a hypothetical protein and is predicted to be membrane bound by 3 transmembrane segments and carry two domains of unknown function DUF421 and DUF1657 (Figure 2C). The hypothetical protein was detected in the spores of 168-HR3, and since the protein is predicted to be in the inner membrane, it is plausible that it is involved in the stability thereof.

Spores of the different B. subtilis strains showed different levels of adaptation to heat, which directly correlated to the number of Tnl546 spoVA operons present in the genome of the strain (Figure 4). The transposon was commonly found integrated in yitF, between yxjA (BSU39020) and yxjB (BSU39010). In addition, a partial duplication of the Tnl546 based spoVA operon was identified. This partial duplication of Tnl546 contained the complete spoVA operon and was flanked by genes encoding mobile genetic element proteins including transposases.

The Tnl546 transposon carrying the spoVA operon was also identified in certain strains of B. amyloliquefaciens and B. licheniformis. The presence of Tnl546 correlated to an increased heat resistance of spores, compared to strains that do not possess the transposon. Tnl546 was identified in 2 out of 9 tested B. amyloliquefaciens strains and 3 out of 9 tested B. licheniformis strains. The difference in B. amyloliquefaciens was more prominent than in B. licheniformis and could be attributed to the presence of at least two Tnl546 copies in B. amyloliquefaciens compared to a single Tnl546 in the B. licheniformis strains. The composition and genomic locations of the Tnl546 transposon are different among the different species. The additional spoVA operon was cloned from B. amyloliquefaciens DSM7 and B. licheniformis 4090 into amyE of B. subtilis 168, and spores displayed an increased heat resistance to heat.

The identified transposon displays high similarity to that of Tnl546 as found in Enterococcus faecium, carrying a transposase, a resolvase and imperfect inverted repeats flanking the transposon (Arthur et al, 1993). The major difference is found in the genes that are carried by the transposon, namely vancomycin resistance genes in E. faecium, and sporulation-related genes in B. subtilis. Genomic analysis of the genes of the transposon also clearly indicates close relatedness to the Tnl546 as found on B. cereus plasmids. However, the order of the genes and exact composition of Tnl546 is different in B. cereus compared to the one identified in the B. subtilis group.

The two distinct spoVA operons belong to the core genome of B. cereus. In a limited number of B. cereus strains, the high spore heat resistance spoVA operon is additionally encoded on plasmids encompassed in a Tnl546 transposon. It is plausible that B. subtilis acquired the spoVA operon that leads to high spore heat resistance by horizontal gene transfer from B. cereus a plasmid carrying Tnl546, that encompasses the spoVA operon as the Tn3 like transposon requires a plasmid intermediate for active transposition (Arthur et al, 1993).

It is a longstanding question how differences in robustness of bacterial spores are mediated (Nicholson et al, 2000; Oomes et al, 2007). It is known that many factors influence the final heat resistance properties of the spores, such as optimal growth temperature, sporulation temperature, pH, salts and sporulation on plates (Cazemier et al, 2001; Baril et al, 2012; Rose et al, 2007). In the present study, an important genomic determinant underlying variation in spore heat resistance was identified. A Tnl546 transposon was identified carrying a spoVA operon, that was proven to be responsible for increased spore heat resistance, by a novel mechanism of adaptation. Possibly food processing selected for spores of those strains that were adapted to different gradations of heat resistance. The limited sequence variation indicated that the adaptations were a result of a recent evolutionary event. The additional spoVA operon was identified in various spore forming Bacilli and the evolutionary relatedness among the different spoVA proteins, indicate distinct evolutionary roles for these proteins in spore formation.

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