WO2015126251A1 - Micro-organismes thermorésistants - Google Patents

Micro-organismes thermorésistants Download PDF

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WO2015126251A1
WO2015126251A1 PCT/NL2015/050110 NL2015050110W WO2015126251A1 WO 2015126251 A1 WO2015126251 A1 WO 2015126251A1 NL 2015050110 W NL2015050110 W NL 2015050110W WO 2015126251 A1 WO2015126251 A1 WO 2015126251A1
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seq
group
polypeptide
polynucleotide
organism
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PCT/NL2015/050110
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Erwin Mathijs BERENDSEN
Maria Henrica Jacoba BENNIK
Oscar Paul Kuipers
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Stichting Top Institute Food And Nutrition
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Publication of WO2015126251A1 publication Critical patent/WO2015126251A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • CBS Cosmetic Base voor Schimmel cultures
  • 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.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • 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).
  • 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).
  • 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.
  • 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.
  • the invention further relates to a method for selecting a batch of a product comprising:
  • 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 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 invention relates to a method for the production of a batch of a microorganism, comprising:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • LDR Ligation Detection Reaction
  • liquid and solid hybridization assays such as Spot blot, Northern blot, Southern blot, and sequencing including whole genome sequencing.
  • 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.
  • 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
  • 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.
  • 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.
  • 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).
  • PCR polymerase chain reaction
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • LDR Ligation Detection Reaction
  • 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 wherein
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 is enhanced expression or reduced expression.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • sequence 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 preferably means that the value may be the given value (of 10) more or less 0.1% of the value.
  • 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.
  • spoVAA polypeptide SEQ ID NO: 47, polynucleotide SEQ ID NO: 54
  • spoVAB polypeptide SEQ ID NO: 48
  • spoVAC polypeptide SEQ ID NO: 49, polynucleotide SEQ ID NO: 56
  • spoVAD polypeptide SEQ ID NO: 50
  • 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
  • spoVAC 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).
  • strains containing three copies of the spoVA operon that mediates heat resistance have spores that are more heat resistance than when two (grey squares) copies or one (black squares) copy of the operon is/are present.
  • Example 1 Investigation of Bacillus strains with spores of high thermal resistance Materials, Methods and Results
  • 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.
  • 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.
  • 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 ⁇ 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 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.
  • 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.
  • CFU/ml colony forming units per milliliter
  • PBS phosphate buffered saline
  • 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 ⁇ , 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.
  • 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.
  • 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
  • the 95% prediction interval (PI) of the logD ref was calculated using the following equation:
  • t DF is the student t-value with degrees of freedom (DF)
  • the residual sum of squares (RSS) is calculated from the data points deviating from the regression line.
  • Table 4 The calculated z-values and logDref for the two groups of spore heat resistance.
  • 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.
  • SpVA2H SpoVA2H hypothetical protein comprising
  • Table 7 Sequence ID of the genes correlated to increased spore heat resistance associated with increased spore heat resistance, with their nucleotide sequences.
  • Table 8 Sequence ID of the polypeptides correlated to the increased spore heat resistance, with corresponding amino acid sequences.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • the reported z-value of 6.1 °C for this strain was similar to the z-values found in this study, i.e. 6.3 °C ( ⁇ 0.7 °C).
  • 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).
  • 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.
  • 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
  • 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 genome sequence was determined previously (Ruckert et al, 2011; Chen et al, 2007). Carry-over of transposon
  • strain 168-HR 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).
  • 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 ⁇ g 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).
  • 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).
  • 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.
  • Tnl546 encoded genes responsible for the increased spore heat resistance 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.
  • DP A dipicolinic acid
  • 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.
  • 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.
  • 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).

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Cette invention concerne un kit et des procédés et des moyens pour déterminer la présence d'organismes thermorésistants. L'invention concerne en outre l'obtention d'organismes chez lesquels la thermorésistance est modulée.
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WO2020264009A1 (fr) * 2019-06-24 2020-12-30 Noxilizer Inc. Procédé de fabrication d'indicateurs biologiques
WO2023214588A1 (fr) * 2022-05-02 2023-11-09 花王株式会社 Procédé de détection d'une bactérie sporogène

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WO2010049489A1 (fr) 2008-10-29 2010-05-06 Check-Points Holding B.V. Procédé pour la détection et l'identification rapides de micro-organismes

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WO2008066931A2 (fr) * 2006-11-29 2008-06-05 Novozymes, Inc. Chromosome de bacillus licheniformis
WO2010049489A1 (fr) 2008-10-29 2010-05-06 Check-Points Holding B.V. Procédé pour la détection et l'identification rapides de micro-organismes

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020264009A1 (fr) * 2019-06-24 2020-12-30 Noxilizer Inc. Procédé de fabrication d'indicateurs biologiques
WO2023214588A1 (fr) * 2022-05-02 2023-11-09 花王株式会社 Procédé de détection d'une bactérie sporogène

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