Method for producing a heat-resistant antibacterial agent having antibacterial activity against gram-positive bacteria, heat-resistant antibacterial agent, and method for imparting resistance to heat-resistant antibacterial substances by resistance gene

Abstract

To provide genes encoding heat-stable antibacterial substances, vectors, transformants, heat-stable antibacterial substances, resistance genes, drug resistance markers and kits.SOLUTION: The disclosure relates to a gene encoding a heat-stable peptidic antibacterial substance having an antibacterial property against gram-positive bacteria. The gram-positive bacteria include bacteria of family Bacillaceae. The disclosure also relates to a heat-stable antibacterial substance encoded by the gene as well as a resistance gene thereto. The resistance gene may be used as a drug resistance marker.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to genes encoding heat-resistant antibacterial substances, vectors, transformed cells, heat-resistant antibacterial substances, resistance genes, drug resistance markers, and kits. [Background technology]

[0002] Non-Patent Document 1 discloses the genome sequencing of the thermophilic bacterium Aeribacillus pallidus PI8. Aeribacillus pallidus PI8 is a bacterium of the phylum Firmicutes, family Bacillaceae, and genus Aeribacillus. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Kyosuke Kita, Atsushi Ishida, Kosei Tanaka, Shu Ishikawa, Ken-ichi Yoshida, ``Complete Genome Sequence of Thermophilic Bacterium Aeribacillus pallidus PI8'', Microbiology Resource Announcements, AMERICAN SOCIETY FOR MICROBIOLOGY,1990, volume 9 Issue 17 Summary of the Invention

[0004] The present inventors have discovered an antibacterial substance produced by Aeribacillus pallidus PI8 and a gene involved in its expression. Furthermore, the present inventors have experimentally discovered a resistance gene to such a heat-resistant antibacterial substance.

[0005] One aspect of the present disclosure is a gene that encodes a thermostable antibacterial substance that has antibacterial activity against Gram-positive bacteria and has peptide properties.

[0006] Another aspect of the present disclosure is a heat-resistant antibacterial substance. The heat-resistant antibacterial substance of the present disclosure has antibacterial activity against gram-positive bacteria.

[0007] Another aspect of the present disclosure is a resistance gene. The resistance gene of the present disclosure confers resistance to Gram-positive bacteria.

[0008] Further details will be described in the following embodiments. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the results of SDS PAGE and Gel-overlay assay of the antibacterial substances. [Figure 2] FIG. 2 shows the gene cluster (pcyn gene cluster) that contributes to the expression of antibacterial substances. [Figure 3] FIG. 3 shows the results of the first verification experiment. [Figure 4] FIG. 4 shows the results of the second verification experiment. DETAILED DESCRIPTION OF THE INVENTION

[0010] <1. Genes encoding heat-resistant antibacterial substances, heat-resistant antibacterial substances, and resistance genes>

[0011] (1) This embodiment relates to a gene consisting of DNA that encodes a heat-stable antibacterial substance having antibacterial activity against Gram-positive bacteria and also having peptidic properties. The gene according to this embodiment preferably consists of any one of the following DNAs (A-1) to (A-5): (A-1) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2; (A-2) DNA consisting of a nucleotide sequence having an identity of 90% or more to the nucleotide sequence represented by SEQ ID NO: 2; (A-3) DNA consisting of a nucleotide sequence having an identity of 30% or more with the nucleotide sequence represented by SEQ ID NO: 2 and contained in the bacteriocin biosynthetic gene cluster of a thermophilic bacterium; (A-4) DNA consisting of the base sequence represented by SEQ ID NO: 2 in which one or more bases have been deleted, substituted, or added; (A-5) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 2.

[0012] The heat-stable antibacterial substance encoded by the gene according to the embodiment has antibacterial activity against Gram-positive bacteria and can therefore be used to control Gram-positive bacteria, for example, for selective cultivation of Gram-positive bacteria. The heat-stable antibacterial substance according to the embodiment has peptide properties. The amino acid sequence of the heat-stable antibacterial substance according to the embodiment may be cyclic or linear. Proteins or peptides with antibacterial activity that are synthesized by bacteria on ribosomes are called "bactericions."

[0013] As used herein, the term "having peptidic properties" refers to a substance that is made up of molecules in which amino acids are linked in a short chain by peptide bonds, and a substance that has peptidic properties is a peptide or a protein.

[0014] In this specification, the identity of nucleotide sequences and amino acid sequences is calculated using an analysis program such as BLAST [J. Mol. Biol., 215, 403 (1990)]. The calculation is performed using the default parameters of the program.

[0015] In the gene encoding the heat-resistant antibacterial substance according to the embodiment, the sequence identity with the base sequence represented by SEQ ID NO: 2 is preferably 30% or more, and may be 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and generally, the greater the identity value, the more preferable. Such a gene preferably consists of a base sequence having an E value of less than 1e-4 with respect to the base sequence represented by SEQ ID NO: 2. 1e-4 is defined as 10 -4 The E value for the base sequence represented by SEQ ID NO: 2 may be any of less than 1e-5, less than 1e-6, less than 1e-7, less than 1e-8, less than 1e-9, and less than 1e-10.

[0016] As used herein, the term "E-value" (expectation value) is an index used to evaluate the homology between a query sequence and another sequence. It refers to the expected number of alignments that will have a score equal to or greater than the query sequence when a sequence database is searched using the query sequence. The E-value is preferably the E-value obtained when a homology search is performed using the BLAST (Basic Local Alignment Search Tool) algorithm as the search algorithm for the sequence database. In this case, the default parameters for the sequence database using the BLAST algorithm are used in the homology search.

[0017] The gene encoding the thermostable antibacterial substance according to the embodiment preferably consists of DNA contained in the genome of a thermophilic bacterium, and more preferably consists of DNA contained in the bacteriocin biosynthesis gene cluster of a thermophilic bacterium. The bacteriocin biosynthesis gene cluster of a thermophilic bacterium has the structure shown in Figure 2 and was identified by the present inventors.

[0018] The bacteriocin biosynthetic gene cluster of a thermophilic bacterium shown in Figure 2 is contained in the genome of a thermophilic bacterium that produces bacteriocin, and consists of the pcynB, pcynA, pcynC, pcynD, pcynE, and pcynF genes arranged in this order from upstream to downstream. In this specification, each gene is defined as described below. The gene cluster consisting of pcynB, pcynA, pcynC, pcynD, pcynE, and pcynF is referred to as the pcyn gene cluster.

[0019] pcynB is a gene consisting of any one of the DNAs (B-1) to (B-5) below. (B-1) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1; (B-2) DNA consisting of a nucleotide sequence having an identity of 90% or more to the nucleotide sequence represented by SEQ ID NO: 1; (B-3) DNA consisting of a nucleotide sequence having an identity of 30% or more to the nucleotide sequence represented by SEQ ID NO: 1; (B-4) DNA consisting of the base sequence represented by SEQ ID NO: 1 in which one or more bases have been deleted, substituted, or added; (B-5) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1.

[0020] The sequence identity with the base sequence represented by SEQ ID NO: 1 is preferably 30% or more, and may be 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, with a larger identity value generally being preferred. The same applies to pcynA, C, D, and E. Such a gene preferably consists of a base sequence having an E value of less than 1e-4 with respect to the base sequence represented by SEQ ID NO: 1. Note that 1e-4 is defined as 10 -4 The E value for the base sequence represented by SEQ ID NO: 1 may be any of less than 1e-5, less than 1e-6, less than 1e-7, less than 1e-8, less than 1e-9, and less than 1e-10. The same applies to pcynA, C, D, and E.

[0021] pcynA is a gene consisting of any one of the DNAs (A-1) to (A-5) described above.

[0022] pcynC is a gene consisting of any one of the DNAs (C-1) to (C-5) below. (C-1) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3; (C-2) DNA consisting of a nucleotide sequence having an identity of 90% or more to the nucleotide sequence represented by SEQ ID NO: 3; (C-3) DNA consisting of a nucleotide sequence having an identity of 30% or more to the nucleotide sequence represented by SEQ ID NO: 3; (C-4) DNA consisting of the base sequence represented by SEQ ID NO: 3 in which one or more bases have been deleted, substituted, or added; (C-5) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3.

[0023] pcynD is a gene consisting of any one of the DNAs (D-1) to (D-5) below. (D-1) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 4; (D-2) DNA consisting of a nucleotide sequence having an identity of 90% or more to the nucleotide sequence represented by SEQ ID NO: 4; (D-3) DNA consisting of a nucleotide sequence having an identity of 30% or more to the nucleotide sequence represented by SEQ ID NO: 4; (D-4) DNA consisting of the base sequence represented by SEQ ID NO: 4 in which one or more bases have been deleted, substituted, or added; (D-5) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 4.

[0024] pcynE is a gene consisting of any one of the DNAs (E-1) to (E-5) below. (E-1) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5; (E-2) DNA consisting of a nucleotide sequence having an identity of 90% or more to the nucleotide sequence represented by SEQ ID NO: 5; (E-3) DNA consisting of a nucleotide sequence having an identity of 30% or more to the nucleotide sequence represented by SEQ ID NO: 5; (E-4) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5 in which one or more nucleotides have been deleted, substituted, or added; (E-5) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 5.

[0025] pcynF is a gene consisting of any one of the DNAs (F-1) to (F-6) described below.

[0026] When extracting candidates for the pcynA gene using a sequence database, a homology search is performed using the sequence shown in SEQ ID NO: 2 as a query sequence, and sequences with a predetermined level of identity, such as 30% or more, are extracted. The sequence extraction condition may be, in addition to or instead of identity, a condition using an E value, such as an E value of less than 1e-4 with respect to the sequence shown in SEQ ID NO: 2. Whether a sequence extracted through a search or experiment is suitable as a candidate for the pcynA gene can be determined by whether the extracted sequence and its surrounding sequences have the organization of the pcyn gene cluster. That is, whether pcynB is present upstream of the extracted sequence and the sequence pcynC, D, E, and F is present downstream of the extracted sequence. Alternatively, whether pcynB is present upstream of the extracted sequence and the sequence pcynC, D, and E is present downstream of the extracted sequence may be determined. Alternatively, whether pcynB is present upstream of the extracted sequence and the sequence pcynC and D is present downstream of the extracted sequence may be determined. Alternatively, the determination may be made based on whether pcynB is present upstream of the extracted sequence and whether pcynC is present downstream of the extracted sequence.

[0027] To determine whether the extracted sequence and its surrounding sequences share the gene arrangement of the pcyn gene cluster, sequences including the extracted sequence are annotated using an annotation tool such as DFAST (DDBJ FAST Annotation and Submission Tool). From the annotated results, candidates for the pcynA gene are identified as described above. If ORFs (Open Reading Frames) corresponding to pcynB, C, D, and E are present around the candidate pcynA gene, it can be confirmed that pcynA and its surrounding genes share the gene arrangement of the pcyn gene cluster.

[0028] Whether a certain nucleotide sequence (e.g., the ORF immediately preceding (upstream of) pcynA in the annotated results) is pcynB or not can be determined by sequence identity, such as whether it has 30% or more identity to the nucleotide sequence set forth in SEQ ID NO: 1. The identity may be 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In addition to or instead of determining sequence identity, determination may also be made using an E value, such as an E value of less than 1e-4. Whether a certain nucleotide sequence is pcynB or not can also be determined by whether the gene product consisting of the amino acid sequence encoded by the nucleotide sequence is a YIP1 family protein.

[0029] Whether a certain sequence (e.g., the ORF immediately (downstream) of pcynA in the annotated results) is pcynC or not is determined by sequence identity, such as whether it has 30% or more identity to the nucleotide sequence shown in SEQ ID NO: 3. The identity may be 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In addition to or instead of determining by sequence identity, determination may also be made using an E value, such as an E value of less than 1e-4. Whether a certain nucleotide sequence is pcynC or not may also be determined by whether a gene product consisting of an amino acid sequence encoded by the nucleotide sequence is a DUF95 family protein.

[0030] Whether a certain sequence (e.g., the ORF immediately (downstream) of pcynC in the annotated results) is pcynD or not is determined by sequence identity, such as whether it has 30% or more identity to the nucleotide sequence shown in SEQ ID NO: 4. The identity may be 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In addition to or instead of determining by sequence identity, determination may also be made using an E value, such as an E value of less than 1e-4. Whether a certain nucleotide sequence is pcynD or not may also be determined by whether a gene product consisting of an amino acid sequence encoded by the nucleotide sequence is a membrane protein for a transporter.

[0031] Whether a certain sequence (e.g., the ORF immediately (downstream) of pcynD in the annotated results) is pcynE or not is determined by sequence identity, such as whether it has 30% or more identity to the nucleotide sequence shown in SEQ ID NO: 5. The identity may be 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In addition to or instead of determining by sequence identity, determination may also be made using an E value, such as an E value of less than 1e-4. Whether a certain nucleotide sequence is pcynE or not may also be determined by whether a gene product consisting of an amino acid sequence encoded by the nucleotide sequence is an ATP-binding domain for a transporter.

[0032] Whether a sequence (e.g., the ORF immediately (downstream) of pcynE in the annotated results) is pcynF or not is determined by sequence identity, such as whether it has 25% or more identity to the nucleotide sequence shown in SEQ ID NO: 6. The identity may be 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In addition to or instead of determining by sequence identity, determination may also be made using an E value, such as an E value of less than 1e-4. Even if the identity to the nucleotide sequence shown in SEQ ID NO: 6 is very low, for example, 0%, it can be pcynF (a resistance gene to heat-stable antibacterial substances encoded by the pcynA gene included in the pcyn gene cluster) as long as it is included in the pcyn gene cluster. In other words, if the presence of pcynB, A, C, D, and E that constitute the pcyn gene cluster is confirmed, the gene sequence (ORF) immediately following pcynE can be determined to be pcynF.

[0033] The bacteriocin biosynthetic gene cluster possessed by the bacteriocin-producing bacterium is preferably a gene cluster for biosynthesis of a cyclic bacteriocin. The thermophilic bacterium that produces bacteriocin is preferably a bacterium of the family Bacillaceae. The bacteriocin-producing bacterium of the family Bacillaceae is preferably a bacterium of the genus Aeribacillus, genus Virgibacillus, genus Alkalihalobacillus, or genus Bacillus, and more preferably a bacterium of the genus Aeribacillus. The bacteriocin-producing bacterium of the genus Aeribacillus is preferably Aeribacillus pallidus. The present inventors have found that the pcyn gene cluster shown in Figure 2 is contained in the genomes of bacterium-producing bacteria of the Bacillaceae family, particularly in the genomes of bacteria of the genera Aeribacillus, Virgibacillus, Alkalihalobacillus, and Bacillus.

[0034] Examples of bacteria of the genus Aeribacillus having the pcyn gene cluster include Aeribacillus pallidus PI8, Aeribacillus pallidus TD1, Aeribacillus pallidus 8, Aeribacillus pallidus 8m3, Aeribacillus pallidus W-12, and Aeribacillus compostii KCTC 33824.

[0035] An example of a bacterium of the genus Virgibacillus that has the pcyn gene cluster is Virgibacillus proomii.

[0036] A bacterium of the genus Alkalihalobacillus that has the pcyn gene cluster is Alkalihalobacillus krulwichiae.

[0037] Bacteria of the genus Bacillus that have the pcyn gene cluster include Bacillus krulwichiae AM31D, Bacillus pumilus CH95a_3T, and Bacillus sp. SLBN-174.

[0038] In the present specification, the number of bases from which one or more bases are deleted, substituted, or added is one or more and is not particularly limited, but is a number that can be deleted, substituted, or added by well-known techniques for deletion, substitution, or addition, and is, for example, one to several tens of bases, preferably 1 to 30 bases, more preferably 1 to 20 bases, even more preferably 1 to 10 bases, even more preferably 1 to 8 bases, and even more preferably 1 to 5 bases.

[0039] As used herein, "DNA that hybridizes under stringent conditions" refers to DNA obtained by colony hybridization, plaque hybridization, Southern blot hybridization, or the like, using a DNA having a nucleotide sequence listed in a public database or a partial DNA fragment thereof as a probe. More specifically, "DNA that hybridizes under stringent conditions" refers to DNA that can be obtained by hybridizing a filter onto which colony- or plaque-derived DNA has been immobilized in the presence of 0.7 to 1.0 M NaCl at 65°C, followed by washing the filter in 0.1 to 2x SSC solution (1x SSC solution is 150 mM NaCl, 15 mM sodium citrate) at 65°C. Hybridization can be performed by well-known methods, such as those described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001). The higher the temperature or the lower the salt concentration, the higher the stringency, allowing for the isolation of polynucleotides with higher homology (sequence identity).

[0040] (2) The Gram-positive bacterium as a bacterium sensitive to the heat-resistant antibacterial substance in (1) above is preferably a bacterium of the family Bacillaceae. The Gram-positive bacterium is more preferably a bacterium of the genus Bacillus, the genus Geobacillus, or the genus Parageobacillus. The Gram-positive bacterium is even more preferably a bacterium of the genus Geobacillus or the genus Parageobacillus.

[0041] (3) The gene (1) is preferably obtained from a bacterium of the family Bacillaceae, and more preferably a thermophilic bacterium. The bacterium of the family Bacillaceae is preferably a bacterium of the genus Aeribacillus, Virgibacillus, Alkalihalobacillus, or Bacillus, and more preferably a bacterium of the genus Aeribacillus. The bacterium of the genus Aeribacillus is preferably Aeribacillus pallidus.

[0042] (4) A vector according to an embodiment of the present invention has the gene according to any one of (1) to (3) introduced therein.

[0043] (5) A transformed cell according to the embodiment contains the vector of (4) above.

[0044] (6) An embodiment relates to a heat-resistant antibacterial substance having antibacterial activity against Gram-positive bacteria. The heat-resistant antibacterial substance according to the embodiment preferably comprises any one of the following (PA-1) to (PA-4). (PA-1) a heat-resistant antibacterial substance consisting of the amino acid sequence of SEQ ID NO: 8; (PA-2) a heat-stable antibacterial substance consisting of an amino acid sequence having an identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 8; (PA-3) A thermostable antibacterial substance derived from a thermophilic bacterium, which consists of an amino acid sequence having an identity of 30% or more with the amino acid sequence represented by SEQ ID NO: 8; (PA-4) A heat-resistant antibacterial substance consisting of an amino acid sequence represented by SEQ ID NO: 8 in which one or more amino acids have been deleted, substituted, or added.

[0045] In the heat-resistant antibacterial substance according to the embodiment, the sequence identity with the amino acid sequence represented by SEQ ID NO: 8 is preferably 30% or more, and may be 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, with a larger identity value being generally preferred. Such an amino acid sequence preferably has an E value of less than 1e-4 with respect to the amino acid sequence represented by SEQ ID NO: 8. 1e-4 is defined as 10 -4 The E value for the amino acid sequence represented by SEQ ID NO: 8 may be any of less than 1e-5, less than 1e-6, less than 1e-7, less than 1e-8, less than 1e-9, and less than 1e-10.

[0046] When extracting candidates for heat-stable antibacterial substances according to the embodiment using a sequence database, a homology search is performed using the sequence shown in SEQ ID NO: 8 as a query sequence, and sequences having a sequence identity of a predetermined value or more, such as 30% or more, are extracted. In addition to or instead of identity, the sequence extraction condition may also be a condition using an E value, such as an E value of less than 1e-4 with respect to the sequence shown in SEQ ID NO: 8.

[0047] Whether a substance consisting of a sequence extracted by search or experiment is suitable as a candidate for a heat-stable antibacterial substance according to an embodiment can be determined by determining whether the gene encoding the extracted sequence (candidate pcynA) and its surrounding genes have the gene organization of the pcyn gene group in the genome of a bacterium having the gene encoding the extracted sequence. That is, the determination is based on whether pcynB is present upstream of the gene encoding the extracted sequence (candidate pcynA) and the sequence pcynC, D, E, and F is present downstream of the gene encoding the extracted sequence (candidate pcynA). Alternatively, the determination may be based on whether pcynB is present upstream of the gene encoding the extracted sequence (candidate pcynA) and the sequence pcynC, D, and E is present downstream of the gene encoding the extracted sequence (candidate pcynA). The determination may also be based on whether pcynB is present upstream of the gene encoding the extracted sequence (candidate pcynA) and the sequence pcynC and D is present downstream of the gene encoding the extracted sequence (candidate pcynA). Alternatively, it may be determined whether pcynB is present upstream of the gene encoding the extracted sequence (candidate pcynA) and whether pcynC is present downstream of the gene encoding the extracted sequence (candidate pcynA).

[0048] The thermostable antibacterial substance according to the embodiment is preferably derived from a thermophilic bacterium. The thermophilic bacterium is preferably a bacterium of the family Bacillaceae. The bacterium of the family Bacillaceae is preferably a bacterium of the genus Aeribacillus, genus Virgibacillus, genus Alkalihalobacillus, or genus Bacillus, and more preferably a bacterium of the genus Aeribacillus. The bacterium of the genus Aeribacillus is preferably Aeribacillus pallidus.

[0049] In the present specification, the number of amino acids in which one or more bases are deleted, substituted, or added is one or more and is not particularly limited, but is a number that can be deleted, substituted, or added by well-known techniques for deletion, substitution, or addition, and is, for example, one to several tens, preferably 1 to 30, more preferably 1 to 20, even more preferably 1 to 10, even more preferably 1 to 8, and even more preferably 1 to 5.

[0050] The heat-stable antibacterial substance according to the embodiment is produced, for example, by introducing the biosynthetic gene cluster (pcyn gene cluster) of the heat-stable antibacterial substance according to the embodiment into a host. The biosynthetic gene cluster of the heat-stable antibacterial substance is introduced into the host using a vector such as a plasmid. The host into which the biosynthetic gene cluster is introduced may be a bacterium that is sensitive to the heat-stable antibacterial substance according to the embodiment (for example, the above-mentioned Gram-positive bacteria), or may be a bacterium that is not sensitive to the heat-stable antibacterial substance according to the embodiment. Examples of bacteria that are not sensitive to the heat-stable antibacterial substance according to the embodiment include Escherichia coli and Bacillus subtilis.

[0051] When the host is not sensitive to the heat-resistant antibacterial substance of the embodiment, the gene group to be introduced may be the entire pcyn gene group (pcynB, pcynA, pcynC, pcynD, pcynE, pcynF) or the gene group excluding pcynF from the pcyn gene group (pcynB, pcynA, pcynC, pcynD, pcynE).

[0052] (7) An embodiment relates to a resistance gene that confers resistance to a heat-resistant antibacterial substance. The resistance gene according to the embodiment preferably comprises any one of the DNAs (F-1) to (F-6) below. (F-1) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 6; (F-2) DNA consisting of a nucleotide sequence having an identity of 90% or more to the nucleotide sequence represented by SEQ ID NO: 6; (F-3) DNA consisting of a nucleotide sequence having an identity of 25% or more with the nucleotide sequence represented by SEQ ID NO: 6 and contained in the bacteriocin biosynthetic gene cluster of a thermophilic bacterium; (F-4) DNA consisting of the base sequence represented by SEQ ID NO: 6 in which one or more bases have been deleted, substituted, or added; (F-5) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 6; (F-6) A resistance gene included in a bacteriocin biosynthesis gene cluster including the gene encoding the heat-resistant antibacterial substance described in (1) (a gene consisting of DNA of any one of (A-1) to (A-5)), which confers resistance to the heat-resistant antibacterial substance encoded by the gene described in (1).

[0053] In the resistance gene according to the embodiment, the sequence identity to the base sequence represented by SEQ ID NO: 6 is preferably 25% or more, and may be 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, with a higher identity value being generally preferred. The resistance gene according to the embodiment preferably comprises a base sequence having an E value of less than 1e-4 with respect to the base sequence represented by SEQ ID NO: 6. The E value with respect to the base sequence represented by SEQ ID NO: 6 may be any of less than 1e-5, less than 1e-6, less than 1e-7, less than 1e-8, less than 1e-9, or less than 1e-10.

[0054] The resistance gene according to the embodiment preferably comprises DNA contained in a bacteriocin biosynthesis gene cluster of a thermophilic bacterium (see FIG. 2). The bacteriocin biosynthesis gene cluster is preferably a cyclic bacteriocin biosynthesis gene cluster.

[0055] The thermophilic bacterium is preferably a bacterium of the family Bacillaceae. The bacterium of the family Bacillaceae is preferably a bacterium of any of the genera Aeribacillus, Virgibacillus, Alkalihalobacillus, and Bacillus, and more preferably a bacterium of the genus Aeribacillus. The bacterium of the genus Aeribacillus is preferably Aeribacillus pallidus.

[0056] By introducing the resistance gene according to the embodiment into, for example, a different species of bacterium, the bacterium is conferred resistance to the heat-stable antibacterial substance according to the embodiment. The resistance gene is introduced using a vector such as a plasmid. Genetically modified bacteria can be selected by transformation to introduce the resistance gene. By introducing the resistance gene according to the embodiment into a bacterium (e.g., the aforementioned Gram-positive bacterium) that is sensitive to the heat-stable antibacterial substance according to the embodiment, the bacterium can be conferred resistance to the heat-stable antibacterial substance according to the embodiment. The Gram-positive bacterium into which the resistance gene according to the embodiment can be introduced is preferably a bacterium of the family Bacillaceae. The Gram-positive bacterium into which the resistance gene according to the embodiment can be introduced is more preferably a bacterium of the genus Bacillus, Geobacillus, or Parageobacillus. The Gram-positive bacterium is even more preferably a bacterium of the genus Geobacillus or Parageobacillus.

[0057] (8) The heat-resistant antibacterial substance encoded by the resistance gene according to the embodiment is preferably the heat-resistant antibacterial substance described in (6) above.

[0058] (9) The drug resistance marker according to the embodiment preferably comprises the resistance gene according to the above (7) or (8). The resistance gene according to the embodiment can be used as a drug resistance marker (selection marker).

[0059] (10) A kit according to the embodiment includes the heat-resistant antibacterial substance described in (6) above and the drug resistance marker described in (9) above.

[0060] Few conventional drug resistance markers are suitable for genetic manipulation of thermophilic bacteria. This has hindered the development of modified breeding of thermophilic bacteria. Conventionally, antibiotics and their resistance genes have been used for genetic modification of thermophilic bacteria. Conventional antibiotics include kanamycin, chloramphenicol, hygromycin, and thiostrepton. Antibiotics other than kanamycin are not common and have the disadvantage of low heat resistance of the antibiotics themselves or the resistance gene products (enzymes, etc.).

[0061] In contrast, the heat-resistant antibacterial substance and its resistance gene provided in the kit according to the embodiment are advantageous in that they are highly stable during high-temperature cultivation.

[0062] <2. Experiment>

[0063] Various experimental examples are shown below. The following experimental examples are non-limiting preferred examples, and the present invention is not limited to these experimental examples.

[0064] <2.1 Antibacterial spectrum>

[0065] Aerobacterium pallidus PI8 was cultured on a culture plate, and bacterial colonies were harvested and suspended in 20 mM phosphate buffer (pH 6.8). The cells were disrupted by sonication (Q500 Sonicator, QSonica, USA) for 5 minutes at 4°C (2 seconds on, 7 seconds off, 20% amplitude). The lysate was centrifuged at 15,000 × g for 20 minutes at 4°C to remove cell debris. The supernatant was sterilized by filtration through a 0.22 μm filter. This is hereafter referred to as CFS (cell-free supernatant). CFS was used for antibacterial spectrum analysis.

[0066] The indicator strain Geobacillus kaustophilus HTA426 was spread on an LB agar plate and cultured overnight at 60°C for 12 hours. The bacteria on the plate were then suspended in LB medium and the OD 600 The pH was adjusted to 1.0. This suspension was mixed with LB agar at a ratio of 1:25 and mixed thoroughly. 25 mL of the resulting cell suspension was poured into a Petri dish and left to solidify. 20 μL of CFS was spotted onto the dish and incubated at an incubation temperature of 60°C for 24 hours. The antibacterial spectrum was determined for the other bacterial strains listed below using the same method. Below, the type of medium and incubation temperature used for each bacterium, including Geobacillus kaustophilus HTA426, are listed next to the strain name.

[0067] Geobacillus kaustophilus HTA426 (LB, 60℃), Geobacillus thermodentrificans K1 (LB, 60℃), Parageobacillus toebii K3 (LB, 60℃), Parageobacillus genomospecies 1 (LB, 60℃), Bacillus megaterium 4-1 (LB, 37℃), Bacillus. megaterium 8-1 (LB, 37℃), Bacillus sp. 7-3 (LB, 37℃), Escherichia coli DH5α (LB, 37℃), Azospirillum brasilense Sp7 (LB, 30℃), Shinorhizobium meliloti 1021 (TY, 30℃).

[0068] Geobacillus kaustophilus HTA426 and Geobacillus thermodenitrificans K1 are Gram-positive bacteria belonging to the family Bacillaceae and genus Geobacillus.

[0069] Parageobacillus toebii K3 and Parageobacillus genomospecies 1 are Gram-positive bacteria belonging to the family Bacillaceae and genus Parageobacillus.

[0070] Bacillus megaterium 4-1, Bacillus megaterium 8-1, and Bacillus sp. 7-3 are Gram-positive bacteria belonging to the family Bacillaceae and genus Bacillus.

[0071] Escherichia coli DH5α, Azospirillum brasilense Sp7, and Shinorhizobium meliloti 1021 are Gram-negative bacteria.

[0072] Table 1 shows the antibacterial spectrum determined by the above-mentioned method. In Table 1, Gram-positive bacteria are represented by G+, and Gram-negative bacteria are represented by G-. Also in Table 1, ++ indicates that the CFS obtained from Aeribacillus pallidus PI8 exhibited strong inhibition against the bacterial strain, + indicates that the CFS exhibited inhibition against the bacterial strain, and - indicates that the CFS did not exhibit inhibition against the bacterial strain. The inhibitory activity, represented by ++, +, and -, indicates the results of observing the periphery of Aeribacillus pallidus PI8 colonies on agar medium, with ++ indicating clear growth inhibition for each test bacterium, + indicating that growth inhibition was unclear but was deemed to have occurred, and - indicating no growth inhibition at all.

[0073] [Table 1]

[0074] As is clear from Table 1, CFS obtained from Aeribacillus pallidus PI8 did not inhibit the growth of Gram-negative bacteria (G-), but inhibited the growth of Gram-positive bacteria. Therefore, it was revealed that CFS is an antibacterial substance that is effective against Gram-positive bacteria. Furthermore, this antibacterial substance is suitable as an antibacterial substance against bacteria belonging to the Bacillaceae family, and more preferably as an antibacterial substance against bacteria belonging to any of the genus Geobacillus, Paradiobacillus, and Bacillus. Furthermore, this antibacterial substance is suitable against thermophilic bacteria.

[0075] According to Table 1, this antibacterial substance exhibited stronger inhibition against bacteria belonging to the genus Geobacillus and Paradiobacillus, and is therefore more suitable as an antibacterial substance against bacteria belonging to the genus Geobacillus or Paradiobacillus.

[0076] <2.2 Stability>

[0077] [Table 2]

[0078] Table 2 shows the results of the stability test of CFS. When CFS was kept at 4°C, its inhibitory activity was maintained and it was stable for more than 7 months. Furthermore, its antibacterial activity was maintained and it was stable both at 80°C for 20 minutes and at 100°C for 20 minutes. Therefore, this antibacterial substance is heat stable. In other words, this antibacterial substance is heat resistant.

[0079] Furthermore, when CFS was subjected to the protease proteinase K, its inhibitory activity was lost. Therefore, it can be determined that this antibacterial substance has peptide properties. In other words, this antibacterial substance can be said to be an antibacterial peptide. Based on the above, it is clear that the antibacterial substance produced by Aeribacillus pallidus PI8 is a heat-stable antibacterial substance (heat-stable antibacterial peptide) that has antibacterial activity against Gram-positive bacteria. This heat-stable antibacterial peptide was named "pallidocyclin." Palidocyclin is a bacteriocin, and is thought to be a cyclic bacteriocin. Palidocyclin secreted into the culture medium by Aeribacillus pallidus PI8 is highly hydrophobic, making isolation difficult. However, the present inventors have succeeded in extracting and analyzing it by mass spectrometry, as described below.

[0080] 2.3 Extraction and molecular weight of palidocycline

[0081] An overnight culture of Aeribacillus pallidus PI8 (hereafter referred to as "PI8") was inoculated onto a single spot on the surface of an LB plate containing 0.8% agar, allowed to soak thoroughly, and then incubated at 60°C for 24 hours. The agar surrounding the PI8 colony was excised, crushed, and submerged in 100% acetonitrile (ACN) and incubated at 4°C for 48 hours to extract palidocycline. The ACN liquid phase was then collected and centrifuged (15,000 xg, 1 minute) to obtain the supernatant. 1 ml of the supernatant was loaded onto an Agilent Bond Elut C18 cartridge (previously washed with 0.3 ml of 100% methanol and 0.3 ml of deionized water). The cartridge was then washed sequentially with 0.3 ml of 30% acetonitrile, 0.3 ml of 30% ethanol, 0.4 ml of 20% isopropyl alcohol (IPA), and 0.4 ml of 40% IPA, and finally palidocycline was eluted with 2 ml of 80% IPA, 0.1% TFA. The eluate was concentrated to dryness by evaporation using a centrifugal concentrator.

[0082] The concentrated dried product was dissolved in SDS sample loading buffer and subjected to Tris-Tricine SDS-PAGE in a 15-20% precast polyacrylamide gel (SuperSep™ Ace, Fujifilm Wako Co., Ltd.). Tricine running buffer solution (Fujifilm Wako Co., Ltd.) was used as the running buffer. Palidocycline was confirmed as a single band by staining with a Silver Stain MS kit (Fujifilm Wako Co., Ltd.). Figure 1(a) shows the results. Figure 1(a) indicates a low molecular weight (3-6.5 kDa). Furthermore, the distribution of the molecular weight over the 3-6.5 kDa range suggests that palidocycline has a cyclic structure.

[0083] The gel after SDS-PAGE was then excised and placed on a plate. Agar was then layered on top of the gel, and the susceptible bacterium Geobacillus caustophilus HTA426 was then sprayed on top of the agar. Figure 1(b) shows the results. Figure 1(b) shows that the growth of Geobacillus caustophilus HTA426 was inhibited in the range P where peptides were detected in SDS-PAGE. From the above, it can be seen that a single antibacterial peptide with a low molecular weight (3 to 6.5 kDa) was detected.

[0084] <2.4 Identification of antibacterial substances and genes>

[0085] After SDS-PAGE and staining, the palidcycline band was excised from the gel and purified by standard trypsin digestion to generate peptides. The partial peptides obtained by trypsin digestion were subjected to mass spectrometry analysis. Specifically, the partial peptides obtained by trypsin digestion were subjected to LC-ESI-MS / MS analysis using a high-performance liquid chromatograph (Paradigm MS2, Michrom BioResources Inc., Auburn, CA, USA), an autosampler (HTS-PAL), and a linear ion trap field-coupled Fourier transform mass spectrometer (Thermo Fisher Scientific LTQ OrbitrapXL / Discovery).

[0086] To identify the detected peptides, the MS / MS data were searched against all peptide sequences predicted from the PI8 genome information using MASCOT software (Matrix Science, Tokyo, Japan). One of the resulting partial peptides matched a peptide that appeared when the protein encoded by the gene predicted from the PI8 genome sequence was digested with trypsin. Therefore, it was suggested that this gene encodes a heat-stable antimicrobial peptide.

[0087] The present inventors further discovered that genes predicted to encode a bacteriocin maturation factor, secretion apparatus, and resistance factor form an operon around the gene (pcynA in Figure 2) in the PI8 genome (see Figure 2). The present inventors also discovered that the gene cluster shown in Figure 2 is commonly distributed among bacteria (preferably thermophilic bacteria) that produce bacteriocin (preferably circular bacteriocin). Bacteria having the gene cluster shown in Figure 2 are, for example, bacteria of the Bacillaceae family. Bacteria of the Bacillaceae family include, for example, bacteria of the genus Aeribacillus, genus Virgibacillus, genus Alkalihalobacillus, and genus Bacillus.

[0088] Figure 2 shows the above-mentioned operon. This operon is referred to as the "pcyn gene cluster." The pcyn gene cluster has pcynA (e.g., SEQ ID NO: 2) located between pcynB (e.g., SEQ ID NO: 1) and pcynC (e.g., SEQ ID NO: 3). In other words, pcynA is located between pcynB on the upstream side and pcynC on the downstream side. The pcyn gene cluster further has pcynD (e.g., SEQ ID NO: 4) following pcynC, pcynE (e.g., SEQ ID NO: 5) following pcynD, and pcynF (e.g., SEQ ID NO: 6) following pcynE.

[0089] In PI8, pcynB is a gene encoding a YIP1 family protein. In PI8, pcynC is a gene encoding a DUF95 family protein. The present inventors discovered that pcynB and pcynC are common domains in the cyclic bactericione biosynthetic gene cluster possessed by bacteria that produce cyclic bactericions, and that a bactericione precursor gene exists between them. Therefore, pcynA, located between pcynB and pcynC, is presumed to be a precursor gene encoding a precursor of pallidocyclin. Furthermore, the peptide encoded by pcynA, sandwiched between pcynB and pcynC, is thought to be a cyclic bactericione. The proteins (gene products) encoded by pcynB and pcynC are thought to be maturation factors that modify the palidocycline precursor. It is thought that the proteins encoded by pcynB and pcynC modify the palidocycline precursor to form a cyclic structure.

[0090] The amino acid sequence of palidocyclin encoded by pcynA (SEQ ID NO: 2) is represented by SEQ ID NO: 8. The amino acid sequence of the protein (gene product) encoded by pcynB (SEQ ID NO: 1) is represented by SEQ ID NO: 7. The amino acid sequence of the protein (gene product) encoded by pcynC (SEQ ID NO: 3) is represented by SEQ ID NO: 9.

[0091] pcynD encodes a transmembrane protein, and pcynE encodes an ATP-binding domain. The transmembrane protein and the ATP-binding domain constitute an ABC transporter, which is a secretion apparatus. The amino acid sequence of the protein encoded by pcynD (SEQ ID NO: 4) is shown in SEQ ID NO: 10. The amino acid sequence of the domain encoded by pcynE (SEQ ID NO: 5) is shown in SEQ ID NO: 11.

[0092] Experiments by the present inventors have revealed that pcynF (SEQ ID NO: 6) is a gene encoding an immunity protein against antibacterial substances. The amino acid sequence of the gene product encoded by the resistance gene represented by pcynF (SEQ ID NO: 6) is represented by SEQ ID NO: 12.

[0093] As described above, this operon (pcyn gene cluster) was presumed to be a gene cluster responsible for the biosynthesis of palidocycline, with pcynA being the gene encoding palidocycline. To experimentally confirm this presumption, this operon (pcyn gene cluster consisting of DNA shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6) was transcriptionally fused downstream of the constitutively and highly expressed rpsI gene on the chromosome of Bacillus subtilis and expressed. As a result, it was confirmed that antibacterial activity similar to that of the antibacterial substance (palidocycline) obtained from PI8 was obtained. Therefore, as previously presumed, this operon was found to be a gene cluster responsible for the biosynthesis of palidcycline, a thermostable antibacterial substance obtained from PI8, and pcynA consisting of DNA shown in SEQ ID NO: 2 was found to be the gene encoding palidocycline.

[0094] For example, even in the genomes of thermophilic bacteria other than PI8, by searching for a gene cluster having a structure similar to the bacteriocin biosynthesis gene cluster shown in Figure 2, it is possible to easily select a gene encoding a heat-stable antibacterial substance having antibacterial activity against Gram-positive bacteria and having peptidic properties, which is DNA consisting of a base sequence having 40% or more identity with the base sequence represented by pcynA (SEQ ID NO: 2). The substance encoded by the gene selected in this way is a heat-stable antibacterial substance having antibacterial activity against Gram-positive bacteria and having peptidic properties.

[0095] 2.5 Immunity proteins

[0096] The immunity protein here is a resistance factor against a heat-stable antibacterial substance according to the embodiment. Bacterial producers have an autoimmune function to protect themselves from the bacterium they produce. The immunity protein is responsible for the autoimmunity. The present inventor hypothesized that the pcynF gene following pcynE in the pcyn gene cluster is an immunity gene encoding an immunity protein against palidocycline. The results of an experiment verifying this hypothesis are shown in Figures 3 and 4.

[0097] Figure 3 shows the results of the first validation experiment. In this experiment, only the pcynF gene of the pcyn gene group was cloned into the pGKE75 plasmid so that its expression was inducible under the control of a maltose-inducible promoter. This plasmid was then introduced into Geobacillus caustophilus, a palidocycline-sensitive bacterium. Geobacillus caustophilus with the pcynF gene introduced in this way was cultured (G. Kaustophilus (pGKE75-pcynF) in Figure 3; 3B, 3D). For comparison, Geobacillus caustophilus was also cultured with the pGKE75 plasmid, which does not contain the pcynF gene (G. Kaustophilus (pGKE75) in Figure 3; 3A, 3C).

[0098] A lysate of PI8 expressing palidocycline was spotted onto the center of cultured Geobacillus caustophilus (Fig. 3A and 3B). Also, PI8 cells expressing palidocycline were spotted onto the center of cultured Geobacillus caustophilus (Fig. 3C and 3D).

[0099] When PI8 lysate was spotted onto Geobacillus caustophilus without the pcynF gene (Fig. 3, 3A), growth of Geobacillus caustophilus was inhibited within the spotted area. Furthermore, when PI8 cells were spotted onto Geobacillus caustophilus without the pcynF gene (Fig. 3, 3C), PI8 cells proliferated within the spotted area, and growth of Geobacillus caustophilus was inhibited around the PI8 cells. Thus, the growth of Geobacillus caustophilus without the pcynF gene was inhibited by palidocycline produced by PI8 (Fig. 3, 3A and 3C).

[0100] On the other hand, when a lysate of PI8 was spotted onto Geobacillus caustophilus into which the pcynF gene had been introduced (Fig. 3B), Geobacillus caustophilus grew within the spotted area. Furthermore, when PI8 cells were spotted onto Geobacillus caustophilus into which the pcynF gene had been introduced (Fig. 3D), PI8 cells grew within the spotted area, but the growth of Geobacillus caustophilus around the PI8 cells was hardly inhibited. This indicates that Geobacillus caustophilus, whose growth is normally inhibited by palidocycline, acquired resistance to palidocycline by introducing the pcynF gene.

[0101] The results of the second validation experiment are shown in Figure 4. In the second validation experiment, an experiment similar to the first validation experiment was carried out on Geobacillus thermodenitrificans, another bacterium susceptible to palidocycline.

[0102] In the second verification experiment, only the pcynF gene of the pcyn gene group was ligated to the pGKE75 plasmid, and this plasmid was introduced into Geobacillus thermodenitrificans, a palidocycline-sensitive bacterium. Geobacillus thermodenitrificans with the pcynF gene introduced in this way was cultured (Figure 4, G. thermodenitrificans (pGKE75-pcynF); 4B, 4D). For comparison, Geobacillus thermodenitrificans was also cultured with the pGKE75 plasmid lacking the pcynF gene (Figure 4, G. thermodenitrificans (pGKE75); 4A, 4C).

[0103] A lysate of palidocyclin-expressing PI8 was spotted onto the center of cultured Geobacillus thermodenitrificans (Figures 4A and 4B), and palidocyclin-expressing PI8 cells were spotted onto the center of cultured Geobacillus thermodenitrificans (Figures 4C and 4D).

[0104] When PI8 lysate was spotted onto Geobacillus thermodenitrificans without the pcynF gene (Figure 4, 4A), growth of Geobacillus thermodenitrificans was inhibited within the spotted area. Furthermore, when PI8 cells were spotted onto Geobacillus thermodenitrificans without the pcynF gene (Figure 4, 4C), PI8 cells proliferated within the spotted area, and growth of Geobacillus thermodenitrificans was inhibited around the PI8 cells. Thus, the growth of Geobacillus thermodenitrificans without the pcynF gene was inhibited by palidocycline produced by PI8 (Figures 4A and 4C).

[0105] On the other hand, when the PI8 lysate was spotted onto Geobacillus thermodenitrificans into which the pcynF gene had been introduced (4B in Figure 4), Geobacillus thermodenitrificans grew within the spotted area. Furthermore, when PI8 cells were spotted onto Geobacillus thermodenitrificans into which the pcynF gene had been introduced (4D in Figure 4), the PI8 cells grew within the spotted area, but the growth of Geobacillus thermodenitrificans around the PI8 cells was hardly inhibited. This indicates that Geobacillus thermodenitrificans, whose growth is normally inhibited by palidocycline, acquired resistance to palidocycline by introducing the pcynF gene.

[0106] The results of the verification experiments shown in Figures 3 and 4 confirmed that the bacteria into which the aforementioned pcynF gene had been introduced had increased resistance to palidocycline, and that the pcynF gene is a resistance gene that confers resistance to palidocycline.

[0107] In the genomes of thermophilic bacteria other than PI8, by searching for gene clusters having a structure similar to that of the bacteriocin biosynthetic gene cluster shown in Figure 2, it is possible to easily determine resistance genes to antibacterial substances biosynthesized by the biosynthetic gene cluster, even if the DNA consists of a base sequence with low identity to the base sequence shown in SEQ ID NO: 6. Furthermore, in the pcyn gene cluster, a gene downstream of pcynE can be determined to be a resistance gene to antibacterial substances encoded by pcynA, regardless of identity to the base sequence shown in SEQ ID NO: 6.

[0108] 2.6 Characteristics of Palidocycline and Immunoproteins

[0109] The isoelectric point (pI value) of palidocycline was 5.97, near neutral. Here, a near-neutral isoelectric point refers to, for example, an isoelectric point in the range of 5 to 8, more preferably in the range of 5 to 7. While cyclic bacterium are generally alkaline, palidocycline is characterized by having a near-neutral isoelectric point. Palidocycline is expected to have a mechanism of action that disrupts the integrity of membrane structure rather than disrupting membrane potential charge.

[0110] The gravy index of palidocycline is 0.9874, a value close to 1. Here, "close to 1" refers to a range of 0.8 to 1.2, more preferably a range of 0.9 to 1.1. Here, the gravy index refers to the Grand Average of Hydropathy. The gravy index is calculated by dividing the sum of the hydrophobicity values ​​of all amino acids by the sequence length. The gravy index can range from - to +, and for most proteins it falls between -2 and +2. The larger the positive value, the more hydrophobic the protein, and the larger the negative value, the more hydrophilic the protein. The gravy index is calculated based on the formula shown in Jack Kyte and Russell F. Doolittle, "A simple method for displaying the hydropathic character of a protein," Journal of Molecular Biology, Volume 157, Issue 1, May 5, 1982, Pages 105-132.

[0111] The immunity protein encoded by the pcynF gene of PI8 (immunity protein for palidocycline) had a near-neutral isoelectric point of 6.7, and a gravy index of 0.257.

Claims

1. A method for producing a heat-resistant antibacterial agent having antibacterial activity against Gram-positive bacteria, which comprises any one of the following DNAs (A-1) to (A-5) and encodes a heat-resistant antibacterial substance having peptidic properties and antibacterial activity against Gram-positive bacteria: (A-1) DNA consisting of the base sequence represented by SEQ ID NO: 2; (A-2) DNA consisting of a base sequence having an identity of 90% or more to the base sequence represented by SEQ ID NO: 2; (A-3) DNA consisting of a nucleotide sequence having an identity of 90% or more with the nucleotide sequence represented by SEQ ID NO: 2 and contained in the bacteriocin biosynthetic gene cluster of a thermophilic bacterium; (A-4) DNA consisting of the base sequence represented by SEQ ID NO: 2 in which 1 to 10 bases have been deleted, substituted, or added; (A-5) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 2; (PA-1) a peptide substance consisting of the amino acid sequence of SEQ ID NO: 8; (PA-2) A substance having peptidic properties consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 8, which has antibacterial activity against Gram-positive bacteria and is heat-resistant; (PA-3) A substance having a peptide property derived from a thermophilic bacterium, which consists of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 8, and which has antibacterial activity against Gram-positive bacteria and is heat-resistant; (PA-4) A substance having peptidic properties consisting of an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 8, which has antibacterial activity against Gram-positive bacteria and is heat-resistant.

2. The Gram-positive bacteria are bacteria of the family Bacillaceae The method of claim 1.

3. The gene is obtained from a bacterium of the family Bacillaceae The method according to claim 1 or claim 2.

4. The heat-resistant antibacterial agent is produced using a transformed cell into which the gene according to claim 1 has been introduced using a vector into which the gene has been introduced.

4. The method according to any one of claims 1 to 3.

5. The heat-resistant antibacterial agent is produced using a transformed cell into which the gene according to claim 1 has been introduced.

4. The method according to any one of claims 1 to 3.

6. A heat-stable antibacterial agent having any one of the following peptide substances (PA-1) to (PA-4), and having antibacterial activity against Gram-positive bacteria: (PA-1) a peptide substance consisting of the amino acid sequence of SEQ ID NO: 8; (PA-2) A substance having peptidic properties consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 8, which has antibacterial activity against Gram-positive bacteria and is heat-resistant; (PA-3) A substance having peptide properties derived from a thermophilic bacterium, which consists of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 8, and which has antibacterial activity against Gram-positive bacteria and is heat-resistant; (PA-4) A substance having peptidic properties consisting of an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 8, which has antibacterial activity against Gram-positive bacteria and is heat-resistant.

7. A method for conferring resistance to a heat-resistant antibacterial substance having antibacterial activity against Gram-positive bacteria to a cell into which the resistance gene has been introduced, by transformation involving introducing a resistance gene consisting of any one of DNAs (F-1) to (F-5) below, the resistance gene conferring resistance to the heat-resistant antibacterial substance, wherein the heat-resistant antibacterial substance is a peptide consisting of the amino acid sequence of SEQ ID NO: 8: (F-1) DNA consisting of the base sequence represented by SEQ ID NO: 6; (F-2) DNA consisting of a nucleotide sequence having an identity of 90% or more to the nucleotide sequence represented by SEQ ID NO: 6; (F-3) DNA consisting of a nucleotide sequence having an identity of 90% or more to the nucleotide sequence represented by SEQ ID NO: 6 and contained in the bacteriocin biosynthetic gene cluster of a thermophilic bacterium; (F-4) DNA consisting of the base sequence represented by SEQ ID NO: 6 in which 1 to 10 bases have been deleted, substituted, or added; (F-5) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 6.

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