Novel strain of the genus Xanthomonas and novel bacteriocin produced therefrom

Abstract

The present invention relates to a novel Xanthomonas citri pv. glycine K2 strain, a bacteriocin produced therefrom, a functional food containing the same, a natural preservative, and an antimicrobial agent. The bacteriocin produced from the novel Xanthomonas citri pv. glycine K2 strain according to the present invention is different from the bacteriocin derived from the existing soybean blistering pathogen and has antimicrobial activity against Gram-positive bacteria as well as the soybean blistering pathogen, and can be usefully used as a functional food, a natural preservative, and an antimicrobial agent.

Description

Technology Field

[0001] The present invention relates to a novel Xanthomonas citrine pv. glycinese K2 ( Xanthomonas citri This invention relates to a pv. glycines K2) strain and a novel bacteriocin produced therefrom. Background Technology

[0003] Although there are various bacterial diseases, bacterial pustule, which primarily affects leaves, is a problem in Korea; since it mainly occurs in soybeans, it is called soybean pustule. Soybean pustule begins to appear around May or June and becomes severe in July and August when temperatures are high and rainfall is frequent. It persists until harvest, and in severe cases, it can reduce the number of seeds per pod and 100-grain weight—key yield components—leading to a 7–15% reduction in yield. In the early stages of infection, small spots appear on the leaves; as the disease progresses, the spots gradually enlarge, turn light brown, and the surrounding area turns yellow. To control this, methods such as cultivating pustule-resistant varieties or spraying fungicides as part of chemical control exist, but their effectiveness is limited.

[0004] Xanthomonas citri pv. glycinese 8ra( Xanthomonas citriP. glycine 8ra) is a representative plant pathogen that accounts for over 80% of domestic races of the soybean blight pathogen and is known to produce a bacteriocin called glycinecin (Fett et al., 1987). A bacteriocin refers to a toxic substance produced and secreted by bacteria to kill their own kind in order to survive in extreme environments. Since these substances are usually composed of proteins, they are broken down by proteolytic enzymes in the digestive tract when ingested; thus, they are non-toxic to humans, leave no residue, and pose no harm to the human body, making them highly valuable as eco-friendly agents. Furthermore, because these bacteriocins specifically kill only their own kind, they cause no harm to beneficial bacteria in the same environment, making them excellent control agents from an environmental perspective. In plants, food additives utilizing these bacteriocins have been commercialized and are used to prevent bacterial contamination in food.

[0005] X. citri The pv. glyciens 8ra strain was confirmed to possess a total of three bacteriocins. Among them, glycinecin A and glycinecin R possess DNase activity and react specifically with Xanthomonas. In particular, glycinecin A is a heterodimer in the form of adjacent glyA (3.9 kDa) and glyB (14 kDa) X. citri One copy of pv. glycines is present in all strains and is believed to be related to the K+ / OH- permeability of the cell membrane. Glycinesin R is approximately 4 kb in size and shows similarity to the Rhs element, and it has been reported that there is more than one copy (Noh., 2008). Glycinesin P has activity similar to bacteriophages and also reacts specifically only with Xanthomonas.

[0007] Against this background, the inventors, among the bacteria recently isolated according to the results of race differentiation of bacterial blight bacteria, X. citri As a result of research efforts to determine whether the pv. glyciens 8ra strain possesses novel bacteriocin activity, that is, antimicrobial activity, X. citri The pv. glyciens K2 strain possesses novel bacteriocins as well as the existing glycinesen A and glycinesen P, and X. citri 106 newly isolated strains of pv. glyciens, X. citri p v. glyciens 8ra strain, homologous Xanthomonas The present invention was completed by confirming that it has antibacterial activity against Gram-positive Staphylococcus species and Gram-positive plant pathogenic bacteria. The problem to be solved

[0009] One object of the present invention is Xanthomonas citri pv. glycinese K2 of accession number KACC92375P ( Xanthomonas citri It is to provide the pv. glycines K2) strain.

[0010] The present invention provides a novel bacteriocin produced by the microorganism.

[0011] Another objective of the present invention is to provide an antimicrobial composition comprising the Xanthomonas citri pv. glycine K2 strain, spores of the strain, culture of the strain, concentrate of the culture, extract and dried culture, or a novel bacteriocin.

[0012] Another objective of the present invention is to provide a food composition comprising a Xanthomonas citri pv. glycine K2 strain, spores of the strain, a culture of the strain, a concentrate of the culture, an extract and a dried product of the culture, or a novel bacteriocin.

[0013] Another objective of the present invention is to provide a feed composition comprising a Xanthomonas citri pv. glycinese K2 strain, spores of the strain, a culture of the strain, a concentrate of the culture, an extract and a dried product of the culture, or a novel bacteriocin. means of solving the problem

[0015] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0017] One aspect of the present invention for achieving the above objective is Xanthomonas citri pv. glycinese K2 of accession number KACC92375P ( Xanthomonas citri Provides a pv. glycines K2) strain.

[0018] In the present invention, the above X. citri Among the 107 strains recently isolated following the race differentiation results of the bacterial blight pathogen, the pv. glycines K2 (Xcg K2) strain is X. citri We selected a bacterium possessing novel bacteriocin activity, i.e., antimicrobial activity, against pv. glycines 8ra, and this X. citri It is designated as pv. glycines K2 and was assigned accession number KACC92357P by the Seed Bank (National Institute of Agricultural Sciences Microbial Bank, KACC) on August 3, 2021.

[0019] In the present invention, the terms "bacteriocin activity," "antimicrobial activity," and "antagonistic power" are used interchangeably with the same meaning and may mean inhibiting, blocking, or controlling the survival, growth, or reproduction of target bacteria.

[0020] According to the present invention X. citri pv. glycines K2 is X. citriIt has strong antibacterial activity against all 111 of the above isolates except pv. glycines K2.

[0021] In addition, according to the present invention X. citri p v. glycines K2 possesses strong antimicrobial activity against Xanthomonas species, Gram-positive Staphylococcus aureus, and plant pathogens. Specifically, regarding the Xanthomonas species, Xanthomonas axonopodis (Xanthomonas axonpodis ), Xanthomonas campestris( Xanthomonas campestris ), and Xanthomonas juvesicatoria ( Xanthomonas euvesicatoria It may include, but is not limited to. In addition, the above-mentioned Gram-positive Staphylococcus and plant pathogens specifically include Staphylococcus caprae ( Staphylococus caprae ), Staphylococcus lentus ( Staphylococus lentus ), Staphilococcus auricularis( Staphylococus auriculalis ), Staphilococcus Areus( Staphylococus aureus ), Staphylococcus hominis( Staphylococus homonis ), Clavibacter michiganensis ( Clavibacter michiganensis ), Pantoea Dispersa( Pantoea dispersa ), Clostridium acetobutylicum ( Clostridium acetobutylicum It may include, but is not limited to.

[0022] The aforementioned Staphylococcus species are known as food poisoning-causing bacteria, and Clavibacter is known as a representative plant pathogenic bacterium; therefore, possessing excellent antimicrobial activity against them X. citri pv. glycines K2 can be used for various purposes in addition to its use against bean wilt.

[0024] One aspect of the present invention for achieving the above objective is, the above X. citri Provides a novel bacteriocin produced by pv. glycines K2.

[0025] In the present invention, the novel bacteriocin is characterized by comprising an amino acid sequence represented by SEQ ID NO. 1 located at a position independent of the existing bacteriocins glycinecine A and glycinecine R. In addition, the present invention is characterized by being encoded by a nucleic acid sequence represented by SEQ ID NO. 2 having bacteriocin activity.

[0026] The term "bacteriocin" in this invention maintains activity at high temperatures, is stable over a wide pH range, and is non-toxic, colorless, and odorless. The difference between bacteriocins and antibiotics is that while antibiotics are secondary metabolites, bacteriocins are biosynthesized from one's own genes; therefore, it is not only easier to maximize production at the molecular level through genetic analysis and manipulation, but also possible to synthesize superior bacteriocins through molecular mutation. Furthermore, although antibiotics have the disadvantage of causing side effects when administered to humans, bacteriocins are degraded by proteolytic enzymes upon ingestion, making them non-toxic and non-residual to the human body. Consequently, their use as biopreservatives for foods and the like, as well as bioregulators for fermented foods, is increasing. For the purposes of this invention, the bacteriocin may refer to a substance that possesses antagonistic activity against the bacterium of the bean blight disease.

[0027] In addition, the present invention X. citri The antibacterial activity shown by pv. glycines K2 is not due to previously known bacteriocins, and specifically, it may mean that it has novel bacteriocins different from glycinesin A, glycinesin R, or glycinesin P.

[0028] In one embodiment of the present invention, through NCBI blast X. citriAs a result of whole-genome analysis to confirm that the novel bacteriocin gene of pv. glycines K2 is located independently of the previously reported glycinesin A and glycinesin R, it was confirmed that it is located at positions 3,134,599–3,136,140 (1542 bp), which are different from the aforementioned existing bacteriocins (Fig. 5).

[0030] One aspect of the present invention for achieving the above objective provides an antimicrobial composition comprising the Xanthomonas citri pv. glycinese K2 strain, spores of the strain, culture of the strain, concentrate of the culture, extract and dried culture, or a novel bacteriocin.

[0031] At this time, the state of the strain may be in a liquid or dry state, but is not limited thereto.

[0032] The above antimicrobial composition may have antimicrobial activity against the bacterium *Symptomia ulmoides*, and specifically, may mean having specific antimicrobial activity against Xanthomonas species. Additionally, it may mean having broad and strong antimicrobial activity against Gram-positive Staphylococcus species or Gram-positive plant pathogenic bacteria, but is not limited thereto.

[0033] The term "spore" in the present invention refers to a reproductive cell such as a bacterium, and for the purposes of this application, the spore may refer to a reproductive cell of Xanthomonas citri pv. glycinese K2.

[0034] The term "culture" in the present invention refers to a product obtained after culturing the strain, and may be a culture stock solution containing cells, or may be a culture strain, or cells obtained by removing or concentrating the culture supernatant. The composition of the culture may additionally include components that act synergistically on the growth of Xanthomonas citri pv. glycinness, in addition to components required for the cultivation of Xanthomonas citri pv. glycinness, and the composition accordingly can be easily selected by a person skilled in the art.

[0035] The term "concentrate" in the present invention means a concentrated form of the culture.

[0036] The term "extract" in the present invention refers to a product extracted from the culture or a concentrate thereof, and is not limited thereto, as long as it is an extract capable of exhibiting a specific antibacterial effect against the bean blight fungus of the present invention, it may include an extract solution, a diluted or concentrated extract solution, a dried product obtained by drying the extract solution, or a modified or purified product thereof, or a fraction obtained by fractionating the same.

[0037] The term "dried material" in the present invention may refer to the culture, its concentrate, its extract, and its fraction that have been dried. Drying methods may include air drying, natural drying, spray drying, and freeze-drying, but are not limited thereto.

[0038] The novelly isolated Xanthomonas citri pv. glycinese K2 of the present invention can be cultured using a conventional culture method for Xanthomonas citri pv. glycinese strains, but is not limited thereto. Natural or synthetic media may be used as the culture medium. Examples of carbon sources for the medium may include glucose, sucrose, dextrin, glycerol, starch, etc., and nitrogen sources may include peptone, meat extract, yeast extract, dried yeast, soybeans, ammonium salts, nitrates, and other organic or inorganic nitrogen-containing compounds, but are not limited to these components. Inorganic salts included in the medium may include magnesium, manganese, calcium, iron, phosphorus, etc., but are not limited thereto. In addition to the components of the carbon source, nitrogen source, and inorganic salts mentioned above, amino acids, vitamins, nucleic acids, and related compounds may be added to the medium.

[0039] The above antimicrobial composition may also include other by-products generated during the process of culturing Xanthomonas citri pv. glycinese K2.

[0040] In the present invention, the antimicrobial composition can be widely used to achieve purposes such as antimicrobial, sterilization, disinfection, and preservation in pesticides, medicines, household goods, food, etc. Specifically, in agriculture, it may be used for purposes such as antimicrobial, sterilization, and disinfection; in medicine, for purposes such as antibiotics or antifungal agents; in food, for preservation or antimicrobial purposes; and in household goods, it may be used in products directly related to microorganisms or for preservation, antimicrobial, or sterilization purposes in cleaning agents, dishwashing agents, etc., but is not limited thereto. In addition, the antimicrobial composition of the present invention may be used as food, food additive, feed, feed additive, antibiotic substitute, or antimicrobial packaging material.

[0041] In one embodiment of the present invention, it was confirmed that the newly isolated Xanthomonas citri pv. glycine K2 and the novel bacteriocin produced therefrom have strong antibacterial activity against Xanthomonas species, including Xanthomonas citri pv. glycine 8ra, which is a pathogen of bean blight, as well as against Staphylococcus species, which cause food poisoning, and Clavibacter, another plant pathogen (Tables 1 to 3).

[0043] Another aspect of the present invention for achieving the above objective provides a food composition comprising the Xanthomonas citri pv. glycinese K2 strain, spores of the strain, culture of the strain, concentrate of the culture, extract and dried product of the culture, or a novel bacteriocin.

[0044] The above spores, cultures, concentrates, extracts, dried products, and bacteriocins are as described above.

[0045] The types of the above food are not particularly limited and may include all foods in the ordinary sense. Non-limiting examples of foods to which the above substance may be added include fermented foods including soy sauce and salted seafood, sauces, meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. When the above composition is used as a food additive, the composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods.

[0046] Specifically, the food composition may include a food-grade acceptable carrier.

[0047] The type of the above carrier is not particularly limited, and any carrier commonly used in the relevant technical field may be used.

[0048] In addition, the above food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, and visual appearance. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

[0049] In addition, the above food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high-grade bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), coloring agents (tar dyes, etc.), colorants (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (sizing agents), coating agents, gum bases, antifoaming agents, solvents, improvers, dispersants, etc. The above additives can be selected according to the type of food and used in appropriate amounts.

[0051] Another aspect of the present invention for achieving the above objective provides a feed composition comprising the Xanthomonas citri pv. glycinese K2 strain, spores of the strain, culture of the strain, concentrate of the culture, extract and dried product of the culture, or a novel bacteriocin.

[0052] The above spores, cultures, concentrates, extracts, dried products, and bacteriocins are as described above.

[0053] The term "feed" in the present invention refers to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest.

[0054] The types of the above feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of the above feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, mineral substances, oils and fats, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0055] The microorganisms or novel bacteriocins of the present invention included in the feed composition of the present invention may vary depending on the purpose and conditions of use of the feed, and, for example, may be included in an amount of 0.01 to 100 weight%, more specifically 1 to 80 weight%, based on the total weight of the livestock feed composition. Effects of the invention

[0057] The novel isolated Xanthomonas citri pv. glycine K2 of the present invention and the novel bacteriocin produced therefrom are characterized by having strong antimicrobial activity against Xanthomonas species, including Xanthomonas citri pv. glycine 8ra, which is a pathogen of bean wilt, as well as against Staphylococcus species that cause food poisoning and Clavibacter, another plant pathogen. Therefore, Xanthomonas citri pv. glycine K2 and the novel bacteriocin produced therefrom can be utilized in various fields such as pesticides, food, medicine, or animal feed. Brief explanation of the drawing

[0059] FIG. 1 shows 107 of the present invention Xanthomonas citriThe existing bacteriocins, glycinesen A and R genes, were identified in the pv. glycines isolate through PCR. Figure 2 is Xanthomonas citri bacteriocins having antibacterial activity against pv. glycines Xanthomonas citri pv. glycines K2 was identified. Fig. 3 is Xanthomonas citri This is the result of a mutant assay conducted to select bacteriocin genes contained in pv. glycines K2. Figure 4 shows the results of the homology investigation of the nucleotide sequence determined through the mutant assay, Xanthomonas citri It was confirmed that pv. glycines K2 is a novel bacteriocin gene produced by pv. glycines K2. Fig. 5 is Xanthomonas citri Whole-genome analysis of pv. glycines K2 confirmed the location of a novel bacteriocin. Figure 6 shows the protein size confirmed after isolating a novel bacteriocin. Specific details for implementing the invention

[0060] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited by these examples.

[0062] Example 1: Xanthomonas citri Isolation of the pv. glycines strain

[0063] The bacteria isolated based on the results of race differentiation of the bacterial blight bacteria were collected.

[0064] Specifically, plants exhibiting bacterial blight symptoms were collected from soybean cultivation areas in Chungcheong-do, Jeolla-do, Gyeongsang-do, Gangwon-do, and Gyeonggi-do. The lesion areas were treated with 75% EtOH for 30 seconds to disinfect the leaf surfaces, after which bacteria were eluted. The eluted solution was then streaked onto TSA medium and cultured. After obtaining a single conlony, the bacteria were identified using the 16S ribosomal RNA (rRNA) gene, DNA gyrase subunit B gene (gyrB), RNA polymerase beta subunit gene (rpoB), and Biolog.

[0065] in result, Xanthomonas citri 107 strains of pv. glycines were obtained.

[0067] Example 2: Identification of glycinecine A and glycinecine R genes

[0068] In order to confirm the bacteriocin genes of strains isolated based on the recent race differentiation results of the bacterium blight, primers capable of diagnosing glyA and glyR were prepared, and the presence of bacteriocin genes in the isolated strains was investigated through PCR.

[0069] As a result, as shown in Fig. 1, of all 107 isolated strains Xanthomonas citri It was confirmed that pv. glycines 8ra possesses glycinecine A and glycinecine R, which are bacteriocins.

[0071] Example 3: Xanthomonas citri Search and Selection of Novel Bacteriocin Activities for pv. glycines

[0072] Representative X. citri pv. glyciens 8ra and isolated strains, which account for more than 80% of domestic strains of the bean blight pathogen, were used as indicator strains, and antagonistic bacteria capable of killing the indicator strains, that is, having bacteriocin activity, were searched for.

[0073] As a result, as shown in Fig. 2, it was confirmed that many bacteria form small clear zones and produce and secrete small amounts of toxic substances; in particular, by confirming that bacterium No. 2 forms a very large clear zone and produces strong toxic substances, Xanthomonas citri It was selected as an antagonist against pv. glycines 8ra and isolated strains. The selected strain is Xcg K2 ( Xanthomonas citri It was named pv. glycines K2) and deposited with the Seed Bank (National Institute of Agricultural Sciences Microbial Bank, KACC), an international microbial depository under the Treaty of Budapest, and was assigned deposit number KACC92357P.

[0075] Example 4: Confirmation of bacteriocin spectrum of Xcg K2 strain

[0076] 1) Same species ( Xanthomonas citri Confirmation of the bacteriocin spectrum of Xcg K2 against pv. glycines) strain

[0077] First, 107 isolates of the same species Xanthomonas citri The antimicrobial activity range of bacteriocins produced and secreted by Xcg K2 against pv. glycines strains was determined, and the results are shown in Table 1 below. The intensity of the antimicrobial activity of Xcg K2 was indicated as "+" when the clear zone range was 0-0.5 cm, "++" when it was 0.5-1 cm, and "+++" when it was 1-2 cm, and "-" when no antimicrobial activity was observed.

[0078]

[0080] As a result, as shown in Table 1 above, Xcg K2 is the same species Xanthomonas citri It was confirmed that pv. glycines exhibited strong antibacterial activity against 106 strains.

[0082] 2) Xanthomonas Confirmation of the bacteriocin spectrum of Xcg K2 against species

[0083] Next, XanthomonasThe antimicrobial activity range of bacteriocins produced and secreted by Xcg K2 against species was determined, and the results are shown in Table 2 below. The intensity of the antimicrobial activity of Xcg K2 was indicated as "++" when the clear zone range was 0.5-1 cm and as "+++" when it was 1-2 cm.

[0084]

[0086] As a result, as shown in Table 2 above, Xcg K2 is Xanthomonas It was confirmed that it exhibits strong antibacterial activity against species as well.

[0088] 3) Xanthomonas Confirmation of the bacteriocin spectrum of Xcg K2 for genera other than the species

[0089] In addition, the bacteriocins produced and secreted by the Xcg K2 strain Xanthomonas We investigated whether it affects other genera besides species.

[0090] Specifically, a total of 5 Staphylococcus species were used, and 2 species Clavibacter , of type 1 Pantoea Xcg K2 toxicity was confirmed against strains, and the results are shown in Table 3 below. The intensity of the antibacterial activity of Xcg K2 was indicated as "++" when the clear zone range was 0.5-1 cm and as "+++" when it was 1-2 cm.

[0091]

[0093] As a result, as shown in Table 3 above, Xcg K2 exhibited toxicity to many Gram-positive bacteria. In particular, Staphylococci Staphylococcus It showed very strong toxicity in 5 species, and Gram-positive bacteria, which are representative plant pathogenic bacteria Clavibacter It was confirmed that all species also exhibited strong toxicity.

[0095] Example 5: Analysis of genes related to bacteriocin activity of Xcg K2

[0096] 1) Construction of tn5 mutation library and mutation library screening

[0097] To select non-pathogenic mutants of the Xcg K2 strain, EZ-Tn5<R6Kγori / KAN-2> A random mutation method using the Tnp Transposome kit (Epicentre Biotechnologies, Madison, WI, USA) was used. This is an effective method for tracking specific unknown genes within a chromosome because the transposome is randomly inserted into the chromosome of the Xcg K2 strain and inhibits gene expression at the insertion site.

[0098] As a result, as shown in Figure 3, 4,740 mutants with randomly inserted Tn5 containing the kanamycin resistance gene were obtained, and bacteriocin activity was performed using the Xcg K2 strain as a control, and 9 out of 17 that lost activity were finally selected.

[0100] 2) Target gene screening and confirmation of bacteriocin activity

[0101] Gene cloning was performed to identify the target gene sequence into which tn5 was inserted, which caused the loss of bacteriocin activity of the Xcg K2 strain.

[0102] Specifically, the gene was obtained through gel extraction after PCR amplification, and transformed after in-fusion cloning. Subsequently, the sequence was confirmed through sequencing after plasmid extraction, and the bacteriocin activity of the sequence was confirmed.

[0103] As a result, a 2190 bp insert sequence containing the target gene was identified, and its bacteriocin activity was finally confirmed.

[0105] Example 6: Construction of target gene construct and verification of bacteriocin activity

[0106] Using the insert sequence confirmed in Example 5 above, a total of six gene constructs containing functional genes having novel bacteriocin activity were constructed, and their bacteriocin activity was confirmed and shown in Table 4 below.

[0107]

[0109] As shown above, bacteriocin activity was confirmed in No. 4, which includes nucleotide sequences from 351 to 1892, and No. 6, which includes nucleotide sequences from 132 to 1892, and No. 4 was selected as the final nucleotide sequence. The amino acid sequence having the above bacteriocin activity was indicated as SEQ ID NO. 1, and the nucleotide sequence encoding it was indicated as SEQ ID NO. 2.

[0111] Example 7: Homology Confirmation

[0112] As a gene with novel bacteriocin activity of the Xcg K2 strain, the final selected nucleotide sequence in Example 5 above was confirmed for homology using NCBL BLAST.

[0113] As a result, as shown in Figure 4, it was confirmed that it is a novel bacteriocin gene with no homology to previously reported bacteriocin genes. Additionally, homology with N-acetylmuramoyl-l-alanine amidase of the above sequence was confirmed, and it was confirmed that this is a gene associated with cell wall permeability and cell wall-targeted antibiotic sensitivity.

[0115] Example 8: Xanthomonas citri Identification of a Novel Bacteriocin via Genome Analysis of pv. glycines K2 Strain

[0116] Selected through the present invention Xanthomonas citri A novel bacteriocin gene possessed by pv. glycines K2, the existing Xanthomonas citriThe whole genome of Xcg K2 was analyzed to confirm that it is independent of glycinecin A and glycinecin R, which are bacteriocins reported in the pv. glycines 8ra strain.

[0117] As a result, as shown in Figure 5A, it was confirmed that the location of the novel bacteriocin gene is located at 3,134,599–3,136,140, ​​which is distinctly different from the locations of glycinecin A and glycinecin R.

[0118] In addition, as shown in FIG. 5B, other Xanthomonas Although four strains of the genus were identified, the novel bacteriocin gene was identified through the discovery of SNPs (Single-nucleotide polymorphisms). Xanthomonas citri It was confirmed that this is a characteristic possessed only by pv. glycines K2.

[0120] Example 9: Isolation of Novel Bacteriocin and Confirmation of Protein Size

[0121] Experiments were conducted to isolate a novel bacteriocin and identify its protein.

[0122] As a result, as shown in Figure 6, the protein size of a novel bacteriocin estimated to be 56 KD was confirmed by confirming the bacteriocin activity in the pellets after centrifugation using 3K and 10K size columns and the supernatant of a 50K size column, respectively.

[0124] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

[0126] Depositing Institution: National Institute of Agricultural Sciences Microbial Bank Accession Number: KACC92357P Date of Deposit: 2021-06-24

Claims

Claim 1 Xanthomonas citri pv. glycines K2 deposited under accession number KACC 92357P ( Xanthomonas citri pv. glycines An antimicrobial composition comprising a strain (K2) or a bacteriocin consisting of the amino acid sequence of SEQ ID NO. 1 produced by said strain, wherein the antimicrobial agent is Xanthomonas citri pv. glycine 8ra ( Xanthomonas citri pv. glycines 8ra), Xanthomonas axonopodis pv. Dieffenbakiae( X. axonpodis pv. diffenbachiae ), Xanthomonas Campestris pv. Dieffenbakiae( X. campestris pv. diffenbachiae ), Xanthomonas juvesicatoria( X. euvesicatoria ) and Xanthomonas Campestris pv. Campestris( X. campestris pv. campestris ), Staphilococcus Kafrae( Staphylococus caprae ), Staphylococcus lentus ( Staphylococus lentus ), Staphilococcus auricularis( Staphylococus auriculalis ), Staphilococcus Areus( Staphylococus aureus ), Staphylococcus hominis( Staphylococus homonis ), Clavibacter michiganensis ( Clavibacter michiganensis ), Pantoea Dispersa( Pantoea dispersa ) or Clostridium acetobutylicum ( Clostridium acetobutylicum Antimicrobial composition for antimicrobial purposes, which is antimicrobial against ). Claim 2 An antibacterial composition according to claim 1, wherein the composition exhibits antibacterial activity against Xanthomonas citri pv. glycine 8ra, a bacterium of bean blight. Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete

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