CN111763248B

CN111763248B - Precursor peptide of thuringiensis with bacteriostatic activity on several pathogenic bacteria

Info

Publication number: CN111763248B
Application number: CN202010369687.1A
Authority: CN
Inventors: 孙明; 信丙越; 郑金水; 彭东海; 阮丽芳
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-03-31
Filing date: 2018-03-31
Publication date: 2022-05-24
Anticipated expiration: 2038-03-31
Also published as: CN109112079B; CN111763248A; CN109112079A

Abstract

The invention belongs to the technical field of agricultural microorganism application, and particularly relates to a precursor peptide of thuringiensis with bacteriostatic activity on several pathogenic bacteria. The precursor peptide sequence is secreted by a separated and screened bacillus thuringiensis XY66, and the preservation number is CCTCC NO: m2018124. The Bacillus thuringiensis of the present invention produces a novel bacteriocin which the applicants have named Bacillus thuringiensis. The molecular weight of the thuringiensis is 4,347.93Da, and the amino acid sequence of the precursor peptide is shown as SEQ ID NO: 1, base positions 1-144. The separated thuringiensis has high-efficiency bacteriostatic activity on various pathogenic bacteria such as streptococcus pneumoniae, listeria monocytogenes and bacillus cereus.

Description

Precursor peptide of thuringiensis with bacteriostatic activity on several pathogenic bacteria

Technical Field

The invention relates to a divisional application with application number of 2018102793082, and the application date is 2018, 03 and 31.

The invention belongs to the field of agricultural microorganism application, and particularly relates to a precursor peptide of bacillus thuringiensis with bacteriostatic activity on several pathogenic bacteria, wherein the bacillus thuringiensis strain is XY66, and the bacillus thuringiensis secretes a bacteriostatic active substance, namely bacillus thuringiensis.

Background

The discovery and use of antibiotics has contributed greatly to the health of humans, while the problem of bacterial resistance caused by abuse of antibiotics has also grown in severity. The search for new, highly effective bacteriostatic substances to combat the problem of antibiotic resistance is imminent (czapewski et al, 2016). Bacteriocins are protein or polypeptide substances with antibacterial activity generated by bacteria through a ribosome synthesis mechanism, have high-efficiency antibacterial activity, are nontoxic to human bodies or animals, can be degraded by proteases in the digestive tracts of the animals, have the characteristics of good selective sterilization effect, easiness in biological modification, strong thermal stability, acid and alkali resistance and the like, and are good antibiotic substitutes (Cotter et al, 2013).

Bacillus thuringiensis (Bt for short) is a gram-positive bacterium which is widely present in soil and has high-efficiency and specific insecticidal activity for various agricultural pests. Bt has now been developed to become the most successful live bacterial biopesticide with the greatest yield and the most widespread use in the world (Bravo et al, 2011). With the progress of research, Bt is found to produce abundant and various secondary metabolites besides insecticidal crystal protein, and bacteriocin is one of the secondary metabolites. Some bacteriocins such as the bacteriocin Thiicin CD found in Bt DPC6431 have high-efficiency and specific bacteriostatic activity on clostridium difficile, do not damage the structure of intestinal flora under a human colon model, and can become new drugs for resisting clostridium difficile infection in human beings in the future (Rea et al, 2010). In addition, the patent applicant also identifies 1 novel two-component lantibiotic Thusin in Bt 4BT1, which has high antibacterial activity on various gram-positive pathogenic bacteria and application potential of becoming a novel antibacterial medicament (Xin et al, 2016).

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and particularly relates to a precursor peptide of thuringiensis with bacteriostatic activity on several pathogenic bacteria, wherein the bacteria secreting the peptide segment is bacillus thuringiensis strain XY66, and the bacteria secretes a novel bacteriocin which is a bacteriostatic active substance, namely the thuringiensis.

The technical scheme of the invention is as follows:

the applicant obtains a Bacillus thuringiensis (Bacillus thuringiensis) strain with bacteriostatic activity on several pathogenic bacteria by separation and screening, the screened strain is named as Bacillus thuringiensis XY66, and the Bacillus thuringiensis XY66 is delivered to China, Wuhan university China Center for Type Culture Collection (CCTCC) for preservation 3 and 13 days 2018, with the preservation number of CCTCC NO: m2018124.

The bacillus thuringiensis XY66 strain had the following properties:

(1) the strain of Bacillus thuringiensis XY66 produces substances with bacteriostatic activity against a variety of pathogenic bacteria, such as Streptococcus pneumoniae (representative of the strain ATCC49619), Listeria monocytogenes (representative of the strain LM201, CMCC54002) and Bacillus cereus (representative of the strain ATCC 14579).

(2) The bacteriostatic active substance produced by the strain of bacillus thuringiensis XY66 is a novel bacteriocin, which is named as thuringiensis by the applicant. The molecular weight of the thuringiensis was 4,347.93Da (determined in monoisotopic mode). The nucleotide sequence synthesized by the precursor peptide of the thuringiensis is shown in a sequence table SEQ ID NO. 1, wherein the sequence from 1 st to 144 th positions of the sequence shown in the SEQ ID NO. 1 is a coding region of the sequence, the corresponding amino acid of the sequence is shown in the base from 1 st to 144 th positions, 47 amino acid residues are coded, and the protein sequence of the precursor peptide of the thuringiensis is shown in the sequence table SEQ ID NO. 2. The nucleotide sequence of the synthetic gene cluster of the thuringiensis is shown as SEQ ID NO. 3.

(3) The thuringiensis has antibacterial activity against a variety of gram-positive pathogenic bacteria (such as Streptococcus pneumoniae ATCC49619, Listeria monocytogenes CMCC54002, Listeria monocytogenes LM201, and Bacillus cereus ATCC 14579).

The microbiological characteristics of the isolated Bacillus thuringiensis (Bacillus cereus) strain XY66 of the present invention are as follows:

the size of the thallus is 1-1.2 μm multiplied by 3-5 μm, the cell is rod-shaped, the spore grows, and gram staining shows positive reaction. On LB solid culture medium, yellowish colony with rough surface, soft texture and irregularity can be formed. The optimum growth temperature is 28 ℃, the pH value is 6-8, the growth is good, and the optimum growth pH value is 7.0-7.2.

The Bacillus thuringiensis strain XY66 can be preserved according to a conventional glycerol tube preservation method.

For a more detailed technical scheme, refer to the description of the detailed description.

Drawings

FIG. 1: colony morphology of bacillus thuringiensis XY66 strain.

FIG. 2: and (3) detecting the bacteriostatic activity of fermentation supernatant of the bacillus thuringiensis XY66 strain grown to the stationary phase on streptococcus pneumoniae, listeria monocytogenes and bacillus cereus. Description of reference numerals: panel A of FIG. 2 shows the results of the measurement of the bacteriostatic activity of Streptococcus pneumoniae ATCC 49619; FIG. 2B is the result of the detection of the bacteriostatic activity of Listeria monocytogenes CMCC 54002; the C picture in figure 2 is the result of the detection of the bacteriostatic activity of Listeria monocytogenes LM 201; FIG. 2D is a graph showing the results of the measurement of the bacteriostatic activity of Bacillus cereus ATCC 14579.

FIG. 3: HPLC analysis of the bacteriocin produced by Bacillus thuringiensis XY66 strain, i.e., thuringiensis.

FIG. 4 is a schematic view of: the mass spectrometry results of the bacteriocin produced by the strain Bacillus thuringiensis XY66, i.e., the thuringiensis.

FIG. 5 is a schematic view of: the N-terminal sequencing analysis result of the bacteriocin produced by the strain of Bacillus thuringiensis XY66, i.e., the thuringiensis.

FIG. 6: schematic representation of the precursor peptide sequence, mature peptide sequence and synthetic gene cluster of thuringiensis. Description of reference numerals: panel A of FIG. 6 is the sequence of the precursor peptide of thuringiensis; panel B of FIG. 6 is the mature peptide sequence of thuringiensis; panel C of FIG. 6 shows the synthetic gene cluster of thuringiensis.

FIG. 7: the detection result of the bacteriostatic activity of the thuringiensis on streptococcus pneumoniae, listeria monocytogenes and bacillus cereus. Description of reference numerals: FIG. 7A is a graph showing the results of the measurement of the bacteriostatic activity of Streptococcus pneumoniae ATCC49619 strain; FIG. 7B is the result of the detection of the bacteriostatic activity of Listeria monocytogenes CMCC 54002; FIG. 7C is the result of the detection of the bacteriostatic activity of Listeria monocytogenes LM201 strain; FIG. 7D is a graph showing the results of the measurement of the bacteriostatic activity of Bacillus cereus ATCC14579 strain.

Detailed Description

Description of sequence listing:

the sequence table SEQ ID NO 1 is the nucleotide sequence of the bacteriocin secreted by the screened Bacillus thuringiensis XY66 strain, namely the precursor peptide of the thuringiensis. The sequence length is 144 bp. Wherein the sequence at positions 1-144 of the sequence shown in SEQ ID NO. 1 is the coding region of the precursor peptide of thuringiensis (i.e., CDS, wherein the three bases TAA at positions 142 and 144 of the sequence do not translate amino acids), and encodes 47 amino acid residues in total.

The sequence table SEQ ID NO 2 is the protein sequence of the precursor peptide of the thuringiensis.

SEQ ID NO 3 is the nucleotide sequence of the synthetic gene cluster of thuringiensis. The sequence length is 6533 bp.

SEQ ID NO. 4 is the 16SrRNA sequence of Bacillus thuringiensis XY66 strain. The sequence length is 1493 bp.

Example 1

Separation and identification of bacillus thuringiensis XY66 strain and detection of bacteriostatic activity

1. Screening of Bacillus having bacteriostatic activity against Streptococcus pneumoniae, Listeria monocytogenes and Bacillus cereus in soil

(1) Separation and screening of bacillus in soil

Weighing 1g of soil of the agricultural university test farm in Huazhong, Wuhan, Hubei province, China, adding the soil into a 250mL triangular flask which is called with LB-NaAc culture medium (formula: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 34.02g of sodium acetate, and supplemented with distilled water to 1L, the pH value is 7.0, and the culture is carried out for 30min at 121 ℃) by shaking at 30 ℃ and 220rpm/min for 4 h. After culturing for 4h, the flask was placed in a water bath at 80 ℃ for 3 min.

After the triangular flask is cooled, 100 mul of culture solution is sucked by a pipette in a super clean bench and coated on Luria-Berta medium (LB) solid culture medium (formula: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 4.5-6g of agar powder, 1L of distilled water is supplemented, pH is 7.0, sterilization is carried out at 121 ℃ for 30min), and the coated plate is placed at 30 ℃ for culture for 12-16 h. Colonies of the colony growth morphology of the Bacillus-like bacteria in the plate were streaked on a T3 solid medium (formulation: peptone 5g, yeast powder 1.5g, manganese chloride 0.05g, 0.05M sodium phosphate buffer, agar powder 4.5-6g, distilled water 1L, pH7.0, sterilized at 121 ℃ for 30min) in a super clean bench and incubated at 30 ℃ for 48 h. Then selecting the single colony of the bacillus, using safranin staining solution (preparation method: 10mL of 2.5% safranin ethanol solution, adding distilled water to dilute to 100mL, filtering and using) to simply stain and then place under a common optical microscope for observation, and can preliminarily judge the bacillus-producing rod-shaped bacteria as the bacillus.

(2) Screening of Bacillus having bacteriostatic activity against Streptococcus pneumoniae, Listeria monocytogenes and Bacillus cereus

Single colonies of the different Bacillus species obtained above were picked and inoculated in 5ml LB liquid medium overnight at 30 ℃. 1mL of the culture was transferred to 1 00mL of LB liquid medium (formulation: peptone 10g, yeast powder 5g, sodium chloride 10g, supplemented with distilled water to 1L, pH7.0, sterilized at 121 ℃ for 30min) was cultured at 30 ℃ for 30h at 220 rpm/min. 2mL of the culture solution was taken out from the super clean bench at 3h intervals from the inoculation, and 10 times of continuous sampling was carried out. Wherein 1mL of the culture was used to determine the OD of the broth at that time₆₀₀The value is obtained. And centrifuging another 1mL of culture solution at 12000rpm/min for 5min, transferring the supernatant into a new centrifuge tube, and measuring the bacteriostatic activity of the fermentation supernatant on the indicator bacteria. The bacteriostatic activity of the fermentation supernatants was tested by conventional agar diffusion (Cintas et al, 1995). An appropriate amount of indicator (after the indicator is derived, it is activated overnight at 37 ℃) was added to the unset agar medium, mixed well, and poured into a plate. Streptococcus pneumoniae ATCC49619 (the strain is from the American type culture Collection), Listeria monocytogenes CMCC54002 (the strain is from the China medical bacterial culture Collection management center), Listeria monocytogenes LM201 (the strain is from the microbiological and immunological laboratory of the animal medical college of agriculture university in China, which is a publicly-exchanged strain) and Bacillus cereus ATCC14579 (the strain is from the American type culture Collection) are selected as indicator bacteria. And (3) punching by using a puncher with the hole diameter of 6mm after the agar culture medium is solidified. Adding about 50 μ L of sample (after mixing) into each well, placing the plate at 4 deg.C for about 2 hr to allow sample to be detected to diffuse sufficiently, culturing at 28 deg.C for 12 hr, and observing antibacterial effect (i.e. observing whether transparent antibacterial zone appears).

3. Identification of Bacillus thuringiensis XY66 Strain

Through the detection of the bacteriostatic activity of fermentation supernatants of multiple strains of bacillus in different growth periods, the applicant finds that one of the bacillus strains can generate substances with bacteriostatic activity on streptococcus pneumoniae ATCC49619, listeria monocytogenes CMCC54002, listeria monocytogenes LM201 and bacillus cereus ATCC14579 in a growth-to-stationary period (the result is shown in figure 2), and the applicant initially names the strain as bacillus XY 66. The applicant subsequently carried out a species identification of the strain Bacillus XY 66. The identification steps are as follows:

(1) extraction of total DNA and whole genome sequencing

A single colony of the Bacillus XY66 strain was picked and inoculated into 5mL of LB liquid medium for overnight activation at 30 ℃ and 0.5mL of the culture was transferred to 50mL of LB liquid medium, cultured under the same conditions for 3 to 4 hours, then treated at 12000rpm for 5min, centrifuged to collect the cells, and the resultant mixture was treated with 5mL of LSTE [ formulation: 0.1mol/L NaCL, 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA (pH 8.0) was washed once, and 3mL solution I [ formulation: 1mol/L Tris-HCl (pH 8.0), 0.5mol/L EDTA (pH 8), 50m mol/L glucose ] and 50. mu.L lysozyme (concentration: 50mg/mL) at 37 ℃ for more than 30 min; 3mL of 10% Sodium Dodecyl Sulfate (SDS) was added in a water bath at 55 ℃ for 30 min: then adding 3.6mL of 5M NaCl, mixing, standing on ice for 10min, processing at 12000rpm/min for 15min, and taking the supernatant and placing in another centrifuge tube; adding equal volume of phenol/chloroform/isoamyl alcohol (at volume ratio of 25: 24: 1), mixing by inversion for 200 times, centrifuging at 12000rpm for 5min, sucking supernatant, and extracting repeatedly for 1-2 times; transferring the upper layer DNA solution to a new centrifuge tube, adding equal volume of 95% ethanol, standing at room temperature for 30min, centrifuging at 12000rpm for 5min, washing the precipitate with 1mL of 70% ethanol once, freezing, draining, and dissolving in 100 mu LTE solution.

The total DNA samples prepared above were subjected to whole genome sequencing (done by Illuminate Hiseq2500 sequencer). The sequencing uses double-end sequencing, the sequencing reading length is 125bp, and the sequencing quantity is 1G. And finally, finishing genome splicing work of XY66 by genome splicing software abys through sequencing data.

(2) PCR amplification of the 16S rDNA sequence of Bacillus XY66

The universal primers 27F and 1492R are designed according to the conserved region of the eubacterium 16S rDNA for PCR amplification.

The DNA sequence of the universal primers is as follows:

27F：5'-AGAGTTTGATCCTGGCTCAG-3',

1492R：5'-GGTTACCTTGTTACGACTT-3'；

PCR amplification reaction procedure: step 1, pre-denaturation at 94 ℃ for 5 min; 2, denaturation at 94 ℃ for 1 min; 3, renaturation at 56 ℃ for 1 min; step 4, extension at 72 ℃ for 1.5 min; step 5 transfers to step 2 to continue running 28 times; step 6, the temperature is extended to 5min at 72 ℃.

(3) Sequencing and analysis of PCR products

After agarose gel electrophoresis of the PCR amplification product, agarose blocks containing the DNA fragments were excised, and the DNA fragments were recovered using a DNA fragment recovery kit manufactured by OMEGA corporation (according to the kit instructions). The obtained DNA fragment was sequenced by Nanjing Kingsler Biotech Co. Through two sequencing reactions, the total length of the PCR amplification product is 1493bp, the sequencing result is input into an NCBI database, and is delivered to a GenBank database by a Blastn program for comparative analysis, so that the sequence is 99.9 percent identical to the sequence of 16S rDNA of a plurality of Bacillus cereus group strains such as Bacillus anthracis FDAARGOS _341, Bacillus cereus JEM-2, Bacillus thuringiensis MPSGA01 strains and the like in the GenBank database. After the genome of the XY66 strain is subjected to predictive analysis of insecticidal crystal proteins by using BtToxin (insecticidal crystal protein prediction software), it is found that the XY66 strain contains 1 complete Cry8Ia1 type insecticidal crystal protein synthesis gene, so that the strain can be judged to belong to Bacillus (Bacillus thuringiensis) of Bacillus (Bacillus) of the family Bacillus, and the applicant names the Bacillus thuringiensis (Bacillus thuringiensis) XY66 strain.

Identification of bacteriocin (thuringiensis) secreted by Bacillus thuringiensis XY66

(1) Whole genome sequencing of bacillus thuringiensis XY66 strain and prediction and analysis of bacteriostatic active substances

The predictive analysis of the bacteriostatic active substance synthetic gene cluster of the strain of the bacillus thuringiensis XY66 is completed by online analysis software of antismat 3.0 and Bagel 3.0. As a result, as shown in panel C of FIG. 6, the genome of the strain Bacillus thuringiensis XY66 has an entire Sactipide-like bacteriocin synthesis genome. The sequence of the bacteriocin precursor peptide in the gene cluster is MEPIQRDDYWGCALKCAGPCLGVCAIDTASPVMDAVGTASGYAGGHG (the sequence is the same as the amino acid sequence shown by 1-144 bases in SEQ ID NO:1, and the visual chart is shown in A chart in figure 6). The sequence of the precursor peptide is analyzed by BlastP, and has no homology with the amino acid sequence of the reported bacteriocin, which indicates that the precursor peptide is a novel Sactipide bacteriocin. Applicants named the secreted product of this synthetic gene cluster as thuringiensis.

(2) Preparation of bacteriocin secreted by Bacillus thuringiensis XY66 strain, i.e. crude product of thuringiensis

A single colony of Bacillus thuringiensis XY66 strain was picked and activated overnight in 5mL LB liquid medium. Transferring the strain to 10 bottles of 200mL LB culture solution according to the inoculation amount of 1% (V/V), culturing the strain at 28 ℃ and 220r/min to obtain OD 600-3.0; centrifuging the fermentation broth (12,000r/min,10min), and adsorbing the obtained 2L supernatant with 200g macroporous adsorbent resin Anborite (Amberlite) XAD-7HP column; the column material after adsorption is firstly treated with 1L ddH ₂Washing with 0.5L 20% ethanol solution; finally, the active substance was eluted with 0.5L of 80% ethanol (pH 2.0); concentrating the eluate at 40 deg.C by rotary evaporator (adjusting pH of the rotary solution during concentration, and keeping pH at about 6.0); spin-drying to about 5mL, collecting and freeze-drying; dissolving the obtained dry powder in 50% acetonitrile (pH 5.0), centrifuging (12,000r/min, 10min), and collecting supernatant as antibacterial substance crude extract.

(3) Preparation of pure thuringiensis rhzomorph

The obtained crude body fluid of the bacteriostatic substance was analyzed by a high pressure liquid chromatograph (Water 1525) system. The main technical parameters are as follows: a chromatographic column: agilent C18 reverse phase column (250 mm. times.4.6 mm, 5. mu.L); mobile phase: ddH₂O (0.1% TFA) and acetonitrile; mobile phase conditions: gradient eluting with 20-80% acetonitrile for 0-15 min; detection wavelength: 220 nm; flow rate: 1 mL/min; collecting the eluate eluted within 15min by stages (once in 1min, 15 times in total), detecting the antibacterial activity of the collected solution by conventional agar diffusion method, and determining the retention time of antibacterial substance. After the retention time of the bacteriostatic substance was determined, it was collected repeatedly, spin-dried, and freeze-dried, and the obtained powder was weighed, dissolved in 20% acetonitrile (pH 5.0), and its purity was checked under the same mobile phase conditions.

(4) First-order mass spectrometric analysis of thuringiensins

The molecular weight of the purified bacteriostatic substance prepared above was determined by using Agilent Technologies 6540UDH Accurate-Mass Q-TOF LC/MS. The primary mass spectrometry conditions were as follows: capillary voltage: 3,500V; spraying pressure: 35lb/in2 gauge; flow rate of drying gas: 9 lites/min; temperature: 350 ℃; Q-TOF scan range: 100-3,000 m/z; data acquisition rate: 1 spectrum/s. The results are shown in FIG. 4, where the active substance secreted by the strain Bacillus thuringiensis XY66 has a molecular weight of 4347.93Da (measured in monoisotopic mode). Whereas when predicted the amino acid sequence of the thuringiensis precursor peptide MEPIQRDDYWGCALKCAGPCLGVCAIDTASPVMDAVGTASGYAGGHG remains after cleavage of the leader MEP: IQRDDYWGCALKCAGPCLGVCAIDTASPVMDAVGTASGYAGGHG (see B in FIG. 6), after cross-linking via-SH of the 4 cysteine side chains to the alpha carbon atoms of the other four amino acids, which occurs during the synthesis of the Sactipide bacteriocin, the mature peptide is formed with a molecular weight of 4347.95Da (measured in monoisotopic peak mode). The molecular weight is almost consistent with the data of 4347.93Da of the active substance secreted by the strain XY66 of the Bacillus thuringiensis, which proves that the active substance secreted by the strain XY66 of the Bacillus thuringiensis is the thuringiensis.

(5) N-terminal sequencing of thuringiensis

And (3) sending the prepared pure active substance to a Beijing agricultural detection center for N-terminal sequencing analysis. Finally, the first five amino acids of the N-terminal of the active substance are respectively detected to be Ile, Glu, Pro, Asp and Asp (see figure 5), and the active substance is matched with the first five amino acids of the thuringiensis, so that the active substance secreted by the strain of the Bacillus thuringiensis XY66 is the thuringiensis.

Thirdly, measuring the bacteriostatic activity and stability of the ceratin

The purified product of the thuringiensis obtained above was prepared into a test sample with a final concentration of 20. mu.g/mL. The agar diffusion method introduced above is used to detect the antibacterial activity of the thuringiensis at the concentration on streptococcus pneumoniae ATCC49619, listeria monocytogenes CMCC54002, listeria monocytogenes LM201 and bacillus cereus ATCC 14579. The results are shown in FIG. 7. The thuringiensis identified by the invention has high-efficiency bacteriostatic activity on several pathogenic bacteria under the concentration of 20 mu g/mL.

Reference to the literature

1.Bravo A,Likitvivatanavong S,Gill SS,Soberon M.Bacillus thuringiensis:a story of a successful bioinsecticide.Insect biochemistry and molecular biology,2011,41:423-31.

2.Cotter PD,Ross RP,Hill C.Bacteriocins-a viable alternative to antibiotics Nature Review of Microbiology,2013,11:95-105.

3.Czaplewski L,Bax R,Clokie M,Dawson M,Fairhead H,Fischetti VA,Foster S,Gilmore BF,Hancock RE,Harper D,Henderson IR,Hilpert K,Jones BV,Kadioglu A,Knowles D,Olafsdottir S,Payne D,Projan S,Shaunak S,Silverman J,et al.Alternatives to antibiotics-a pipeline portfolio review.Lancet Infectious Diseases,2016,16:239-51.

4.Rea MC,Sit CS,Clayton E,O'Connor PM,Whittal RM,Zheng J,Vederas JC,Ross RP,Hill C.Thuricin CD,a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile.Proceedings of the National Academy of Sciences of the United States of America,2010,107:9352-7.

5.Xin B,Zheng J,Liu H,Li J,Ruan L,Peng D,Sajid M,Sun M.Thusin,a novel two-component lantibiotic with potent antimicrobial activity against several Gram-positive pathogens.Frontiers in microbiology,2016,7:1115。

Sequence listing

<110> university of agriculture in Huazhong

<120> thuringiensis precursor peptides with bacteriostatic activity against several pathogenic bacteria

<141> 2020-05-05

<160> 4

<170> SIPOSequenceListing 1.0

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<211> 144

<212> DNA

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<220>

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<222> (1)..(144)

<220>

<221> CDS

<222> (1)..(144)

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atg gaa cca att caa cgt gat gac tat tgg ggt tgt gct tta aaa tgt 48

Met Glu Pro Ile Gln Arg Asp Asp Tyr Trp Gly Cys Ala Leu Lys Cys

1 5 10 15

gcg ggc ccg tgt ctc gga gtt tgt gct att gat aca gca agt cca gta 96

Ala Gly Pro Cys Leu Gly Val Cys Ala Ile Asp Thr Ala Ser Pro Val

20 25 30

atg gat gca gtt gga aca gca tca gga tat gct gga gga cat ggt taa 144

Met Asp Ala Val Gly Thr Ala Ser Gly Tyr Ala Gly Gly His Gly

35 40 45

<210> 2

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<212> PRT

<213> Bacillus thuringiensis (Bacillus thuringiensis)

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Met Glu Pro Ile Gln Arg Asp Asp Tyr Trp Gly Cys Ala Leu Lys Cys

1 5 10 15

Ala Gly Pro Cys Leu Gly Val Cys Ala Ile Asp Thr Ala Ser Pro Val

20 25 30

Met Asp Ala Val Gly Thr Ala Ser Gly Tyr Ala Gly Gly His Gly

35 40 45

<210> 3

<211> 6533

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<213> Bacillus thuringiensis (Bacillus thuringiensis)

<220>

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<222> (1)..(6533)

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atggaaccaa ttcaacgtga tgactattgg ggttgtgctt taaaatgtgc gggcccatgt 60

ctcggagttt gtgctattga tacagcaagt ccagtaatgg atgcagttgg aacagcatca 120

gggtatgctg gaggacatgg ttaagccgat ttataaaata atattgttta accctaacta 180

aaacaaagat ttagccaaaa ctaaattgaa ggagtggttt atatggaacc aattcaacgt 240

gatgactatt ggggttgtgc tttaaaatgt gcgggcccgt gtctcggagt ttgtgctatt 300

gatacagcaa gtccagtaat ggatgcagtt ggaacagcat caggatatgc tggaggacat 360

ggttaaaccg atttactaat tatattcact aaaattgtta aataattact aaaacaaaga 420

tttagctaaa accaaattga gggagtggtt ttttatggaa ccaattcaac gtgatgacta 480

ttggggttgt gctttaaaat gtgcgggccc atgtctcgga gtttgtgcta ttgatacagc 540

aagtccagta atggatgcag ttggaacagc atcaggatat gctggaggac atggttaaac 600

cgatttatca attttgtttc tttataaaaa gccatccatt atttagtagg atggctttta 660

ttgaaatcaa taaaataagg aagtgtctaa aatggaggaa agaggatttt attttaaaac 720

ttctaaaaac acatatttct ataatgatgt ttcgggcaac gtaggattag cagatagttc 780

taaatcagaa aaattaattt ttggtaaaga aagagtaact cccaaaacaa ttgacaaaaa 840

atctttgagt gaatttattg ataaaaatgg ttttagtcag cttatcttga tagttaccga 900

aagttgtaac ttaagatgta aatactgtct ttactcaggg gaatataata ataatcgaac 960

acatgattat tttaagatgc aaactggtac tgctaaagaa gctgtgttta agtacttggc 1020

aagtgtgaca aaaattaaac aagacaaacc ttttgttatt ccaatgattg gattctatgg 1080

gggagagcca ttattaaact atcaacttat taaagaaatt gttgattatg caaaagaagt 1140

atacgaaggt aaaatctatt ttaatatcac aacaaatgca actcttttaa ctaaagaaaa 1200

gatacgcttt tttatagaaa acaacttctt tctatctata agtttaaatg ggtacaaaga 1260

agaaaatgac cgaatgagag tatttgcaaa taataaaggg acatttgaag ttataatgag 1320

taaattacgc tttattaaag agaattatcc ggattatttt agaacaaaac tacaaataat 1380

cgatgtattt gatattggta cggacttata taaattaaaa gatttttatc aatcaaatga 1440

attagtaaaa aacaaaatat caattttgtt acaagtttca gatgtaggaa ctgattggta 1500

tgatcagtat acggaaaaag ataaagaaag gtttaataat cagatacata ctctaagaga 1560

agaatttaaa gaaaaagtaa ttgctggtga gaaattagat ccactttcta gattgttgtt 1620

tgctttacca ctatatgaaa ttattaatag accagttaat gcacctttaa aagaattaaa 1680

gccagaattt ttacctttta ccggcacttg tgtaccagga actaaagttg ctgttgatac 1740

taaaggggta ttacatagtt gcgaaaaagt gaatgataaa atgcctatcg gtacagttag 1800

aggatggatt gatattgata aaatagcaga aagcttagaa gagtataatc aatatatggg 1860

tactcaatgt gttaattgtc ctattcaacg tctatgtcca acatgtttta gaaatttcat 1920

tgataatgag ggtggttttg atagaaaatt aagcaaacct tgtaaagaat ttattaatga 1980

gaaaaagaat gcatttaaaa ccacctatac tctgttagaa caaggcataa aactagatga 2040

tatattaaat atgtaattta taaaaagggg cagatgttca tgaataaata ctttaattta 2100

tactctcatt gcgttcctgt taatggacaa tttcgtagca tattgtatga ctttaataaa 2160

gaaacgttat ttcacctcaa agaaaaagaa agtattatta taaatctaat tactaatggt 2220

aaagaaatta aagagattaa gaagaatatt gatgggaacg ttgtagatga gtttataaat 2280

ttacttatag ataaaaatat gggggaatat tcgaattact ttcttcctag agaaacttat 2340

cgaaaaggag caataaaaaa tttattaaaa gatgaaaata ttcccatgca aaatttcttt 2400

atagaactac catgtgaatg caataaaaat tgtatacatt gcgataatac taaaataaac 2460

ggttgctata gttgtagaaa agccatacaa actgatagta aagaattagg cttttactat 2520

aatttaatac gcgatattgt atccctgaag gttaagaacc tttattttca tggaggtgat 2580

ccattacttg aatgggagtt tacaaaagag atattacact ttacatcttt gttgactcca 2640

aatgaacaaa atatattttt acagacaaat ggcagcatgt tagataatga aaaggctcga 2700

tttatgctag aatataatat tacaccaatt attaacattg attttacggg gaataattta 2760

gaaaaagcta tgcaaataat acaaactatg aaaacaatag ttcaaaactg tgataaaaat 2820

gataagatta ttatagttaa tgtgttgttt agtacggaaa atttaaaaaa ttataaatta 2880

atatatgaga aattaaaaca gatcggtatt cagcagatta ttccttcagt tcttattaaa 2940

agtattgatg ataaagtagt ttaccatcaa aatttgcctg tttttacaaa agatattaat 3000

tctttttcgt atttaggaga atatcatcct tgtttggcag gtactttggc aattggtgca 3060

gataaaaaag tatacccttg tccaagaatg catgaagaac cgttaatgga tttaggacgt 3120

aaagaaagat ttttagattt atttgatgaa aaagaggata tgttaaaata ttggaaatta 3180

agtctggaaa aaatagaacc atgtagtaaa tgtgaattta gaaggatgtg tggggactgt 3240

agagcagcag aatgggaatt ttcaaacgat tttacaaaaa aatcattatg ctcttatgct 3300

taatataggg gaagcgagat attgagaaag gaggtggaat gtatggaata tatcaggtta 3360

gaaaatgtta ataaaggatt tgggaataat gaacttatat taaaaaatat caatttaaca 3420

tttacagctg gtgagagagt tggaatcgtt ggctcaatag gatcaggaaa aacaacatta 3480

ctaagagtca ttatgggaat ttatcggccg acatctggtg aagtagttca ttatataaat 3540

aaaaatcaaa taggatattt accagcatcc aaaggggtaa ctgatgaatt aacggtattg 3600

ggaaatttaa tgttttgggc taaggcttat aataaatcaa ttgaagctgt gaaagaagta 3660

gtatccaatt taaaaatgga atctatggtt gagaaaaagg tatctcaatt atcctccgga 3720

atgaaacaaa agctagcgtt tgcatgtgct atcattcata agcctaaact tttaatattg 3780

gatgaaccta cagttaacct cgatgtagag aatagaatac tacttaagaa tataataaaa 3840

aattatcttc ctgaaagctg tattattatt acttcacata acttcgatga tattgaaaat 3900

ctttgtgagc gaattttgtt aatttccgaa ggggaaataa aaattcaaaa gcaattagag 3960

actttaaaaa cagagtataa aagttcaaaa gtacatataa ggctattaga agatatgtca 4020

gaaaaacaaa gattaaaaat acaacaaaca tttgatcagt ctgtaataaa taagaatgag 4080

attgtcattg accgagatac ctataattta aatgcagtat tattaatatt aattcaatta 4140

cgaattaata ttagggatgt tgtggaaagt gatgtaatgt tggaagaaat atatttaaat 4200

atcaataaga aagagggata gaaaagggat gaaatcttta atttggaaag agttacgact 4260

tgtaagaagt cgttggcaaa aaacaatctt catacttttc atattagcag ctctatcttt 4320

atatgctaca tttagtttga aaactctctc tattccgatg aaatttaatt ttatacttgg 4380

agtaacatca attattttta tgtcagaaat aatatttgag tctattaaat cagataaaaa 4440

taataaaact ttagaaaaac tattaccatt atttagttta gggaaaataa taatggccaa 4500

gacaattttt ggcatggtga cagcaagtct tgtgagtatt atatactctt cagtgttctt 4560

tatttatttt gttttattaa gcagttatcc tcattttgaa atattaattt ctatttcttt 4620

attaccgatc atcaacctgt tatttggaaa tattttcact atattagtcc ttttgattga 4680

taacatgttt gtgaataaac tcttcactat gactggaaca atactatata tactattatt 4740

ctcaaatata aaagatttga aatttgtttt tatgttagtt ggtatactgc taataattgt 4800

atttttactg aatatcgcaa taaagaaacg atccgcagaa aatattttat agtgtatact 4860

acctatattg atagaaggtg agagtattga aagcttttat aatatatttt aaaaagagtt 4920

tgatgacaat tatattttta ggatttataa ctgttttaat tgctattatg acaaatgcta 4980

ttccaatttt gacgcaaaaa gtatttgatg agggaatatt aaagagagat attaatagta 5040

tagtaatatt tacagctgta cttattatta tatatttaag tcgaagtata ttaaactata 5100

tgagtgactt ccttttagca aaaacttcat ctaaagtaat tgctgatata aaaactgata 5160

tgatacataa aactatgaat atgccaatgt cattttttga tagcaaatct actgcttata 5220

tattatcaag aataaatgaa gccaactctt tgtcttcgat atttacacct acagtatttg 5280

tttttttttc atcctccata tcgatgatcg gggctttaat atatatattt agtaaaagta 5340

ttctaatatt tattatttgt atagtattta taccactagt ttatataatt tctaatggtt 5400

cactaagtgt aataaataaa tattctaaag aaatgttcga aacaaatgct agaacaaata 5460

ataaaattca ttcaactttt gaaggaatag taactttaaa acagctaaat caggaaaaaa 5520

atgttaatga aatcatttca aaagaagtat ttagtttagc tgaaaaaaca gtaaagcaaa 5580

gtaaaacaat aagtaaaagt tctcaattgc ttaatgtagt aatattaatc attcaatctt 5640

taattattgg tgtaattgcg tatttaatta caaaagaaag attaaaaata ggggattatg 5700

tggccttgac ccagtacgtt ggtatggtgt atgtcccaat tacaatgttt caaggtttta 5760

aaattactat acaacctgcg ttagccgcta tttctcgatt aaatagttta attgaaaaaa 5820

catttcttga aaacaaagga ataaaggtga atgaaatcaa aaccattgta cttaaaaatg 5880

tatcttttaa atatgaaact gcacaaaaag aaatattaaa aaatatcaat ttagagataa 5940

atactgggga aaaactagct cttattggta gcaatgggtc tggtaaaact actattgtaa 6000

aactgttatt aggattttat caaaattact taggaaatat atatataaat ggaatagaat 6060

tgaaaaaaat aaatataagg gaattaagag atagaattgg gattatacct caaaatatat 6120

atttatttga aaatacaatt gctgataata ttaaaatagg aaatactaag attacagaga 6180

aagaatttca acaaaaaatc aatttactta aaaaacaagg cattttagca gatctagatt 6240

taaacaaaaa aattattgaa aatggaaaga atttatctaa aggtcagatt caacaaattg 6300

cttttgcgag ggctttcatg aaaaattttg atgtactgat atttgatgaa gctacatcta 6360

atatggataa aaatgcgaga aaagccttta aagagatttt atctcaggaa ttttcaaata 6420

aaatatgtat tttcattagt catgataatg agttaagtaa ttttattgat aaaaaatttg 6480

tactactaga ggacttcaat gatgaaggcg agaaggaggg tggattaaga tga 6533

<210> 4

<211> 1493

<212> DNA

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<220>

<221> gene

<222> (1)..(1493)

<400> 4

ggttaccttt gttacgactt caccccaatc atctgtccca ccttaggcgg ctggctccaa 60

aaaggttacc ccaccgactt cgggtgttac aaactctcgt ggtgtgacgg gcggtgtgta 120

caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc gattccagct 180

tcatgtaggc gagttgcagc ctacaatccg aactgagaac ggttttatga gattagctcc 240

acctcgcggt cttgcagctc tttgtaccgt ccattgtagc acgtgtgtag cccaggtcat 300

aaggggcatg atgatttgac gtcatcccca ccttcctccg gtttgtcacc ggcagtcacc 360

ttagagtgcc caactaaatg atggcaacta agatcaaggg ttgcgctcgt tgcgggactt 420

aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtca ctctgctccc 480

gaaggagaag ccctatctct agggttgtca gaggatgtca agacctggta aggttcttcg 540

cgttgcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc aattcctttg 600

agtttcagcc ttgcggccgt actccccagg cggagtgctt aatgcgttaa cttcagcact 660

aaagggcgga aaccctctaa cacttagcac tcatcgttta cggcgtggac taccagggta 720

tctaatcctg tttgctcccc acgctttcgc gcctcagtgt cagttacaga ccagaaagtc 780

gccttcgcca ctggtgttcc tccatatctc tacgcatttc accgctacac atggaattcc 840

actttcctct tctgcactca agtctcccag tttccaatga ccctccacgg ttgagccgtg 900

ggctttcaca tcagacttaa gaaaccacct gcgcgcgctt tacgcccaat aattccggat 960

aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt ggctttctgg 1020

ttaggtaccg tcaaggtgcc agcttattca actagcactt gttcttccct aacaacagag 1080

ttttacgacc cgaaagcctt catcactcac gcggcgttgc tccgtcagac tttcgtccat 1140

tgcggaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg 1200

tggccgatca ccctctcagg tcggctacgc atcgttgcct tggtgagccg ttacctcacc 1260

aactagctaa tgcgacgcgg gtccatccat aagtgacagc cgaagccgcc tttcaatttc 1320

gaaccatgcg gttcaaaatg ttatccggta ttagccccgg tttcccggag ttatcccagt 1380

cttatgggca ggttacccac gtgttactca cccgtccgcc gctaacttca taagagcaag 1440

ctcttaatcc attcgctcga ctgcatgtat aggcaccccc gccactcccc ggt 1493

Claims

1. The application of the thuringiensis precursor peptide in preparing medicines, food additives or feed additives for resisting streptococcus pneumoniae, listeria monocytogenes and bacillus cereus is disclosed, wherein the amino acid sequence of the precursor peptide is shown as SEQ ID NO. 2.