Quorum sensing quenching strain Stone spring sea-source bacterium derived from hydrothermal solution and application of quorum sensing quenching strain Stone spring sea-source bacterium

Abstract

The invention provides a hydrothermal-source quorum sensing quenching strain Stone spring sea-source bacterium and application thereof, and the screened strain can inhibit pathogenic bacteria and is used for preparing antibacterial drugs; the preservation number of the screened stone spring sea source bacteria is CCTCC (China Center For Type Culture Collection) NO: M 20251224. The invention also provides an AHL degrading enzyme Aii937 protein separated from the YESM3 strain, and the AHL degrading enzyme Aii937 protein is characterized in that the amino acid sequence of the protein is SEQ ID NO: 2. According to the YESM3 strain disclosed by the invention, a QS channel of pathogenic bacteria can be blocked through AHL lactonase Aii937, and secretion of virulence factors of the pathogenic bacteria is reduced; the strain YESM3 has no pathogenicity. The AHL lactonase does not have broad spectrum, is high in substrate specificity, and only degrades C6-HSL and 3OC-C14-HSL.

Description

Technical Field

[0001] The present invention belongs to the technical field of beneficial bacteria screening and application, and particularly relates to a hydrothermal-derived quorum induction quenching strain Shiquan Haiyuan bacteria and an application thereof. Background Art

[0002] The rapid development of aquaculture has led to the continuous expansion of industrialized and intensive aquaculture models. Although this model can provide considerable economic benefits using limited space, it has also encountered considerable problems. The threat of various aquaculture animal diseases caused by viruses and pathogens is increasing. For example, Vibrio spp. ( Vibrios ) bacteria, which can infect turbot ( Scophthalmus maximus ) and other aquatic animals, causing hemorrhagic disease, fin rot, gastroenteritis and sepsis; Pseudomonas putida ( Pseudomonas putida ) can infect large yellow croaker ( Larimichthys crocea ) makes its movements slow and white nodes visible in its kidneys, all of which cause great harm to the aquaculture industry.

[0003] Traditionally, bacterial infections are treated with antibiotics. While the use of antibiotics to prevent and treat aquatic animal diseases has a positive short-term effect, in the long term, as bacterial resistance increases, the demand for antibiotics further increases, which can easily lead to the problem of antibiotic abuse. Antibiotic resistance in pathogens has become a major research topic in the 21st century. In recent years, with the increasing resistance of pathogens, the demand for traditional antibiotics has increased and their efficiency has decreased. Therefore, there is an urgent need for a new, highly effective antibacterial product that can metabolize antibiotics and be used to treat diseases caused by pathogens, providing strong support for the healthy development of my country's aquaculture industry.

[0004] Quorum sensing (QS) is a population density-based communication mechanism in microorganisms that regulates the expression of specific genes. Bacteria can use QS to control processes such as biofilm formation, virulence factor expression, or genetic material transfer (conjugation and transformation). N -Acylhomoserine lactones ( N -acyl-l-homoserine lactones (AHLs) are the most common bacterial QS signaling molecules. The chemical structure of AHLs consists of two parts, a homoserine lactone ring (HSL) and an acyl side chain. According to the length of the acyl side chain, the signaling molecules are divided into two types: short-chain AHLs (C4-C8) and long-chain AHLs (C 10 -C 18 Clinical drug-resistant pathogens such as Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Aeromonas ( Aeromonas) all use AHLs as signaling molecules. Quorum quenching (QQ) refers to a mechanism that can inhibit QS regulation. It inhibits the QS pathway by interfering with the production, release, accumulation, or response of signaling molecules. Microorganisms contain a variety of QQ substances, which can be simply divided into small-molecule QS inhibitors (QS inhibitors, QSIs) and large-molecule QQ enzymes (QQEs) based on their molecular weight. Small-molecule QS inhibitors mainly inhibit the synthesis or recognition process of QS signaling molecule synthases or receptor proteins, ultimately blocking signal transmission throughout the QS pathway and achieving the purpose of reverse regulation of target gene expression. Large-molecule QQ substances mainly achieve the purpose of blocking the QS pathway by degrading the QS signaling molecules themselves.

[0005] QQ enzyme is one of the main means of destroying microbial QS and hindering their collective behavior. Currently, the most studied QQ is the QQ enzyme that can change the structural characteristics of AHL-type signaling molecules, thereby making them lose their biological activity. Among them, the most common are AHL lactonase and AHL acyltransferase. Quenching enzymes targeting AHLs signaling molecules help to treat and prevent many bacterial infections. Compared with traditional treatments, the QQ process aims to inhibit the expression of virulence without killing bacteria. Therefore, QQ technology is considered to be an environmentally friendly disease prevention and control measure. This measure can reduce dependence on antibiotics, thereby delaying the evolution of drug resistance and reducing the generation of super bacteria. QQ enzymes are being increasingly explored as potential antimicrobial agents targeting pathogenic bacteria. To date, they have been used in bacteria including Bacillus ( Bacillus ), Chryseobacterium ( Chryseobacterium ), Enterobacter spp. ( Enterobacter ), Microbacterium ( Microbacteirum ), Agrobacterium spp. ( Agrobacterium ), Kurtia spp. ( Kurthia ), Brucella spp. ( Brucella ), Arthrobacter spp. ( Arthrobacter ), Pseudomonas spp. ( Pseudomonas ), soil Bacillus spp. ( Solibacillus ) and Rhodococcus ( Rhodococcus Over 50 enzymes that degrade AHLs have been identified from over 30 bacterial genera, including ( ). These discoveries provide a vast resource of enzymes for the future treatment of bacterial diseases, with broad market potential. Further development of novel QQ enzymes with excellent environmental stability, high enzymatic activity, and strong substrate specificity, as well as investigation of suitable methods to ensure stable expression of these enzymes, are crucial for promoting their practical application.

[0006] The ocean covers 71% of the Earth's surface and hosts a diverse array of complex habitats, including deep-sea trenches, hydrothermal vents, and ocean blue holes. Over the past few decades, marine bacteria have emerged as a rich reservoir of novel bioactive compounds and enzymes. Due to the extreme variations in marine environmental factors (such as temperature, salinity, and osmotic pressure), these bacteria have evolved unique metabolic pathways to produce a variety of bioactive compounds, enabling them to survive in specific niches and compete with other microorganisms, thereby maximizing their adaptability. Consequently, marine microorganisms produce compounds with remarkable structural and bioactive diversity, with some even exceeding the frequency of terrestrial microorganisms. For example, the proportion of marine bacteria with QQ activity has been reported to be much higher than that in agricultural soils or crop rhizospheres. Consequently, marine bacteria possess a richer reservoir of novel QQ enzymes. To date, numerous novel QQ enzymes with unique bioactivities and biochemical properties have been identified and characterized from various marine sources, including seawater, sediments, and marine organisms. However, few studies have examined microorganisms from deep-sea hydrothermal regions. Deep-sea hydrothermal areas, oases of life, harbor close connections between microorganisms in their high-temperature, high-pressure environments, potentially harboring novel QQ enzymes with unique physical and chemical properties. Therefore, these areas may harbor a rich reservoir of QQ enzymes awaiting discovery, offering promising development prospects as a novel, highly effective antibacterial drug for the treatment of bacterial diseases. Summary of the Invention

[0007] The purpose of the present invention is to provide a hydrothermal-derived quorum sensing quenching strain Shiquan Haiyuan bacteria and its application. The screened strain can inhibit pathogenic bacteria and be used for preparing antibacterial drugs.

[0008] The present invention first provides a Shiquan Haiyuan fungus ( Idiomarina fontislapidosi ) YESM3 strain was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan City, Hunan Province on May 29, 2025, with the deposit number CCTCC NO: M 20251224.

[0009] The culture temperature range of the Shiquanhaiyuan bacteria YESM3 screened by the present invention is 4-45° C., and the optimum culture temperature is 28-32° C.; the growth pH range is 5-10, and the optimum culture pH is 7-8.

[0010] The strain of the invention can be used to prepare a bacterial preparation for quenching the density sensing system of aquaculture pathogens and reducing the secretion of virulence factors thereof.

[0011] The bacterial preparation contains live bacteria of Shiquan Haiyuan bacteria YESM3.

[0012] The bacterial preparation is a feed additive or a breeding water additive.

[0013] The present invention also provides an AHL degrading enzyme Aii937 protein isolated from the YESM3 strain, characterized in that the protein amino acid sequence is SEQ ID NO: 2.

[0014] The present invention also provides a nucleotide, characterized in that the nucleotide encodes Aii937 protein; the nucleotide sequence is SEQ ID NO: 3.

[0015] The AHL degrading enzyme Aii937 protein is used in the preparation of aquatic feed additives or aquaculture water additives.

[0016] The YESM3 strain of the present invention can block the QS pathway of pathogenic bacteria through AHL lactonase Aii937, reducing the secretion of virulence factors of pathogenic bacteria; strain YESM3 is not pathogenic. AHL lactonase does not have a broad spectrum, but has strong substrate specificity, and only degrades C6-HSL and 3OC-C 14 -HSL. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Activity diagram of AHLs signal molecules degradation by strain YESM3, where 1: positive control (MomL protein), 2: negative control (2216E), 3: YESM3; Figure 2 :Activity diagram of AHLs signal molecules degradation by Aii937, including 1: C6-HSL, 2: C8-HSL, 3: 3OC-C8-HSL, 4: 3OH C8-HSL, 5: C 10 -HSL, 6:3OC-C 10 -HSL, 7:C 12 -HSL, 8:3OH-C 12 -HSL, 9:3OC-C 12 -HSL, 10:3OH-C 14 -HSL, 11:3OC-C 14 -HSL, 12:C 14 -HSL.

[0018] Figure 3 : QQ enzyme encoding gene used in the present invention Aii937 Heterologous expression agarose gel electrophoresis verification diagram, where A: Aii937 Gel electrophoresis of gene clones, B: Screening E. coil JM109 / pET-24a(+) / YESM3- Aii937 Gel electrophoresis diagram, C: screening E. coil BL21 / pET-24a(+) / YESM3- Aii937Gel electrophoresis diagram. In the figure, M represents marker. Figures A and B are 2000 bp markers. The sizes from top to bottom in Figure A are 2000, 1000, 750, 500, 250, 100. The sizes from top to bottom in Figure B are 2000, 1500, 1000, 750, 500, 250, 100. Figure C is a 5000 bp marker. The sizes from top to bottom are 5000, 3000, 2000, 1500, 1000, 500, 250, 100. Figure 4 : SDS-PAGE electrophoresis of crude enzyme solution of QQ enzyme Aii937, where M: protein marker, 1: E. coli BL21 / pET-24a(+) supernatant, 2: E. coli BL21 / pET-24a(+) / Aii937 supernatant, 3: QQ enzyme Aii937 after elution with imidazole. DETAILED DESCRIPTION

[0019] The strain of the present invention is described in detail below with reference to the examples and accompanying drawings.

[0020] Example 1: Isolation of target strains In June 2018, marine bacteria were isolated and cultured from sediment samples collected from the hydrothermal area of ​​the Okinawa Trough at station R11 (E122.34°, N25.03°) during the 2016 HOBAB4 cruise. First, the samples were diluted to 10% with 0.85% (w / v) saline. -1 , 10 -2 , 10 -3 Then, 100 μL of the dilutions of different concentrations were spread onto 2216E plates, the plates were incubated at 28°C, and the plate streaking method was used to perform 3-4 streaking operations on the 2216E plates to obtain purified colonies.

[0021] The target strain was obtained by screening the AHL degradation efficiency and named YESM3. The strain YESM3 of the present invention was confirmed to be Shiquan Haiyuan bacteria ( Idiomarina fontislapidosi ). It was deposited in the China Center for Type Culture Collection at 299 Bayi Road, Wuchang District, Wuhan City, Hunan Province on May 29, 2025, with the accession number CCTCC M 20251224.

[0022] The 16S rRNA sequence of this bacterium is as follows:

[0023] Example 2: AHL degradation activity of Shiquanhaiyuan bacteria YESM3 First, strain YESM3 was cultured overnight until the logarithmic growth phase. C6-HSL (final concentration 5 μM / L), PIPES (1 mM, pH = 6.73) buffer (to stabilize the pH and prevent acid or alkaline hydrolysis of the signal molecule) and the bacterial suspension were mixed and incubated at 28°C in the dark for 24 h. After incubation, the supernatant was obtained by centrifugation at 4°C and 6000 rpm for 10 min and filtered through a 0.22 μm filter. The supernatant was mixed with Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) A136 X-gal assay solution was mixed in a 96-well plate and incubated at 28°C for an additional 24 h. All experiments were repeated 3-4 times. The results were compared with the negative control and the t-test was used to determine whether the strain had QQ activity ( Figure 1 ).

[0024] Example 3: Amplification of the QQ enzyme gene Aii937 sequence Whole-genome sequencing of strain YESM3 and RAST annotation analysis of the assembled genome revealed a potential QQ enzyme-encoding gene, Aii937, which showed 31.73% similarity to known QQ enzymes. Primers were then designed to incorporate appropriate restriction sites upstream and downstream of the target gene, including EcoRI and HindIII. YESM3 DNA was then amplified using the strain's DNA as a template.

[0025] The primer sequences are as follows: Aii937F: 5′-CGGAATTCATGGCAGAGTCAGAATTAC-3′, Aii937R: 5′-CCCAAGCTTAAGACGACCTTGGCT-3′.

[0026] The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0027] Table 1: PCR reaction system Table 2: PCR reaction schedule

[0028] The PCR products were detected by agarose gel electrophoresis to verify whether the target band size was correct ( Figure 3 A). Use a gel excision and recovery kit to remove nonspecific amplified bands and purify the PCR product to obtain the purified target gene fragment. Sequencing shows that the nucleotide sequence of the encoding gene is as follows: Atggcagagtcagaattactgcattacgaaaccatgggtgacaagcagaaccccgccgttattatcattcacggtttgttcggagatggagataacctaaagagtcttgctcgcgatttaacgcctgactatttctgcgtactccccgatgcacgcaatcacggtgaatctcctcatcgtgaaagtatgacttacacggaaatggcagacgatatcgtggcccttgctgatgaattagctttagaacacttttcattagttggtcattccatgggcggcaaaatcgccatggaagtggctatgcgctatgaagatcgcgtacaagcagctgttttcgctgatattgcgcccgtcgcttatcctgcccatcacaatggcattctagatgccctagcaggtttggacctagaccaaataggcagccgcaccgaagccgataaacaactgagttcagctataaaagaaaaaggcgtacgccaatttttactgaagaacctccgcaaggacggtgaccatttcgcgtggcggctcaacttaactgcgattactgagcgataccaacagatcgctgacggtgtgagcgatggtcattattcaggcccctgtttgtttattaagggcggaaattcagactacctaaccgaacagcacagagcgcaagtgactcagcgttttagcaatacacaggtcaaagttgttgaaaacacaggccattggttacacgctgaaaaaccacgtatttttaaccgtttggtcaaggattttctagcgagccaaggtcgtctttaa (SEQ ID NO:3).

[0029] The amino acid sequence of the encoded protein is as follows: MAESELLHYETMGDKQNPAVIIIHGLFGDGDNLKSLARDLTPDYFCVLPDARNHGESPHRESMTYTEMADDIVALADELALEHFSLVGHSMGGKIAMEVAMRYEDRVQAAVFADIAPVAYPAHHNGILDALAG LDLDQIGSRTEADKQLSSAIKEKGVRQFLLKNLRKDGDHFAWRLNLTAITERYQQIADGVSDGHYSGPCLFIKGGNSDYLTEQHRAQVTQRFSNTQVKVVENTGHWLHAEKPRIFNRLVKDFLASQGRL (SEQ ID NO:2).

[0030] Analysis showed that Aii937 possesses a signal peptide sequence and belongs to the α / β hydrolase family. Among the identified AHL lactonases, Aii937 shares the highest amino acid sequence similarity with Aii810 (31.73% identity).

[0031] Example 4: Cloning and transformation of QQ enzyme gene Aii937 The target gene fragment and pET-24a(+) plasmid were double-digested (Table 3). After double-digestion at 37℃ metal bath for 2 hours, the target gene and vector were detected by agarose electrophoresis. The target gene and plasmid after double-digestion were ligated at 16℃ overnight (4) to construct the recombinant plasmid. E. coli For competent JM109, plate onto LB plates containing kanamycin (final concentration 50 μg / mL) and incubate overnight at 37°C. Pick a single colony and inoculate it into 5 mL of LB liquid medium containing kanamycin (final concentration 50 μg / mL) and incubate at 37°C, 170 rpm for 8-12 hours. Once the bacterial solution becomes turbid, verify positive recombination using PCR. PCR products were detected by 1.0% (w / v) agarose gel electrophoresis. The target band was approximately 800 bp, consistent with the target gene Aii937 ( Figure 3 B). The positive recombinant bacterial solution was sequenced and aligned by Qingdao BGI to ensure sequence accuracy, and the plasmid was extracted using the Omega kit.

[0032] Table 3: Enzyme digestion system The extracted recombinant plasmid was transferred into E. coliIn the BL21 (DE3) competent cell culture, 600 μL of LB liquid medium, which had been pre-bathed on ice, was added to the above system and cultured at 37°C and 170 rpm for 1-2 h. After removal, the culture was centrifuged at 4000 rpm for 5 min, 400 μL of supernatant was aspirated, and the remainder was resuspended. 200 μL of bacterial solution was spread on a resistance plate (kanamycin, final concentration of 50 μg / mL), allowed to dry, and then incubated inverted in a 37°C incubator for 12-16 h. Recombinants were picked and transferred to LB medium containing kanamycin at a final concentration of 50 μg / mL. When the bacterial solution became turbid, positive recombinants were verified by bacterial solution PCR. The PCR product was detected by 1.0% (w / v) agarose gel electrophoresis. The target band was approximately 800 bp, which was consistent with the size of the target gene Aii937 ( Figure 3 C). The bacterial cultures identified as positive for recombination were sent to Qingdao BGI Genomics Co., Ltd. for sequencing and alignment to further verify the correctness of the sequences.

[0033] Table 4: Connection system table Pick E. coli BL21 / pET-24a(+) / Aii937 was cultured in 5 mL LB liquid medium (final kanamycin concentration 50 μg / mL) at 37°C, 170 rpm, with shaking to prepare seed solution. The seed solution was inoculated into 300 mL LB medium (final kanamycin concentration 50 μg / mL) at a 1% inoculum and cultured at 37°C, 170 rpm, with shaking until the OD 600 The concentration of the protein was 0.4-0.6, and then IPTG was added (final concentration was 0.1 mM / L), and the culture was shaken at 16°C and 170 rpm for 12-16 h to induce expression.

[0034] After induction of expression, the cells were collected by centrifugation at 4°C, 6000 rpm, and part of the fermentation supernatant was retained for activity testing; Binding Buffer was added to the cells to resuspend the cells, and the cells were concentrated 10 times and mixed by pipetting; the cells were ultrasonically disrupted under the following conditions: working voltage 300 V, power 25%, disruption time 5 s, interval time 10 s, disruption 200-300 times, and operation on ice; after disruption, centrifuge at 4°C, 12000 rpm for 15 min, collect the supernatant (crude enzyme solution), and resuspend the precipitate with Binding Buffer and retain it for subsequent enzyme activity testing.

[0035] The crude enzyme solution was added to the equilibrated nickel column and the sample loading was repeated 3 times. The nickel column was washed with different concentrations of imidazole in sequence and the effluent was collected for SDS-PAGE electrophoresis detection. The obtained high-purity protein was placed in a dialysis bag, immersed in the dialysate, and dialyzed at 4°C to remove salt ions. The dialysate was replaced every 8 hours. After dialysis for 32 hours, the high-purity target protein ( Figure 4 ).

[0036] Agrobacterium tumefaciens A136 was used as a reporter strain to detect the activity of the recombinant Aii937 protein. The results showed that the recombinant Aii937 protein could degrade C6-HSL and 3OC-C 14 -HSL molecules, verifying the AHL signaling molecule degradation activity of Aii937 ( Figure 2 ).

[0037] Example 5: Enzymatic properties of Aii937 When an AHL molecule is hydrolyzed by lactonase, a proton (H + , produced by electrolysis of the carboxyl groups after hydrolysis), and the accumulated protons can cause pH changes. When a pH indicator is added to a reaction system with weak buffering capacity, the absorbance change can be used to measure AHL lactonase activity. This method is called pH-indicating continuous spectrophotometry. This present invention adopts Tang Kaihao's research method to detect enzymatic properties.

[0038] The enzymatic properties of Aii937 were tested using the above method, and it was found that Aii937 has the following properties: 1) The optimal reaction temperature for Aii937 is 30°C.

[0039] 2) The results showed that after treatment at 60°C for 30 min, the degradation activity decreased to about 45.5% of the control.

[0040] 3) The optimal reaction pH is 9.

[0041] From the above results, it can be seen that Aii93 obtained in the present invention is a new type of marine AHL lactonase, and its high AHL degradation activity and substrate specificity indicate that it has value for further application and development.

Claims

1. A Shiquan Haiyuan fungus, characterized in that The Shiquan Haiyuan bacteria is deposited with CCTCC NO: M20251224.

2. The Shiquan Haiyuan bacteria according to claim 1, characterized in that The sequence of 16S rRNA of the Shiquanhaiyuan bacteria is SEQ ID NO:

1.

3. The Shiquan Haiyuan bacteria according to claim 1, characterized in that The culture temperature range of the Shiquan Haiyuan bacteria is 4-45° C., and the optimum culture temperature is 28-32° C.; the growth pH range is 5-10, and the optimum culture pH is 7-8.

4. Use of the Shiquan Haiyuan bacteria according to claim 1 in the preparation of products for quenching aquaculture pathogens or reducing the virulence factors of aquaculture pathogens.

5. A bacterial preparation for killing pathogenic bacteria in aquaculture, characterized in that: The bacterial preparation contains the live bacteria of the Shiquan Haiyuan bacteria according to claim 1.

6. The bacterial preparation according to claim 5, characterized in that The bacterial preparation is a feed additive or a breeding water additive.

7. A kind of bacteria separated from the Shiquanhaiyuan bacteria according to claim 1 N -acylhomoserine lactone AHL-degrading enzyme, characterized in that The amino acid sequence of the degradation enzyme is SEQ ID NO:

2.

8. A gene, characterized in that The gene encodes the degradation enzyme according to claim 7.

9. The gene according to claim 8, wherein The nucleotide sequence of the gene is SEQ ID NO:

3.

10. Use of the degradative enzyme according to claim 7 in the preparation of an aquatic feed additive or an aquaculture water additive.

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