Hydrothermal source of quorum sensing quenching strain shiquania flava and application thereof

Abstract

The application provides a hot liquid source quorum quenching strain Shiquan Haeyuan bacteria and application thereof, the strain screened can inhibit pathogenic bacteria and is used for preparing bacteriostatic drugs; the preservation number of the Shiquan Haeyuan bacteria screened is CCTCC NO: M 20251224.The application also provides an AHL degradation enzyme Aii937 protein separated from the YESM3 strain, characterized in that the amino acid sequence of the protein is SEQ ID NO: 2.The YESM3 strain of the application can block the QS path of pathogenic bacteria through AHL lactonase Aii937, reduce the secretion of virulence factors of pathogenic bacteria; the strain YESM3 has no pathogenicity.AHL lactonase does not have broad spectrum, has strong substrate specificity, and only degrades C6-HSL and 3OC-C 14 -HSL.

Description

Technical Field

[0001] This invention belongs to the field of beneficial bacteria screening and application technology, specifically involving a hydrothermal vent-derived quorum sensing quenching strain, *Synthetium lappa*, and its application. Background Technology

[0002] The rapid development of aquaculture has led to the continuous expansion of industrialized and intensive aquaculture models. While this model can provide considerable economic benefits within limited space, it has also encountered significant problems. The threat of various diseases in farmed aquatic animals caused by viruses and pathogens is increasing, such as Vibrio species, which account for a considerable proportion of pathogens in marine aquaculture infections. Vibrios Bacteria can infect turbot ( Scophthalmus maximus This disease can cause hemorrhagic disease, fin rot, gastroenteritis, and septicemia in aquatic animals, including *Pseudomonas putida*. Pseudomonas putida ) can infect large yellow croaker ( Larimichthys crocea These effects, such as slowed movement and visible white nodules on the kidneys, pose a significant threat to the aquaculture industry.

[0003] Traditionally, bacterial infections are treated with antibiotics. While antibiotics have a positive effect on the prevention and treatment of diseases in aquatic animals in the short term, the increasing resistance of bacteria in the long run leads to a greater demand for antibiotics and a risk of antibiotic overuse. 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 while their effectiveness has decreased. Therefore, there is an urgent need for a highly effective, new antibacterial product that can metabolize antibiotics 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 can regulate the expression of specific genes. Bacteria can use QS to regulate processes such as biofilm formation, virulence factor expression, or genetic material transfer (binding and transformation). N -Acylhomoserine lactones ( N α-acyl-l-homoserine lactones (AHLs) are the most common bacterial QS signaling molecules. The chemical structure of an AHL consists of two parts: a homoserine lactone ring (HSL) and an acyl side chain. Based on the length of the acyl side chain, signaling molecules are classified into two types: short-chain AHLs (C4-C8) and long-chain AHLs (C4-C8). 10 -C 18 Clinically resistant pathogens such as Pseudomonas aeruginosa ( ). Pseudomonas aeruginosa Aeromonas ( AeromonasAll studies used AHLs as signaling molecules. Quorum quenching (QQ) refers to a mechanism that inhibits QS regulation by interfering with the production, release, accumulation, or response of signaling molecules, thereby suppressing the QS pathway. Microorganisms contain a variety of QQ substances, which can be broadly classified into small-molecule QS inhibitors (QSIs) and large-molecule QQ quenching enzymes (QQEs) based on their molecular weight. Small-molecule QS inhibitors mainly block the synthesis or recognition of QS signaling molecules by specifically binding to the synthases or receptor proteins of QS signaling molecules, ultimately blocking the signal transduction of the entire QS pathway and achieving the purpose of reverse regulation of target gene expression. Large-molecule QQ substances mainly block the QS pathway by degrading the QS signaling molecules themselves.

[0005] QQ enzymes are one of the main means of disrupting the QS (Qualitative Signaling System) of microorganisms and inhibiting their collective behavior. Currently, the most researched QQ enzymes are those that can alter the structural properties of AHL-like signaling molecules, thereby rendering them biologically inactive. The most common of these are AHL lactones and AHL acyltransferases. Quenching enzymes targeting AHL signaling molecules help treat and prevent many bacterial infections. Compared to traditional treatments, the QQ process aims to inhibit virulence expression without killing bacteria. Therefore, QQ technology is considered an environmentally friendly disease control measure that can reduce dependence on antibiotics, thereby delaying the evolution of drug resistance and reducing the emergence of superbugs. QQ enzymes are increasingly being explored as potential antimicrobial agents targeting pathogenic bacteria. To date, enzymes from species including Bacillus (…) have been identified. Bacillus ), Chlorella ( Chryseobacterium ), Enterobacteriaceae ( Enterobacter Microbacteria ( Microbacteirum Agrobacterium spp. Agrobacterium ), Kurtella spp. Kurthia Brucella ( ) Brucella Arthrobacter spp. Arthrobacter ), Pseudomonas spp. Pseudomonas ), Bacillus spp. ( Solibacillus ) and Rhodococcus spp. ( Rhodococcus More than 50 enzymes degrading AHLs have been identified from over 30 bacterial genera, including [list of genera]. These findings provide a wealth of enzyme resources for the future treatment of bacterial diseases and have broad market application potential. Further development of novel QQ enzymes with good environmental stability, high enzyme activity, and strong substrate specificity, and research into suitable methods to ensure the stable expression of these enzymes, are crucial for promoting their practical application.

[0006] The ocean covers 71% of the Earth's surface and contains a variety of complex habitats, such as deep-sea trenches, hydrothermal vents, and blue holes. Over the past few decades, marine bacteria have served as a rich repository of novel bioactive substances 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 wide range of bioactive compounds to help them survive in specific ecological niches and compete with other microorganisms, thus maximizing their adaptation to the environment. Therefore, marine microorganisms can produce compounds with extremely diverse structures and bioactivities, with some bioactive substances appearing even more frequently than in terrestrial microorganisms. For example, it has been reported that the proportion of marine bacteria with QQ activity is much higher than in agricultural soils or crop rhizosphere soils. Therefore, marine bacteria possess a richer resource of novel QQ enzymes. To date, many novel QQ enzymes with unique bioactivities and biochemical properties have been identified and characterized from various marine sources, such as seawater, sediments, and marine organisms. However, few studies have focused on microorganisms from deep-sea hydrothermal vents. Deep-sea hydrothermal vents, as oases of life, possess a high-temperature, high-pressure environment where microorganisms interact closely, potentially harboring novel QQ enzymes with unique physicochemical properties. Therefore, deep-sea hydrothermal vents may hold abundant QQ enzyme resources awaiting exploration, and they hold broad development prospects as novel and highly effective antibacterial drugs for treating bacterial diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a hydrothermal quorum induction quenching strain, *Synthetium lappa*, and its application. The screened strain can inhibit pathogenic bacteria and can be used to prepare antibacterial drugs.

[0008] This invention first provides a *Stone Spring Marine Source Bacterium* (Stone Spring Marine Source Bacterium) Idiomarina fontislapidosi The YESM3 strain was deposited on May 29, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University in Wuhan, Hunan Province, with accession number CCTCC NO: M 20251224.

[0009] The culture temperature range of the *Salvia miltiorrhiza* YESM3 screened in this invention is 4-45℃, with the optimal culture temperature being 28-32℃; the growth pH range is 5-10, with the optimal culture pH being 7-8.

[0010] The strains of this invention can be used to prepare bacterial preparations for quenching density sensing systems of aquaculture pathogens and reducing their secretion of virulence factors.

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

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

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

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

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

[0016] The YESM3 strain of this invention can block the QS pathway of pathogenic bacteria through the AHL lactonease Aii937, thereby reducing the secretion of pathogenic virulence factors; strain YESM3 is non-pathogenic. The AHL lactonease is not broad-spectrum, but has strong substrate specificity, degrading only C6-HSL and 3OC-C. 14 -HSL. Attached Figure Description

[0017] Figure 1 : Activity diagram of AHLs degradation signaling molecules of strain YESM3, where 1: positive control (MomL protein), 2: negative control (2216E), 3: YESM3;

[0018] Figure 2 Aii937 activity diagram of AHL degradation signaling molecules, where 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.

[0019] Figure 3 The QQ enzyme encoding gene used in this invention Aii937 Agarose gel electrophoresis verification image of heterologous expression, where A: Aii937 Gel electrophoresis image of gene clones, B: screening E. coil JM109 / pET-24a(+) / YESM3- Aii937 Gel electrophoresis image, C: screening E. coil BL21 / pET-24a(+) / YESM3- Aii937The images show gel electrophoresis results. In the figures, M represents the marker. Figures A and B show 2000 bp markers. The sizes of markers in Figure A from top to bottom are 2000, 1000, 750, 500, 250, 100. The sizes of markers in Figure B from top to bottom are 2000, 1500, 1000, 750, 500, 250, 100. Figure C shows a 5000 bp marker, with sizes of 5000, 3000, 2000, 1500, 1000, 500, 250, 100.

[0020] Figure 4 SDS-PAGE electrophoresis image of crude QQ enzyme Aii937 solution, where M: protein marker, 1: E. coli BL21 / pET-24a(+) fragmented supernatant, 2: E. coli BL21 / pET-24a(+) / Aii937 lysate supernatant, 3: QQ enzyme Aii937 eluted with imidazole. Detailed Implementation

[0021] The strains of the present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0022] Example 1: Isolation of the target strain

[0023] In June 2018, marine bacteria were isolated and cultured from sediment samples obtained at station R11 (E122.34°, N25.03°) during HOBAB4 expedition in 2016. First, the samples were diluted to 10⁻⁶ ppm with 0.85% (w / v) physiological saline. -1 10 -2 10 -3 Then, 100 μL of different concentration dilutions were spread onto 2216E plates, and the plates were incubated at 28°C. The plates were then streaked 3-4 times to obtain purified colonies.

[0024] The target strain was obtained by screening for AHL degradation activity efficiency and named YESM3. 16S rRNA gene sequencing and alignment confirmed that strain YESM3 of this invention is *Bacterium sarcodactylis* (also known as *Bacterium sarcodactylis*). Idiomarina fontislapidosi It was deposited on May 29, 2025, at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hunan Province, with accession number CCTCC M 20251224.

[0025] The 16S rRNA sequence of this bacterium is as follows:

[0026]

[0027] Example 2: AHL degradation activity of *Bacterium sarcodactylis* YESM3

[0028] First, strain YESM3 was cultured overnight to the logarithmic growth phase. C6-HSL (final concentration 5 μM / L), PIPES (1 mM, pH=6.73) buffer (to stabilize pH and prevent acid or alkaline degradation of signal molecules) was mixed with the bacterial culture and incubated at 28°C in the dark for 24 h. After incubation, the supernatant was obtained by centrifugation at 4°C, 6000 rpm for 10 min and filtered through a 0.22 μm filter. The supernatant was then mixed with *Agrobacterium tumefaciens* (…). Agrobacterium tumefaciens The A136 X-gal assay solution was mixed in a 96-well plate and incubated at 28°C for 24 h. All experiments were repeated 3-4 times. Results were compared with the negative control, and the presence of QQ activity of the strain was confirmed by t-test. Figure 1 ).

[0029] Example 3: Amplification of the QQ enzyme gene Aii937 sequence

[0030] Whole-genome sequencing of strain YESM3 and RAST annotation analysis of the assembled genome revealed the potential QQ enzyme encoding gene Aii937, which showed a similarity of 31.73% with known QQ enzymes. Primers were then designed, and appropriate restriction enzyme sites were added upstream and downstream of the target gene: EcoRI and HindIII, respectively. YESM3 DNA was then amplified using strain YESM3 as a template.

[0031] The primer sequences are as follows:

[0032] Aii937F: 5′-CGGAATTCATGGCAGAGTCAGAATTAC-3′,

[0033] Aii937R: 5′-CCCAAGCTTAAGACGACCTTGGCT-3′.

[0034] The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0035] Table 1: PCR Reaction System

[0036] Table 2: PCR reaction procedure

[0037]

[0038] The PCR products were detected by agarose gel electrophoresis to verify whether the target band size was correct. Figure 3A) Non-specifically amplified bands were removed using a gel extraction kit, and the PCR product was purified and recovered to obtain the purified target gene fragment. Sequencing revealed the following nucleotide sequence of the encoding gene:

[0039] (SEQ ID NO:3).

[0040] The amino acid sequence of its encoded protein is as follows:

[0041] MAESELLHYETMGDKQNPAVIIIHGLFGDGDNLKSLARDLTPDYFCVLPDARNHGESPHRESMTYTEMADDIVALADELALEHFSLVGHSMGGKIAMEVAMRYEDRVQAAVFADIAPVAYPAHHNGILDALAG LDLDQIGSRTEADKQLSSAIKEKGVRQFLLKNLRKDGDHFAWRLNLTAITERYQQIADGVSDGHYSGPCLFIKGGNSDYLTEQHRAQVTQRFSNTQVKVVENTGHWLHAEKPRIFNRLVKDFLASQGRL (SEQ ID NO:2).

[0042] Analysis showed that Aii937 possesses a signal peptide sequence and belongs to the α / β hydrolase family. Among the identified AHL lactoneases, Aii937 and Aii810 showed the highest amino acid sequence similarity (31.73% identity).

[0043] Example 4: Cloning and transformation of the QQ enzyme gene Aiii937

[0044] The target gene fragment was double-digested with pET-24a(+) plasmid (Table 3). After double digestion in a metal bath at 37℃ for 2 h, the digestion was detected by agarose gel electrophoresis. The target gene and vector in the agarose gel were recovered. The double-digested target gene and plasmid were ligated overnight at 16℃ (4) to construct the recombinant plasmid. The plasmid was then transformed into pET-24a(+) using the heat shock method. E. coli JM109 competent cells were plated onto LB agar plates containing kanamycin (final concentration 50 μg / mL) and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing kanamycin (final concentration 50 μg / mL) and incubated at 37°C and 170 rpm for 8–12 h. Once the bacterial culture became turbid, positive recombination was verified by bacterial PCR. The PCR product was detected by 1.0% (w / v) agarose gel electrophoresis; the target band was approximately 800 bp, consistent with the size of the target gene Aii937. Figure 3 B). The positive recombinant bacterial culture was sequenced and compared by BGI Genomics in Qingdao to ensure the correctness of the sequence, and plasmids were extracted using the omega kit.

[0045] Table 3: Enzyme digestion system

[0046] The extracted recombinant plasmid was transferred into E. coliIn BL21(DE3) competent cells, 600 μL of LB liquid medium (pre-chilled on ice) was added to the above system, and the cells were incubated at 37°C with shaking at 170 rpm for 1-2 h. After incubation, the cells were centrifuged at 4000 rpm for 5 min, and 400 μL of supernatant was collected, with the remainder resuspended. 200 μL of the bacterial culture was spread on an antibiotic plate (kanamycin, final concentration 50 μg / mL), and after drying, it was incubated upside down at 37°C for 12-16 h. Recombinants were picked and added to LB medium containing kanamycin at a final concentration of 50 μg / mL. After the bacterial culture became turbid, positive recombinants were verified by bacterial PCR. The PCR product was detected by 1.0% (w / v) agarose gel electrophoresis. The target band was approximately 800 bp, consistent with the size of the target gene Aii937. Figure 3 C). The bacterial culture identified as positive for recombination was sent to BGI Genomics in Qingdao for sequencing and comparison to further verify the correctness of the sequence.

[0047] Table 4: Connection System Table

[0048] Pick E. coli BL21 / pET-24a(+) / Aii937 was cultured in 5 mL of LB liquid medium (final kanamycin concentration 50 μg / mL) at 37°C with shaking at 170 rpm to prepare a seed culture. The seed culture was then inoculated into 300 mL of LB medium (final kanamycin concentration 50 μg / mL) at a 1% inoculum and cultured at 37°C with shaking at 170 rpm until OD (open-circuit retrieval). 600 The concentration was 0.4-0.6, then IPTG (final concentration 0.1 mM / L) was added, and the mixture was incubated at 16°C with shaking at 170 rpm for 12-16 h to induce expression.

[0049] After induction of expression, the bacterial cells were collected by centrifugation at 4°C and 6000 rpm, and a portion of the fermentation broth supernatant was retained for activity testing. The bacterial cells were resuspended in Binding Buffer and concentrated 10-fold, then mixed by pipetting. The bacterial cells were then sonicated under the following conditions: operating voltage 300 V, power 25%, disruption time 5 s, interval time 10 s, disruption 200-300 times, operated on ice. After disruption, the cells were centrifuged at 4°C and 12000 rpm for 15 min, and the disruption supernatant, i.e., crude enzyme solution, was collected. At the same time, the precipitate was resuspended in Binding Buffer and retained for subsequent enzyme activity testing.

[0050] Add the crude enzyme solution to the equilibrated nickel column and repeat the loading process three times. Wash the nickel column with different concentrations of imidazole sequentially, and collect the eluent for SDS-PAGE electrophoresis. Place the obtained high-purity protein in a dialysis bag, immerse it in dialysis solution, and dialyze at 4°C to remove salt ions. Change the dialysis solution every 8 hours and dialyze for 32 hours to obtain the high-purity target protein. Figure 4 ).

[0051] The activity of recombinant Aii937 protein was detected using Agrobacterium tumefaciens A136 as a reporter strain. The results showed that recombinant Aii937 protein could degrade C6-HSL and 3OC-C. 14 -HSL molecules were used to verify the AHL signaling molecule degradation activity of Aii937. Figure 2 ).

[0052] Example 5: Enzymatic properties of Aii937

[0053] When an AHL molecule is hydrolyzed by a lactone enzyme, it produces a proton (H). + (The carboxyl groups produced after hydrolysis are electrolyzed), and the accumulated protons can cause a change in pH. When a pH indicator is added to a reaction system with weak buffering capacity, the activity of AHL lactonease can be reflected by detecting changes in absorbance. This method is called pH-indicated continuous spectrophotometric analysis. This invention uses Tang Kaihao's research method to detect enzymatic properties.

[0054] Using the above method to detect the enzymatic properties of Aii937, the following properties were found:

[0055] 1) The optimal reaction temperature for Aii937 is 30°C.

[0056] 2) It was shown that after treatment at 60°C for 30 min, the degradation activity decreased to about 45.5% of the control.

[0057] 3) The optimal reaction pH is 9.

[0058] The results above indicate that the Aii93 obtained by this invention is a novel marine AHL lactonease. Its high AHL degradation activity and substrate specificity suggest that it has value for further application and development.

Claims

1. A type of *Stone Spring Marine Source Bacterium*, characterized in that, The *Stone Spring Marine Source Bacterium* was preserved with the accession number CCTCC NO: M20251224.

2. The *Salix viminalis* strain as described in claim 1, characterized in that, The 16S rRNA sequence of *S. shiquanhaiyuan* is SEQ ID NO:

1.

3. The *Salix viminalis* as described in claim 1, characterized in that, The culture temperature range of the *Stone Spring Marine Source Bacterium* is 4-45℃, with the optimal culture temperature being 28-32℃; the growth pH range is 5-10, with the optimal culture pH being 7-8.

4. The use of the *Stone Spring Marine Source Bacterium* as described in claim 1 in the preparation of products that quench aquatic pathogens or reduce the virulence factors of aquatic pathogens.

5. A bacterial preparation for quenching pathogens in aquaculture, characterized in that, The bacterial preparation contains live bacteria of the *Stone Spring Sea Source Bacterium* as described in claim 1.

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

7. A method isolated from the *Salvia miltiorrhiza* as described in claim 1. N -Acylhomoserine lactone AHL-degrading enzyme, characterized in that... The amino acid sequence of the degrading enzyme is SEQ ID NO:

2.

8. A gene characterized by, The gene encodes the degrading enzyme of claim 7.

9. The gene as described in claim 8, characterized in that, The nucleotide sequence of the gene is SEQ ID NO:

3.

10. The application of the degrading enzyme according to claim 7 in the preparation of aquatic feed additives or aquaculture water additives.

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