CN113106111B - N-acyl homoserine lactone acyltransferase encoding gene aigC and application thereof - Google Patents
N-acyl homoserine lactone acyltransferase encoding gene aigC and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biological control, and discovers that N-acyl homoserine lactone acyltransferase coding genes are cloned by molecular biological technology on the basis that Pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the chain length of C4-C14 acyl. In the previous research, the invention discovers that the coding gene of the N-acyl homoserine lactone acyltransferase is cloned by a molecular biology technology on the basis that the Pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the chain length of C4-C14 acyl. The gene has broad-spectrum and high-efficiency quenching activity on AHLs with different side chain lengths and different side chain substituent bands. The N-homoserine lactone quenching gene expressed in pathogenic bacteria depending on AHLs can obviously weaken the motility, biofilm formation and the production of virulence factors such as extracellular enzyme of the pathogenic bacteria, and obviously weaken the pathogenicity of the pathogenic bacteria to host plants.
Description
Technical Field
The invention relates to the technical field of molecular biological control, in particular to an N-acyl homoserine lactone acyltransferase encoding gene aigC and application thereof.
Background
The use of pesticides and antibiotics is the most common method for preventing and treating pathogenic bacteria at present, however, long-term abuse of pesticides and antibiotics poses threats to environmental safety and human and animal health, and even causes drug resistance of microorganisms. Therefore, a new and environmentally friendly antimicrobial approach is urgently needed.
Quorum sensing is an intercellular communication mechanism, cells synthesize and secrete a small molecule compound signal, the number of populations is determined by sensing the concentration of signal molecules diffusing to the outside of the cells, and when the concentration of the signal molecules reaches a certain threshold, the cells activate the expression of downstream target genes through a series of signal transduction. Bacteria regulate their own various physiological and biochemical functions through quorum sensing signal molecules, such as: plasmid transfer, drug resistance, motility, bioluminescence, biofilm formation, drug resistance, production of extracellular enzymes, etc., to adjust self-adaptability to the environment and viability. In particular, many gram-negative pathogens utilize quorum sensing systems to regulate the production and virulence of their own virulence factors in order to obtain a greater infectivity of the host.
N-acyl-homoserine lactones (AHLs) are the most widely existing quorum sensing signal molecules in gram-negative pathogenic bacteria and are responsible for regulating and controlling the generation and pathogenicity of virulence factors in various gram-negative pathogenic bacteria, such as: motility, formation of biofilm, and production of extracellular enzymes, extracellular polysaccharides, toxins, and the like. The first AHLs signal molecule was found in the bioluminescence study of the marine bacteria Vibrio fischeri (Vibrio fischeri) and Vibrio harveyi (Vibrio harveyi). More and more AHLs are identified later, and the molecular structures of the AHLs are conservative and mainly consist of an N-acyl homoserine lactone ring and acyl chains with 4 to 18 different carbon atoms and different substituent modifications at the carbon position 3.
Quorum quenching is a way to block or destroy quorum sensing systems, and can be achieved mainly in three ways: inhibiting quorum sensing signal synthetase activity, inhibiting quorum sensing signal receptor protein activity, and modifying or enzymatically hydrolyzing quorum sensing signal molecules with a quenching enzyme. The discovery and application of group quenching genes or quenching enzymes has become one of the research hotspots for quenching of the current group. The first AHLs quencher enzyme identified was AHL lactonase AiiA found in bacillus thuringiensis, which is capable of destroying the lactone bond of the lactone ring in AHLs molecules, thereby inactivating AHL signal molecules. The expression of AiiA in pathogenic bacteria can obviously weaken the yield of pathogenic bacteria virulence factors and the virulence to host plants, and the expression of AiiA in plants can effectively improve the disease resistance of host plants to pathogenic bacteria. After the identification of AHL lactonase, AHL acyltransferase capable of cleaving amide bond between AHL lactone ring and acyl chain and AHL oxidoreductase acting on side chain hydrogen atom were also successively found.
Researches show that the expression of most of the identified AHLs quenching genes in pathogenic bacteria can weaken the toxicity of the pathogenic bacteria, the expression of the AHLs quenching genes in host plants can improve the resistance of the host to the pathogenic bacteria, and AHLs quenching enzymes, which are coded products of the AHLs quenching genes or the AHLs quenching genes, are widely and effectively applied to the aspects of biological prevention and control of agriculture and aquaculture industry and a biofilm reactor for sewage treatment. Therefore, the excavation, identification and application of the AHLs quenching genes lay a foundation for enriching AHLs colony quenching preparation resources, and have great practical application value for biological control of colony quenching ways.
Disclosure of Invention
The present invention has been made to overcome the above-mentioned problems occurring in the prior art, and it is a first object of the present invention to provide a gene encoding N-acyl homoserine lactone acyltransferase.
It is a second object of the present invention to provide a recombinant vector containing a gene encoding N-acylhomoserine lactone acylase.
The third object of the present invention is to provide a recombinant bacterium containing the recombinant vector.
The fourth purpose of the invention is to provide the application of the recombinant vector.
The fifth purpose of the invention is to provide the application of the recombinant bacterium.
The purpose of the invention is realized by the following technical scheme:
an N-acyl homoserine lactone acyltransferase encoding gene aigC, the nucleotide sequence of which is shown as SEQ ID NO:1 is shown.
In the previous research, the invention discovers that the coding gene of the N-acyl homoserine lactone acyltransferase is cloned by a molecular biology technology on the basis that the Pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the chain length of C4-C14 acyl.
It is understood that the above-mentioned recombinant vector containing the gene encoding the N-acyl homoserine lactone acyltransferase is also within the scope of the present invention; recombinant bacteria containing the recombinant vector are also within the scope of the present invention.
As a preferred embodiment, the recombinant vector is obtained by inserting the gene encoding N-acyl homoserine lactone acyltransferase into a broad host vector pBBR1 for heterologous expression of the gene encoding N-acyl homoserine lactone acyltransferase.
As another preferred embodiment, the recombinant vector can also be a recombinant vector for prokaryotic expression of the N-acyl homoserine lactone acyltransferase protein, which is obtained by inserting the gene encoding the N-acyl homoserine lactone acyltransferase into a prokaryotic protein expression vector pET32 a.
As a preferred embodiment, the recombinant bacterium is preferably obtained by introducing the recombinant vector expressing the gene encoding N-acyl homoserine lactone acyltransferase in the broad host vector pBBR1 into Escherichia coli DH5 alpha.
In another preferred embodiment, the recombinant bacterium is obtained by introducing the recombinant vector expressing the gene encoding N-acylhomoserine lactone acyltransferase in the broad-host vector pBBR1 into pathogenic bacteria which depend on AHL to cause diseases.
As another preferred embodiment, the recombinant bacterium is obtained by introducing the recombinant vector for expressing the N-acyl homoserine lactone acyltransferase in a prokaryotic protein expression vector pET32a into Escherichia coli BL21 (DE 3).
The invention also provides N-acyl homoserine lactone acyltransferase which is named AigC, and the amino acid sequence of the N-acyl homoserine lactone acyltransferase is shown as SEQ ID NO:2, respectively.
The invention also provides a preparation method of the N-acyl homoserine lactone acyltransferase, which is characterized in that a recombinant bacterium for expressing the N-acyl homoserine lactone acyltransferase in a protein prokaryotic expression vector pET32a is fermented and cultured, and a fermented and cultured recombinant bacterium strain is crushed and separated and purified to obtain the N-acyl homoserine lactone acyltransferase protein with the His label.
The invention also provides application of the recombinant bacterium or the N-acyl homoserine lactone acyltransferase in degradation of AHLs signal molecules.
Preferably, the AHLs signal molecules include C4-HSL, C6-SHL, 3-O-C6-HSL, C8-HSL, 3-OH-C8-HSL, C10-HSL, 3-OH-C10-HSL, C12-HSL, 3-O-C12-HSL, 3-OH-C14-HSL; the N-acyl homoserine lactone acyltransferase has a broad spectrum of quenching activity for different AHLs signal molecules, and the reactivity of different substrates and N-acyl homoserine lactone acyltransferases may be different.
The invention also provides an application of the coding gene of the N-acyl homoserine lactone acyltransferase, the recombinant vector, the recombinant bacterium or the N-acyl homoserine lactone acyltransferase of claim 4 in preventing and treating pathogenic bacteria which depend on AHLs.
Preferably, the AHLs-dependent pathogenic bacteria include Pectinophytrium carotovorum subspecies, burkholderia cepacia, pseudomonas aeruginosa, laurella solanacearum, agrobacterium tumefaciens, rhizoctonia solani, erwinia europaea, and bacterial wilt of maize.
As a preferred embodiment, when the gene encoding N-acyl homoserine lactone acyltransferase is used, the gene encoding N-acyl homoserine lactone acyltransferase is expressed in AHLs mediated pathogenic bacteria, and recombinant pathogenic bacteria successfully expressing the N-acyl homoserine lactone acyltransferase are screened.
As another preferred embodiment, when the recombinant vector is used, the recombinant vector is introduced into AHLs-mediated pathogenic bacteria, and recombinant pathogenic bacteria that successfully express N-acylhomoserine lactone acyltransferase are selected.
As still another preferred embodiment, when the recombinant bacterium is used, a recombinant pathogen is selected which successfully expresses N-acylhomoserine lactone acyltransferase.
The screening of the recombinant pathogenic bacteria successfully expressing the N-acyl homoserine lactone acyltransferase obviously weakens the yield and pathogenicity of virulence factors of the pathogenic bacteria.
Preferably, the pathogenic bacteria are Burkholderia cepacia (Burkholderia cenocepacia) H111 which depends on C8-HSL to cause disease, and Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 which depends on C4-HSL and 3-OH-C12-HSL to cause disease.
In a preferred embodiment, the recombinant pathogenic bacterium that successfully expresses N-acylhomoserine lactone acyltransferase comprises:
(1) The yield of AHLs is reduced; and/or the presence of a gas in the gas,
(2) The sports type is reduced; and/or the presence of a gas in the gas,
(3) Reduces the formation of biofilm; and/or the presence of a gas in the gas,
(4) The production amount of protease is reduced; and/or the presence of a gas in the atmosphere,
(5) The pathogenic force is weakened.
Compared with the prior art, the invention has the following beneficial effects:
in the previous research, the invention discovers that the coding gene of the N-acyl homoserine lactone acyltransferase is cloned by a molecular biology technology on the basis that the Pseudomonas nitroreducens HS-18 can efficiently degrade AHLs signal molecules with the chain length of C4-C14 acyl. The gene has broad-spectrum and high-efficiency quenching activity on AHLs with different side chain lengths and different side chain substituent bands. The N-homoserine lactone quenching gene expressed in pathogenic bacteria depending on AHLs can obviously weaken the motility, biofilm formation and the production of virulence factors such as extracellular enzyme of the pathogenic bacteria, and obviously weaken the pathogenicity of the pathogenic bacteria to host plants.
Drawings
FIG. 1 is a diagram showing the AHLs degradation effect of recombinant E.coli DH5 alpha (aigC) (FIG. 1B) expressing N-acyl homoserine lactone acyltransferase encoding gene in broad host vector pBBR1 and E.coli DH5 alpha (pBBR 1) (FIG. 1A) containing empty vector after culturing for 36h according to the present invention;
FIG. 2 is an AigC phylogenetic tree analysis of the N-acyl homoserine lactone acyltransferase protein according to the present invention;
FIG. 3 is the expression of AigC protein; m: a protein Marker;1: expressing the total protein expression condition in the AigC protein recombinant bacteria; 2: BL21 (DE 3) total protein expression with empty vector pET32 a; 3: expressing the intracellular protein expression condition in the AigC protein recombinant bacteria; 4: intracellular protein expression in BL21 (DE 3) containing empty vector pET32 a;
FIG. 4 shows the effect of expression of the gene aigC encoding N-acyl homoserine lactone acyltransferase of the present invention in Burkholderia cepacia H111, a AHL-dependent pathogen, on H111 growth, self-produced AHL production and virulence factors; FIG. 4A shows a Burkholderia cepacia growth curve; FIG. 4B shows intracellular C8-HSL production; FIG. 4C shows Burkholderia cepacia motility; FIG. 4D shows the formation of Burkholderia cepacia biofilm; FIG. 4E shows Burkholderia cepacia protease production;
FIG. 5 shows the effect of expression of gene aigC encoding N-acyl homoserine lactone acyltransferase of the present invention in H111 pathogenicity of AHL dependent pathogenic bacterium Burkholderia cepacia H111;
FIG. 6 shows the effect of expression of the gene aigC encoding N-acyl homoserine lactone acyltransferase of the present invention in P.aeruginosa PAO1, a AHL dependent pathogen, on growth, self-produced AHL production and virulence factors of PAO 1; FIG. 6A shows a Pseudomonas aeruginosa growth curve; FIG. 6B shows intracellular C4-HSL production of P.aeruginosa; FIG. 6C shows the intracellular 3-O-C12-HSL production of P.aeruginosa; FIG. 6D shows Pseudomonas aeruginosa motility; FIG. 6E shows Pseudomonas aeruginosa protease production;
FIG. 7 shows the influence of expression of the gene aigC encoding N-acyl homoserine lactone acyltransferase of the present invention in pathogenic bacteria Pseudomonas aeruginosa PAO1 depending on AHL pathogenicity on the pathogenicity of PAO1, FIG. 7A shows the influence on the pathogenicity of lettuce, and FIG. 7B shows the influence on the pathogenicity of cabbage.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
MM inorganic salt medium: k 2 HPO 4 ,10.5g/L;KH 2 PO 4 ,4.5g/L;(NH 4 ) 2 SO 4 ,2.0g/L;MgSO 4 ·7H 2 O,0.2g/L;FeSO 4 ,0.005g/L;CaCl 2 ,0.01g/L;MnCl 2 0.002g/L; 2.0g/L of glycerol; mannitol, 2.0g/L; the pH value is 6.8-7.2, and the sterilization is carried out for 20min at the temperature of 121 ℃.
LB culture medium: trypton 10.0g/L, yeast extract 5.0g/L, naCl 10.0g/L, pH 6.8-7.2, sterilizing at 121 ℃ for 15-25 min.
PBS phosphate buffer, X-gal and the reagents needed in the culture medium are all purchased from biological reagent companies such as Guangzhou Qixiang, dingguo, etc., and the AHLs for detecting degradation activity in the invention are purchased from Shanghai Youder chemical technology Co., ltd and Sigma-Aldrich.
The pseudomonas nitroreducens HS-18 is separated from soil samples polluted by oil for a long time near southern China agricultural university in Guangzhou city, and is preserved in the China center for type culture Collection in 2017, 5 months and 12 days, wherein the preservation number is CCTCC NO: m2017257. HS-18 was cultured at 30 ℃ using LB medium.
Example 1 acquisition and identification of AHLs degradation genes
According to the whole genome sequencing result of P.nitroreducens strain HS-18 in the previous work, the gene aigC which possibly codes N-acyl homoserine lactone acyltransferase is discovered by using genome annotation and bioinformatics alignment. The amino acid sequences of AigC and the currently known N-acyl homoserine lactone acyltransferase were aligned using the software MEGA 5.10, and ClustalX1.8.3, the adjacent approach was usedAnalyzing phylogenetic evolution and constructing an evolutionary tree. The results are shown in FIG. 2, where AigC is compared with the known AHLs acyltransferase HacB in P.aeruginosa PAO1 PAO1 With a high degree of amino acid similarity (76.6%).
Designing primers for respectively amplifying and constructing primers of aigC inserted broad host vector pBBR1 and protein prokaryotic expression vector pET32a recombinant vector, wherein the sequences of the primers are as follows:
pBBR1-aigC-F:gtcgacggtatcgataagcttGCAGAATCGCCGCATAACA
pBBR1-aigC-R:cgctctagaactagtggatccTCAGCGACGGACGGGGAT
pET32a-aigC-F:gccatggctgatatcggatccATGAAACGCACTTTGACTGTCCT
pET32a-aigC-R:ctcgagtgcggccgcaagcttTCAGCGACGGACGGGGAT
example 2 detection of AHLs quenching Activity by recombinant Strain DH5 alpha (aigC)
AHLs degradation system setting: OD was obtained by overnight culturing the successfully constructed DH 5. Alpha. (aigC) in LB liquid medium 600 Adding equal volume of fresh LB liquid culture medium, exogenous AHLs with different carbon chain lengths and different substituents and MOPS with the final concentration of 50mM into the seed liquid with the final concentration of 10-50 mu M to prepare a degradation system (different AHLs have proper concentrations according to different intensities of the color development of the report strain), culturing for 36h in a constant temperature shaking table at 37 ℃ and 200rpm, and taking DH5 alpha (pBBR 1) bacterial liquid and LB liquid culture medium without the bacterial liquid as controls. Next, the culture broth was extracted with an equal volume of ethyl acetate, and the content of AHLs remaining in 10. Mu.l of ethyl acetate extract was measured using a reporter strain.
Quantitative detection of short-chain AHLs (C4-C6): short-chain AHLs (C4-C6) are detected by using a reporter strain purple bacillus CV026. First, CV026 was activated on LB solid plates and cultured overnight on an LB liquid medium in a constant temperature shaker at 28 ℃ and 200 rpm. An LB solid plate is prepared in a square culture dish of 13cm × 13cm, and cut into 0.8cm wide alternating agar strips, 10 μ l of ethyl acetate extract of a sample to be detected is loaded at the upper end of the agar strips, and a row of report strain liquid drops with similar sizes are continuously spotted below the loading position of the sample. The area to which AHLs diffuse induces violacein production by Violaceous bacillus CV026, rendering the bacteria purple. After the sample on the agar strip is dried, culturing for 16h in a constant temperature incubator at 28 ℃, and observing and counting the distance of bacteria presenting purple CV026. The results from FIG. 1 show that DH 5. Alpha. (aigC) can significantly degrade short-chain AHLs for detection (C4-HSL, C6-HSL, 3-O-C6-HSL) with high efficiency.
And detecting the medium-long chain AHLs (C8-C14) by using a report strain Agrobacterium tumefaciens NT1. First, NT1 was activated by LB solid plate, and NT1 was cultured overnight in an LB liquid medium supplemented with 50. Mu.g/ml kanamycin at 28 ℃ in a constant temperature shaker at 200 rpm. Preparing MM solid plates in a square culture dish of 13cm × 13cm, cutting the MM solid plates into alternate agar strips with the width of 0.8cm, loading 10 μ l of ethyl acetate extract of a sample to be detected at the upper ends of the agar strips, and continuously dropping a row of reporter strain drops with similar sizes below the sample loading position. The area where AHLs diffuse can induce Agrobacterium tumefaciens NT1 to produce beta-galactosidase, which decomposes X-gal to make the thallus appear blue. The distance of the agrobacterium tumefaciens NT1 producing blue is proportional to the content of AHLs to be detected. After the sample on the agar strip is dried, the agar strip is cultured in a constant temperature incubator at 28 ℃ for 16h, and the distance of NT1 showing blue color is observed and counted. The results in FIG. 1 show that DH 5. Alpha. (aigC) pairs of medium-and long-chain AHLs (C8-HSL, 3-OH-C8-HSL, C10-HSL, 3-OH-C10-HSL, C12-HSL, 3-O-C12-HSL, 3-OH-C14-HSL) were used for the detection. Therefore, the aigC has high-efficiency and broad-spectrum quenching activity on AHLs with different chain lengths and different substituents of C4-C14 to be detected.
Example 3 prokaryotic expression of AigC protein
The successfully constructed recombinant strain BL21 (DE 3) (pET 32 a-aigC) was cultured overnight in LB liquid medium supplemented with ampicillin at a final concentration of 100. Mu.g/ml, and cultured in a constant temperature shaker at 37 ℃ and 200rpm to obtain a seed solution. And then mixing the seed liquid in a proportion of 1:100 to a fresh LB liquid medium containing ampicillin at a final concentration of 100. Mu.g/ml, was incubated at 37 ℃ on a 200rpm constant temperature shaker to OD 600 = 0.6-0.8, and was induced by adding IPTG to a final concentration of 0.5mM and cultured overnight in a constant temperature shaker at 200rpm at 18 ℃. Overnight cultured cells were collected, disrupted, and the expression of AigC was identified by SDS-PAGE electrophoresis. The results in FIG. 3 show that AigC with His tag can proceedNormally expressed in pronuclei, the size is about 106.54kDa.
Example 4 Effect of AigC expression on AHL-mediated growth of pathogen H111 and intracellular AHL production
Detecting the growth condition of H111: culturing the successfully constructed recombinant bacterium H111 (aigC) seed liquid to OD overnight 600 =0.5, measured in a 1: adding LB liquid medium containing kanamycin to a final concentration of 50. Mu.g/ml at a ratio of 100, culturing at 30 ℃ in a constant temperature shaker at 200rpm, and measuring OD every 2h 600 H111 (pBBR 1) was used as a control. Growth curve results as shown in fig. 4A, the expression of aigC in H111 did not affect the growth of H111.
Detection of intracellular AHL production in H111: seed fluid was cultured overnight to OD 600 =0.5, measured in a ratio of 1: adding the seed solution into an LB liquid culture medium containing 50 mu g/ml kanamycin at a final concentration of 100 proportion, culturing for 17h in a constant-temperature shaking table at 30 ℃ and 200rpm, taking an equal-volume ethyl acetate extract bacterial solution, evaporating an ethyl acetate extract organic phase by spinning, adding a report strain NT1, culturing for 8h, crushing the thalli of the culture solution, and detecting the activity of beta-galactosidase generated by the AHL induced NT1 in the supernatant after cell crushing. As can be seen in FIG. 4B, the expression of aigC in H111 significantly reduced the production of C8-HSL in H111.
Example 5 Effect of AigC expression on AHL mediated biological phenotype of pathogenic bacteria H111
Detection of motility: a semi-solid medium (0.8% tryptone,0.5% glucose,0.3% agarose) was prepared for measuring the motility of H111. The recombinant bacteria H111 (aigC) activated on the LB plate and the control H111 (pBBR 1) are dipped by toothpicks in the center of a motility culture medium plate, and after standing culture is carried out for 17H in a 30 ℃ constant temperature incubator, the motility diameter is observed and counted. The results are shown in fig. 4C, where aigC expression significantly attenuated H111 motility.
And (3) detecting a biological membrane: the overnight-cultured seed solution was adjusted to OD 600 =0.5, measured in a ratio of 1:100 portions were added to 100. Mu.l of LB liquid medium containing 50. Mu.g/ml kanamycin in a 96-well plate, and cultured on a 200rpm constant temperature shaker at 30 ℃ for 9 hours for biofilm measurement, 8 replicates of each treatment. First, OD was measured 600 Then carefully using the pipetteSucking away bacteria liquid, discarding, carefully cleaning each well with sterile water for 3 times, adding 150 μ l of 0.1% crystal violet, standing and dyeing at room temperature for 15min, cleaning each well with sterile water for 3 times, air drying, adding 300 μ l of 95% ethanol, standing for 10min, measuring absorbance at 595nm, and calculating OD 595 /OD 600 Biofilm formation by the strain was quantified. As a result, as shown in fig. 4D, the expression of aigC significantly attenuated the biofilm formation of H111.
And (3) detecting the protease activity: the overnight-cultured seed solution was adjusted to OD 600 20 μ l of the suspension was added to each of the wells punched out by a puncher on an LB +1% mounted mill plate, and 3 replicates were set for each well. The plate was subjected to static culture in an incubator at 30 ℃ for 17 hours, and the size of a transparent circle generated around the well by the strain was measured to quantitatively detect the protease activity. Results as shown in fig. 4E, expression of aigC significantly attenuated protease production of H111.
Example 6 Effect of AigC expression on AHL mediated pathogenicity of pathogen H111
Culturing the recombinant strain seed liquid overnight, and resuspending the strain liquid to OD by using PBS buffer solution 600 =1.0. Equally dividing fresh onion into four parts, peeling onion sections to serve as inoculation tissues, slightly stabbing wounds in the center of the inner sides of the onion sections by using a sterile small gun head, adding 20 mu l of bacterial suspension to the wounds, repeating the steps in each treatment setting, performing moisture-preserving culture at 30 ℃ for 3 days, and observing and counting the sizes of the scabs. The results are shown in fig. 5, where aigC expression significantly attenuated H111 pathogenicity.
Example 7 Effect of AigC expression on AHL mediated growth of the pathogenic bacterium Pseudomonas aeruginosa PAO1 and intracellular AHLs production
Growth condition detection of PAO 1: culturing the successfully constructed recombinant bacterium PAO1 (aigC) seed liquid to OD overnight 600 =0.5, measured in a 1: adding 100 proportion of seed solution into LB liquid culture medium containing final concentration of 50 μ g/ml gentamicin, culturing at 37 deg.C and 200rpm constant temperature shaking table, measuring OD once every 2h 600 PAO1 (pBBR 1) was used as a control. Growth curve results as shown in fig. 6A, the expression of aigC in PAO1 did not affect the growth of PAO1.
Detection of intracellular AHLs produced in PAO 1: for treatingSeed fluid was cultured overnight to OD 600 =0.5, measured in a 1: adding the seed solution into LB liquid culture medium containing gentamicin with final concentration of 50 mug/ml in proportion of 100, culturing for 17h in a constant temperature shaking table at 37 ℃ and 200rpm, and taking equal volume of ethyl acetate to extract bacterial liquid. PAO1 can produce both C4-HSL and 3-O-C12-HSL AHLs.
Detection of C4-HSL production: after ethyl acetate extracts were evaporated to dryness, reporter strain CV026 was added and cultured overnight. And (3) centrifuging to obtain thalli, taking lysate to resuspend the thalli and lyse cells, taking 200 mu l of lysed supernatant, and detecting the light absorption value of violacein induced by AHL in CV026 at 545nm absorption wavelength.
Detection of 3-O-C12-HSL production: and (3) after ethyl acetate extraction liquid is evaporated by rotary evaporation, adding a report strain NT1, carrying out overnight culture, then crushing thalli, and detecting the activity of beta-galactosidase generated by the NT1 induced by AHL in a crushed supernatant. As can be seen in FIGS. 6B and 6C, the expression of aigC in PAO1 significantly reduced the production of C4-HSL and 3-O-C12-HSL in PAO1.
Example 8 Effect of AigC expression on the biological phenotype of the AHL-mediated pathogenic bacterium, PAO1
Detection of cluster motility: a semi-solid medium (1% tryptone,0.5% NaCl,0.35% agarose) for detecting the motility of PAO1 clusters was prepared, the recombinant bacteria PAO1 (aigC) activated on LB plate and the control PAO1 (pBBR 1) were dipped with toothpicks, spotted on the center of the plate of the motility medium, and after static culture in a 37 ℃ incubator for 17 hours, the diameter of the motility was observed and counted. The results are shown in fig. 6D, where aigC expression significantly attenuated PAO1 motility.
And (3) detecting the protease activity: seed culture to OD overnight 600 20 μ l of the suspension was added to each of the wells punched out by a puncher on an LB +1% mounted mill plate, and 3 replicates were set for each well. The plate was subjected to static culture in an incubator at 37 ℃ for 17 hours, and the size of a transparent circle generated around the well by the strain was measured to quantify the protease activity. Results as shown in fig. 6E, expression of aigC significantly impaired the protease production of PAO1.
Example 9 Effect of the expression of aigC on the pathogenicity of the AHL-mediated pathogenic bacterium, PAO1
Overnight culture of recombinant strain seedsResuspending the bacterial suspension to OD with PBS buffer 600 =1.0. Taking 2cm × 4cm fresh lettuce stalk part and 5cm × 4cm fresh Chinese cabbage stalk part, slightly pricking wound at the center of the surfaces of the lettuce stalk and the Chinese cabbage stalk by using a sterile small gun head, taking 20 mul of bacterial suspension to be added on the wound, repeating three times for each treatment, carrying out moisture-preserving culture at 30 ℃ for 3 days, and observing and counting the size of lesion spots. The results are shown in FIG. 7, where aigC expression significantly attenuated the pathogenicity of PAO1.
Sequence listing
<110> southern China university of agriculture
<120> N-acyl homoserine lactone acyltransferase encoding gene aigC and application thereof
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accgaccgca tgttccgtag cctgcgcatc cgcgaccacg cggcggagta tgtgaagacg 360
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ttccgtaccg aaccggtgct gacctacgtg cgcgaccaac tgggcgcgga ttacctgaaa 600
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gactggcagg acatgagcgc catcgcccag ctcagccacg cggccctgga gaaggccggc 720
ctgccgcagt tcgaaggcag caacgcctgg gccgtgtccg gcagccgcac caagagcggc 780
aagccgctgc tggcgggtga cccacacatc cgcttcgccg tgccggcagt gtggtacgag 840
atgcaggcca gcgcgccggg gttcgagctg tacggccact accaggcgct caacccgttc 900
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aagatcaccc tgcgcagctc gccccacggt ccgctggtca atgacgcgct gggcactgcc 1140
gcgggcaaga caccggtggc catgtggtgg gccttcctgg aaacccagaa cccgatcctc 1200
gatgccttct acgagctgaa ccgcgccgat acccttgcca aggcccgcac ggcggcttcg 1260
aagatccagt cgccgggcct caacgtggta tgggccaacg cccgcggcga catcggctgg 1320
tgggcatcgg cgcaattgcc ggtacgcccg gacggggtca acccgaactt cctgctcgac 1380
ggcgccagcg gccaggccga caagaccggc ttctacccct tcagcgagaa cccgcaggaa 1440
gaaaacccgg cgcgcggcta catcgtctcg gccaacttcc agccggtacc ggccaacggt 1500
cgcccggtgc cgggctacta caacctgccc gaccgcggtc agcaactgaa caagcgcctg 1560
tccgacgatt cggtgaaatg ggacctgcag aacagccagg cgctgcaact ggacaccgcc 1620
accggctatg gcccgcgctt cctcaagccg ctgctgccga tcctgcgtga agccgcggca 1680
accgacgaag agaaggcgct ggtggagagc ctggccaact ggcagggcga ccatccgctg 1740
gactccgtga ccgccacgct gttcaaccag ctcctctacc aggtggcaga cggcgccatg 1800
cgcgacgaga tgggcgacgc cttcttcgac aacctgctgt ccacccgcgt gctcgacgtg 1860
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cacggccacc cgctgggcca gcagaagccg ctggatcgcc tcttcaatgt cggcccattc 2100
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caggccgaag cctacatcga aggccagtac ctgccgcagc actacgatga aaacgaggtg 2340
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Arg Ala Ile Gly Tyr Val His Ala Gln Asp Arg Leu Phe Gln Met Glu
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His Ala Ala Glu Tyr Val Lys Thr Gln Asp Lys Asn Ser Pro Ala Trp
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Asn Gly Val Leu Thr Pro Ser Pro Leu Ala Ala Ala Asp Trp Gln Asp
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Thr Lys Ser Gly Lys Pro Leu Leu Ala Gly Asp Pro His Ile Arg Phe
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Glu Leu Tyr Gly His Tyr Gln Ala Leu Asn Pro Phe Ala Ser Leu Gly
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His Asn Leu Gln Phe Gly Trp Ser Leu Thr Met Phe Gln Asn Asp Asp
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Val Asp Leu Val Ala Glu Lys Val Asn Pro Asp Asn Pro Asn Gln Val
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Claims (8)
1. An N-acyl homoserine lactone acyltransferase encoding gene aigC, which is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.
2. A recombinant vector comprising the gene encoding N-acyl homoserine lactone acyltransferase of claim 1.
3. A recombinant bacterium comprising the recombinant vector of claim 2.
4. An N-acyl homoserine lactone acyltransferase characterized in that its amino acid sequence is as shown in SEQ ID NO:2, respectively.
5. The recombinant bacterium of claim 3, or the use of the N-acylhomoserine lactone acyltransferase of claim 4 for degrading AHLs signal molecule, wherein the AHLs signal molecule is C4-HSL, C6-SHL, 3-O-C6-HSL, C8-HSL, 3-OH-C8-HSL, C10-HSL, 3-OH-C10-HSL, C12-HSL, 3-O-C12-HSL, 3-OH-C14-HSL.
6. Use of the gene encoding an N-acyl homoserine lactone acyltransferase of claim 1, or the recombinant vector of claim 2, or the recombinant bacterium of claim 3, or the N-acyl homoserine lactone acyltransferase of claim 4 for the control of AHLs-dependent pathogens such as burkholderia cepacia and pseudomonas aeruginosa.
7. The use according to claim 6, wherein when the gene encoding N-acyl homoserine lactone acyltransferase of claim 1 is used, the gene encoding N-acyl homoserine lactone acyltransferase is expressed in AHLs-dependent pathogenic bacteria, and recombinant pathogenic bacteria are selected for successful expression of N-acyl homoserine lactone acyltransferase; when the recombinant vector of claim 2 is used, the recombinant vector is introduced into AHLs-dependent pathogenic bacteria, and recombinant pathogenic bacteria that successfully express N-acylhomoserine lactone acyltransferase are selected; when the recombinant bacterium according to claim 3 is used, a recombinant pathogen is selected which successfully expresses N-acylhomoserine lactone acylase.
8. Use according to claim 7, wherein the recombinant pathogen that successfully expresses the N-acyl homoserine lactone acyltransferase:
(1) The yield of AHLs is reduced; and/or the presence of a gas in the atmosphere,
(2) The motility is reduced; and/or the presence of a gas in the gas,
(3) The formation of a biological film is reduced; and/or the presence of a gas in the gas,
(4) The production amount of protease is reduced; and/or the presence of a gas in the gas,
(5) The pathogenic force is weakened.
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