Gene Cloning,Expression And Function Of Quorum Quenching Gene YtnP From Bacillus Licheniformis T-1

The main pathogenic bacteria of aquatic animals are gram-negative bacteria,N-acyl-homoserine lactones(AHLs),which are the key signal molecules in pathogenic response system of Gram-negative bacteria.When AHLs accumulate to a certain concentration in the bacterial flora,they can activate the expression of pathogenic genes and produce pathogenicity.Herien,reducing the concentration of pathogenic bacteria AHLs will be the key to prevent bacterial diseases.N-acyl homoserine lactonase is a class of metalloproteolytic enzymes that specifically degrade AHLs and is widely present in many microorganisms.In recent years,N-acyl homoserine lactonase has become a research hotspot for quorum quenching strategies.The aim of this study was to clone and express the ytnP gene of Bacillus licheniformis T-1 and to study its function.To explore the new stratege from the point of view of quorum sensing inhibition and the path provides technical support for the healthy and sustainable development of the aquaculture industry.1.Full-length cloning and biological analysis of ytnP geneIn this study,the full-length sequence of ytnP gene was obtained from the whole genome sequencing of Bacillus licheniformis T-1.The target gene ytnP was amplified by PCR and ligated into the cloning vector pGEM-T Easy Vector.The positive clones were screened and further identified by sequencing.The full-length 900 bp sequence of ytnP was obtained.According to the bioinformatic analysis of ytnP.Coding region of ytnP was770 bp and encoded 256 amino acids with a molecular weight of 29 kD and an isoelectric point of 5.62.It contained S phosphorylation sites.The lactamase family conserved domain HH-DH--,--GH-H—was found in ytnP gene;According to the hydrophobicity analysis and transmembrane signal peptide prediction,YtnP is an acidic hydrophilic protein,not an extracellular secretory protein;And according to the three-dimensional structure prediction,YtnP protein contains?-helix,?-sheet and irregular coil structure accounting for 37.11%,22.27%and 40.62%,respectively,the predicted three-dimensional structure of YtnP and reported N-acyl homoserine lactone The enzyme homology is as high as 98%and contains active centers and active sites of the same family.2.Prokaryotic expression and purification of ytnPIn this study,the recombinant plasmid pEASY-Blunt E1-ytnP was successfully constructed and transferred into E.coli to obtain the BL21(DE3)-pEASY-Blunt E1-ytnP bacterium;then IPTG(isopropylthiogalactoside,Isopropyl?was obtained.-D-Thiogalactoside)induces engineering bacteria and successfully expresses ytnP.And optimized expression conditions,obtained ytnP gene in 0.2 mM IPTG,induced at 37°C for 8 h,the concentration of YtnP protein was highest.Purification of YtnP protein was used by chelation of nickel ions and recombinant histidine-tagged tags to obtain an optimal elution condition with an imidazole concentration of 150 mM,filler volume 3mL and controlling the flow rate to 30 mL/h.Concentration of YtnP was 2.17?g/?L.Through the combination of dialysis and glutathione redox system,YtnP was successfully refolded and the protein concentration after refolding was 0.81?g/?L and the refolding rate was 36.8%.Chromobacterium Violaceum ATCC 12472 and C.Violaceum CV026were used as reporter strains,the quorum-induced quenching effect of the constructed engineering bacteria and renatured proteins was successfully verified by the"H"streaking method and the Oxford cup method,respectively.The degradation of C6-HSL signaling molecules by active YtnP protein was confirmed and the YtnP activity was determined to be 1.97 U/mL.3.Effect of YtnP on biofilm formation of Aeromonas hydrophilaStudies have shown that the formation of Aeromonas hydrophila biofilm is regulated by QS.In this experimental study,by measuring the effect of different inoculum size on the biofilm formation of A.hydrophila,the optimal concentration of A.hydrophila for biofilm formation was obtained when the inoculum volume was 10~7 CFU/mL;YtnP enzyme was added to the Aeromonas co-culture showed that when the concentration of YtnP was 80 ng/?L,the inhibition rate of the biofilm was 68%and the concentration of YtnP was 20 ng/?L,compared with the control group.The inhibition rate of its biofilm was still higher than 50%(p<0.05).In order to study the effect of the combination of YtnP and common aquatic antibiotics on the minimal inhibitory concentration(MIC)of A.hydrophila,under the synergistic effect of YtnP and thiamphenicol and doxycycline hydrochloride,the MIC values of A.hydrophila decreased from 4?g/mL and 0.5?g/mL to 1?g/mL and 0.125?g/mL respectively and the sensitivity is significantly increased.4.Effect of YtnP on virulence factor expression of A.hydrophilaThe QS system with AHLs as signal molecules exists in A.hydrophila and regulates the expression of virulence factors of A.hydrophila.In this experiment,the virulence gene expression of A.hydrophila was quantified after adding YtnP by means of fluorescence quantitative methods.The results showed that the gene expression of the enterotoxin gene ast was significantly down-regulated compared to the control group within 20-36 h,and was down-regulated by 20 fold,15 fold,and 7 fold(p<0.05)respectively.hem gene expression the amount of down-regulation at 8-12 h and 30-36 h was very significant,both down 5-20 times(p<0.01).The expression level of aerA enterotoxin gene was very low at 4-8 h,20-24 h and 36-60 h.Significant,the highest down 80-fold(p<0.01).The expression of extracellular protease gene ep was significantly down-regulated at 12-20 h and 24-48 h(p<0.01).Using blood plates to detect the hemolytic activity of the broth after addition of YtnP and the broth without YtnP,it was found that the addition of YtnP made the A.hydrophila.non-hemolytic within 4-12 h.According to the results of the protective experiment on Carassius auratus infected with A.hydrophila,the recombinant enzyme YtnP is safe and non-toxic.After adding YtnP,the cumulative mortality rate of the carp decreased from 78%to 27%within 96 h(p<0.05).This study successfully cloned and expressed the quorum-sensing suppressor gene ytnP from B.licheniformis for the first time.The active protein was first studied in the biological control of A.hydrophila.YtnP can significantly reduce the biofilm formation of A.hydrophila.Inhibiting the expression of its virulence factors,thereby reducing the host's infection and toxicity of pathogenic A.hydrophila.The study also laid a theoretical foundation for its application to the biological control of pathogenic A.hydrophila.