Cloning, Expression And Function Analysis Of Quorum Sensing Genes HalI/R Of Hafnia Alvei

Hafnia alvei is a rod-shaped,Gram-negative and representative bacterium of the family Enterobacteriaceae.Due to its preference for psychrophilic,anaerobic and humid environments,it is often the Specific Spoilage Organisms?SSO?in low temperature storage foods.Studies have shown that its spoilage characteristics are regulated by the lux-type quorum sensing system mediated by N-acyl-homoserine lactones?AHLs?.Therefore,this paper studied the strain,Hafnia alvei Ha-01,which was isolated and purified from the Scophthalmus maximus.The lux-type quorum sensing regulatory genes halI/R in its strains was mainly studied by genetic engineering and bioinformatics.The functional properties of the HalI/R proteins encoded by these two genes and whether they could form a quorum sensing system to regulate biofilm formation in strain Ha-01 were investigated.The research aims to explore the mechanism of fish decay regulated by quorum sensing system,and provides theoretical basis for establishing new technology for aquatic product preservation based on targeted control of the quorum sensing system.1.According to the halI/R sequences registered in GenBank,the primers were designed to carry out PCR reaction to obtain the target genes.The cloning vectors pMD-halI and pMD-halR were constructed by TA cloning technology and introduced into the host E.coli JM109.Amplification,screening and extraction of positive clone plasmids using the sequencing universal primer M13 to obtain the complete sequence of the target gene.The results of BLAST alignment in the NCBI database showed that the sequence similarities between the fragmentes obtained by cloning and sequencing in Ha-01 and the registered quorum sensing regulatory genes halI/R were 100%and 99%,respectively,indicating that the quorum sensing genes halI and halR are indeed present in the Ha-01 strain.2.Based on the amino acid sequences encoded by halI/R genes,the basic properties of HalI/R protein were predicted.The HalI size was 25.06 KDa,the theoretical isoelectric point was 5.46,the HalR size was 27.97 KDa,and the theoretical isoelectric point was 6.98.Sexual analysis showed that both were hydrophilic proteins.No signal peptide and transmembrane region indicated that both were non-secreted proteins,which may be soluble in the cells.Functional region prediction showed that they respectively have the characteristic structure of autoinducer synthase and HTH characteristic structure of LuxR type protein.Secondary and stereo structure prediction results showed that they have obvious conformational differences,and the quality of the predicted three-dimensional models are good for subsequent molecular docking research.Protein interaction results showed that theose two proteins can constitute a full quorum sensing system and has the regulatory effects on bacterial biofilm formation,and the prediction results were in a high confidence interval.On the other hand,the codon usage bias of the halI/R genes were analyzed based on the nucleic acid sequences,and the results indicated that they are preferable to use the Escherichia coli expression system.3.Codon optimization of the target genes was completed to improve the theoretical expression efficiency.The recombinant expression plasmids pET22-halI and pET28-halR were successfully constructed by the In-fusion cloning technology,and transformed into host E.coli BL21?DE3?for expression.The induction condition was 1 mM IPTG for 4 h at 37°C.The results of SDS-PAGE and Western Blotting showed that both of HalI/R proteins could be highly expressed in E.coli BL21?DE3?,and some were intracellular soluble expression.4.The results of biosensor strain CV026 chromogenic assay and GC-MS analysis showed that HalI protein can indeed synthesize AHL-like signal molecules C₆-HSL,which functions as an autoinducer synthase.The in vitro binding experiments and molecular docking results indicated that the HalR protein mainly binds to C₆-HSL by hydrogen bondings,which functions as a receptor protein.The results of biofilms assay indicated that the HalI/R quorum sensing system has a significant regulatory effect on the biofilms formation in Hafnia alvei Ha-01.