Isolation Of Quorum-quenching Bacteria And Cloning Genes From Bacterium WX14

Quorum sensing?QS?is a kind of signal molecule transfer mechanism between bacterial cells,which can coordinate related gene expression induced by bacterial autoinducer molecule itself?autoinducer,AI?concentrations such as N-cylhooerine lactones?AHLs?in order to accommodate the changing natural environment.Many plant bacterial pathogens regulate the expression of virulence genesthrough quorum sensing system,so quorum sensing system is a potential target for plant disease control.The interference and destruction of QS regulation mechanism was called quorum quenching?QQ?.In this study,we used cushin ring and gamma-butyrolactone plateto isolate AHL-degrading bacterial strains from different soil and identify these bacteria according to analysis of 16S rDNA gene sequences.The results revealed 13 candidate strains were obtained from 566 soil isolates based on cushin ring method.The analysis of 16S rDNA gene revealed that 6 candidate strains are Pseudomonas spp.,5 candidate strains are Acinetobacter spp.,1 candidate strain is Proteus sp and Rheinheimera esophila.14 candidate strains were obtained from 828 soil 1 candidate strain is isolatesbased on gamma-butyrolactone plate.The analysis of 16S DNA gene revealed 6 candidate strains are Pseudomonas spp.,6 candidate strains are Arthrobacterium spp.And 2 candidate strains are Bacillus spp.,The results showed that less number of bacteria were isolated using cushin ring,but it is easier to get some new strains.Strain WX14 was isolated from Wuxi soil sample nd was designated Pseudomonas fluoresens according to the 16S rDNA gene sequence identity.This strain was chosen for further study due to both the supernatant and WX14 cells responsible for AHL-degrading activity.The genes,termed hacA and h.acB,responsible for AHL-degrading werecloned,and the lengths of hacA and hacB were 2286 bp and 2370 bp,repectively.The analysis of DNAMAN revealed the hacA gene encodes a protein of 761 amino acids with a predicted molecular mass of 83.7 kDa.hacB gene encodes a protein of 781 amino acids with a predicted molecular mass of 86.8 kDa.The BLAST search revealed that HacA from strain WX14 showed 62.74%and 54.32%sequence identity with Psyr₁971 from Pseudomonas syringae and PvdQ from Pseudomonas aeruginosa,respectively.HacB from strain WX14 showed 71.13%and 68.25%sequence identity with Psyr₄858 from Pseudomonas syringae and PA0305 from Pseudomonas aeruginosa,respectively.Moreover,analysis of sequence similarity showed that HacA and HacB may undergo post-translational processing resulting in ? and ?-subunits.And catalytic serine is the first amino acid of the ?-subunit,suggesting that both HacA and HacB are members of the N-terminal nucleophile hydrolase superfamily.