The Identification Of AiiK From KURTHIA HUAKUII And Its Application On Quenching Pathogens

Quorum sensing（QS）is a common and important cell-to-cell communication mechanism in bacteria,coordinates the expression of specialized structural gene sets triggered by specific receptors that can sense signal molecules reaching a threshold concentration when the bacteria at high cell densities.N-Acyl homoserine lactones（AHLs）acted as the key QS signal molecules in gram-negative bacteria,which coordinates gene expression and then activates various processes,such as swimming ability,virulence factors production,biofilm formation,and so on in many pathogens.Quorum quenching（QQ）,the interference or disruption of QS signaling,encompasses the main mechanisms by degrading or modifying the signal molecules by enzymes（mainly AHL lactonase）.Compared with bactericidal or bacteriostatic strategies,QQ does not generate selective pressure on the survival of bacteria,nor does it directly affect the growth of bacteria,which does not induce the development of resistance on bacteria.Therefore,QQ achieved by AHL lactonase represents a promising strategy to prevent and control these pathogens.In this study,an AHL lactonase gene（aii K）was cloned from K.huakuii LAM0618^T,expressed by E.coli BL21（DE3）,and the AiiK was purified with Ni-NTA for the further study.Considering that purification was complicated and purified AiiK had a poor resistance to environment,and the cost for purification was high.Thus,we constructed plasmids p ELX1-aii K,p ELSH-aii K,and p ELCW-aii K based on plasmids p ELX1 and p ELSH,and transformations were carried out.The results were as follows:1.AiiK shares 26%and 42%identity with AHL lactonases Aii A and Aii B,respectively.Multiple sequence alignment result and acidified hydrolysis assay suggested that AiiK is an AHL lactonase.In short,AiiK is a new AHL lactonase.AiiK can degrade C₆-HSL,3-Oxo-C₆-HSL,C₁₀-HSL,and C₁₄-HSL.The total hydrolytic efficiency of AiiK for C₁₀-HSL is 3.9 s^-1?m M^-1,and 4μg/m L AiiK can degrade 97%of C₁₀-HSL（48.5μM）within 30 min at 25°C.The optimal temperature and optimal p H of AiiK for degrading C₁₀-HSL are 45°C and 7.5,respectively.The activity of AiiK was enhanced by 1m M Co²⁺,Mn²⁺,Mg²⁺,and Ni²⁺,and it maintained over 100%of activity after AiiK was incubated with proteases.2.When the application amount of purified AiiK achieved 10μg/m L,the inhibition ratios of biofilm formation,extracellular proteolytic activity and pyocyanin production on Pseudomonas aeruginosa PAO1 were 40.78%,47.30%,and 71.30%,and the AiiK did not kill the pathogen at the same time,which indicates the potential application of AiiK as a biocontrol agent.3.The crude enzyme extracts from recombinant strains p ELX1-aii K/L.casei MCJΔ1,p ELSH-aii K/L.casei MCJΔ1,and p ELCW-aii K/L.casei MCJΔ1（LCAiiK）all exhibited AHL-degrading ability.The crude enzyme extracts from LCAiiK exhibited the second highest activity and the easiest preparation method among the three.The plasmid stability of p ELCW-aii K was over 50%after continuous passage culture in MRS for 15 generations under the nonselective condition.4.The Lc AiiK exhibited obvious QQ ability on Aeromonas hydrophila AH-1 and AH-4 by attenuating their swimming motility,haemolytic activity,extracellular proteolytic activity and biofilm formation instead of killing them,which indicates the potential application of Lc AiiK as a biocontrol agent.In our study,an autoinducer inactivation gene is the firstly identified from the genus Kurthia,and AiiK was identified as a new AHL lactonase.We firstly expressed the AHL lactonase by BAC（L.casei MCJΔ1）.The purified AiiK and the Lc AiiK exhibited obvious QQ ability on P.aeruginosa PAO1 and A.hydrophila,respectively,which representing a biocontrol agent or an anti-pathogenic drug of AiiK to control AHL-mediated QS pathogens.