申氏杆菌37-1中群体感应淬灭内酯酶AiiS的功能分析

许多植物病原细菌通过群体感应（quorum sensing，QS）系统调控相关毒性因子的表达，而群体感应淬灭（quorum quenching，QQ）是通过干扰QS系统，达到防治植物细菌性病害的重要策略之一。本研究利用原位培养法分离得到2000多株不同菌株形态的植物根围细菌，结合QS系统信号分子检测平板筛选到7株具有QQ活性的候选细菌，其中菌株37-1可完全降解信号分子。16S rDNA序列分析表明，菌株37-1属于Shinella sp.。全基因组序列分析发现菌株37-1中存在一个可能的QQ降解酶编码基因aiiS（autoinducer inactivation gene from Shinella sp.）。系统发育分析表明AiiS属于α/β水解酶家族蛋白。液相色谱-串联质谱分析进一步表明AiiS可水解N-乙酰高丝氨酸内酯（N-acylhomoserine lactone，AHL）类QS信号分子中的内酯健，生成酰基高丝氨酸，因此AiiS属于AHL内酯酶。将aiiS基因导入胡萝卜软腐果胶杆菌Pectobacterium carotovorum subsp.carotovorum Z3-3中，可显著降低该菌AHL信号分子和果胶酸盐裂解酶的产生及其在白菜、马铃薯和胡萝卜上的致病性。以上结果表明菌株37-1中AiiS蛋白是一种AHL内酯酶；病原细菌中异源表达aiiS基因可有效干扰相关病原细菌中QS系统的调控功能，表明AiiS蛋白具备开发为潜在新型生防制剂的价值。

Characterization the Function of Quorum-quenching N-Acylhomoserine Lactonase AiiS from Shinella sp. 37-1

The expression of multiple virulence factors is regulated by quorum sensing (QS) in many phytopathogenic bacteria. Quorum quenching (QQ), which influences the function of QS by either disruption of QS signals or the process of signal production and perception, is a promising strategy for attenuating the pathogenicity of these bacteria. In this study, we isolated and screened rhizobacteria with QQ activity. One candidate 37-1 was obtained and identified as a Shinella sp. strain based on 16S rDNA gene analysis. Bioinformatics and genetic analyses indicated that aiiS gene (autoinducer inactivation gene from Shinella sp.) was responsible for the degradation of N-acylhomoserine lactone (AHL). High-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) analysis further demonstrated that AiiS was an AHL lactonase that hydrolyzed the ester bond of the homoserine lactone ring. In addition, the production of AHLs, the activity of pectate lyases, and the virulence on host plants were significantly attenuated when overexpression of aiiS gene in Pectobacterium carotovorum subsp. carotovorum Z3-3. These results suggested that AiiS was an AHL lactonase, and pathogenic bacteria harboring aiiS gene could interfere efficiently with quorum-sensing-regulated functions, suggesting that AiiS could be a potential biocontrol agent for developing quorum-quenching procedures.