海洋黄杆菌科细菌中新型海洋N-酰基高丝氨酸内酯酶的多样性及其生理学功能

Quorum sensing (QS) is a population-density dependent signal pathway, and ubiquitous in microorganisms. Quorum quenching (QQ) is defined as the process which interferes with the QS signaling pathways. The applicant of this project found a novel marine-derived QQ enzyme MomL which can effectively degrade QS signaling molecules—N-acyl homoserine lactones (AHLs). MomL homologues are prevalent in marine Flavobacteriaceae bacteria as well as in many unculturable marine bacteria. Although it has been proposed that the production of QQ enzymes is one of the adaptive strategies utilized by bacteria to compete against QS, experimental evidences supporting this hypothesis are still very limited. Furthermore, numerous studies have demonstrated that QS regulates the expression of various genes and plays an important role in the marine ecological system, however, the physiological functions of QQ enzymes have not been extensively studies. In this project, the QQ activities of MomL homologues from different marine Flavobacteriaceae bacteria will be determined by A136 liquid X-gal assay, and their distribution pattern in the ecosystem will be analyzed. The activation of momL expression in representative Flavobacteriaceae bacteria will be checked by quantitative RT-PCR in various environmental conditions as well as in the presence or the absence of AHLs. The role of MomL homologues in terms of biofilm formation, resistance to marine relevant stressors and competitive advantages in the mixed-culture system of representative Flavobacteriaceae bacteria with QS strains will be investigated using the momL gene knockout strains combined with complementation studies. The aim of the above studies is to discover the physiological functions of novel marine-derived AHL lactonases in marine Flavobacteriaceae bacteria, thus providing important insights into a better understanding of the cooperation and competition among marine bacteria.

密度感应（QS）是微生物依赖种群数量的信号传导通路。密度感应淬灭（QQ）指阻断QS通路的过程。虽然QQ酶被认为是微生物抵抗QS的一种竞争机制，但这一假设尚缺乏确切的证据。QS在海洋生态系统中具有重要的作用，但关于QQ酶生理功能的研究还很少。项目申请人前期研究发现一种新型海洋QQ酶—MomL特异性水解QS信号分子N-酰基高丝氨酸内酯（AHL）。MomL普遍存在于海洋黄杆菌科细菌中，但其同源蛋白具有不同的QQ活性。本项目拟以海洋黄杆菌科细菌MomL为研究对象，采用微生物学、分子生物学和生物信息学的方法和技术，检测不同MomL的活性，分析其物种分布特点；检测不同海洋环境胁迫条件下momL基因的表达以及是否受AHL的激活；通过基因敲除技术研究MomL对生物被膜、海洋环境抗逆性以及在混合群体中竞争优势的影响。通过上述研究揭示新型海洋AHL内酯酶的生理学功能，进而加深对海洋微生物协作和竞争关系的认识。

密度感应（QS）作为细菌之间沟通交流和调控多种生物学过程的重要通路已经得到充分的研究和认识。密度感应淬灭（QQ）指阻断QS通路的过程。但是目前对于QQ酶的功能和生态学作用研究非常有限。本项目围绕海洋密度感应淬灭酶的多样性和功能展开研究：（1）鉴定到多个海洋QQ酶，发现其中N-酰基高丝氨酸内酯酶MomL具有活性差异的两种类型并分布于特定的海洋黄杆菌中，MomL同源蛋白广泛存在于变形菌门和拟杆菌门中；（2）明确代表性黄杆菌科细菌中MomL编码基因组成型表达规律，发现外源信号分子能够调控海洋黄杆菌转运蛋白基因的表达；（3）揭示MomL的定位于外膜和分泌特性，能大大降低玫瑰杆菌的生物膜形成能力，阐明MomL能够赋予海洋黄杆菌获得竞争优势的功能；（4）进一步发现密度感应淬灭菌具有其他功能，包括分泌抑菌物质和参与宿主的氮循环等。这些结果扩展了细菌之间竞争机制的多样性，为菌群的调控提供了新思路。本项目开展过程中发表了3篇SCI论文，1篇中文核心论文。项目执行期内负责人汤开浩晋升为副研究员，协助培养硕士研究生2名。