厚壳贻贝附着过程中天然化学诱因调控的分子机理研究

Mussels are typical members of fouling communities worldwide. Although chemical cues play a crucial role in larval settlement of many species of marine invertebrates, the molecular genetic basis of natural chemical cues regulating mussel settlement remains little known. In the present study, the effects of natural chemical cues derived from the Pseudoalteromonas biofilms on the larval settlement and metamorphosis of the mussel Mytilus coruscus will be investigated. In order to clarify whether all tested Pseudoalteromonas biofilms could impact the larval settlement and metamorphosis, different species of the genus Pseudoalteromonas were isolated and the biofilm-settlement inducing activity of these bacteria were tested. In addition, interaction between the bacterial density, phylogenetic relationship and biofilm-settlement inducing activity will be examined. Confocal laser scanning microscopy will be used to investigate the exopolysaccharides, lipids and proteins of the biofilms to find the key driver that regulate the larval settlement. To determine whether the single-domain PilZ protein PA4608 functions as a trans-acting adaptor protein by binding to the intended protein target of the bacterial messenger cyclic diguanylate monophosphate (c-di-GMP), bacterial two-hybrid screening using the BacterioMatch II Two-Hybrid system with PA4608 as the bait to probe for the prey protein will be conducted. Coimmunoprecipitation assays will be carried for the purpose of clarifying the chemotaxis methyltransferase CheR-PA4608 interaction and how c-di-GMP affects protein-protein interaction. In vitro methylation assays were conducted to investigate whether PA4608 and c-di-GMP affect the CheR-catalyzed methylation of the chemoreceptor. The variance of contents of the c-di-GMP, cellulose and alginate, and the expression of extracellular polymeric substance genes (e.g. BcsA and Alg44) will determined between the wild-type and ΔPA4608 mutant. To clarify the key driver of the extracellular polymeric substance genes on biofilm-settlement inducing activity, in-frame deletion mutants (ΔBcsA, ΔAlg44 and ΔCapD) were constructed to test the effects of biofilms formed by these mutants on larval settlement. The findings will provide the novel insight to clarify the mechanism of mussel settlement, and are also beneficial in the field of biofouling and antifouling researches and aquaculture industry, and subsequently develop more environmentally friendly antifouling technologies.

贻贝作为代表性海洋大型污损生物，其附着的分子机制尚不清楚。本课题以厚壳贻贝为研究对象，开展天然化学诱因对幼虫附着影响研究，查清假交替单胞菌属细菌生物被膜的膜厚、细菌密度、胞外产物与幼虫附着的相互关系,筛选高效诱导活性菌株；利用细菌双杂交技术验证结构域蛋白PilZ是否直接作用于趋化性甲基转移酶CheR，通过免疫共沉降技术确定PilZ和CheR蛋白互作；验证第二信使c-di-GMP存在时，PA4608能否抑制CheR对甲基化受体趋化蛋白的甲基化作用；分析敲除PA4608基因前后c-di-GMP水平、胞外产物纤维素、藻多糖等量的变化以及BcsA、Alg44等胞外产物基因表达的变化；敲除BcsA、Alg44、CapD，确定调控附着的关键基因。本项目的顺利实施有助于解析贻贝附着分子机制，为后续调控海洋无脊椎动物附着提供新的思路，最终为实现研发控制海洋大型污损生物附着的生态友好型防污新技术奠定基础。

针对天然化学诱因诱导厚壳贻贝附着机制尚不清楚的问题，本项目开展了假交替单胞菌微生物被膜形成、胞外产物产生和微生物被膜诱导厚壳贻贝附着变态三者之间关系及其分子作用机制的研究。研究发现，不同生境分离和来源的假交替单胞菌生物被膜对厚壳贻贝幼虫附着变态均有诱导作用，胞外产物在幼虫附着变态发育过程中发挥重要介导作用。筛选出高效诱导活性的Pseudoalteromonas marina，构建了集全基因组测序、基因编辑，蛋白相互作用，生物成像，动物学实验、分子生物学分析的研究平台。我们研究发现，趋化性基因调控胞内c-di-GMP水平、细菌运动性、微生物被膜形成和膜胞外产物产生，同时趋化性基因缺失改变生物被膜胞外产物产生从而影响幼虫附着变态。c-di-GMP和结构域蛋白PilZ特异性结合，pilZ缺失不影响c-di-GMP的产生，但影响与趋化性紧密相关的细菌运动性、生物被膜形成和幼虫附着变态，初步验证了PilZ、c-di-GMP和趋化性通路三者之间的相互作用关系。c-di-GMP缺失导致胞内c-di-GMP水平降低，被膜胞外产物变化，并显著降低了幼虫附着变态诱导活性。通过探索细菌胞外产物合成相关基因作用与幼虫附着变态的关系，我们发现多糖合成基因、藻酸盐合成基因、鞭毛合成蛋白基因等可能通过c-di-GMP调节生物被膜形成及胞外产物产生，这些基因的缺失导致幼虫附着变态率的降低。鞭毛蛋白、脂肪酸、脂多糖和藻酸盐能有效促进幼虫变态发育，且纤维素、脂肪酸、藻酸盐和脂多糖能通过调控被膜胞外产物的分泌进而影响厚壳贻贝幼虫的附着变态。本项目初步解析了天然化学诱因介导幼虫附着变态的分子作用机制，为后续研究海洋无脊椎动物附着变态机制和生态种群演变奠定基础，最终为研发海洋生物防污新技术提供技术支持。