利用气单胞菌作用模式系统研究水华微囊藻合成胞外多糖的信号调节机制

Microcystis aeruginosa is the most notorious bloom-forming cyanobacterium reported in fresh waters. Synthesis of extrocellular polysaccharide (EPS) in algal cells plays an important role in protecting them from adverse environmental factors and forming colonies as buoyant surface blooms. The annual cycle of M. aeruginosa reveals the caprice of algal blooming, which relates to the change of colony size and EPS synthesis. Mechanism for EPS synthesis in cyanobacterial cells remains unclear with respect to the signaling molecules termed second messengers and their signaling pathways. On the other hand, cyanobacterial polysaccharides have received considerable attention due to their potential applications. However, there are limited studies on their structure, physic-chemical properties and biosynthesis..The ubiquitous Microcystis rely on complex signal transduction systems to adapt to aquatic habitats. Yet the complexity of the signal web makes the attempt in vain during clarifying a signaling pathway for a special stimulus or a specific output. In the proposed research, a model system for elucidation of signaling pathway during EPS synthesis in M. aeruginosa was utilized, in which the cyanobacterium is treated with extracellular substances of an Aeromonas sp., while the alga synthesizes and releases EPS within a few hours. The research involves structure and physic-chemical properties of EPS released by M. aeruginosa in the model system. The presumed second messenger, i.e., bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP) will be tested and confirmed for EPS synthesis control in the model system. For this purpose, enzymes regulating the level of c-di-GMP in algal cells will be examined, including diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) or proteins harboring GGDEE and EAL/HD-GYP domains. Further, genes involving the c-di-GMP signaling pathway will be screened by genomic sequence analysis and transcriptome analysis, and functionally confirmed for correlating with EPS synthesis. Thereby, regulation of EPS synthesis dependent on c-di-GMP signalling will be elucidated. The proposed study will provide knowledge for harmful algal blooming control and application of cyanobacterial polysaccharides.

微囊藻胞外多糖的合成与其水华的发生机制有密切关系，目前国际上未见关于蓝藻细胞调节胞外多糖合成的第二信使及其信号途径的研究报道。另一方面，蓝藻多糖具有潜在的工业化应用前景，而关于微囊藻多糖的结构、理化特征以及生物合成的基础研究相对匮乏。本项目拟采用气单胞菌胞外物质处理铜绿微囊藻而引起后者合成胞外多糖这一模式系统，研究调节微囊藻胞外多糖合成的第二信使及其信号途径。研究内容包括：考察微囊藻胞外多糖的结构及理化特征；分析胞内双鸟苷酸环化酶与磷酸二酯酶蛋白活性及其对微囊藻胞外多糖合成的影响，确认环 状 双 鸟 苷 酸 （c-di-GMP）调节胞外多糖合成；同时通过基因组序列分析与转录组分析手段筛选c-di-GMP信号途径相关成分并确认其功能，由此归纳微囊藻合成胞外多糖的信号调节机制。本研究可为蓝藻水华的有效控制及蓝藻多糖的生产利用提供理论依据。

天然水体中微囊藻通过分泌胞外多糖包裹多数细胞而形成群体，与水华的发生机制有密切关系，也对有效除藻及水华控制有重要影响。在蓝细菌中胞外多糖合成的信号调控机制鲜有研究报道，而在蓝细菌中c-di-GMP的调控作用缺乏实验证据。本项目在气单胞菌胞外物质处理铜绿微囊藻的作用条件下，对铜绿微囊藻中多糖合成及c-di-GMP代谢开展研究。项目首先优化了气单胞菌胞外物质的制备条件，并对主要其活性作用成分做了分析，确定了其中氨基酸组分中L-赖氨酸的抑藻作用。另外针对铜绿微囊藻中编码双鸟苷酸环化酶（Diguanylate cyclases）中GGDEF结构域、磷酸二酯酶（Phosphodiesterases）的EAL结构域，以及c-di-GMP的重要受体PilZ结构域的基因设计兼并引物通过PCR筛选两株铜绿微囊藻，进行了基因组全序列测序分析。通过对测序结果进行基因注释及保守结构域分析，获得了其含GGDEE保守结构域的蛋白编码基因序列，及含EAL或HD-GYP保守结构域的蛋白编码基因序列。另外，以含PilZ结构域的多糖合成基因为重点，分析获得了菌株NaRea975及CHAOHU 1326中参与多糖合成的基因序列。在此基础上，提取铜绿微囊藻RNA，通过逆转录PCR确认了上述参与c-di-GMP代谢与多糖合成的基因的转录活性；并选择代表性的c-di-GMP代谢基因与含PilZ结构域的多糖合成基因，通过实时荧光定量PCR检测其在气单胞菌胞外物质活性组分（L-赖氨酸）作用后转录活性的变化，发现上述基因的转录活性均上调，暗示了 c-di-GMP可能参与了多糖合成调控。另外，对选择的含GGDEF/EAL结构域的双功能酶基因及含PilZ结构域的多糖合成酶基因进行了克隆，并在大肠杆菌BL21(DE3) 、Rosetta gamiTM2 (DE3) plysS中进行异源表达。本项目通过对铜绿微囊藻中c-di-GMP代谢酶及含c-di-GMP作用靶点的多糖合成酶开展研究，可为c-di-GMP参与蓝细菌多糖合成调控提供实验证据。