深海细菌及其分泌的M23家族蛋白酶在深海肽聚糖再循环利用中的作用及机制研究

Peptidoglycan is important component of organic matter in the deep sea. In this project, with the deep-sea bacterium Pseudoalteromonas sp. CF6-2 and its extracellular M23 family protease, pseudoalterin, as study objects, the ability of Pseudoalteromonas sp. CF6-2 to lyse and utilize cells of variously typical marine bacterial strains as carbon and energy sources for growth will be firstly examined, and the leading role of pseudoalterin in the lysis effect of Pseudoalteromonas sp. CF6-2 to marine bacterial strains will be demonstrated through the gene knockout and complementation experiments; then the crystal structure of pseudoalterin will be solved, and based on the structural and mutational analysis, the molecular mechanism of pseudoalterin to absorb peptidoglycan will be illustrated; finally, based on the above analysis combined with the observation using atomic force microscopy (AFM) and the analysis of the cleavage sites on the peptide chain of peptidoglycan by pseudoalterin, the degradation mechanism of pseudoalterin to peptidoglycan will be elucidated. Results from this project will reveal the role and mechanism of deep-sea bacteria and their extracellular M23 family proteases in the releasing and recycling of deep-sea peptidoglycan, providing experimental and theoretical basis for comprehensively elaborating the degradation mechanism of deep-sea organic matter.

肽聚糖是深海有机质的重要成分。本项目以深海细菌Pseudoalteromonas sp. CF6-2及其分泌的新型蛋白酶pseudoalterin为研究对象，将首先检测Pseudoalteromonas sp. CF6-2对典型海洋细菌的裂解及利用能力，并基于基因敲除及互补实验阐明蛋白酶pseudoalterin在Pseudoalteromonas sp. CF6-2裂解海洋细菌中的主导作用；进一步利用晶体学方法解析pseudoalterin的晶体结构，并基于晶体结构分析及突变验证阐明pseudoalterin吸附肽聚糖的分子机制；最终结合原子力显微镜（AFM）观察及酶切位点分析结果，阐明pseudoalterin对肽聚糖的降解机制。本项目将揭示海洋细菌及其分泌的M23家族蛋白酶在深海肽聚糖释放，再循环利用中的作用及作用机制，为全面阐述深海有机质降解机制提供实验及理论依据。

肽聚糖是深海有机质的重要成分。本项目以深海细菌Pseudoalteromonas sp. CF6-2及其分泌的新型蛋白酶pseudoalterin为研究对象，首先利用培养技术检测了海洋细菌Pseudoalteromonas sp. CF6-2对典型海洋细菌的裂解利用能力，并基于基因敲除及回补实验证实了Pseudoalteromonas sp. CF6-2通过分泌蛋白酶pseudoalterin裂解利用肽聚糖及海洋革兰氏阳性细菌，同时阐明了pseudoalterin诱导，成熟和分泌机制；进而通过解析pseudoalterin的晶体结构并结合分子模拟及突变分析，阐明了pseudoalterin吸附及降解肽聚糖的分子机制，并最终提出了蛋白酶介导的海洋细菌间新型捕食-被捕食相互作用及营养物利用新机制模型，由此揭示了海洋细菌及其分泌的蛋白酶在海洋环境肽聚糖释放，再循环利用过程中的作用及生态意义。另外，本项目还研究了海洋细菌代谢D型氨基酸机制，海洋细菌胞外蛋白酶诱导效应，家族水平多样性和新型蛋白酶的特性。同时本项目还资助了利用海洋细菌蛋白酶制备胶原蛋白寡肽，海洋细菌鉴定，基因组分析及其新型有机质降解酶的特性等项研究。项目已资助发表SCI论文12篇。