嗜冷古菌R15“进化”为中温菌的糖蛋白组学研究

Abstract:Methanolobus psychrophilus R15 was isolated from a cold wetland, Zoige wetland at Tibetan Plateau, which exhibits the optimal growth at 18°C and not grows above 25℃.After subculturing at 18°C in trimethylamine for several years, strain R15 becomes mesophilic and grows optimally at 30°C.However,it just weakly grows in methanol at 30°C.Our previously SPN analysis between the psychrophilic parental and the mesophilic generations showed that the majority of the mutation sites were located in genes coding cell surface proteins and those for methane production from trimethylamine and some were located in glycosylation consensus sequeon.The 30°C growth of strain R15 was severely inhibited by the glycosylation inhibitor tunicamycin but not the growth at 18°C.This strongly suggests that proteins glycosylation can contribute to the thermal ”evolution”. Therefore, this proposed project will focus on that mechanism of glycosylation of cell surface proteins by perform four works as follows.1) By using proteomics method, differential cell surface protein abundances will be quantified for strain R15 grown at 18°C,30°C and using trimethylamine or methanol as the sole carbon source, respectively.2) Specific glycosylation sites for cell surface proteins associated thermal adaptation will be clarified. 3) Differential transcription of the oligosaccharyltransferase will be quantified for strain R15 grown at 18°C and 30°C by using trimethylamine or methanol as substrate, respectively. By using isotope tracing, contribution of trimethylamine to the cell surface proteins will be probed .This research will give new insights into clarifying the mechanism of thermal adaption for psychrophilic archaea.

摘要：嗜冷甲烷叶菌R15是分离自若尔盖高寒湿地的罕见嗜冷古菌，最适生长温度18℃，高于25℃不生长。但在以三甲胺为底物、18℃传代数年后，菌株R15的最适生长温度提高到30℃，但在甲醇上30℃微弱生长。比较“进化”前后菌株基因组SNP发现突变位点主要集中在细胞表面蛋白及三甲胺产甲烷相关蛋白的基因，有些突变位点位于保守的糖基化motif。前期研究发现糖基化抑制剂衣霉素抑制嗜冷古菌R1530℃生长，但对18℃生长影响小，暗示蛋白糖基化对其“进化”为中温生长重要。本研究拟从四个方面探讨细胞表面蛋白糖基化对嗜冷古菌R15适应中温的作用：１）比较中温、低温、甲醇和三甲胺培养物的细胞表面蛋白差异；２）比较上述四种培养物的细胞表面蛋白糖基化水平差异；３）探讨上述四种培养物中细胞糖基转移酶的表达差异；４）探讨三甲胺对细胞表面蛋白合成的贡献。该研究试图揭示低温古菌“进化”为中温菌与细胞表面蛋白糖基化相关。

通过研究两种底物（三甲胺和甲醇），以及中低温（18°C和30°C）S-layer的N-糖基化水平变化，主要包括：N-糖基化位点和N-糖链组成分析，以期揭示嗜冷甲烷叶菌R15中温适应机制；2）通过比较基因组学方法，研究嗜冷古菌R15与其它两株中温甲烷菌KZC,GZC（与R15菌株16S rRNA identity 99%）基因组core-pan分析，COG聚类差异分析;以及差异基因转录水平差异分析，以期在基因组及转录水平揭示嗜冷古菌R15中温适应机制。3）研究青藏高原不同盐碱湖优势产甲烷途径及功能产甲烷菌。以青藏高原不同盐度和不同湖滨植被的4个盐碱湖：公珠错（高山柳等小灌木）、昆仲错（阔叶灌木丛）、班公错和兹格塘错的沉积物为研究对象，通过高通量测序和定量PCR（qPCR）定量古菌16S rRNA，并分析其古菌多样性和优势古菌类群；模拟原位环境盐浓度及pH，比较沉积物从不同产甲烷底物（甲醇、三甲胺、乙酸和H2/CO2）的产甲烷速率，以分析其中的优势产甲烷途径；通过添加氯仿抑制所有的代谢过程及产甲烷特异抑制剂2-溴乙烷磺酸盐（BrES），检测沉积物中产甲烷底物的积累，确定产甲烷途径。目的是探讨青藏高原不同类型盐碱湖中的优势产甲烷菌群和优势产甲烷代谢途径。