冷休克蛋白调控腾冲嗜热厌氧杆菌冷应激反应的机制

Studies on the function and regulation mechanism of cold shock response (Csp) is very important for revealing adaption mechanism of microbes to various environments and for preventing or controlling of pathogenic microorganisms. Over the past decades, althouth the biological functions of some Csps are well studied, that of Csp in thermophiles is scarcely investigated. Based on our previous finding that Csp and cold shock response is exsisted in Thermoanaerobacter tengcongensis, this proposal is to study the function and thermo-stability of Csp in Thermoanaerobacter tengcongensis using methods of EMSA and Circular Dichroism, to detect mobile or differential expression of proteins using Real-time PCR and Western blot, and to reveal the difference in protein expression under normal and cold-shock conditions by using two-dimensional gel electrophoresis and further also identify interations between proteins and mRNAs by the combination of MALDI-TOF Mass Spectrometry, Co-Immunoprecipitation (Co-IP), and RNA-Binding Protein Immunoprecipitation (RIP) respectively. The physiological functions will be assayed with respect to the differences in survival rate, growth rate and phenotype of mutant strains in comparison with those of wild strain. This research project is aimed to clarify the regulatory mechanism of cold shock proteins in Thermoanaerobacter tengcongensis and to provide essential data for clarifying adaptive mechanism of thermophiles in low-temperature condition.

冷休克蛋白功能的研究在生物环境适应机制揭示、病原微生物防控等方面有重要意义，目前多个常温菌的冷休克蛋白已得到研究，但极端嗜热菌的冷休克蛋白的研究鲜有报道。本项目在发现极端嗜热菌- - 腾冲嗜热厌氧杆菌具有冷应激反应机制的基础上，拟用EMSA、变温圆二色谱法测定冷休克蛋白的生化特性及热稳定性；以Real-time PCR、Western blot监测冷应激反应中冷休克蛋白的动态表达；以双向凝胶电泳和质谱鉴定在冷应激和正常条件下差异表达的蛋白，确定参与冷应激反应的蛋白；以免疫共沉淀和RNA结合蛋白免疫共沉淀法分析冷休克蛋白的互作蛋白及作用靶点，阐明其调控机制；观察冷休克基因缺失菌株和野生菌株在正常条件和冷应激中生长速率和表型差异，评价冷休克蛋白的生理功能。本项目旨在揭示腾冲嗜热厌氧杆菌中冷休克蛋白的功能及其调控机制，为阐明极端嗜热菌低温环境的适应机制提供重要依据。

腾冲嗜热厌氧杆菌Thermoanaerobacter tengcongensis MB4是含且仅含一个冷休克蛋白基因的微生物，其存在典型的冷应急反应。圆二色谱法测定了大肠杆菌异源表达的T. tengcongensis MB4冷休克蛋白CspC的热稳定性，表明CspC在85℃时仍有良好的稳定性；凝胶阻滞实验结果进一步证实CspC能结合RNA和单链DNA。以上结果与相关文献报道的嗜热微生物的冷休克蛋白特征一致，即T. tengcongensis MB4冷休克蛋白可能通过结合特定基因的mRNA进而防止mRNA在低温时形成稳定的二级结构，促进冷休克相关基因在低温时表达，进而对提高低温环境胁迫的适应性。. T. tengcongensis MB4于75℃ 培养至OD600 约0.4~0.5后转移至50℃进行冷激处理，冷休克蛋白基因csp转录水平在冷激60分钟时最高（提高了8倍）；之后csp转录水平逐渐降低，表明冷休克蛋白参与了T. tengcongensis MB4冷休克反应。进一步研究发现，在最适生长温度（75℃）时，cspC转录丰度为所有转录基因中第7位，冷应激后cspC的转录丰度最高，表明了冷休克蛋白对于宿主冷应激反应乃至正常生长均发挥重要的生理功能。.环境温度的迅速下降导致了T. tengcongensis MB4 70个转录因子的34个发生了显著差异性表达，暗示了环境温度变化对T. tengcongensis MB4生理活性的造成了全局性影响。低温引起了与细胞壁组分肽聚糖和细胞膜组分脂肪酸合成相关的基因转录水平改变，其中不饱和脂肪酸和支链脂肪酸合成相关基因的转录水平得以提高，表明了T. tengcongensis MB4通过重构细胞壁和细胞膜来适应低温环境；另外，T. tengcongensis MB4还通过增强与芽孢形成相关基因转录以及重塑能量代谢等途径来适应低温环境。值得注意的是，Cas系统和sRNA也通过特定机制参与了T. tengcongensis MB4对不同温度环境的适应性。. 冷休克蛋白基因缺失导致宿主即使在正常生长温度下不能生存，说明cspC为T. tengcongensis MB4必需基因。，同时也暗示了T. tengcongensis MB4冷休克蛋白具有全局调控的作用，冷休克蛋白调控多个基因的转录和表达。