酸土脂环酸芽孢杆菌响应酸热胁迫的分子调控机制研究

Alicyclobacillus acidoterrestris is a genus of spoilage bacteria causing contamination in low pH fruits due to its acidophilic and thermophilic resistance, which led to economic losses to fruit juice industry, but its acid-resistant thermostable enzymes have the great potential application in the food industry. Therefore, to investigate the molecular regulatory mechanism how A. acidoterrestris responded to heat and acid stress is the key to hazard control and effective utilization of this microbe. In this study, a Label-free quantification will be used to analyze differentially expressed proteins in A. acidotresstris at the heat and acid stress conditions and bioinformatics analysis will be used to determine the molecular function of the differential protein and metabolic pathways involved; And then, qRT-PCR will be performed to screen key regulating proteins responding to heat and acid stress from main pathways which enriched most differential protein. Gene overexpression, gene knockout and retro-complementation of key proteins will be used to further verify the regulation function of the key proteins in the thermo-acidophilic resistance of A. acidoterrestris; Co-Immunoprecipitation will be used to identify interacting proteins with key proteins, establish the key protein regulation network and the model of proteins interaction to further illuminate the function of key proteins and molecular regulatory mechanism in responding to heat and acid stresses; The results will fully reveal the physiological adaptation mechanism of A. acidoterrestris from the protein level, which will provide a theoretical and experimental basis for acidic beverages preservation, microbial resistance research and the development of special industrial strains resisting the heat and acid conditions.

酸土脂环酸芽孢杆菌具有嗜酸耐热的双重生理特性，能引起巴氏灭菌果汁腐败变质，给果汁加工业造成经济损失，但其所含的耐酸热稳酶在食品工业中应用潜力巨大，解析该菌响应酸热胁迫的分子调控机制是其危害控制和有效利用的关键。本课题利用Label-free技术筛选酸土脂环酸芽孢杆菌在酸热胁迫下的差异表达蛋白并进行生物信息学分析，研究差异蛋白的分子功能及其参与的代谢途径，进而利用qRT-PCR从富集大量差异蛋白的主要通路中筛选响应酸热胁迫的关键调控蛋白；通过关键蛋白基因过表达、基因敲除、基因回补，验证关键蛋白调控该菌嗜酸耐热特性的作用；利用免疫共沉淀技术筛选关键蛋白的互作蛋白，构建关键蛋白调控网络、预测互作模式，进一步阐明关键蛋白的功能及其在酸热应答中的分子调控机制，从蛋白水平上全面揭示酸土脂环酸芽孢杆菌嗜酸耐热生理适应机制，为食品保藏、微生物抗逆特性研究及抗高温高酸的特殊工业菌研发等提供理论和实验依据。

嗜酸耐热酸土脂环酸芽孢杆菌是果汁工业中关键的质量安全因子，该菌污染引起的营养损失、风味劣变、出现沉淀等质量问题是制约果蔬加工及出口贸易的瓶颈。本研究利用蛋白质组学技术和分子生物学技术系统研究了该菌嗜酸耐热特性的分子调控机理，解析了该菌嗜酸耐热关键蛋白的功能及互作网络，探明了该菌在酸性条件下的生长机理，为酸性果蔬制品中该菌危害控制新技术开发奠定了理论基础。主要研究结果如下：.（1）利用定量蛋白质组学技术，筛选获得了酸土脂环酸芽孢杆菌热、酸应激差异表达蛋白和磷酸化差异蛋白。通过生物信息学分析，发现热应激差异蛋白主要与蛋白质、DNA、细胞壁等生物大分子保护和修复有关，表明酸土脂环酸芽孢杆菌耐热关键蛋白可能通过蛋白、DNA和RNA分子的结构和构象变化感知、调控热应激反应。酸应激差异表达蛋白主要与DNA修复和氨基酸代谢通路相关，尤其是精氨酸酶表达量显著上调，提示酸土脂环酸芽孢杆菌中可能存在一个类似乳酸菌中组氨酸依赖型的精氨酸耐酸系统，从蛋白水平揭示了酸土脂环酸芽孢杆菌响应热、酸胁迫的关键蛋白调控作用及机制，为筛选高效特异性抑菌剂提供了理论依据。.（2）利用Stitch数据搜索嗜酸耐热关键蛋白的靶点信息，构建热、酸应激关键蛋白互作网络并进行分析，发现热应激相关蛋白主要参与压力应激响应通路，酸应激相关蛋白主要参与氨基酸代谢、DNA代谢及压力应激响应通路。通过热、酸应激关键蛋白互作网络预测与分析，筛选得到了关键调控蛋白DnaK。以DnaK为诱饵蛋白，利用酵母双杂交技术筛选获得了DnaK的互作蛋白，揭示了关键蛋白调控该菌嗜酸耐热特性的分子机制。.（3）利用定量蛋白质组学技术鉴定出芽孢萌发相关差异蛋白98个，分析发现芽孢萌发差异蛋白与芽孢萌发过程中孢子皮层水解和细胞壁生物合成的调控和信号转导有关，从蛋白水平揭示了酸土脂环酸芽孢杆菌芽孢在低pH条件下萌发的分子调控机制，为研发芽孢污染控制新方法、突破果蔬制品中芽孢污染难以控制的技术瓶颈奠定基础。