脂环酸芽孢杆菌嗜酸耐热的分子机理研究

Alicyclobacillus spp is the typical genus of acidophilic and thermophilic bacteria with strong resistance to heat and high acid. During apple juice processing, it can survive the pasteurization treatment, causing some off-flavor in apple juice and decreasing apple juice quality. Based on our early studies, it was found that DnaK plays an important role in its acid and heat shock resistance. Molecular method was used to knock out DnaK chaperone to construct auxotroph strain and its retro-complementary strain. And iTRAQ technology was applied to screen differential protein related to DnaK chaperone under heat and acid stress, Western-blot and MRMc technology were performed to detect the target protein function, bacterial two-hybrid system and GST pulldown technology were used to confirm the cross reaction between DnaK and target proteins. The purpose of this project is to attempt to identify and explain the physiological and molecular mechanism of heat and acid resistance, laying a solid foundation for its control and application.

脂环酸芽孢杆菌（Alicyclobacillus spp）是嗜酸耐热菌的典型代表，具有嗜酸耐热双重生理特性，该菌能经受较强的杀菌作用而存活，极大影响果汁的质量。前期研究发现DnaK分子伴侣蛋白与该菌耐酸、热胁迫能力关系密切，基于此，本研究拟构建该菌的分子伴侣DnaK基因缺陷型及缺陷回补突变体，并通过施加不同的酸、热胁迫，利用iTRAQ技术检测野生型、DnaK基因缺陷型、DnaK基因缺陷回补型在正常、酸、热胁迫条件下表达蛋白的差异，寻求与DnaK分子伴侣蛋白相关联的目标蛋白，并利用MRM技术验证所获关键蛋白的功能表现，进一步利用细菌双杂交及GST pull-down技术探究分子伴侣DnaK蛋白与这些关键蛋白之间的互作关系，从分子水平上揭示脂环酸芽孢杆菌嗜酸耐热的生理特性和分子机制，为控制和开发该类菌奠定良好的理论基础

Alicyclobacillus acidoterrestris具有独特的嗜酸耐热特性，可导致巴氏杀菌果汁品质劣变。深入理解该菌在高酸环境下能耐受巴氏杀菌而存活的机理是获得既高效又不影响果汁品质的防控措施的前提。由于课题组未能成功获得基因突变株，所以本研究在明确酸胁迫适应性响应对A. acidoterrestris耐高温和酸胁迫影响的基础上，围绕该菌的嗜酸耐热机制，从基因组水平出发开展了相关研究，并取得了大量研究成果。研究发现，非致死条件的酸适应性胁迫处理可诱导A. acidoterrestris产生酸胁迫适应性响应，从而提高对酸胁迫的耐受性，并产生对高温胁迫的交叉保护作用。酸适应性胁迫处理不会破坏该菌的细胞膜电位稳定性和胞内pH稳态，但会降低胞内ATP浓度、增加细胞膜中ω-环状脂肪酸含量以及诱导不饱和脂肪酸形成；pH为3.2和3.0的胁迫条件可诱导芽孢形成。RNA-seq测定结果显示，该菌中共517个基因在酸适应性胁迫下显著差异表达；且与表型特性相对应，菌体在响应酸适应性胁迫时上调了胞内pH稳态调节、细胞膜修饰及芽孢形成相关通路的基因，并激活了核苷酸补救途径。基于TMT标记的定量蛋白组学技术和PRM靶向蛋白组学技术测定结果显示共100个蛋白在酸适应性胁迫中显著差异表达。RNA-seq测定结果显示共911个基因在高温胁迫下显著差异表达，且菌体高温胁迫响应策略为：通过修饰肽聚糖和脂肪酸组成维持整体形态结构完整；通过降低糖降解、抑制核糖体从头合成以及诱导核糖体休眠来实施低能量消耗的策略；通过上调分子伴侣和Clp蛋白酶保护和修复蛋白；此外，MarR家族转录调控因子和好氧呼吸链中末端氧化酶表达相关基因（cydAB、cydCD及qoxBC）显著上调，且高温胁迫使细胞产生了活性氧、超氧化物歧化酶水平升高，表明高温胁迫会同时诱导产生氧气胁迫。