基于群体感应通路解析保加利亚乳杆菌自溶的分子机制

Autolysis of Lactic acid bacteria (LAB) plays a vital role in the process of flavour during cheese ripening while restricts the proliferation of cell in yogurt fermentation and probiotics production.Therefore,It is necessary to clarify the mechanism of autolysis for the different requirement of various products.Because the bacteria autolysis is often accompanied by cell lysis, the previous research on cell autolysis mostly around the autolytic enzyme. But so far, mechanism of lactic acid bacteria autolysis is still no clear. With bacterial quorum-sensing phenomenon was found, revealing the bacterial flora has social attribute, the researchers found that many physiological activities including biofilm formation, antibiotic synthesis, competent cell formation had intimate relationships with the quorum sensing system. But for the autolysis of lactic acid bacteria and quorum-sensing signal molecules secreted whether there is a correlation, no related research in this area by now. Therefore, this study focuses on the research of lactic acid bacteria quorum-sensing system, degeneracy primers will be designed by using biological information, using the degeneracy primers, gene sequences of the lactic acid bacteria QS signal molecules will be cloned. Analysis of related genes of QS signal transduction pathway will be studied by using transcriptome sequencing method. Objective is to find the key regulatory gene of lactic acid bacteria autolysis, and will lay the theoretical foundation for directional control and selection of different autolytic lactic acid bacterias.

乳酸菌自溶会导致乳酸菌的死亡和胞内酶的释放，对发酵乳制品的风味形成、品质特性及生产周期等具有重要影响。阐明乳酸菌自溶的机理，为实现今后定向筛选和构建不同自溶度的乳品工业用菌株具有重要意义。本课题组在前期的研究过程中发现乳酸菌发生自溶的程度与发酵液中菌体密度呈正相关，并以此为切入点从保加利亚乳杆菌Ljj-6基因组中克隆出了群体感应（quorum-sensing）相关的双组分信号转导系统（TCS）和QS信号分子基因全序列，通过菌体培养液中添加人工合成信号分子实验，证实了周围培养液中信号分子浓度的累积是导致保加利亚乳杆菌菌体发生自溶的重要因素。但是菌体感知环境密度信号后，通过哪条通路触发了菌体的自溶过程是下一步需要解决的关键问题。本研究以上述实验为基础，采用转录组测序分析手段对QS信号分子传导通路的相关基因进行研究，筛选出乳酸菌自溶过程中的起主导作用的关键调控基因，为阐释乳酸菌自溶机理奠定基础。

乳酸菌自溶会导致乳酸菌的死亡和胞内酶的释放，对发酵乳制品的风味形成、品质特性及生产周期等具有重要影响。阐明乳酸菌自溶的机理，并根据需要对其进行定向选育或调控已成为目前乳制品发酵工业亟待解决的关键问题，对实现定向筛选和构建不同自溶度的乳品工业用菌株具有重要意义。以保加利亚乳杆菌基因组编码的一种主要自溶酶基因lytM为靶基因，利用基因敲除技术和超量表达技术对其功能进行了验证，结果表明lytM基因所编码的一种肽聚糖水解酶在保加利亚乳杆菌自溶过程中发挥着重要的作用。为了进一步解析lytM基因的调控网络，开展保加利亚乳杆菌在不同生长环境下的培养实验，并提取菌体总RNA进行转录组测序，分析保加利亚乳杆菌在添加群体感应信号分子和未添加群体感应信号分子两种条件下，保加利亚乳杆菌在RNA转录层面的差异，筛选出具有明显差异的基因，通过GO聚类和KEGG聚类对表达差异的基因进行聚类分析，对聚类出的代谢通路进行分析，通过基因敲除的手段对六条疑似的具有自溶调控功能的代谢通路进行验证，发现vicKR通路参与了自溶信号的传导。本项目研究结果表明，乳酸菌在生长过程中会向周围环境中分泌一种信号肽分子，当周围环境中信号肽的浓度达到或超出阈值的时，位于细胞壁上的组氨酸蛋白激酶vicK会启动磷酸化反应，将反馈调节蛋白vicR磷酸化，磷酸化的vicR会与RNA聚合酶结合，定位到lytM、ssaA、atlA三个基因的转录起始位点，上调这3个基因的表达，表达的肽聚糖水解酶加速了细胞壁的破裂，本项目首次解析了乳酸菌自溶的分子机制。