双组分系统在德氏乳杆菌保加利亚亚种与嗜热链球菌共生关系中的作用研究

Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are necessary for starter cultures for yogurt manufacturing, and widely used in yogurt production in the world. The two species are synergistic, and they stimulate each other's growth and acid production during the milk formation. The mechanism of protocooperation between L. delbrueckii subsp. bulgaricus and S. thermophilus is complex and not very clear at present. The two component systems (TCSs) are one of the most important mechanisms for external environmental sensing and signal transduction for the majority of Gram-positive and Gram-negative bacteria. TCSs play an important role in the protocooperation between two species. In our previous study, L. delbrueckii subsp. bulgaricus CH3 and S. thermophilus S4 were isolated from yogurt in traditional pasturing area, and showed potential used as yogurt starter culture. They were used in this study. The aim of this study is to identify the function of TCSs in the protocooperation between two species; clarify the signal regulation mechanism of protocooperation; lay a theoretical basis for consummating the protocooperation mechanism, and improving the protocooperation effect of two species; and accelerate the development of high-efficiency yogurt starter cultures. The study including three contents: to analyze the TCSs of L. delbrueckii subsp. bulgaricus CH3 and S. thermophilus S4 strains in monoculture and mixed culture by means of real-time PCR technology, and get differentially expressed TCSs; to construct key TCSs mutants and determine functions of key TCSs in protocooperation; to reveal the related proteins which be regulated by key TCSs, to construct a network of protocooperation regulated by TCSs.

德氏乳杆菌保加利亚亚种与嗜热链球菌是构成酸奶发酵剂的必要菌种，在乳发酵过程中彼此促进生长和酸化。两菌共生机制复杂多样，备受关注，但尚未完全明确。双组分系统（TCS）作为细菌感受外界环境变化和应答的重要机制，在微环境不断变化的两菌共生体系中，更占有极其重要地位。本研究拟以前期获得的优良酸奶发酵剂菌株-德氏乳杆菌保加利亚亚种CH3与嗜热链球菌S4为材料，探讨TCSs在二者共生关系中的作用，明确两菌共生的信号调控机制，进而从信号转导角度，揭示两菌的共生机制，为完善二者共生机制和提高两菌共生效能提供理论依据，进而加速高效酸奶发酵剂研发。本研究包括三部分：首先以实时定量PCR技术分析CH3与S4单菌发酵和混菌发酵酸奶过程中TCSs的表达变化，确定关键TCSs；然后构建关键TCSs突变体，研究其在两菌共生中作用；确定关键TCSs调节的蛋白，最终建立由关键TCSs调控的共生调控网络。

德氏乳杆菌保加利亚亚种与嗜热链球菌是构成酸奶发酵剂的必要菌种，二者共生，相互促进。两菌共生机制复杂，备受关注，但尚未完全明确。双组分系统（TCS）作为细菌感受外界环境变化和应答的重要机制，在微环境不断变化的两菌共生体系中，更占有极其重要地位。本研究以前期获得的优良酸奶发酵剂菌株-德氏乳杆菌保加利亚亚种与嗜热链球菌为材料，探讨TCSs在二者共生关系中的作用，明确两菌共生的信号调控机制，进而从信号转导角度，揭示两菌的共生机制，为完善二者共生机制和提高两菌共生效能提供理论依据，进而加速高效酸奶发酵剂研发。本研究包括：首先以实时定量PCR技术分析单菌发酵和混菌发酵酸奶过程中TCSs的表达变化，确定关键TCSs；然后构建关键TCSs突变体，研究其在两菌共生中作用；确定关键TCSs调节的蛋白，最终建立由关键TCSs调控的共生调控网络。通过对单菌及共生菌株发酵脱脂乳的生长曲线和pH值的测定，明确了这两种菌株单独发酵和共生发酵脱脂乳时菌株生长的四个时期（迟缓期、对数期、稳定期、衰退期），并选取了包括四个生长时期在内的关键的时间点进行菌体的收集，RNA的提取和cDNA的合成。采用RT-qPCR技术研究单菌和两菌共生发酵脱脂乳的过程中，各自TCSs基因表达量的差异，从而明确S. thermophilus和L．delbrueckii subsp. bulgaricus两菌共生对S. thermophilus和L．delbrueckii subsp. bulgaricus菌株各自TCSs基因表达量情况发生明显变化的关键TCSs基因。结果表明，S. thermophilus中的8个TCSs中hpk2/rr2、hpk4/rr4、hpk7/rr7为关键的TCSs；L. delbrueckii subsp. bulgaricus中的7个TCSs中hpk1/rr1、hpk2/rr2、hpk3/rr3、hpk7/rr7为关键的TCS。关键TCS突变株与野生型菌株共生发酵脱脂乳，对生长性能、产风味物质乙醛含量、产黏度等比较分析发现，突变后的菌株各发酵特性都有不同程度的减弱，其中突变菌株CH3-R测定的上述三种指标的发酵性能最差。结果表明TCS参与菌株生长、风味物质合成、多糖合成等多种代谢调节。