异源表达德氏乳杆菌保加利亚亚种TCS1调控酸适应基因提高乳酸乳球菌NZ9000耐酸性

Expression of genes regulated by acid adaptation related TCS1 of Lactobacillus delbrueckii subsp. bulgaricus improved acid tolerance of Lactococcus lactis subsp. cremoris NZ9000

摘要:

[目的] 在乳酸乳球菌NZ9000中异源表达德氏乳杆菌保加利亚亚种中由双组分系统TCS1（JN675228/JN675229）调控的与酸适应相关基因，进而探究德氏乳杆菌保加利亚亚种应对酸胁迫的机制。[方法] 通过逆转录聚合酶链式反应和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳验证由德氏乳杆菌保加利亚亚种TCS1调控的与酸适应相关基因中腺嘌呤磷酸核糖转移酶（aprt）、D-丙氨酸-D-丙氨酸连接酶（ddl）、寡肽ABC转运蛋白（oppDII）和延伸因子Ts（tsf）在乳酸乳球菌NZ9000中的表达情况。酸处理实验验证基因表达对宿主菌酸胁迫耐受能力的影响。并采用酵母双杂交验证双组分系统TCS1与表达的酸适应相关基因之间的互作关系及具体的互作部位。[结果] 结果表明，乳酸乳球菌NZ9000中成功表达了aprt、ddl、oppDII和tsf。aprt、ddl基因使重组菌对酸胁迫的抗性分别提高了75倍和114倍。oppDII和tsf基因的表达对重组菌株的耐酸能力没有明显影响。酵母双杂交实验表明TCS1中的组氨酸蛋白激酶HPK1与Ddl之间存在相互作用，且HPK1-C结构域是二者相互作用的关键区域。[结论] aprt和ddl过表达菌株酸刺激的适应能力显著高于对照菌株，该研究结果可为德氏乳杆菌保加利亚亚种及类似菌株耐酸性特性的获得策略提供参考。

Abstract:

[Objective] We investigated the acid tolerance mechanisms of Lactobacillus delbrueckii subsp. bulgaricus by heterologous expression of genes related to acid adaptation regulated by two-component system TCS1 (JN675228/JN675229) of Lb. bulgaricus in Lactococcus lactis subsp. cremoris NZ9000. [Methods] We used reverse transcription polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to verify the expression of acid adaptation related genes, such as adenine phosphoribosyltransferase (aprt), D-alanine-D-alanine ligase (ddl), oligopeptide ABC transporter (oppDII) and elongation factor Ts (tsf), regulated by TCS1 of Lb. bulgaricus, in Lc. lactis NZ9000. We validated acid treatment experiment to verify the effect of gene expression on acid stress tolerance of host bacteria. We confirmed the interactions between TCS1 and the expressed genes related to acid adaptation by yeast two hybrid. [Results] Our results show that aprt, ddl, oppDII and tsf were successfully expressed in Lc. lactis NZ9000. The expression of aprt and ddl genes increased the resistance of recombinant strains to acid stress by 75 and 114 times. The expression of oppDII and tsf genes had no significant effect on the acid resistance of the recombinant strains. Yeast two hybrid system showed that there was interaction between histidine protein kinase (HPK1) of TCS1 and Ddl, and HPK1-C domain was the key region of the interaction. [Conclusion] The acid tolerance of strains expressing Aprt and Ddl was significantly higher than that of the control strain. The results of this study can provide references for the strategies of obtaining acid resistance of Lb. bulgaricus and other similar strains.