甘氨酸甜菜碱和分子伴侣dnaK在嗜盐四联球菌耐盐机制中的作用  

The role of glycine betaine and molecular chaperone dnaK from Tetragenococcus halophilus on salinity adaptation

作　　者：卢良坤[1] 罗立新[1] 黎攀[1] 成丽丽[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《中国酿造》2013年第2期25-32,共8页China Brewing

基　　金：国家自然科学基金资助项目(31271924)

摘　　要：能耐高盐的嗜盐四联球菌CICC 10469是从传统的酱料中分离得到。为研究耐盐特性与相容性溶质积累和分子伴侣dnaK特性的关系,使用核磁共振来检测其主要的相容性溶质。不同盐浓度对耐盐的嗜盐四联球菌和不耐盐的乳酸乳球菌MG1363的生长的影响通过测量OD600来获得。盐浓度对分子伴侣dnaK表达的影响通过实时荧光定量RT-PCR来检测。相容性溶质和可溶性蛋白随盐浓度的变化通过HPLC和酶标仪来检测。结果显示嗜盐四联球菌以甘氨酸甜菜碱作为主要的相容性溶质。适合的盐浓度能够促进菌体的生长,在一定盐浓度范围内,甘氨酸甜菜碱,可溶性蛋白和分子伴侣dnaK随盐浓度的增加而升高,然而超过一定盐浓度范围后,表达量将会减少。这表明细菌能够根据外部环境做出适当的调整以维持细胞的正常生理功能。而乳酸乳球菌MG1363很少有相容性溶质和分子伴侣dnaK以耐受外部盐浓度。这表明相容性溶质和分子伴侣dnak在嗜盐四联球菌生长中起重要的作用。总之,中度嗜盐菌的主要耐盐机制是通过快速合成和释放甘氨酸甜菜碱及分子伴侣dnaK高活性表达来完成。Tetragenococcus halophilus CICC 10469 with a high-salinity resistance was isolated from the traditional soy sauce qcoji＇. To explore the correlation between salt-tolerance and accumulation of compatible solute as well as the property of molecular chaperone dnaK, naturally-abundant 1H andl 3C-nuclear magnetic resonance （NMR） was used to probe its main compatible solute. The effects of salinity on growth of lactic acid bacteria T. halophilus （salt-tolerant） and Lactococcus lactis MG1363 （salt-sensitive） were monitored by measuring OD600 at specific intervals. The influence of salinity on the expression of molecular chaperone dnaK was detected by real-time Quantitative PCR. The variation of compatible solute and soluble proteins changed in salinities were analysed by HPLC and microplate reader. Results showed that T. halophilus CICC 10469 produced glycine betaine as the main compatible solute. Appropriate NaC1 concentration stimulated the growth of bacteria. Within a certain range of NaC1 concentration, the synthesis of compatible solutes betaine, expression of molecular chaperones dnaK and production of soluble proteins increased with salt concentration. However, excess NaC1 （concentration higher than 20%） caused a decline in the amounts of all these substances, indicating that, depending on the environment, the bacteria could adjust for maintaining the normal physiological functions. Nevertheless, T. halophilus, L. lactis MG1363 could not or rarely produced compatible solutes betaine and the activity of its molecular chaperone dnaK, which led a low tolerance to NaC1. From these observations, it can be inferred that compatible solute betaine and molecular chaperone dnaK played a very important role in T. halophilus＇ halophilic process. In conclusion, rapid synthesis and release of compatible solutes as well as strong activity of molecular chaperone dnaK may be the main halophilic mechanism for moderately halophilic bacteria.

关 键 词：嗜盐四联球菌 分子伴侣dnaK 相容性溶质 核磁共振 高效液相色谱 耐盐 

分 类 号：Q939.97[生物学—微生物学]