不同生长阶段青枯菌的高效离子交换色谱表征

Characterization of Ralstonia solanacearum in different growth phases by high performance ion exchange liquid chromatography analysis

摘要:

采用高效离子交换色谱和激光光散射仪对不同生长阶段的青枯菌进行色谱表征。青枯菌经过色谱分离得到3个特征峰。通过对延滞期、对数生长期和稳定期青枯菌细胞浓度、发酵液pH值、细胞表面黏附的胞外酸性多糖(EPSⅠ)含量与青枯菌色谱行为的关系研究,发现在8h时,青枯菌运动能力强,细胞表面EPSⅠ少,吸附力弱,绝大多数菌体直接随流动相流出形成峰1;随着培养时间延长,细胞表面黏附的EPSⅠ量增多,与树脂吸附力增强,保留时间延长。因此分析3个色谱峰的形成与青枯菌的运动性、以及细胞表面EPSⅠ与阴离子交换树脂的吸附作用有关。并通过比较3个色谱峰青枯菌细胞表面EPSⅠ含量的差异和观察青枯菌经过4%甲醛固定处理后色谱行为的变化加以进一步验证。高效离子交换色谱为细菌完整细胞的研究提供了一种新的分析方法。

Abstract:

High performance ion exchange liquid chromatography and laser light scattering instrument were employed to characterize Ralstonia solanacearum in different growth phases. The pure culture of Ralstonia solanacearum was successfully separated into three characteristic fractions. Chromatographic behaviors of Ralstonia solanacearum in lag phase, logarithmic phase and stationary phase were carefully investigated, and their relationships to the cell concentration, pH of fermentation broth and extracellular polysaccharide (EPSⅠ) content of cell surface were analyzed. It was found that the majority of Ralstonia solanacearum cells obtained at 8h culture time could not be absorbed to the resin due to the strong motility of the cells which could apparently overcome the electrostatic interaction. Furthermore, when the mobility of the cells was destroyed by 4% formaldehyde treatment, the prolonged retention time was recorded due to the stronger adsorbility to the resin. On the other hand, the EPSⅠ content of cell surface was determined to increase with the culture time, indicating an increasing interaction between the cells and the resin. EPSⅠ content of three characteristic chromatographic fractions was determined, and it was found that the higher EPSⅠ content led to the longer retention time of the fraction, which confirmed the above-mentioned observation. As a result, it is concluded that the formation of three chromatographic fractions of the pure culture of Ralstonia solanacearum is attributed to bacterial motility and the interaction of EPSⅠ of cell surface with the anionic exchange resin, and the novel method of ion exchange chromatography of the intact bacterial cells can be a useful tool in bacteriological study.