Cover W H, Baerwald R J, Levens T
Department of Biological Sciences, University of New Orleans, Lakefront, Louisiana 70148.
Microbios. 1991;67(274):23-32.
Osmotically stable spheroplasts were produced from Escherichia coli ML-35 and W7-M5 using either 1 min exposure to polymyxin B or 10 min exposure to Tris/EDTA, followed by 1 to 3 h incubation with lysozyme. Spheroplast membrane permeability studies were conducted using paired radioactive probes with E. coli ML-35. Experiments with 14C-sucrose-16 kD 3H-dextran indicated that the outer membrane had lost its barrier to 16 kD dextran. Parallel experiments with 81 kD 3H-dextran indicated that the outer membrane was impermeable to the larger dextran. EDTA treated cells also showed outer membrane permeability to 16 kD dextran. Cytoplasmic membrane integrity was confirmed using 14C-sucrose and 3H2O before and after exposure to polymyxin B and EDTA. Scanning electron microscopy showed that a rough surface on polymyxin B produced spheroplasts while Tris/EDTA spheroplasts showed the same smooth surface as control cells.
通过以下两种方法从大肠杆菌ML - 35和W7 - M5中制备渗透稳定的原生质球：一是用多粘菌素B处理1分钟，二是用Tris/EDTA处理10分钟，然后用溶菌酶孵育1至3小时。使用成对的放射性探针与大肠杆菌ML - 35进行原生质球膜通透性研究。用14C - 蔗糖 - 16 kD 3H - 葡聚糖进行的实验表明，外膜对16 kD葡聚糖失去了屏障作用。用81 kD 3H - 葡聚糖进行的平行实验表明，外膜对较大的葡聚糖是不可渗透的。经EDTA处理的细胞对外膜对16 kD葡聚糖也具有通透性。在暴露于多粘菌素B和EDTA之前和之后，使用14C - 蔗糖和3H2O确认细胞质膜的完整性。扫描电子显微镜显示，多粘菌素B处理产生的原生质球表面粗糙，而Tris/EDTA原生质球与对照细胞表面一样光滑。
