BACTERIAL PROTOPLASTS INDUCED BY PENICILLIN
BACTERIAL PROTOPLASTS INDUCED BY PENICILLIN
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DOI：
10.1073/pnas.42.9.574
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发表时间：
1956-01-01
影响因子：
11.1
通讯作者：
LEDERBERG, J
中科院分区：
文献类型：
--
作者：
LEDERBERG, J
细菌原生质体被认为是被剥夺了刚性细胞壁的细胞单位。“因此，它们的区别在于它们的球形形状（在杆状物种中）和对低渗培养基中细胞溶解的敏感性。几位作者认为，原生质体可能是研究病毒和酶的生物合成的有用材料，因为它比完整细胞更简单或更容易进行外部修饰。到目前为止，只有对溶菌酶敏感的革兰氏阳性细菌被用于此类研究。现在已经发现了一种从肠道细菌如大肠杆菌和鼠伤寒沙门氏菌有效生产原生质体的方法，这些细菌已经是熟悉的生理学和遗传学对象。该技术主要包括将生长细胞暴露于含有青霉素、蔗糖和Mg++的培养基中。原生质体的制备大肠杆菌菌株K-12及其各种突变亚株用于大多数实验。青霉素和溶菌酶作为溶解剂之间的可能类比以及高渗蔗糖会中断溶菌酶对巨大芽孢杆菌的溶菌作用的发现表明青霉素和蔗糖的使用。此外，在青霉素的其他应用中，球形体也被偶然地注意到，许多作者强调了它在L型生产中的用途。[5]下面的程序是在经验试验之后采用的，毫无疑问可以进一步改进。使细菌在具有10 ml.肉汤（Difco Penassay培养基），在旋转器上。样品3 ml。将10 yL生长的培养物（约2 × 109个细胞/ml）直接加入到10 ml中。加入青霉素1，000 u/ml、蔗糖20%、硫酸镁0.2%的肉汤，2-3小时内细胞定量转化为球形。在此期间，光密度（在科尔曼14分光光度计中在650 m1 A处测量）增加约50%，但总计数（在Petroff-Hausser室中估计的球体或棒）保持恒定。当悬浮液在蒸馏水中稀释时，球体迅速溶解，因此它们被认为是“原生质体”。“所示的补充物是大量过量的，用5%的蔗糖，100 u.青霉素和0.1%的硫酸镁。高镁需要量可能部分取决于与青霉素制剂中所用柠檬酸钠的结合
Bacterial protoplasts are believed to be cellular units that have been deprived of their rigid cell wall.'Accordingly, they are distinguished by their spherical shape (in bacilliform species) and their sensitivity to cytolysis in hypotonic media. Several authors'-'have suggested that protoplasts might be useful material for the study of biosynthesis of viruses and enzymesin a system simpler or more accessible to externalmodification than the intact cell. So far, only gram-positive bacteria, which are susceptible to lysozyme, have been used for such studies. A method has now been found for the efficient production of protoplasts from enteric bacteria such as Escherichia coli and Salmonella typhimurium, which are already familiar physiological and genetic subjects. The technique consists essentially of the exposure of growing cells to a medium containing penicillin, sucrose, and Mg++. Preparation of Protoplasts.-Escherichia coli strain K-12 and a variety of its mutant substrains are used in most of the experiments. The use of penicillin and sucrose was suggested by the possible analogy between penicillin and lysozyme as lytic agents and by the finding that hypertonic sucrose would interrupt bacteriolysis of Bacillus megaterium by lysozyme.'In addition, spherical bodies had been casually noted in other applications of penicillin, 4 and many authors have emphasized its use in the production of L-forms. 5 The following procedure was adopted after empirical trials and can doubtless be further improved. The bacteria were grown overnight in tubes with 10 ml. of broth (Difco penassay medium) at 370 C., on a rotator. Samples of 3 ml. of the grown culture (about 2 X 109 cells/ml) were added directly to 10 ml. of broth supplemented with penicillin, 1,000 u/ml, sucrose 20 per cent, and magnesium sulfate 0.2 per cent. In 2-3 hours the cells were quantitatively converted into spheres. During this interval the optical density (measured at 650 m1A in a Coleman 14 spectrophotometer) increased about 50 per cent, but the total count (spheres or rods, estimated in a Petroff-Hausser chamber) remained constant. The spheres promptly lysed when the suspension was diluted in distilled water, and they are therefore regarded as" protoplasts." The indicated supplements are in substantial excess, and nearly-optimal yields of protoplasts can be obtained with 5 per cent sucrose, 100 u. penicillin, and 0.1 per cent MgSO4. The high magnesium requirement may depend partly on binding with the sodium citrate used in the compounding of the penicillin preparation