The synthesis of tabtoxin peptide bond in Pseudomonas syringae pv. tabaci.
The synthesis of tabtoxin peptide bond in Pseudomonas syringae pv. tabaci.
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丁香假单胞菌中标签毒素肽键的合成。
DOI：
10.1042/bst026s383
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发表时间：
1998
影响因子：
3.9
通讯作者：
L. Larous
中科院分区：
文献类型：
--
作者：
D. Harzallah;L. Larous
丁香假单胞菌烟粉虱（tabaci）是烟草（Nicotiana spp.）它产生的tabtoxin也由至少两种其他植物病原性细菌密切相关的假单胞菌属物种产生（1，2）。以前的工作已经解决了调节tabtoxin生物合成的问题，以预测它将产生的环境条件（3，4）。这种调节tabtoxin合成的复杂性部分取决于其生物合成步骤的数量。已经表明，tabtoxinine-β-内酰胺是通过天冬氨酸、甘油和甲硫氨酸的生物合成途径产生的（5）。在阐明tabtoxin的生物合成过程中要解决的下一个问题是，肽键是在核糖体上合成的，还是在酶上合成的。其他肽类抗生素（如短杆菌肽）中的肽键不是在核糖体上合成的（6），但没有证据表明假单胞菌肽类抗生素的合成位点。这项工作描述了一个实验，其中在存在和不存在抑制C-苏氨酸掺入蛋白质的浓度的氯霉素的情况下，测量通过C-苏氨酸掺入到tabtoxin中确定的tabtoxin中的肽键的合成。这些实验的补料时间为10- 15分钟持续时间，并且使用积极合成tabtoxin的恒化培养物。掺入标记到TCA不溶性沉淀的氯霉素处理的文化发生在5%的掺入到TCA不溶性沉淀的控制率。显然，氯霉素治疗大大减少了蛋白质合成。可溶性的。I4 C从苏氨酸中迅速消失，到15分钟时，约90%出现在HPLC柱空隙体积中洗脱的化合物中。在对照组和氯霉素治疗组中，约10%的标记物出现在tabtoxin中。标记物从苏氨酸中消失的时间过程及其在空隙体积中的出现如图1所示;在上清液和TCA不溶性沉淀物中定量回收了以苏氨酸形式添加的I4 C，相当于0.74 × 106 dpm/ml（图2）。
Pseudomonas syringae pv. tabaci is the causal agent of the wildfire disease of tobacco (Nicotiana spp.) It produces tabtoxin which is also produced by at least two other phytopathogenic bacteria closely related Pseudomonas species (1, 2). Previous work has addressed the problem of the regulation of tabtoxin biosynthesis in order to predict the environmental circumstances under which it will be produced (3, 4). The complexity of this regulatory contro: I on tabtoxin synthesis will be determined in part by the number of steps in its biosynthesis. It has been shown that the tabtoxinine-&lactam is produced on a biosynthetic pathway fed by aspartic acid, glycerol and methionine (5). The next question to tackle in eluciditing the biosynthesis of tabtoxin is whether the peptide bond is synthesised on ribosome, or whether it is synthesised on a enzyms. The peptide bond in other peptide antibiotics, such as gramicidin, are not synthesised on ribosomes (6) but there exist no evidence for the site of synthesis of the Pseudomonas peptide antibiotics. This work describes an experiment in which the synthesis of the peptide bond in tabtoxin, determined by the incorporation of'C-threonine into tabtoxin, was measured in the presence and absence of chloramphenicol at concentrations that inhibited'C-threonine in incorporation into protein. The feeding time for these experiments were of 10-1 5 minutes duration, and chemostat cultures were used which were actively synthesising tabtoxin. Incorporation of label into TCA-insoluble precipitate of the chloramphonicol-treated culture occured at 5% of the rate of incorporation into the TCA-insoluble precipitate in the control. Clearly, the treatment with chloramphenicol had substantially reduced protein synthesis. The soluble. I4C disappeared rapidly out of threonine and by 15 minutes, about 90% appeared in compounds that eluted in the void volume of the HPLC column. Approximately 10% of the label appeared in tabtoxin in both the control and the chloramphenicol treatment. The time-course of disappearance of label from threonine and its appearance in the void volume is shown in (Figure 1); The I4C added as threonine, equivalent to O, 74x1O6 dpm I ml was quantitativety recovered in the supernatant and TCA-insoluble precipitate (Figure 2).