Scupham A J, Triplett E W
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA.
J Appl Microbiol. 2006 Mar;100(3):500-7. doi: 10.1111/j.1365-2672.2005.02803.x.
The first aim was to determine those amino acid residues required for the biological activity of the potent peptide antibiotic, trifolitoxin (TFX). The second aim was to determine the concentrations of TFX1 and TFX2 that cause 50% inhibition of bacterial growth (Ki), the two predominant isomeric forms of TFX made by Rhizobium.
Site-directed mutagenesis of tfxA was used to produce strains that made mutant TFX peptides. The mutant tfxA genes were placed on a vector and inserted in Rhizobium leguminosarum b. trifolii Tn54A112, a tfxA mutant of strain T24 that lacks trifolitoxin activity. Our standard bioassay was used to assess the activity of these mutants. TFX1 and TFX2 were purified by reverse phase chromatography. Several concentrations of each peptide were assayed for biological activity to determine Ki. The unmodified TFX peptide (DIGGSRQGCVA) was synthesized and was found to lack any biological activity. Four of the 11 amino acid residues in ribosomally synthesized, post-translationally modified peptide were required for TFX activity. These required amino acids include arginine (R37), glutamine (Q38), glycine (G39) and cysteine (C40). S36T and S36Y mutants showed reduced TFX activity. The numbering system is based on the 42-amino acid TfxA peptide that is post-translationally modified to form the active TFX peptide. The Ki of TFX2 was determined to be 10-fold lower than TFX1.
The post-translational modifications of the TfxA peptide are required for biological activity. TFX2 is far more active than TFX1.
The sequence of the TfxA peptide appears to have been optimized for maximum activity through the course of evolution. Even conservative changes to any of the amino acid residues required for activity results in a complete loss of activity. The understanding of the action of this peptide is critical for its proposed action as a control agent for crown gall disease.
第一个目的是确定强效肽抗生素三叶因子毒素（TFX）生物活性所需的氨基酸残基。第二个目的是确定导致细菌生长受到50%抑制（Ki）的TFX1和TFX2的浓度，它们是根瘤菌产生的两种主要的异构体形式。
采用tfxA的定点诱变技术构建产生突变型TFX肽的菌株。将突变的tfxA基因置于载体上，并插入到缺少三叶因子毒素活性的T24菌株的tfxA突变体豆科根瘤菌三叶亚种Tn54A112中。我们使用标准生物测定法评估这些突变体的活性。通过反相色谱法纯化TFX1和TFX2。测定每种肽的几种浓度的生物活性以确定Ki。合成了未修饰的TFX肽（DIGGSRQGCVA），发现其没有任何生物活性。核糖体合成的、翻译后修饰的肽中的11个氨基酸残基中有4个是TFX活性所必需的。这些必需氨基酸包括精氨酸（R37）、谷氨酰胺（Q38）、甘氨酸（G39）和半胱氨酸（C40）。S36T和S36Y突变体显示TFX活性降低。编号系统基于翻译后修饰形成活性TFX肽的42个氨基酸的TfxA肽。确定TFX2的Ki比TFX1低10倍。
TfxA肽的翻译后修饰是生物活性所必需的。TFX2比TFX1活性高得多。
在进化过程中，TfxA肽的序列似乎已被优化以实现最大活性。即使对活性所需的任何氨基酸残基进行保守改变也会导致活性完全丧失。对这种肽作用的理解对于其作为冠瘿病控制剂的预期作用至关重要。
