Determination of the amino acid residues required for the activity of the anti-rhizobial peptide antibiotic trifolitoxin

Abstract

Aims: 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. Methods and Results: 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. Conclusions: The post‐translational modifications of the TfxA peptide are required for biological activity. TFX2 is far more active than TFX1. Significance and Impact of the Study: 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.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

“…Only those LAPs with a fully elucidated chemical structure are discussed here, but it is probable that trifolitoxin and some nitrile hydratase/nif11-derived natural products are also LAPs. 3,175,176 Goadsporin is produced by Streptomyces sp. TP-A0584 and contains two thiazoles, four (methyl)oxazoles, two dehydroalanines, and an acetylated N-terminus (Fig.…”

Bacteriocins from plant pathogenic bacteria

“…Trifolitoxins are peptide bacteriocins produced by Gram‐negative species such as Agrobacterium tumefaciens and R. leguminosarum . These bacteriocins consist of eleven amino acids carrying several (partly unknown) posttranslational modifications (Scupham & Triplett, 2006). Several studies confirm the role of these compounds in competition with closely related species (Robleto et al , 1998; Herlache & Triplett, 2002).…”

Low‐molecular‐weight post‐translationally modified microcins

“…The McbC dehydrogenase is highly similar to TfxC, a protein involved in biosynthesis of trifolitoxin, a peptide antibiotic produced by some strains of Rhizobium leguminosarum bv. trifolii (Scupham and Triplett, 2006). Like MccB17, trifolitoxin contains a fused thiazole–oxazole heterocycle and is produced from a longer ribosomally synthesized precursor.…”