Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic

Benjamin J Lethbridge,Graham P Jones,Max E Tate,Jodie V Johnson,Eric W Triplett,Victor Rumjanek,James J Sims,Laura S Bailey,Brenda T Breil,Robert E Asenstorfer

doi:10.1038/s41429-021-00497-0

Abstract

Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.

Highlights

The eleven C-terminal amino acid sequence (DIGGSRQGCVA) of the 42 amino acid residue structural gene product of TfxA [10] is consistent with the reported degradative amino acid sequence of purified, blue fluorescent, trifolitoxin (DIGGSRXGCVA) [6] post-translational modification of the predicted 42mer prepeptide
High resolution mass spectra (HR-MS) of TFX1 & TFX2 were obtained by Fast-atom bombardment mass spectrometry (FAB-MS) using a ZAB-ET tandem mass spectrometer (VG Analytical Ltd, Manchester, UK)
NMR data for TFX1 and TFX2 were collected on a Bruker

Summary

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Trifolii T24 in the nodulation of clover was reported [1] It was subsequently named Trifolitoxin (TFX) by Triplett and Barta [2]. The eleven C-terminal amino acid sequence (DIGGSRQGCVA) of the 42 amino acid residue structural gene product of TfxA [10] is consistent with the reported degradative amino acid sequence of purified, blue fluorescent, trifolitoxin (DIGGSRXGCVA) [6] post-translational modification of the predicted 42mer prepeptide. Conservative substitution of amino acids RQGC by sitedirected mutagenesis eliminated toxicity [11] indicating further post-translational modification or involvement of these amino acids in the activity or synthesis of TFX. Post-translational modifications of amino acids and adjacent peptide backbones of the eleven C-terminal amino acids of the pre-peptide produces a blue fluorescent trifolitoxin

Methods

Results

Discussion

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