Identification of extracellular siderophores and a related peptide from the endophytic fungus Epichloë festucae in culture and endophyte-infected Lolium perenne

Abstract

A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three N(δ)-trans-anhydromevalonyl-N(δ)-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.

Graphical abstract

Fig. 1

Structures of siderophores and a related peptide from Epichloë festucae strain Fl1, and related siderophores.

Fig. 2

HRMS² spectrum of the protonated cyclic octapeptide 1 ion cyclo-[GGGGQ(AMHO)₃H]⁺, using Collision Induced Dissociation with different amounts of energy. Product ions are assigned as acylium species ([M+H−H₂O]⁺) of the peptides shown in the annotation. Inset is a subsection of the spectrum, showing losses of anhydromevalonyl (AM) and AMHO plus glycine.

Fig. 3

HRMS³ spectrum of the m/z 357 product ion of loss of three AMHO moieties from the protonated octapeptide 1 ion cyclo-[GGGGQ(AMHO)₃H]⁺. Annotated ions are acylium species.

Fig. 4

Schematic model showing the proposed peptide ring conformation of 1 in the vicinity of the C14-glutamine and adjacent C17-AMHO moieties, and key NOESY correlations within one of the accessible side-chain rotamers.

Fig. 5a

HRMS² spectrum of 4. Annotated ions are acylium species. Inset is subsection of the spectrum, showing the losses of serine corresponding to glycine losses in 1.

Fig. 5b

HRMS² spectrum of 5. Annotated ions are acylium species.

Fig. 6

HRMS² spectrum of the protonated heptapeptide 2 ion cyclo-[GGGQ(AMHO)₃H]⁺, using Collision Induced Dissociation with different amount of energy. Annotated ions are acylium species. Inset is a subsection of the spectrum, showing the loss of AM plus glycine.

Fig. 7

HRMS³ spectrum of the m/z 923 product ion of loss of an anhydromevalonyl moiety from the protonated octapeptide 3 ion cyclo-[GGGGQ(AMO)₃H]⁺. Annotated ions are acylium species.