Isotope-Assisted Screening for Iron-Containing Metabolites Reveals a High Degree of Diversity among Known and Unknown Siderophores Produced by Trichoderma spp

Abstract

Due to low iron availability under environmental conditions, many microorganisms excrete iron-chelating agents (siderophores) to cover their iron demands. A novel screening approach for the detection of siderophores using liquid chromatography coupled to high-resolution tandem mass spectrometry was developed to study the production of extracellular siderophores of 10 wild-type Trichoderma strains. For annotation of siderophores, an in-house library comprising 422 known microbial siderophores was established. After 96 h of cultivation, 18 different iron chelators were detected. Four of those (dimerum acid, fusigen, coprogen, and ferricrocin) were identified by measuring authentic standards. cis-Fusarinine, fusarinine A and B, and des-diserylglycylferrirhodin were annotated based on high-accuracy mass spectral analysis. In total, at least 10 novel iron-containing metabolites of the hydroxamate type were found. On average Trichoderma spp. produced 12 to 14 siderophores, with 6 common to all species tested. The highest number (15) of siderophores was detected for the most common environmental opportunistic and strongly fungicidic species, Trichoderma harzianum, which, however, did not have any unique compounds. The tropical species T. reesei had the most distinctive pattern, producing one unique siderophore (cis-fusarinine) and three others that were present only in T. harzianum and not in other species. The diversity of siderophores did not directly correlate with the antifungal potential of the species tested. Our data suggest that the high diversity of siderophores produced by Trichoderma spp. might be the result of further modifications of the nonribosomal peptide synthetase (NRPS) products and not due to diverse NRPS-encoding genes.

Trichoderma Research in the Genome Era

“…On average, Trichoderma spp. produced 12-14 siderophores (74). The genomes of T. virens and T. reesei contain a gene for extracellular siderophore production (SidD ortholog) in addition to the functional NPS6 cluster that is conserved across the three species (92).…”

The Siderophore Metabolome of Azotobacter vinelandii

“…A recent development in the discovery of siderophores is the use of high-resolution liquid-chromatography electrospray ionization mass spectrometry (HR-LC-MS) methods that exploit the characteristic 54 Fe- 56 Fe isotope pattern associated with organic Fe chelates (18)(19)(20). Data mining techniques are available for filtering the relevant Fe isotope patterns associated with Fe complexes even at low abundances and in highly complex matrixes, as well as for detecting the corresponding apo siderophores (18).…”

Molecular determination of marine iron ligands by mass spectrometry

“…When Fe is incorporated into an organic compound, two isotopologues that reflect the mass difference and relative intensity of the major Fe isotopes are detected. This approach has been used successfully in culture to identify novel siderophores produced by the filamentous fungus Trichoderma and from the nitrogen fixing bacteria Azotobacter vinelandii [Lehner et al, 2013;Baars et al, 2014Baars et al, , 2015. Isotope-assisted screening has also been used to detect putative novel Fe binding siderophores directly from seawater extracts .…”

“…However, ICPMS provides no information on the structure of the organic complex. Electrospray ionization mass spectrometry (ESIMS) can be used to detect isotopologues of the intact metal complexes Lehner et al, 2013;Baars et al, 2014;Waska et al, 2015]. For metals such as iron that have multiple isotopes, computer algorithms can search LC-ESIMS mass spectra and identify features that match a metal's characteristic isotope pattern [Lehner et al, 2013;Baars et al, 2014;.…”