Method: Gas Chromatography-Mass Spectrometry

Gas chromatography-mass spectrometry (GC-MS) was carried out with Hewlett Packard GC (HP6890) and MS (HP5973) instrumentation. Samples (extracted as for HPLC analysis) were resuspended in 50 ?L of 1:1 methanol:chloroform and one microlitre of this extract was injected onto the GC column at an injection temperature of 250?C (Varian CP-SIL 5 CB Low bleed/MS 0.25 mm ? 0.25 ?m ? 25 m). The carrier gas was helium. The temperature profile consisted of 2 min at 150?C, then an increase of 6?C per min to a maximum of 320?C, with a total run length of 30.33 min. Samples were analysed in scanning mode. GC-MS profiles were compared to a standard preparation of gliotoxin (Sigma) and that published elsewhere [17, 18].

Supplementary figure 1: Domain architecture of the non-ribosomal peptide synthetase (NRPS), GliP. GliP has two complete modules with an additional phosphopantetheine binding domain. Its domain structure is identical the sirodesmin NRPS, SirP.

Supplementary figure 2: RP-HPLC chromatograms of gliotoxin (10 ?g) (a) and culture filtrate extract of Aspergillus fumigatus isolate 293 grown in Czapek Dox media for 48 h at 37?C with agitation (b). The major peak in b (1) has an identical retention time to that in a.

Supplementary figure 3: Gas chromatography-mass spectrometry of Aspergillus fumigatus culture filtrate grown for 48 h at 37?C with agitation. (a) Gas chromatogram with two major peaks eluting at 11.65 and 12.25 min. Both these peaks were observed in crude culture filtrates, authentic gliotoxin and eluates from the HPLC column. (b) Mass spectrum of the peak eluting at 11.65 min. The identity of this peak is unknown, however its mass spectrum shares a number of ions in common with the spectrum in c (M/Z = 75, 88, 115, 143, 207 and 214) and is therefore likely to be a degradation product of gliotoxin. (c) Mass spectrum of the peak eluting at 12.25 min. The mass spectrum of this molecule matched (99%) that of desthiogliotoxin (structure is shown) in the Wiley275 mass spectrum database.

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