Ferrichrysin [M-H2+Fe]

Siderophore-mediated acquisition of iron has been shown to be indispensable for the virulence of several fungal pathogens, the siderophore transporter Sit1 was found to mediate uptake of the novel antifungal drug VL-2397, and siderophores were shown to be useful as biomarkers as well as for imaging of fungal infections. However, siderophore uptake in filamentous fungi is poorly characterized. The opportunistic human pathogen Aspergillus fumigatus possesses five putative siderophore transporters. Here, we demonstrate that the siderophore transporters Sit1 and Sit2 have overlapping, as well as unique, substrate specificities. With respect to ferrichrome-type siderophores, the utilization of ferrirhodin and ferrirubin depended exclusively on Sit2, use of ferrichrome A depended mainly on Sit1, and utilization of ferrichrome, ferricrocin, and ferrichrysin was mediated by both transporters. Moreover, both Sit1 and Sit2 mediated use of the coprogen-type siderophores coprogen and coprogen B, while only Sit1 transported the bacterial ferrioxamine-type xenosiderophores ferrioxamines B, G, and E. Neither Sit1 nor Sit2 were important for the utilization of the endogenous siderophores fusarinine C and triacetylfusarinine C. Furthermore, A. fumigatus was found to lack utilization of the xenosiderophores schizokinen, basidiochrome, rhizoferrin, ornibactin, rhodotorulic acid, and enterobactin. Taken together, this study characterized siderophore use by A. fumigatus and substrate characteristics of Sit1 and Sit2.

Based on studies using laboratory strains, the efficiency of gene disruption in Aspergillus oryzae, commonly known as koji mold, is low; thus, gene disruption has rarely been applied to the breeding of koji mold. To evaluate the efficiency of gene disruption in industrial strains of A. oryzae, we produced ferrichrysin biosynthesis gene (dffA) disruptants using three different industrial strains as hosts. PCR analysis of 438 pyrithiamine-resistant transformants showed dffA gene disruption efficiency of 42.9%-64.1%, which is much higher than previously reported. Analysis of the physiological characteristics of the disruptants indicated that dffA gene disruption results in hypersensitivity to hydrogen peroxide. To investigate the industrial characteristics of dffA gene disruptants, two strains were used to make rice koji and their properties were compared to those of the host strains. No differences were found between the dffA gene disruptants and the host strains, except that the disruptants did not produce ferrichrysin. Thus, this gene disruption technique is much more effective than conventional mutagenesis for A. oryzae breeding.

A liquid chromatography with electrospray ionization mass spectrometry method was developed to quantitatively and qualitatively analyze 13 hydroxamate siderophores (ferrichrome, ferrirubin, ferrirhodin, ferrichrysin, ferricrocin, ferrioxamine B, D1 , E and G, neocoprogen I and II, coprogen and triacetylfusarinine C). Samples were preconcentrated on-line by a switch-valve setup prior to analyte separation on a Kinetex C18 column. Gradient elution was performed using a mixture of an ammonium formate buffer and acetonitrile. Total analysis time including column conditioning was 20.5 min. Analytes were fragmented by applying collision-induced dissociation, enabling structural identification by tandem mass spectrometry. Limit of detection values for the selected ion monitoring method ranged from 71 pM to 1.5 nM with corresponding values of two to nine times higher for the multiple reaction monitoring method. The liquid chromatography with mass spectrometry method resulted in a robust and sensitive quantification of hydroxamate siderophores as indicated by retention time stability, linearity, sensitivity, precision and recovery. The analytical error of the methods, assessed through random-order, duplicate analysis of soil samples extracted with a mixture of 10 mM phosphate buffer and methanol, appears negligible in relation to between-sample variations.