Analysis of ferrichrome biosynthesis in the phytopathogenic fungus Ustilago maydis: cloning of an ornithine-N5-oxygenase gene.

By using a non-enterobactin-producing enb-7 mutant of Salmonella typhimurium LT2 as a biological indicator, a novel screening method was developed for identifying mutants of Ustilago maydis defective in the biosynthesis of the siderophores ferrichrome and ferrichrome A. Two classes of siderophore mutations, both recessive, were isolated after mutagenesis of haploid cells of the corn smut fungus. Class I mutants no longer produced ferrichrome while retaining the ability to produce ferrichrome A; class II mutants were defective in the production of both ferrichrome and ferrichrome A. Genetic and biochemical data suggest that class II mutants are defective in the ability to hydroxylate L-ornithine to delta-N-hydroxyornithine, the first step in the biosynthesis of these siderophores. A genomic library of wild-type U. maydis DNA was constructed in the cosmid transformation vector pCU3, which contains a dominant selectable marker for hygromycin B resistance. Two cosmids, pSid1 and pSid2, were identified in this library by their ability to complement class II siderophore auxotrophs. The production of both siderophores was concomitantly restored in the majority of the resultant transformants. Transforming DNA could be recovered from the fungal, cosmid-containing transformants by in vitro packaging with lambda bacteriophage extracts. Alternatively, the clones could be identified by a sib selection procedure. Cotransformation was found to occur at a high frequency in the fungus and was used to determine that a 2.5-kilobase HindIII-NruI fragment in pSid1 was responsible for complementing the class II siderophore biosynthetic mutation.

Malonichrome, a new iron chelate from Fusarium roseum.

The predominant iron chelates, or siderochromes, produced by the fungus, Fusarium roseum during culture periods up to seven days are the ester type fusarinine compounds. During longer periods of incubation, the fusarinine compounds completely disappear from the culture medium and are replaced by a new siderochrome. The new compound has been isolated, purified, and its structure determined. It is a cyclic hexapeptide containing one residue of L-alanine, two residues of glycine and three residues of delta-N-hydroxyornithine. The hydroxylamino groups of the ornithine residues are acylated with 3 mol of malonic acid to form a negatively charged ferrichrome type chelate. The circular dichroism spectrum indicates that the stereochemistry about the iron is lambda-cis. This compounds, which we name malonichrome, is not an efficient iron donor to F. roseum nor does it show growth factor activity towards Arthrobacter flavescens.

[Incorporation of glycine and serine in sideramines of the ferrichrome type with fungi of the genus Aspergillus in vivo and in vitro (author's transl)]. / Einbau von Glycin und Serin in Sideramine vom Ferrichrom-Typ bei Pilzen der Gattung Aspergillus in vivo und in vitro.

A 1.6-11.8% incorporation of labelled glycine and serine into ferrichrome, ferricrocin and ferrichrysin has been achieved with four strains of Aspergillus in vivo. Cell-free extracts of Aspergillus fumigatus and A. quadricinctus capable of cyclohexa-peptide biosynthesis have been prepared.