Characterization of theUstilago maydis sid2Gene, Encoding a Multidomain Peptide Synthetase in the Ferrichrome Biosynthetic Gene Cluster

Abstract

Ustilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation ofl-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase.sid1encodesl-ornithineN⁵-oxygenase, which catalyzes hydroxylation ofl-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis inU. maydis. In this report we characterizesid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed thatsid2is located 3.7 kb upstream ofsid1and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulatedsid2transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption ofurbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis inUstilago. Multiple GATA motifs are present in the intergenic region betweensid1andsid2, suggesting bidirectional transcription regulation byurbs1of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation ofsid1, altered the differential regulation ofsid2by iron.