Characterization of the Ustilago maydis sid2 gene, encoding a multidomain peptide synthetase in the ferrichrome biosynthetic gene cluster

Ustilago maydis, the causal agent of corn smut disease, acquires and transp orts ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likel y proceeds by hydroxylation and acetylation of L-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase. sid1 encodes L-ornithine Ns-oxygenase, wh ich catalyzes hydroxylation of L-ornithine, the first committed step of fer richrome and ferrichrome A biosynthesis in U. maydis, In this report we cha racterize sid2, another biosynthetic gene in the pathway, by gene complemen tation, gene replacement, DNA sequence, and Northern hybridization analysis . Nucleotide sequencing has revealed that sid2 is located 3.7 kb upstream o f sid1 and encodes an intronless polypeptide of 3,947 amino acids with thre e iterated modules of an approximate length of 1,000 amino acids each. Mult iple motifs characteristic of the nonribosomal peptide synthetase protein f amily were identified in each module. A corresponding iron-regulated sid2 t ranscript of 11 kb was detected by Northern hybridization analysis. By cont rast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption of urbs1, a zinc finger, GATA family transcrip tion factor previously shown to regulate siderophore biosynthesis in Ustila go, Multiple GATA motifs are present in the intergenic region between sid1 and sid2, suggesting bidirectional transcription regulation by urbs1 of thi s pathway. Indeed, mutation of two of these motifs, known to be important t o regulation of sid1, altered the differential regulation of sid2 by iron.