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 transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely 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 N(5)-oxygenase, which catalyzes hydroxylation of L-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. In this report we characterize sid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed that sid2 is located 3.7 kb upstream of sid1 and 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-regulated sid2 transcript 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 of urbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis in Ustilago. Multiple GATA motifs are present in the intergenic region between sid1 and sid2, suggesting bidirectional transcription regulation by urbs1 of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation of sid1, altered the differential regulation of sid2 by iron.
Yuan
W M
WM
Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Gentil
G D
GD
Budde
A D
AD
Leong
S A
SA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D005291:Ferrichrome / Q000378:metabolism*; D005656:Fungal Proteins; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D008040:Genetic Linkage; D007501:Iron / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016296:Mutagenesis; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014588:Ustilago / Q000201:enzymology / Q000235:genetics*
D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D005291:Ferrichrome; D007501:Iron; D006899:Mixed Function Oxygenases; C059419:ornithine N5-oxygenase; D010453:Peptide Synthases; C430341:Sid2 protein, Ustilago maydis