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 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.

Identification ofHistoplasma capsulatumTranscripts Induced in Response to Reactive Nitrogen Species

“…Siderophores are small molecules secreted by microbes to scavenge iron from the environment. In Ustilago maydis, the l-ornithine monooxygenase and nonribosomal peptide synthetase required for siderophore production are grouped together in the genome (Yuan et al, 2001). Our iron acquisition cluster also contains a nonribosomal peptide synthetase (H.c. NPS1), which, similar to U. maydis, is grouped together in the genome with H.c. LOM1.…”

Siderophore-Based Iron Acquisition and Pathogen Control

“…They are distinguished from all other known fungal GATA factors by the presence of two zinc fingers and a conserved intervening cysteine-rich region, which might represent the most probable binding site for the direct sensing of iron by binding iron or an iron-sulfur cluster (124). While Urbs1 appears to be the exclusive siderophore biosynthesis regulator in U. maydis that represses the siderophore biosynthesis genes sid1 and sid2 during iron repletion (351), additional regulatory factors seem to be present in N. crassa and A. nidulans (124). In A. nidulans, SreA deficiency leads not only to the derepression of siderophore biosynthesis but also to the deregulation of siderophore-mediated iron uptake (236).…”

“…This has been reported for coelichelin of S. coelicolor (52,182), the pyoverdins of fluorescent Pseudomonas spp. (2,224), the exochelins from nonpathogenic mycobacteria (350,360), and the ferrichromes/ferricrocins from various fungi (84,295,333,351) and can be anticipated for alterobactin of Alteromonas luteoviolacea, the pseudobactins of Pseudomonas spp., the azotobactins of Azotobacter spp., and the ornibactins of Burkholderia spp.…”

The genome sequence of the filamentous fungus Neurospora crassa

“…By phylogenomic analysis, one NRPS gene is orthologous to an Aureobasidium pullulans NRPS. The most closely related NRPS of known function is sid2 of Ustilago maydis, which is responsible for production of hydroxamate siderophores 59 . The remaining two are of unknown function, although one is orthologous to an NRPS in Magnaporthe grisea, and the other is orthologous to an NRPS found in all other filamentous ascomycetes with genome sequence (see Methods).…”