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

Abstract

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 8-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, pSidl 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 pSidl was responsible for complementing the class II siderophore biosynthetic mutation.

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

“…Mutagenesis of U. maydis with nitrosoguanidine treatment previously led to the isolation of several classes of siderophore auxotrophic mutants (42). The ferrichrome Ϫ (Fc Ϫ ) phenotype was assigned to the mutant S1, a derivative of strain 518 (a2b2), because no ferrichrome was detected by high-pressure liquid chromatography in the culture supernatants when the mutant was grown in low iron medium for at least 4 days (42). To determine the genetic basis of the siderophore-nonproducing phenotype, S1 was independently crossed with the compatible wild-type strain 521 (a1b1) and the auxotrophic mutant 288 (a1b1 pan1-1 inos1-3 nar1-1 rec 1-1) and siderophore production of the basidiospore segregants was examined by assessing their ability to support the growth of the enterobactin-deficient Salmonella serovar Typhimurium LT2 strain enb-7 on E medium.…”

“…Mutagenesis of U. maydis with nitrosoguanidine treatment previously led to the isolation of several classes of siderophore auxotrophic mutants (42). The ferrichrome Ϫ (Fc Ϫ ) phenotype was assigned to the mutant S1, a derivative of strain 518 (a2b2), because no ferrichrome was detected by high-pressure liquid chromatography in the culture supernatants when the mutant was grown in low iron medium for at least 4 days (42).…”

“…Minimal, complete, and plate media used in these studies were adapted from Holliday (22) as previously described (41). Low-iron medium, E medium, and EM medium were previously described (42). Escherichia coli DH5alpha (Bethesda Research Laboratories, Bethesda, Md.)…”

“…Production of ferrichrome by U. maydis colonies was routinely assessed by the ability of colonies to cross feed the Salmonella serovar Typhimurium LT-2 mutant, as described by Wang (42). To confirm the production of ferrichrome, an XAD-4 resin-based extraction procedure was used.…”

“…Two ferrichrome biosynthesis-related genes, sid1 and urbs1, encoding L-ornithine N 5 -oxygenase and a GATA family transcription factor, respectively, have been previously identified and characterized. sid1 is responsible for the first committed step, the hydroxylation of L-ornithine, in ferrichrome and ferrichrome A biosynthesis (28,42). Northern hybridization analysis has shown that the accumulation of sid1 transcript is negatively affected by iron concentration in the growth medium (28).…”