|VON DOHREN, H.|
Submitted to: Industrial Microorganisms International Symposium
Publication Type: Proceedings
Publication Acceptance Date: 11/1/1996
Publication Date: N/A
Technical Abstract: Regulation of cellular iron homeostasis requires a careful balance between uptake of adequate iron for metabolism and restriction of iron uptake such that deleterious iron-catalyzed free radicals are not generated in vivo. In response to iron starvation Ustilago maydis produces the cyclic peptide siderophores ferrichrome and ferrichrome A(Budde,A.D.and S.A.Leong,1989, Mycopathologia 108)125-133).sid1 and sid2, two genes required for ferrichrome siderophore biosynthesis have been cloned.sid1 encodes ornithine-Nx10**5-oxygenase, while sid2 encodes a ferrichrome peptide synthetase. Regulation of sid1 has been studied by GUS reporter analysis and in vitro electrophoretic gel mobility shift analysis. As expected, GUS activity was regulated by the availability of iron. Sequences required for iron-mediated regulation of sid1 were identified by deletion and site- directed mutagenesis. Two GATA motifs located 1.6kb upstream of the transcription initiation site were required to mediate repression of sid1. Previous studies have led to the cloning of urbs1, a putative repressor of sid1. Electrophoretic gel mobility shift analysis using these motifs as well as GAT mutant derivatives as DNA probes indicated that Urbs 1 binds specifically to the GATA sequences of sid1. Mutation of the C-terminal finger motif but not the N-terminal finger motif of Urbs1 significantly reduced DNA binding activity. Efforts are underway to purify Urbs1 from U. maydis and to determine how Urbs1 senses intracellular levels of iron in cells. In vitro DNA binding and invivo reporter gene analysis has revealed that siderophores are not corepressors of Urbs1. As with the bacterial homologue Fur, we anticipate that iron may interact directly with Urbs1 to mediate repression of target genes.