The second finger of Urbs1 is required for iron-mediated repression of sid1 in Ustilago maydis

Abstract

The urbs1 gene encodes a transcriptional regulator of siderophore biosynthesis in Ustilago maydis. Biological and DNA-binding activities of the two putative zinc-finger motifs of Urbs1 were studied by analyzing mutants containing altered finger domains. The mutated urbs1 alleles from three previously described N'-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutants were mapped and cloned by a gap-repair procedure. Sequence analyses revealed single amino acid substitutions in two of the NTG mutants. Both mutations (G-507 to D in urbs1-1 and P-491 to L in urbs1-3), which are located in the Urbs1 C-terminal finger domain, reduced DNA-binding activity by 10-fold and were sufficient to confer a urbs1-minus phenotype. The third NTG urbs1 mutant (urbs1-2) also contained a mutation in one of the conserved amino acids (P-518 to S) in the C-terminal finger domain, but this mutation alone was not sufficient to confer a urbs1-minus phenotype. A second frame shift mutation was identified in urbs1-2 and is necessary for the urbs1-minus phenotype. In an analysis of the function of the N-terminal finger of Urbs1, the conserved amino acid Arg-350 was mutated to leucine. A Urbs1 protein with this mutation complemented a urbs1 null mutant strain. By contrast, a similar mutation in the C-terminal domain abolished the ability of Urbs1 to regulate siderophore biosynthesis and greatly reduced its ability to bind target DNA.

Figure 1

(A) urbs1 locus of U. maydis. Horizontal arrow represents the urbs1 open reading frame (ORF). Restriction sites: A, AsuII; B, BamHI; BI, BglII; H, HindIII; P, PstI; S, StuI; X, XbaI. (B) Epitope-tagged urbs1 alleles. pAN15 contains the wild-type allele. pAN15–1, pAN15–2, and pAN15–3 contain the NTG-induced urbs1 mutations contained in the 2-kb StuI fragment from UMC002, UMC005, and UMC007, respectively. pAKR494L and pAKR350L contain single amino acid mutations introduced via site-directed mutagenesis.

Figure 2

Detection of epitope-tagged wild-type (pAN15) and mutant (pAN15–1, pAN15–2, and pAN15–3) Urbs1 by Western blotting. UMC015 is a urbs1 disruption mutant. pSC3 carries the wild-type urbs1 gene. pAN15 carries the urbs1-tag gene. pAN15–1, pAN15–2, and pAN15–3 carry the tagged mutant urbs1 alleles from UMC002 (urbs1–1), UMC005 (urbs1–2), and UMC007 (urbs1–3), respectively. Assay conditions are as previously described.

Figure 3

DNA-binding assay of the 0.3-kb DNA fragment with whole cell protein extracts of UMC015, UMC015/pSC3, UMC015/pAN15, UMC015/pAN15–1, UMC015/pAN15–2, and UMC015/pAN15–3. Strain UMC015 and various plasmids are described in Fig. 1 and in the text. Assay conditions are as previously described.

Figure 4

(A) DNA-binding assay of the 0.3-kb DNA fragment with whole cell protein extracts of UMC015, UMC015/pAN15, UMC015/pAKR350L, and UMC015/pAKR494L. Strain UMC015 is described in the text. pAKR350L and pAKR494L carry the Arg-350 to leucine mutation in the N-terminal finger domain and the analogous Arg-494 to leucine mutation in the C-terminal finger domain, respectively. Assay conditions are as previously described. (B) Detection of epitope-tagged wild-type (pAN15) and mutant (pAKR350L and pAKR494L) Urbs1 by Western blotting. Conditions are as in Fig. 2.