Role of a GATA-type transcriptional repressor Sre1 in regulation of siderophore biosynthesis in the marine-derived Aureobasidium pullulans HN6.2.

The GATA-type transcriptional repressor structural gene SRE1 was isolated from both the genomic DNA and mRNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RACE. An open reading frame (ORF) of 1,002 bp encoding a 334 amino acid protein (a calculated isoelectric point: 8.6) with a calculated molecular weight of 35.1 kDa was characterized. The corresponding gene had one single intron of 51 bp, and in its promoter two putative 5'-HGATAR-3' sequences could be recognized. The deduced protein from the cloned gene contained two conserved zinc-finger domains [Cys-(X2)-Cys-(X17)-Cys-(X2)-Cys)], nine sequences of Ser(Thr)-Pro-X-X which was characteristics of the regulator, and one cysteine-rich central domain which was located between the two zinc fingers. The SRE1 gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase gene into the ORF of the SRE1 gene using homologous recombination. Two hundreds of the disruptants (Δsre1) (one of them was named R6) obtained still synthesized both intracellular and extracellular siderophores in the presence of added Fe(3+) and the expression of the SidA gene encoding L-ornithine N(5)-oxygenase in the disruptant R6 was also partially derepressed in the presence of added Fe(3+). The colonies of the disruptant R6 grown on the iron-replete medium with 1.5 and 2.0 mM Fe(3+) and also with 1.5 mM Fe(2+) became brown. In contrast, A. pullulans HN6.2 could not grow in the iron-replete medium with 1.5 mM and 2.0 mM Fe(3+). The brown-colored colonies of the disruptant R6 also had high level of siderophore and iron.