Role of a GATA-type transcriptional repressor Sre1 in regulation of siderophore biosynthesis in the marine-derived Aureo
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Role of a GATA-type transcriptional repressor Sre1 in regulation of siderophore biosynthesis in the marinederived Aureobasidium pullulans HN6.2 Zhe Chi • Xing-Xing Wang • Qian Geng Zhen-Ming Chi
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Received: 14 January 2013 / Accepted: 21 August 2013 / Published online: 30 August 2013 Ó Springer Science+Business Media New York 2013
Abstract 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 50 -HGATAR-30 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 (Dsre1) (one of them was named R6) obtained still synthesized both intracellular and extracellular siderophores in the presence of added Fe3? and the expression of the SidA gene encoding 5 L-ornithine N -oxygenase in the disruptant R6 was
Z. Chi X.-X. Wang Q. Geng Z.-M. Chi (&) UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China e-mail: [email protected]
also partially derepressed in the presence of added Fe3?. The colonies of the disruptant R6 grown on the iron-replete medium with 1.5 and 2.0 mM Fe3? and also with 1.5 mM Fe2? became brown. In contrast, A. pullulans HN6.2 could not grow in the iron-replete medium with 1.5 mM and 2.0 mM Fe3?. The browncolored colonies of the disruptant R6 also had high level of siderophore and iron. Keywords The transcriptional repressor Marine-derived yeasts SRE1 gene Gene disruption Derepression
Introduction Iron can be used in many biochemical processes such as the synthesis of deoxyribonucleotides, respiration, the tricarboxylic acid cycle, and the synthesis of amino acids, lipids and sterols (Philpott 2006). It has been shown that both reductive iron assimilation (ferroxidation/permeation) and siderophore-mediated iron uptake (or non-reductive iron assimilation) are involved in iron uptake in fungi (Johnson 2008). Siderophores are lowmolecular weight, high affinity ferric iron-specific chelators. It has been well known that under iron starvation, most fungi excrete at least one type of siderophore in order to solubilize environmental iron and supply iron to the cells. Conversely, excess iron or incorrect storage of iron in the cells is deleterious as free iron catalyses the production of cell-damaging reac
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