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Dual regulation of catecholate siderophore biosynthesis in Azotobacter vinelandii by iron and oxidative stress

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9¹

Author for correspondence: William J. Page. Tel: +1 780 492 4782. Fax: +1 780 492 2216. e-mail: bill.page{at}ualberta.ca

Azotobacter vinelandii forms both catecholate and azotobactin siderophores during iron-limited growth. Azotobactin is repressed by about 3 µM iron, but catecholate siderophore synthesis continues up to a maximum of 10 µM iron. This suggests that catecholate siderophore synthesis is regulated by other factors in addition to the ferric uptake repressor (Fur). In this study the first gene required for catecholate siderophore biosynthesis, which encodes an isochorismate synthase (csbC), was isolated. The region upstream of csbC contained a typical ⁷⁰ promoter, with an iron-box overlapping the -35 sequence and a Sox-box (Box 1) overlapping the -10 sequence. Another Sox-box was found further upstream of the -35 sequence (Box 2). Also upstream, an unidentified gene (orfA) was detected which would be transcribed from a divergent promoter, also controlled by an iron-box. The activity of csbC and a csbC::luxAB fusion was negatively regulated by iron availability and upregulated by increased aeration and by superoxide stress. The iron-box in the csbC promoter was 74% identical to the Fur-binding consensus sequence and bound the Fur protein of Escherichia coli with relatively high affinity. Both Box 1 and Box 2 were in good agreement with the consensus sequence for binding the SoxS protein of E. coli and Box 1 was in very good agreement with the Sox-box found in the fpr promoter of A. vinelandii, which is also regulated by superoxide stress. Both Sox-boxes bound a protein found in A. vinelandii cell extracts, with Box 1 exhibiting the higher binding affinity. The Sox protein identified in this assay appeared to be constitutive, rather than inducible by superoxide stress. This indicates that the Sox response in A. vinelandii is different from that in E. coli. These data support the hypothesis that catecholate siderophore biosynthesis is under dual control, repressed by a Fur–iron complex and activated by another DNA-binding protein in response to superoxide stress. The interaction between these regulators is likely to account for the delay in ferric repression of catecholate siderophore production, since these siderophores have an additional role to play in the protection of iron-limited cells against oxidative damage.

Keywords: Azotobacter vinelandii, catecholate siderophores, isochorismate synthase, Fur, superoxide stress

Abbreviations: CFX, cell-free extract; FdI, ferredoxin I; Fpr, ferredoxin:NADPH reductase; Fur, ferric uptake regulator protein; IRP, iron-repressible promoter; Lux, bioluminescence; MV, methyl viologen; PDHE1, pyruvate dehydrogenase E1 subunit; SOD, superoxide dismutase; Sox, superoxide stress

The GenBank accession number for the sequence reported in this paper is AF238500.

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