Author's personal copy Unravelling the regulatory function of FurA in Anabaena sp. PCC 7120 through 2-D DIGE proteomic analysis Andrés González, M. Teresa Bes, M. Luisa Peleato, María F. Fillat ⁎ Department of Biochemistry and Molecular and Cell Biology, University of Zaragoza, 50009 Zaragoza, Spain Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, 50009 Zaragoza, Spain ARTICLE INFO ABSTRACT Article history: Received 27 December 2010 Accepted 2 February 2011 Available online 22 February 2011 The Anabaena sp. PCC 7120 ferric uptake regulator FurA controls iron homeostasis and appears implicated in a broad regulatory network, whereas failures to eliminate wild-type copies of furA gene from the polyploid genome suggest essential functions. In the present study, we comparatively analyzed the proteomes of a furA-overexpressing strain and its parental wild-type in conjunction with subsequent semi-quantitative RT-PCR and electrophoretic mobility shift assays, in order to identify direct transcriptional targets and unravel new biological roles of FurA. The results of such approach drove us to find 10 novel direct targets belonging to different functional categories including photosynthesis, energy metabolism, oxidative stress defences, redox regulation, signal transduction mechanisms, DNA replication, thiamine biosynthesis and heterocyst differentiation. Two peroxiredoxins and the thioredoxin reductase exhibited the most significant changes in both mRNA level and protein abundance under a FurA overexpression background, indicating a connection between iron metabolism, redox signalling and oxidative stress defences. A FurA box consensus sequence was ill-defined. The results suggest that particular DNA structures rather than a defined sequence govern FurA regulation of its target genes. Overall, the results provide new insights into the FurA regulon in Anabaena sp. PCC 7120. © 2011 Elsevier B.V. All rights reserved. Keywords: Anabaena Ferric uptake regulator 2-D DIGE Gene regulation Iron boxes 1. Introduction Iron is crucial for many major cellular processes, but also potentially toxic due to its tendency to catalyse free radical generation. Bacteria tightly regulate their iron metabolism in response to iron availability by a predominant regulatory system orchestrated by members of a family of proteins known as ferric uptake regulator (Fur). The classical model describes Fur as a dual function protein that senses intracel- lular free iron level and, under iron-rich conditions, binds to Fe 2+ and acts as a transcriptional repressor by binding to “iron boxes” located in the promoter regions of iron-responsive genes. Under iron-restricted conditions, the complex Fur-Fe 2+ is dissociated and the repressor becomes inactive, leading to transcription of target genes [1]. Several studies have shown that Fur works as a global regulator that control not only the expression of iron acquisi- tion and storage systems, but also a large number of genes and operons involved in different cellular processes [2–4]. In some microorganisms Fur has been also described acting as a positive regulator in controlling gene expression [5,6], while cross-talk with other transcriptional regulators [7], as well as iron-independent mechanisms of transcriptional repression [8] have been documented. In the filamentous nitrogen-fixing cyanobacterium Ana- baena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), the JOURNAL OF PROTEOMICS 74 (2011) 660 – 671 ⁎ Corresponding author at: Department of Biochemistry and Molecular and Cell Biology, University of Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain. Tel.: +34 976 76 12 82; fax: +34 976762123. E-mail address: fillat@unizar.es (M.F. Fillat). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.02.001 available at www.sciencedirect.com www.elsevier.com/locate/jprot