RESEARCH ARTICLE Summary. The nrdDG promoter regulates transcriptional expression of the anaerobic ribonucleotide reductase of Escherichia coli, an essential enzyme required to supply the building blocks for DNA synthesis. In this work, binding of the pleiotropic FNR (f umarate and n itrate r eduction) transcriptional regulator to the nrdDG promoter region and the effects of binding on transcription were investigated. Gel retardation analysis with purified FNR* demonstrated FNR interaction at two FNR sites, termed FNR-2 and FNR-1, while studies with altered FNR boxes indicated that the upstream FNR-2 site was essential for anaerobic activation of the nrdDG promoter. Although the FNR-1 site was not absolutely required, it allowed maximal expression of this promoter. These results suggest that the two sites have an additive effect in coordinating nrdDG expression in response to shifting oxygen concentrations. [Int Microbiol 2008; 11(1):49-56] Key words: Escherichia coli · fumarate and nitrate reduction (FNR) · ribonucleotide reductase · gene nrd Introduction Balanced de novo synthesis of deoxyribonucleotides (dNTPs) is an essential requirement of all DNA-based organ- isms. This function is supplied by the ubiquitous enzyme ribonucleotide reductase (RNR), whose allosteric regulation and unique chemistry provide the cell with a balanced pool of all four dNTPs by reducing the corresponding ribonu- cleotides (NTPs) [21]. Life on Earth has evolved to prolifer- ate under oxic and anoxic environments, an ability due in part to the evolution of three classes of RNRs, enzymes that carry out DNA synthesis and repair [29]. Class I RNRs con- tain a stable tyrosil radical and an oxygen-linked diferric center required for radical generation. Since this process requires oxygen, class I enzymes are only functional under oxic conditions. Class II enzymes require S-adenosylcobal- amine as a radical generator and do not depend on oxygen. Class III RNRs contain a free radical located on a glycyl residue. This radical is generated with the aid of S-adenosyl- methionine (SAM) together with an iron-sulfur cluster. Thus, class III enzymes are only active under strictly anoxic condi- tions. It seems reasonable that a strict aerobe should contain a class I enzyme, a strict anaerobe a class III enzyme, and facultative organisms RNRs of class II, but the situation is actually more complex. For example, some facultative microorganisms bear both class I and class III enzymes, but INTERNATIONAL MICROBIOLOGY (2008) 11:49-56 DOI: 10.2436/20.1501.01.44 ISSN: 1139-6709 www.im.microbios.org *Corresponding author: I. Gibert Genètica Molecular Bacteriana Institut de Biotecnologia i de Biomedicina Universitat Autònoma de Barcelona 08193 Bellaterra, Spain Tel. +34-935812050. Fax +34-935812011 E-mail: Isidre.Gibert@uab.cat ¶ Present address: Genes and Disease Program, Center for Genomic Regulation (CRG), Barcelona, Spain Present address: § Institute for Bioengineering of Catalonia (IBEC), Barcelona Scientific Park, Barcelona, Spain Ignasi Roca, Ester Ballana, ¶ Anaïs Panosa, Eduard Torrents, § Isidre Gibert* Bacterial Molecular Genetics Group, Institute of Biotechnology and Biomedicine, and Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, Spain Received 23 December 2007 · Accepted 24 February 2008 Fumarate and nitrate reduction (FNR) dependent activation of the Escherichia coli anaerobic ribonucleotide reductase nrdDG promoter