The 11th Nitrogen Cycle Meeting 2005 185 N-oxide sensing and denitrification: the DNR transcription factors S. Rinaldo 1 , G. Giardina, M. Brunori and F. Cutruzzol ` a Department of Biochemical Sciences ‘A. Rossi Fanelli’ and Istituto di Biologia e Patologia Molecolari – CNR, University of Rome La Sapienza, P. le A. Moro 5, 00185 Rome, Italy Abstract All denitrifiers can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels by controlling the expression of denitrification gene clusters by redox signalling through transcriptional regulators belonging to the CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulator) superfamily. Introduction Nitric oxide (NO)-responsive regulators belong to three dif- ferent subgroups of the CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulator) superfamily [FNR, DNR (dissimilative nitrate respiration regulator) and NnrR] and can control N-oxide homoeostasis under both anaerobic and aerobic conditions. The FNR-type share a cysteine-rich motif involved in the formation of an iron– sulphur cluster as a redox centre, which is not present in the other two subgroups. Most of the regulators involved in the regulation of denitri- fication, belonging to the DNR and NnrR subgroups, regu- late nitrite reductase (nir), NO reductase (nor) and nitrous oxide reductase (nos) gene expression. The NO dependence of the transcriptional activity of promoters regulated by these transcription factors has suggested that these factors may act as NO sensors in vivo. To date, no structural information and little biochemical data are available on this class of regulators. In order to gain insights into the molecular and structural basis of NO-dependent regulation, we have recently ex- pressed in Escherichia coli and partially characterized the DNR protein from Pseudomonas aeruginosa. NO-responsive elements belonging to the CRP/FNR superfamily of transcription factors The expression of the denitrification gene clusters is tightly controlled by redox signalling through a cascade of oxygen- responsive regulators activating the N-oxide-responsive genes. These regulators control NO homoeostasis, maintain- ing the steady-state concentrations of nitrite and NO below cytotoxic levels; as a consequence, the free NO concentration is in the nanomolar range. Key words: dissimilative nitrate respiration regulator (DNR), denitrification, cAMP receptor protein/fumarate and nitrate reductase regulator superfamily (CRP/FNR superfamily), nitric oxide, Pseudomonas aeruginosa, transcriptional regulation. Abbreviations used: ANR, anaerobic regulation of arginine deaminase and nitrate reduction; CRP, cAMP receptor protein; DNR, dissimilative nitrate respiration regulator; FNR, fumarate and nitrate reductase regulator. 1 To whom correspondence should be addressed (email serena.rinaldo@uniroma1.it). The denitrification pathway is transcriptionally regulated by redox-linked transcription factors mostly belonging to the CRP/FNR superfamily [1], structurally related to the CRP protein from E. coli [2]. The CRP/FNR proteins are similar in size with approx. 230–250 amino acid residues, with the first 150–170 residues corresponding to the effector domain [1]. These regulators respond to both extracellular and intra- cellular signals by binding the allosteric effector either directly (as for cAMP in CRP from E. coli) or through a prosthetic group (as for the iron–sulphur cluster of FNR from E. coli) [3]. All members of this superfamily bind DNA via a C-terminal helix–turn–helix domain which inter- acts with the major groove of target DNA sequence, the FNR box (TTGATN 4 ATCAA) [1]. Multiple members of these regulators, belonging to differ- ent subgroups, can either coexist in the same host or regulate the same metabolic pathway in different organisms [1]. This is the case for the regulators of denitrification and in general for NO-responsive components which belong to three differ- ent subgroups of the CRP/FNR superfamily (FNR, DNR and NnrR) and can control N-oxide homoeostasis under both anaerobic and aerobic conditions [1]. To date, no struc- tural information and limited biochemical data are available for the last two subgroups involved in the regulation of denitrification, while the first one is well characterized. In E. coli, as an example, nitrosative stress induces expression of the flavohaemoglobin protein, encoded by the hmp gene, which acts as a NO scavenger. The hmp gene expression requires the FNR protein, which is a repressor under normal conditions; in the presence of NO, the [4Fe-4S] 2+ cluster is converted into the [2Fe-2S] 2+ state, inducing monomer formation in the FNR protein [4]. The FNR form modified by NO binds the hmp promoter with lower affinity, inducing flavohaemoglobin expression. The DNR-type of transcription regulators All members of the DNR subgroup share the same motif (EXXSR) involved in recognition of the binding site on DNA, while most members of the NnrR subgroup contain C  2006 Biochemical Society