The Global Response Regulator RegR Controls Expression of Denitrification Genes in Bradyrhizobium japonicum Maria J. Torres 1 , Montserrat Argandon ˜a 2 , Carmen Vargas 2 , Eulogio J. Bedmar 1 , Hans-Martin Fischer 3 , Socorro Mesa 1 , Marı´a J. Delgado 1 * 1 Estacio ´ n Experimental del Zaidin, Consejo Superior de Investigaciones Cientı ´ficas (CSIC), Granada, Spain, 2 Departamento de Microbiologı ´a y Parasitologı ´a, Universidad de Sevilla, Sevilla, Spain, 3 ETH Zurich, Institute of Microbiology, Zurich, Switzerland Abstract Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544) or cy 2 (bll2388), and genes involved in nitric oxide detoxification (blr2806-09) and copper homeostasis (copCAB), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK-like gene (bll3466), and bll4130, which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC. P1 matched with the previously mapped 59end of norC mRNA which we demonstrate in this work to be under FixK 2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c-staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS. Citation: Torres MJ, Argandon ˜ a M, Vargas C, Bedmar EJ, Fischer H-M, et al. (2014) The Global Response Regulator RegR Controls Expression of Denitrification Genes in Bradyrhizobium japonicum. PLoS ONE 9(6): e99011. doi:10.1371/journal.pone.0099011 Editor: Szabolcs Semsey, Niels Bohr Institute, Denmark Received March 27, 2014; Accepted April 24, 2014; Published June 20, 2014 Copyright: ß 2014 Torres et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The microarray data are available in the NCBI Gene Expression Omnibus database (GEO; http://www.ncbi.nlm.nih.gov/geo) under GEO Series accession number GSE56668. Funding: Support was provided by Fondo Europeo de Desarrollo Regional (FEDER)-cofinanced grants AGL2010-18607, AGL2011-23383 and BIO2011-22833 from Ministerio de Economı ´a y Competitividad (Spain) (to MJD CV SM); Spanish National Network on Extremophilic Microorganisms (BIO2011-12879-E) (to CV); Junta de Andalucı ´a to Group BIO-275 and CVI-7293 (to EJB CV); Consejo Superior de Investigaciones Cientificas I3P Programme (to MJT); Grants from ETH Zurich and Functional Genomics Centre of ETH Zurich and University of Zurich (FGCZ) (to H-MF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: mdelgado@eez.csic.es Introduction The Rhizobiales order of a-Proteobacteria include Gram- negative nitrogen-fixing soil bacteria collectively named rhizobia which have the unique ability to establish N 2 -fixing symbioses with legume roots and stems of some aquatic legumes, leading to the formation of new plant organs called nodules. Expression of nitrogen fixation and other symbiosis-related genes requires low- oxygen conditions [1–2]. To cope with oxygen limitation prevailing under microoxic free-living conditions or in so-called bacteroids existing within plant cells of nodules, rhizobial species express the high-affinity cbb 3 oxidase encoded by the fixNOQP operon [3]. Moreover, some rhizobial species are able to use nitrate as final electron acceptor to support respiration under microoxic or anoxic conditions [4–6]. The switch from oxygen to nitrate respiration leads to a reduction in the ATP yield, yet it allows bacteria to survive and multiply under oxygen-limiting conditions [7]. Denitrification has been defined as the dissimilatory reduction of nitrate (NO 3 2 ) or nitrite (NO 2 2 ) to N 2 via the gaseous intermediates nitric oxide (NO) and nitrous oxide (N 2 O) with concomitant ATP generation [8]. This process requires four separate enzymatic reactions catalyzed by nitrate-, nitrite-, nitric oxide-, and nitrous oxide reductases, encoded by nar/nap, nir, nor, and nos genes, respectively [9–11]. In recent years, it has emerged that many rhizobial species have denitrification genes [4–6]. Among them, the soybean symbiont Bradyrhizobium japonicum is considered the model organism for studying rhizobial denitrification. In this bacterium, denitrification depends on the napEDABC, nirK, norCBQD, and nosRZDYFLX genes that encode a periplasmic nitrate reductase (Nap), a copper- containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), a nitrous oxide reductase (Nos), respectively [4]. Similar to PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99011