A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori Beth M. Carpenter1, Hanan Gancz1, Reyda P. Gonzalez-Nieves1, Abby L. West2, Jeannette M. Whitmire1, Sarah L. J. Michel2, D. Scott Merrell1* 1 Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America, 2 Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland, United States of America Abstract Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter. Citation: Carpenter BM, Gancz H, Gonzalez-Nieves RP, West AL, Whitmire JM, et al. (2009) A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori. PLoS ONE 4(4): e5369. doi:10.1371/journal.pone.0005369 Editor: Niyaz Ahmed, University of Hyderabad, India Received August 25, 2008; Accepted March 27, 2009; Published April 28, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: Research in the laboratory of D. Scott Merrell is made possible by grants R073LA from USUHS and AI065529 from the NIAID. Contents of this manuscript are the sole responsibility of the authors and do not necessarily represent the official views of the NIH or the DOD. The funders has 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. * E-mail: dmerrell@usuhs.mil Introduction Helicobacter pylori is an important human pathogen that infects over 50% of the world’s population [2]. While infection is predominantly asymptomatic, this bacterium is associated with development of gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. Infection often occurs early in childhood and persists throughout a person’s lifetime unless they are treated with specific antibiotics [3]. Given its propensity for chronic colonization and the substantial number of infected individuals, H. pylori imposes a significant disease burden worldwide. This microaerophilic, Gram negative bacterium is interesting in that it colonizes and survives within the gastric mucosa of the human stomach. H. pylori is well suited to life within this niche and has many factors that enable it to thrive there [2,4]. One such factor, the Ferric uptake regulator (Fur), functions as a transcrip- tional regulator that is involved in maintaining iron homeostasis [5]. Iron is essential for bacterial survival and is a co-factor in a variety of proteins; however, iron is redox active and can promote oxidative damage making it imperative that intracellular iron levels are tightly controlled. One particularly deleterious reaction that free iron can promote is reaction with reactive oxygen species (ROS) to form highly reactive hydroxyl radicals via Fenton chemistry. Hydroxyl radicals cause DNA and cellular damage that eventually lead to cell death. Thus, cells must strive to maintain a balance between insufficient and excess iron. Fur is involved in preserving this fine balance in H. pylori, and consequently, it is not surprising that fur has been shown to be critical for colonization in both gerbil and murine models of infection [6,7]. Fur is conserved in a wide variety of bacterial species and functions similarly in all of them by repressing gene expression under conditions of sufficient cellular iron. When Fur is bound to its iron (Fe2+) co-factor, it binds to specific regions in iron- regulated promoters called Fur Boxes and blocks the binding of RNA polymerase. Genes regulated in this manner are often associated with iron acquisition and are repressed under iron replete conditions to prevent the harmful effects of iron overload. While H. pylori Fur has been found to repress a set of genes in its iron-bound state, it has also uniquely been found to repress an additional set of genes in the absence of the iron cofactor, i.e. when Fur is in its apo form. apo-Fur regulation involves repression of an iron storage gene and occurs under iron limited conditions [8]. apo-Fur regulation has not been described for other bacterial species, and given that Fur plays a role in global gene regulation in response to environmental stressors and enhances the fitness of H. pylori as a pathogen, functional studies of Fur in H. pylori are of particular interest. One gene known to be repressed by apo-Fur in H. PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5369