Crystal Structures of Two Transcriptional Regulators from Bacillus cereus Define the Conserved Structural Features of a PadR Subfamily Guntur Fibriansah 1¤a ,A ´ kos T. Kova ´ cs 2¤b , Trijntje J. Pool 1 , Mirjam Boonstra 2 , Oscar P. Kuipers 2 , Andy- Mark W. H. Thunnissen 1 * 1 Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands, 2 Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Abstract PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively). BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206–BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206–BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1) is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR- like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction. Citation: Fibriansah G, Kova ´cs A ´ T, Pool TJ, Boonstra M, Kuipers OP, et al. (2012) Crystal Structures of Two Transcriptional Regulators from Bacillus cereus Define the Conserved Structural Features of a PadR Subfamily. PLoS ONE 7(11): e48015. doi:10.1371/journal.pone.0048015 Editor: Eric Cascales, Centre National de la Recherche Scientifique, Aix-Marseille Universite ´ , France Received June 27, 2012; Accepted September 20, 2012; Published November 26, 2012 Copyright: ß 2012 Fibriansah 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. Funding: Funding was provided by the University of Groningen. Work was supported in part by an Ubbo Emmius Bursary (University of Groningen) awarded to GF. 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. * E-mail: a.m.w.h.thunnissen@rug.nl ¤a Current address: Laboratory of Virus Structure and Function, Duke-NUS Graduate Medical School, Singapore, Singapore ¤b Current address: Terrestrial Biofilms, Institute of Microbiology, Friedrich Schiller University of Jena, Jena, Germany Introduction Members of the PadR-like protein family (accession no. Pfam PF03551) form a large and widespread group of bacterial transcription factors that regulate diverse processes including multi-drug resistance, virulence and detoxification. They are named after the phenolic acid decarboxylation repressor, which is a transcription factor that represses genes associated with the phenolic acid stress response in gram-positive bacteria such as Bacillus subtilis, Pediococcus pentosaceus, and Lactobacillus plantarum [1,2]. Besides the founding member, only a few of the PadR-like proteins have been or are currently under investigation. These include AphA, which activates virulence gene expression in Vibrio cholerae [3], LmrR and LadR, which are repressors of genes encoding a multi-drug resistance pump in Lactococcus lactis and Listeria monocytogenesis, respectively [4,5], and Pex, a regulator of circadian rhythms in Synechococcus elongates [6]. Crystal structures have been reported of AphA, LmrR and Pex, confirming that these proteins share a common fold consisting of two domains: a highly conserved N-terminal winged helix-turn-helix (wHTH) domain of approximately 80–90 amino acid residues, involved in DNA-binding, and a variable C-terminal domain of one or more a-helices, involved in dimerization [7–9]. PadR-like proteins are related to members of the multiple antibiotic resistance regulator (MarR) protein family, which have similar wHTH DNA-binding domains but significantly larger and distinct C-terminal dimeriza- tion domains [10]. The size of the C-terminal domain is also a major discriminator among different PadR-like proteins, which have been classified into two distinct subfamilies [4]. PadR-like proteins of subfamily 1 (hereafter abbreviated as PadR-s1, approximately 180 amino acids), which include AphA, PadR and LadR, have relatively large C-terminal domains of about 80– 90 amino acids containing multiple a-helices, while subfamily 2 PadR-like proteins (PadR-s2, approximately 110 amino acids), which includes LmrR, have significantly smaller C-terminal domains of about 20–30 amino acids forming a single a-helix. PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e48015