Structure and regulon of Campylobacter jejuni ferric uptake regulator Fur define apo-Fur regulation James Butchera,1, Sabina Sarvana,1, Joseph S. Brunzelleb, Jean-François Couturea,2, and Alain Stintzia,2 aDepartment of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada, K1H 8M5; and bDepartment of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 Edited* by Kenneth N. Raymond, University of California, Berkeley, CA, and approved April 10, 2012 (received for review November 24, 2011) The full regulatory potential of the ferric uptake regulator (Fur) genes encoding proteins involved in iron acquisition, oxidative family of proteins remains undefined despite over 20 years of stress defense, flagellar biogenesis, and energy metabolism (4, 13). study. We report herein an integrated approach that combines However, these studies failed to discriminate direct from indirect both genome-wide technologies and structural studies to define gene-regulatory mechanisms. the role of Fur in Campylobacter jejuni (Cj). CjFur ChIP-chip assays The structural basis underlying the regulation of gene expression identified 95 genomic loci bound by CjFur associated with functions by Fur proteins has been extensively documented (14–22). Fur as diverse as iron acquisition, flagellar biogenesis, and non-iron ion proteins consistently fold into two domains consisting of an transport. Comparative analysis with transcriptomic data revealed N-terminal DNA-binding domain (DBD) linked by a hinge region that CjFur regulation extends beyond solely repression and also to a C-terminal dimerization domain (DD). In contrast to the includes both gene activation and iron-independent regulation. common fold of these proteins, the coordination, number, and Computational analysis revealed the presence of an elongated geometry of the metal binding sites within Fur-family proteins di- holo-Fur repression motif along with a divergent holo-Fur activa- verge (2, 14, 15, 20, 22, 23). Despite these studies, the structural tion motif. This diversity of CjFur DNA-binding elements is sup- determinants controlling the divergent mode of regulation of gene ported by the crystal structure of CjFur, which revealed a unique expression by this family of proteins have remained unresolved. conformation of its DNA-binding domain and the absence of metal We herein provide the full extent of the C. jejuni Fur regulon in the regulatory site. Strikingly, our results indicate that the apo- using a chromatin immunoprecipitation and microarray analysis CjFur structure retains the canonical V-shaped dimer reminiscent of approach (ChIP-chip). Our results establish that CjFur regulates previously characterized holo-Fur proteins enabling DNA interac- 95 transcriptional units and report the presence of apo- and tion. This conformation stems from a structurally unique hinge do- holo-CjFur gene repression and activation in C. jejuni. Corre- main that is poised to further contribute to CjFur’s regulatory spondingly, crystallographic studies reveal that apo-CjFur adopts functions by modulating the orientation of the DNA-binding do- the canonical V-shaped dimer characteristic of holo-Fur pro- main upon binding of iron. The unique features of the CjFur crystal teins, with two zinc ions per protomer. However, comparative structure rationalize the binding sequence diversity that was un- analysis of apo-CjFur with other known Fur proteins reveals that covered during ChIP-chip analysis and defines apo-Fur regulation. apo-CjFur’s DBD is rotated by 180° compared with other known Fur structures, a structural rearrangement stemming from a gene regulation | metal regulation | transcription factor | DNA-protein reorientation of the apo-CjFur hinge region. Overall, our results interactions | structural characterization highlight the structural diversity of the Fur family of proteins and rationalize the consensus binding sequences revealed by our ron is critical to many fundamental biological processes, in- ChIP-chip and transcriptomics data. Icluding DNA synthesis, respiration, and the tricarboxylic acid cycle. Unfortunately, although iron is essential for life, it can also Results be toxic under physiological conditions. Its ability to undergo Identification of in Vivo Genome-Wide CjFur-Regulatory Targets. facile oxidation–reduction can catalyze the production of noxious Transcriptomic analysis of a Δfur mutant in the presence or ab- radical species by Fenton or Haber–Weiss chemistry (1). Most sence of iron established that CjFur may activate and repress gene Gram-negative bacteria maintain a remarkably precise control expression in both holo and apo forms (4). Given that tran- over cytoplasmic iron levels through the transcriptional regulator scriptomic approaches cannot distinguish direct from indirect reg- Fur (ferric uptake regulator) (1, 2). In the classical Fur regulation ulation, we sought to identify CjFur-binding regions on a genomic paradigm, Fur binds ferrous ions and the dimeric Fe2+–Fur scale using ChIP-chip experiments. We identified 95 high-confi- complex (holo-Fur) recognizes target sequences upstream of dence CjFur-regulated transcriptional units (Table S1); however, iron-regulated genes and represses their transcription (2). In due to the high gene density in the C. jejuni genome, we could not addition to regulating genes involved in iron homeostasis, Fur has distinguish between intergenic and intragenic binding. Fig. 1A been shown to play an important role in the modulation of bac- shows a schematic representation of the CjFur binding sites along terial virulence, acid, nitrosative, and oxidative resistances, and redox metabolism (2–9). In some bacterial species, Fur has been reported to directly Author contributions: J.B., S.S., J.-F.C., and A.S. designed research; J.B. and S.S. performed activate gene expression, establishing a significant departure from research; J.S.B. contributed new reagents/analytic tools; J.B., S.S., J.-F.C., and A.S. analyzed data; and J.B., J.-F.C., and A.S. wrote the paper. the classical model of Fur regulation (6, 7, 10). In Helicobacter fl MICROBIOLOGY pylori (Hp), apo-HpFur represses transcription of the bacter- The authors declare no con ict of interest. ioferritin-like gene pfr and the superoxide dismutase gene sodB, *This Direct Submission article had a prearranged editor. resulting in transcriptional activation of these genes in the pres- Database deposition: Crystallographic data, atomic coordinates, and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org ence of iron (10). Also, work on Bradyrhizobium japonicum Fur (PDB ID code 4ETS). protein (BjFur) and the Fur homolog BosR in Borrelia burgdorferi 1J.B. and S.S. contributed equally to this work. have demonstrated that certain Fur-family proteins can recognize 2To whom correspondence may be addressed. E-mail: [email protected] multiple consensus binding sequences (11, 12). Recent tran- or [email protected]. scriptomic analyses have revealed that Campylobacter jejuni Fur This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (CjFur) regulates, either positively or negatively, more than 60 1073/pnas.1118321109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1118321109 PNAS | June 19, 2012 | vol. 109 | no. 25 | 10047–10052 Downloaded by guest on September 26, 2021 leuC rpsJ Cj1583c ispA chuA A rrc B thyX Energy production and conversion (C) tupB Cell cycle control and mitosis (D) trxB Amino acid metabolism and transport (E) cfbpA Nucleotide metabolism and transport (F) ctsD Carbohydrate metabolism and transport (G) kpsT zupT Coenzyme metabolism (H) hddC Lipid metabolism (I) modB Translation (J) serC Replication and repair (L) fldA uvrA Cell wall/membrane/envelope biogenesis (M) Cell motility (N) ceuB Post-translational modification, chaperones (O) flaB Inorganic ion transport and metabolism (P) * Cj0415 pseA Secondary Structure (Q) fabG Signal Transduction (T) Cj1287c Intracellular trafficking and secretion (U) Cj0485 General Functions (R) Function Unknown (S) Cj1237c 02468101214 proS Number of Transcriptional Units C neuA1 holo-CjFur Repression holo-CjFur Activation Cj0644 ftsH uvrB * pyrB 2.0 2.0 kdtA Cj0742 1.0 bits 1.0 hisS bits purU C A glnH T AC C fbp dsbB T T A AT A T A T T T C G A T T GTGG CTA G A TTA T AAATA AAT A CT TA GAC AC TA G 0.0 TGA TAT A 0.0 T G TT TG T 5101520 5 10 15 secA Cj1005c Cj0980 Fig. 1. (A) Genomic map of CjFur-enriched transcriptional units overlaid with C. jejuni’s transcriptional response to iron limitation. The outer ring lists all of the transcriptional units that were enriched under Fur ChIP (≥1.5 enrichment, P ≤ 10−4). Blue denotes the gene that was found to be enriched under Fur ChIP. The inner ring displays the transcriptional response of each gene to iron limitation, with green denoting iron-repressed genes and red denoting iron-activated − genes (≥1.5 fold change, P ≤ 10 4). The figure was made using Circos version 0.54 (34). (B) COG functional groups present in CjFur ChIP-enriched tran- scriptional units. CjFur ChIP-enriched genes encompass a diverse range of COG functional categories, indicating that CjFur plays regulatory roles beyond iron metabolism. The COG functional category inorganic ion transport and metabolism was found to be statistically overrepresented (*). (C) Consensus sequences for CjFur binding sites. (Left) Holo-CjFur repression