Characterization of the Bacteroides fragilis bfr Gene Product Identifiesa Bacterial DPS-Like Protein and Suggests Evolutionary Linksin the Ferritin Superfamily

George H. Gauss,a,b Michael A. Reott,c Edson R. Rocha,c Mark J. Young,a,d,e Trevor Douglas,a,b C. Jeffrey Smith,c

and C. Martin Lawrencea,b

Thermal Biology Institutea and Departments of Chemistry and Biochemistry,b Microbiology,d and Plant Sciences and Plant Pathology,e Montana State University,Bozeman, Montana, USA, and Department of Microbiology and Immunology, East Carolina University, Brody School of Medicine, Greenville, North Carolina, USAc

A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical in-fections, is the bacterium’s extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess forma-tion. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNAlevels are increased in stationary phase or in response to O2 or iron. In addition, bfr null mutants exhibit reduced aerotolerance,and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein witha dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cys-teine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however,this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminaltail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interiorsurface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delin-eate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotationsfor B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin super-family that connects dodecameric DPS to the (bacterio)ferritin 24-mer.

Bacteroides fragilis is a strict anaerobe that comprises approxi-mately 1 to 2% of the normal intestinal flora in humans. While

normally present as a commensal bacterium in this reducing en-vironment, it is also the pathogenic anaerobe most frequently iso-lated from intra-abdominal infections, abscesses, and blood (27,57, 58). Factors contributing to the pathogenesis of B. fragilis in-clude resistance to oxidative stress and extreme aerotolerance,each of which is an important virulence factor for extraintestinalinfections (61).

A significant pathway for the production of reactive oxygenspecies (ROS) is dependent upon the presence of free Fe2�, whereFe2� and O2 react to form the toxic superoxide anion (19, 37).Biologically, superoxide is then converted to H2O2 and O2 by theaction of superoxide dismutase (69) or superoxide reductase (43).Then, via the Fenton reaction, the hydrogen peroxide may reactwith remaining Fe2� to produce the hydroxyl radical (HO·), themost toxic of all ROSs. Management of the intracellular iron poolis thus an important facet in the control of oxidative stress invirtually all organisms.

One strategy for limiting the availability of free iron is adoptedby ferritin, bacterioferritin, and DNA protection in nutrient-starved cells (DPS) protein (31, 37, 64). These members of theferritin superfamily utilize oxygen or hydrogen peroxide forthe oxidation of Fe2�, producing an insoluble iron oxide withinthe hollow core of these oligomeric proteins. By sequestering ironin the core, these proteins protect the cell from oxidative damageby simultaneously lowering the cellular levels of Fe2� and eitherH2O2 or O2. For these reasons, the roles of ferritin (ftnA) and DPSin the B. fragilis oxidative stress response have been investigated indetail (33, 49–52, 61). However, in addition to ferritin and DPS, B.

fragilis has a bacterioferritin-related gene, bfr, and a recent expres-sion microarray study shows that expression of the bfr gene issignificantly induced by exposure to air, suggesting that it mightalso help protect against ROS (61).

Further interest in B. fragilis bfr and its gene product stemsfrom a study suggesting that it might actually be more closelyrelated to the archaeal DPS-like (DPSL) proteins and that the bfrgene product is a member of the newly identified miniferritin thatis structurally distinct from both the larger ferritin and bacterio-ferritin 24-mers and dodecameric DPS (18, 28). Like DPS, thearchaeal DPSL proteins are expressed in response to oxidativestress, adopt the overall fold of the DPS subunit, and form dodeca-meric (12-mer) cage-like particles (28, 40, 47, 67). However, un-like DPS, where the ferroxidase site is located at the subunit inter-face, DPSL proteins contain a di-iron carboxylate binding siteburied within the four-helix bundle of each subunit, much likeferritin and bacterioferritin. Archaeal DPSL proteins are furtherdistinguished by a pair of conserved cysteine residues, apparentlyunique to the DPSL proteins, that are juxtaposed to thebacterioferritin-like dimetal binding site. This cysteine pair and

Received 9 May 2011 Accepted 12 October 2011

Published ahead of print 21 October 2011

Address correspondence to C. Jeffrey Smith, smithcha@ecu.edu, or C. MartinLawrence, lawrence@chemistry.montana.edu.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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the bacterioferritin-like ferrioxidase center, all housed within adodecameric oligomer, are hallmarks of the archaeal DPSL pro-teins and can be observed in the primary sequence as the thiofer-ritin motif (28). Together, these features differentiate DPSL as anew member of the ferritin superfamily.

Interestingly, the DPSL thioferritin motif is also found in everygreen sulfur bacterial genome sequenced to date and in severalspecies of Bacteroides, including the B. fragilis bacterioferritin-related, or bfr, gene product (15, 28). The original bfr annotationappears to stem from the similarity between the bacterioferritinand DPSL dimetal binding sites. However, this bacterioferritin-related protein sequence lacks the conserved methionine residuesthat coordinate the heme iron in bacterioferritins. All other pro-teins displaying the DPSL motif, i.e., conserved domain cd01052,also lack these methionines (28, 42). This suggests that the bfr geneproduct may in fact be a bacterial counterpart to the archaealDPSL proteins and that the occurrence of this newly describedminiferritin might extend into the bacterial domain of life.

Finally, there is a paucity of information on the oxidative stressresponse of anaerobic bacteria, and nearly nothing is known of themechanisms that they use to manage their iron pools. For thesereasons, we have explored the role of bfr and its associated geneproduct, which we designate B. fragilis DPSL (BfDPSL), in the B.fragilis oxidative stress response. We have characterized bfr mRNAlevels in response to O2, H2O2, inorganic iron, and growth stateand show that bfr null mutants displayed reduced aerotolerance.In addition, we show that BfDPSL has DNA protection activity invitro and present the findings of crystallographic studies that con-firm a DPS-like protein structure, but one that shows importantdifferences in comparison to the archaeal DPSL proteins.

MATERIALS AND METHODSConstruction of deletion mutant strains. B. fragilis strain 638R was thewild type (WT) strain used for expression and mutational analyses.Strains were grown in brain heart infusion (BHIS) broth that was supple-mented with cysteine (1 g/liter), hemin (5 mg/liter) or protoporphyrin IX(5 mg/liter), NaHCO3 (24 mM), and in some cases, FeSO4 (100 �M), asdescribed in the text (52). Mutant strains were constructed by allelic ex-change (see Fig. S1 in the supplemental material) as described previouslyusing the suicide vector pFD516 and the PCR primers described in TableS1 in the supplemental material (53). For the construction of mutantBER63, 400 bp from the central portion of the ftnA gene was deleted andreplaced with a 2.2-kb tetQ gene fragment. The BER74 mutant was con-structed by deletion of the bfr gene from bp 152 of the coding region to 48bp downstream of the C terminus of the coding region, and this wasreplaced by a 2.1-kb cefoxitin resistance gene, cfxA. The double mutant,BER75, was constructed by mobilization of the BER74 mutation constructinto BER63 with selection on cefoxitin followed by subsequent screeningfor erythromycin sensitivity.

Oxidative stress sensitivity assays. Disk diffusion assays to test forsensitivity to oxidative stress were performed by spreading 100 �l of anovernight culture on BHIS plates (without cysteine [BHIS-cys]), allowingthe plates to dry, and then adding a sterile 6-mm filter disk to the center ofthe plate. Ten microliters of 2 M diamide or 10% hydrogen peroxide wasadded to the disk, and then the plates were exposed to air (at 37°C) for 6 hprior to anaerobic incubation. Following overnight incubation, the diam-eters of the zones of growth inhibition were measured, and the results arethe averages from three independent experiments done in triplicate. Asignificant difference in inhibition of mutants compared to WT was de-termined by two-tail t test.

For the aerobic survival assays, overnight cultures were grown inBHIS-cys until they reached an optical density at 550 nm (OD550) of 0.5.Each strain was then serially diluted into BHIS-cys, and 5 �l was spotted

onto fresh BHIS-cys agar plates and placed in an aerobic incubator at37°C. At each time point, one plate was removed and placed into ananaerobic incubator at 37°C for at least 48 h until sufficient growth on theplate was observed.

RNA isolation and cDNA synthesis. RNA was harvested fromexponential- and stationary-phase cultures grown in BHIS with proto-porphyrin IX and FeSO4, as indicated in the text. Cultures were either keptanaerobic or exposed to air with shaking for 1 h. RNA was isolated usingthe hot acid-phenol method (53). Fifty micrograms of total RNA wasprecipitated with ethanol, and contaminating DNA was removed by treat-ment with Turbo DNA-free DNase (Ambion). For synthesis of cDNA,0.75 �g of RNA was added to reaction mixtures containing 10 ng/�lrandom hexamers, 0.5 mM deoxynucleoside triphosphates, first-strandbuffer (Invitrogen, Carlsbad, CA), and 1 �l Superscript II RNase H reversetranscriptase I; reaction mixtures were incubated at 42°C for 50 min; andSuperscript II was heat inactivated by incubating the reaction mixtures at70°C for 15 min.

Quantitative RT-PCR. Real-time reverse transcription-PCR (RT-PCR) was performed as described previously using a Bio-Rad iCyclerinstrument with real-time PCR detection (Bio-Rad) (48). Reaction mix-tures contained 12.5 �l 2� iQ Sybr green Supermix, 1.5 �l of 5 �Mforward primer, 1.5 �l of 5 �M reverse primer, 8.5 �l H2O, and 1 �l ofcDNA template (diluted 1/100) per well. Samples were run in triplicate,and RNA samples without the addition of reverse transcriptase were runas controls to monitor for genomic DNA contamination. Relative expres-sion values were calculated using the Pfaffl method (46). Fold inductionrelative to the wild type under anaerobic conditions, as indicated in thetext, was determined for each gene using 16S rRNA as a reference.

Cloning, expression, and purification. Genomic DNA from B. fragilisNCTC 9343 was obtained from the American Type Culture Collection.The BfDPSL gene (locus tag BF3271) of NCTC 9343 is identical to thegene from strain 638R and was amplified by PCR using the primersghg180 and ghg181. The BfDPSL gene was cloned into pDONR201 (In-vitrogen) after att sites were added in a second round of PCR using prim-ers 1-3 and attB2 (see Table S1 in the supplemental material). The se-quence of the pDONR201-BfDPSL clone was confirmed by sequencing.The BfDPSL gene was transferred into the expression vector pDEST14(Invitrogen).

The pDEST14-BfDPSL vector was transformed into Escherichia coliBL21(DE3)RIL (Stratagene) for expression of wild-type untagged BfDPSLprotein. Cells were grown to an OD600 of 2.0, and BfDPSL protein expres-sion was induced by addition of isopropyl-�-D-thiogalactopyranoside(IPTG) to a final concentration of 500 �M. At 4 to 6 h after induction, cellswere pelleted and frozen at �80°C. Growth medium contained 100 �g/mlampicillin, 34 �g/ml chloramphenicol, 3.5 g/liter KH2PO4, 5 g/literK2HPO4, 3.5 g/liter (NH4)2SO4, 0.5 g/liter MgSO4 · 7H2O, 5 g/liter yeastextract, 30 g/liter dextrose, 5.9 �M FeCl3, 0.84 �M CoCl2, 0.6 �M CuCl2,1.5 �M ZnCl2, 0.8 �M NaMoO4, and 0.8 �M H3BO3.

Cells were resuspended (0.2 g/ml) in lysis buffer (1 M NaCl, 20 mMbis-Tris HCl, pH 6.5, 100 �M phenylmethylsulfonyl fluoride [PMSF])and lysed with a microfluidizer (Microfluidics). Cellular debris was pel-leted (20 min, 30,000 � g, 4°C), and the supernatant was heat treated (10min at 65°C). The sample was centrifuged again (20 min, 30,000 � g, 4°C),and 106 g/liter (NH4)2SO4 was added to the supernatant. After centrifu-gation (20 min, 30,000 � g, 4°C), the supernatant was bound to phenylSepharose (GE Healthcare). After washing with 800 mM (NH4)2SO4, 20mM bis-Tris HCl, pH 6.5, protein was eluted with an 800 to 0 mM(NH4)2SO4 gradient. Fractions were analyzed on 10% SDS Tris-Tricinegels. Fractions containing BfDPSL were pooled and diluted 1:5 into 0.15M NaCl, 20 mM bis-Tris HCl, pH 6.5, and loaded onto a HiTrap Qcolumn (GE Healthcare). The column was washed with 0.15 M NaCl, 20mM bis-Tris HCl, pH 6.5, and eluted with a gradient of 0.15 to 0.6 MNaCl. Fractions containing BfDPSL were pooled, concentrated by ultra-filtration to 5 mg/ml, and applied to a Superdex 200 column (GE Health-care) equilibrated in 0.3 M NaCl, 20 mM bis-Tris HCl, pH 6.5. BfDPSL

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was concentrated to 15 mg/ml and stored at 4°C or frozen in liquid nitro-gen and stored at �80°C. Protein concentrations were determined withthe Bradford assay using bovine serum albumin as a standard (9).

E. coli DPS (EcDPS) was overexpressed from pET-EcDPS inBL21(DE3)RIL cells by IPTG induction, using conditions similar to thoseused for BfDPSL overexpression. Cells were lysed by a French press in 50mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.5 mM EDTA, 0.1 mM PMSF.Insoluble material was pelleted (20 min, 30,000 � g, 4°C), ammoniumsulfate was added to the supernatant (390 g/liter), and the mixture wasstirred 30 min at 4°C. Precipitated protein was pelleted as described aboveand resuspended in 50 mM Tris-HCl, pH 8, 2 M NaCl, 0.5 mM EDTA.The supernatant was dialyzed overnight at 4°C against 20 mM Tris HCl,pH 8.0, 0.5 mM EDTA, which causes DPS to precipitate. The DPS pelletwas resuspended in 50 mM Tris-HCl, pH 7.8, 300 mM NaCl, 0.5 mMEDTA. After a clarifying spin, the protein was bound to a Mono S column(GE Healthcare) equilibrated with 20 mM bis-Tris, pH 6.5, 250 mM NaCl,and eluted with a 250 mM to 1 M NaCl gradient. Fractions containingDPS were pooled, concentrated, and applied to a Superdex 200 column(GE Healthcare) equilibrated with 20 mM bis-Tris, pH 6.5, 300 mM NaCl.The peak fraction was concentrated to 11 mg/ml and stored at 4°C orfrozen in liquid nitrogen and stored at �80°C.

Antisera and Western hybridizations. Imgenex (San Diego, CA) wassupplied with purified BfDPSL for the production of rabbit anti-BfDPSLantiserum that was used for Western blotting. Samples for Western blot-ting were obtained from exponential- and stationary-phase culturesgrown in BHIS supplemented as described in the text and either keptanaerobic or exposed to atmospheric oxygen with aeration for an hour.Cultures were centrifuged and stored at �80°C. Protein concentrationwas determined using Bio-Rad Better Bradford assay reagents. Sampleswere adjusted to equal amounts of protein, and then each sample wasadded to loading buffer containing dye in a total volume of 20 �l, boiledfor 10 min, allowed to quickly cool on ice, and then loaded onto a 12%SDS-polyacrylamide gel. Proteins were then transferred to a polyvi-nylidene difluoride membrane, probed with primary antibody, and thenlabeled with an alkaline phosphatase secondary antibody, and bands werevisualized using chromogenic detection with nitroblue tetrazolium and5-bromo-4-chloro-3=-indolylphosphate. Digital densitometric analysis ofphotographic images was performed using ImageQuant software (Amer-sham Bioscience) to estimate relative band intensity. For these analyses,SDS-polyacrylamide gels in which the total protein concentration of sam-ples was equalized over a range of from 0.8 to 25 �g of protein were tested.Following chromogenic development of the blots, densitometric mea-surements from at least 3 independent gels were obtained.

Particle characterization. The oligomeric state was routinely esti-mated using calibrated Superdex 75 HR10/30 and Superdex 200 HR 10/30columns (GE Healthcare) run at 25°C on a fast-performance liquid chro-matography system with a UV monitor. The molecular mass of the dimerpeak was verified by size exclusion chromatography (SEC; Wyatt WTC-0305S column) with inline multiangle light scattering (MALS; WyattHELIOS 8) and refractive index (Wyatt Optimus T-rEX) detectors (seeFig. S2 in the supplemental material). Samples were run at 25°C in 20 mMbis-Tris HCl, pH 6.5, 300 mM NaCl. The mass was calculated with theASTRA (version 5.3.14) software package (Wyatt Technology) using adn/dc value of 0.185 (7). Using the concentrated material, the particle sizewas estimated by transmission electron microscopy (TEM; Leo 912ABTEM; Oberkochen, Germany) with a microscope operating at 120 keV.Samples for TEM were stained with 2% uranyl acetate.

DNA protection assay. Assays were performed as described previ-ously (8) with the following modifications. BfDPSL and EcDPS were ex-changed by SEC into 20 mM bis-Tris HCl, pH 7.0, 300 mM NaCl. DNAbinding reaction mixtures contained 36 �M protein (subunit basis), 18nM plasmid, 20 mM bis-Tris HCl, pH 7.0, 50 mM NaCl, and were incu-bated for 30 min at 22°C. For DNA protection, 144 �M FeSO4 and 10 mMH2O2 were added 5 min after mixing protein and DNA. The ratio of Fe2�

to BfDPSL was thus 4 atoms per subunit, or 48 atoms per dodecamer.

Where indicated, protein/DNA complexes were disrupted by addition ofSDS to 2% and incubation at 80°C for 3 min. Samples were run on 0.8%agarose gels in TAE (Tris-acetate-EDTA) buffer and stained withethidium bromide.

Mineralization assay. Iron was added in small aliquots [0.5 �l of 0.1M (NH4)2Fe(SO4)2 (FAS) dissolved in deaerated H2O] to 22 �l of protein(1.2 � 10�8 moles of subunit) in 0.1 M bis-Tris, pH 6.5, 0.3 M NaCl at22°C. E. coli DPS was used as a positive control, and bovine serum albu-min (BSA) was used as a negative control. Nine aliquots of FAS wereadded over 90 min, for a total iron loading of 4.5 � 10�7 moles or 450 Featoms per dodecamer. For mineralization reactions with H2O2 as oxidant,aliquots (0.5 �l of 50 mM H2O2) were added 2 to 3 min after each FASaddition; and the reaction was performed within a Coy anaerobic glovebox using deaerated solutions. For assays using O2 as oxidant, aliquots ofdeaerated FAS were added to protein under standard atmospheric condi-tions. Ten minutes after the final FAS addition, 1.5 �l of 1.3 M sodiumcitrate, pH 6.0, was added to chelate residual iron. Mineralized proteinswere then visualized by Prussian blue stain, as shown by Allen et al. (1).Briefly, reaction mixtures were loaded onto a nondenaturing 0.8% aga-rose gel (20 �g protein per lane) and electrophoresed at 15°C. The elec-trophoresis buffer and gel contained 0.1 M Tris-acetate, pH 8.5, 0.3 MNaCl, 1 mM EDTA, and 20% (wt/vol) glycerol. The gel was first stainedwith Prussian blue (2.5% [vol/wt] K4Fe(CN)6 · H2O, 10% [vol/vol] HCl)to visualize iron and then stained with Coomassie blue to visualize pro-tein.

Crystallization and data collection. BfDPSL was crystallized by vapordiffusion in hanging drops at 22°C. Two microliters of protein solution(13 mg/liter) was added to 2 �l of crystallization solution containing 0.1M Tris HCl, pH 8.5, 0.25 M MgCl2, 16% polyethylene glycol 4000, 2%(wt/vol) benzamidine HCl, and 20% glycerol. The drop was placed over 1ml of well solution that was identical in composition to the crystallizationsolution, except that benzamidine HCl was omitted. Crystals appearedwithin 2 to 4 days. Crystals were then frozen by plunging into liquidnitrogen. A single data set to 2.3-Å resolution was collected at the peakwavelength of the zinc K edge (1.282 Å) on Stanford Synchrotron radia-tion light source (SSRL) beam line 9-2 (Table 1). The data were integratedand reduced in space group P213 using the HKL2000 package (44).

Structure determination and refinement. Initial phases to 3.0 Å werefound by molecular replacement using the COMO program (35) with asearch model derived from Sulfolobus solfataricus DPSL (SsDPSL). TheCHAINSAW program (21) was used to modify the SsDPSL subunit (Pro-tein Data Bank [PDB] accession number 2CLB), and the successful searchmodel contained four copies of the modified subunit. The four subunits inthe asymmetric unit lie along a crystallographic 3-fold axis and in con-junction with two adjacent asymmetric units form a dodecameric particlein the crystal lattice. Phases were extended to 2.3 Å using the RESOLVEprogram (63) with noncrystallographic symmetry averaging, solvent flat-tening, and histogram matching. The resulting phases yielded an easilyinterpretable electron density map with two strong peaks for the metalpositions in the dimetal binding site. These positions were modeled asiron atoms, consistent with inductively coupled plasma mass spectrome-try (ICP-MS) data (Energy Laboratories, Billings, MT) from the purifiedprotein that indicated 1.2 Fe atoms per BfDPSL subunit, although zincwas also present in smaller amounts (0.3 Zn atoms per subunit). A thirdputative metal position on the inside surface of the particle (site C) wasassigned to Mg, as MgCl2 is present in the crystallization solution andgenerally adopts the observed octahedral coordination. Anomalous dif-ference maps provided no evidence for iron or zinc at this position. Incontrast to Mg, placement of water at this position resulted in a significantresidual difference density. Iterative rounds of manual model rebuildingwith the COOT program (21) and refinement with the REFMAC5 pro-gram (21) yielded a final BfDPSL model with Rwork equal to 19.5% andRfree equal to 22.3%. Noncrystallographic restraints were used in refine-ment. The MOLPROBITY server was used for structure validation (23).Molecular graphics figures were prepared with the PyMOL tool (24). Elec-

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trostatic surface potential was calculated with the adaptive Poisson-Boltzmann solver (APBS) using a temperature of 310 K, protein and sol-vent dielectric constants of 2 and 80, and 150 mM monovalent saltconcentration (6). Metal ions were excluded from the APBS calculations.

Protein structure accession number. Atomic coordinates and struc-ture factors for BfDPSL have been deposited with the Protein Data Bankunder accession number 2VZB.

RESULTSOxidative stress and growth phase control of bfr. The bfr geneproduct, which we refer to as BfDPSL, is one of three potentialiron storage proteins encoded within the B. fragilis genome (bfr,ftnA, dps). In a previous study on the B. fragilis transcriptome, itwas observed that while expression of bfr is not induced by H2O2,it was induced following exposure to air for 1 h, suggesting a rolein managing oxidative stress (61). In order to begin to understandits role in iron homeostasis and aerotolerance, bfr transcriptionalregulation was examined in more detail and was compared toexpression of ftnA, whose role in B. fragilis oxidative stress hasbeen previously characterized (52, 61). In log-phase cultures, bothgenes were induced after exposure to air. Air exposure increasedbfr and ftnA expression by 35- and 8-fold, respectively (Fig. 1).When cultures were grown in the presence of excess iron (added tomedium in the ferrous form) and then exposed to air, there was afurther 15-fold increase in bfr mRNA (relative to wild type), whilea more modest 4-fold increase was observed for ftnA. There was

no noticeable effect on expression of either gene when excess ironwas added to anaerobic cultures (data not shown). Aerobic induc-tion of bfr was also observed at the protein level by comparinganaerobic and aerobic cultures using polyclonal anti-BfDPSL an-tibody. As shown in the Western blot in Fig. 2, there was a 1.8(�0.2)-fold increase in the amount of BfDPSL when cultures ofthe wild-type strain were exposed to air compared to that whenthe cultures were anaerobic. The increase in BfDPSL protein didnot match the larger increase in transcription, but this may be dueto posttranscriptional control or the limited availability of re-

TABLE 1 Data collection and refinement statistics

Parameter Value(s)

Data collection statisticsSpace group P213Unit cell parameters, a, b, c (Å) 129.695Wavelength (Å) 1.2822Resolution range (Å)a 50.00–2.30 (2.38–2.30)Total no. of reflections 117,520No. of unique reflections 32,431Total no. of possible unique reflections 32,561Redundancy 3.6Data coverage (%) 99.6 (99.4)Mean I/sigma I 17.5 (2.9)Rsym (%)b 7.7 (24.4)

Refinement statisticsResolution range (Å) 37.00–2.30Rcryst (%)/Rfree (%)c 19.5/22.3Coordinate error (Å)d 0.170Real space CCe 0.951RMSDf from ideality, bonds (Å)/angles (°) 0.011/1.134Ramachandran plot, favored/outliers (%)g 98.6/0.0Avg residual B values (Å2) 34.822PDB accession no. 2VZB

a Numbers in parentheses refer to the highest-resolution shell.b Rsym � 100 · �h�i |Ii(h) � �I(h)�|/�hI(h), where Ii(h) is the ith measurement of thehth reflection, and �I(h)� is the average value of the reflection intensity.c Rcryst � S||Fo| � |Fc||/S|Fo|, where Fo and Fc are the structure factor amplitudes fromthe data and the model, respectively. Rfree is calculated similarly, using 5% of thestructure factors held back as a test set.d Based on maximum likelihood.e Correlation coefficient (CC) is between the model and the electron density mapcalculated in the conventional way using sigma A weighted Fourier coefficients(2mFo � DFc).f RMSD, root mean square deviation.g Calculated using the MOLPROBITY server (24).

FIG 1 Effect of aerobic exposure on bfr and ftnA expression in the WT andftnA mutant strains. Exponential-phase cultures were exposed to air for 1 h,and total RNA was extracted and used in quantitative RT-PCRs with primersfor the bfr and ftnA genes. Fold induction was determined from baseline valuesobtained for an exponential-phase, anaerobic culture of the wild-type straingrown in BHIS with protoporphyrin IX. �Fe, supplementation of mediumwith FeSO4 (100 �M). bfr (striped bars) and ftnA (gray bars) expression wasdetermined by triplicate assays from two independent RNA samples.

FIG 2 Growth phase and aerobic exposure modulate expression of BfDPSL.Cultures of the WT and bfr mutant (A) or WT and ftnA mutant (B) were grownanaerobically to mid-logarithmic phase (An), exposed to air for an hour (Air),or allowed to grow anaerobically for 24 h into stationary phase (Stationary).Growth medium was supplemented with protoporphyrin IX and FeSO4. Thecells were then harvested, equal amounts of protein (2.5 �g [A] or 1.25 �g [B])were electrophoresed on SDS-polyacrylamide gels, and Western hybridizationwas performed using BfDPSL polyclonal antibody. The arrow to the left of theblot indicates the BfDPSL monomer at 19.6 kDa. The molecular mass markershows the 26- and 17-kDa proteins in the first lane.

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sources for protein synthesis under the growth-restrictive, aerobicconditions.

Growth phase is known to be important for the control ofboth bacterioferritin and DPS in many bacteria, although it isnot a major regulator of the archaeal Sulfolobus solfataricusDPSL (SsDPSL). Results in Fig. 2B indicate that there was a 3.8(�1.4)-fold increase in the amount of BfDPSL present instationary-phase cultures of B. fragilis. The levels of bfr mRNA inthese stationary-phase cultures increased slightly, but statistically,they were not significantly greater than those in the mid-logarithmic-phase anaerobic cultures (data not shown). Thus, theaccumulation of BfDPSL in stationary phase may suggest that theprotein is relatively stable and not subject to rapid degradation.

The bfr gene product protects against oxidative stress. Theresults from studies on transcriptional regulation of bfr thus sug-gested that it has a role in protection against oxidative stress. Wemade bfr, ftnA, and double bfr ftnA mutants and examined theirphenotypes in several oxidative stress assays. Since both genes aremonocistronic (see Fig. S1 in the supplemental material), there islittle likelihood that mutations will be polar on the downstreamgenes. As shown in the disk diffusion assays, Fig. 3A, neither singlemutation resulted in an obvious phenotype with H2O2 or di-amide, but we did note that the bfr and the double mutant hadsomewhat greater sensitivity to cumene hydroperoxide. The dou-ble mutation in the bfr ftnA mutant strain resulted in a significantincrease in sensitivity to diamide but not to H2O2 (Fig. 3A). Theweak phenotype against H2O2 may be due to the fact that thecatalase in these strains is highly active and may mask the pheno-type. A second measure of resistance to oxidative stress was ob-tained in the long-term aerobic survival experiment, in whichstrains were exposed to aerobic conditions for up to 84 h (Fig. 3B).The wild-type parental strain was able to survive for more than 84h, but the bfr and ftnA mutants lost viability much sooner andthere was a difference of 2 orders of magnitude in survival afterabout 60 h of aerobic exposure. The double ftnA bfr mutant wassomewhat more sensitive to these conditions than either singlemutant strain in this assay.

The observation that a double mutant lacking both ftnA and bfrhad a more pronounced phenotype in some of the oxidative stressassays suggested that there is complex interplay between the rolesof the different ferritin family proteins in B. fragilis. As shown inFig. 1, there was a tendency for an increase (about 8- to 10-fold) inbfr expression in the ftnA mutant strain background relative to thewild-type strain, but this increase was not observed at the proteinlevel. Results in Fig. 2B suggest that levels of BfDPSL in the ftnAmutant are similar to those in the wild-type strain during stress.Likewise, there was no change in the accumulation of BfDPSL inthe wild-type strain compared to the ftnA mutant in stationary-phase cultures. These results suggest that the ftnA mutant did notdirectly compensate for the loss of FtnA with increased synthesisof BfDPSL during aerobic exposure or stationary phase. It is alsoworth noting that there was no evidence for increased transcrip-tion of the ftnA gene in the bfr mutant (data not shown).

Biochemical characterization. Recombinant BfDPSL was ex-pressed in E. coli and purified to homogeneity. The purified pro-tein behaves as a dimer on the size exclusion column, and this hasbeen confirmed with SEC-MALS (see Fig. S2 in the supplementalmaterial). However, at higher concentrations (13 mg/ml), spher-ically shaped particles with diameters of 95 Å were seen bynegative-stain transmission electron microscopy (see Fig. S3 in

the supplemental material), suggesting assembly of a dodecamericparticle like that expected for DPS or DPSL, as opposed to the24-mer common to ferritin and bacterioferritin. While membersof the ferritin superfamily are often found as stable oligomers,their assembly states can also be dependent upon changes in pH,salt concentration, metal ions, and temperature (8, 12, 14, 20, 30).However, despite surveying a variety of protein concentrationsand buffer conditions, we were unable to identify conditions thatgive a stable dodecamer on the SEC column. This suggests thatwhile BfDPSL is present in solution predominantly as a dimer, it isin dynamic equilibrium with higher-order oligomers, includingthe dodecamer, and is capable of rapid assembly and disassembly.

Many members of the ferritin superfamily sequester iron as aninsoluble oxide inside the oligomeric particle. We were thus inter-ested in whether BfDPSL possessed a similar activity. While ourpositive control, E. coli DPS, displayed robust mineralization ac-tivity using H2O2 as the oxidant (and minimal activity using O2),we did not observe mineralization activity with BfDPSL using ei-

FIG 3 Sensitivity of the wild-type, ftnA, bfr, and ftnA bfr strains tooxidative stress. (A) Growth inhibition was measured on agar plates as thediameter of the growth inhibition zone around the stress agent. Asterisks, astatistically significant difference from WT (P � 0.001). (B) Sensitivity ofstrains to exposure to air was tested by diluting cultures as indicated andspotting 5 �l of each dilution on a plate. The plates were then exposed to air ina 37°C incubator for 0, 60, 72, or 84 h, followed by anaerobic incubation for 2days to determine growth.

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ther O2 or H2O2 as the oxidant (see Fig. S4 in the supplementalmaterial). The lack of mineralization activity might be a conse-quence of the primarily dimeric form of DPSL that would be pres-ent in these assays. Alternatively, this may be a consequence of thedisulfide or the nonnative metals or mixed metal occupancy at thedimetal binding site, perhaps due to heterologous expression ofBfDPSL in E. coli. ICP-MS analysis of the purified protein shows1.2 Fe atoms and 0.3 Zn atom per subunit. Attempts to remove thecopurifying Zn have, to date, resulted in the production of insol-uble protein.

We next examined the ability of the predominantly dimericBfDPSL to protect DNA from strand cleavage in vitro in the pres-ence of Fe2�/H2O2. As shown in Fig. 4A, the protective activity ofBfDPSL is comparable to that of E. coli DPS, which was used as apositive control, and greater than that of BSA, which was used as anegative control. As expected, E. coli DPS formed a complex withDNA that prevented it from entering the gel (11), but BfDPSL didnot appear to bind DNA under the conditions of the DNA protec-tion assay (Fig. 4B).

Structural similarities between BfDPSL and SsDPSL. Thepurified BfDPSL was crystallized, with the crystals diffracting to2.3-Å resolution. Consistent with the particles seen by electronmicroscopy (EM), self-rotation functions suggested the presenceof a dodecameric DPS-like assembly. We thus used the SsDPSLprotein as the molecular replacement search model to successfullyphase the structure. As hypothesized, the resulting structure ofBfDPSL (PDB accession number 2VZB) revealed a dodecamericassembly that is in many ways similar to SsDPSL. Superposition ofthe BfDPSL and SsDPSL subunits yields 155 equivalent C-� posi-tions with a root mean square deviation of 1.2 Å. Like SsDPSL, the

BfDPSL subunit folds into a four-helix bundle that is elaboratedupon by the addition of the BC and N-terminal helices (Fig. 5A).Consequently, at the quaternary level, BfDPSL forms a hollowdodecameric shell (external diameter, 95 Å; Fig. 5B) with a 23-point group symmetry that encloses a negatively charged centralcavity approximately 50 Å in diameter.

Structural similarity in the dimetal site. Like SsDPSL,BfDPSL contains two metal atoms, A and B, at the center of eachsubunit that are coordinated by 2 histidine and 4 glutamate resi-dues (His-65, His-149, Glu-29, Glu-62, Glu-114, and Glu-146) ina classical bacterioferritin-like dimetal binding motif (Fig. 5C; seeFig. S5 in the supplemental material). Distances between the twoiron atoms range from 4.0 to 4.2 Å. Two waters, W1 and W2, arepositioned 2.3 to 2.5 Å from the iron atoms, where they occupy the6th ligand position and complete the approximate octahedral co-ordination geometry for each iron atom (see Fig. S5 in the supple-mental material).

Further similarity to SsDPSL is seen in a solvent-filled channelthat leads from the dimetal binding site to the exterior surface ofthe dodecamer (Fig. 5D). This channel may allow water, hydrogenperoxide, oxygen, or iron to access the putative ferroxidase center.The channel is slightly larger in BfDPSL than in SsDPSL and con-tains eight ordered water molecules, including the two coordi-nated by the iron atoms. Also similar to SsDPSL, the two cysteineresidues unique to DPSL proteins, Cys-93 and Cys-116, are juxta-posed between the exterior of the dodecameric shell and the chan-nel leading into the dimetal binding site. There is clear electrondensity for a bridging disulfide bond in the B. fragilis crystal struc-ture.

The structure of B. fragilis DPSL thus shows greater similarityto the archaeal DPSL proteins than it does to true bacterioferritinsand provides definitive evidence for the existence of DPSL mini-ferritins in the bacterial domain of life. Hence, we chose to refer tothe B. fragilis bfr gene product as B. fragilis DPSL, or BfDPSL. Toour knowledge, BfDPSL is the first bacterial DPSL to be charac-terized and thus provides the opportunity to compare and con-trast structural features of the bacterial and archaeal DPSL pro-teins.

Unique features of bacterial DPSL. (i) A third metal bindingsite. In addition to the dimetal binding site discussed above, athird metal binding site is found in BfDPSL. This C-site metal,which is not found in SsDPSL, is located on the inner surface of thedodecamer, approximately 7 Å from where the major pore (dis-cussed below) opens into the interior of the particle (Fig. 5D).Fo � Fc difference maps (Fig. 5C and D) show strong (10 �) elec-tron density at this position, suggestive of a cation with octahedralcoordination by His-58, Glu-54, Asp-152, and 3 water molecules.An Mg2� ion was modeled at this position because the crystalliza-tion solution contained 0.25 M MgCl2 and anomalous differencemaps do not indicate any iron or zinc content at this site. Inter-estingly, these three C-site residues are strictly conserved in 44bacterial DPSL-type sequences, while they are uniformly absentfrom the archaeal DPSL-type sequences (Fig. 6, green residues; seeFig. S6 in the supplemental material). Notably, the relative loca-tion of the bacterial C-site metal seen in BfDPSL is reminiscent ofthe C-site iron position in E. coli ferritin (32, 37, 59) and the thirdiron site on the inner surface of bacterioferritin (22). Although theanomalous difference data rule out Fe at this site in the BfDPSLcrystal structure, iron might access the site, in vivo, in several ways.The most obvious route is via the major pores in the BfDPSL

FIG 4 DNA protection by BfDPSL. BfDPSL protects DNA from hydroxylradical cleavage but does not retard the mobility of DNA under the sameconditions as EcDPS. (A) DNA protection. BfDPSL was evaluated for DNAprotection activity by incubation with supercoiled plasmid prior to exposureto FeSO4 and H2O2. Purified EcDPS was used as a positive control, and BSAwas used as a negative control. DNA integrity was assayed by electrophoresisafter DNA/protein interactions were disrupted by SDS/heat treatment. With-out protein protection, the DNA is degraded and appears as a smear on the gel.(B) BfDPSL was evaluated for DNA binding activity by gel retardation assay,with EcDPS as a positive control. After addition of BfDPSL or EcDPS to su-percoiled plasmid, samples were analyzed by electrophoresis without furthertreatment (B) or after DNA/protein interactions were disrupted by SDS/heattreatment (C). In panel B, ethidium bromide staining is visible in the well,indicating that the EcDPS/DNA complex does not migrate into the gel. Inpanel C, the DNA migrates at the expected mobility after the complex is dis-rupted. Bf, BfDPSL; Ec, EcDPS; �, no protein added; M, molecular massstandard.

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dodecamer. Alternatively, as suggested by Macedo et al. (41) andothers (10) for bacterioferritin, concerted movement of severalside chains might allow iron to move between the B and C sites. Ineither event, the C site might serve to facilitate movement of ironthrough the protein shell and/or as a nucleation center for ironmineralization.

(ii) Conformation of the C-terminal tail. In the DPS andSsDPSL dodecamers, the N-terminal ends of three subunitscome together at one end of the 3-fold axis in a set of interac-tions that are similar to the 3-fold interactions in ferritin. Incontrast, interactions at the opposite end of the 3-fold axisinvolve the C-terminal ends of three subunits, giving rise to

FIG 5 Structure of BfDPSL subunit. (A) Stereo panel. BfDPSL (PDB accession number 2VZB, red/magenta) and SsDPSL (PDB accession number 2CLB, yellow)have been superimposed (36). The structures show significant similarity from the N-terminal helix through helix A, helix B, the BC loop with its short BC helix,helix C, and much of helix D. The structures show significant differences, however, at their C termini. Also shown are the positions of the metals in the BfDPSLdimetal binding site (orange spheres). (B) The BfDPSL dodecamer is shown colored by subunit. The red subunit is in the same orientation as in panel A. (C) TheBfDPSL metal binding sites. Relative to panel A, the orientation in panel C has been rotated 90° about the y and z axes. Two iron atoms (A and B) are bound ina bacterioferritin-like dimetal binding site. Two conserved cysteine residues, present as a disulfide, are juxtaposed between the solvent channel (D) and theexterior of the particle. W1 and W2, two of the eight ordered waters in the channel, which are bound to iron A and iron B, respectively, and positioned 7.7 Å fromthe disulfide. At the lower right, a single magnesium atom (green, labeled C) is bound to the inner surface of the subunits facing the central cavity of thedodecamer (toward the bottom). The distance from metal B to metal C is 8.7 Å. The Fo � Fc electron density omit map is contoured at 4.0 �. Three residues,His-58, Glu-54, and Asp-152, and three water molecules (blue) surround the magnesium in approximate octahedral geometry. (D) Cross section throughdodecamer shell showing the relative positions of the metal binding sites, the channel to the dimetal binding site, and the major, or NS3S, pore. The channel tothe dimetal binding site is immediately above the iron atoms (orange spheres) and connects to the exterior surface. The NS3S pore is to the right of the dimetalbinding site, running at a 45° angle, and connecting the interior and exterior surfaces of the particle. Site C is shown on the inner surface of the dodecameradjacent to the NS3S opening. To highlight the pore, the orientation is rotated slightly relative to panel C (30° about the y and x axes). The electrostatic surfaceis contoured from �15 kT/electron (red) to �15 kT/electron (blue). In the absence of metals, the channel to the dimetal binding site, the major pore (NS3S), andthe interior of the particle all exhibit strong negative potential.

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3-fold symmetric interactions that are unique to the DPS andDPSL dodecamers.

It is here, in the C-terminal residues, that the structure ofBfDPSL differs significantly from that of SsDPSL. The B. fragilisand S. solfataricus protomers superimpose well only up to Thr-158in the D helix (Tyr-168 in SsDPSL). After this point they adoptvery different conformations. In BfDPSL, the last 12 residues(Asp-159 through Lys-170) are used to extend the D helix (Fig.5A), with all but the last C-terminal residue visible in the electrondensity. The BfDPSL D helix is thus significantly longer than the Dhelix in SsDPSL and runs along the exterior surface of the particle,toward the 3-fold axis, where it interacts with the D helices insymmetry-related subunits to form a 3-fold symmetric cone ortepee-shaped structure (Fig. 7A; see Fig. S7 in the supplementalmaterial). At the analogous position in SsDPSL, we find the en-

trance to the major pore (the C-terminal 3-fold pore) allowingaccess to the interior of dodecameric shell (Fig. 7B) (28). InBfDPSL, however, the symmetry-related D helices come togetheron the outside surface of the particle to cap the C-terminal 3-foldaxis, preventing access to the interior of the particle via theC-terminal 3-fold pore.

This BfDPSL cap is stabilized by a central core of buried hydro-phobic side chains, with polar and charged residues projectingoutward (Fig. 7A; see Fig. S7 in the supplemental material). Eachhelix contributes 3 residues to this hydrophobic core: Ile-160,Met-163, and Phe-167. This hydrophobic core is well conservedamong bacterial DPSLs. Using PSI-BLAST (2, 56) seeded withprotein sequences containing the DPSL thioferritin motif (the twoconserved cysteines plus the dimetal site residues), we identified74 DPSL proteins in the current sequence database. Figure 6

FIG 6 Representative DPSL sequence alignment. A representative DPSL sequence from each of 12 different phyla was aligned with ClustalW (34, 65). Thesecondary structure elements in BfDPSL and SsDPSL are shown above and below the sequences, respectively, followed by ferritin (EcFtn), bacterioferritin(DdBfr), and DPS (HsDPS) sequences, where minor adjustments were made in order to align known secondary structural elements. The DPSL sequences clearlydivide into two groups: one containing a short C-terminal tail and one containing a long C-terminal tail. The short-tailed sequences predominate in mesophilicbacteria, while long-tailed DPSLs predominate in archaea and thermophilic bacteria. Both groups share the conserved thioferritin motif; the 2 cysteines are in red,and the 6 residues in the dimetal binding site are in blue. The short-tailed DPSLs contain two additional distinguishing motifs. First, residues comprising metalbinding site C are strictly conserved in this group (green). Second, a pattern of hydrophobic residues consistent with an amphipathic helix (3/4 spacing) is foundin the short C-terminal tail; these residues, along with Met-47 from the AB loop, form a hydrophobic core that stabilizes the cap over the C-terminal 3-fold axis(magenta). In addition, Arg-48 (or Lys) forms an interchain salt bridge with Asp-159 that also stabilizes the cap (magenta). In contrast, the long-tailed DPSLscontain the conserved PSGH motif (yellow) that functions to transit the protein shell, and in place of the hydrophobic Met-47, they prefer a glutamate residue(E; yellow), which in SsDPSL is found at the opening of the 3-fold pore. Asterisks, conserved residues in the 74 proteins containing the thioferritin motif (see Fig.S6 in the supplemental material). Ferroxidase active-site residues are colored blue (EcFtn, DdBfr) or orange (HsDPS). The number of DPSL sequences in eachphylum is in parentheses, followed by the range of amino acid identity with BfDPSL for each phyla. The abbreviations used are followed by organism andGenBank gene identifier: BfDPSL, Bacteroides fragilis gi60682741; Ct3511, Chloroherpeton thalassium gi193215108; Dt1699, Desulfurobacterium thermolithotro-phum gi325294845; St6192, Spirochaeta thermophila gi307719855; GM21, Geobacter sp. strain M21 gi253701127; Tpe, Thermoanaerobacter pseudethanolicusgi167036853; Ip2456, Ilyobacter polytropus gi310780231; Ac2261, Aminobacterium colombiense gi294101498; Tm, Thermotoga maritima gi15643326; Gv, Gloeo-bacter violaceus gi37523861; Pf, Pyrococcus furiosus gi18977565, SsDPSL, Sulfolobus solfataricus gi15898865; DdBfr, Desulfovibrio desulfuricans (PDB accessionnumber 1NFV) gi220904655; EcFtn, Escherichia coli (PDB accession number 1EUM) gi15802314; HsDPS, Halobacterium salinarum (PDB accession number1TKO) gi15791220; CFB; Bacteroidetes, GSB, Chlorobi; aquif, Aquificae; spiro, Spirochaetes; prote, Proteobacteria; firmi, Firmicutes; fusob, Fusobacteria; syner,Synergistetes; therm, Thermotogae; cyano, Cyanobacteria; eurya, Euryarchaeota; crena, Crenarchaeota.

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shows a representative alignment of sequences containing thethioferritin motif from 12 different phyla (a complete alignmentof all sequences containing the thioferritin motif is provided inFig. S6 in the supplemental material). The sequence alignmentshows that mesophilic bacterial sequences retain hydrophobic res-idues at these positions, whereas the archaeal proteins do not.

The cap appears to be further stabilized by Ala-46, Met-47, andArg-48, which are contributed by the A-B loop (see Fig. S7 in thesupplemental material). Ala-46 and Met-47 lie at the base of thecap, where they join the hydrophobic core, while Arg-48 forms anintersubunit salt bridge with Asp-159. In contrast, SsDPSL and theother archaeal proteins do not utilize the intersubunit salt bridgeand generally substitute Glu or Asp for Met-47, which adds anadditional ring of negative charge to the external opening of their3-fold C-terminal pores. In all, the conserved features of thesebacterial sequences, coupled with the role of these residues inBfDPSL, suggest that the extended D helix and the cap at theC-terminal 3-fold axis are common features of these bacterialDPSL proteins.

While the long D helix in BfDPSL forms a cap, the D helix ofSsDPSL is shorter and is followed by a 21-residue tail (28). Thisshorter D helix and extended C-terminal tail appear to be charac-teristic features of the archaeal DPSL proteins (Fig. 5 and 6; see Fig.S6 in the supplemental material). In SsDPSL, the first seven resi-dues of the C-terminal tail adopt a random coil structure thatturns away from the 3-fold axis and passes through the proteinshell into the interior of the dodecamer (Fig. 7B), where the last 14C-terminal residues are disordered. As the tail extends into theinterior, four ordered residues, Pro-Ser-Gly-His, are involved intransiting the protein shell (yellow highlights in Fig. 6 and 7B).Strikingly, these residues are conserved in the archaeal-type pro-teins but are absent from the bacterial sequences. This longerC-terminal tail with the embedded Pro-Ser-Gly-His sequencethus appears to be a hallmark of proteins with an internal tail.

(iii) Number and location of major pores. The different con-formations of the C-terminal tail in the SsDPSL and BfDPSL sub-units result in a major difference in the structures of their respec-tive dodecameric particles. While the major pores in SsDPSL lie atthe C-terminal 3-fold, the C-terminal tail of the BfDPSL subunitcaps these C-terminal 3-fold pores. However, as the 12 D helicesare extended in the BfDPSL dodecamer to close the four, 3-foldC-terminal pores, 12 alternate pores are created (Fig. 7). This isbecause the paths taken by the 12 C-terminal tails that transit theshell in the SsDPSL dodecamer are now vacant. This is demon-strated by superposition of the S. solfataricus and B. fragilis DPSLdodecamers; the C-terminal residues in archaeal SsDPSL (yellow)superimpose on the hydrophilic pores of the bacterial BfDPSLdodecamer (Fig. 7C). These positions appear to be the major poresin BfDPSL and are located at a nonsymmetric junction of threesubunits (Fig. 7A). We thus designate these nonsymmetric three-subunit (NS3S) pores. Two of the three subunits forming this poreare related by a 2-fold rotation axis, while the third subunit is

FIG 7 Conformation of the C-terminal tail, pore number, and location inDPSL subtypes. (A) Six subunits of bacterial BfDPSL are viewed looking downthe C-terminal 3-fold axis. The extended D helices (magenta) come together atthe center to cap the 3-fold axis. The locations of the nonsymmetric three-subunit (NS3S) pores are indicated by the three black circles surrounding theC-terminal 3-fold axis. With 4 C-terminal 3-fold axes per particle, this gives atotal of 12 NS3S pores. The locations of the A and B metal sites are shown asorange spheres within the red subunits, and metal site C is shown as a greensphere. (B) Archaeal SsDPSL in the same orientation. The C-terminal tail(yellow) is in a markedly different conformation and transits the protein shellat a location equivalent to that of the NS3S pores in bacterial DPSL. While thisopens a single pore at the C-terminal 3-fold, it plugs the 3 potential NS3S poressurrounding the 3-fold axis. The net result is only 4 major pores in the archaealSsDPSL, as opposed to 12 pores in the bacterial BfDPSL. (C) Cross section

from a surface representation of the BfDPSL particle colored by electrostaticpotential as in Fig. 5D. Two NS3S pores transit the protein shell (top left andtop right), connecting the interior and exterior surfaces of the particle. The tailof a single superpositioned SsDPSL subunit (yellow) transits the protein shell,demonstrating how a C-terminal tail in this conformation would plug anypotential NS3S pore (top right of panel C).

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positioned against the end of the first two, resulting in a pore witha minimal diameter of 4.3 Å (Fig. 7A and viewed in cross section inFig. 5D and 7C). The relative positions of the C-terminal 3-fold,the NS3S pores, and the channels to the dimetal binding sites areshown in a surface representation of the BfDPSL dodecamer inFig. S8 in the supplemental material. Importantly, compared tothe four C-terminal 3-fold pores in SsDPSL, there are now 12NS3S pores in the BfDPSL dodecamer (Fig. 7).

Interestingly, a similar set of NS3S pores is seen for at least onemember of the DPS family, the dodecameric DpsA from Halobac-terium salinarum (70). Analysis of the iron-loaded DpsA structureidentifies 3 iron atoms within the H. salinarum NS3S pore (PDBaccession number 1TKO), suggesting that this pore allows iron toenter the central cavity of the DpsA dodecamer. In addition, de-spite assembling to form a 24-mer, analogous pores are found in abacterioferritin and several bacterial ferritins. More specifically,the NS3S pore in BfDPSL is equivalent to the B (37, 41) or MC (10)pore in the bacterioferritins from Desulfovibrio desulfuricans andAzotobacter vinelandii (38, 62) and similar pores in the ferritinsfrom E. coli (59) and Thermotoga maritima (PDB accession num-ber 1Z4A; S. Steinbacher et al., unpublished data).

Similar to H. salinarum DpsA, residues lining the NS3S pore inBfDPSL are largely hydrophilic and several ordered water mole-cules are found within the pore (Fig. 5D). Additional electrondensity within the pore is also consistent with the presence of amolecule of bis-Tris, the buffer used during protein purification.Surrounding the opening to the pore on the exterior surface of thedodecamer are Lys-3, Glu-77, and Val-79 from subunit A; Glu-44and Phe-101 from subunit B; and Thr-158 from subunit C. Ahigher density of charged side chains surrounds the openingwhere it exits into the interior of the dodecamer: Gln-67, Asp-71,and Glu-75 from subunit A; Arg-48, Gln-52, and Glu-56 fromsubunit B; and Glu-54, Glu-148, and Asp-152 from subunit C. Inconjunction with the main-chain carbonyl groups that line thewall of the pore, there is a strong net negative charge that mayfunction to draw ferrous iron through the pore (25). Importantly,metal binding site C is present on the interior surface of the do-decamer, only 7 Å from the central axis of the NS3S pore (Fig. 5Dand 7A and C).

DISCUSSIONRegulation of bfr expression. The structural similarity betweenSsDPSL and BfDPSL suggests that they may have analogous func-tions in vivo. In this regard, an important role in the oxidativestress response is supported by transcriptional analysis of bothgenes. The SsDPSL transcript is upregulated in response to H2O2,whereas the BfDPSL transcript is increased 35-fold during expo-sure to air. Notably, there was no increase in BfDPSL mRNA dur-ing H2O2 exposure, but this may be due to the rapid induction ofcatalase, which is a part of the OxyR regulon in B. fragilis. Consis-tent with this idea, a previous expression microarray study showeda modest 4-fold induction of bfr by H2O2 in an oxyR mutant whichhas greatly reduced catalase activity (61). In contrast to oxidativestress, there did appear to be a difference in the response to ironlevels. In S. solfataricus, the DPSL gene is induced by iron limita-tion. In contrast, B. fragilis bfr expression increased during growthwith excess iron levels during oxidative stress, but this was notfound in anaerobic cells.

The difference in the response to iron does not necessarily in-dicate mechanistic differences between SsDPSL and BfDPSL. The

presence of 3 members of the ferritin family in the B. fragilis ge-nome (dps, ftnA, and bfr), combined with differences in gene reg-ulation, may simply allow the optimal deployment of a set of re-lated activities under a variety of different conditions. Consistentwith this is the observation that the bfr and ftnA mutants and thebfr ftnA double mutant displayed somewhat different phenotypes,depending on the type of oxidative stress tested in the assays (Fig.3). In addition, the bfr and ftnA genes are regulated differently,and deletion of the ftnA gene did not directly affect expression ofBfDPSL under conditions of aerobic stress or stationary phase.Differential expression of ferritin superfamily members has alsobeen observed in other organisms, including Bacillus subtilis (5,16, 26), Bacillus anthracis (3, 39, 45), Salmonella enterica serovarTyphimurium (66), and Pseudomonas putida (17). Thus, as the B.fragilis dps gene is extremely responsive to peroxide or oxygen (49,61), B. fragilis may hold the activity of BfDPSL in reserve for ironstorage and/or DNA protection during the transition to stationaryphase or for prolonged O2 exposure. This would be consistentwith the phenotype observed for the bfr mutant in the extendedaerobic exposure assay (Fig. 3B).

DNA binding, protection, and mineralization. While we areunable to offer a satisfying explanation for DNA protection byBfDPSL, we note that E. coli DPS is thought to protect DNA in twoways. First, as discussed in the introduction, it can sequester ironoxide inside the dodecamer, reducing the concentration of bothiron and reactive oxygen species. Second, as it binds and seques-ters DNA, it may provide a physical barrier that limits damage. Inthis light, we note that dimeric forms of DPS from two otherorganisms have demonstrated DNA protection activity. In thefirst example, the dimeric form of DPS from Mycobacterium smeg-matis protects DNA without apparent DNA binding (14). Ceci etal. (14) attributed the DNA protection to iron oxidation at the twoferroxidase sites which remain intact at the subunit interface ofdimeric Mycobacterium DPS. Similarly, the intact ferroxidase siteswithin each subunit of the dimeric form of BfDPSL may provideprotection in our assay. Alternatively, in the unassembled form,the negatively charged inner surface of the dimer might act tochelate a significant portion of the iron, reducing the productionof reactive oxygen species. In a second example, dimeric DPS fromDeinococcus radiodurans is able to protect DNA in a similar assay,but in this case the protection may be facilitated by DNA binding(30). Thus, we also consider the possibility of protective BfDPSL-DNA interactions that are not apparent in our assay; these inter-actions might be disrupted during electrophoresis and are there-fore not visible on the gel. On the other hand, protective activity inthe absence of DNA binding has been observed for several dodeca-meric DPS proteins, for example, DPS proteins from Listeria in-nocua, Agrobacterium tumefaciens, and Streptococcus mutans (13,60, 68).

In the E. coli DPS dodecamer, DNA binding activity is medi-ated by positively charged residues present on a flexible extensionat the N terminus of the subunits (11). While the BfDPSL subunithas three lysines near the N terminus and two near the C terminus,these residues are present in helix N, the �N-�A loop, and theextension to helix D. The N and D helices in the BfDPSL dodeca-meric assembly do not appear to be sufficiently exposed to insertwithin the DNA major groove. In addition, the electrostatic sur-face of the dodecamer (see Fig. S8 in the supplemental material)does not appear to be sufficiently positive to promote strong elec-trostatic interactions with the ribose phosphate backbone. These

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observations may explain the lack of DNA binding activity. Alter-natively, BfDPSL may lack DNA binding activity under our assayconditions due to its predominantly dimeric state.

Quaternary structure. Interestingly, while BfDPSL was pre-dominantly a dimer in solution, it was found to assemble into adodecamer both within the crystal and on the EM grids. Based onits similarity to other ferritins and its importance in the B. fragilisoxidative stress response, we presume that the dodecameric as-sembly is physiologically relevant. Importantly, recent literaturefor several other members of the ferritin superfamily also indicateseasy dissociation into dimers or monomers in vitro (4, 8, 12, 14,20, 30). One possible explanation for BfDPSL is that assemblymight be facilitated by other proteins in vivo. For example, Sul-folobus DPSL is reported to be a part of a larger supramolecularcomplex (40) that also contains superoxide dismutase (SOD) andperoxiredoxin, two proteins upregulated under oxidative stress inSulfolobus. Perhaps a similar complex may occur in Bacteroides,where SOD and several peroxidases are also upregulated followingexposure to oxygen (61).

DPSL subclasses and role of C-terminal tail in ferritin super-family. Significant structural differences between SsDPSL andBfDPSL include differences in sequence and structure at the Cterminus, the number and location of the major pores, differingsequence motifs within the A-B loop, and the presence or absenceof the C-site metal binding residues. Considered together, it be-comes clear that there are at least two distinct structural sub-classes among DPSL proteins. We note that the division be-tween subclasses falls largely along the boundary between thearchaeal (SsDPSL) and bacterial (BfDPSL) domains, althoughDPSLs from the thermophilic bacteria group with the archaealsequences.

Role of C-terminal residues in ferritin superfamily. One ofseveral examples of the role of the C-terminal residues in modu-lating the assembly and activity of ferritins is the distinctly differ-ent conformations of the C-terminal tail in SsDPSL and BfDPSL.In the 24-subunit ferritins and bacterioferritins, the C-terminalresidues are present as an additional � helix that mediates inter-actions at the 4-fold axis (31, 41). In dodecameric DPS proteins,this short C-terminal helix is lost, with the C-terminal tail adopt-ing a random coil arrangement on the outside of the particle (29),where, along with the N-terminal tail, it may play a role in mod-ulating nonspecific interactions with DNA (54). In addition, theC-terminal tail in DPS has also been shown to play a critical role instabilizing the dodecamer (54, 55). Now, in the SsDPSL andBfDPSL dodecamers, we see that the C-terminal tail can adopt twoadditional conformations. In the case of SsDPSL, the C-terminaltail extends through the NS3S pore into the interior of the particle.In contrast, the C-terminal residues in BfDPSL extend the D helixand cap the C-terminal 3-fold pore. In both cases, theseC-terminal tails are involved in intersubunit interactions and arethus likely to stabilize their dodecameric assemblies.

Relationships in ferritin superfamily. It is also interesting toconsider the position of bacterial DPSL within the ferritin super-family from an evolutionary point of view. It is relatively easy toimagine the loss of the disulfide and dimetal binding sites withinthe four-helix bundle, coupled with the appearance of metal bind-ing sites at the subunit interfaces of the dodecameric assembly,giving rise to a bacterial DPS. Alternatively, small changes in thesubunit interfaces and loss of the disulfide could give rise to aferritin-like 24-mer, as opposed to the dodecameric DPSL. In

turn, the ferritin subunit interface could be further modified toaccommodate the intersubunit heme found in bacterioferritin.The DPSL structure thus seems to be a logical evolutionary inter-mediate within the ferritin superfamily, one that seemingly con-nects the bacterial DPS proteins to the larger (bacterio)ferritins(see Fig. S10 in the supplemental material). It is not clear, how-ever, in which directions these evolutionary events might haveproceeded.

In summary, we have examined the expression of the B. fragilisbfr transcript in response to oxidative stress and show that dele-tion of the bfr gene decreases oxygen tolerance. We have alsoshown that the bfr gene product, BfDPSL, protects DNA againstoxidative stress in vitro. In addition, our structural studies showthat BfDPSL contains many of the structural elements of the ar-chaeal DPSL protein, including a dodecameric assembly that har-bors the thioferritin motif. This extends the observation of thisnew class of miniferritin to the bacterial domain, where it is foundin at least seven different phyla. However, the structure also high-lights significant differences between the short-tailed bacterialDPSL proteins and their longer-tailed archaeal homologs. Impor-tantly, these structural differences are clearly manifested in theirrespective primary sequence motifs, and these may be used todifferentiate the two structural subclasses. This should facilitateproper reannotation of these bacterial DPSL proteins, which arefrequently mistaken for bacterioferritins. Combined, these studiesconfirm a role for BfDPSL in the oxidative stress response of B.fragilis, the most commonly isolated anaerobic human pathogen.As resistance to oxidative stress is a virulence factor for B. fragilis,BfDPSL might be an attractive target for development of antibi-otics against B. fragilis and other members of the Bacteroidetes,particularly since DPSL proteins have not been found in eukary-otic organisms.

ACKNOWLEDGMENTS

We thank the R. Gerlach, B. Bothner, and J. Peters laboratories for assis-tance with ICP-MS, liquid chromatography/MS, and anaerobic tech-niques. The assistance of Anita Parker with the Western blotting is grate-fully acknowledged.

This work was supported by grants from the Public Health Service(AI40599 to C.J.S. and AI079183 to E.R.R.), by the Human Frontier Sci-ence Program (RGP61/2007 to T.D.), and by the National Institutes ofHealth (GM084326 to C.M.L.). Portions of this research were carried outat the Stanford Synchrotron Radiation Laboratory (SSRL), a national userfacility operated by Stanford University on behalf of the U.S. Departmentof Energy, Office of Basic Energy Sciences. The SSRL Structural MolecularBiology Program is supported by the U.S. Department of Energy, Office ofBiological and Environmental Research, and by the National Institutes ofHealth, National Center for Research Resources, Biomedical TechnologyProgram, and the National Institute of General Medical Sciences. TheMacromolecular Diffraction Laboratory at Montana State University wassupported, in part, by a grant from the Murdock Foundation.

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