Siderophore-controlled Iron Assimilation in the Enterobacterium Erwinia chrysanthemi
2008; Elsevier BV; Volume: 283; Issue: 52 Linguagem: Inglês
10.1074/jbc.m807749200
ISSN1083-351X
AutoresDominique Expert, Aïda Boughammoura, Thierry Franza,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoThe intracellular fate of iron acquired by bacteria during siderophore-mediated assimilation is poorly understood. We investigated this question in the pathogenic enterobacterium Erwinia chrysanthemi. This bacterium produces two siderophores, chrysobactin and achromobactin, during plant infection. We analyzed the distribution of iron into cytosolic proteins in bacterial cells supplied with 59Fe-chrysobactin using native gel electrophoresis. A parental strain and mutants deficient in bacterioferritin (bfr), miniferritin (dps), ferritin (ftnA), bacterioferredoxin (bfd), or iron-sulfur cluster assembly machinery (sufABCDSE) were studied. In the parental strain, we observed two rapidly 59Fe-labeled protein signals identified as bacterioferritin and an iron pool associated to the protein chain-elongation process. In the presence of increased 59Fe-chrysobactin concentrations, we detected mini-ferritin-bound iron. Iron incorporation into bacterioferritin was severely reduced in nonpolar sufA, sufB, sufD, sufS, and sufE mutants but not in a sufC background. Iron recycling from bacterioferritin did not occur in bfd and sufC mutants. Iron depletion caused a loss of aconitase activity, whereas ferric chrysobactin supplementation stimulated the production of active aconitase in parental cells and in bfr and bfd mutants. Aconitase activity in sufA, sufB, sufD, sufS, and sufE mutant strains was 10 times lower than that in parental cells. In the sufC mutant, it was twice as low as that in the parental strain. Defects observed in the mutants were not caused by altered ferric chrysobactin transport. Our data demonstrate a functional link between bacterioferritin, bacterioferredoxin, and the Suf protein machinery resulting in optimal bacterial growth and a balanced distribution of iron between essential metalloproteins. The intracellular fate of iron acquired by bacteria during siderophore-mediated assimilation is poorly understood. We investigated this question in the pathogenic enterobacterium Erwinia chrysanthemi. This bacterium produces two siderophores, chrysobactin and achromobactin, during plant infection. We analyzed the distribution of iron into cytosolic proteins in bacterial cells supplied with 59Fe-chrysobactin using native gel electrophoresis. A parental strain and mutants deficient in bacterioferritin (bfr), miniferritin (dps), ferritin (ftnA), bacterioferredoxin (bfd), or iron-sulfur cluster assembly machinery (sufABCDSE) were studied. In the parental strain, we observed two rapidly 59Fe-labeled protein signals identified as bacterioferritin and an iron pool associated to the protein chain-elongation process. In the presence of increased 59Fe-chrysobactin concentrations, we detected mini-ferritin-bound iron. Iron incorporation into bacterioferritin was severely reduced in nonpolar sufA, sufB, sufD, sufS, and sufE mutants but not in a sufC background. Iron recycling from bacterioferritin did not occur in bfd and sufC mutants. Iron depletion caused a loss of aconitase activity, whereas ferric chrysobactin supplementation stimulated the production of active aconitase in parental cells and in bfr and bfd mutants. Aconitase activity in sufA, sufB, sufD, sufS, and sufE mutant strains was 10 times lower than that in parental cells. In the sufC mutant, it was twice as low as that in the parental strain. Defects observed in the mutants were not caused by altered ferric chrysobactin transport. Our data demonstrate a functional link between bacterioferritin, bacterioferredoxin, and the Suf protein machinery resulting in optimal bacterial growth and a balanced distribution of iron between essential metalloproteins. Iron is necessary for most forms of life, being required for the catalytic activity of essential proteins mediating electron transfer and redox reactions. The importance of this metal relies on its electronic structure, which can undergo changes through several oxidation states differing by one electron. The ferric form of iron predominates in aerobic environments, but its bioavailability is severely compromised by its poor solubility (1Neilands J.B. Biol. Met. 1991; 4: 1-6Crossref PubMed Scopus (48) Google Scholar). Ferrous iron is present at significant levels in the cell but can be toxic as a consequence of its participation in "Fenton-type" redox chemistry (2Pierre J.-L. Fontecave M. BioMetals. 1999; 12: 195-199Crossref PubMed Scopus (239) Google Scholar, 3Imlay J.A. Annu. Rev. Microbiol. 2003; 57: 395-418Crossref PubMed Scopus (1693) Google Scholar). Thus, iron acquisition, utilization, and storage are subject to different levels of homeostatic regulation. Microorganisms have developed powerful iron acquisition systems based on production of siderophores, which are selective ferric ion chelators secreted in response to iron deficiency (4Faraldo-Gomez J.D. Sansom M.S. P. Nat. Rev. Mol. Cell. Biol. 2003; 4: 105-116Crossref PubMed Scopus (291) Google Scholar). Once loaded with iron, the siderophore is specifically imported into the cell. This requires active transport which, in Gram-negative bacteria, is achieved by a TonB-dependent outer membrane receptor and a permease belonging to the ABC transporter family (5Krewulak K.D. Vogel H.J. Biochim. Biophys. Acta. 2008; 1778: 1781-1804Crossref PubMed Scopus (381) Google Scholar). Regulation of siderophore production and uptake involves the metalloprotein Fur 3The abbreviations used are:Furferric uptake regulationIsciron-sulfur clusterSufsulfur utilization factorFtnferritinBfrbacterioferritinDpsDNA protein from starved cellsBfdbacterioferredoxinFMPfast migrating proteins or functional analogs, acting as transcriptional repressors of iron-responsive genes (6Lee J.W. Helmann J.D. BioMetals. 2007; 20: 485-499Crossref PubMed Scopus (339) Google Scholar). Although major advances have improved understanding of the processes involved in ferric siderophore uptake, there is still little information on the intracellular fate of iron, once released from its ligand. Enzymatic degradation of the ferric siderophore complex and/or enzymatic reduction to the ferrous state are effective mechanisms for iron removal (7Miethke M. Marahiel M.A. Microbiol. Mol. Biol. Rev. 2007; 71: 413-451Crossref PubMed Scopus (1184) Google Scholar). It remains to be determined how the cell prioritizes its intracellular iron utilization so that iron-containing proteins preferentially receive iron, at the same time avoiding toxic side reactions. Of the various classes of iron-containing proteins, iron-sulfur proteins and ferritins are of particular interest. ferric uptake regulation iron-sulfur cluster sulfur utilization factor ferritin bacterioferritin DNA protein from starved cells bacterioferredoxin fast migrating proteins Biosynthesis of iron-sulfur proteins involves complex protein machineries that build iron-sulfur clusters from cytosolic iron and sulfur sources and transfer them to their cognate protein acceptors. Studies in various bacterial species, including Escherichia coli, Erwinia chrysanthemi, and cyanobacteria have led to the identification of the Isc and Suf machinery, which have homologs in eukaryotes (8Barras F. Loiseau L. Py B. Adv. Microb. 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The proteins encoded by the isc operon appear to mediate a general pathway for the assembly of a variety of iron-sulfur proteins, whereas those encoded by the suf operon play a role in iron-sulfur cluster biosynthesis under conditions of iron deficiency or oxidative stress (13Nachin L. El Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (158) Google Scholar, 14Outten F.W. Djaman O. Storz G. Mol. Microbiol. 2004; 52: 861-872Crossref PubMed Scopus (350) Google Scholar, 15Yeao W.-S. Lee J.-H. Lee K.-C. Roe J.-H. Mol. Microbiol. 2006; 61: 206-218Crossref PubMed Scopus (167) Google Scholar). The iron donation step for cluster assembly in vivo is unknown (16Layer G. Ollagnier de Choudens S. Sanakis Y. Fontecave M. J. Biol. Chem. 2006; 281: 16256-16263Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17Ding H. Yang J. Coleman L.C Yeung S. J. Biol. Chem. 2007; 282: 7997-8004Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In eukaryotes, the mitochondrial frataxin protein has been proposed to act as an iron donor for assembly of iron-sulfur clusters (18Gerber J. Mühlenhoff U. Lill R. EMBO Rep. 2003; 4: 906-911Crossref PubMed Scopus (315) Google Scholar, 19Foury F. Pastore A. Trincal M. EMBO Rep. 2007; 8: 194-199Crossref PubMed Scopus (73) Google Scholar) and a similar function was assigned to the bacterial homolog CyaY for the Isc machinery, although this still needs to be confirmed (16Layer G. Ollagnier de Choudens S. Sanakis Y. Fontecave M. J. Biol. Chem. 2006; 281: 16256-16263Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The ability of the Suf pathway to function when iron is limiting suggests not only that Suf is involved in iron-sulfur cluster assembly but also that Suf may act as an iron trap. Ferritins are iron storage proteins that sequester iron in a nonreactive form, protecting the cell from iron-induced toxicity (20Matzanke B.F. Winkelmann G. Carrano C.J. Transition Metals in Microbial Metabolism. Harwood Academic Publishers GmbH, Amsterdam, The Netherlands1997: 117-158Google Scholar, 21Andrews S.C. Robinson A.K. Rodriguez-Quinones F. FEMS Microbiol. Rev. 2003; 27: 215-237Crossref PubMed Scopus (1917) Google Scholar). In bacteria, these proteins are present in the same compartment as other iron-requiring proteins falling into three categories: heme-free ferritins (Ftn), found in prokaryotes and eukaryotes; heme-containing bacterioferritins (Bfr), found only in bacteria; and Dps proteins, also called mini-ferritins, present only in prokaryotes (22Chiancone E. Ceci P. Ilari A. Ribacchi F. Stefanini S. BioMetals. 2004; 17: 197-202Crossref PubMed Scopus (117) Google Scholar, 23Smith J.L. Crit. Rev. Microbiol. 2004; 30: 173-185Crossref PubMed Scopus (112) Google Scholar). Ferritins and bacterioferritins are composed of 24 identical subunits, and Dps proteins contain 12 identical subunits. These subunits assemble to make a spherical protein shell surrounding a central cavity able to hold up to between 2,000 and 3,000 ferric iron atoms for ferritins and 500 atoms for mini-ferritins (21Andrews S.C. Robinson A.K. Rodriguez-Quinones F. FEMS Microbiol. Rev. 2003; 27: 215-237Crossref PubMed Scopus (1917) Google Scholar, 24Carrondo M.A. EMBO J. 2003; 22: 1959-1968Crossref PubMed Scopus (238) Google Scholar). Ferritins have a binuclear di-iron center constituting the ferroxidase center, which is involved in the oxidation of the ferrous iron (25Le Brun N.E. Andrews S.C. Guest J.R. Harrison P.M. Moore G.R. Thomson A.J. Biochem. J. 1995; 312: 385-392Crossref PubMed Scopus (72) Google Scholar, 26Ilari A. Stefanini S. Chiancone E. Tsernoglou D. Nat. Struct. Biol. 2000; 7: 38-43Crossref PubMed Scopus (234) Google Scholar, 27Stillman T.J. Connolly P.P. Latimer C.L. Morland A.F. Quail M.A. Andrews S.C. Hudson A.J. Treffry A. Guest J.R. Harrison P.M. J. Biol. Chem. 2003; 278: 26275-26286Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 28Liu X. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8557-8562Crossref PubMed Scopus (88) Google Scholar). Ferritins can act as acceptors and donors of ferrous ions, but their precise contribution in bacterial iron metabolism is not well understood. An appropriate system physiologically relevant and representative of the bacterial world is needed to address these unresolved issues. We have developed E. chrysanthemi (Dickeya dadantii) as a model plant pathogen to investigate the role of iron during infection (29Expert D. Annu. Rev. Phytopathol. 1999; 37: 307-334Crossref PubMed Scopus (137) Google Scholar). The genome sequence of this bacterium is available and like many other bacterial species pathogenic to mammals, E. chrysanthemi requires powerful iron transport routes to obtain iron from host tissues. Notably, this bacterium produces two siderophores, chrysobactin and achromobactin, which are important for its virulence (30Franza T. Mahé B. Expert D. Mol. Microbiol. 2005; 55: 261-275Crossref PubMed Scopus (149) Google Scholar). Successful infection also requires a functional Suf system (31Nachin L. Loiseau L. Expert D. Barras F. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (227) Google Scholar). The E. chrysanthemi FtnA ferritin and Bfr bacterioferritin have different roles in vivo. Like in E. coli, expression of the cognate ftnA gene is positively controlled by iron and the Fur repressor through a mechanism involving the small antisense RNA RyhB (32Boughammoura A. Matzanke B. Böttger L. Reverchon S. Lesuisse E. Expert D. Franza T. J. Bacteriol. 2008; 190: 1518-1530Crossref PubMed Scopus (39) Google Scholar). The E. chrysanthemi bfr gene is clustered into an operon with the bfd gene that encodes a 64-amino acid peptide (Bfd). This peptide shows 70% of sequence identity with the bacterioferritin-associated [2Fe-2S] ferredoxin from E. coli K-12 (33Andrews S.C. Harisson P.C. Guest J.R. J. Bacteriol. 1989; 171: 3940-3947Crossref PubMed Google Scholar, 34Garg P.R. Vargo C.J. Cui X. Kurtz D.M. Biochemistry. 1996; 35: 6297-6301Crossref PubMed Scopus (55) Google Scholar). The operon is expressed at a basal level and up-regulated at the stationary phase of growth by the sigma S factor (32Boughammoura A. Matzanke B. Böttger L. Reverchon S. Lesuisse E. Expert D. Franza T. J. Bacteriol. 2008; 190: 1518-1530Crossref PubMed Scopus (39) Google Scholar). The E. chrysanthemi genome sequence also revealed the existence of a gene (dps) encoding a putative mini-ferritin (Dps). In this study, we investigated the distribution of iron bound to cytosolic proteins from E. chrysanthemi mutant cells defective in siderophore biosynthesis, supplied with 59Fe via chrysobactin. The addition of iron stimulates growth and leads to global metabolic recovery in iron-deprived cells. We compared mutants deficient in bacterioferritin, bacterioferredoxin, ferritin, mini-ferritin, various Suf proteins, and Fur protein. We demonstrated a functional link between Bfr, Bfd, and the Suf protein machinery, which results in the balanced distribution of iron between essential proteins. Bacterial Strains, Plasmids, and Microbiological Techniques—The bacterial strains and plasmids used are described in supplemental Table S1. The cbsE-1, cbsC-1, acsA-37, acsF::Ω and dps::Ω mutations were introduced into the appropriate bfr, ftnA, suf, and fur deficient mutants by transduction using phage φEC2 as described previously (35Franza T. Enard C. Van Gijsegem F. Expert D. Mol. Microbiol. 1991; 5: 1319-1329Crossref PubMed Scopus (25) Google Scholar). The rich media used were L broth and L agar (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Tris medium was used as a low iron minimal medium (37Franza T. Sauvage C. Expert D. Mol. Plant-Microbe Interact. 1999; 12: 119-129Crossref PubMed Scopus (67) Google Scholar). Glassware for Tris medium was treated as described previously (37Franza T. Sauvage C. Expert D. Mol. Plant-Microbe Interact. 1999; 12: 119-129Crossref PubMed Scopus (67) Google Scholar). For iron-replete conditions, Tris medium was supplemented with ferric chrysobactin at the concentrations indicated. Ferric chrysobactin was prepared by adding FeCl3 to chrysobactin in 0.1 m Tris, pH 7.5, at a ligand/iron ratio of 3:1. Glucose (2 g/liter) was used as carbon source. The antibacterial agents and chemicals were used as reported previously (35Franza T. Enard C. Van Gijsegem F. Expert D. Mol. Microbiol. 1991; 5: 1319-1329Crossref PubMed Scopus (25) Google Scholar, 37Franza T. Sauvage C. Expert D. Mol. Plant-Microbe Interact. 1999; 12: 119-129Crossref PubMed Scopus (67) Google Scholar) unless otherwise specified. Construction of the dps and bfd Deficient Mutants and General DNA Methods—A genomic fragment from the dps locus was amplified by PCR with the primers 5′ sense dps1s (5′-attatcgcctcgctggga-3′) and 3′ reverse dps1r (5′-catcaaaacgtccttcctc-3′) and cloned using the pGEM-T Easy vector. This fragment was then subcloned into the pBC plasmid using appropriate restriction enzymes to introduce a unique EcoRV restriction site into the dps gene. The Ω-Spec interposon from pHP45Ω hydrolyzed with SmaI was inserted into the EcoRV site to obtain plasmid pAB12 (38Résibois A. Colet M. Faelen M. Schoonejans E. Toussaint A. Virology. 1984; 137: 102-112Crossref PubMed Scopus (96) Google Scholar, 39Prentki P. Krisch H.M. Gene (Amst.). 1984; 29: 303-313Crossref PubMed Scopus (1353) Google Scholar). To introduce a nonpolar mutation in the bfd gene, the apha-3 cassette, obtained by SmaI digestion of pUC18K, was inserted into the T4 Polymerase blunted NsiI site of pAB3 (40Ménard R. Sansonetti P. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (619) Google Scholar). Plasmid transformation and marker exchange recombination with the chromosome were performed as described previously (37Franza T. Sauvage C. Expert D. Mol. Plant-Microbe Interact. 1999; 12: 119-129Crossref PubMed Scopus (67) Google Scholar). Correct recombination was confirmed by PCR and Southern blot hybridization experiments. For construction of plasmid pAB14, a genomic fragment from the bfd-bfr encoding locus was amplified by PCR with the primers sense bfr1s (5′-ggtcgtgtagagcggca-3′) and reverse bfdr (5′-gtagaagcggtcaacacagag-3′) and inserted into the pGEM-T Easy vector. This fragment was subcloned into the ApaI and SpeI sites of the pBC plasmid. DNA manipulation techniques (chromosomal DNA isolation, cloning, and electrophoresis) were described previously (35Franza T. Enard C. Van Gijsegem F. Expert D. Mol. Microbiol. 1991; 5: 1319-1329Crossref PubMed Scopus (25) Google Scholar). Plasmids were extracted using the QIAprep spin miniprep kit (Qiagen). All of the cloning experiments were performed in the DH5α strain of E. coli. PCR was performed in a DNA thermocycler (Hybaid PCR Express System) with denaturation at 94 °C for 60 s, annealing at 52 °C for 75 s, an extension at 72 °C for 90 s, and a final extension at 72 °C for 10 min. PCR products were cloned using the plasmid pGEM-T Easy, according to the manufacturer's instructions. Nucleotide sequences of PCR products and plasmids were obtained from Genome Express. In Vivo Labeling of Bacterial Cultures, Preparation of Whole Cell Extracts, and Analysis of Protein-bound 59Fe Iron—An overnight culture in L broth of the strain to be studied was diluted 1:40 in 5 ml of Tris medium supplemented with glucose placed in a 50-ml Erlenmeyer flask and incubated with shaking until A600 nm = 0.4 was reached. Iron labeling was started by adding a ferric chrysobactin complex solution prepared with 59FeCl3 (3-20 mCi/mg iron in 0.5 m HCl; GE Healthcare). Samples (1 ml) were taken at the indicated times. Excess unlabeled ferric chrysobactin was added to each sample. Bacterial cells were spun down in a microcentrifuge, at 7000 RPM at 4 °C. The cells were washed in a solution containing 50 mm potassium phosphate, pH 7.8, 0.1 mm EDTA, and 10 mm MgCl2 and harvested by centrifugation. The bacterial pellets were resuspended in 20-40 μl of the same solution with DNase I and lysozyme added at final concentrations of 0.1 and 0.2 mg/ml, respectively, and incubated for 30 min at 4 °C. The cells were lysed by six freeze/thaw cycles. The extracts were centrifuged at 15,000 × g for 5 min, and supernatant fluids were kept at -20 °C. To analyze the fate of iron during bacterial growth, labeled cells were centrifuged at 3000 × g at 4 °C for 15 min. The cells were washed twice in Tris medium; washing fluids were eliminated by centrifugation. The washed cells were resuspended in Tris medium with glucose and left to grow for 2 h. The samples (1 ml) were taken at the indicated times, and bacterial cells were treated as described above. Whole cell extracts (25 μg protein) were analyzed by native PAGE in 10% acrylamide and Tris-glycine buffer. Protein concentration was determined by the Bradford method (41Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (221990) Google Scholar). The dried gels were autoradiographed at -80 °C, for 48 h, using KODAK BioMax XAR films. For each bacterial strain, six independent time course experiments were performed. The autoradiograms shown are representative of one experiment. For each time point, the amount of 59Fe bound to relevant proteins was determined by scintillation counting of the excised bands from the gels. The results are given as the relative percentages of total counts measured on the gels, for each lane. The data reported are the averages of six independent experiments, and the standard deviations are indicated. Immunodetection of the Bfr Protein—Proteins (25 μg) were loaded and run onto 15% polyacrylamide 0.1% SDS denaturating gels. The proteins were transferred onto nitrocellulose membranes (Protran BA 83; Whatman) at 350 mA for 75 min in 30 mm Tris, 192 mm glycine, 0.025% SDS, 20% v/v methanol, pH 8.3, using the Bio-Rad mini-Trans-Blot electrophoretic transfer cell. Bfr antiserum was used at a dilution of 1/4000. Antibody binding was detected with goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase. Analysis of FMP Iron and Mass Spectrometry—Bands corresponding to the 59Fe-labeled proteins doublet detected by autoradiography of native gels were cut out (Fig. 5A, panel 1) and analyzed by SDS-PAGE in 10% acrylamide and Tris-glycine buffer (Fig. 5A, panel 3), as described by Schägger and Von Jagow (42Schägger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1937) Google Scholar). The gels were stained with Coomassie Blue. For control experiments, the protein bands excised from the gel were analyzed a second time on native PAGE (Fig. 5A, panel 2) to check for the presence of a 59Fe spot corresponding to the protein doublet detected on the first gel. Individual spots visualized after SDS-PAGE were excised, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin overnight at 37 °C. Tryptinized peptides were extracted from the gel pieces with 50% acetonitrile, 0.1% trifluoroacetic acid, concentrated, desalted, using a ZipTip (Millipore, Molsheim, France), and spotted onto a steel target with α-cyano-4-hydroxycinnamic acid as a matrix. The peptide mass fingerprint was acquired after external calibration with ions from des-Arg1-Bradykinin, Angitensin I, Glu1-Fibrinopeptide B, and neurotensin, on a 4800 TOF-TOF spectrometer (Applied Biosystems) equipped with a YAG-200 Hz laser (355 nm). Operating parameters for the Reflectron included 1500 laser shots/spectrum. Monoisotopic masses were used with a maximum deviation of ± 100 ppm for mass assignment. For protein identification, the peptide mass fingerprint was searched against the ASAP data base, using Mascot (Matrix Science, London, UK). Protein hits were accepted if the Mascot score was greater than the significance threshold. This procedure was carried out in six independent samples isolated from cytosolic extracts of parental cells supplied with 59Fe-chrysobactin at concentrations of 0.25 μm, for 40 min. Transport Assays—Bacterial cultures were grown in the same conditions used for in vivo labeling experiments. Transport assays were carried out as described by Rauscher et al. (43Rauscher L. Expert D. Matzanke B.F. Trautwein A.X. J. Biol. Chem. 2002; 277: 2385-2395Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), with the addition of 59Fe-labeled chrysobactin to the transport medium at a concentration of 0.25 μm. Enzyme Assays—Aconitase activity was assayed in bacterial pellets from 100-ml cultures grown in the conditions indicated and stored at -80 °C for 24 h, as described by Gardner (44Gardner P.R. Methods Enzymol. 2002; 349: 9-23Crossref PubMed Scopus (253) Google Scholar). The protein concentration was determined by the Bradford method. Intracellular Distribution of Iron in Cells Supplied with 59Fe-Chrysobactin—Previous studies have shown that the ferric chrysobactin complex is dissociated inside the cell through a rapid reductive process, making iron available for metabolic needs (43Rauscher L. Expert D. Matzanke B.F. Trautwein A.X. J. Biol. Chem. 2002; 277: 2385-2395Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Thus, we used 59Fe-chrysobactin to try to identify intracellular protein targets of iron in E. chrysanthemi cells. We used a double mutant strain deficient in biosynthesis of both chrysobactin and achromobactin (PPV20) to avoid iron exchange between both ligands. In the low iron minimal Tris medium, the doubling time of this mutant was 160 min. The addition of ferric chrysobactin to the medium stimulated the growth rate, the doubling time being 100 min. Cytosolic protein extracts were analyzed by PAGE on nondenaturing gels, and protein-bound iron was visualized by autoradiography as described under "Experimental Procedures." We first tested a concentration range of 0.06-1 μm 59Fe-chrysobactin added over a period of 40 min (Fig. 1A). We detected a continuum of bands increasing in intensity with higher 59Fe concentrations, which could correspond to the banding region revealed by Coomassie Blue staining. In addition, two strong 59Fe signals (designated as 1 and 2 on Fig. 1A), a slowly migrating protein species and a quickly migrating protein doublet, were apparent. A third signal (designated as 3 on Fig. 1A) became visible with concentrations of 59Fe higher than 0.125 μm. We then performed a time course experiment. Protein-bound iron was probed at 10-min intervals over a 40-min period after adding 0.25 μm 59Fe-chrysobactin (Fig. 1B). We observed the same 59Fe signals increasing in intensity with time. Thus, iron binds a wide variety of proteins. Those referred to as signals 1 and 3 could be ferritin-like compounds. To test this possibility, we performed similar experiments with bfr, dps, and ftnA mutant strains carrying defects in the production of Bfr, Dps, or FtnA protein (PPV51, PPV52, and PPV53), respectively. Iron-deprived bacterial cells were supplied with 0.25 μm 59Fe-chrysobactin for 40 min, and protein-bound iron was analyzed (supplemental Fig. S1). Signal 1, identified in the parental strain as a slowly migrating protein, was absent from the bfr mutant but present in dps and ftnA mutants, suggesting that this protein could be Bfr. Signal 3 (Fig. 1, A and B), another slowly migrating protein identified in the parental strain, was present in the ftnA mutant but absent from the dps mutant. This signal was therefore likely to correspond to Dps. A fur mutant, which displays a low level of transcriptional activity of the bfd-bfr operon, showed a particularly strong Dps signal (supplemental Fig. S1). To confirm these data, we increased the concentration of 59Fe-chrysobactin in the medium to 1 μm and compared the signal patterns obtained from mutant strains to those from the parental strain (Fig. 1C). In the parental strain, the signals corresponding to Bfr and Dps were strong, whereas they were absent from the respective mutants. Both signals were absent from the bfr dps double mutant (PPV54) (Fig. 1C). The doublet corresponding to the quickly migrating protein species was present in all of the mutant strains. Its signal intensity increased over time, as observed for the parental strain (supplemental Fig. S1). Thus, some of the iron released from chrysobactin is sequestered by Bfr, Dps, and a set of proteins unrelated to ferritins, designated FMP. The amounts of 59Fe bound to these various protein species were quantified (supplemental Table S2). In parental cells supplied with 0.25 μm 59Fe-chrysobactin for 40 min, iron bound to Bfr represented ∼5%, Dps iron represented ∼1%, and FMP iron 16% of total 59Fe detected on the gels. In the bfr mutant, levels of FMP iron were similar to those of the parent strain, whereas they reached 20% in the dps mutant. In the fur mutant, iron bound to Dps and FMP represented 16 and 20% of total 59Fe signal, respectively. We did not detect iron bound to FtnA, probably because the ftnA gene is not expressed in iron-deprived cells (32Boughammoura A. Matzanke B. Böttger L. Reverchon S. Lesuisse E. Expert D. Franza T. J. Bacteriol. 2008; 190: 1518-1530Crossref PubMed Scopus (39) Google Scholar). Release of Iron from Bacterioferritin Requires Bacterioferredoxin—The roles of bacterioferritin and bacterioferredoxin in iron metabolism are not well understood. As in E. coli, E. chrysanthemi bacterioferritin is not involved in long term iron storage (21Andrews S.C. Robinson A.K. Rodriguez-Quinones F. FEMS Microbiol. Rev. 2003; 27: 215-237Crossref PubMed Scopus (1917) Google Scholar, 32Boughammoura A. Matzanke B. Böttger L. Reverchon S. Lesuisse E. Expert D. Franza T. J. Bacteriol. 2008; 190: 1518-1530Crossref PubMed Scopus (39) Google Scholar). To gain insight into the roles of these proteins, we constructed a nonpolar bfd-negative mutant (PPV55) and examined the distribution of iron in this mutant
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