FhuD1, a Ferric Hydroxamate-binding Lipoprotein in Staphylococcus aureus
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m409793200
ISSN1083-351X
AutoresM. Tom Sebulsky, Craig D. Speziali, Brian H. Shilton, David R. Edgell, David E. Heinrichs,
Tópico(s)Metal-Catalyzed Oxygenation Mechanisms
ResumoStaphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us to examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments. Staphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us to examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments. With few exceptions, all bacteria have an absolute requirement for iron (1Posey J.E. Gherardini F.C. Science. 2000; 288: 1651-1653Crossref PubMed Scopus (401) Google Scholar, 2Weinberg E.D. Perspect. Biol. Med. 1997; 40: 578-583Crossref PubMed Scopus (126) Google Scholar). The amount of free, biologically relevant iron, however, is negligible at physiological pH (10-18m) (3Braun V. Hantke K. Köster W. Met. Ions Biol. Syst. 1998; 35: 67-145PubMed Google Scholar). This is primarily caused by the rapid formation of iron(III)-hydroxy precipitates that are highly insoluble. In response to the stress generated from a low iron environment, many microorganisms secrete low molecular weight iron chelating compounds termed siderophores. Siderophores commonly bind ferric iron with extremely high affinities (4Byers B.R. Arceneaux E.L. Sigel A. Sigel H. Iron Transport and Storage in Microorganisms, Plants, and Animals. 35. Marcel Dekker, Inc., New York1998: 37-66Google Scholar) and serve to solubilize iron from the biologically inert iron(III)-hydroxy precipitates. Typically, ferric-siderophores are mobilized across the cell envelope of Gram-negative bacteria expressing cognate outer membrane receptors, periplasmic-binding proteins, and associated ABC 1The abbreviations used are: ABC, ATP-binding cassette; SAXS, small angle x-ray scattering; MBP, maltose-binding protein.-type transporters (5Köster W. Res. Microbiol. 2001; 152: 291-301Crossref PubMed Scopus (213) Google Scholar, 6Ratledge C. Dover L.G. Annu. Rev. Microbiol. 2000; 54: 881-941Crossref PubMed Scopus (1176) Google Scholar). In Gram-positive bacteria, ferric siderophores are captured by lipoproteins that function as high affinity receptors and subsequently feed ligand to the ABC transporter in the cytoplasmic membrane. The lipid group on these receptors acts as a tether to anchor the receptor protein to the external face of the cell membrane (7Sutcliffe I.C. Russell R.R.B. J. Bacteriol. 1995; 177: 1123-1128Crossref PubMed Scopus (332) Google Scholar). Whereas Staphylococcus aureus isolates produce endogenous siderophores (8Courcol R.J. Trivier D. Bissinger M-C. Martin G.R. Brown M. R. W Infect. Immun. 1997; 65: 1944-1948Crossref PubMed Google Scholar, 9Dale S.E. Doherty-Kirby A. Lajoie G. Heinrichs D.E. Infect Immun. 2004; 72: 29-37Crossref PubMed Scopus (167) Google Scholar, 10Drechsel H. Freund S. Nicholson G. Haag H. Jung O. Zähner H. Jung G. BioMetals. 1993; 6: 185-192Crossref PubMed Scopus (95) Google Scholar, 11Konetschny-Rapp S. Jung G. Meiwes J. Zähner H. Eur. J. Biochem. 1990; 191: 65-74Crossref PubMed Scopus (134) Google Scholar), S. aureus utilizes others that are produced by other microorganisms (so-called xenosiderophores) for growth under conditions of iron deprivation. One group of xenosiderophores that S. aureus can utilize is the hydroxamate-class of siderophore, including aerobactin, coprogen, ferrioxamine B (Desferal™), ferrichrome, and rhodotorulic acid (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar). In our previous studies, we showed that S. aureus strain RN6390 possessed at least five different iron-regulated genes whose products were involved in the ferric hydroxamate uptake process (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar, 13Sebulsky M.T. Hohnstein D. Hunter M.D. Heinrichs D.E. J. Bacteriol. 2000; 182: 4394-4400Crossref PubMed Scopus (110) Google Scholar, 14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). A three gene operon, fhuCBG, encodes a classical traffic ATPase (for a review of traffic ATPases, see Ref. 15Ames G.F. Mimura C.S. Holbrook S.R. Shyamala V. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 1-47PubMed Google Scholar) while a fourth gene, fhuD2, codes for a lipoprotein (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar) that functions as a high affinity receptor for aerobactin, coprogen, Desferal™, ferrichrome, and rhodotorulic acid (14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The fifth gene involved in this transport system, fhuD1, is predicted to encode a protein with 50% total similarity to FhuD2. We showed that the fhuD1 gene product could partially compensate for the loss of the fhuD2 gene product (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar). In this communication, we show that the presence and location of fhuCBG and fhuD2 are strictly conserved in several S. aureus genomes. In contrast, the fhuD1 gene is mobile: it is absent in some strains and appears on a genomic island in others. The unusual occurrence of the fhuD1 gene warranted a detailed investigation of the function of the FhuD1 protein. Bacterial Strains and Growth Media—S. aureus RN6390 (16Peng H.L. Novick R.P. Kreiswirth B. Kornblum J. Schlievert P. J. Bacteriol. 1988; 170: 4365-4372Crossref PubMed Scopus (420) Google Scholar) served as the wild-type staphylococcal strain in this study, and derivatives H430 (RN6390 fhuD1::Km), H364 (RN6390 fhuD2::Tet), and H295 (RN6390 fur::Km) have been previously described (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar, 13Sebulsky M.T. Hohnstein D. Hunter M.D. Heinrichs D.E. J. Bacteriol. 2000; 182: 4394-4400Crossref PubMed Scopus (110) Google Scholar). Escherichia coli DH5α (Promega) was used for routine cloning and recombinant protein overexpression. For the routine cultivation of S. aureus, tryptic soy broth (Difco) was the chosen growth medium, and Luria-Bertani broth (Difco) was the selected medium for E. coli. Solid media were obtained by the addition of 1.5% (w/v) Bacto agar (Difco). S. aureus was grown in Tris-minimal succinate (TMS) medium supplemented with either FeCl3 (50 μm) or ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA) (1 μm) to create iron-replete and iron-restricted growth conditions, respectively. TMS was made as follows: 40 ml of a 25× Tris minimal salts stock (in 1 liter was dissolved 145 g of NaCl, 92.5 g of KCl, 27.5gofNH4Cl, 3.55 g of Na2SO4, 6.8gofKH2PO4) was added to 12.1 g of Tris base, 16.6 g of succinate, and 50 ml 20% casamino acids in ∼800 ml of water, and the pH was adjusted to 7.4 prior to autoclaving. After autoclaving, 2 ml of tryptophan (10 mg/ml) and 1-ml volumes of cysteine (22 mg/ml), thiamine (16.9 mg/ml), nicotinic acid (1.23 mg/ml), pantothenic acid (0.5 mg/ml), biotin (0.01 mg/ml), MgCl2 (95.3 mg/ml), and CaCl2 (11.1 mg/ml) were incorporated and the volume adjusted to 1 liter with sterilized MilliQ water. When required, kanamycin (50 μg/ml) and tetracycline (4 μg/ml) were included in S. aureus growth medium. Ampicillin (100 μg/ml), when appropriate, was incorporated into medium for the growth of E. coli. Protein Overexpression and Purification—The fhuD1 gene, without the predicted signal sequence codons, was PCR-amplified as a 1.0-kb DNA fragment, digested with BamHI and EcoRI (sites were incorporated into the oligonucleotides) and then cloned into BamHI- and EcoRI-digested pGEX-2T-TEV (14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The resulting plasmid, pMTS78, from which a translational fusion between glutathione S-transferase (GST) and FhuD1 is encoded, was introduced into E. coli strain DH5α to generate strain H659. Strain H659 was grown to an approximate OD600 of 0.8, and, following the addition of isopropyl-1-thio-β-d-galactopyranoside (0.4 mm), growth was allowed to continue for 3 h before the cells were lysed. The resulting supernatant was centrifuged at 40000 × g to remove insoluble cellular components and then passed across a 5-ml GSTrap column (Amersham Biosciences) for purification. Once purified, the GST-FhuD1 fusion protein was cleaved by the TEV protease (4 h at 23 °C in 50 mm Tris-Cl, pH 8.0) before it was applied to an 8-ml Mono-S column (Amersham Biosciences) and recovered using a 0–1 m NaCl gradient. Generation of Polyclonal Antisera to FhuD1—The purified FhuD1 was used to generate polyclonal antisera in two New Zealand White rabbits (Charles River). Briefly, 100 μg of protein in Titer Max Gold adjuvant (Cedarlane) was injected subcutaneously into each rabbit. The rabbits were boosted with 50 μg of protein at 2–4 weeks, and at 5 weeks, the rabbits were sacrificed, and sera were collected. Sera were adsorbed three times over polyvinylidene difluoride-immobilized DH5α (pGEX-2T-TEV) cell lysate, induced to express glutathione S-transferase. Detergent Extraction and Phase Partitioning—S. aureus cells in late log-phase were collected, washed three times with 0.9% NaCl (w/v), and then equilibrated to an OD600 of 1.0. The cell wall was digested using lysostaphin (50 μg), followed by sonication to lyse cells. Triton X-114 phase partitioning was performed as previously described (14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Before electrophoresis on SDS-polyacrylamide gels, the detergent pellets were diluted 1:1 with water followed by the addition of sample loading buffer. Immunoblot Analysis—Following SDS-polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose membranes. Washing and detection were performed using the ECL Plus Western blotting detection system (Amersham Biosciences) as per the manufacturer's instructions. In all cases, the primary antibody was adsorbed polyclonal antisera raised against FhuD1 (typically diluted 1:15,000), and the secondary antibody was horseradish peroxidase-conjugated goat α-rabbit (Amersham Biosciences) used at a 1:5000 dilution. FhuD1 Dissociation Constant (KD) Determinations—Fluorescence titration experiments were conducted using a Flourolog 3 spectrofluorometer (ISA Instruments.) Purified FhuD1 protein was used at a concentration of 8–16 nm, the reaction buffer was 2 ml of 10 mm sodium phosphate buffer, pH 7.5, and the temperature was held constant at 23 °C. The excitation and emission slits were set at 1 and 10 nm, respectively, with excitation and emission wavelengths set at 283 and 348 nm, respectively. Dissociation constants (KD) were calculated as previously described (14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Proteinase K Digests—The FhuD1 protein (2 μg) was mixed with ferric-siderophore and incubated at 55 °C for 30 min in the presence of proteinase K (2 μg per reaction). The reaction buffer was 30 mm sodium phosphate buffer, pH 7.5. Samples were heated to 95 °C in sample loading buffer for 5 min and then examined by SDS-PAGE as described previously (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Siderophores—Ferrichrome was purchased from Sigma, desferrioxamine B (used as Desferal™ [Novartis]) was obtained from the London Health Sciences Centre, and aerobactin and coprogen were purchased from EMC microcollections GmbH (Tübingen, Germany). Pyoverdine was prepared by methods previously described (18Meyer J.M. Stintzi A. De Vos D. Cornelis P. Tappe R. Taraz K. Budzikiewicz H. Microbiology. 1997; 143: 35-43Crossref PubMed Scopus (213) Google Scholar). Siderophore Plate Bioassays—Siderophore plate bioassays were performed essentially as previously described (13Sebulsky M.T. Hohnstein D. Hunter M.D. Heinrichs D.E. J. Bacteriol. 2000; 182: 4394-4400Crossref PubMed Scopus (110) Google Scholar). 5-μl volumes of ferric siderophore solutions, at concentrations as indicated in Table II, were placed onto sterile 6-mm paper discs before being placed on agar plates containing impregnated bacteria. Bacteria were incorporated into the plates at a concentration of 1 × 104 cells/ml. Results were scored after incubation at 37 °C for 48 h followed by 24 h at room temperature.Table IIGrowth promoting ability of hydroxamate siderophores on S. aureusa Values in gray bars indicate concentration (in μm) of siderophore.b Represents mean diameter (in mm) of growth halo; discs are 6 mm. Open table in a new tab a Values in gray bars indicate concentration (in μm) of siderophore. b Represents mean diameter (in mm) of growth halo; discs are 6 mm. Small Angle X-ray Scattering (SAXS)—SAXS measurements for FhuD2 were carried out as previously described (14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Measurements for FhuD1 were made at the Advanced Photon Source, Beamline ID-18, as follows: the sample temperature was 20 °C, and the protein solution was moved continuously through a 1-mm quartz capillary during the course of the measurement to minimize the effects of radiation damage. Data were collected at 1370 mm using a CCD detector, with X-rays at a wavelength of 1.03 Å, to cover the momentum transfer range 0.027 nm-1 < S < 0.60 nm-1, where S is defined as 2sinθ/λ (2θ is the scattering angle, while λ is the wavelength of the radiation). Note that Q, used in the Guinier plots, is defined as 4πsinθ/λ. For each sample, 5 10-s exposures were recorded that consisted of 3 measurements from the protein solution bracketed by 2 measurements of the buffer solution. Data were integrated using Fit2D (19Hammersley, A. P. (1998) ESRF Internal Report, ESRF97HA02T, Grenoble, FranceGoogle Scholar, 20Hammersley, A. P. (1997) ESRF Internal Report, ESRF97HA02T, Grenoble, FranceGoogle Scholar), and exported into a spreadsheet program. The three protein solution curves and two background buffer curves were inspected and averaged, and the background buffer curve was subtracted, with no correction, from the protein solution curve to yield scattering from the hydrated protein. In our previous work that described the properties of the FhuD2 protein in detail (14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), we showed that expression of FhuD2 was iron-regulated, that FhuD2 was amphiphilic and present in the cell membrane, and that purified FhuD2 bound ferric hydroxamates with dissociation constants in the nanomolar to micromolar range. The data suggested that the protein acts as a high affinity receptor for hydroxamate siderophores at the external face of the cytoplasmic membrane in S. aureus (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar, 14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In our initial genetic studies, however, we also identified a second gene, fhuD1, that was predicted to encode a protein with ∼50% similarity to FhuD2 (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar). Examination of the phenotype of fhuD1 and fhuD2 knockout strains demonstrated that the fhuD1 gene product could partially compensate for the loss of fhuD2 in terms of the transport of hydroxamate siderophores. The following studies were undertaken to further characterize the function of FhuD1. Variable Occurrence of fhuD1 in S. aureus—The observation that FhuD1 is more similar in sequence to FhuD2 than to other FhuD homologs encoded from the genomes of other Gram-positive bacteria suggests that they arose from a gene duplication event at some point in the evolution of S. aureus. Seven S. aureus genome sequencing projects (either completed or in the process of completion) were analyzed for the presence of fhu genes: S. aureus NCTC 8325 (www.genome.ou.edu/staph.html), MRSA252 (21Holden M.T. Feil E.J. Lindsay J.A. Peacock S.J. Day N.P. Enright M.C. Foster T.J. Moore C.E. Hurst L. Atkin R. Barron A. Bason N. Bentley S.D. Chillingworth C. Chillingworth T. Churcher C. Clark L. Corton C. Cronin A. Doggett J. Dowd L. Feltwell T. Hance Z. Harris B. Hauser H. Holroyd S. Jagels K. James K.D. Lennard N. Line A. Mayes R. Moule S. Mungall K. Ormond D. Quail M.A. Rabbinowitsch E. Rutherford K. Sanders M. Sharp S. Simmonds M. Stevens K. Whitehead S. Barrell B.G. Spratt B.G. Parkhill J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9786-9791Crossref PubMed Scopus (733) Google Scholar), MSSA476 (21Holden M.T. Feil E.J. Lindsay J.A. Peacock S.J. Day N.P. Enright M.C. Foster T.J. Moore C.E. Hurst L. Atkin R. Barron A. Bason N. Bentley S.D. Chillingworth C. Chillingworth T. Churcher C. Clark L. Corton C. Cronin A. Doggett J. Dowd L. Feltwell T. Hance Z. Harris B. Hauser H. Holroyd S. Jagels K. James K.D. Lennard N. Line A. Mayes R. Moule S. Mungall K. Ormond D. Quail M.A. Rabbinowitsch E. Rutherford K. Sanders M. Sharp S. Simmonds M. Stevens K. Whitehead S. Barrell B.G. Spratt B.G. Parkhill J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9786-9791Crossref PubMed Scopus (733) Google Scholar), COL (TIGR), N315 (22Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumura H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar), Mu50 (22Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumura H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar), and MW2 (23Baba T. Takeuchi F. Kuroda M. Yuzawa H. Aoki K. Oguchi A. Nagai Y. Iwama N. Asano K. Naimi T. Kuroda H. Cui L. Yamamoto K. Hiramatsu K. Lancet. 2002; 359: 1819-1827Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar). As illustrated in Fig. 1, A and B, fhuCBG and fhuD2 are present in the same genomic locations in all seven genomes. In contrast, fhuD1 is only present in five genomes (MW2, Mu50, NCTC8325, MSSA476, and COL) (Fig. 1C). In four of these genomes (MW2, COL, NCTC8325, and MSSA476), the fhuD1 gene is in a similar genomic location, while in the Mu50 genome the fhuD1 coding sequence was found on a genomic island referred to as SaGIm (22Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumura H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar). The fhuD homolog identified by Kuroda et al. (22Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumura H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar) was designated as SAV0812. However, our analyses showed that SAV0812 encodes a secreted Von Willebrand factor-binding protein. The fhuD1 coding region is SAV0803, still within the SaGIm island. It is interesting to note that the presence of fhuD1 is correlated with the presence of the mw1944 homolog, encoding a predicted 283 amino acid protein with similarity to a methyl-transferase involved in tetracenomycin synthesis. The two genes are inserted between mw1943 and mw1946 homologs in MW2, NCTC8325, MSSA476, and COL, and are both absent from the equivalent genomic location in N315, Mu50, and MRSA252. Interestingly, mw1944-fhuD1 (between mw1943 and mw1946) is between a pathogenicity island, SaPIn1 (N315)/SaPIm1 (Mu50), and a bacteriophage, ΦN315/ΦMu50A, in the genomes of N315 and Mu50 (22Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumura H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar). The unusual location/occurrence of the fhuD1 gene within the genomes of S. aureus warranted a functional characterization of the fhuD1 gene product. S. aureus fhuD1 Encodes an Iron-regulated Lipoprotein— Late log phase cells of S. aureus RN6390 and derivatives were harvested by centrifugation after culture in either iron-replete or iron-restricted medium. Cells were subjected to Triton X-114 phase partitioning, a technique that separates cell components into three fractions: insoluble, aqueous, and detergent soluble (membrane-associated proteins are present within the latter fraction). In Fig. 2, we show that FhuD1 partitions with the detergent soluble fraction, indicating that the protein is amphipathic and associated with the membrane. Similar results using Triton X-114 fractionation have been obtained for several other lipoproteins (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar, 24Cockayne A. Hill P.J. Powell N.B.L. Bishop K. Sims C. Williams P. Infect. Immun. 1998; 66: 3767-3774Crossref PubMed Google Scholar, 25Tai S.S. Yu C. Lee J.K. FEMS Microbiol. Lett. 2003; 220: 303-308Crossref PubMed Scopus (37) Google Scholar). This result is consistent with the presence of a lipobox motif (15LTAC18) in FhuD1 (26Sutcliffe I.C. Harrington D.J. Microbiology. 2002; 148: 2065-2077Crossref PubMed Scopus (141) Google Scholar). Our results confirm that FhuD1, although primarily hydrophilic, resides within the detergent soluble membrane component of the cell. Expression of FhuD1 in the cell membrane was regulated by exogenous iron concentrations, since FhuD1 is only detected in cells grown in iron-starved medium, and this regulation is mediated by the activity of the Fur protein, because a fur mutant-expressed FhuD1 in the membrane of cells grows under iron-replete conditions (Fig. 2). No band is observed in the lane loaded with protein extracted from H430 (RN6390 fhuD1::Km) indicating that the antibody is specific for FhuD1 and does not cross-react with FhuD2. FhuD1 Binds Hydroxamate-type Siderophores but with Less Affinity Than FhuD2—Our previous studies identified a role for fhuD1 in ferric hydroxamate uptake since a fhuD2 knockout strain that expressed fhuD1 could still utilize ferric hydroxamates, albeit to a lesser extent than wild-type RN6390 (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar). Moreover, since FhuD1 shares 50% total similarity with FhuD2 (12Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (87) Google Scholar, 14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), a protein that our group has characterized as a high affinity receptor for ferric hydroxamates (14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), we reasoned that FhuD1 is also a receptor for ferric hydroxamates. To investigate this further, and as an initial attempt to demonstrate that ferric hydroxamates physically interact with FhuD1, we examined in SDS-PAGE the reaction products of FhuD1 treated with proteinase K after incubation with various ferric siderophores (Fig. 3). We showed that the slowest migrating form of FhuD1 was protected from proteolysis when preincubated with either coprogen, Desferal™ or ferrichrome (Fig. 3, lanes 6–8, respectively), but not with aerobactin (Fig. 3, lane 5) or the pseudomonal siderophore pyoverdine (Fig. 3, lane 9); the latter siderophore does not promote the growth of S. aureus in biological assays. The identity of the faster migrating band in these experiments was not confirmed but is likely partially degraded FhuD1 as a result of incomplete proteolysis in the presence of some siderophores. In the presence of an appropriate ligand, it is assumed that FhuD1 adopts a conformation that is resistant to proteolytic degradation. As a more appropriate measure of ligand binding, we then used fluorescence spectroscopy to determine the affinity of FhuD1 for ferric hydroxamates, and the results are presented in Table I. Compared with FhuD2, FhuD1 has a marginally lower affinity for ferrichrome, Desferal™ and coprogen and a much lower affinity for aerobactin. The low affinity of FhuD1 for aerobactin is consistent with our observation that aerobactin does not protect FhuD1 from proteolysis (see Fig. 3).Table IDissociation constants of FhuD1 for ferric hydroxamate siderophoresProteinKDAerobactinDesferal™CoprogenFerrichromeμmFhuD1400.96.70.05FhuD2aS. aureus FhuD2.0.30.051.70.02FhuDbE. coli FhuD values have been previously reported (14, 31).0.4350.31.0a S. aureus FhuD2.b E. coli FhuD values have been previously reported (14Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 31Rohrbach M.R. Braun V. Köster W. J. Bacteriol. 1995; 177: 7186-7193Crossref Pu
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