Identification and Characterization of a Membrane Permease Involved in Iron-Hydroxamate Transport inStaphylococcus aureus

ABSTRACT Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An S. aureusmutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn917-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis. DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG, all of which show significant similarities to ferric hydroxamate uptake (fhu) genes in B. subtilis. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the S. aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S. aureus. This regulation is attributable to Fur in S. aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream offhuCBG are recognized by the Escherichia coliFur protein.

[1]  S. Payne,et al.  Detection, isolation, and characterization of siderophores. , 1994, Methods in enzymology.

[2]  G. Archer,et al.  The Staphylococci in Human Disease , 1996 .

[3]  J. H. Crosa,et al.  Aerobactin iron transport genes commonly encoded by certain ColV plasmids occur in the chromosome of a human invasive strain of Escherichia coli K1 , 1984, Infection and immunity.

[4]  G. Jung,et al.  Isolation and characterization of staphyloferrin A, a compound with siderophore activity from Staphylococcus hyicus DSM 20459. , 1990, FEMS microbiology letters.

[5]  B. Modun,et al.  The Staphylococcal Transferrin-Binding Protein Is a Cell Wall Glyceraldehyde-3-Phosphate Dehydrogenase , 1999, Infection and Immunity.

[6]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[7]  J. R. White,et al.  Iron enhances the bactericidal action of streptonigrin. , 1982, Biochemical and biophysical research communications.

[8]  P. Williams.  Novel iron uptake system specified by ColV plasmids: an important component in the virulence of invasive strains of Escherichia coli , 1979, Infection and immunity.

[9]  J. Heesemann,et al.  Virulence of Yersinia enterocolitica is closely associated with siderophore production, expression of an iron‐repressible outer membrane polypeptide of 65 000 Da and pesticin sensitivity , 1993, Molecular microbiology.

[10]  D. Heinrichs,et al.  Pyoverdine-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein. , 1991, FEMS microbiology letters.

[11]  D. Heinrichs,et al.  Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein , 1991, Infection and immunity.

[12]  A Danchin,et al.  Codon usage and lateral gene transfer in Bacillus subtilis. , 1999, Current opinion in microbiology.

[13]  J. Porter,et al.  Long-term outcome of continuous 24-hour deferoxamine infusion via indwelling intravenous catheters in high-risk β-thalassemia , 2000 .

[14]  J. Brock,et al.  The effect of desferrioxamine on the growth of Staphylococcus aureus, Yersinia enterocolitica and Streptococcus faecalis in human serum: Uptake of desferrioxamine-bound iron , 1983 .

[15]  H. Rogers.  Iron-Binding Catechols and Virulence in Escherichia coli , 1973, Infection and immunity.

[16]  P. Williams,et al.  Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein , 1994, Infection and immunity.

[17]  J. Kornblum,et al.  Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus , 1988, Journal of bacteriology.

[18]  D. Hohnadel,et al.  Cepabactin from Pseudomonas cepacia, a new type of siderophore. , 1989, Journal of general microbiology.

[19]  I. Tessman,et al.  THYMIDINE-REQUIRING MUTANTS OF PHAGE T4. , 1963, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Sparling,et al.  Isolation by streptonigrin enrichment and characterization of a transferrin-specific iron uptake mutant of Neisseria meningitidis. , 1987, Microbial pathogenesis.

[21]  K. Hantke,et al.  Fur regulon in gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a fur titration assay. , 1994, Journal of molecular biology.

[22]  J. R. White,et al.  Iron requirement in the bactericidal mechanism of streptonigrin , 1982, Antimicrobial Agents and Chemotherapy.

[23]  G. Jung,et al.  Staphyloferrin A: a structurally new siderophore from staphylococci. , 1990, European journal of biochemistry.

[24]  R. Courcol,et al.  Siderophore production by Staphylococcus aureus and identification of iron-regulated proteins , 1997, Infection and immunity.

[25]  J. H. Crosa.  A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron-sequestering system , 1980, Nature.

[26]  C. D. Cox.  Effect of pyochelin on the virulence of Pseudomonas aeruginosa , 1982, Infection and immunity.

[27]  P. Youngman,et al.  Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions , 1990, Journal of bacteriology.

[28]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[29]  R. Vellanoweth.  Translation and Its Regulation , 1993 .

[30]  V. Singh,et al.  Molecular characterization of the ferric-uptake regulator, fur, from Staphylococcus aureus. , 2000, Microbiology.

[31]  Robert L. Jones,et al.  Effects of iron chelators and iron overload on Salmonella infection , 1977, Nature.

[32]  J. Neilands,et al.  Universal chemical assay for the detection and determination of siderophores. , 1987, Analytical biochemistry.

[33]  S. Foster,et al.  Isolation and characterization of Staphylococcus aureus starvation-induced, stationary-phase mutants defective in survival or recovery. , 1998, Microbiology.

[34]  W. Goebel,et al.  Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene , 1992, Journal of bacteriology.

[35]  P. Stragier,et al.  Antibiotic-resistance cassettes for Bacillus subtilis. , 1995, Gene.

[36]  A. Neely,et al.  Pyoverdin is essential for virulence of Pseudomonas aeruginosa , 1996, Infection and immunity.

[37]  C. Edmiston,et al.  IRON AND INFECTION: MOLECULAR, PHYSIOLOGICAL AND CLINICAL ASPECTS, 2ND EDITION , 1999 .

[38]  A. A. Miles,et al.  Microbial iron-chelators and their action on Klebsiella infections in the skin of guinea-pigs. , 1978, British journal of experimental pathology.

[39]  R. Losick,et al.  Bacillus Subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics , 1993 .

[40]  X. Nassif.  Iron and infection. Molecular, physiological and clinical aspects, Bullen J.J., Griffiths E.. John Wiley & Sons, Chichester, New York (1987), Prix: £38.00 , 1988 .

[41]  P. Williams,et al.  Isolation and characterisation of Haemophilus influenzae type b mutants defective in transferrin-binding and iron assimilation. , 1991, FEMS microbiology letters.

[42]  R. Novick.  Genetic systems in staphylococci. , 1991, Methods in enzymology.

[43]  H. Nikaido,et al.  Overview of bacterial ABC transporters. , 1998, Methods in enzymology.

[44]  A. Kotoujansky,et al.  Systemic virulence of Erwinia chrysanthemi 3937 requires a functional iron assimilation system , 1988, Journal of bacteriology.

[45]  J. Rabinowitz,et al.  1 – Translational Specificity in Bacillus subtilis , 1985 .

[46]  B. Ratcliff.  Iron and Infection. Molecular, Physiological and Clinical Aspects , 1988, The Yale Journal of Biology and Medicine.

[47]  W. Köster,et al.  Transmembrane topology of the two FhuB domains representing the hydrophobic components of bacterial ABC transporters involved in the uptake of siderophores, haem and vitamin B12. , 1998, Microbiology.

[48]  M. Hanson,et al.  Identification and Characterization of SirA, an Iron-Regulated Protein from Staphylococcus aureus , 1999, Journal of bacteriology.

[49]  K. Hantke,et al.  Iron‐hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding protein‐dependent transport system , 1993, Molecular microbiology.

[50]  Haruo Watanabe,et al.  Penicillin-Binding Protein 1 ofStaphylococcus aureus Is Essential for Growth , 1998, Journal of bacteriology.

[51]  P. Hill,et al.  Molecular Cloning of a 32-Kilodalton Lipoprotein Component of a Novel Iron-Regulated Staphylococcus epidermidis ABC Transporter , 1998, Infection and Immunity.

[52]  T. Riley,et al.  Staphylococcal iron requirements, siderophore production, and iron-regulated protein expression , 1994, Infection and immunity.

[53]  M. O'Reilly,et al.  The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage , 1983, Nature.

[54]  V. Braun,et al.  Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism for iron(III) dicitrate in Escherichia coli , 1989, Journal of bacteriology.

[55]  W Köster,et al.  Bacterial iron transport: mechanisms, genetics, and regulation. , 1998, Metal ions in biological systems.