Browsing by Author "Xiao, Rong"

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    Fluorescent Pseudomonad Pyoverdines Bind and Oxidize Ferrous Ion
    (Applied and Environmental Microbiology, 1998) Xiao, Rong; Kisaalita, William S.
    Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P. aeruginosa ATCC 15692 (Pa-C), and P. putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe2+-pyoverdine than for Fe3+-pyoverdine. At pH 7.4, about 90% of Fe3+was bound by pyoverdine Pa-C after 24 h whereas Fe2+was bound by the pyoverdine completely in only 5 min. The possibility that Fe2+ underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe2+concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe2+ with pyoverdine in the presence of 8-hydroxyquinoline, an Fe3+-specific chelating agent, resulted in the formation of a Fe3+-hydroxyquinoline complex, suggesting that the iron in the Fe2+-pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.
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    Iron acquisition from transferrin and Iactoferrin by Pseudornonas aeruginosa pyoverdin
    (Microbiology, 1997) Xiao, Rong; Kisaalita, William S.
    Pseudomonas aeruginosa is an opportunistic pathogen which is frequently found in clinical specimens from burns, surface wounds, urinary tract, ear and eye infections, and is commonly isolated from the lungs of patients with cystic fibrosis (CF)(Doggett et al., 1966; Reynolds et al., 1976). In response to iron deprivation P. aeruginosa produces two unrelated siderophores, pyoverdin and pyochelin, as well as membrane receptors for binding the corresponding iron-siderophore complexes. Both of these siderophores promoted the growth of P. aeruginosa when added to medium with iron-transferrin or human sera as the iron sources (Ankenbauer et al., 1985). Because of the higher iron-pyoverdin binding constant (lo3,)(Demange et al., 1990), in comparison to that of iron-pyochelin (lo5)(Cox & Graham, 1979), pyoverdin was considered to be more effective. It has been reported that a pyochelin-deficient mutant (Pvd+ Pch-) strain grew as well as the parent strain whereas a pyoverdin-deficient mutant (Pvd-Pch+) exhi-Abbreviations: CF, cystic fibrosis; ICP, inductively coupled plasma. bited severely retarded growth (Ankenbauer et al., 1985).
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    Purification of Pyoverdines of Pseudomonas fluorescens 2-79 by Copper-Chelate Chromatography
    (Applied and environmental microbiology, 1995) Xiao, Rong; Kisaalita, William S.
    Three pyoverdines, Pf-A, Pf-B, and Pf-C, were purified with copper-chelate Sepharose and Sephadex G-15 columns from Pseudomonas fluorescens 2-79, and the yields (per 100 ml of culture supernatant) were 2.8, 21.6, and 3.2 mg, respectively. The absorption and fluorescence spectra of these pyoverdines were strongly pH dependent. Characteristic changes in the maximal absorbance wavelengths were observed when Fe(sup3+) or Cu(sup2+) was added. The addition of Cu(sup2+) shifted the pyoverdine Pf-B absorbance spectrum so that it exhibited a single peak at 410 nm but did not give rise to a new absorbance maximum at approximately 460 nm, which appeared when Fe(sup3+) was added. Fluorescence quenching experiments revealed that the forward reaction rate constant with pyoverdines was much higher with Cu(sup2+) (10(sup4) to 10(sup5) M(sup-1) s(sup-1)) than with Fe(sup3+) (10(sup2) M(sup-1) s(sup-1)). However, Cu(sup2+)-pyoverdine complexes were completely dissociated by EDTA at a low concentration (0.1 mM), while the level of Fe(sup3+)-pyoverdine complex dissociation at the same EDTA concentration was relatively low. The dissociation of Fe(sup3+)-pyoverdine complexes was EDTA concentration dependent. Formation of free pyoverdine was observed when the three types of Fe(sup3+)-pyoverdine complexes were incubated separately with P. fluorescens 2-79 cells, thus demonstrating that pyoverdines Pf-A, Pf-B, and Pf-C mediate iron transport.