Langmuir 2007, 23, 7189-7195
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Adsorption to Metal Oxides of the Pseudomonas aeruginosa Siderophore Pyoverdine and Implications for Bacterial Biofilm Formation on Metals Hamish G. Upritchard,† Jing Yang,‡ Philip J. Bremer,§ Iain L. Lamont,† and A. James McQuillan*,‡ Departments of Biochemistry, Chemistry, and Food Science, UniVersity of Otago, P.O. Box 56, Dunedin, New Zealand ReceiVed February 11, 2007. In Final Form: April 5, 2007 The initiation of biofilm formation is poorly understood, and in particular, the contribution of chemical bond formation between bacterial cells and metal surfaces has received little attention. We have previously used in situ infrared spectroscopy to show, during the initial stages of Pseudomonas aeruginosa biofilm formation, the formation of coordinate covalent bonds between titanium dioxide particle films and pyoverdine, a mixed catecholate and hydroxamate siderophore. Here we show using infrared spectroscopy that pyoverdine can also form covalent bonds with particle films of Fe2O3, CrOOH, and AlOOH. Adsorption to the metal oxides through the catechol-like 2,3-diamino-6,7dihydroxyquinoline part of pyoverdine was most evident in the infrared spectrum of the adsorbed pyoverdine molecule. Weaker infrared absorption bands that are consistent with the hydroxamic acids of pyoverdine binding covalently to TiO2, Fe2O3, and AlOOH surfaces were also observed. The adsorption of pyoverdine to TiO2 and Fe2O3 surfaces showed a pH dependence that is indicative of the dominance of the catechol-like ligand of pyoverdine. Infrared absorption bands were also evident for pyoverdine associated with the cells of P. aeruginosa on TiO2 and Fe2O3 surfaces and were notably absent for genetically modified cells unable to synthesize or bind pyoverdine at the cell surface. These studies confirm the generality of pyoverdine-metal bond formation and suggest a wider involvement of siderophores in bacterial biofilm initiation on metals.
Introduction Bacterial attachment to metal surfaces leading to the formation of communities of bacterial cells (biofilms) is a major problem in many diverse settings. Biofilms are a significant contributor to infections associated with surgical implants,1,2 are a source of contamination in food processing environments,3 and can enhance corrosion and/or fouling in industrial and aquatic environments.4,5 Interactions between bacteria and metals are also important for microbial virulence6 and in mineral deposition or recycling.7,8 Understanding the factors that influence the attachment of bacteria to surfaces is critical for the development of strategies to control biofilms. Biofilm formation generally occurs through an initial reversible association with a surface of freely moving bacteria followed by their irreversible attachment, growth, and reproduction. The development of biofilms following surface colonization is reasonably well characterized.9,10 In contrast, the initiation of biofilms and, in particular, the contribution of chemical interactions between bacterial cells and surfaces are * Corresponding author. E-mail:
[email protected]. Phone: 64-3-479-7928. Fax: 64-3-479-7906. † Department of Biochemistry. ‡ Department of Chemistry. § Department of Food Science. (1) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284, 1318. (2) Darouiche, R. O. Healthcare Epidemiology 2001, 33, 1567. (3) Kumar, G. C.; Anand, S. K. Int. J. Food Microbiol. 1998, 42, 9. (4) Costerton, J. W.; Cheng, K. J.; Geesey, G. G.; Ladd, T. I.; Nickel, J. C.; Dasgupta, M.; Marrie, T. J. Annu. ReV. Microbiol. 1987, 41, 435. (5) Hamilton, W. A. Biofouling 2003, 19, 65. (6) Ratledge, C.; Dover, L. G. Annu. ReV. Microbiol. 2000, 54, 881. (7) Konhauser, K. O. Earth-Sci. ReV. 1998, 43, 91. (8) Kraemer, S. M. Aquat. Sci. 2004, 66, 3. (9) Stoodley, P.; Sauer, K.; Davies, D. G.; Costerton, J. W. Annu. ReV. Microbiol. 2002, 56, 187. (10) O'Toole, G. A. J. Bacteriol. 2003, 185, 2687.
poorly understood. Bacterial attachment has generally been described using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory,11 which is based on van der Waals attractive forces and electrical double layer interactions. The theory describes reversible attachment associated with an energy minimum in which bacteria reside before irreversible attachment. Whereas the initial DLVO model has been expanded to include steric and hydrophobic interactions,12 other interactions such as covalent bonding have yet to be included. Most metal surfaces in contact with the atmosphere or in aqueous environments, including titanium, iron, aluminum, and stainless steel, are covered with a thin film of metal oxide. Therefore, contact between bacteria and metal surfaces generally involves interaction with metal oxides. The interfacial chemistry of bacteria and aqueous particle metal oxide films can be studied in situ using attenuated total reflection infrared (ATR-IR) spectroscopy.13-15 In ATR-IR spectroscopy, the total internal reflection of infrared light at the crystal-solution interface sets up an evanescent wave in the solution. Infrared-active vibrations of molecules present in the path of the wave absorb energy at characteristic frequencies, resulting in infrared spectra. As the wave decays exponentially with distance from the crystalsolution interface, the resulting spectra are biased toward molecules near that interface.13,14 Coating the crystal surface with a metal oxide particle film with a thickness less than the penetration depth of the evanescent wave (∼2 µm) enables infrared spectra of species adsorbed on the metal oxide to be measured. When the metal oxide particles are very small (e10 nm), the high surface area gives enhanced spectral sensitivity for (11) Hermansson, M. Colloids Surf., B 1999, 14, 105. (12) van Oss, C. J. Cell Biophys. 1989, 14, 1. (13) Harrick, N. J. Internal Reflection Spectroscopy; Wiley: New York, 1967. (14) Mirabella, F. J. Appl. Spectrosc. ReV. 1985, 21, 45. (15) McQuillan, A. J. AdV. Mater. 2001, 13, 1034.
10.1021/la7004024 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/26/2007
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adsorbed species. Furthermore, only the infrared absorptions of molecules adsorbed to the high-area metal oxide surfaces are detected when solution concentrations are sufficiently low (typically e10-4 mol L-1). Among the many biofilm-forming bacteria is Pseudomonas aeruginosa, a notable opportunistic human pathogen isolated from ubiquitous environments. Its ability to survive and grow in diverse habitats is dependent in part on an efficient ironacquisition system centered on a siderophore, pyoverdine. Siderophores that are secreted by many bacteria are organic chelators with a very high specific affinity for iron(III) (association constants K > 1020 L mol-1).16,17 The resulting ferrisiderophore molecules reenter the bacteria via specific cell-surface receptor proteins. Siderophores are chemically very diverse, but most bind Fe3+ ions through hydroxamate, catecholate, or hydroxycarboxylate groups.16,18 Pyoverdines, represented schematically in Figure 1, coordinate iron(III) via a catecholate and two hydroxamate bidendate ligands. Common to all strains is a catechol-like chromophore that is a derivative of 2,3-diamino6,7-dihydroxyquinoline, a strain-specific octapeptide chain attached to the chromophore and an acyl side chain (either dicarboxylic acid or amide) attached via a peptide bond to the amino group of the chromophore.19 For P. aeruginosa strain PAO1 (ATCC 15692), iron(III) is coordinated by the catechollike ligand of the chromophore and two hydroxamate ligands provided by two L-N5-formyl-N5-hydroxyornithine residues that form part of the octapeptide (Figure 1). As well as being secreted, pyoverdine without Fe3+ (apopyoverdine) is also held at the bacterial cell surface by a FpvA receptor protein that is required for the transport of ferripyoverdine.20 We have previously used ATR-IR spectroscopy to show that pyoverdine that is present at the surface of cells of P. aeruginosa may contribute to the attachment of these bacteria to TiO2 and Fe2O3 surfaces.21 We have also recently shown22 that simple hydroxamic acids, including ones that are similar to those found in pyoverdines, adsorb strongly to TiO2 as bidentate ligands. The role of siderophores in iron-containing mineral dissolution is a related area of research also involving the adsorptive coordination of ligands to metal ions at mineral surfaces. Holmen et al.23,24 found using ATR-IR spectroscopy that acetohydroxamic acid, a siderophore analogue, adsorbs to Fe(III) ions at the goethite surface as a bidentate ligand. Sposito et al.25-28 have studied the influence of the trihydroxamate siderophore desferrioxamine B on the goethite dissolution rate and have established that adsorption is a precursor to dissolution. Kraemer8 has recently (16) Abdallah, M. A. In CRC Handbook of Microbial Iron Chelates; Winkelman, G., Ed.; CRC Press: Boca Raton, FL, 1991; p 139. (17) Boukhalfa, H.; Brickman, T. J.; Armstrong, S. K.; Crumbliss, A. L. Inorg. Chem. 2000, 39, 5591. (18) Dertz, E. A.; Raymond, K. N. In ComprehensiVe Coordination Chemistry II; McCleverty, J. A., Meyer T. J., Eds.; Elsevier Ltd: Amsterdam, 2003; p 141. (19) Budzikiewicz, H. In Progress in the Chemistry of Organic Natural Products; Herz, W., Falk, H., Kirby, G. W., Eds.; Springer: Vienna, Austria, 2004; Vol. 87, p 81. (20) Schalk, I. J.; Hennard, C.; Dugave, C.; Poole, K.; Abdallah, M. A.; Pattus, F. Mol. Microbiol. 2001, 39, 351. (21) McWhirter, M. J.; Bremer, P. J.; Lamont, I. L.; McQuillan, A. J. Langmuir 2003, 19, 3575. (22) Yang, J.; Bremer, P. J.; Lamont, I. L.; McQuillan, A. J. Langmuir 2006, 22, 10109. (23) Holmen, B. A.; Casey, W. H. Geochim. Cosmochim. Acta 1996, 60, 4403. (24) Holmen, B. A.; Tejedor-Tejedor, M. I.; Casey, W. H. Langmuir 1997, 13, 2197. (25) Kraemer, S. M.; Cheah, S. F.; Zapf, R.; Xu, J.; Raymond, K. N.; Sposito, G. Geochim. Cosmochim. Acta 1999, 63, 3003. (26) Cocozza, C.; Tsao, C. C. G.; Cheah, S.-F.; Kraemer, S. M.; Raymond, K. N.; Miano, T. M.; Sposito, G. Geochim. Cosmochim. Acta 2002, 66, 431. (27) Cervini-Silva, J.; Sposito, G. Environ. Sci. Technol. 2002, 36, 337. (28) Cheah, S.-F.; Kraemer, S. M.; Cervini-Silva, J.; Sposito, G. Chem. Geol. 2003, 198, 63.
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Figure 1. Schematic structure of pyoverdine PaA produced by P. aeruginosa strain PAO1. The structure shown was adapted from Tzou et al.51 A catechol-like group forming part of the quinolinetype chromophore together with the hydroxamate groups from L-N5formyl-N5-hydroxyornithine residues provides three bidentate coordination ligands for the Fe3+ ion. For pyoverdine utilized in this work, the dicarboxylic acid succinate is attached as a side chain to the quinoline-type chromophore as shown. Amino acids are abbreviated with the conventional three-letter code except for L-N5formyl-N5-hydroxyornithine (L-foOHOrn).
reviewed the geochemical aspects of the role of siderophores in biological iron acquisition. In the present research, we have used ATR-IR spectroscopy to investigate the generality of pyoverdine binding to a number of metal surfaces relevant to medical and industrial environments. Pertinent metal oxides for some of these metals are titanium dioxide (TiO2), iron(III) oxide (Fe2O3), boehmite (γ-AlOOH), and chromium(III) oxide hydroxide (CrOOH). The pH dependence of adsorption of pyoverdine to films of TiO2 and Fe2O3 was also investigated. We also examined the abilities of wildtype bacteria and mutant bacteria that are unable to hold pyoverdine at the cell surface to form covalent bonds with metal oxide surfaces. Materials and Methods Materials. Pyoverdine was isolated from P. aeruginosa PAO1 and purified via chromatography, as has previously been described.29 N-Methylformohydroxamic acid (Fmha) was prepared from morpholine and N-methylhydroxylamine.30 HCl (BDH, AR), NaOH (Merck, AR), and KCl (BDH, AR) were used as received. All water for solutions and cell washing was deionized (Millipore, Milli-Q). Solution pH measurements were made with a Mettler-Toledo MP220 meter to a precision of (0.1. Preparation of Biomass. P. aeruginosa PAO1 (ATCC15692) and mutant strains used in this study were cultivated at 37 °C with good aeration in Vogel Bonner minimal medium31 as described previously.32 Cells were harvested when they reached the early stationary growth phase (16 h) by centrifugation at 10 000 g for 10 min, and were then washed three times with water and resuspended in water or 0.03 mol L-1 KCl, pH 8. Suspensions used in flow cell experiments had cell concentrations of ∼109 cfu mL-1 as determined by viable counts with cells plated onto Luria-Bertani agar.33 To investigate the function of pyoverdine during bacterial adhesion to metal oxide surfaces, a mutant was constructed lacking FpvA FpvB protein receptors that are reported to bind pyoverdine at the (29) Meyer, J.-M.; Stintzi, A.; De Vos, D.; Cornelis, P.; Tappe, R.; Taraz, K.; Budzikiewicz, H. Microbiology 1997, 143, 35. (30) Gate, E. N.; Threadgill, M. D.; Stevens, M. F. G.; Chubb, D.; Vickers, L. M.; Langdon, S. P.; Hickman, J. A.; Gescher, A. J. Med. Chem. 1986, 29, 1046. (31) Vogel, H.; Bonner, D. J. Biol. Chem. 1956, 218, 97. (32) McWhirter, M. J.; McQuillan, A. J.; Bremer, P. J. Colloids Surf., B 2002, 26, 365. (33) Sambrook, J.; Fritsch, E. F.; Maniatis. T. In Molecular Cloning: A Laboratory Manual; Nolan, C., Ferguson, M., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989; Vol. 3, p A.1.
Adsorption to Metal Oxides of P. aeruginosa outer membrane.20,34 Briefly, to construct the fpVB mutant, a 1.4-kb PstI fragment containing the kanamycin-resistance cassette from pNRE135 was cloned into the fpVB gene. The resulting fpVB::kan construct was cloned into pEX18Gm,36 and this suicide plasmid was introduced into P. aeruginosa PAO1 by conjugation from E. coli strain S17-1.37 Isolates in which the wild-type fpVB gene had been replaced with the fpVB::kan construct were identified by standard methods.36 Plasmid pEX18Gm::fpVA was constructed by cloning an internal 828-bp EcoRI-SalI fragment of fpVA into pEX18Gm. Similarly, pEX18Gm::fpVA was transformed into E. coli strain S17-1 and transferred into a P. aeruginosa fpVB minus recipient by conjugation, resulting in the inactivation of both fpVA and fpVB genes. Confirmation of the intended mutations was determined by polymerase chain reaction (PCR) and Southern blotting. Preparation of Metal Oxides. Thin particle films of titanium dioxide (TiO2), iron(III) oxide (Fe2O3), boehmite (γ-AlOOH), and chromium(III) oxide hydroxide (CrOOH) were used to model the surfaces of titanium, mild steel, aluminum, and stainless steel. Titanium(IV) oxide sol (0.01 mol L-1) was prepared by hydrolysis of TiCl4 (Riedel de Haen) in water as previously reported.38 The TiO2 in these particle films was amorphous, with an isoelectric point of pH ∼5 and a high surface area, providing good sensitivity for IR spectroscopic studies.39 An iron(III) oxide sol (0.01 mol L-1) was prepared by slowly adding 2 mL of a 30% w/v solution of iron(III) chloride (BDH) to 500 mL of stirred boiling water.40 Boehmite (γ-AlOOH) sol (0.01 mol L-1) was prepared by suspension of 10 mg of the powder (Disperal P2, Sasol) in 10 mL of deionized water. The sol was sonicated for 10 min, and the pH was adjusted to 7.0 with NaOH. Chromium(III) oxide hydroxide sol (0.001 mol L-1) was prepared with a 2:1 Cr3+/SO42- by heating an aqueous solution containing Cr(NO3)3‚9H2O and K2SO4 to 100 °C for 2 h.41 CrOOH particles were collected with filter paper and rinsed extensively with NaOH before suspension and sonication for several cycles in deionized water. Particles were later resuspended in 1 × 10-3 mol L-1 perchloric acid (Reidel de Haen) to stabilize the 0.01 mol L-1 sol. ATR-IR Spectroscopic Measurements. Infrared spectra were recorded using a Harrick FastIR ATR accessory with a 45° singlereflection ZnSe prism. Thin metal oxide particle films were prepared by placing 200 µL of the particular sol on the ZnSe prism and drying it in a desiccator under water pump vacuum for 25 to 60 min. Immediately before the drying procedure, the sols were sonicated for 5 min to break down any particle agglomerations. A flow cell with an internal volume of 0.22 mL and a surface contact area of 2.2 cm2 was clamped onto the sample surface of the internal reflection element and sealed via a nitrile rubber gasket. The prism was mounted inside a Digilab FTS 4000 spectrometer fitted with a DTGS detector, and the IR data were analyzed using Merlin 3.4 software (Digilab, Cambridge, MA). The ZnSe prism was cleaned prior to film deposition by polishing with 0.015 µm Al2O3 on a wet microcloth (Buehler), followed by rinsing in water. All spectra were calculated from 64 scans at 4 cm-1 resolution and were not corrected for the wavelength dependence of absorbance in ATR-IR spectra compared with transmission spectra. Noise levels in spectra were typically 5 × 10-5 absorbance. All pyoverdine solution spectra were recorded using water on the bare prism as a background. Spectra of adsorbed species were recorded when equilibrium had been established, typically after solutions were flowed over films for 30-60 min in (34) Ghysels, B.; Dieu, B. T.; Beatson, S. A.; Pirnay, J. P.; Ochsner, U. A.; Vasil, M. L.; Cornelis, P. Microbiology 2004, 150, 1671. (35) Berryman, T. A. In The Application of Gene Recombinant DNA Technology in the Analysis of Candida albicans; University of Otago: Dunedin, New Zealand, 1994; p 15. (36) Hoang, T. T.; Karkhoff-Schweizer, R. R.; Kutchma, A. J.; Schweizer, H. P. Gene 1998, 212, 77. (37) Simon, R.; O’Connell, M.; Labes, M.; Puhler, A. Methods Enzymol. 1986, 118, 640. (38) Connor, P. A.; Dobson, K. D.; McQuillan, A. J. Langmuir 1995, 11, 4193. (39) Dobson, K. D.; Connor, P. A.; McQuillan, A. J. Langmuir 1997, 13, 2614. (40) Everett, D. H. Basic Principles of Colloid Science; Royal Society of Chemistry: London, 1988; p 211. (41) Degenhardt, J.; McQuillan, A. J. Chem. Phys. Lett. 1999, 311, 179.
Langmuir, Vol. 23, No. 13, 2007 7191 replicate experiments. A constant solution flow rate of 1 mL min-1 from a Masterflex peristaltic pump was maintained for all experiments. Pyoverdine Adsorbed to Metal Oxide Particle Films. The adsorption of pyoverdine to TiO2, Fe2O3, boehmite, and CrOOH films deposited on a ZnSe prism was examined in separate experiments. Deposited particle films were thoroughly cleaned with 0.01 mol L-1 NaOH and later with water purged with nitrogen. For subsequent spectra, a background single-beam spectrum was recorded following the in situ water washing of the bare metal oxide film. Subsequently, a solution of 3 × 10-5 mol L-1 pyoverdine that had also been purged with nitrogen was flowed over the surface until equilibrium had been established. Material not adsorbed was removed by in situ washing with nitrogen-purged water for another 20 min. Influence of pH on Pyoverdine Adsorption to TiO2 and Fe2O3. The pH dependence of pyoverdine’s adsorption to metal oxides was recorded by flowing a series of pyoverdine solutions with increasing pH over films of TiO2 or Fe2O3. Each pyoverdine solution had a concentration of 1 × 10-4 mol L-1, and the pH was adjusted from low to high using small volumes of 0.1 mol L-1 HCl and 0.1 mol L-1 NaOH solutions. Competition for TiO2 Adsorption by Fmha and Catechol. A series of solutions containing Fmha and catechol at the same 1 × 10-4 mol L-1 concentration were flowed across a ZnSe prism coated with TiO2. The solution containing Fmha and catechol was adjusted from low to high pH using small volumes of 0.1 mol L-1 HCl and 0.1 mol L-1 NaOH solutions. Biofilm Formation of P. aeruginosa on TiO2 and Fe2O3 Particle Films. Experiments were conducted under constant flow conditions. The suspension containing bacterial cells was flowed over the surface of an ATR-IR ZnSe prism coated with either TiO2 or Fe2O3 for 60 min. During this procedure, a thin layer of bacterial cells attached to the metal oxide surface. The flow of the bacterial suspension was then replaced for 30 min with a flow of water to remove suspended and loosely attached cells. The background single-beam spectrum was recorded with water or 1 × 10-3 mol L-1 KCl, pH 8 flowing over the metal oxide surface prior to deposition of the bacteria.
Results and Discussion IR Spectra of Pyoverdine in Solution and Adsorbed to TiO2. In considering the adsorption of pyoverdine to metal oxide surfaces, the net charges of pyoverdine and of the metal oxide surface are potentially important influences, both of which vary with pH. Pyoverdine from ATCC 15692 (PAO1) has an isoelectric point at pH 8.829 indicating that in neutral solution (pH 7) pyoverdine has a net positive charge. TiO2 has a negative charge at pH 7 so that at this pH the adsorption of pyoverdine to TiO2 should be electrostatically favorable. Electrostatic factors are often important in promoting the close approach of ligands to surfaces, creating conditions that facilitate coordinative adsorption. Spectrum a in Figure 2 is that of pyoverdine in aqueous solution. The pyoverdine solution spectrum was recorded at different pH values (data not shown) and showed very little change in the pH 4-10 range. The pyoverdine structure is dominated by an octapeptide chain (Figure 1); therefore, the prominent bands at 1644 and 1544 cm-1 in the spectrum contain major contributions from amide I (mainly the CdO stretch) and amide II (mainly the N-H bend) modes of the peptide bonds. A strong band at 1389 cm-1 most probably arises from the in-plane δ-OH deformation mode of the dihydroxyquinoline ligand because a corresponding δ-OH absorption at 1374 cm-1 exists for catechol in acidic solutions.42,43 A sharp absorption at 1594 cm-1 is probably due to in-plane ring-stretching vibrations of the dihydroxyquinoline (42) Greaves, S. J.; Griffith, W. P. Spectrochim. Acta 1991, 47A, 133. (43) Lana-Villarreal, T.; Rodes, A.; Perez, J. M.; Gomez, R. J. Am. Chem. Soc. 2005, 127, 12601.
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Figure 2. ATR-IR spectra of pyoverdine in aqueous solution and adsorbed to TiO2. Shown are IR spectra of (a) 1 × 10-2 mol L-1 pyoverdine, pH 7.0 in aqueous solution and (b) 1 × 10-4 mol L-1 pyoverdine, pH 7 adsorbed on a particle film of TiO2.
ligand. The weaker absorption at 1287 cm-1 is similar to that exhibited by aqueous catechol solutions.38 Spectrum b in Figure 2 is that of pyoverdine adsorbed to TiO2 from a 10-4 mol L-1 aqueous solution at pH 7. The prominent absorptions related to the amide I and amide II modes of the peptides are readily recognized with peaks at 1633 and 1549 cm-1, but their relative intensities and band shapes are altered as a consequence of the adsorption. These changes with adsorption may arise from several influences, including the distortion of the amide I band due to an imbalance in the compensation of the water absorption at 1640 cm-1 using water on the TiO2 film as background and changes in the configuration of the pyoverdine octapeptide chain similar to those observed for bulk proteins.44 Major changes are evident in the 1500-1200 cm-1 region of the spectrum upon pyoverdine adsorption. Spectrum b of Figure 2 shows the emergence of strong, sharp absorptions at 1495 and 1290 cm-1 as well as weak bands at ∼1730, 1364, 1339, and 1314 cm-1. The strong bands at 1496 and 1291 cm-1 are characteristic of the aromatic ring C-C stretching vibrations and C-O stretching vibrations of catecholate-like ligands coordinatively bound to metal ions38 and clearly indicate that the dihydroxyquinoline part of pyoverdine has become bound to the TiO2 surface, as has previously been reported.21 Accompanying the appearance of the 1495 and 1290 cm-1 bands in the adsorbed pyoverdine spectrum is the loss of the strong band at 1389 cm-1 seen in the pyoverdine solution spectrum and attributed to a δ-OH absorption. These spectral changes provide direct confirmation of deprotonation of the dihydroxyquinoline ligand on the adsorption of pyoverdine to TiO2. Furthermore, the bidentate nature of the coordination is supported by the close similarity in appearance of the observed spectrum to that of catechol coordinatively adsorbed on TiO2 as the catecholate anion.38 The small peak at 1339 cm-1, which is clearly evident above noise levels in repeated measurements, may arise from the formohydroxamic ligand bonded to TiO2. A peak at the same wavenumber, attributed to a CH deformation mode, is observed in the spectrum of N-methylformohydroxamic acid adsorbed to the same TiO2 substrate.22 There are also minor peaks at 1380, 1364, and 1339 cm-1 that could arise from the adsorption of pyoverdine’s hydroxamic acid ligands. The appearance of a small peak at ∼1730 cm-1 may arise from the CdO stretch absorption of an undissociated carboxylic acid, such as from the succinate group, but this is not normally observed for carboxylic acids in neutral solutions. However, Parikh and Chorover have recently suggested,45 from IR studies of interactions between colloidal (44) Vedanthum, G.; Sparks, H. G.; Sane, S. U.; Tzannis, S.; Przybycien, T. M. Anal. Biochem. 2000, 285, 33. (45) Parikh, S. J.; Chorover, J. Langmuir 2006, 22, 8492.
Figure 3. Effect of pH on the competitive adsorption of catechol and Fmha ligands to TiO2. Spectra were collected using a series of solutions containing both catechol and Fmha ligands at a concentration of 10-4 mol L-1. The solution containing the ligand mixture was adjusted from low to high pH with 0.1 mol L-1 HCl and 0.1 mol L-1 NaOH solutions.
iron oxides and several bacteria, that such observations are due to the protonation of carboxylate groups through interactions with the hydroxyl groups at the oxide surface. The spectral data in Figure 2 provide clear evidence for the involvement of the dihydroxyquinoline ligand in the adsorption to TiO2 and also evidence that hydroxamate ligands are active in the adsorption. The mode of adsorption of pyoverdine to a metal oxide surface at a particular pH is likely to be determined by the relative affinities of the catechol-like and hydroxamate ligands and their pH-dependent adsorption propensities. In addition, the adsorptive binding of a siderophore to a metal oxide surface has different conformational constraints than the chelation of a single metal ion by three chelate groups. However, the ligand-ending arms of the solution-free pyoverdine molecule are likely to be sufficiently flexible to accommodate each of the bidentate ligands binding independently to different sites at a metal oxide surface. IR Spectra from the Adsorption of a Catechol and an N-Methylformohydroxamic (Fmha) Acid Mixture on TiO2. To clarify the relative adsorption propensities as a function of the pH of catechol-like and hydroxamic acid ligands on TiO2, competitive adsorption experiments were carried out with solutions containing a mixture of the simple ligands Nmethylformohydroxamic (Fmha) and catechol. These compounds were chosen to model the adsorption behavior of the formohydroxamic and dihydroxyquinoline ligands within the pyoverdine molecule. The spectra were recorded in sequence from high to low pH. The adsorption behavior of catechol is shown in Figure 3 most clearly in the characteristic sharp bands at 1482 and 1258 cm-1, which appear weakly at pH 11.0 and reach a maximum absorbance (Figure 4) at pH 6.2. The adsorbed hydroxamate ligand gives much weaker absorptions (1333 and 1206 cm-1) that reach their maximum absorbance at pH 5.2. These data show clearly that the adsorbed hydroxamate signals are considerably weaker than those of the adsorbed catechol ligand. Both the hydroxamate and catechol ligands on TiO2 have comparable Langmuir adsorption constants of about 104 L
Adsorption to Metal Oxides of P. aeruginosa
Figure 4. Influence of pH on the competitive absorption of catechol and Fmha ligands to TiO2. Spectra were collected using a series of solutions containing both catechol and Fmha ligands at a concentration of 10-4 mol L-1. The solution containing the catechol and Fmha ligands was adjusted from low to high pH during the experiment. Plotted are major absorption bands (Figure 3) related to adsorbed catechol and adsorbed Fmha.
mol-1,22,46 indicating similar surface affinity or strength of coordinative bonds to the TiO2 surface. The weaker IR signals of adsorbed hydroxamate ligands, relative to those of adsorbed catecholate-like ligands, may account for their relatively weak signals during the adsorption of pyoverdine to TiO2. Alternatively, the weaker hydroxamate signal may reflect a lower hydroxamate surface affinity under the conditions of competitive adsorption. In a study of hydroxamate ligands adsorbed to TiO2, Yang et al.22 have shown that the maximum signal for Fmha adsorbed alone onto TiO2 occurs at pH 3. The data for competitive adsorption (Figure 4) generally indicates that Fmha is adsorbed to a greater extent at lower pH whereas catechol-like ligands show maximum adsorption at higher pH. IR Spectra of Pyoverdine Adsorbed to Fe2O3, AlOOH, and CrOOH. The adsorption of pyoverdine to these metal oxide surfaces and the pH dependence of this adsorption were examined to explore the generality of pyoverdine-metal oxide bond formation. It might therefore be expected that the pH dependence of pyoverdine adsorption would reflect the pH dependence of its component catecholate and hydroxamate ligands. Figure 5 shows the ATR-IR spectra of pyoverdine adsorbed on particle films of TiO2, Fe2O3, AlOOH, and CrOOH. The most prominent features are from the amide I and amide II absorptions of the peptide chain of pyoverdine with peaks at 1640 and 1550 cm-1, respectively. Although the amide II band is similar in all adsorbate spectra, there are some variations in the amide I band shape that may reflect conformational differences on these substrates. The appearance in all spectra of absorption bands at 1491-1503 cm-1 due to C-C ring modes and at 1284-1292 cm-1 due to the C-O stretching modes indicates that the catechol-like 2,3diamino-6,7-dihydroxyquinoline part of the pyoverdine molecule (Figure 1) binds covalently as a catecholate-like ligand on all four metal oxide surfaces. The variation over the different metal oxides in observed peak wavenumber for these bands has been previously observed for catechol adsorbed on several metal oxides.38 Accompanying the adsorption of pyoverdine to TiO2, Fe2O3, and AlOOH, there is an absorbance increase in spectral (46) Araujo, P. Z.; Mendive, C. B.; Rodenas, L. A.; Morando, P. J.; Regazzoni, A. E.; Blesa, M. A.; Bahnemann, D. Colloids Surf., A 2005, 265, 73.
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Figure 5. ATR-IR spectra of pyoverdine on a single-reflection ZnSe prism coated with (a) TiO2, (b) Fe2O3, (c) CrOOH, and (d) AlOOH with a 3 × 10-5 mol L-1 aqueous solution of pyoverdine flowing across the surface for 60 min. The pH of the pyoverdine solution was adjusted with NaOH to pH 7.1 for the TiO2 surface and pH 8.5 for Fe2O3, CrOOH, and AlOOH.
bands from 1358-1364 and 1336-1339 cm-1 (Figure 5) that may be an indication of hydroxamate groups coordinating to surface metal ions, as discussed in the previous section. Adsorption of N-methylformohydroxamic acid (Fmha) on TiO2 showed CH2 deformations at 1355 and 1334 cm-1 and a combination of C-H bending and CdO stretching modes at 1339 cm-1.22 Pyoverdine contains two formohydroxamic acid residues, and slight differences in the absorption frequencies of the adsorbed hydroxamates could reflect differences in the covalent linkages of these residues to the octapeptide. Solution pH, which determines both the degree of protonation of catechol-like and hydroxamic acid groups and also the chemical nature of metal oxide surfaces, is likely to influence the pH dependence of the adsorption of pyoverdine to metal oxide surfaces. The IR spectra of pyoverdine adsorbed on TiO2 and on Fe2O3 were measured over pH ranges applicable to the different substrates. The absorbances of prominent spectral bands corresponding to amide II (1551/1549 cm-1), vibrations related to the C-C ring of pyoverdine’s chromophore (1491/1496 cm-1), and the C-O stretch of the catechol-like end group (1291/1284 cm-1) were monitored and are shown in Figure 6. The amount of adsorbed pyoverdine on TiO2 and Fe2O3 showed similar pH dependences despite a difference in the isoelectric point (IEP) of these two substrates (IEPs of 5 for TiO239 and 8 to 9 for Fe2O347). Thus, it appears for these oxides that a propensity for coordinative adsorption is dominant over interfacial charge factors. The pH dependence of adsorption was also similar to that observed with the catechol and Fmha mixture on TiO2 (Figure 4). Adsorption was less below about pH 8 on both surfaces. The similarity in behavior on different surfaces indicates that analogous coordination chemistry of the pyoverdine-metal complex occurs with both TiO2 and Fe2O3 surfaces. The Figure 6 data indicate that the amount of pyoverdine adsorbed to the metal oxide surface increases up to pH 8. They are also consistent with previous data on the pH dependence of catechol-like ligands.48 Spectral bands in the 1400-1300 cm-1 region that relate to the hydroxamate ligands of pyoverdine binding the respective metals were also observed for the entire pH ranges monitored (47) Arai, Y.; Sparks, D. L. J. Colloid Interface Sci. 2001, 241, 317. (48) Martin, S. T.; Kesselman, J. M.; Park, D. S.; Lewis, N. S.; Hoffmann, M. R. EnViron. Sci. Technol. 1996, 30, 2535.
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Figure 7. ATR-IR spectra of P. aeruginosa cells (a) PAO1 and (b) PAO1 fpVA fpVB attached to TiO2 coated on a ZnSe prism. Spectra were collected following 30 min of bacterial flow across the surface. The bacteria were suspended in H2O. Approximate IR band positions are indicated.
Figure 6. Effect of pH on pyoverdine adsorption to (a) TiO2 and (b) Fe2O3. Data collected from single-reflection ATR-IR spectra. A 1 × 10-4 mol L-1 aqueous pyoverdine solution was adjusted from low to high pH with 0.1 mol L-1 HCl and 0.1 mol L-1 NaOH solutions. Plotted are major absorption bands (Figure 2) related to adsorbed pyoverdine.
(data not shown). The intensity of these absorptions appeared to increase with increasing pH, and this is consistent with these ligands coordinating to metal ions rather than arising from pyoverdine-surface electrostatic interactions.22 Siderophore Metal Bond Formation during the Attachment of P. aeruginosa to TiO2 and Fe2O3. Pyoverdine is bound at the surface of P. aeruginosa cells by the FpvA protein20 and is held in a binding pocket forming part of a barrel structure inserted into the membrane.49 To test the hypothesis that pyoverdine at the cell surface forms covalent bonds with metal oxides, we examined the attachment of P. aeruginosa cells to TiO2 and Fe2O3. A comparison was made of the attachment to TiO2 and to Fe2O3 films of wild-type bacteria and a mutant strain PAO1 fpVA- fpVB- that synthesizes a reduced amount of pyoverdine and does not have the FpvA or FpvB proteins20,34 to potentially bind pyoverdine at the cell surface. The resultant IR spectra are shown in Figures 7 and 8. The absorption bands were generally typical of bacteria adsorbed on ATR-IR crystals and have been previously assigned to functional groups of bacterial components.50 Prominent bands from bacterial cell proteins are amide I, which is mainly due to CdO stretching of the peptide bond (1652/1642 cm-1), and amide II, which is mainly due to N-H bending (1545/1548 cm-1). Spectra from wild-type P. aeruginosa cells showed bands at 1497, 1314, and 1288 cm-1 on TiO2 (Figure 7, spectrum a) and 1493 and 1283 cm-1 on Fe2O3 (Figure 8, spectrum a). These absorptions (C-C ring vibrations and C-O stretch of the chromophore) corresponded to those for pyoverdine forming covalent bonds with TiO2 and Fe2O3 surfaces (Figure 5). However, (49) Cobessi, D.; Celia, H.; Folschweiller, N.; Schalk, I. J.; Abdallah, M. A.; Pattus, F. J. Mol. Biol. 2005, 347, 121. (50) Jiang, W.; Saxena, A.; Song, B.; Ward, B. B.; Beveridge, T. J.; Myneni, S. C. B. Langmuir 2004, 20, 11433. (51) Tzou, D. L.; Wasielewski, E.; Abdallah, M. A.; Kieffer, B.; Atkinson, R. A. Biopolymers 2005, 79, 141.
Figure 8. ATR-IR spectra of P. aeruginosa cells (a) PAO1 and (b) PAO1 fpVA fpVB attached to Fe2O3 coated on a ZnSe prism. Spectra were collected following 60 min of bacterial flow across the surface. The bacteria were suspended in a 0.03 mol L-1 KCl solution, pH 8. Approximate IR band positions are indicated.
the corresponding bands were consistently not obtained for the PAO1 fpVA- fpVB- mutant strain on TiO2 (Figure 7, spectrum b) and on Fe2O3 (Figure 8, spectrum b) in replicate experiments. These data show that pyoverdine that is bound at the cell surface by the FpvA protein (and potentially FpvB) can adsorb to TiO2 and Fe2O3 surfaces. The likely presence of hydroxamate ligands on the metal oxide surfaces as indicated by bands around 1355 and 1335 cm-1 for pyoverdine (Figure 2) was not evident with experiments using wild-type P. aeruginosa cells on TiO2 films (Figure 7). On Fe2O3 films when wild-type P. aeruginosa was attached, there was a small absorption at 1338 cm-1 that may suggest the involvement of the hydroxamate ligand(s) on this substrate. Absorption bands associated with bacteria-surface interactions may have masked hydroxamate-associated bands that were evident when pyoverdine was adsorbed on TiO2.
Summary and Conclusions Our data show that pyoverdine binds covalently through the catechol-like ligand of the pyoverdine molecule to particle films of TiO2, Fe2O3, CrOOH, and AlOOH. Although the catecholate ligand appears to be the major contributor in binding, experiments also indicated that the hydroxamate ligands of pyoverdine bind covalently to TiO2, Fe2O3, and AlOOH surfaces. These observations are of major significance given that these oxide surfaces are widely represented in medical and industrial environments. They are consistent with the hypothesis that P. aeruginosa cells bind to metal oxide surfaces via pyoverdine that can be bound by a receptor protein at the cell surface. Pyoverdine-iron binding would enhance the opportunities for iron solubilization and would
Adsorption to Metal Oxides of P. aeruginosa
allow the nutrient requirements of bacteria for iron to be met. The formation of bonds between pyoverdine and TiO2 or Fe2O3 surfaces showed a pH dependence consistent with bonding being favored by deprotonation of the catechol-like ligand. The basis of the adsorption is therefore primarily covalent bonding to the surface because generally the adsorption of an anion will decrease dramatically at a pH higher than the isoelectric point of the metal oxide if an electrostatic interaction predominates. The similarity of adsorption behavior by pyoverdine on different surfaces indicates that analogous coordination chemistry occurs. Small changes in the spectral band wavenumber did, however, suggest subtle differences in the pyoverdine-metal complexes. Wild-type P. aeruginosa cells able to bind pyoverdine at the cell surface when attached to metal oxide surfaces had infrared
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absorption bands consistent with the catecholate-like ligand of pyoverdine forming bonds with the metal oxide surfaces. These bands were not seen with bacteria that lack the FpvA receptor protein. Collectively, the data presented here suggest a general mechanism by which pyoverdine at the surface of P. aeruginosa cells forms covalent bonds with the metal oxide surface during the initiation of biofilms on metal surfaces. The importance of siderophore chemical bonding in the adhesion to metal oxides of other bacteria will constitute further studies. Acknowledgment. This research was supported by grant UOO 203 from the New Zealand Marsden Fund. LA7004024
