Langmuir 2003, 19, 3575-3577
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Siderophore-Mediated Covalent Bonding to Metal (Oxide) Surfaces during Biofilm Initiation by Pseudomonas aeruginosa Bacteria Michael J. McWhirter,† Phil J. Bremer,‡ Iain L. Lamont,§ and A. James McQuillan*,† Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand, New Zealand Institute for Crop & Food Research, Department of Food Science, University of Otago, Dunedin, New Zealand, and Department of Biochemistry and Centre for Gene Research, University of Otago, Dunedin, New Zealand Received November 18, 2002. In Final Form: March 11, 2003 Bacterial biofilms are of major importance in many infectious diseases and also in a wide range of industrial settings. The initiation of biofilm formation is poorly understood and, in particular, formation of covalent bonds between bacterial cells and surfaces has not been identified as a contributor to this process. Using in situ infrared spectroscopy, we have shown that pyoverdine, a siderophore produced by Pseudomonas aeruginosa, can be firmly bound to the bacterial cell surface and simultaneously bind covalently to TiO2 and to iron(III) oxide. Siderophores may play a role in attachment of bacteria to metal and mineral surfaces.
Introduction Bacterial biofilms on metals can enhance fouling and corrosion in industrial and aquatic environments and may act as foci of infection or contamination in medical and food processing settings.1-4 Interactions between bacteria and metals (especially iron) are also important in microbial virulence and growth5 and in mineral deposition/recycling.6 Biofilm formation involves initial attachment of free-moving bacteria to a surface followed by their growth and reproduction. The development of biofilms following surface colonization is increasingly well described,7 but understanding of the initial attachment of cells to abiotic surfaces is limited to predictive models.4,8 Bacterial attachment to surfaces is generally considered as occurring in a first stage, where the bacteria are loosely associated with a surface, followed by a second stage where exopolysaccharide production causes irreversible attachment.8 The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory,9 based on van der Waals attractive forces and electrical double layer repulsive interactions, is frequently used to model the first stage of bacterial attachment. Although the DLVO theory has been extended to include steric and hydrophobic interactions,10 there are * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: +64 3 479 7928. Fax: +64 3 479 7906. † Department of Chemistry, University of Otago. ‡ New Zealand Institute for Crop & Food Research, Department of Food Science, University of Otago. § Department of Biochemistry and Centre for Gene Research, University of Otago. (1) Fenchel, T. Science 2002, 296, 1055. (2) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284, 1318. (3) Davey, M. E.; O’Toole, G. A. Microbiol. Mol. Biol. Rev. 2000, 64, 847. (4) Bos, R.; van der Mei, H. C.; Busscher, H. J. FEMS Microbiol. Rev. 1999, 23, 179. (5) Ratledge, C.; Dover, L. G. Annu. Rev. Microbiol. 2000, 54, 881. (6) Konhauser, K. O. Earth Sci. Rev. 1998, 43, 91. (7) O’Toole, G.; Kaplan, H. B.; Kolter, R. Annu. Rev. Microbiol. 2000, 54, 49. (8) Hermansson, M. Colloids Surf., B 1999, 14, 105. (9) Israelachvili, J. N. Intermolecular Surface Forces; Academic Press: San Diego, CA, 1992.
many examples11-13 where it does not explain observed attachment behavior. Recently charge transfer has been measured during bacterial adhesion to indium tin oxide14 and titanium oxynitride15 coatings suggesting that chemical reactions may occur during adhesion. Bacteria-surface covalent bonds have not been considered in DLVO models, and this is an important omission. Identification of bacteria-surface bonds is of critical importance in understanding and controlling bacterial attachment. Ironsequestering siderophore molecules secreted by many bacteria16 are among the candidates to form covalent bonds between bacteria and the metal oxides at metal surfaces. At least some siderophores remain associated with the cell surface17,18 and could be involved in bacterial attachment. Almost all metal surfaces, in contact with the atmosphere and aqueous environments, are covered with thin metal oxide films. Consequently, contact between bacteria in aqueous solutions and metals generally involves interaction with metal oxide surfaces. In the present work bacterial attachment to metal oxide surfaces was monitored in situ using attenuated total reflection infrared (ATR-IR) spectroscopy.19 In this form of spectroscopy, total internal reflection of infrared light at a crystal-solution interface creates an evanescent wave extending into the solution. Infrared-active vibrations of molecules present in the path of the wave absorb energy at characteristic frequencies, allowing their identification from analysis of (10) van Oss, C. J. Cell Biophys. 1989, 14, 1. (11) Ong, Y.; Razatos, A.; Georgiou, G.; Sharma, M. M. Langmuir 1999, 15, 2719. (12) McWhirter, M. J.; McQuillan, A. J.; Bremer, P. J. Colloids Surf., B 2002, 26, 365. (13) Ohmura, N.; Kitamura, K.; Saiki, H. Appl. Environ. Microbiol. 1993, 59, 4044. (14) Poortinga, A. T.; Bos, R.; Busscher, H. J. J. Microbiol. Methods 1999, 38, 183. (15) Poortinga, A. T.; Bos, R.; Busscher, H. J. Biophys. Chem. 2001, 91, 273. (16) Drechsel, H.; Winkelmann, G. Transition Met. Microb. Metab. 1997, 1. (17) Schalk, I. J.; Kyslik, P.; Prome, D.; van Rorsselaer, A.; Poole, K.; Abdallah, M. A.; Paltus, F. Biochemistry 1999, 38, 9357. (18) Granger, J.; Price, N. M. Limnol. Oceanogr. 1999, 44, 541. (19) Mirabella, F. J. Appl. Spectrosc. Rev. 1985, 21, 45.
10.1021/la020918z CCC: $25.00 © 2003 American Chemical Society Published on Web 04/02/2003
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Figure 1. Schematic flow cell arrangement for ATR-IR spectroscopy of attached bacteria. The flow cell had an internal volume of 0.22 mL, had a surface contact area of 2.2 cm2, and was sealed to the ZnSe surface via an O-ring. All solutions were flowed at 2 mL min-1.
the resulting infrared spectra. These spectra are biased toward molecules near the crystal-solution interface because the wave decays exponentially with distance into the solution phase.19 Coating the crystal surface with a metal oxide film, of thickness less than the pentration depth of the evanescent wave (∼2 µm), enables spectra of species adsorbed on the metal oxide to be observed. For solutions with concentrations below 10-3 M, only the infrared absorptions of the surface (adsorbed) molecules are detected. The intensity of spectra from adsorbed molecules can be greatly enhanced by depositing a porous particle film with a high surface area on the ATR crystal as this increases the number of adsorbed molecules within the evanescent wave.20 In this paper we have used ATR-IR spectroscopy to detect covalent interactions between cell surface-located pyoverdine of Pseudomonas aeruginosa and a metal oxide film to which the cells of this bacterium adhere. Materials and Methods Pseudomonas aeruginosa (ATCC27853) cells were cultured and maintained as described previously.12 To prepare the cells for addition to the flow cell, a 24 h culture was centrifuged (7520g, 10 min, 4 °C) and resuspended in water (pH ) 6.3) three times. Plate counts showed that numbers of viable bacteria did not decrease during the washing process. A suspension of 1010 cfu mL-1 of P. aeruginosa cells12 was flowed over either a 13 reflection ZnSe crystal (Harrick) or the ZnSe crystal covered with a ∼50 nm thick coating of TiO2 particles as shown in Figure 1. Experiments were repeated with P. aeruginosa PAO1, and similar results were obtained. Pyoverdine was purified as described previously.21 Before experiments the pyoverdine was passed through a 10000 MWCO filter (Millipore). High surface area TiO2 particle films were formed by dipcoating a bare ZnSe crystal with a TiO2 sol.12 Iron(III) oxide films were deposited in a similar fashion from a iron(III) oxide sol prepared by slowly adding 2 mL of a 30% solution of iron(III) chloride (BDH, GPR) to 500 mL of stirred boiling MilliQ water. Infrared spectral measurements were made with a BioRad FTS60 spectrometer using Win-IR software. Spectra were constructed from 64 scans taken at a resolution of 4 cm-1. In each experiment the surface was washed in situ for 30 min with 10-2 M NaOH (Merck, pure) to ensure the surface was clean.20 Water was passed over the surface to provide a background spectrum for the subsequent bacterial spectra (pH ) 6.3). Spectra were obtained at intervals during the first 30 min of suspension flow over the surfaces and compared with the background spectrum. The baseline was corrected by setting the absorbance at 1800 and 900 cm-1 to zero and assuming a straight line between these wavenumbers. Spectral noise at about 1640 cm-1 is due to total water absorption from the multiple internal reflections. (20) McQuillan, A. J. Adv. Mater. 2001, 13, 1034. (21) Meyer, J.-M.; Stintzi, A.; De Vos, D.; Cornelis, P.; Tappe, R.; Taraz, K.; Budzikiewicz, H. Microbiology 1997, 143, 35.
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Figure 2. Infrared spectra of Pseudomonas aeruginosa attached to (a) ZnSe crystal and (b) TiO2-coated ZnSe crystal obtained after 30 min of adhesion from flowing aqueous bacterial suspensions.
Results and Discussion The spectra after 30 min of bacterial flow over ZnSe and TiO2 are shown in Figure 2. The number of cells that attach to the surface increased with time as observed in previous such experiments.12,15 The spectral peak intensities increased throughout the 30 min period, but the relative peak intensities remained constant (data not shown). The peaks in the spectrum from the bare ZnSe crystal (Figure 2a) are typical of bacteria attached to ATRIR crystals and have been assigned previously to vibrations of bacterial components.22 The most prominent features in this spectrum are peaks at 1650, 1550, and 1240 cm-1, arising from amide vibrations, and the peak at 1080 cm-1, which is partly due to carbohydrate polymers. With the TiO2 film (Figure 2b), additional peaks are observed at 1491, 1314, and 1286 cm-1. Very similar additional peaks were observed when the experiment was repeated with a ZnSe crystal coated with particles of iron(III) oxide. There are no absorption bands of TiO2 or iron(III) oxide in this spectral region. When the bacteria were removed from the washed cell suspension by centrifugation (15300g, 15 min, 4 °C) and filtration (0.45 µm then 0.2 µm filters) and the cell-free solution was flowed over a fresh TiO2 surface, none of the peaks shown in the Figure 2b spectrum was obtained. This result indicates that all these peaks were due to molecules bound to the bacteria and did not arise from molecules in the surrounding solution. The extra peaks detected with the TiO2 and iron(III) oxide surfaces are similar to those at 1481, 1333, and 1258 cm-1 in the infrared spectrum of the bidentate ligand catechol bound to TiO2.23 The 1481 cm-1 band arises from the benzene ring vibrations while the 1258 cm-1 band is associated with the C-O stretch.24 When catechol binds covalently to TiO2 as the catecholate anion, the intensities of these two absorptions increase by a factor of 5 relative to those of the other catechol bands and become by far the strongest absorptions of the bound catechol ligand.25 The spectra of the naturally occurring catechol-containing species L-dopa, chlorogenic, and caffeic acids26 adsorbed to TiO2 (data not shown) contained two strong peaks, but their peak wavenumbers did not match those in Figure 2b at 1491 and 1286 cm-1. Pseudomonas aeruginosa (22) Geesey, G. G.; Suci, P. A. In Biofilms: Recent Advances in Their Study Control; Evans, L. V., Ed.; Harwood Academic: Amsterdam, 2000; Chapter 15, p 253. (23) Connor, P. A.; Dobson, K. D.; McQuillan, A. J. Langmuir 1995, 11, 4193. (24) Wicklund, P. A.; Brown, D. G. Inorg. Chem. 1976, 15, 396. (25) Martin, S. T.; Kesselman, J. M.; Park, D. S.; Lewis, N. S.; Hoffmann, M. R. Environ. Sci. Technol. 1996, 30, 2535. (26) Hansen, D. C.; McCafferty, E. J. Electrochem. Soc. 1996, 143, 114.
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Figure 3. Infrared spectrum of pyoverdine adsorbed to a TiO2coated ZnSe crystal from a 3 × 10-5 M aqueous pyoverdine solution.
produces a catechol-containing siderophore called pyoverdine.16 Pyoverdine is secreted into solution by the bacteria, but some of it also remains bound to a cell surface protein.17 When a 3 × 10-5 M aqueous solution of pyoverdine was flowed over the high surface area TiO2 substrate, the spectrum in Figure 3 was obtained. The prominent bands at 1650 and 1550 cm-1 are due to the amide absorptions of the pyoverdine molecule while the spectrum also includes distinct bands at 1491, 1314, and 1286 cm-1. No spectral bands were observed when the same solution was flowed over a bare ZnSe surface. Therefore the bands at 1491,1314, and 1286 cm-1 in Figure 3 are from pyoverdine bound to the TiO2 surface. The match between the three marked peaks in Figures 3 and 2b clearly identifies adsorbed pyoverdine as the reason for the differences between the spectra of P. aeruginosa cells attached to ZnSe and to TiO2 (Figure 2a and Figure 2b). Under the prevaling (neutral) solution conditions, deprotonation of catechol-like ligands is highly unfavored and can only occur through a chemical reaction, such as when the ligand coordinates to a metal ion to form a covalent bond. These results suggest that pyoverdine mediates covalent bonding interactions between the bacterial cell surface and a TiO2 surface and may thus play a role in adhesion of the cell to this substrate. Pyoverdine was detected on TiO2 (Figure 2b and Figure 3) and iron(III) oxide particle films but not on bare ZnSe (Figure 2a) most likely because the TiO2 and iron(III) oxide particle films have a much higher surface area and greater capacity to adsorb pyoverdine than the bare ZnSe. A schematic illustration of the covalent bonds formed during bacterial attachment is shown in Figure 4. Catechol forms coordination complexes having high stability constants with many metal ions including those of aluminum, zinc, titanium, manganese, and iron.27 Pyoverdine is known to chelate a variety of metal ions,28 and it is therefore likely that pyoverdine establishes covalent interactions that participate in the attachment of P. aeruginosa cells to many metal and mineral surfaces. Interestingly, a catechol-containing molecule, L-dopa, is involved in the adhesion of sea mussels to surfaces,28 showing that similar molecules perform similar roles in nature. Pyoverdine also employs hydroxamic acid bidentate ligands in chelating iron, and these ligands may also (27) Abdallah, M. A. In CRC Handbook of microbial iron chelates; Winkelman, G., Ed.; CRC Press: Boca Raton, FL, 1991; pp 139-153. (28) Suci, P. A.; Geesey, G. G. Langmuir 2001, 17, 2538.
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Figure 4. Schematic of covalent bonds formed when cells of Pseudomonas aeruginosa in suspension (a) attach to a TiO2 surface (b). Linkage of the catechol end group of pyoverdine to the bacterial cell is indicated by ≡.
contribute to binding to the metal oxide surfaces. To test this possibility, we prepared N-methylhydroxamic acid (N-hydroxy-N-methylformamide),29 and a spectrum of it adsorbed to a TiO2 particle film gave prominent absorptions at 1636 and 1335 cm-1. The spectra of P. aeruginosa attached to TiO2 (Figure 2b) and of pyoverdine adsorbed to TiO2 (Figure 3) show no distinct absorption in the 1335 cm-1 region, while the strong amide I and water absorptions around 1650 cm-1 would obscure any possible peak around 1636 cm-1. This suggests that hydroxamate ligands do not contribute to the spectrum of the attached bacteria and are not involved in the initial binding of the siderophore to the oxide surface in this system, but further experiments will be required to fully examine this question. In further research we have begun to assess the role of pyoverdine’s adsorptive binding to metal (oxide) surfaces in relation to the rate and strength of Pseudomonas aeruginosa adhesion to metals. Conclusions We have shown that, during bacterial attachment, pyoverdine bound to P. aeruginosa cells binds covalently to TiO2 and ferric oxide surfaces via a catechol ligand. Thus the catechol group may participate in the bacterialsurface attachment process. This observation is of broad significance as many bacteria produce catechol-containing siderophores and potentially many or all of these could contribute to the initiation of biofilm formation. The chemical bonds involved in bacterial attachment have not been identified previously. Our use of ATR-IR spectroscopy with high-surface-area particle films enabled the identification of covalent bonds formed between bacteria and metal surfaces. This new knowledge and the technique by which it was obtained will be important in further analyzing bond formation and bacteria-metal interactions in ongoing efforts to control biofilm formation. Acknowledgment. This work was supported by the New Zealand New Economy Research Fund Grant No. CO8X9903: Functional Interfaces and Materials. We thank Ian Monk and Angela Steel for their help in the laboratory and Paul Beare for providing the pyoverdine. LA020918Z (29) Gate, E. N.; Threadgill, M. D.; Stevens, M. F.; Chubb, D.; Vickers, L. M.; Langdon, S. P.; Hickman, J. A.; Gescher, A. J. Med. Chem. 1986, 29, 1046.
