Combined Spectroscopic and Topographic Characterization of

Daad Saffarini, and H. Peter Lu*,‡. Fundamental Science Directorate, Pacific Northwest National Laboratory, Richland, Washington 99352. ReceiVed May...
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Langmuir 2007, 23, 1333-1338

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Combined Spectroscopic and Topographic Characterization of Nanoscale Domains and Their Distributions of a Redox Protein on Bacterial Cell Surfaces Vasudevanpillai Biju,† Duohai Pan,† Yuri A. Gorby, Jim Fredrickson, Jeff McLean, Daad Saffarini, and H. Peter Lu*,‡ Fundamental Science Directorate, Pacific Northwest National Laboratory, Richland, Washington 99352 ReceiVed May 11, 2006. In Final Form: August 28, 2006 Redox protein nanoscale domains on the cell surface of a bacterium, Shewanella oneidensis MR1, grown in the absence and presence of electron acceptors, is topographically characterized using combined atomic force microscopy (AFM) and confocal surface enhanced Raman scattering (SERS) spectroscopy. The protruding nanoscale domains on the outer membrane of S. oneidensis were observed, as was their disappearance upon exposure to electron acceptors such as oxygen, nitrate, fumarate, and iron nitrilotriacetate (FeNTA). Using SERS spectroscopy, a redox heme protein was identified as a major component of the cell surface domains. This conclusion was further confirmed by the disappearance of Raman vibrational frequencies, characteristic of heme proteins, upon exposure of the cells to electron acceptors. Our experimental results from our AFM imaging and SERS spectroscopy, consistent with the literature, suggest the protruding nanoscale surface domains as heme-containing secretions. Our results on the distributions of redox proteins on microbial cell surfaces will be helpful for a mechanistic understanding of the behaviors of surface proteins and their interactions with redox environments.

Introduction Shewanella oneidensis, a gram negative bacterium, has been known to consume a variety of anaerobic terminal electron acceptors, such as fumarate, dimethyl sulfoxide (DMSO), nitrate, trimethylamine N-oxide (TMAO), iron, manganese, and uranium for energy conservation and metabolism.1-15 The early studies * Corresponding author: [email protected]. † These authors contributed equally to this work. ‡ Current address: Bowling Green State University, Department of Chemistry, Center for Photochemical Sciences, Bowling Green, OH 43403. (1) Beliaev, A. S.; Saffarini, D. A. Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction. J. Bacteriol. 1998, 180 (23), 6292-6297. (2) Carpentier, W.; Sandra, K.; De Smet, I.; Brige, A.; De Smet, L.; Van Beeumen, J. Microbial reduction and precipitation of vanadium by Shewanella oneidensis. Appl. EnViron. Microbiol. 2003, 69 (6), 3636-3639. (3) Cooper, D. C.; Picardal, F. W.; Schimmelmann, A.; Coby, A. J. Chemical and biological interactions during nitrate and goethite reduction by Shewanella putrefaciens 200. Appl. EnViron. Microbiol. 2003, 69 (6), 3517-3525. (4) Das, A.; Caccavo, F. Adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to crystalline Fe(III) oxides. Curr. Microbiol. 2001, 42 (3), 151-154. (5) Glasauer, S.; Langley, S.; Beveridge, T. J. Intracellular iron minerals in a dissimilatory iron-reducing bacterium. Science 2002, 295 (5552), 117-119. (6) Liu, C. X.; Gorby, Y. A.; Zachara, J. M.; Fredrickson, J. K.; Brown, C. F. Reduction kinetics of Fe(III), Co(III), U(VI), Cr(VI) and Tc(VII) in cultures of dissimilatory metal-reducing bacteria. Biotechnol. Bioeng. 2002, 80 (6), 637649. (7) Lloyd, J. R. Microbial reduction of metals and radionuclides. FEMS Microbiol. ReV. 2003, 27 (2-3), 411-425. (8) Lloyd, J. R.; Lovley, D. R. Microbial detoxification of metals and radionuclides. Curr. Opin. Biotechnol. 2001, 12 (3), 248-253. (9) Lowe, K. L.; Straube, W.; Little, B.; Jones-Meehan, J. Aerobic and anaerobic reduction of Cr(VI) by Shewanella oneidensis effects of cationic metals, sorbing agents and mixed microbial cultures. Acta Biotechnol. 2003, 23 (2-3), 161-178. (10) Myers, C. R.; Myers, J. M. Cell surface exposure of the outer membrane cytochromes of Shewanella oneidensis MR-1. Lett. Appl. Microbiol. 2003, 37 (3), 254-258. (11) Myers, C. R.; Nealson, K. H. Respiration-Linked Proton Translocation Coupled to Anaerobic Reduction of Manganese(IV) and Iron(III) in Shewanella putrefaciens Mr-1. J. Bacteriol. 1990, 172 (11), 6232-6238. (12) Neal, A. L.; Rosso, K. M.; Geesey, G. G.; Gorby, Y. A.; Little, B. J. Surface structure effects on direct reduction of iron oxides by Shewanella oneidensis. Geochim. Cosmochim. Acta. 2003, 67 (23), 4489-4503. (13) Semple, K. M.; Doran, J. L.; Westlake, D. W. S. DNA Relatedness of Oil-Field Isolates of Shewanella putrefaciens. Can. J. Microbiol. 1989, 35 (10), 925-931.

of the mechanism of dissmilatory iron and manganiese oxide reduction demonstrated that Shewanella contains abundant heme proteins distributed among inner membrane, periplasm, and outer membrane cell fractions.16-19 Progress over the past 20 years supports a hypothesis that multiheme cytochromes located in association with and possibly on the outer surface of the outer membrane of these gram negative bacteria catalyze the reduction of solid-phase iron oxide.10,17,20,21 The cell membranes play important roles in respiratory redox reactions or electron-transfer flux by regulating the interactions of electron-accepting organic or inorganic oxidants with electron donor cytochrome proteins.22-25 Different mechanisms, such as (14) Viamajala, S.; Peyton, B. M.; Apel, W. A.; Petersen, J. N. Chromate reduction in Shewanella oneidensis MR-1 is an inducible process associated with anaerobic growth. Biotechnol. Prog. 2002, 18 (2), 290-295. (15) Fredrickson, J. K.; Kota, S.; Kukkadapu, R. K.; Liu, C. X.; Zachara, J. M. Influence of electron donor/acceptor concentrations on hydrous ferric oxide (HFO) bioreduction. Biodegradation 2003, 14 (2), 91-103. (16) Gorby, Y. A.; Lovley, D. R. Electron-Transport in the Dissimilatory Iron Reducer, Gs-15. Appl. EnViron. Microbiol. 1991, 57 (3), 867-870. (17) Myers, C. R.; Myers, J. M. Localization of Cytochromes to the OuterMembrane of Anaerobically Grown Shewanella putrefaciens Mr-1. J. Bacteriol. 1992, 174 (11), 3429-3438. (18) Myers, C. R.; Myers, J. M. Ferric Reductase Is Associated with the Membranes of Anaerobically Grown Shewanella putrefaciens Mr-1. FEMS Microbiol. Lett. 1993, 108 (1), 15-22. (19) Seeliger, S.; Cord-Ruwisch, R.; Schink, B. A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreducens acts as a ferric iron reductase and as an electron carrier to other acceptors or to partner bacteria. J. Bacteriol. 1998, 180 (14), 3686-3691. (20) Beliaev, A. S.; Saffarini, D. A.; McLaughlin, J. L.; Hunnicutt, D. MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1. Mol. Microbiol. 2001, 39 (3), 722-730. (21) Magnuson, T. S.; Isoyama, N.; Hodges-Myerson, A. L.; Davidson, G.; Maroney, M. J.; Geesey, G. G.; Lovley, D. R. Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens. Biochem. J. 2001, 359, 147-152. (22) Bamford, V.; Dobbin, P. S.; Lee, S. C.; Reilly, A.; Powell, A. K.; Richardson, D. J.; Hemmings, A. M. Crystallization and preliminary X-ray crystallographic analysis of a periplasmic tetrahaem flavocytochrome c(3) from Shewanella frigidimarina NCIMB400 which has fumarate reductase activity. Acta Crystallogr., Sect. D 1999, 55, 1222-1225. (23) Butt, J. N.; Thornton, J.; Richardson, D. J.; Dobbin, P. S. Voltammetry of a flavocytochrome c(3): The lowest potential heme modulates fumarate reduction rates. Biophys. J. 2000, 78 (2), 1001-1009.

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the involvement of protein systems that can transfer electrons from the cytoplasmic components to the outer membrane by shuttling through the periplasm or the synthesis of cytochrome proteins on the outer membrane, have been proposed to account for the reduction of insoluble oxidants.26 Eventually, iron oxides received electrons from a select subset of mutiheme cytochromes. Although some experimental results10 suggest that the cytochromes must be located on the outer surface of the outer membrane to facilitate direct electron transfer to relatively insoluble oxide minerals, the distributions and identifications of these redox proteins and the transport of electrons across the periplasm to the inner cytoplasmics membrane are less understood. The involvement of outer membrane proteins in the use of electron acceptors such as iron oxide was recently demonstrated in biological force microscopic investigations using AFM.27,28 However, the biophysical and biochemical redox properties of the outer membrane proteins are still largely unknown, e.g., their distributions and identifications, the mechanism of interactions between cell surface and terminal electron acceptors, and the transport of electrons across the periplasm to the inner cytoplasmic membrane. Raman spectroscopy and microscopy have been widely applied to biology and biophysics, since Raman spectra provide detailed vibrational and compositional analyses of the samples of interest. We recently developed a correlated confocal Raman microscopy and atomic force microscopy (AFM) technique.29,30 This technique allows us to obtain chemical information to be coupled to nanometer-scale topographic information, which is ideally used to probe the biological samples such as single cells and tissue samples, providing a more complete characterization of sample morphology and chemistry on the nanometer scale. In this paper, we use confocal surface-enhanced Raman spectroscopy (SERS) combined with AFM to examine the topographic distributions and spectroscopic identifications of cell-surface redox proteins as well as the biophysical changes of the outer membrane of S. oneidensis under electron-acceptor limited vs exceeded conditions. AFM analysis shows that the cell-surface domains disappeared upon releasing the stress of electron-acceptor limitation through exposure to oxygen, fumarate, nitrate, or iron oxide. SERS spectra indicate that the cell-surface domains were predominately the contributions of heme proteins. The results of our AFM imaging and confocal SERS spectroscopic analyses most likely suggest the expression of heme proteins as secretions on the outer membrane of S. oneidensis in conditions of electronacceptor limitation. Materials and Methods Cells of S. oneidensis (wild type) were grown for 24 h at 25 °C under anaerobic (100% N2 atmosphere) conditions in a defined growth medium, consisting of a mixture of electrolyte solutions (see Supporting Information). The pH of the medium was adjusted to 7.5 (24) Leys, D.; Tsapin, A. S.; Nealson, K. H.; Meyer, T. E.; Cusanovich, M. A.; Van Beeumen, J. J. Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1. Nat. Struct. Biol. 1999, 6 (12), 1113-1117. (25) Newman, D. K.; Kolter, R. A role for excreted quinones in extracellular electron transfer. Nature (London) 2000, 405 (6782), 94-97. (26) Pitts, K. E.; Dobbin, P. S.; Reyes-Ramirez, F.; Thomson, A. J.; Richardson, D. J.; Seward, H. E. Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA. J. Biol. Chem. 2003, 278 (30), 27758-27765. (27) Lower, S. K.; Hochella, M. F.; Beveridge, T. J. Bacterial recognition of mineral surfaces: Nanoscale interactions between Shewanella and R-FeOOH. Science 2001, 292 (5520), 1360-1363. (28) Sadana, A. Protein Adsorption and Inactivation on Surfaces - Influence of Heterogeneities. Chem. ReV. 1992, 92 (8), 1799-1818. (29) Hu, D. H.; Micic, M.; Klymyshyn, N.; Suh, Y. D.; Lu, H. P. Correlated topographic and spectroscopic imaging by combined atomic force microscopy and optical microscopy. J. Lumin. 2004, 107 (1-4), 4-12.

Biju et al. using a HEPES buffer (10 mM). Electron-acceptor limited samples were recovered from reactors using anaerobic technique to maintain electron-acceptor limitation.31 These samples were immediately fixed using an anaerobic solution of glutaraldehyde (2% final concentration). For investigating the outer membrane surface features under oxygen-limited conditions, samples were withdrawn from the growth chamber in a nitrogen atmosphere and then fixed with glutaraldehyde in a HEPES buffer. Sufficient care was taken to prevent the samples from coming in contact with oxygen before fixing. To prepare the samples under electron-acceptor exceeded conditions, the stress of the cells due to electron-acceptor limitation was relieved by treating with one of the electron acceptors, 10 mM FeNTA, 10 mM nitrate, or 10 mM fumarate, for 5-10 min before fixing. Control samples were prepared by withdrawing electron-acceptor limited cell samples and saturating with oxygen by bubbling air. The samples were then fixed with glutaraldehyde. The procedures of bacterial growth and sample preparation, including fixation, are essentially standard, and detailed descriptions have been reported in the literature.31 The samples for AFM measurements were prepared by incubating the cell samples on clean glass coverslips for 30 min, followed by repeated washing with water. Coverslips (Fisher) were thoroughly cleaned by sonication in an aqueous NaOH solution (0.1 M), deionized water, and acetone and deionized water, respectively, and dried in a jet of nitrogen gas. Samples for SERS measurements were prepared by first sputter-coating a thin film (∼15 nm) of silver on a clean glass coverslip, followed by incubating the cell samples on the silvercoated glass surface. Control samples for SERS spectroscopy were prepared by incubating a phosphate buffer (pH 7.5) solution of bovine heart cytochrome c proteins (Sigma) on a glass coverslip coated with silver film. In our AFM imaging and Raman spectroscopic measurements, only the fixed cells were used because of the advantages of avoiding the following complications: (i) possible responses and interactions of live cell surface features with AFM cantilever tips, (ii) cell movements during measurements, (iii) possible complex biological interactions between the live cells and silver surfaces during SERS measurements that might alter the nature of biologically relevant domains,28 and (iv) interactions with possible trace amount of diffused oxygen. Overall, the use of fixed cells in AFM and SERS measurements provided us the advantage of cell-surface features being maintained as they were before fixation in the growth medium under either electron-acceptor limited or exceeded conditions. All AFM measurements were done with a Nanoscope III microscope (Digital Instruments, Santa Barbara, CA). Tapping-mode AFM images were collected in air, using ultrasharp (radius of curvature 10-15 nm, cone side angle ∼17°) silicon nanoprobes (Digital Instruments, Santa Barbara). The cantilevers used were ∼120 µm long with a spring constant of ∼20-100 N/m and a resonance frequency of 356 kHz. SERS spectroscopy of cell surfaces was conducted using a correlated AFM-confocal optical microscope.29,30 The correlated AFM-confocal optical microscopy setup was capable of characterizing cell surfaces and recording the Raman spectra of cell surfaces. A schematic presentation of the experimental setup is shown in Figure 1. For SERS measurements, a 514.5 nm laser beam line (500 nW) of an Ar+ laser was used. The laser beam was reflected by a dichroic beam-splitter and focused by a high numerical aperture objective (1.4 NA, 63×, Zeiss) on the sample surface. Energy transfer between the outer membrane molecules and the silver film was effective in reducing autofluorescence from cells,30 while the silver film also provided surface enhancement to the Raman signals.32,33 (30) Micic, M.; Hu, D. H.; Suh, Y. D.; Newton, G.; Romine, M.; Lu, H. P. Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells. Colloids Surf., B 2004, 34 (4), 205-212. (31) Kieft, T. L.; Fredrickson, J. K.; Onstott, T. C.; Gorby, Y. A.; Kostandarithes, H. M.; Bailey, T. J.; Kennedy, D. W.; Li, S. W.; Plymale, A. E.; Spadoni, C. M.; Gray, M. S. Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate. Appl. EnViron. Microbiol. 1999, 65 (3), 1214-1221. (32) Moskovits, M. Surface-Enhanced Spectroscopy. ReV. Mod. Phys. 1985, 57 (3), 783-826. (33) Otto, A. Surface-enhanced Raman scattering: “classical” and “chemical” origins. In Light scattering in solids IV; Springer-Verlag: Berlin, 1984; p 289.

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Figure 2. AFM image of ensemble of anaerobically grown S. oneidensis cells, adsorbed on a cover glass, showing the strong hydrophilic interaction between the glass surface and the cell surfaces. Figure 1. A schematic presentation of the experimental setup used for correlated AFM-SERS measurements. APD is a photon-counting avalanche photodiode detector. Raman signals were collected with the same objective, transmitted by the beam-splitter, and detected using a liquid-nitrogen-cooled back-illuminated charge-coupled device (CCD, Roper Scientific, Trend, NJ), which was coupled to a holographic spectrograph (HoleSpec f/1.8i, Kaiser Optical Systems, Inc.). Raman spectra were corrected for the wavelength dependence of the spectrometer efficiency by using white light and calibrated with a mercury lamp. The reported frequencies were accurate to (2 cm-1. Raman signals from molecules on a silver surface are both chemically and electromagnetically enhanced.32,33 Typically, a chemical enhancement occurs when a molecule has direct chemical contact with the silver surface, and the enhancement factor is on the order of 10-100. On the other hand, an electromagnetic near-field enhancement can be as high as 104-1010 and exponentially decay to the background within about 200 nm from the surface.32-36 In our experiments, S. oneidensis cells were deposited on a silver-coated glass slide, which provided direct contact between cell-surface domains on the outer membrane and the silver surface. Consequently, the enhanced Raman signals from the cell surfaces dominated the Raman signals from the interior of the cells. Therefore, SERS spectroscopy was selectively sensitive to the surface domains based on the chemical enhancement in our measurements. Similar SERS spectroscopic techniques have been widely applied to study the structures, dynamics, and reaction mechanisms of several biological systems.37-43 We used SERS to examine the cell-surface domains for two essential reasons: (i) The enhancement of Raman signals (34) Michaels, A. M.; Jiang, J.; Brus, L. Ag nanocrystal junctions as the site for surface-enhanced Raman scattering of single Rhodamine 6G molecules. J. Phys. Chem. B 2000, 104 (50), 11965-11971. (35) Nie, S. M.; Emery, S. R. Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 1997, 275 (5303), 1102-1106. (36) Yang, W. H.; Schatz, G. C.; Vanduyne, R. P. Discrete Dipole Approximation for Calculating Extinction and Raman Intensities for Small Particles with Arbitrary Shapes. J. Chem. Phys. 1995, 103 (3), 869-875. (37) Cotton, T. M.; Parks, K. D.; Vanduyne, R. P. Resonance Raman-Spectra of Bacteriochlorophyll and Its Electrogenerated Cation Radical - Excitation of the Soret Bands by Use of Stimulated Raman-Scattering from H-2 and D2. J. Am. Chem. Soc. 1980, 102 (21), 6399-6407. (38) Hildebrandt, P.; Stockburger, M. Cytochrome-C at Charged Interfaces. 1. Conformational and Redox Equilibria at the Electrode-Electrolyte Interface Probed by Surface-Enhanced Resonance Raman-Spectroscopy. Biochemistry 1989, 28 (16), 6710-6721. (39) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. Protein:Colloid Conjugates for Surface Enhanced Raman Scattering: Stability and Control of Protein Orientation. J. Phys. Chem. B 1998, 102 (47), 9404-9413. (40) Premasiri, W. R.; Moir, D. T.; Klempner, M. S.; Krieger, N.; Jones, G.; Ziegler, L. D. Characterization of the Surface Enhanced Raman Scattering (SERS) of Bacteria. J. Phys. Chem. B 2005, 109 (1), 312-320.

is specific to the very outer surface of cells and the domains in close proximity to the metal surface due to the spatial confinement of surface plasmon and (ii) the autofluorescence signals from the cells are effectively quenched due to energy transfer with metal surfaces.44

Results and Discussion We examined the cell surface of S. oneidensis using tappingmode AFM imaging. Figure 2 shows a typical AFM image of S. oneidensis grown under anaerobic conditions and deposited on a glass surface. Under high-resolution AFM imaging, we observed rather smooth cell surfaces of S. oneidensis exposed to oxygen in the air. Figure 3A shows an AFM image of a portion of the cell surface of S. oneidensis grown anaerobically and bubbled with air for 5-10 min before fixation. The AFM image in Figure 3A was one of the roughest surfaces obtained for cells exposed to air. Although a high density of nanoscale domains clearly was not present, a few domains at a significantly lower density were observed for cells exposed to air before fixing. In contrast, we observed high-density distributions of domain structures on the outer membrane of anaerobically grown S. oneidensis, fixed before exposure to air. Figure 3B shows a typical AFM image of a portion of the cell surface of S. oneidensis grown anaerobically and under electron-acceptor limited conditions. The domains on the outer membrane of S. oneidensis were found to be independent of the scan angles, scan rates, and scan ranges in the AFM imaging experiments, confirming the reliability and accuracy of the AFM topographic characterization. Furthermore, zoom-in imaging was repeated for several areas on different cells and reconfirmed the cell-surface domains. A highresolution image of a portion of the cell surface of S. oneidensis grown anaerobially and under electron-acceptor limited conditions is shown in Figure 3C. The height of the structures varied from 18 to 25 nm and the lateral dimension from 35 to 50 nm. The lateral resolution was limited by the AFM tip convolution. On the basis of our AFM imaging experiments of many S. oneidensis (41) Smulevich, G.; Spiro, T. G. Surface Enhanced Raman-Spectroscopic Evidence That Adsorption on Silver Particles Can Denature Heme-Proteins. J. Phys. Chem. 1985, 89 (24), 5168-5173. (42) Suh, Y. D.; Schenter, G. K.; Zhu, L. Y.; Lu, H. P. Probing nanoscale surface enhanced Raman-scattering fluctuation dynamics using correlated AFM and confocal ultramicroscopy. Ultramicroscopy 2003, 97 (1-4), 89-102. (43) Xu, H. X.; Bjerneld, E. J.; Kall, M.; Borjesson, L. Spectroscopy of single hemoglobin molecules by surface enhanced Raman scattering. Phys. ReV. Lett. 1999, 83 (21), 4357-4360. (44) Hu, D. H.; Micic, M.; Klymyshyn, N.; Suh, Y. D.; Lu, H. P. Correlated topographic and spectroscopic imaging beyond diffraction limit by atomic force microscopy metallic tip-enhanced near-field fluorescence lifetime microscopy. ReV. Sci. Instrum. 2003, 74 (7), 3347-3355.

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Figure 4. AFM images of the cell surface of S. oneidensis grown anaerobically after treating with different electron acceptors for ∼5 min before fixing: (A) 10 mM fumarate; (B) 10 mM nitrate; and (C) 10 mM FeNTA. The outer membranes of S. oneidensis cells in samples treated with electron acceptors were smooth, and no characteristic domains were observed. (D) A high density of characteristic cell-surface domains retained in the case of samples treated with HEPES, a nonelectron-accepting buffer.

Figure 3. AFM images of the cell surface of S. oneidensis grown anaerobically and without any electron acceptor. (A) Recorded after bubbling air through the sample for ∼5 min. The surface features in (A) were probably from a few nanoscale domains that remained even after exposure to electron-accepting oxygen. The AFM image (A) was one of the roughest surfaces obtained for cells exposed to oxygen. (B) Recorded without exposing the sample to air. (C) A zoom-in and scanned image of a cell surface without exposure to air, confirming the structures observed in (B).

cells, we found that the high density of surface domains was general and inherent in cells grown under electron-acceptor limited conditions. This feature is likely related to a respiratory stress of an electron-acceptor limitation. The absence of dissolved oxygen or other electron acceptors in the growth medium and periplasm might have stimulated the S. oneidensis cells to express the surface domains for a possible interfacial electron transfer to insoluble electron acceptors in the environment if such were available.1,7,10,20,26,45-51 This attribute was further confirmed when we observed the disappearance or considerable decrease in the density of the cell(45) Harada, E.; Kumagai, J.; Ozawa, K.; Imabayashi, S.; Tsapin, A. S.; Nealson, K. H.; Meyer, T. E.; Cusanovich, M. A.; Akutsu, H. A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis. FEBS Lett. 2002, 532 (3), 333-337. (46) Louro, R. O.; Pessanha, M.; Reid, G. A.; Chapman, S. K.; Turner, D. L.; Salgueiro, C. A. Determination of the orientation of the axial ligands and of the magnetic properties of the haems in the tetrahaem ferricytochrome from Shewanella frigidimarina. FEBS Lett. 2002, 531 (3), 520-524. (47) Myers, C. R.; Myers, J. M. MtrB is required for proper incorporation of the cytochromes OmcA and OmcB into the outer membrane of Shewanella putrefaciens MR-1. Appl. EnViron. Microbiol. 2002, 68 (11), 5585-5594. (48) Myers, J. M.; Myers, C. R. Role for outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in reduction of manganese dioxide. Appl. EnViron. Microbiol. 2001, 67 (1), 260-269. (49) Myers, J. M.; Myers, C. R. Overlapping role of the outer membrane cytochromes of Shewanella oneidensis MR-1 in the reduction of manganese(IV) oxide. Lett. Appl. Microbiol. 2003, 37 (1), 21-25. (50) Schwalb, C.; Chapman, S. K.; Reid, G. A. The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella. Biochem. Soc. Trans. 2002, 30, 658-662. (51) Schwalb, C.; Chapman, S. K.; Reid, G. A. The tetraheme cytochrome CymA is required for anaerobic respiration with dimethyl sulfoxide and nitrite in Shewanella oneidensis. Biochemistry 2003, 42 (31), 9491-9497.

surface domains when the stress of the electron-acceptor limitation was relieved, i.e., when we exposed the cells to electron acceptors such as dissolved oxygen, nitrate, fumarate, and iron. We sampled the anaerobically grown S. oneidensis cells under electronacceptor limited conditions and exposed them to different soluble electron acceptors. The electron acceptors tested in our experiments included dissolved oxygen (partial pressure ∼0.24 psi), fumarate (10 mM), nitrate (10 mM), and FeNTA (10 mM). The anaerobically grown cells were exposed to fumarate, nitrate, and FeNTA in the absence of oxygen. Typical AFM images of S. oneidensis grown anaerobically and treated with fumarate, nitrate, and FeNTA are shown in Figure 4A-C, respectively. The cell samples were treated following the same procedure for anaerobic samples used previously in AFM imaging (Figure 3B). AFM images were obtained for cells treated with electron acceptors and showed no characteristic cell-surface domains in the presence of electron acceptors (Figures 3A, 4A-C). The domains had likely disappeared or were considerably reduced upon exposure to electron acceptors. We have also demonstrated that the disappearance of the surface domains was not simply due to the electrolyte condition changes. In a control experiment in which a sample was treated with degassed HEPES buffer, a nonreactive electrolyte, we confirmed the expression of surface domains on the outer membrane of S. oneidensis grown anaerobically and under conditions of electron-acceptor limitation. The cells treated with HEPES buffer retained cell-surface features (Figure 4D) that were essentially similar to the cells grown under anaerobic conditions (Figure 3B). Our observation of cell-surface domains supports the view that the domains expressed on the outer membrane are strongly related to the anaerobic respiration of S. oneidensis. The disappearance or significant decrease of the outer membrane domains was presumably due to withdrawal of heme proteins from the outer membrane in the presence of electron acceptors. This hypothesis and literature reports1,7,10,20,26,45-51 correlated the relation between the expressed heme-based domains on the outer membrane and the kind of electron acceptor. Soluble electron acceptors such as oxygen, fumarate, and nitrate were widely known to diffuse inside the outer membrane and involve

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Figure 6. SERS spectra of (A) the cell surface of S. oneidensis grown anaerobically. The spectrum was taken from a single cell under a focused laser excitation. The size of the laser focus spot is about 300 nm diameter. (B) Identical sample as in (A) except that it was bubbled with O2 for 5 s to relieve electron acceptor limitation before the cell was fixed. (C) pure bovine heart cytochrome c. (D) a MR-1 gspD mutant cultivated under electron acceptor limited condition. The inset of (A) is a far-field Raman spectroscopic image of anaerobically grown cells, obtained by recording the Raman signals in the region from 1350 to 1600 cm-1.

Figure 5. Diagram illustrating four characteristic vibrational bands of the porphyrin skeletal structures.

redox reactions with cytochromes in the cytoplasmic membrane. Therefore, it was not surprising that, in the presence of the soluble electron acceptors, redox protein domains disappeared from the outer membrane. AFM imaging provided unambiguous topographic identification of the existence of the high-density surface domains of the S. oneidensis cells under electron-acceptor limited conditions. To identify the chemical composition of the domains expressed on the outer membrane, we have performed SERS measurements on the surface domains. Raman spectroscopic analysis is particularly selective to identify heme proteins, as there are four characteristic Raman bands of the porphyrin molecule (Figure 5), a chromophore of the heme proteins. These vibrational fingerprints provide information on the redox and ligation states of heme proteins.52,53 A typical Raman spectrum of the outer membrane of S. oneidensis cells under electron-acceptor limited conditions is shown in Figure 6A. A far-field optical image was obtained for an anaerobically grown S. oneidensis cell (inset of Figure 6A). The optical image was obtained by raster-scanning the sample and recording the Raman signals in the region 1350 cm-1600 cm-1 by using a photon-counting avalanche photodiode (Figure 1). To examine a possible contribution of heme proteins to the surface domains expressed on the outer membrane under electronacceptor limited conditions, we compared the Raman spectrum (Figure 6A) of cell-surface structures with the Raman spectrum of oxidized forms of cytochrome c from bovine heart (Figure 6C). The Raman spectra in Figure 6A-D were recorded under similar experimental conditions. In Figure 6C, the band at 1371 (52) Hu, S. Z.; Spiro, T. G. The Origin of Infrared Marker Bands of Porphyrin π-Cation Radicals - Infrared Assignments for Cations of Copper(II) Complexes of Octaethylporphine and Tetraphenylporphine. J. Am. Chem. Soc. 1993, 115 (25), 12029-12034. (53) Spiro, T. G.; Li, X. Y. Resonance Raman Spectroscopy of Metalloporphyrins; Wiley-Interscience Publication: New York, 1988; pp 1-37.

cm-1 was assigned to the iron-porphyrin ring CR-N breathing mode (Figure 5A), ν4, which was known to be sensitive to the redox state change of heme proteins. The intense ν4 band is characteristic of a strong Franck-Condon factor, a result of selective resonance Raman enhancement of the Q-band, sensitive to the excitation wavelength selected (514.5 nm), of porphyrin chromophore.53,54 The 1492 cm-1 band corresponds to a highspin iron marker (ν3), and the 1568 and 1643 cm-1 bands were assigned to ν2 and ν10, respectively (Figure 5). All of these Raman band assignments are consistent with the literature,52,53 suggesting that the reference sample, cytochrome c, was predominately in the oxidized state (Figure 6C). Interestingly, the Raman spectrum of the outer membrane (Figure 6A) was essentially identical to that of oxidized cytochrome c (Figure 6C). By analogy with the Raman spectrum of cytochrome c, the band at 1368 cm-1 (Figure 6A) of the outer membrane was assigned to the porphyrin skeletal mode, ν4. Furthermore, the vibrational bands at 1489, 1569, and 1639 cm-1 of the cell-surface domains (Figure 6A) were attributed to ν3, ν2, and ν10 modes, respectively (Figure 5). The frequency of the oxidation-state marker (Figure 6A) strongly suggests that the cell-surface domains included oxidized heme proteins as a primary component.55,56 At this stage, using SERS spectroscopy and AFM imaging, we were unable to identify explicitly the type of heme proteins, such as single- or multiple-cytochrome proteins, that are composite in the domains. Figure 6B shows a typical Raman spectrum of a S. oneidensis cell from a sample exposed to air before fixation. Figure 6D presents the Raman spectrum of a MR-1 gspD mutant (see Supporting Information) under electron-acceptor limited conditions. The gspD mutants of MR-1 do not produce outer-membrane porin-forming proteins that (54) Champion, P. M.; Albrecht, A. C. Single-Mode Versus Multimode Calculations of Raman Intensities of Cytochrome-C. J. Chem. Phys. 1981, 75 (7), 3211-3214. (55) Ma, J. G.; Laberge, M.; Song, X. Z.; Jentzen, W.; Jia, S. L.; Zhang, J.; Vanderkooi, J. M.; Shelnutt, J. A. Protein-induced changes in nonplanarity of the porphyrin in nickel cytochrome c probed by resonance Raman spectroscopy. Biochemistry 1998, 37 (15), 5118-5128. (56) Parthasarathi, N.; Hansen, C.; Yamaguchi, S.; Spiro, T. G. Metalloporphyrin Core Size Resonance Raman Marker Bands Revisited - Implications for the Interpretation of Hemoglobin Photoproduct Raman Frequencies. J. Am. Chem. Soc. 1987, 109 (13), 3865-3871.

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presumably allow the cytchrome proteins to be expressed on cell surfaces. Interestingly, vibrational bands characteristic of heme proteins were not observed in the Raman spectra for cells under both electron-acceptor limited and exceeded conditions for gspD mutant MR-1 cells. Furthermore, AFM images (data not shown) of mutant gspD also clearly demonstrated the absence of surface protrusions under both electron-acceptor limited and electronacceptor exceeded conditions. These AFM imaging and Raman spectroscopic observations support a possible existence of an outer-membrane redox protein secretion pathway. Considering the topographic characterization and spectroscopic identification, we concluded that the nanoscale surface domains expressed on the outer membrane of S. oneidensis were most likely rich in heme proteins, cytochrome c-type proteins: (i) The nanoscale protein domains were present only on the outer membrane under electron-acceptor limited conditions, (ii) the SERS spectral features of the surface domains were characteristic of heme proteins, and (iii) our observations are consistent with the hypotheses of c-type cytochrome protein expression and secretion on the outer membrane and related biological verifications.1,7,10,20,26,45-51

Conclusion We observed expression of nanoscale domains on the outer membrane of S. oneidensis under a stress due to electron-acceptor limitation. The expression was not observable in the presence of a variety of electron acceptors, such as oxygen in the air, fumarate, nitrate, and FeNTA. The predominant contribution of heme protein to the cell-surface domains was determined by using SERS spectroscopy. Our direct experimental results of cell-surface structures using correlated AFM-optical measurements substantiated and supported the mechanism of secretion of an outer-membrane heme protein during the anaerobic

Biju et al.

respiration of S. oneidensis. Our new approach of correlated topographic and Raman spectroscopic analyses would be highly useful for a detailed understanding of the respiratory mechanism of S. oneidensis in particular and gram negative bacteria in general which involves complex and inhomogeneous electron-transfer dynamics and protein-mineral interactions. We anticipate that our unique approach using correlated AFM-confocal optical spectroscopic technique will be informative for further investigation of the interactions between cell surfaces and environmental oxidants, the topographic and spectroscopic characteristics of the outer membrane, and eventually the mechanisms of interfacial electron-transfer flux across the cell membrane of microbial cells. In addition, beyond this reported static topographic and spectroscopic characterizations, a dynamic study with time-dependent measurements will provide insights on the mechanism and dynamics of the accumulation and disappearance of the heme protein domains on the bacterial surface under electron-acceptor limited and exceeded conditions, respectively. Acknowledgment. We thank Dehong Hu for his support on correlated AFM-optical instrumentation. This work was supported by the Microbial Cell Program of the Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy (DOE) and by the Laboratory Directed Research Development funding at the Pacific Northwest National Laboratory. The Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for DOE under the contract DEAC06-76RLO1830. Supporting Information Available: Descriptions of the culture conditions, O2 limited growth condition, and construction of the gspD mutant. This material is available free of charge via the Internet at http://pubs.acs.org. LA061343Z