Project 3: Mechanisms of biofilm formation by Mycobacterium avium PI: Timothy Ford, Vice President for Research and Dean of Graduate Studies. Specific aims: (1) To characterize the mechanisms of biofilm formation by Mycobacterium avium and other bacteria found in drinking water, (2) to examine interactions between biofilms and their underlying substrata, and the role of these interactions in biocorrosion.

Brief background and significance: Dr. Ford’s research group has examined Mycobacterium avium, an opportunistic pathogen commonly found in drinking water biofilms. Early research using fluorescently labeled antibodies suggested that M. avium clusters close to the biofilm substratum interface in model systems of mixed species biofilmi (Fig 3a). More recent research from Ford’s group has focused on characterizing the attachment characteristics of M. avium, and in particular, attachment by a white transparent morphotypic variation of the bacteria that is dominant amongst drinking water isolates. Our research suggests that this morphotypic variation has a greater propensity to form biofilms and resist disinfection more than other morphotypes. We have examined the role of cell wall glycopeptidolipid in surface adherence of these morphotypesii, and most recently have been examining the role of cell-signaling molecules in biofilm formationiii. Geier et al.’s work has shown that increased concentrations of autoinducer-2 trigger an oxidative stress response in M. avium, determined by microarray analysis and Real-time PCR of up-regulated genes, leading to biofilm formation (Fig 3b).

Fig. 3a: Confocal micrograph of a mixed biofilm of Pseudomonas aeruginosa and Mycobacterium avium. Biofilms were incubated with rabbit polyclonal antibody to Erdman lipoarabinomannan, a Mycobacterium cell-wall lipopolysaccharide, followed by goat anti-rabbit antibody conjugated to rhodamine. M. avium (brightly stained) appears to be clustered close to the biofilm substratum interface.17 b: Confocal image of Influence of Quorum Sensing Signal Autoinducer-2 on M. avium Biofilm Formation. FM 1-43 membrane stain, excitation at 510nm. The negative control (A) shows the smallest amount of biofilm, whereas increasing the AI-2 concentration to 2.5 µM (B) or 25 µM (C) resulted in increased biofilm formation.19 c: SPM image of initiation of polysaccharide matrix formation, similar to control growth seen in b. N.B. matrix trail left by motile bacteria. Vertical height scale from dark to light is 500nm, with ±1nm vertical resolution.

The optical imaging was conducted in Ford’s laboratory at Montana State University in collaboration with the confocal microscopy facility at MSU. The Center for Biofilms Engineering at MSU has developed selective staining techniques that allow for distinguishing bacteriaiv (M avium through GFP expression) from their matrixv (BODIPy 630/650-X SE). Now that Ford has recently moved to the University of New England, it will not be possible to continue this work without a confocal microscope facility to image M. avium. A number of further questions remain that Ford’s research group is interested in pursuing, including separating the roles of cell to cell signaling from the oxidative stress response in inducing biofilm formation, both for M. avium and other human pathogens.

Ford is also interested in the influence of biofilms on underlying materialsvi,vii,viii,ix and will be proposing to use a combination of both spectral confocal microscopy and scanning probe microscopy to examine mechanisms of biocorrosionx under drinking water biofilms. Biofilm formation is well-suited for analysis by SPM because of the high z-scale sensitivity in which the early stages of biofilm creation can be easily observed (Fig. 3c). This complimentary information would allow determination of the biocorrosion source between action of the pathogens cells or a matrix associated process. Furthermore the temperature-controlled purfusion incubation stage will enable time studies to be undertaken under optimal growth conditions. Transparent substrates consisting of thin layers of AISI 316 stainless steel sputter-coated onto glass, for biocorrosion SCM/SPM imaging, will be provided by our collaborators at the University of Maine, Dr. Scott Collins and Dr. Rosemary Smith.

Project 4: Growth of Metallized Quaduplex DNA PI: James Vesenka, Department of Chemistry and Physics Specific aims: To characterize the mechanism of metallization enhancement of G20 G-wire DNA. Brief background and significance: Attempts to improve the conductive properties of DNA have involved "metallization", a process in which the DNA is used as a template for the adhesion of conductive metals like silverxi. Another approach is to use three or four stranded DNA with more dense base stacking than duplex DNA to improve electron hopping. Quadruplex DNA is particularly appealing because it is composed of stacks of guanine-quartet DNA and contains a natural pore (Figs. 4a&b) in which metal ions stabilize the DNAxii, and might improve its electrical characteristics. Quadruplex DNA has been found to have substantially greater polarizability compared to duplex DNAxiii and thus a greater potential for electron transfer. Dr. Vesenka's research group has previously targeted the guanine-rich oligonucleotide (GRO) sequence comprised of repeated G4T2G4 DNA naturally found in the telomeric (end of chromosome) regions of the ciliated water beetle Tetrahymena thermophila. In the presence of monovalent cations, such as potassium or sodium, this GRO spontaneously forms linear quadruplex "G-wire" DNAxiv. Vesenka has recently discovered that synthetic GRO consisting of G20 is easily metallized by Copper (II) (Cu2+) or silver (Ag+) cation substitution (Figs. 4c&d).

Experimental plan: Naturally occurring forms of GRO quadruplex DNA terminate G-wires when mismatches occurxv. The G20 sequence appears to have incomplete quadruplex formation allowing for strong ionic bonding between the negative phosphate backbone and free metal cations. The free ends disrupt the oligonucleotide sequence from forming a pure G-wire, but with sufficient concentrations of oligo to continue a chain at the core of the hybrid cation/G-wire molecule. The proposed experiments will involve both high-resolution SPM (using Veeco's patented new PeakForce technology) and electric force microscopy to detect molecular polarizability. The integrated "Nano-man" manipulation software in the Bioscope Catalyst SPM will also allow us to move metallized-G-wire clusters in solution over microfabricated gold contacts on transparent glass substrates, using the SCM as a fluorescent optical navigator, to directly detect conductivity. These substrates will be made by our collaborators at the University of Maine, Dr. Scott Collins and Dr. Rosemary Smith.

The proposed equipment would solve a pressing problem, finding low density metallized DNA, which tend to strongly cluster, even after mechanical separation. SPM imaging of metallized DNA is limited to regions near optically identifiable structures that spread non-uniformly over the substrate (Fig. 5d). Our current optical configuration, with a low-resolution long-working-distance lens, only identifies these larger clusters. However silver enhancement has been shown to provide a strong autofluorescence signalxvi (Fig. 4e) and can be identified by the confocal microscope (optical navigation) and then imaged by the combined scanning probe microscope. The Leica system we have chosen is a full functioning epi-fluorescence microscope with an integrated long-life metal-halide lamp, with broad excitation UV illumination, specifically designed for quick fluorescence viewing. Lastly the specimen holder is attached to a large sample stage manipulator on the combined microscopes, increasing the region that can be viewed by a million-fold compared to our old microscope (current view area about 10mm2).

Fig 4a:

Guanine-rich

oligonucleotide

sequences can hydrogen-bond on two sides to generate a tetrad motif called the "G-quartet". b: G-quartets base stack into helical four-stranded structure, a.k.a. "G-wires", with a channel parallel to the long axis. G-wires are stabilized with cations in solution, commonly Na+ and K+ incorporated into the central pore and Mg2+ and Ca2+ on the periphery. We have found ionic copper and silver can metallize G20 DNA. c: 10mM CuCl2 was mixed with 15mM G20 in pure water and the G-wires grew from 2nm to 5nm in diameter. This suggests metal salts must bind to the perimeter of the G-wire DNA. d: Silver metallized G-20 DNA. The structures made under these conditions ranged from 5-8nm in diameter. The silver-G-wire clusters, in which this image was captured, was found under the far-field-working lens of our old SPM and clusters must be large (micrometers) for detection. e:

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Silver enhanced G-wire DNA autofluoresces under ultraviolet light illumination (390nm in the image above) that is enhanced 1000-fold in solution by the presence of co-adsorbed silver (same sample as in d), enabling the identification of otherwise diffraction limited clusters, not all visible (right pointing arrows) or distinguished from detritus (left pointing arrows) in the bright field image. Other users: Cell Project: Co-localization of insulin receptors and caveolae, Amy Davidoff, Dept. of Basic Sciences Specific aims: (1) To evaluate the cell surface distribution of receptors (e.g., insulin receptor) relative to caveolin in several cell lines derived from striated and smooth muscle cells. (2) To determine whether stressors (e.g., ROS, cytokines) alter the co-localization of these receptors and diminish signal transduction.

Background and research: The overall goal of this collaboration is to understand the physiological consequences of cell surface receptor co-localization (and dispersion) with caveolae (specialized regions of lipid rafts). In many examples of membrane transduction systems, the cell surface receptor is located in or near caveolae, and the intracellular targets are thought to be anchored to the caveolae. It has been suggested that under certain conditions, the surface receptors migrate away from the caveolae resulting in reduced signaling. We propose to develop protocols to ‘visualize’ cell surface receptors utilizing the scanning probe microscope (SPM) in a manner that minimally perturbs the integrity of cells (i.e., ultimately working with living cells). This project requires combining the imaging capability of an SPM with that of a SCM. The latter is necessary in order to focus in on caveolar regions (with reflecting or fluorescent probes) so that the SPM can be precisely positioned in that region. The strategy is to label two proteins with their respective antibodies, conjugated with different sized gold nanoparticles (e.g., 5 and 15 nm particles), and utilize the SPM force modulus detection to determine their relative proximity to one another. The Bioscope's temperature controlled fluid cell we have included in this proposal will provide the thermal stability needed to make a much more accurate topographical map of the cell surface improving the resolution about 50 times greater than traditional immunohistochemistry.

Fig. 5a show immunohistochemistry of cells probed with an insulin receptor antibody and Fig. 5b illustrates a caveolin 3 antibody, staining H9C2 cells that have been chemically fixed and non-permeabilized. Fig. 5c highlights the fluorescence of caveolin 3 on the cell surface in living cells. To date, we have only been able to blindly position the SPM probe and image cell surfaces without antibodies (see Figs. 5d & e). A number of preliminary studies will be conducted to ensure specificity of each antibody to its receptor, including running western blots using antibody concentrations that are appropriate for immunohistochemistry and SPM. Specific aim 2 will involve exposing cells to various stressors (e.g., cytokines), as well as insulin, to evaluate whether these effectors change the distribution of the receptor relative to the caveolae. We anticipate that the insulin receptor will migrate away from the caveolae and that will coincide with reduced insulin signaling (e.g., insulin-stimulated glucose uptake). Dr. Davidoff has had more than 19 years experience with cell culture, and basic cellular physiology (including Ca2+ regulation). Successful development of these protocols in this pilot study could lead to methods for understanding the basic biology of many cell surface receptors (e.g., Notch receptors) relative to their proximity to caveolae, and in turn, lay the foundation for following intracellular trafficking as proposed by Dr. Small.

Fig 5a&b: H9c2 cells chemically fixed, non-permeabilized and stained with DAB (brown; ABC Kit from Vector) and hematoxylin (blue). a: Insulin receptor alpha subunit antibody from Abcam (1:50). b: Caveolin-3 antibody from Stanta Cruz (1:100). c: Live H9c2 cells epifluorescent images in physiological saline. Caveolin-3 primary antibody from Stanta Cruz (1:1000). Image was digitally altered for illustrative purposes. d: Tapping AFM phase images of a hydrated H9c2 cell that was fixed with 4% paraformaldehyde and imaged in dH2O. e: Increased resolution of same sample as depicted in d. Drift is evident in these fluid cell images even though the sample had been allowed to equilibrate for over a day.

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Collaborative Project: Freshwater iron oxidizing bacteria, David Emerson, Bigelow Laboratories Specific aim: Correlation of cell motility and sheath production.

Background and research: Iron oxidizing bacteria (FeOB) can convert iron in solution to tube-like "sheaths" in which they live and reproduce. Dr. Emerson is collaborating with Dr. Vesenka on preliminary studies to analyze the ultrastructure of Leptothrix ochracea using a variety of SPM techniques. Optical micrographs (Fig. 6a&b) indicate that these FeOB construct and reside inside sheaths of iron oxide. SPM images of FeOB sheaths from L. ochracea indicate the tubes are about 1.6µm in diameter and have a sheath thickness ranging from 35-40nm, confirming electron microscopy (EM) analysis. Fig. 6c is an SPM image with three presentations of FeOB sheaths: native, collapsed "bilayer" and collapsed "monolayer" forms. The height of the native sheath and the width of the collapsed bilayer sheath have consistent dimensions of 1.6µm diameter. The collapsed monolayer was half the width of the collapsed bilayer. The SPM provide new information unable to be obtained by EM techniques: the grain size analysis of a high-resolution image from the native sheath indicates an average size of 1760nm2, or about 42nm in diameter and were aligned parallel to the sheath's axis (Fig. 6d). This data suggests the sheath is comprised of single grains (a monolayer) of iron oxides. Though a number of other SPM experiments are in the works (including electric and magnetic force imaging) the more demanding questions about how the sheaths are formed and the motility of the bacteria cannot be addressed without the combined SCM/SPM imaging system. The specific experiment we envision is similar to that used by Pelling on yeast cellsxvii, using the SPM probe in order to monitor vibrations produced by cell motion in the sheath. The scanning confocal microscope is essential for optically locating live cells fluorescently labeled in the transparent sheaths.

Figs 6a&

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contrast image, b is the same region in epi-fluorescence showing the filaments of cells inside the sheaths, N.B. many of the sheaths are empty. c: Three dimensional SPM image of a L. ochracea sheath whole (1.6µm tall) and a pair of collapsed sheaths (about 35-40nm thick and 2.5µm wide). The 5µm scale bar, which is very close in length to the optical images, a&b, may appear to provide seemingly contradictory evidence on the sheath dimensions. A collapsed sheath will appear about circumference/2 = πr in width, or about 2.5µm, which is consistent with the scale bar in the SPM image. d: High pass filter image from a high resolution SPM image of the top of the whole sheath (10nm lateral resolution) indicates the iron oxide grows in rectangular plates that tend to orient parallel to the axis of the sheath. A grain size analysis indicates the diameters of the grains to be about 40nm, about the same thickness as the sheath. The striations appearing at either side (below or above) of the sheath in d are SPM tip artifacts due to the sheath imaging the side of the probe. i Ford, TE. Microbiological Safety of Drinking Water: United States and Global Perspectives. (1999) Environ Health Perspect 107(Suppl 1):191-206. PMCID: 1566363 ii Freeman R., Geier H, Weigel KM, Do J, Ford TE, Cangelosi GA. Roles for cell wall glycopeptidolipid in surface adherence and planktonic dispersal of Mycobacterium avium. (2006) Appl Environ Micro 72: 7554-7558. PMC1694245 iii Geier H, Mostowy S, Cangelosi GA, Behr MA, Ford TE. Autoinducer-2 triggers the oxidative stress response in Mycobacterium avium leading to biofilm formation. (2008) Appl Environ Microbiol 74:1798-1804. PMC2268301

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iv Parker, A.E., Bermudez L.E. Expression of the green fluorescent protein (GFP) in Mycobacterium avium as a tool to study the interaction between Mycobacteria and host cells. (1997) Microbial Path 22, 192-198. v Pitts, B., Staining bacterial biofilms: new uses for classic fluorescent dyes. (2007) Bioprobes 53, 2-3. vi Gu J-D, Ford TE, Mitchell R. Microbial degradation of materials: general processes. Revie W, ed. 2nd edition, The Uhlig Corrosion Handbook, John Wiley & Sons, New York 2010a (in press). vii Gu J-D, Ford TE, Mitton B, Mitchell R. Microbial degradation of polymeric materials. Revie W, ed. 2nd edition, The Uhlig Corrosion Handbook, John Wiley & Sons, New York 2010b (in press). viii Gu J-D, Sand W, Ford TE, Mitchell R. Microbial degradation of concrete. Revie W, ed. 2nd edition, The Uhlig Corrosion Handbook, John Wiley & Sons, New York 2010c (in press). ix Gu J-D, Ford TE, Mitchell R. Microbial corrosion of metals. Revie, W, ed. 2nd edition, The Uhlig Corrosion Handbook, John Wiley & Sons, New York 2010d (in press). x Beech IA, Smith J.R., Steele A.A., Penegar I., Campbell S.A. The use of atomic force microscopy for studying interactions of bacterial biofilms with surfaces. (2002) Colloids Surf B: Biointerfaces 23, 231-247. xi E. Braun, Y. Eichen, U. Sivan, and G. Ben-Yoseph, “DNA-Templated Assembly and Electrode Attachment of a Conducting Silver Wire.” (1998) Nature. 391, pp 775-778. xii D. Sen & W. Gilbert, "Novel DNA superstructures formed by telomere-like oligomers." (1992) Biochem. 31: 65. xiii H. Cohen, T. Sapir, N. Borovok, T. Molotsky, R. Di Felice, A.B. Kotlyar, D. Porath, "Polarizability of G4-DNA Observed by Electrostatic Force Microscopy Measurements " (2007) Nano Lett., 7: 981-986 xiv T. Marsh, J. Vesenka, & E. Henderson, "A new DNA nanostructure, the G-wire, imaged by scanning probe microscopy." (1995) Nucleic Acids Res. 23, 696. xv Personal conversation with Alexander Kotlyar at DNA Electronics Conference, May 2008, Jena Germany. xvi J.R. Lakowicz, B. Shen, Z. Gryczynski, S. D’Auria, & I. Gryczynski, (2001) Biochem. and Biophys. Res. Com. 286, 875-879 xvii A.E. Pelling, S. Sehati, E.B. Gralla, J.S. Valentine, J.K. Gimzewski "Local Nanomechanical Motion of the Cell Wall of Saccharomyces cerevisiae" (2004) Science 305, 1147-1150.