The development of a novel approach to the design of microdevices
Submitted in total fulfillment of the requirements for the degree of
Doctor of Philosophy
by
Yulia Alekseeva
Faculty of Life and Social Sciences
Swinburne University of Technology
December 2011 Abstract
The effectiveness of protein-based microdevices depends on the ability of their surfaces to provide spatial immobilization and maintain protein bioactivities. Although methodologies for the construction of microdevices for biomedical applications have been developed, the manufacturing of microdevices remains expensive due to the high cost of materials and fabrication processes. As the surfaces display structural uniformities which restrict protein-surface interactions and consequently protein immobilization, innovative approaches to the design of surfaces are required. The approaches need to allow for the minimization of fabrication costs via efficient amplification and spatial immobilization of multiplex proteins so that the bioactivity of protein-based microdevices (e.g., microarrays) can be retained. A novel approach to the design of surfaces for microdevices has been developed and evaluated in this work. This approach is based on micro/nanostructures fabricated via laser ablation of a thin metal layer deposited on a transparent polymer. The structures of a 100 nm-range are represented by „combinatorialized‟ micro/nano- channels that allow amplified protein immobilization in a highly controlled manner. The relationship between the properties of the micro/nano-channel surface topography, physico-chemistry, and protein immobilization, for five, molecularly different proteins, i.e., lysozyme, myoglobin, alpha-chymotrypsin, human serum albumin, and human immunoglobulin has been investigated. Using quantitative fluorescence measurements and atomic force microscopy, protein immobilization on microstructures has been characterized. It has been found that the combinatorial nature of the micro/nano-channels allowed a 3 to 10- fold amplification of protein adsorption, as compared to the protein adsorption on flat, chemically homogenous polymeric surfaces. An improved methodology allowing in vitro assembled micron- and nano- scale tracks of proteins (i.e., actin) which support unidirectional translocation of beads functionalized with motor proteins (i.e., myosin) was also developed. The nanotracks composed of aligned F-actin/gelsolin bundles were formed by electrostatic condensation of F-actin/gelsolin with Ba2+.
ii The prospects for employment of bacterial ATP producers as replacements for the energy source, and prokaryotic actin homologues as replacements for eukaryotic actin in microdevices based on molecular motor-based systems, have been explored. A search for ATP producers among 86 environmental strains of 17 genera, including 4 species of 3 genera described in this thesis has been performed. Bacteria belonging to the genera Sulfitobacter, Marinobacter and Staleya and/or Planococcus and Kocuria have been found to be promising producers of extracellular and intracellular ATP, respectively. Substitution of eukaryotic actin with inherently stable prokaryotic actin- related proteins, i.e., MreB or FtsA, may point the way to the development of the next generation of microdevices for biomedical applications.
iii Acknowledgments
This thesis resulted from research supported by the Australian Research Council (ARC) and partially supported by the Defense Advanced Research Projects Agency (DARPA).
I acknowledge the great support of the Research Higher Degrees Committee (RHDC) of my alma mater.
I enjoyed working on my project. I had the great honor and pleasure of being supervised by a person with a very strong interdisciplinary vision, Professor Elena Ivanova. I acknowledge her incredible support and encouragement. I thank Professor Michael Gilding for his kind words of encouragement and support. I acknowledge the support provided by Professor Pam Green. I thank Professor Russell Crawford for his support.
I also wish to thank Professor Dan V. Nicolau for his support; Dr Vlado Buljan and Dr Murat Kekic (the University of Sydney) for sharing their experience in molecular motor protein extraction and handling; Dr Igor Sbarski, Dr Gregory M. Demyashev, Dr Luisa Filipponi, Dr Andrea Viezzoli, Dr Dan V. Nicolau, Jr., Dr Jonathan P. Wright, Dr Duy K. Pham, Marjan Ilkov, Dr Hans Brinkies, Anya Ilkova, Dr Natasa Mitik-Dineva for assisting in laboratory experiments and data analysis. I thank all LSS and IRIS staff members who supported this study. I thank my student teammates who have shared their thoughts with me. I enjoyed a friendly scientific atmosphere of our group meetings.
I acknowledge the support of Professor Tomoo Sawabe and Dr Karin Hayashi (Hokkaido University), Professor Richard Christen (the University of Nice Sophia Antipolis), Dr Nataliya I. Kalinovskaya, Dr Natalia V. Zhukova, Dr Galina M. Frolova, Professor Valery V. Mikhailov, Dr Nataliya M. Gorshkova, Dr Valeriya V. Kurilenko, Dr Olga I. Nedashkovskaya (Pacific Institute of Bioorganic Chemistry)
iv and Arkady Kurilenko (Pacific Oceanological Institute). I thank Professor Victor P. Chelomin (Pacific Oceanological Institute) for fruitful scientific discussions.
I would also like to thank Maryna Mews for her editorial assistance.
Last but not least, I would like to express my greatest appreciation to my darling parents, Lyubov & Vladimir, for participating in my son‟s upbringing and supporting this study. Special thanks are extended to all those who helped me in gaining sole custody of my child. And lastly, I would love to thank my son, Maxim, for being a good guy.
v
This thesis is dedicated to my son, Maxim.
vi DECLARATION
I certify that the work presented in the thesis contains no material which has been submitted for another degree of any other university. To the best of my knowledge it does not contain any material previously published or written by another person except where due reference is made in the text.
Contributions of the respective researchers to this study: Professor Elena Ivanova supervised the research project; Professor Dan V. Nicolau organized the project; Dr Vlado Buljan and Dr Murat Kekic (the University of Sydney) assisted in protein extraction and handling; Dr Igor Sbarski, Dr Gregory M. Demyashev, Dr Luisa Filipponi, Dr Andrea Viezzoli, Dr Dan V. Nicolau, Jr., Dr Jonathan P. Wright, Dr Duy K. Pham, Marjan Ilkov, Dr Hans Brinkies, Anya Ilkova, Dr Natasa Mitik-Dineva assisted in laboratory experiments and data analysis; Professor Tomoo Sawabe and Dr Karin Hayashi (Hokkaido University), Professor Richard Christen (the University of Nice Sophia Antipolis), Dr Nataliya I. Kalinovskaya, Dr Natalia V. Zhukova, Dr Galina M. Frolova, Professor Valery V. Mikhailov, Dr Nataliya M. Gorshkova, Dr Valeriya V. Kurilenko, Dr Olga I. Nedashkovskaya (Pacific Institute of Bioorganic Chemistry), Professor Victor P. Chelomin and Arkady Kurilenko (Pacific Oceanological Institute) assisted in microbiology-related experiments and data analysis.
I declare that this thesis has been partially professionally copyedited and proofread by Maryna Mews, however, the editorial assistance did not affect its substantive content.
Yulia Alekseeva
vii List of Publications
Book chapter
1. Critical aspects in microfluidic systems design. Alekseeva YV, Crawford RJ, Ivanova EP. In: Advances in Chemistry Research 15, Nova Publishers (NY), 2012.
Journal articles
2. Protein immobilisation on micro/nanostructures fabricated by laser microablation. Nicolau DV, Ivanova EP, Fulga F, Filipponi L, Viezzoli A, Dobroiu S, Alekseeva YV, Pham DK. Biosensors and Bioelectronics 26(4):1337- 1345, 2010.
3. “Pseudoalteromonas januaria" SUT 11 as the source of rare lipodepsipeptides. Kalinovskaya NI, Dmitrenok AS, Kuznetsova TA, Frolova GM, Christen R, Laatsch H, Alexeeva YV, Ivanova EP. Current Microbiology 56(3):199-207, 2008.
4. ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces. Ivanova EP, Alexeeva YV, Pham DK, Wright JP, Nicolau DV. International Microbiology 9(1):37-46, 2006.
5. A comparative study between the adsorption and covalent binding of human immunoglobulin and lysozyme on surface-modified poly( tert-butyl methacrylate). Ivanova EP, Wright JP, Pham DK, Brack N, Pigram P, Alekseeva YV, Demyashev GM, Nicolau DV. Biomedical Materials 1(1):24-32, 2006.
6. Characterization of unusual alkaliphilic gram-positive bacteria isolated from degraded brown alga thalluses. Ivanova EP, Wright JP, Lysenko AM, Zhukova NV, Alexeeva YV, Buljan V, Kalinovskaya NI, Nicolau DV, Christen R, Mikhailov VV. Microbiological Journal 68(4):10-20, 2006.
7. Controlling the covalent and noncovalent adsorption of proteins on polymeric surfaces in aqueous liquids. Ivanova EP, Pham DK, Alekseeva YV, Demyashev GM, Nicolau DV. Chinese Journal of Light Scattering 17(3):234-236, 2005.
8. Presence of ecophysiologically diverse populations within Cobetia marina strains isolated from marine invertebrate, algae and the environments. Ivanova EP, Christen R, Sawabe T, Alexeeva YV, Lysenko AM, Chelomin VP, Mikhailov VV. Microbes and Environments 20(4):200-207, 2005.
9. Controlled self-assembly of actin filaments for dynamic biodevices. Alexeeva YV , Ivanova EP, Pham DK, Buljan V, Sbarski I, Ilkov M, Brinkies HG, Nicolau DV. Nanobiotechnology 1(4):379-388, 2005.
10. Bacillus algicola sp. nov., a novel filamentous organism isolated from brown alga Fucus evanescens. Ivanova EP, Alex eeva YV, Zhukova NV, Gorshkova NM, Buljan V, Nicolau DV, Mikhailov VV, Christen R. Systematic and Applied Microbiology 27(3):301-307, 2004. viii 11. Brevibacterium celere sp. nov., isolated from degraded thallus of a brown alga. Ivanova EP, Christen R, Alexeeva YV, Zhukova NV, Gorshkova NM, Lysenko AM, Mikhailov VV, Nicolau DV. International Journal of Systematic and Evolutionary Microbiology 54:2107-2111, 2004.
12. Formosa algae gen. nov., sp. nov., a novel member of the family Flavobacteriaceae. Ivanova EP, Alexeeva YV, Flavier S, Wright JP, Zhukova NV, Gorshkova NM, Mikhailov VV, Nicolau DV, Christen R. International Journal of Systematic and Evolutionary Microbiology 54(3):705-711, 2004.
13. Low-molecular-weight, biologically active compounds from marine Pseudoalteromona s species. Kalinovskaya NI, Ivanova EP, Alexeeva YV, Gorshkova NM, Kuznetsova TA, Dmitrenok AS, Nicolau DV. Current Microbiology 48(6):441-446, 2004.
14. Sulfitobacter delicatus sp. nov. and Sulfitobacter dubius sp. nov., respectively from a starfish (Stellaster equestris) and sea grass (Zostera marina). Ivanova EP, Gorshkova NM, Sawabe T, Zhukova NV, Hayashi K, Kurilenko VV, Alexeeva Y, Buljan V, Nicolau DV, Mikhailov VV, Christen R. International Journal of Systematic and Evolutionary Microbiology 54(2):475-480, 2004.
15. Impact of cultivation conditions on haemolytic activity of Pseudoalteromonas issachenkonii KMM 3549T. Alexeeva YV. Kalinovskaya NI, Kuznetsova TA, Ivanova EP. Letters in Applied Microbiology 38(1):38-42, 2003.
16. Ecophysiological variabilities in ectohydrolytic enzyme activities of some Pseudoalteromonas species, P. citrea, P. issachenkonii, and P. nigrifaciens. Ivanova EP, Bakunina IY, Nedashkovskaya OI, Gorshkova NM, Alexeeva YV, Zelepuga EA, Zvaygintseva TN, Nicolau DV, Mikhailov VV. Current Microbiology 46(1):6- 10, 2003.
17. Marinobacter excellens sp. nov., isolated from sediments of the sea of Japan. Gorshkova NM, Ivanova EP, Sergeev AF, Zhukova NV, Alexeeva Y, Wright JP, Nicolau DV, Mikhailov VV, Christen R. International Journal of Systematic and Evolutionary Microbiology 53):2073(6 –2078, 2003.
18. Optimization of glycosidases production by Pseudoalteromonas issachenkonii. Alex eeva YV, Ivanova EP, Bakunina IY, Zvaygintseva TN, Mikhailov VV. Letters in Applied Microbiology 35(4):343-346, 2002.
19. Pseudoalteromonas issachenkonii sp. nov., a bacterium that degrades the thallus of the brown alga Fucus evanescens. Ivanova EP, Sawabe T, Alexeeva YV, Lysenko AM, Gorshkova NM, Hayashi K, Zukova NV, Christen R, Mikhailov VV. International Journal of Systematic and Evolutionary Microbiology 52(1):229-234, 2002.
20. Two species of culturable bacteria associated with degradation of brown algae Fucus evanescens. Ivanova EP, Bakunina IY, Sawabe T, Hayashi K, Alexeeva YV, Zhukova NV, Nicolau DV, Zvaygintseva TN, Mikhailov VV. Microbial Ecology 43(2):242-249, 2002. ix Referred Conference proceedings
1. Amplification of protein adsorption on micro/nanostructures for microarray applications. Ivanova EP, Alekseeva YV, Pham DK, Filipponi L, Nicolau DV. NSTI Proceedings 1:95-98, 2004.
2. Microlithographically fabricated bar-coded microarrays. Ivanova EP, Pham DK, Alekseeva YV, Filipponi L, Nicolau DV. SPIE Proceedings 5328:49-55, 2004.
3. Actin nanotracks for hybrid nanodevices based on linear protein molecular motors. Watson GS, Cahill C, Blach J, Myhra S, Alexeeva Y, Ivanova EP, Nicolau DV. MRS Proceedings 820:25-35, 2004.
4. Immobilization of multiple proteins in polymer microstructures fabricated via laser ablation. Ivanova EP, Viezzoli A, Alekseeva YV, Demyashev GM, Nicolau DV Jr, Filipponi L, Pham DK, Nicolau DV. SPIE Proceedings 4966:37- 49, 2003.
Poster presentations with published abstracts
1. Protein adsorption on micro/nano-structures fabricated by laser microablation. Nicolau DV, Ivanova EP, Alexeeva YV, Viezzoli A, Pham DK, Dobroiu S et al. 20th Anniversary World Congress on Biosensors, P2.1.127, 2010.
2. Variations in ATP levels in heterotrophic bacteria during biofilm formation. Ivanova EP, Alexeeva YV, Pham DK, Wright JP, Nicolau DV. “ASM 2004 Sydney National Conference”, 2004.
3. Controlled self-assembly of actin filaments for nanobiotechnological devices. Alexeeva YV, Ivanova EP, Buljan V, Nicolau DV. “ASM 2004 Sydney National Conference”, 2004.
4. The optimization of fermentation processes of Pseudoalteromonas issachenkonii. Alexeeva YV, Ivanova EP, Bakunina IY, Zvyagenseva TN, Mikhailov VV. Conference “Marine Bio Shizuoka-2001”, p. 98, 2001.
5. Microbial community of Pseudoalteromonas issachenkonii and Halomonas marina degrade the tallus of brown algae Fucus evanescens. Ivanova EP, Alexeeva YV, Bakunina IY, Zvyagenseva TN, Mikhailov VV. 9th Int Symposium on Microbial Ecology, Amsterdam, p. 314, 2001.
6. Convertible energy of living organisms. Alexeeva YV, Burceva RА. II regional conference “Problems of marine biology, ecology and biotechnology”, Vladivostok, FESU, p. 6, 1999. ______*My surname Alekseeva can be spelled either with “x” or with “ks”.
x TABLE OF CONTENTS
Abstract ii Acknowledgments iv Declaration vii List of publications viii List of abbreviations xix List of tables xxiii List of figures xxv List of schemes xxviii
Chapter 1. Introduction 1 1.1. Overview 2 1.2. Aim of the study 5 1.3. Organization of the thesis 5
Chapter 2. Literature review 7 2.1. Overview 8 2.2. Concept and benefits of biosensors 8 2.3. Types of biosensors 11 2.4. Microfluidic devices 13 2.4.1. Overview 13 2.4.2. Advantages of microfluidic devices 14 2.4.3. Critical aspects of microfluidic devices 17 2.4.3.1. Overview 17 2.4.3.2. Surface properties 17 2.4.3.3. Microfluidic device geometry 20 2.4.3.4. Fluid properties 21 2.4.4. Types of microfluidic devices 23 2.4.4.1. Overview 23 2.4.4.2. Droplet system 23 2.4.4.3. Continuous system 26 xi 2.4.5. Control of microfluidic devices 28 2.4.5.1. Overview 28 2.4.5.2. Control of fluidic movement 28 2.4.5.3. Control of fluidic interactions 30 2.4.5.4. Immobilization of proteins 33 2.4.5.4.1. Overview 33 2.4.5.4.2. Physical adsorption 33 2.4.5.4.3. Covalent binding 35 2.4.5.4.4. Self-assembled monolayers (SAMs) 37 2.5. Concept of protein molecular motors 39 2.5.1. Overview 39 2.5.2. Eukaryotic actin 39 2.5.3. Prokaryotic actin related proteins 42 2.5.3.1. Overview 42 2.5.3.1.1. MreB 43 2.5.3.1.2. FtsA 46 2.5.4. Evolution/Phylogeny of bacterial actin homologues 48 2.5.4.1. Overview 48 2.5.4.2. Evolutionary/Phylogenetic comparison of MreB with FtsA 48 2.5.4.3. Use of 16S rRNA as a molecular chronometer 50 2.5.5. Classification of protein molecular motors 52 2.5.5.1. Overview 52 2.5.5.2. Linear molecular motors 53 2.5.5.3. Rotary molecular motors 55 2.5.6. Native functions of molecular motors 57 2.5.6.1. Overview 57 2.5.6.2. Cytoskeleton 58 2.5.6.3. Cellular metabolism 60 2.5.6.4. Flagella-based motion 62 2.5.6.5. Tactics of enteric pathogens 65 2.5.6.5.1. Overview 65
xii 2.5.6.5.2. Common tactics of enteric pathogens 65 2.5.6.5.3. Listeria as a regulator of actin assembly 67 2.5.6.6. Bacterial ATP generation 70 2.5.6.7. Use of MreB and FtsA proteins by bacteria 72
Chapter 3. Methodology 76 3.1. Overview 77 3.2. Methods used to study protein-surface interactions 78 3.2.1. Protein preparation for immobilization on polymeric surfaces 78 3.2.2. Polymeric film preparation 78 3.2.3. Preparation of microfabricated structures 79 3.2.4. Protein adsorption on surfaces 80 3.2.4.1. Protein adsorption on flat surfaces 80 3.2.4.2. Protein adsorption on micro/nano-fabricated structures 80 3.2.5. Protein covalent binding onto surfaces 80 3.2.6. Detection and quantification techniques 81 3.2.6.1. Fluorescence spectroscopy of adsorbed proteins 81 3.2.6.2. X-ray photoelectron spectroscopy 81 3.2.6.3. Goniometry 82 3.2.6.4. Ellipsometry 82 3.2.6.5. Atomic force microscopy (AFM) 83 3.2.6.6. Calculation of protein-surface parameters 83
3.3. Methods of actin/myosin preparation 84 3.3.1. Actin and heavy meromyosin (HMM) preparation 84 3.3.2. Preparation of the electrostatically condensed actin bundles 85 3.3.3. Preparation of the polymeric surfaces 85 3.3.4. Protein immobilization on the polymeric surfaces in the flow cell 86 3.3.5. Beads functionalization 86 3.3.6. Fluorescence microscopy 87 3.3.7. Scanning electron microscopy (SEM) 87
xiii 3.3.8. X-ray photoelectron spectroscopy 87 3.3.9. Rheological measurements 88
3.4. Methods of bacterial taxonomy 88 3.4.1. Bacterial isolation 88 3.4.1.1. Isolation of gram-negative bacteria 88 3.4.1.1.1. Isolation of Marinobacter excellens 88 3.4.1.1.2. Isolation of Sulfitobacter delicatus and Sulfitobacter dubius 89 3.4.1.2. Isolation of gram-positive bacteria 90 3.4.1.2.1. Isolation of Planococcus maritimus 90 3.4.2. Bacterial characterization 90 3.4.2.1. Phenotypic analysis 90 3.4.2.1.1. General phenotypic tests 91 3.4.2.1.1.1. Microscopic examination 91 3.4.2.1.1.2. Utilization of organic substrates 92 3.4.2.1.1.3. Degradation of macromolecules 92 3.4.2.1.1.4. Cytotoxic and antibacterial activities 93 3.4.2.1.1.5. Susceptibility to antibiotics 93 3.4.2.1.2. Species-specific phenotypic tests 93 3.4.2.2. Chemotaxonomic methods 94 3.4.2.2.1. Polar lipid (PL) analysis 94 3.4.2.2.2. Fatty acid (FA) analysis 94 3.4.2.3. Genotypic analysis 95 3.4.2.3.1. DNA GC content determination 95 3.4.2.3.2. DNA hybridization 95 3.4.2.4. Phylogenetic analysis 96 3.4.2.4.1. 16S rRNA gene analysis 96
3.5. Methods used to assess ATP production by bacteria 97 3.5.1. Bacterial strains 97 3.5.2. Polymeric surface preparation 100
xiv 3.5.3. Contact angle measurements 101 3.5.4. Bacterial growth and sample preparation 101 3.5.5. Bioluminescence assay for ATP determination 102 3.5.6. Cell-surface characterization by AFM 102
3.6. Methods used to assess MreB and FtsA proteins 103 3.6.1. Analysis of mreB and ftsA genes 103 3.6.2. Computation of MreB and FtsA protein parameters 104
Chapter 4. Immobilization of proteins on flat surfaces 105 4.1. Overview 106 4.2. Results and discussion 106 4.2.1. PtBMA film characterization 106 4.2.2. Adsorption and covalent binding of selected HIgG on PtBMA surface 110 4.2.2.1. X-ray photoelectron spectroscopy analyses 110 4.2.2.2. Ellipsometry analysis 114 4.2.2.3. AFM analysis 116 4.3. Conclusion 119
Chapter 5. Advantage of immobilization of proteins in microchannels 121 5.1. Overview 122 5.2. Results and discussion 123 5.2.1. Characterization of poly(methyl methacrylate) polymeric films 123 5.2.2. Fabrication of microstructures in Au-deposited PMMA films 123 5.2.3. Impact of molecular descriptors on protein adsorption on microstructures 130 5.2.4. Characterization of thickness of polymeric films and attached proteins 134 5.2.5. Protein adsorption in PMMA-based channels and on native PMMA films 137 5.2.6. Characterization of adsorption properties of selected proteins 139 5.3. Conclusion 142
xv Chapter 6. Control of self-assembly of actin filaments for dynamic microdevices 144 6.1. Overview 145 6.2. Results and discussion 146 6.2.1. Polymeric surface characterization 146 6.2.2. Effectiveness and stability of G-actin self-assembly 146 6.2.2.1. Adsorption and self-assembly of G-actin on selected polymeric surfaces 146 6.2.2.2. Evaluation of covalent bonding of G-actin on selected polymeric surfaces 149 6.2.2.3. Covalent bonding and self-assembly of G-actin on selected polymeric surfaces 150 6.2.3. Alignment of self-assembled actin filaments along fabricated microstructures 151 6.2.4. Fabrication of electrostatically self-assembled actin filaments bundles 152 6.3. Conclusion 158
Chapter 7. Characterization of potential ATP, MreB and FtsA producers 160 7.1. Overview 161 7.2. Results and discussion 162 7.2.1. Phenotypic and chemotaxonomic classification 162 7.2.1.1. Gram-negative marine bacteria belonging to the genera Sulfitobacter and Marinobacter 162 7.2.1.1.1. Phenotypic and chemotaxonomic properties of Sulfitobacter delicatus 162 7.2.1.1.2. Phenotypic and chemotaxonomic properties of Sulfitobacter dubius 165 7.2.1.1.3. Phenotypic and chemotaxonomic properties of Marinobacter excellens 166 7.2.1.2. Gram-positive bacteria belonging to the genus Planococcus 168 7.2.1.2.1. Phenotypic and chemotaxonomic properties of Planococcus maritimus 168
xvi 7.2.2. Genotypic and phylogenetic characterization 173 7.2.2.1. Gram-negative marine bacteria belonging to the genera Sulfitobacter and Marinobacter 173 7.2.2.1.1. Genotypic and phylogenetic characterization of Sulfitobacter delicatus and Sulfitobacter dubius 173 7.2.2.1.2. Genotypic and phylogenetic characterization of Marinobacter excellens 175 7.2.2.2. Gram-positive bacteria belonging to the genus Planococcus 177 7.2.2.2.1. Genotypic and phylogenetic characterization of Planococcus maritimus 177 7.3. Conclusion 179 7.3.1. Classification of gram-negative marine isolates 179 7.3.2. Classification of gram-positive marine isolates 180
Chapter 8. Characterization of ATPases activities of marine bacteria 182 8.1. Overview 183 8.2. Results and discussion 184 8.2.1. Levels of ATP detected in heterotrophic bacteria of different taxa 184 8.2.2. Pattern of bacterial growth on surfaces 186 8.2.3. Effect of polymeric surfaces on intracellular ATP generation 188 8.2.4. Variation in extracellular ATP generation 189
8.2.5. AFM investigation of bacterial surface ultrastructure 190 8.3. Conclusion 196
Chapter 9. Evaluation of MreB, FtsA proteins and actin 198 9.1. Overview 199 9.2. Results and discussion 200 9.2.1. Comparison/Evaluation of predicted physicochemical properties of MreB proteins of selected bacterial taxa and actin 200 9.2.1.1. Stability of MreB proteins and actin 200 9.2.1.2. Isoelectric point (pI) and grand average of hydropathicity (GRAVY) of MreBs and actin 203
xvii 9.2.1.3. Phylogenetic relationships of MreB producers 205 9.2.2. Comparison/Evaluation of predicted physicochemical properties of FtsA proteins of selected bacterial taxa and actin 209 9.2.2.1. Stability of FtsA proteins and actin 209 9.2.2.2. Isoelectric point (pI) and grand average of hydropathicity (GRAVY) of FtsAs and actin 213 9.2.2.3. Phylogenetic relationships of FtsA producers 215 9.3. Conclusion 218
Chapter 10. Conclusions and further work 219 10.1. Conclusions 220 10.1.1. Overview 220 10.1.2. Protein immobilization in „combinatorialized‟ micro/nano-channels 220 10.1.3. Controlled self-assembly of actin filaments along microchannels in a continuous-flow system 221 10.1.3.1. Search for bacterial ATP producers to be used as replacements for the energy source in microdevices 222 10.1.3.2. Evaluation of prokaryotic actin-related proteins, MreB and FtsA, as possible replacements for eukaryotic actin 223 10.2. Future work 224 10.2.1. Advancements of surface modification 224 10.2.2. Incorporation of ATP-producers into microdevices 224 10.2.3. Study of MreB and FtsA proteins in vitro 224
List of References 225
xviii LIST OF ABBREVIATIONS
ABP Actin-binding protein AChE Acetylcholinesterase ADP Adenosine diphosphate AFM Atomic force microscopy AI Aliphatic index AIEC Adherent invasive Escherichia coli Arp 2/3 Actin-related protein 2/3 complex ATP Adenosine triphosphate ATPase Adenosine triphosphatase BIONJ An advanced version of the neighbor joining (NJ) algorithm BLAST Basic local alignment search tool BSA Bovine serum albumin BW Biological warfare Cc Critical concentration CFB Cytophaga–Flavobacterium–Bacteroides CIP Collection of the Pasteur Institute DEP Dielectrophoresis DNA Deoxyribonucleic acid DSM German Collection of Microorganisms DTT Dithiothreitol ECM Extracellular matrix EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EHD Electrohydrodynamics Ena/VASP Enabled/vasodilator-stimulated phosphoprotein EPEC Enteropathogenic Escherichia coli ESI-MS Electrospray ionization mass spectrometry F-actin Filamentous actin Fn Fibronectin G-actin Globular (or monomeric) actin
xix GLS Gelsolin GLUT Glucose transporter GRAVY Grand average of hydropathicity GTP Guanosine triphosphate HBP Heparin-binding peptide HDMS Hexamethyldisilazane HGF Hepatocyte growth factor HIgG Human immunoglobulin G HMM Heavy meromyosin HSA Human serum albumin HPAEC Human pulmonary artery endothelial cell Hsp Heat shock protein iDEP Insulator-based DEP IMF Ion motive force II Instability index KMM Collection of Marine Microorganisms LOC Lab-on-a-Chip LPS Lipopolysaccharide LYZ Lysozyme MAP Multiphoton absorption polymerization MCJ Multicellular junction MCM Mini-chromosome maintenance MF Motive force MHD Magnetohydrodynamics ML Maximum-likelihood MP Maximum-parsimony MTS Membrane targeting sequence NC Nitrocellulose NCIMB National Collection of Industrial and Marine Bacteria NJ Neighbor-joining OP Organophosphate
xx OTS Octadecyltrichlorosilane PAG Photoacid generator PARP Prokaryotic actin-related protein PBS Phosphate buffered saline PC Polycarbonate PCR Polymerase chain reaction PDB Protein data bank pDEP Positive dielectrophoresis PDMS Polydimethylsiloxane PE Polyethylene PETG Poly(ethylene terephthalate glycol) PGMEA Propylene glycol methyl ether acetate PHYLIP Phylogeny Inference Package pI Isoelectric point PMF Proton motive force PMMA Poly(methyl methacrylate) PS Polystyrene PSMA Poly(styrene-maleic acid) PtBMA Poly(tert-butyl methacrylate) rRNA Ribosomal ribonucleic acid RSA Random sequential adsorption RT Room temperature PVC Poly(vinyl chloride) SAM Self-assembled monolayer SCOEW Single-sided continuous optoelectrowetting SCS Specialty coating systems SCVs Small-colony variants SE Secondary electron SEM Scanning electron microscopy SERS Surface-enhanced Raman spectroscopy Si Silicon
xxi SMA Shape memory alloy STR Short tandem repeat Sulfo-NHS N-hydroxysulfosuccinimide TCA Trichloroacetic acid TEA Trapezoidal electrode array Tg Thyroglobulin THF Tetrahydrofurane TIGER Triangulation identification for genetic evaluation of risks TTF Triphenylsulfonium triftalate UV Ultraviolet VASP Vasodilator-stimulated phosphoprotein VR Virtual reality WD Working distance XPS X-ray photoelectron spectroscopy
xxii LIST OF TABLES
Table 1. Strains and environmental (marine) bacterial isolates used in the 99 study. Table 2. Atomic concentration ratios (determined by XPS) obtained for 108 adsorbed and covalently immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers on activated P(tBMA) surfaces. Table 3. Elemental compositions (determined by XPS) obtained for 110 adsorbed and covalently immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers on activated P(tBMA) surfaces. Table 4. Ellipsometric measurements obtained for adsorbed and covalently 114 immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers on activated P(tBuMA) surfaces. Table 5. Ellipsometric measurements of thicknesses of adsorbed proteins 137 and correspondent PMMA polymeric films. Table 6. Characteristics of selected proteins. 140 Table 7. Characteristics that differentiate Sulfitobacter delicatus KMM 164 3584T and Sulfitobacter dubius KMM 3554T from phylogenetically related species. Table 8. Characteristics that differentiate Marinobacter excellens from 167 phylogenetically related species. Table 9. Differential phenotypic characteristics of Planococcus maritimus 170 and other species of the genera Planococcus and Planomicrobium. Table 10. Levels of extracellular adenosine triphosphate (ATP) detected in 185 heterotrophic bacteria of different taxa. Table 11. Comparison of theoretical stability parameters (AI, II and half- 202 life) of MreB proteins of γ-Proteobacteria (1), α-Proteobacteria (2), Firmicutes (3), Thermotogae (4) and rabbit actin (5).
xxiii Table 12. Comparison of theoretical pI and GRAVY of MreB proteins of γ- 205 Proteobacteria (1), α-Proteobacteria (2), Firmicutes (3), Thermotogae (4) and rabbit actin (5). Table 13. Comparison of theoretical stability parameters (AI, II and half- 211 life) of FtsA proteins of γ-Proteobacteria (1), α-Proteobacteria (2), CFB group (3), Firmicutes (4), Thermotogae (5) and rabbit actin (6). Table 14. Comparison of theoretical pI and GRAVY of FtsA proteins of γ- 214 Proteobacteria (1), α-Proteobacteria (2), CFB group (3), Firmicutes (4), Thermotogae (5) and rabbit actin (6).
xxiv LIST OF FIGURES
Figure 1. Representative surface topography of fluorescence images of human 109 immunoglobulin (HIgG) adsorbed (top, left) and covalently immobilized (top, right) and lysozyme (LYZ) adsorbed (bottom, left) and covalently immobilized (bottom, right) on UV-irradiated PtBMA surfaces. Similar images were obtained in different regions of at least two different samples. Figure 2. XPS spectra of PtBMA+COOH surfaces: (a) typical C1s; (b) high- 111 resolution N 1s spectra of samples „activated‟ by treatment with EDC and NHS; and (c) sample following covalent protein attachment; (d) high-resolution S 2p spectra of samples „activated‟ by treatment with EDC and NHS; and (e) of samples following covalent protein attachment. Figure 3. Representative surface topography images and their corresponding 116 line profile analyses of human immunoglobulin (HIgG) adsorbed (top) and covalently immobilized (bottom) on UV-irradiated PtBMA surfaces. Similar images were obtained in different regions of at least two different samples. Figure 4. Representative surface topography images and their corresponding 117 line profile analyses of lysozyme (LYZ) adsorbed (top) and covalently immobilized (bottom) on the UV-irradiated PtBMA surface. Figure 5. Fabrication of micro/nano-structures for protein arrays using 124 microablation and directed deposition. Figure 6. AFM mapping of the ablated microchannels. 125 Figure 7. AFM topographical (top left) and lateral force (top right) image of a 126 channel fabricated via the ablation of a 30 nm Au layer on top of PMMA. Figure 8. Possible pyrolysis pathways of PMMA localized in micro-regions 128 leading to the observed lateral distribution of hydrophobicities.
xxv Figure 9. General concept of the probing of molecular surface of proteins. 129 Figure 10. Modulation of the amplification of protein adsorption in 132 micro/nano-channels vs. the molecular surfaces of the respective protein. Figure 11. Correlation between nanothickness and refractive index of PMMA 134 on glass surface treated with HMDS. Figure 12. Correlation between nanothickness and refractive index of HSA in 136 double nanolayered sandwich of HSA/PMMA on glass-surface treated with HMDS. Figure 13. Protein adsorption in microstructured PMMA surface. 138 Figure 14. Protein adsorption in the channels of thin gold layer deposited on a 139 poly(methyl methacrylate) film and on poly(methyl methacrylate) films. Figure 15. Adsorption and polymerization of F-actin (23 mM) after 1.5 h in the 147 continuous flow with the flow rate of 0.06 mL min–1 on polymeric surfaces: (A) NC, (B) PSMA, (C) PMMA (exposed), (D) P(tBuMA) (exposed).
Figure 16. Estimation of the working buffer viscosity with and without BaSO4 149 (108 mM). Figure 17. Covalent bonding and polymerization of F-actin (23 mM) after 1.5 h 150 in the continuous flow with the flow rate of 0.06 mL min-1 on polymeric surfaces: (A) PSMA, (B) PMMA (exposed), (C) P(tBuMA) (exposed). Figure 18. Binding of self-assembled F-actin (23 nM) on functionalized PSMA 152 polymeric surfaces. Figure 19. Fluorescence images of (A) 2-μm actin filaments (23 nM) with their 154 barbed ends blocked by gelsolin; and (B) their bundles condensed with Ba2+ (108 mM) during 45 min. Fluorescence images of electrostatically condensed and aligned actin-filament bundles assembled from 2-μm actin filaments (23 nM) (C) after 1.5 h; (D) after 3 h; and intact F-actin filaments: after 1.5 h (E) and 3 h (F) in
xxvi the continuous flow system with the flow rate of 0.06 mL min-1. Figure 20. SEM images of F-actin/gelsolin bundles formed from 156 electrostatically condensed F-actin filaments. Figure 21. Translocation of the antiHMM–HMM bead along the bundle formed 158 from 2-μm-actin Alexa 488-phalloidin–labeled filaments (23 nM) and condensed with Ba2+ (108 mM). Figure 22. High-resolution AFM topographical images of Planococcus 172 maritimus F 90 cells and a close-up of the area on the cell surface (non-contact mode, top) revealing dark spots/pores. Figure 23. Phylogenetic position of Sulfitobacter delicatus KMM 3584T and 174 Sulfitobacter dubius KMM 3554T according to 16S rRNA gene sequence analysis. Figure 24. Phylogenetic position of Marinobacter excellens according to 16S 176 rRNA gene sequence analysis. Figure 25. Phylogenetic position of Planococcus maritimus KMM 3738 based 178 on 16S rRNA gene sequence. Figure 26. Kinetics of adenosine triphosphate (ATP) production by 186 Sulfitobacter mediterraneus ATCC 700856T during attachment on poly (tert-butyl methacrylate) (PtBMA) and mica. Figure 27. Kinetics of ATP production by Planococcus maritimus F 90 during 187 attachment on PtBMA and mica. Figure 28. High-resolution atomic-force microscopy (AFM) topographical 191 images of Staleya guttiformis DSM 11458T cells and a close-up of an area on the cell surface revealing dark spots/porous features. Figure 29. AFM of cells of Formosa algae KMM 3553T. 193 Figure 30. High-resolution AFM topographical images of Marinobacter 194 excellens KMM 3809T cells and a close-up of the area on the cell surface revealing dark spots/porous features. Figure 31. Protein neighbor-joining phylogenetic tree shown is based on MreB 208 sequences from heterotrophic bacteria using Thermotoga maritima as outgroup.
xxvii Figure 32. Protein neighbor-joining phylogenetic tree shown is based on FtsA 217 sequences from heterotrophic bacteria using Thermotoga maritima as outgroup.
LIST OF SCHEMES
Scheme 1. Reaction scheme for the formation of sulfo-N-hydroxysuccinimide 107 (sulfo-NHS) activated poly(tert-butyl methacrylate) (PtBMA).
xxviii
CHAPTER 1 INTRODUCTION
1 1.1. Overview
Protein-based microdevices are the focus of intensive research (Rajamani and Sayre, 2011, Zavgorodniy et al., 2010, Lelyveld et al., 2010, Sankaran et al., 2011, Li et al., 2011a, Campbell et al., 2011). In respect of the rapidity and cost of biosensing, protein-based microdevices offer an attractive alternative to existing methods allowing rapid (Chou et al., 2010), efficient (Chandra et al., 2011, Doerr, 2010), and quantitative (Washburn et al., 2010) protein detection. For example, the detection of molecularly different proteins, e.g., biomarkers of diseases (Natesan and Ulrich, 2010, Boja et al., 2011) in multiplex protein-based microdevices as well as in the rapid detection of pathogens (West et al., 2009, Laue and Bannert, 2010) for ensuring food safety (Mandal et al., 2011) are the areas of potential application of motor protein- based microdevices. Although methods for the construction of protein-based microdevices for proteomic (Treitz et al., 2008, Cosnier et al., 2009, Kotz et al., 2010), and immunoassay (Bremer et al., 2009, Li et al., 2011, Chiriaco et al., 2011) analyses have been developed, the manufacturing of new microdevices remains expensive (Shim et al., 2011) due to the high cost of materials and fabrication processes (Yan et al., 2011, Lee et al., 2011). Novel approaches to the design of surfaces are required. These need to allow for the minimization of the fabrication costs via efficient amplification and spatial immobilization of multiplex proteins so that the bioactivity of such proteins (e.g., microarrays, protein-based microdevices) can be retained. The surface design posed considerable technological challenges arising from (i) the large variety of proteins that needed to be immobilized on the surface; (ii) increased density of laterally defined areas with precise immobilization for specific proteins, required by high-throughput analysis; and (iii) the ever present complexities of protein-surface and protein-protein interactions, reflected in complex fabrication and function, respectively. Most technologies for fabrication of protein microdevices have to ensure the confinement of molecularly different proteins in laterally-defined, either flat 2D; or profiled ―2D+‖ micro-areas. The profiled features have the advantage of minimization of inter-spot contamination and the drawback of more difficult access
2 of the recognition of biomolecules (e.g., antigens for antibody microarrays) in a micro- confined area. An optimal shallow profile feature would take advantage of the benefits of the former and mitigate the latter (Han and Yoon, 2009). Among the enabling technologies for the above biomolecule micropatterning methods, laser beams are capable, depending on the exposure energy and sensitivity or absorbance of the exposed material, of enabling both photolithography (Wang et al., 2009) and photo-assisted etching (Gudymovich and Vanifat'eva, 2009). Also, focused laser beams can solve, in principle, a critical fabrication and operating problem of the protein chips better than most other alternative methods, i.e. the controlled and confined variation of the surface properties of the micro-areas where different proteins are deposited (Uemura et al., 2010, Park and Cho, 2011). Proteins present extremely complex surfaces (e.g., hydrothilic or hydrophobic; acidic or basic; neutral or charged) that interact with the surface via electrostatic forces, hydrogen-bonding, van der Waals or hydrophobic interactions (Heo et al., 2010, Pleskova et al., 2011, Gokarn et al., 2011). This variety of molecular surface – microdevice surface interactions, can lead to large variations in protein surface concentrations as well as the possibility of important changes to protein bioactivity and its denaturation (Hnaien et al., 2011, Alvarez et al., 2011). Building on this, advanced biomolecule immobilization on the surfaces has to be developed. One logical approach would result in the spatial immobilization of multiplexed proteins in micro/nano-channels. This approach will not only require a very accurate control of the surface properties at the micro- and/or nano -scale level, but also a iprior knowledge regarding the nature of the deposited proteins and their interactions with surfaces. Employment of molecular motors in microdevice construction received a significant amount of attention by the following researchers (Agarwal and Hess, 2010, Fujita et al., 2011, Fiasconaro et al., 2009). The motor proteins possess many of the characteristics required to power nanomachines, e.g., generation of force (Linari et al., 2009), and ability to transport specific cargoes over appropriate substrates (Takatsuki et al., 2010). The rate of motor protein action can be controlled by the direct application of in vitro motility assays (Valentine et al., 2006). While motility assays are easily reproducible, to achieve directional motility is not a simple task. This
3 is due to the fact that motor proteins tend to attach onto the target surfaces at random locations and consequently move in random directions. A significant number of studies have focused on solving this problem by modification of surface topography (van der Meer et al., 2010) and/or surface chemistry (Park et al., 2010b) to fabricate microstructures of certain geometries for directional movement of molecular motors. While the latter direction has emerged quite recently, the former has been intensively explored over several years (Takahashi et al., 2011, Chen et al., 2011). A number of different methodologies have been applied to align the motility of filaments through a variety of techniques including protein (myosin) guiding (Butt et al., 2009), magnetic field (Kaur et al., 2010), electric field (Wigge et al., 2010), UV lithography (Yamamoto et al., 2008). However, these techniques are not suitable for the fabrication of aligned proteins (actin) tracks, which can support unidirectional bead translocation in vitro, due to the lack of precise control over them at the level of either individual or bundled linear assemblies. In case of microdevices based on protein molecular motors, there are a few other challenging aspects that have to be resolved. One of these aspects is the protein lifetime and in particular the lifetime of actin tracks which would sustain robust microdevice functioning (Phung et al., 2011, Liu et al., 2010, Pagan and Griebenow, 2010, Oguchi et al., 2010). The actin prokaryotic homologue MreB (Bean and Amann, 2008, Ikeuchi et al., 1990) was found to be a more mechanically robust protein (Popp et al., 2010b, Shaevitz and Gitai, 2010). It is capable of assembling filaments across a wide range of temperatures (Bean and Amann, 2008), pH values (Cabeen and Jacobs- Wagner, 2010), and ionic concentrations (Popp et al., 2010b), and hence MreB proteins may be useful and replace actin. Functional properties of motor proteins are directly correlated with the energy suppliers, namely, ATP (Oiwa et al., 1990). Therefore, a reusable source of ATP such as ATP-producing bacteria may be an attractive alternative.
4 1.2. Aim of the study
The aim of this study was to develop an approach in the design of advanced surfaces which can be used for construction of protein-based microdevices, the surfaces that would be suitable for efficient multiplexed spatial immobilization of proteins which will be able to retain their bioactivity. The evaluation of the suitability of the employment of bacterial ATP producers and prokaryotic actin-related proteins as replacements for the energy source and eukaryotic actin, respectively, in construction of the next generation of microdevices was also anticipated.
1.3. Organization of the thesis
Chapter 2 starts with a general overview of microdevices followed by an outline of the various types of biosensors. It looks/reveals several critical aspects of microfluidic systems before discussing how they can be controlled. The chapter also explores the prospect of applications of both prokaryotic and eukaryotic molecular motor proteins and includes an evolutionary/phylogenetic comparison of MreB and FtsA homologues of eukaryotic actin. Since an understanding of the functions of bacterial molecular motors is essential for incorporation of prokaryotic motor proteins in biosensors, the chapter ends by describing the most useful functions, from the microdevice design point of view. The following chapters are based on results of this study already published in peer-reviewed journals, with the exception of Chapter 9. Chapter 3 contains a description of the methods used for: microdevice surface design and fabrication; the study of protein-surface interactions; protein handling and immobilization. It continues with a description of the methods used in bacterial taxonomy, namely, phenotypic, chemotaxonomic, genotypic, and phylogenetic analyses. It ends with the methods used to assess MreB and FtsA proteins. While Chapter 4 covers immobilization of proteins on flat surfaces, Chapter 5 presents comparative immobilization of proteins in micro/nano structures. The latter describes a newly developed approach for surface design applicable to microdevices.
5 The approach is based on spatial multiplex immobilization of proteins in micro/nano- channels fabricated via laser ablation. Chapter 6 is focused on an approach to the design of the surfaces of microdevices based on self-assembled actin bundles as model protein structures that retain their bioactivity, i.e., they can support unidirectional movement of cargo particles. Chapter 7 describes phenotypic, chemotaxonomic, genotypic and phylogenetic properties of potential ATP, MreB, and FtsA producers. Chapter 8 presents a characterization of ATP motor activities discovered in environmental bacteria. Chapter 9 describes a comparative analysis and evaluation of MreB and FtsA proteins detected in selected bacterial taxa. The final chapter of the thesis draws conclusions from the results presented in this thesis and discusses future work.
6
CHAPTER 2
LITERATURE REVIEW
7 2.1. Overview
This chapter consists of five subsections. It starts with an overview of the concept and benefits of biosensors (subsection 2.2.), and is followed by a description of different types of biosensors (subsection 2.3.). As the main goal of this thesis is to develop an approach to the design of surfaces for biosensors based on motor proteins (see chapter 6), subsection 2.4. is devoted to microfluidic devices. This subsection covers the general aspects of microfluidic devices highlighting the importance of surface properties, geometry and fluid properties (see subsections 2.4.3.1.–2.4.3.4. and chapters 4–6). Subsection 2.4. continues with an overview of the types of microfluidic systems (see subsection 2.4.4.) and ends with a description of methods of control of microfluidic devices (see subsection 2.4.5.). Subsection 2.5. describes the concept of protein molecular motors including the evolutionary/phylogeny of prokaryotic actin- related proteins, namely, MreB and FtsA (see subsections 2.5.2.–2.5.4. and chapters 6–9). Classification of molecular motors based on the mode of operation is given in subsection 2.5.5. Also, the chapter describes native functions of molecular motor proteins, which can be used for the development of surfaces for biosensors based on motor proteins (see subsection 2.5.6. and chapters 6, 8 and 9).
2.2. Concept and benefits of biosensors
The unification of science and technology as a distinct nanotechnology area has been acclaimed as one of the most important events in the history of science and has deeply influenced ideas on the manipulation of molecules at the micro/nano-scale level (Chen et al., 2011). Nanotechnology can be best defined as the ability to understand and control matter of a small size (Ramsden, 2009). The growth of nanotechnology has led to the fabrication of advanced microdevices (Jiang et al., 2011, Reedy et al., 2011). In general, it is accepted that an analytical microdevice composed of a biological sensing component (e.g., bacteria, spores, proteins), a physico-chemical signal transducer (discussed in the next subsection) and a signal processing unit (Wildeboer et al., 2010, Lee et al., 2010a), can be considered a
8 biosensor. The common view is that the role of a sensing element is to recognize and react with a target analyte; the transducer serves as a converter, transferring a countable output signal from analyte concentration. The history of biosensor creation dates back to the early 1960s when Leland C. Clark described oxygen electrode. Continued technological development of this type of biosensor led to the manufacture of new generations of biosensors for different applications. Biosensors have been developed for a number of applications ranging from biomedical monitoring (Bachand et al., 2009) to the detection of biological warfare (Gooding, 2006). Society can benefit in multiple ways from nanotechnologically improved products. For example, glucose biosensors are being used in glucose monitoring technology (Chu et al., 2009), in the food industry (Bordonaba and Terry, 2009), and other biotechnological areas (Gramsbergen et al., 2003). The construction of a glucose sensor was initially based on an amperometric enzyme electrode (Pandey et al., 1992). However, during the past decades, much new research on the improvement of biosensor performance has been done (Ducloux et al., 2010). Li et al. (2007) have reviewed the development of implantable electrochemical devices for the management of diabetes mellitus. Their results indicated that sensors suffer from aging issues that need to be overcome.
Lyandres et al. (2008) have demonstrated that there was potential for detection of glucose using surface-enhanced Raman spectroscopy (SERS). This optical technique has been successfully employed for real-time analysis of diverse chemical substances (Rae and Khan, 2010). Moreover, biosensors based on SERS have been incorporated into a virtual reality (VR) image-guided surgery unit for accurate targeting of surgical margins of cancer cells (Reisner et al., 2007). However, there are two major drawbacks to this system with regard to the precise positioning of the Raman probe and steady-state scanning. It is believed that the ability to assess the health risk of different chemicals is an essential ingredient for preventing life-threatening illnesses (Kovacic and Somanathan, 2009). Much of the commercial success of environmental biosensors is attributed to a growing human demand for advanced environmental control. These sensors offer a simple way of detecting many different compounds. In recent years, significant progress has been made in constructing cholinesterase (AChE) biosensors
9 that selectively react with hazardous toxins (Pohanka et al., 2009), organophosphate (OP) pesticides (Istamboulie et al., 2010), and other compounds (Woznica et al.,
2010). A bacterium-based NO2 biosensor that has been applied to the analysis of freshwater, marine and oxic-anoxic wastewater utilizes Stenotrophomonas nitritireducens coupled to an electrochemical NO2 sensor to detect nitrite (Nielsen et al., 2004). Although preferred bacterial species that can be used as bioelements were clearly identified, the biosensor possessed disadvantages such as a narrow operating temperature and salinity ranges due to the non-psychrophilic physiology of microbial candidates. Nowadays, progress has been made in building optical biosensors for detection of enteric pathogens such as Escherichia coli (Day et al., 2010). The development of biosensors based on linear molecular motors (see subsection 2.5.5.2.) allows not only detection of analytes but also their transport to specific compartments of biosensors, for example, detection units. Of two ways of biosensor miniaturization, such as microfluidic and ―smart dust‖ approaches, the former was used by Martinez-Neira et al. (2005) for the construction of a biosensor based on linear motors (actin and myosin). In so doing, actin and myosin were used as biosensors for the detection of toxic cations. The latter approach was employed by Fischer (2009) for building hybrid biosensors based on linear (kinesin and tubulin) molecular motors ; the energy was provided by caged ATP. Despite unification of transport and energy supply systems in one microdevice, signal accuracy and strength remained key issues. Since the terrorist attacks on America in 2001, significant public concern regarding the possibility of a large-scale bioterrorism event has resulted in the development of technology for rapid detection and identification of biological warfare (BW). To accomplish this goal, Hofstadler et al. (Hofstadler et al., 2005) developed TIGER (Triangulation Identification for Genetic Evaluation of Risks); biosensors based on electrospray mass spectrometric (ESI-MS) detection of nucleic acids. Although the TIGER biosensor can be used for identification of various groups of pathogenic microorganisms, it does not allow real-time analysis due to time- consuming procedures, e.g. incubation of microorganisms, isolation and purification of genomic DNA and purification of PCR products.
10 2.3. Types of biosensors
Biosensors can be classified into various groups according to the biological recognition mechanisms. On the basis of the signal transduction, biosensors may be divided into four groups: electrochemical, thermal, mass-sensitive and optical sensors. Of these biosensors, the most publicized are electrochemical, which include amperometric, potentiometric and conductimetric. Electrochemical biosensors can respond to the concentration of analytes as small as 10-6 M (Turek et al., 2007). This detection limit is sufficient for measurement of carbohydrates (glucose, galactose and fructose), polyphenols, amino acids (glutamate), metabolites (urea and lactate), cholesterol, and drugs. However, recognition of analytes in the 10-9 M concentration range remains a problem (Jubete et al., 2008). Thus, this type of biosensor is not sufficiently sensitive and accurate for the detection of hormones and other serum components. In 2000, Romani developed the first amperometric biosensor with a measurement range of 20 to 80 µM polyphenols (Romani et al., 2000). This sensor could be used for purposes such as the screening of plant materials for polyphenols. A major drawback of the amperometric kind of sensor is its limitation in the analysis of biological samples in which endogenous components are present. Chronopotentiometric ion biosensor with a detection range of 0.1–1 µg/mL for avidin has been developed by Xu and Bakker (2009). However, this type of biosensor is dependent on the charge density of the analyte. Therefore, the potential problem is the inability of potentiometric biosensors to analyze complex biological solutions. Though conductimetric biosensors measure only small changes in electrical resistance, they may have applications in the qualititative analysis of chemicals. Thermal biosensors can be applied to measuring changes in heat and can be used for detection of different analytes. However, sensitivity of the biosensor remains a major problem and is associated with the dissipation of heat to the surroundings. Mass- sensitive biosensors provide a way to detect changes in mass. In this case, optical and electrical methods can be used to measure the deflection of cantilevers. Compared to optical techniques, piezo-resistive cantilevers are unaffected by the optical artefacts and can operate in non-transparent samples.
11 Optical biosensors offer considerable promise for obtaining optical information in a highly selective, fast and reproducible manner. Many recent advances in optical technology can be traced to the development of different techniques such as fluorescence, ellipsometry, bioluminescence, chemiluminescence, phosphorescence, rotation and polarization for the measurement of analytes. The advantage of using the fluorescence method is that continuous images from fluorescent groups can be produced. In addition to providing the fluorescence required to directly observe biomolecules, the fluorescence approach also allows for quantitative analysis of molecular motion. The main drawback of this approach is photodestruction of the biomolecule and of other moieties of the biomolecule such as enzyme sites. This leads to harmful effects on the performance of the biosensor, apart from decreasing the reproducibility of results. A number of in vitro motility experiments with assays containing prokaryotic actin- and tubulin-related proteins were reported in the 2000s, which formed the foundation for device-oriented research in this area. According to the biological recognition mechanism, biosensors can be classified into biocatalytic, bioaffinity and whole-cell systems. In 1962, Clark developed the first biocatalytic biosensor based on a layer of the glucose oxidase enzyme ―GOD‖ which was deposited close to the surface of the oxygen electrode (Clark and Lyons, 1962). Glucose sensors may be used for self-monitoring of the concentration of glucose in drops of fresh blood. A measurement range of 1x10-6M to 1.5x10-3M for glucose was reported (Ding et al., 2010). The use of enzymes in biosensing opens the door for a large number of environmental applications. Enzymatic biosensors have been developed for the detection of environmental pollutants, such as phenols (Li et al., 2010), cyanide (Mak et al., 2005), nitrate (Quan et al., 2005), and uranyl (Liu et al., 2007). However, this kind of biosensor suffers from limitations related to the necessity of using artificial mediators (Kosela et al., 2002), and substrates (Ges and Baudenbacher, 2010). Bioaffinity sensors, such as nucleic acid and immunosensors, offer rapidity, simplicity, and selectivity. It has been shown that DNA recognition layers can be fabricated for frequent utilization (Wang, 2000). Owing to its selectivity, a DNA-based biosensor can be used for targeting of particular bacterial species (Ng et al., 2008). A sensitive enzyme immunosensor for
12 the detection of Vibrio parahaemolyticus, the food-borne pathogen, with a detection limit of 6.9 x 103 cfu/ml has been recently developed by Zhao et al. (2010). Although the main disadvantage of immunosensors is their stability, this biosensor exhibited only slightly decreased firmness after a week of storage. For whole cell biosensors, use of living prokaryotic and eukaryotic cells can be beneficial. In the middle of the 1970s, Divies (1975) suggested that a bacterial cell could be employed as the sensing element in microbial electrodes for the estimation of alcohol concentration from the target analyte. Recently, Elad et al. (2008) have developed a bioluminescence-based bacterial sensor for the detection of toxicants. These authors indicated that bacterial bioreporter cells were capable of recognizing analytes within a 30-minute detection time. Even though there is a major drawback in using bacterial bioreporters, such as the dependence of microbial productivity on the physiological state, some bioreporters have the potential for becoming a part of biosensors.
2.4. Microfluidic devices
2.4.1. Overview
The use of microfluidic technology is crucial for the utilization of biological recognition and/or transporting elements, such as molecular motors, in biosensors. Microfluidic devices can be constructed in three formats: microarray, droplet and continuous-flow systems. Furthermore, the growing demand for miniaturization of large-scale devices along with integration of the sample preparation steps with biosensing procedures has led to the development of ―Lab-on-a-C hip‖ (LOC) systems. In doing so, the proposed prototype of biodevice has a high potential for becoming an essential part of a future LOC. Since the function of this biological motor-based cargo delivery system mainly depends on a microfluidic environment, the overview of available formats, droplet and continuous-flow, is given below. It is important to note that the microarray approach has been used in this study only for the specific purpose
13 of identifying microorganisms. Owing to their distinct applications, DNA as well as Biolog phenotype microarrays are briefly mentioned in chapter 3.
2.4.2. Advantages of microfluidic devices
Microfluidic technology is an essential instrument-granting transformation to biosensing in the scale range from pico-to micrometer level. Success in this technology results from the fact that fluid can be precisely controlled at a micro level. Early on, scientists realized that the commercialization of microdevices would lead to financial rewards and so took the first steps to the development of applications. The ubiquitousness of microfluidics has been witnessed in its utilization in different applications, with specialists in nanotechnology expanding the borders. Nowadays, with applications including printing, biomedical analysis and defence, the biodevice has become the centre of profound scientific attention. The story of microfluidics began nearly half a century ago with Mack J. Fulwyler‘s report of his construction of a prototype device (Fulwyler, 1965). His prototype device consisted of a volume sensor and an ink-writing oscillograph (Robinson, 2005). Ink-jet type printing in its mature form has evolved from microfluidic technology. Microfluidics has enabled clinical trials to deliver very accurate results. The microfluidic device has been successfully used for targeting serum thyroglobulin (Tg), a cancer biomarker, by loosely adsorbed Immunoglobulin G (IgG); the latter was evicted from the occupied microfluidic territory by the former through competitive adsorption (Choi and Chae, 2009). Researchers emphasized that the method used allowed for the elimination of an antibody probe. However, actual clinical samples may contain a few different important markers which should be analyzed concurrently. Murphy et al. (2009) have showed that both metabolic and enzymatic markers can be detected at the same time. The author‘s findings demonstrated the possibility of a combination of competitive and non-competitive assay formats. In the case of lightweight substances, i.e., haptens, such as steroids, the way in which antibodies identify haptens in immunometric format needs to be ameliorated (Kobayashi et al., 2007). In addition to giving an overview of new approaches for
14 targeting haptens, the researchers presented an immunometric assay which was capable of detecting attomolar quantities of a model hapten. It has been found that the creation of interchanged poly(methyl methacrylate) (PMMA) – polycarbonate and nanocapillary coats in devices can help separate a mixture of amino acids (Kim et al., 2009a). Although improved separation of complex analytes was achieved, the proposed device, as noted by the authors, needed to be upgraded with a cooling element, new channels and/or optimized fluids. The applicability of a polymer microfluidic chip for biochemical analysis has been demonstrated by Yang et al. (2010). It was concluded that the unification of two methods, such as affinity chromatography and electrophoresis, was necessary for accurate measurement of biomarkers in biochemically heterogeneous samples. Despite progress in microfluidics, the need for miniaturization of microfluidic devices, e.g., for drug discovery applications still exists (Upadhyaya and Selvaganapathy, 2010) Recently, it has been found that the density of spots (cell layers) on the modified nanomembrane of a microfluidic structure can be enhanced by means of an electrical field (Upadhyaya and Selvaganapathy, 2010). The importance of shear stress in microfluidics has recently been studied by means of computational (Cioffi et al., 2010) and experimental (van der Meer et al., 2010) methods. For example, shear-stress was used to examine the interplay of either plain particles or aptamer-decorated particles and human cells in a microfluidic system by Farokhzad et al. (2005). These researchers have further developed their approach for cancer treatment (Farokhzad et al., 2006, Dhar et al., 2011). The authors have described the use of nanoparticle-aptomer bioconjugates for hunting down malignant prostate cells. As microfluidics has been extending its borders, novel approaches need to be developed. Fundamental methods for restoring various types of mammalian tissues have been reviewed by Lalan et al. (2001). A new approach to investigating implant-associated bacterial diseases has been proposed by Lee et al. (2010b). They used a microfluidic system to examine how the presence of an implant-associated diplococcus affects in vitro behavior of osteoblast cells. The researchers concluded that osteoblasts were strongly influenced by the settlement of small-colony variants (SCVs) (Singh et al., 2009), which was described as a biofilm producing phenotype of
15 Staphylococcus epidermidis (Al Laham et al., 2007). A method with potential applications in cardiovascular tissue engineering has been developed by Suzuki et al. (2010). The method allowed differentiation of multipotent cells into muscle ones by means of laminin (LM), a basement-membrane protein. By incorporating different steps of biochemical analysis performed in individual devices into one procedure carried out in a single laboratory-on-chip (LOC) system, researchers moved microfluidics forward. Remarkably, the architecture of a LOC format can be modified to meet the user‘s needs (Shaikh et al., 2005). For example, the usefulness of the multicomponent construction for the detection of lead has been demonstrated (Shaikh et al., 2005). It should be noted that a microfluidic PCR (polymerase chain reaction) can be used for detection of various clinical and environmental bacterial species. The most striking thing about the utilization of the microfluidic chip for PCR is the fact that different procedures, such as amplification and extraction of nucleic acids from suspicious samples, can be run in one device (Kim et al., 2010). Recently, a microfluidic device has been developed for forensic DNA analysis (Aboud et al., 2010). This study has focused on enabling pentameric short tandem repeat (STR) - based separation in a miniaturized microfluidic channel. The issue of chemical contamination of food motivated researchers to build devices for the screening of toxic chemicals in dairy foods. Using a microfluidic approach along with ultraviolet (UV) radiation detection, Zhai et al. (2010) have developed a strategy for detecting melamine in milk samples. Nowadays, concerns regarding the threat of uncontrolled use of biological weapons come from their increased production (Coleman and Zilinskas, 2010). In this regard, it has been demonstrated that biomotors can be successfully employed for the detection of one of the biological weapon agents, namely, Staphylococcal enterotoxin B in a microfluidic device (Soto et al., 2008). In fact, microfluidic devices based on molecular motor proteins have different potential applications (Korten et al., 2010), ranging from molecular assembly to drug delivery and biosensing (Fischer et al., 2009). These include military use. Although biomolecular motors may offer a new microfluidic
16 paradigm, a major disadvantage of their in vitro employment lies in their insufficient stability and poor longevity.
2.4.3. Critical aspects of microfluidic devices
2.4.3.1. Overview
Microfluidic devices provide opportunities for their users to manage fluid behavior at microscale level. Since the beginning of device manufacturing, substantial progress has been made in the development of high-grade microfluidic systems. In order to understand fluid behavior in real conditions for devices, one needs to know the physical aspects of fluid dynamics. As mentioned above, the success of in vitro biomolecular productivity depends on being able to provide contact for surfaces in an optimum environment.
2.4.3.2. Surface properties
Surface preparation is known to play a crucial role in the endurance of all types of microfluidic systems. Various techniques for fabricating surfaces with specific properties have been worked out. However, the choice of a method relies on the suitability of the substrate chemistry for a particular physical and/or chemical treatment and subsequent application. For example, microfluidic devices can be constructed from glass (Thiele et al., 2010, Pjescic et al., 2010), or silicon (Si) (Retterer et al., 2010, Bienvenue et al., 2010). Material features that make glass a valuable candidate substance are its optical and mechanical qualities (Bach and Neuroth, 1995, Bach and Neuroth, 1998). In fact, owing to its mechanical properties, glass remains a widely used construction material. Morever, Bach and Neuroth (1998) noted that these properties are highly dependent on the composition of glass. Like glass, quartz is considered to have outstanding optical as well as thermostable properties (Spassov et al., 2008). However, since quartz usually contains various admixtures that may affect microdevice performance, the purity of this material
17 should be evaluated by means of gamma-ray technique prior to its utilization (Komov et al., 1994). In addition to its impressive mechanical features, silicon, a quite popular electronic material, is considered to have valuable electrical features (Fukuda and Menz, 2001) as well as biocompatible (Martinez et al., 2009, Erogbogbo et al., 2010) properties. However, this substrate is known to be brittle (Fukuda and Menz, 2001) and so is not reliable. Moreover, even though non-carbon based substrates have valuable properties, they expand more than organic ones. Growth in demand for disposable devices led to the integration of new techniques for construction of microfluidic systems. Nowadays, scientific efforts directed at the utilization of organic substrates have focused on testing living tissues and synthetic polymers. The engineering of three-dimensional (3D) structures such as tissues is based on building controllable scaffolds which can support their applicable cells not only physically but also nutritionally (Chao et al., 2010, Sharifpoor et al., 2010). The success of this research depends on the degree of availability of degradable scaffolds for cells. Taylor et al. (2007) have indicated that scaffolds should be in a very close biological relationship with the cells deposited onto them. As for polymeric substrates, some of them, namely, poly(styrene-maleic acid) (PSMA), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), polystyrene (PS), poly(tert-butyl methacrylate) (PtBMA), polycarbonate (PC) and polyethylene (PE) are known to offer user-oriented physical and chemical properties that can be useful for constructing cheaper microfluidic systems. A major advantage of using polymeric materials is that native attributes of polymers can be effectively modulated by suitable methods. Broadly, these fabrication techniques may fall into the following groups: replication methods (Becker and Gartner, 2000) and direct methods (Liu et al., 2005a). The master templates can be fabricated using a variety of tools, such as micromachining (Entcheva and Bien, 2005), electroplating (Burek and Greer, 2010), embossing (Gan et al., 2010), injection moulding (Liu et al., 2009), and casting (Gitlin et al., 2009). Replication technologies can be used for the repetitive fabrication of identical devices in huge quantities. Direct technologies have been found to be useful for the construction of individual devices. One of the most widely used methods for the treatment of polymeric substrates is laser ablation. It includes the use of radiation
18 to break down polymer bonds and takes away the demolished portion of polymer from the ablated spot/pattern of the surface. The success of any ablation depends largely on optimizing its parameters, mainly energy delivery and polymer removal speed. By adjusting laser pulse (Gonzalez et al., 2007) and fluency (Amer et al., 2005), researchers have taken the first step toward minimizing the size of the heat-affected zones (HAZs) surrounding the ablation area. However, the chemical nature of the polymers has been known to play a key role in the final morphology of the channel. Pugmire et al. (2002) have studied surface properties of the channels using laser ablation under different pressures. The results showed that there was no difference between a PMMA workpiece and its native film. When poly(ethylene terephthalate glycol) (PETG), poly(vinyl chloride) (PVC), and polycarbonate (PC) ablated under different atmospheres, they showed remarkable changes in chemical composition. To make a particular pattern on polymeric surface, one can utilize the power of lithography. With techniques adopting SU-8 (an epoxy-based negative photoresist) (Gao et al., 2008) and similar resists, thick-film technology has attracted a worldwide attention in the field of microtechnology. Thus, a photosensitive epoxy (SU-8) has been explored in creation of microchannels of different depths (Edwards et al., 2000). However, SU-8 fabrication methods include time-consuming multiple step procedures (Yu et al., 2006). The studies of protein-surface interactions have demonstrated that lithographically decorated polymeric films can be used as substrates for exclusive immobilization of nonmotor (Nicolau et al., 2010b) as well as motor proteins, such as microtubules, and proteins (Turner et al., 1995). Also, see chapters 4 and 5. These describe immobilization of nonmotor proteins. See chapter 6 for immobilization of motor proteins on polymer surfaces. The successful deposition of molecular motors on favourable surfaces and control over their movements, for example, by patterning of channels for microtubule-based movement (Hiratsuka et al., 2001) is considered to be a crucial part of the microdevice fabrication process.
19 2.4.3.3. Microfluidic device geometry
Adaptation of any type of fluid in a microfluidic channel, a key part of the device, depends largely on its having an appropriate geometry. The principal role of a channel is to provide guidance for a micro-scale fluid flow to the detector. Once constructed, the system may allow accommodation of specific analytes under optimum biochemical conditions. Since there is a great range of liquids and analytes, cross- sections of microchannels should be designed to suit particular microfluidic tasks. Some fundamental aspects of microfluidic dynamics through channels have been discussed by Morini (2004). The author used the ―Obot-Jones‖ method to estimate the value of a Reynolds number for a flow-regime transition. This method was chosen from a range of channel parameters as its shape is the most crucial. A cross-sectional profile has been identified as a factor that affects the stability of condensation processes in microchannels (Wu et al., 2010). For example, application of circular channels is limited due to the lack of sharp edges (Rahmat and Hubert, 2006). Channels with elliptical profiles, which can be built by means of a thermal technique, have been found to be useful for nanofluidic flow modeling (Czaplewski et al., 2003). Also, other researchers have concluded that such a channel shape provides an advantage in stabilizing the flow (Huang et al., 2006b). Recently, attention has been drawn to the distinctive features of fluid flow through a channel with a triangular profile (Park et al., 2010a). It was noted that the local flow rate was a critical parameter for the control of solute deposition. A microchannel with a trapezoidal profile has been designed by Jindal et al. (2005). The authors used selective filling to introduce polymers onto distinctly specified areas in channels. A rectangular-shaped channel has been successfully used for alignment of bacterial cells by means of an electric force (Choi et al., 2010) . The feasibility of using rectangular silicon channels for detection of fluorescently labeled material has been demonstrated (Kutchoukov et al., 2004). Generation of various channel profiles in a single polydimethylsiloxane microfluidic device became possible with the use of multiphoton absorption polymerization (MAP) (Kumi et al., 2010). To accomplish it, the authors used a photoacid generator (PAG) instead of laser beam. Even though this
20 technique has been demonstrated to be appropriate for SU8, it cannot be employed for all materials because of their different chemistries. A microchannel system has been designed and then etched by Hirst et al. (2005) for production of aligned macromolecular assemblies. Improvement of interaction between aqueous samples and channel walls was achieved by increasing hydrophilicity of the sidewalls. Although the functionality of self-assemblies has not been demonstrated, the guided assemblies in the titanium channel have been presented. It should be noted that flow rate, a crucial parameter of the fluid flux, may change channel geometry. For example, relatively elastic PDMS channels with low aspect ratio (AR) geometry (width is much greater than depth) can become deformed easily (Gervais et al., 2006). Consequently, it may affect spatial fluid velocity distributions within a channel. Importantly, the authors suggested that deformation may be reduced by using more rigid material than PDMS (Gervais et al., 2006). In fact, Attia and Alcock (2010) have preferred using PMMA to employing elastic materials owing to the low-shrinkage and rigidity of PMMA. As a result, they created a structure that was capable of handling flow rate without leakage.
2.4.3.4. Fluid properties
A fluid has three important properties: viscosity, compressibility, and density. Of these, viscosity is considered the most critical because it reflects a force of the fluid drag. The analyte-induced viscosity of a fluid can be determined by means of measurement by the microdevice (Kamholz et al., 1999). Different analytes have been detected in T-sensor by a competitive immunoassay based on the quantitative evaluation of molecular diffusion (Hatch et al., 2001). Theoretical estimation of fluid parameters and hence prognostication of its behavior has been accomplished by using an analytical model (Kamholz et al., 1999). The transverse diffusion of flow in microfluidic channels can be analyzed by means of T-sensors (Wang et al., 2005). It was demonstrated that diffusion relied on channel geometry and dimension. Since a concentration gradient can be used to control biosensors, researchers have developed approaches such as distinctive inlet channel profiles (Yang et al., 2007), chemical
21 density differences (Kong et al., 2010), diffusive mixing (Englert et al., 2010), etc., to build up the gradient. The control of the gradient can be tested by means of computational fluid dynamics (CFD) simulations (Yang et al., 2007). The fluid interface location in the microchannel can be adjusted by manipulating the volumetric flow rates of the two fluid streams (Stiles and Fletcher, 2004). Chung et al. (2009) have recently reported evidence of viscosity ratio maximization for droplets in viscoelastic fluids. The authors assumed that it was due to stress build-up in the area between the droplets and the channel wall. D‘Avino et al. (2010) have studied the distinctive features of a single particle journeying through confined geometries. It was demonstrated that a sphere was not always cruising within a main stream. There was a possibility that it could get off the route and hence change its direction. Moreover, in the case of continuous flow, a sphere moved fast only at the start of its journey due to the primary accrual stress. Another parameter characterizing fluid behavior is compressibility. It is important to note that if the device is started up properly it may help prevent the compressibility caused by an air bubble in the microfluidic channel (Cabral and Hudson, 2006). Townsend et al. (2006) have utilized acoustic radiation to stimulate bacteria to get closer to the sensor element. The radiation needed for this procedure depended on the values of the compressibility and density of the particles in fluid. Wang et al. (2007) have presented a PDMS multilayer device for mammalian cell- based screening. To optimize density, the cells were captured by sieving, cultivated, and screened. In this process, one device was used to perform high-density screening of cytotoxic analytes. In general, the compressibility of biomolecules is affected by density. Kalinin et al. (2010) have reported the effects of interdevice bacterial cell density on the chemotactic strategy of Escherichia coli. Amazingly, the activity of the bacterium depended heavily on the balance between chemotaxis receptors to judge surrounding conditions; the relationship between receptors depended on cell density. Ghodssi et al. (2010) have conducted studies to examine microbial reactions to various stimuli. The device used analyzed biological samples of high density, for example, products of bacterial metabolites. Owing to the necessity of supplying some biosensors with
22 energy, microbial fuel cell (MFC) research has become more mature. Recently, a very small MFC device has been constructed for electric current generation (Qian et al., 2009). The authors presented evidence showing that some electricity-producing bacteria, such as Shewanella oneidensis, can be employed as bioenergy suppliers. It is noted that biofilm was suggested as being in charge of biofuel production. As biofilm was formed only on the gold anode, it seems that enhancement of cell-anode contact can shorten the device start-up. Choi et al. (2007) have constructed a system ensuring viability of high-cell density structures. The authors adapted a perfusion system with a PMMA pipe to provide fluid flow through microchannels located in microneedles. Gottschamel et al. (2009) have presented microdevices for prolonged analysis of variation in the population dynamics of fungi. The device was successfully used to assess utilization of monosaccharides by Candida albicans. Huang et al. (2007) have developed a three-tier microfluidic device for kinesin-based transport of microtubules. Decapitated kinesin was used in order to optimize its density in enclosed microstructures. However, this system lacked sufficient ATP energy.
2.4.4. Types of microfluidic devices
2.4.4.1. Overview
Nowadays, continuous and droplet-based technologies are explored to construct microfluidic systems. In continuous-flow-based systems, liquids are treated as steady flows in appropriate channels. This system can be used for certain applications, such as biomolecular transport. In droplet-based microfluidic devices, liquids are treated as discrete droplets on microarray surfaces. This system can help to improve the efficiency of screening for various analytes, such as different proteins.
2.4.4.2. Droplet system
Droplet-based microfluidic devices provide system scalability and dynamic reconfigurability. They offer a unique opportunity to support reconfiguration for faulty
23 tolerance. For example, biomolecules in devices can be reconfigured to alter their properties to go around defective cells. A number of different techniques enable droplet-based devices to scale down to handy dimensions while increasing the throughput rate and efficiency of analysis. Electrowetting and dielectrophoresis have been proven to be the most effective droplet techniques for fast control and handling of fluid dynamics on a nano/micro-meter scale. Zeng et al. (2004) have studied the operating principles of electrowetting on dielectric and dielectrophoresis. The authors demonstrated the applicability of two techniques for droplet generation and manipulation. Paik et al. (2003) have used electrowetting techniques to mix microliters of fluids. This mixing process can be configured to fit particular system needs, resulting in improved performance. A one-dimensional oscillator model has been proposed by Baret and co-workers, in which the appropriate intrinsic and extrinsic physical characteristics of the fluid were used to describe drop oscillations (Baret et al., 2007). The authors employed electrowetting techniques to study the aqueous phase drops in oil phase surroundings. Park et al. (2010d) have proposed a single-sided continuous optoelectrowetting (SCOEW) that allowed prolonged active control over the droplet (e.g. splitting, mixing). Moreover, this technique can be used over a comparatively wide range of microvolumes. Morimoto et al. (2006) have fabricated a microfluidic system which had a single pH-sensing site and protease sites. Furthermore, a gold electrode was joined with an electrowetting-based valve. The authors performed a trypsin assay, using a bovine serum albumin (BSA), and applied an enzyme-containing solution to the sensing sites in a micro channel. Nashida et al. (2007) have presented a microdevice composed of glass and a poly(dimethylsiloxane) (PDMS) substrates. The authors employed direct electrowetting to operate the working electrodes of a microdevice constructed for immunoassay analysis. Ohgami et al. (2007) have shown the use of a micro-electrochemical sensor with Y-shaped PDMS microfluidic channels to examine the activities of two enzymes. The authors indicated that the sensitivity of a chip to enzyme concentration depended on the dimension of the channel. Bahadur et al. (2007) have explained how electrowetting could simulate droplet shape on rough substrates. The major advantage of the proposed method is that it can predict the contact angle of a small volume of fluid.
24 Nowadays, numerous studies have focused on the re-evaluation of available methods as well as the development of new ones. The droplet-based approach was used for protein immobilization as described in chapters 4 and 5. Recently, Park et al. (2010c) have provided a method for the electrowetting-controlled transport of droplets. It is important to note that the benefits of utilization of single-plate configuration were well explained by theory. In some cases, such as degradation and high-speed situations, and electrowetting-force values can be used instead of contact- angle values for characterization of the electrowetting process (Crane et al., 2010). However, to make an accurate calculation of electrowetting, compensation methods may need to be applied. In fact, although droplet dynamics can be ignored in this case, evaporation should be taken into consideration. As for parallel plate microchannels, disregarding the dynamics of electrowetting in them has been proven to affect assessment of the basic parameters, such as contact angle and droplet velocity (Keshavarz-Motamed et al., 2010). An alternative method for droplet transfer control is dielectrophoresis (DEP). Non-uniform electric force can be used to polarize and localize the micro- (or sub- micro-) particles, such as proteins and DNA molecules, by DEP. Yantzi et al. (2007) have presented a multi-electrode setup for controlled bio-particle locating and clustering. Scientists employed DEP and AC electrokinetic techniques to control the positions of particles in solution. Wiklund et al. (2006) have organized physical competition between dielectrophoretic, ultrasonic and viscous drag forces for translocation of bioparticles. This device was constructed to provide precise handling of single particles or structural units of particles. The micro-fluidic sequence of liquid droplet collisions with a substrate cavity has been described by Chau and co-workers (2004). The effects of fluid properties have been discussed in terms of microfluidic characteristics for specific tasks in the droplet deposition process. Taff et al. (2005) have presented a positive dielectrophoretic (p-DEP) array for controlled manipulation of single beads. The researchers used unique ―ring-dot‖ p-DEP trap geometry to achieve single cell capture. Choi et al. (2005) have developed microfluidic systems for separation of dielectric particles. Trapezoidal electrode array (TEA) was used to provide dielectrophoretic force for running reactions. Recently, Moncada-Hernandez
25 et al. (2010) have demonstrated concentration and separation of a bacteria-yeast blend by using insulator-based DEP (iDEP). Although negative dielectrophoretic trapping was found to be suitable for microbial as well as fungal manipulation, yeast gave a better result. Over the last decade enormous progress has been made in all aspects of dielectrophoresis (Pethig, 2010). DEP is considered a promising area due to elimination of needs for biochemical tags and surfaces.
2.4.4.3. Continuous system
This system supports translocation of continuous flow through a channel network. External components that can be used to gain precise control of devices include pumps, valves, and mixers. In addition, various electrokinetic techniques can be utilized to operate devices. Depending on application, any of basic electrokinetic forces, namely, electrothermal, electrowetting, electroosmotic and/or electrophoretic can be employed for actuating fluids. Since electrothermal flow induces a temperature gradient that runs through the body of the device, proteins are put at risk of conformational change into a ―soup‖ of molecular debris. On the other hand, electrothermal flow has been employed to mix landing biomolecules in order to improve their ability to bind to functionalized substrates (Sigurdson et al., 2005). The electrowetting technique may be used to successfully manipulate tiny quantities of liquid. Electroosmosis is based on an electrical field having a guiding effect on the motion of microfluids along a charged substrate. With technology involving electroosmotic flow control, information on channel conditions can be gained. Electrophoretically guided movement of a conductive fluid, or small particles enclosed in fluid are important for many applications of microfluidics. It has been demonstrated that continuous flow devices offer enormous possibilities for applying motor proteins for carrying out technological tasks. Jia et al. (2004) have presented a transport system that used electric forces to effectively guide microtubules through kinesin-coated microfabricated channels. The researchers have provided evidence that kinesin motors can convey nanowire over relatively long distances. However,
26 unidirectional transport of wire has not been demonstrated. Heuvel et al. (2005) have developed nanofabricated structures for bringing microtubules to a kinesin-covered wharf. They improved the microtubule transport rate by applying voltage pulses to the gold surface. The contribution of the second kinesin head to motion has been evaluated by Berliner et al. (1995). It was demonstrated that decapitated kinesin was not able to support uninterrupted movement. On the other hand, headless kinesin can compete with headed kinesin for the spot on the surface of a microchannel controlling adsorption (Huang et al., 2007). Kim et al. (2007) have demonstrated controlled alignment of microtubules in channels using an electric field. The unique neck domain and K-loop of the Neurospara crassa kinesin was shown to allow single-headed kinesins to walk in procession (Lakamper et al., 2003). Kim and co-workers (2007) have shown that different kinesins can perform similarly to each other. What they suggested is that a weak electric field can cause inefficient alignment. Li et al. (2005) have fabricated an electrophoretic cell for the analysis of individual fluorescently labeled macromolecules. Their work has shown that an approach based on measurement of the electrophoretic properties of biomolecules was appropriate for filamentous actin. Huang et al. (2006a) applied an electric field to align actin filaments in a certain direction. The researchers used gelsolin to produce specifically polarized actin filaments. By using a dielectrophoretic procedure, a study of the alignment behavior of actin filaments has been carried out (Asokan et al., 2003) Although short filaments failed to align under dielectrophoretic conditions, long ones responded well. In addition, the effect of DEP on biomolecules has been employed to pattern filamentous actin (Asokan et al., 2003). Making polarized actin filaments and then depositing them in microstructures, Lee and co-workers (2009) constructed a system that allowed for cargo translocation. The authors coated the channel with the bacterial protein streptavidin to control the directionality of flow.
27 2.4.5. Control of microfluidic devices
2.4.5.1. Overview
Incorporation of fluidic components into continuous devices has been widely used to deliver a better analysis. The appropriate components are generally used to guide a sample through a channel. Actions that can be accomplished by means of different microactuators include opening/closing, positioning, separating, and controlling. Although microactuators may be driven by different energy sources (Sankaranarayanan and Bhethanabotla, 2009, MacDonald et al., 2004), they are often powered by thermal, electromagnetical and/or mechanical means. Utilizing technological knowledge of microactuators, one can control fluidic interactions at the microscale level.
2.4.5.2. Control of fluidic movement
Although it has been generally accepted that fluid molecules cruise along paths in a laminar state that lets fluids pass through the device without mixing, their final destination depends on their properties. While bigger molecules are likely to go on traveling until the end, smaller ones can either follow their initial route, or diffuse away. Understanding the behavior of fluids in microstructures is the primary step towards constructing proper devices. The behavior patterns of fluids can be influenced by use of micropumps, valves, and mixers. Micropumps are known to be essential elements in many devices due to necessity of maintaining controllable flows of fluids. As their fundamental parameters such as head pressure and flow rate can be optimized, micropumps may provide solutions to flow control. Based on the presence or absence of moving parts, micropumps can be subdivided into two groups: non- mechanical and mechanical. Non-mechanical micropumps that rely on properties of fluids include: electrokinetic, magnetohydrodynamic (MHD), bubble based, and ultrasonic pumps. Since the transition of electrical energy into energy by pumping by electrokinetic pumps can occur in different ways, this kind of actuator can be further
28 subdivided into two categories: electroosmotic or electrohydrodynamic. Xu and co- workers (2010) have described experiments that indicated that cell perfusion was regulated by means of a device with a double chamber micropump. It resulted in the establishment of a steady-state flow. Electrohydrodynamic (EHD) means themselves have been recommended as being quite manageable for microfabrication. Kazemi and co-workers (2009) have successfully introduced an asymmetry in the electrode of the EHD micropump in order to improve its flow rate. Qian and Bau (2009) have described advantages and disadvantages of non-mechanical micropumps based on magnetohydrodynamic principles. Pan et al. (2009) have used numerical simulation to study a micronozzle-diffuser pump based on the principle of thermal bubble nucleation. Masini and co-workers (2010) have presented a micropump actuated by the surface-acoustic-wave (SAW) mechanism. An accurate relocation of the fluids as well as its splitting was accomplished. Even though it seems easy to impose certain conditions on microfluids, some fluids can be driven only by means of mechanical pumps. The workings of different mechanisms that construct micropumps, which include, but are not limited to, reciprocating and peristaltic movements, result in different types of fluid flows. The various types of actuation that deliver reciprocating movement can be classified as follows: thermopneumatic (Pol van de et al., 1990), electrostatic (Nakano et al., 2005), electromagnetic (Zordan et al., 2010), shape memory alloy (SMA) (Benard et al., 1997), and piezoelectric (Wang et al., 2006). Tai et al. (2007) have used a pneumatic micropump to push cells ceaselessly through microfluidic channels. Jun and co-workers (2007) have presented a thermopneumatic micropump that used surface tension to take in fluid. Yun et al. (2002) have employed the continuous electrowetting (CEW) phenomenon to move a mercury drop in an electrolyte-filled microchannel. Yamahata et al. (2005a) have reported diaphragm micropumps based on the electromagnetic actuation principle. Guo et al. (2006) have developed a prototype micropump that used a solenoid actuator to provide a motion. Rocha et al. (2009) have presented a micropumping system for potential LOC applications.
29 There are numerous application-related conditions that can affect the working performance of micropumping systems. While pneumatic actuation needs improved flow control (Yang and Hsiung, 2008), thermopneumatic actuation requires efficient heating to induce vibration of the diaphragm (Pol van de et al., 1990). It has been demonstrated that one of the hallmarks of electrostatic actuation is a fast response time. Yet, actuation by electrostatic means appears to have a driving force that works over short distances (Woias, 2005). In contrast, electromagnetic pumps have a relatively strong driving force; nevertheless they run on high power consumption (Yamahata et al., 2005b, Shen et al., 2008). Although piezoelectric and SMA-driven peristaltic micropumps are also power demanding, they can deliver great actuation forces (Graf and Bowser, 2008). Because the majority of peristaltic pumps have advanced programmable and flow control features, they can be relied upon (Graf and Bowser, 2008). These kinds of pumps may be utilized for transporting heterogeneous fluids (Hsu and Lee, 2009) including bacterial suspensions (Zhu et al., 2010a). The method does not require incorporation of valves or mixers. As mentioned above, some fluidic tasks can be accomplished by using either passive or active micromixers. Since passive ones lack outer fields, they depend on the geometrical or chemical properties of channels. Active mixers explore various phenomena, including electrokinetic (Oddy et al., 2001), electromagnetic (Mohebi and Evans, 2002), magnetic (Wei et al., 2010), electroosmotic (Jain et al., 2009), thermal (Kim et al., 2009b), and ultrasonic (Monnier et al., 2000). As active mixers can contain moving components, such as stir bars or diaphragms, they may be used to either homogenize or dissolve samples.
2.4.5.3. Control of fluidic interactions
The theoretical insights that promote our understanding of fluid flow phenomena are essential for design and construction of microdevices. Heterogeneous fluids containing mixtures of either single biomolecules or self-assembled structures can make fluid-fluid and/or fluid-solid boundary layers. Mechanical and electromagnetic interactions have been known to control behavior of particles in
30 complex flows. Freer et al. (2004) have demonstrated the use of interfacial shear and dilatational deformations to investigate the rheology of proteins at fluid/fluid interfaces. Du and co-workers (2010) have studied the dynamics of tension for particles in a fluid/fluid system so as to determine the parameters of the adsorption process. The effect of the salt content of the particle environment and the structure of the oil phase on the energetic parameters of particles has been demonstrated. Rathman et al. (2005) have achieved a synthesis of biocompatible protein films at fluid/fluid interfaces. The researchers proposed a mechanism of biomolecule self-assembly at the interface. While the properties of fluid/fluid interfaces can be modified by addition of different electrolytes (Mellema and Isenbart, 2004), their stability, can be modified by applied potential (Thaokar and Kumaran, 2005). The leaning of some molecules can be affected by interfacial curvature. In the case of acetonitrile, it may be due to the necessity of protruding methyl groups of acetonitrile in a vapor phase (Partay et al., 2009). It should be noted that there is a substantial difference between the dynamic response of Newtonian and non-Newtonian samples (Torralba et al., 2005). In later experiments the researchers observed the formation of nonsymmetric molecular arrangements at very high amplitudes in the complex fluid (Torralba et al., 2007). Helton et al. (2007) have reported the flow rate related instabilities of the interface between the viscoelastic fluid and the Newtonian buffer. Molecular dynamic studies of the fate of the interchannel bubble by Xie and Liu (2009) have indicated that bubbles existed in the central part of hydrophilic channels. Furthermore, their velocities were slightly lower than a single bubble of the main flow. In order to create a suitable fluid-surface interface, one should identify best choice surfaces relevant to applications. Using computer simulations, Voronov and his colleagues (2006) have shown that the hydrophobicity of interfaces can be modified by adjusting energy and size characteristics of fluid/surface couples. They reported that drag lessening on hydrophobic substrates can be accomplished by selecting fluid/substrate couples that have both low energetic and a high size parameter values. The utilization of the surface nanostructures to achieve the friction control of interchannel flows was presented by Cao et al. (2006). Pal and co-workers (2005a) have studied the effect of surface topography on interfacial energy. It is interesting
31 that a higher energy was attributed to the structural features of the surface. In another study these researchers have shown the use of pits for enhancement of substrate hydrophobicity (Pal et al., 2005b). Later on, Setny et al. (2006) have reported that the deportment of water molecules towards the surface in the neighbourhood depends on the crookedness of the surface. Priezjev et al. (2007) have presented evidence that the size of a fluid slip in a flow basically depends on local density and temperature. Guo et al. (2005) have demonstrated that the impact of temperature on the velocity slip can be eliminated. Iliescu et al. (2007a) have explored effects of both mechanical and dielectrophoretic forces to transport suspension of round particles through a filter device. Choi and Park (2007) have described a method of creation of pressure fields for polystyrene particle manipulation in a microfluidic channel. Shevkoplyas and his colleagues (2007) have studied the dynamic behavior of superparamagnetic particles in a microchannel under an applied magnetic field. Cheng et al. (2009) have indicated that conformations of DNA molecules depend on their positions in the curvilinear fluid flow. Zhu and co-workers (2010b) have highlighted the role of the interchannel flow parameters in the enhancement of microbial detection. Wu et al. (2009) have presented a new microfluidic method for purification of human erythrocytes from the sample contaminated with Escherichia coli. The authors used asymmetrical flow in a suitable microchannel to fend off bigger specks. Understanding the procedure for recruiting bacteria for power generation work under microfluidic conditions is essential for the development of economical devices. According to a study conducted by Kaehr and Shear (2009), motile bacteria, for example Escherichia coli, can be in charge of microtransport in customized microdevices. Over the past decade, many attempts have been made to use aligned biopolymers in devices. Asokan et al. (2003) have utilized electrical forces to align actin filaments during motility. The authors reported that the difference in response of filaments to DEP torque was related to length of filaments. Recently, Kaur and co- workers (2010) have provided evidence that actin alignment behavior can be controlled by applying a weak magnetic field. They noted that filaments should be deposited onto the surface prior to magnetic treatment. Importantly, Meer et al. (2010)
32 have successfully performed and analyzed the alignment of actin filaments in microchannels controlled simply by shear stress.
2.4.5.4. Immobilization of proteins
2.4.5.4.1. Overview
There are two main types of relationships between proteins and surfaces that can be defined: specific and non-specific. A target protein may selectively attach to a surface either through specific or non-specific interactions. Although surface modification techniques can be used to control protein adsorption behavior, undesirable deposition may take place, when, for example, the protein undergoes surface-triggered conformational changes. Non-specific immobilization may occur via weak forces such as electrostatic, Van der Waals or hydrophobic. A specific kind of immobilization can be implemented by the formation of covalent bonds between the molecule and the channel wall (see chapters 4 and 5). It relies on complementarities of proteins to surface functional groups. The SAM (self-assembled monolayer) method can be used to deposit proteins in precise location at certain degrees of adsorption.
2.4.5.4.2. Physical adsorption
Physical adsorption of biomolecules on water-insoluble interfaces is a general method of non-covalent attachment. The amount of adsorbed protein depends on several factors including the kind of protein used, its concentration and environment, and the contact performance of a material surface. A single molecule has the unique ability to form both specific and non-specific bonds with available surface sites. The diffusion of protein molecules from a liquid phase towards a surface is a set of reversible and irreversible processes (Vroman and Adams, 1969, Vroman and Adams, 1986). Depending on the size and structure of the molecule, protein can become a permanent or a temporary resident of polymeric surface. Erban and Chapman (2007) have utilized the random sequential adsorption (RSA)-driven approach for the simulation of irreversible adsorption of objects in real physical time. In the RSA
33 model, the arriving molecule tends to occupy the empty spot on the surface. After touching down at the surface, the molecule blocks the access to its surface area for the landing adsorbent (Evans, 1993). However, a later arriving molecule can compete for a piece of surface with smaller and relatively less attractive settlers. Krishnan et al. (2003) have highlighted the fact that protein competition for space at the liquid-vapor interface is based on differences in molecular weight. Upon arrival at the surface, pliable human immunoglobulin G (HIgG) can be met by early-arriving albumin (BSA) and delivered to the surface via the Vroman‘s phenomenon (Sahin and Burgess, 2003). Once attached, a protein may retain or change its native conformation and bioactivity. For example, protein from almonds was capable of retaining its enzymatic activity after irreversible adsorption at the water/organic medium boundary (Hickel et al., 2001). Bower et al. (1999) have studied the effect of net electric charge on the enzymatic activity of lysozyme at an interface. The study showed that electrostatic force had a crucial effect on the functional activity of protein at a colloidal silica substrate. Moulton and co-workers (2003) have demonstrated that electrostatic protein attraction to an electrode surface is highly affected by an electrode charge. The authors proved that human serum albumin (HSA) had a better blocking effect on the electron transfer between an electrical conductor and an electroactive group than immunoglobulin G (IgG). Kim and Yoon (Kim and Yoon, 2002) have indicated that protein concentration in a solution affects the fractional surface coverage of polymer particles. Final surface coverage depends on the protein properties and surface conditions. Brewer et al. (2005) have explored adsorption of BSA on both citrate- coated and bare gold substrates. Comparison of two surfaces indicated that protein binding favors the bare gold one. This implies that protein spreads out on gold surfaces upon unfolding. In the late 1990s, Cho et al. (1997) have shown that the BSA protein has slow unfolding kinetics at the air-water boundary. The entire unfolding journey of a BSA molecule can take on average over 20 hours (Cho et al., 1997). Lu et al. (1999) have examined effects of BSA concentration and pH on the formation of a protein layer at the air-water interface. It was found that a high protein concentration at pH5 causes formation of a thicker adsorption layer due to low electrostatic repulsion. The authors have noticed that BSA molecules tend to lie on the upper water
34 layer rather than stand on it. In addition to pH, salt concentration and surface hydrophobicity can be used to control adsorption (Chang et al., 2010a). Noinville et al (2002) have analyzed interfacial behavior of fungal lipase, BSA, lysozyme and α- chymotrypsin on hydrophobic surface. This study demonstrates that α-chymotrypsin exhibits greater attraction toward hydrophobic substrate than BSA and lysozyme. Changes in the conformation of three model proteins upon adsorption were observed. Lu et al. (1998) have examined morphology of the adsorbed layer of lysozyme at the hydrophobic surface. It was concluded that the protein underwent irreversible structural alteration. Su et al. (1998) have indicated that adsorbed lysozyme remains safe in a low ionic environment on the hydrophilic surface. With increasing ionic strength (above 0.5M), all protein molecules are forced to shift from the surface. Special attention needs to be paid to ―working‖ conditions for self-assembled proteins and their molecular partners. Numerous studies have demonstrated the influence of magnesium (Gicquaud et al., 2003) and potassium on globular actin polymerization (Senger and Goldmann, 1995, Goldmann, 2002). Rioux and Gicquaud (1985) have noticed that negatively charged actin filaments produce self-assembled structures on positively charged lipid substrates. St-Onge and Gicquaud (1990) have suggested that the initial electrostatic attraction between actin and lipids alleviates subsequent hydrophobic interactions. This type of protein adsorption may occur anywhere on an appropriate substrate. Recently, Albet-Torres and co-workers (2010) have proposed different mechanisms to account for heavy meromyosin (HMM) adsorption. In doing so, the authors compared HMM interaction with two kinds of substrates: SiO2 and positively-charged ones. It should be noted that despite considerable research efforts, control of protein interfacial behavior remains incomplete.
2.4.5.4.3. Covalent binding
Covalent immobilisation of biomolecules onto polymeric surfaces allows for more precise application and operation of the coating layer. Covalently attached molecules have a number of advantages over adsorbed ones (see chapters 4 and 5).
35 These advantages include stability, accessibility and security. Biomolecules can offer a range of external surface groups (e.g., sulfhydryls, phosphates, amines, carboxyles, etc.) for covalent immobilization. Using surface properties, one can guide a target molecule toward a supportive matrix. Comparison of the various surface chemistries such as aldehyde, amino-silane and carboxylic acid-modified glass surfaces revealed high affinity binding of aminated DNA probes to aldehyde groups (Zammatteo et al., 2000). These interactions were shown to happen without the participation of a crosslinking agent. The employment of the crosslinking couple, 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) with N-hydroxysulfosuccinimide (sulfo- NHS) helped antibodies bind securely to carboxyl substrates (Pei et al., 2010). Assessment of recruitment of a crosslinking team for immobilization of human immunoglobulin G (HIgG) and lysozyme (LYZ) on surface-modified poly(tert-butyl methacrylate) PtBMA has been provided by Ivanova (2006c). In most cases the efficiency of coupling may be facilitated by the binding environment. There is enough evidence to show the influence of pH (Weber et al., 1996, Cisneros and Dunlap, 1990), and temperature (Cisneros and Dunlap, 1990, Kao et al., 2010) on the binding ratios of biomolecules. Although most of the biomolecules can be linked via various groups, the systems produced by such interactions may become retarded or even become disabled. Oriented towards substrate, some molecules cannot explore their active sites. Lu et al. (1996) have demonstrated that the key to improving the binding capacity of antigen is its orientation. Accordingly, immobilized molecules should meet their binding partners at the right spot. It has been shown that secure positioning of DNA molecules on the surface is crucial for obtaining a good DNA-DNA hybridization yield (Zammatteo et al., 1997). Later studies have proven that one DNA probe should have enough room on the surface to capture target DNA without colliding with DNA probe neighbours (Zammatteo et al., 2000). Moreover, a coupled DNA or protein probes should be able to resist exposure to severe conditions (e.g., temperature, pH). A comparative study of immobilization techniques has answered the question of how much temperature change affects both the kinetics and the efficiency of hybridization (Hakala and Lonnberg, 1997). The authors demonstrated that rising
36 temperature leads to a decrease in the efficiency of oligonucleotide hybridization. To stay attached securely, the DNA molecule should be supported by a suitable linker. Azo-linkers have been developed which can assist in the short-time UV-irradiation treatment of DNA (Wanga et al., 2008). The bifunctional crosslinker EDC has been employed to demonstrate that oligonucleotides can be successfully covalently introduced to the polystyrene-co-maleic acid (PSMA) polymeric surface (Ivanova et al., 2002c). The same crosslinker has been utilized to support stable binding of DNA molecules to NucleoLink under DNA-DNA hybridization conditions (Christensen et al., 2000). Our study provides evidence that the EDC linker not only anchors G-actin on a PSMA surface, but also allows subsequent self-assembly of the actin filament (Alexeeva et al., 2005).
2.4.5.4.4. Self-assembled monolayers (SAMs)
Self-assembly of supramolecular structures can be used as a means towards the fabrication of novel nanomaterials. Controlled chemistry of SAMs allows the optimization of surface properties that are employed in the construction of biosensors (see chapter 6). A broad range of subunits (e.g., alkanethioles, proteins, peptides, nucleotides, lipids) can be successfully utilized for SAM creation. Molecular interactions in SAMs are mediated by the formation of noncovalent bonds between building components. Although connection via weak forces is sufficient for SAM growth, it is not secure for its accommodation. Ataka and Heberle (2008) have noted that some bulky non-polar molecules tend to dislocate easier than small polar ones. By applying covalent immobilization, researchers could provide a higher level of SAM stability for eliminating molecular rearrangements (Darain et al., 2009, Min et al., 2010). This ―bottom-up‖ approach, in which novel functional structures (actin bundles, muscle fibres, ATPase, etc.) are produced from suitable building blocks, is used by all living organisms. In cell dynamics of major self-assembled networks, such as pro- and eukaryotic cytoskeletons, it is regulated by both extra- and intracellular (e.g., temperature, pressure, ion concentration) conditions. Understanding that natural
37 processes can be employed for the production of biomimetic materials, researchers study the ability of biomolecules to perform nanotechnological tasks on various artificial surfaces; accordingly, they have molecular candidates checked for ―biomolecular – surface‖ biocompatibility. Baujard-Lamotte et al. (2008) have demonstrated the effect of SAMs properties on the adsorption of fibronectin (fn), extracellular glycoprotein. A conformational change from normal to β-sheet-modified state of fn accompanies its adsorption onto the hydrophobic substrate. The researchers found that protein concentration acts on SAM capacity to accommodate protein, letting a certain number of native fn molecules contact SAM. Sagnella et al. (2005) have developed a system with the surface properties of the extracellular matrix. The authors created a biomimetic substrate of poly(vinylamine) with adhesive components on the octadecyltrichlorosilane (OTS) SAM and seeded human pulmonary artery endothelial cells (HPAEC) on the resulting polymer. Although the stimulator of adhesion, HBP (heparin-binding peptide), was able to interact with endothelial cells to provide bonding, the lack of sufficient support for HPAEC adhesion caused untimely cell cytoskeletal damage and cell-surface detachment. Meanwhile, other researchers have demonstrated that muscle cells had survived on the cysteamine surface without anchorage by the extracellular matrix (ECM) regulating components (Coletti et al., 2009). Furthermore, the study of the cultivation of skeletal muscle cells on surfaces showed that myotube monolayer morphology was more uniform on cysteamine coated with gold than when myotubes were on bare gold. However, the ability of muscle cells grown on the cysteamine SAM to display their differentiation potential was only about 70 %. Studying the physiology of muscle cells on SAM can help researchers create hybrid devices that contain rotary motors, ATPase-like muscle cells (Montemagno and Bachand, 1999), but function much longer. In fact, changes in local environment (e.g., pH, temperature) may influence ATPase performance. To assemble a more controllable rotary motor, Tao et al. (2009) combined ATPase with self- assembled structures. Obviously, ATPase is a promising candidate for implementation of power management in microdevices. In addition to the ―bottom-up‖ approach, there is another type of approach, the ―top-down‖ that is used to take away some of the material to create a structure. As the
38 size of any kind of destruction goes up, the potential issues of the method, such as physical barriers and cost may arise. Despite the potential limits of 3-D structure construction (e.g., channels of different sizes and geometries), the ―top-down‖ approach may be used to complement the ―bottom-up‖ method. As discussed in previous subsections, an optimized microfluidic system is able to guide self-assembly of molecular motor proteins.
2.5. Concept of protein molecular motors
2.5.1. Overview
Molecular motors are protein machines driven by energy coupled to nucleotide (ATP, GTP) hydrolysis (Liu et al., 2005b), ion motive (IMF) (Bai et al., 2009) or proton motive forces (PMF) (Nakanishi-Matsui et al., 2010). They can produce mechanical force and torque and transport cargoes over specific substrates, while the character and rate of their action can be controlled (Wang and Manesh, 2010, Bustamante et al., 2001). Although the term motor refers primarily to cytoskeletal motors like myosin, kynesin or dynein, their partners (actin, tubulin) also produce force (extend/shrink motion) and may be described as distinct types of motors (Kueh and Mitchison, 2009). Unlike cytoskeletal motors, the transmembrane F0 motor is unable to shift membrane. It requires either nucleotide hydrolysis or motive force (MF) to produce rotary torque. In addition to eukaryotic motors, there are bacterial nucleotidases such as actin homologues (MreB, FtsA, ParM) that have the structural and dynamic properties of molecular motors.
2.5.2. Eukaryotic actin
Self-assembly is crucial characteristic of monomeric actin (G-actin) that consumes ATP energy produced by rotary motors to form a double-stranded, polar, helical, filamentous actin (F-actin). The polymerization goes though three stages of development: nucleation, elongation and equilibration and needs sufficient
39 concentration of G-actin (Grintsevich et al., 2010, Husson et al., 2010). Actin in its monomeric form (G-actin) is composed of two similar domains (Kabsch and Holmes, 1995). Each of the domains consists of two subdomains; two upper subdomains, also known as the ―barbed end‖, have greater affinity for globular actin, and the two bottom subdomains (the ―pointed end‖) have lower exchange rates for actin (Southwick, 2000). The phosphate moiety of a nucleotide (ATP or ADP) sits on the interdomain clift (Kabsch et al., 1990). Polymeric forms of actin, or thin filaments (or f-actin) are helical polymers which have 13 actin molecules (42 kDa) arranged on six left-handed turns repeating every 36 nm. The thermodynamic properties of the self- assembly/disassembly of actin have been described by Oosawa and Asakura (1975). The authors suggested that the rate-limited step for polymerization altered with the joining of the third protomer. Further support for this came when Purich and Allison (1999) compared kinetic properties of microtubules and actin filaments. The authors concluded that the third G-actin-ATP protomer takes an important role in the nucleation process that induces a more stable polymerization ―nucleus‖. Once the three-meric ―nucleus‖ is built, a thermodynamically favoured elongation stage begins. Woodrum et al. (1975) studied the growth of F-actin and reported that bidirectional polymerization occurred from both sides of actin filaments. Moreover, authors highlighted that topologically distinct ends of the nucleus had different growth potentials. Thus, the ―barbed‖ tip of an actin trimer grew faster than the ―pointed‖ one. Pollard (1986b) has demonstrated the importance of the critical concentration of ATP- actin in a local medium for filament assembly. Thus, as long as it remains above critical value, both sides of the filament elongate. A decrease in ATP-actin concentration to a critical concentration is associated with entering the equilibration stage of polymerization. At equilibrium, the addition of actin subunits at the barbed tip is in balance with the loss of actin subunits at the pointed tip of the filament. As a result, F-actin ends are in a state of subunit flux (Kirschner, 1980); it maintains constant average polymer size. The destiny of actin polymer (detachment, branching, fragmentation, crystallization, lifetime, etc.) at any stage of assembly is strongly affected by numerous factors: temperature, pH, protein, salt and ATP concentration, the presence
40 of actin-binding protein (ABP), etc. Thus, polymerization can be accelerated by increasing temperature up to room temperature (Grazi and Trombetta, 1985), adjusting pH to 7.0 (Lin et al., 1997, Wang, 1989), adding ATP (Pollard, 1986b, Fujiwara et al., 2007), including the most effective bivalent ions Ca++, Mg++ (Bergeron et al., 2010, Carlier et al., 1994), and joining actin binding proteins (ABP) (Pollard, 1986a). Wachsstock et al. (1993) have realized that the factors that guided actin alignment were affinity of ABP, F-actin length and protein concentrations. In the author‘s model, diffusion can mislead actin filaments. Thus, the formation of aligned actin bundles does occur, but the behavior of actin in the highly viscous fluid state is so unpredictable that the influence of diffusion is strong enough to reorganize pre- bundled filaments. For generating electrostatic association between actin filaments, the polymerizing medium must contain sufficient amount of divalent cations. Angelini et al. (2003) have used high (Z) counter ions Ba++ for F-actin bundling. Their later work continued on the issue of the bundling mechanism, particularly its dynamics (Angelini et al., 2006). The remarkable fact that has been noticed is that Ba++ bound between filaments caused acoustic–based dispersion. Furthermore, when divalent metal ions interacted with nearest-neighbour cages, they produced a liquid-like correlation phase and dynamic system response. However, both ion and coion behavior need to be evaluated regarding various areas of the heterogeneous actin surface. Undoubtedly, a two-way actin assembly may include interactions with various members of ABP group. The work reported in this thesis is mainly focused on ABP representatives from three different families, such as actin, gelsolin and PARPs. Gelsolin (GLS) is an actin-serving protein that affects both assembly (e.g., capping, nucleation) and disassembly (e.g., fragmentation) of actin. It contains six structurally similar domains that differ in regard to actin and calcium affinity (Burtnick et al., 1997). A schematic model was proposed by Way et al. (1989) to explain the contributions of individual GLS domains to interactions with actin. It demonstrates that only GLS 1 remains in touch with actin after the calcium shifts. The authors further concluded that the GLS 1 segment is responsible for a stable capping of actin
41 after calcium withdrawal. As for segments 2 to 6, GLS 2-3 serve and subsequently cap actin filaments, while GLS 2-6 support actin nucleation. So the GLS 1 segment has a specific function. Utilizing the properties of the gelsolin domain (GLS 1), the length of the actin filament can be controlled via stabilization of the conformation of the actin‘s domains. In doing so, the length parameter of F-actin can be adjusted (Janmey et al., 1986). While actin polymerization in the cell (Meindl et al., 1994), in the solution (Flanagan and Lin, 1979), and at the surface (Gadasi et al., 1974) was intensively investigated for its role in a variety of important cellular processes (see sections 2.5.2. and 2.5.6), the paramount importance of actin assembly along a topographically patterned surface was realized only in 1990s. For example, it was not until 1995 that the orientation of kidney fibroblasts at the grooved surface was studied (Wojciak- Stothard et al., 1995). By exploring patterned substrates – such as poly(methyl methacrylate) (Suzuki et al., 1997), titanium and silicon (Hirst et al., 2005) – the researchers have become convinced of the need for development of polymeric surfaces with built-in channels (Nicolau et al., 1999), and achievement of a better surface molecular motor alignment (discussed in previous subsections). However, under device-realistic conditions, eukaryotic actin can stay fit enough to perform its nanotechnological tasks only for a relatively short period of time. Theoretically, the best alternative possible is one of PARPs, namely, the MreB or FtsA that the majority of bacterial species use to stay in a particular body shape and other purposes (discussed in the following chapter).
2.5.3. Prokaryotic actin related proteins
2.5.3.1. Overview
In the last fifteen or so years, the attention of researchers has been drawn to the importance of non-molecular motor-based motions. Since then, several families of actin-related proteins have been defined in organisms as different as humans, yeast, plants and bacteria; with each ARP assigned to a family on the basis of its primary
42 amino acid sequence identity compared to conventional actin. Actin-related proteins share 11 to 60 % identity (Schafer and Schroer, 1999, Carballido-Lopez, 2006). Many bacterial actin relatives share significant sequence similarity but have limited functional homology and ligand binding specificity. Based on homology in amino acid sequences, several bacterial proteins are believed to belong to the actin family. These proteins include heat shock proteins, sugar kinases, and the following proteins synthesised in microorganisms: the bacterial chaperon DnaK [heat shock protein (Hsp70)] (Wetzstein et al., 1992, Martinez-Alonso et al., 2010, Rhee et al., 2009), the cell division protein FtsA (Strahl and Hamoen, 2010); the plasmid-encoded ParM (Popp et al., 2010a) as well as a constituent of bacterial cytoskeleton Mbl, MreBH (Schirner and Errington, 2009), and MreB (Wang et al., 2010). In term of the structural and functional similarity, four bacterial nucleotidases, namely, actin (MreB, FtsA, ParM) and tubulin (FtsZ) homologues own unique properties of eukaryotic molecular motors.
2.5.3.1.1. MreB
Among all proteins of the Hsp70/actin/sugar kinases superfamily, MreB (murein cluster e) protein is the most similar to actin. This actin homologue has a similar 3D structure and the ability to undergo actin-like polymerisation. It has been reported that MreB and actin monomers showed significant resemblance at their atomic level (van den Ent et al., 2001). Both of them had two domains with binding pockets for ATP molecules. Also, each domain consisted of two subdomains that are structurally more identical with actin than any other actin-related proteins (the following subsection provides an example of the FtsA protein structure). The MreB protein assembles into one-dimensional protofilaments with smaller subunit spacing (51 Ǻ) than actin (55 Ǻ). Futhermore, in vivo MreB monomers can treadmill (Biteen and Moerner, 2010) in a directional manner and hence generate polarized assembly (Kim et al., 2006). Although MreB is structured similarly to actin, it is capable of self- organizing into straight filaments (Vollmer, 2006, Allard and Rutenberg, 2009), bundles (Srinivasan et al., 2007, Jiang and Sun, 2010), sheets (Popp et al., 2010b) and
43 ring-shaped structures (Esue et al., 2005). Like actin, MreB polymerization can be affected by such factors as critical concentration (Cc), the availability of a source of energy (ATP or GTP), the presence of bivalent cations (Mg++) and temperature (Mayer and Amann, 2009). On the basis of a report by Esue et al. (2005), there has been ample evidence that under the same experimental conditions the critical concentration (Cc) for MreB can be as low as 0.003µM in comparison with 0.25µM for actin. This means that prokaryotic MreB is roughly 83 times more efficient than its eukaryotic homologue. In discussing their paper, the authors offered the suggestion that MreB monomers have not only a much greater affinity for each other but also a remarkably better affinity for the MreB filament than actin. In this work, MreB sets phosphate free (Pi) at a rate of 0.10 Pi per actin per min. (Esue et al., 2005), which is very similar to F-actin (approximately 0.16 times slower). It should be noted that, in contrast with actin preferences, where ATP is a more potent player than GTP, MreB catalyses ATP and GTP hydrolysis equally well (Esue et al., 2006). The fact that some bacteria, namely, Bacillus subtilis owns three isoforms of the MreB protein (Mbl, MreBH and MreB) (Schirner and Errington, 2009), while other bacteria, for example, Thermotoga maritima (Popp et al., 2010b) and/or Escherichia coli (Varma and Young, 2009) hold only single MreBs suggests that all three isoforms could play important roles in growth and morphogenesis of only some of the bacteria, such as Bacillus subtilis (Kawai et al., 2009a). Additionally, MreB does not act alone; it collaborates with other actin orthologs, namely, MreC and MreD (Defeu Soufo and Graumann, 2005). It is important to emphasize that MreB protein does not have a protein partner, molecular motor (Vats et al., 2009), so the eukaryotic mechanism of force generation is unlikely to work in bacteria (Erickson, 2001). However, one protein, RodZ (YfgA), discovered by Bendezu et al. (2009), was found to be part of the MreB spiral-like structure (van den Ent et al., 2010). The functional role of the important prokaryotic player MreB in bacteria is discussed in chapter 2.5.6. The discovery that bacteria contain ―single‖ actin homologues raised new issues, especially in respect to assembly of MreBs from various bacteria. It has been demonstrated that MreB works in both gram-positive, namely, Bacillus subtilis (Mayer and Amann, 2009), Listeria monocytogenes (van der Veen et al., 2007), and
44 gram-negative bacteria, for example, Thermotoga maritima (Esue et al., 2005), Vibrio parahaemolyticus (Chiu et al., 2008). However, some pathogenic bacteria, such as Listeria monocytogenes, do not waste their own MreBs on travelling through the host cells. They would rather explore the host‘s actin than invest in their own material. The pathogenic strategy of enteric bacteria is discussed in detail in subsection 2.5.6.5. Studies on the polymerization of Thermotoga maritima MreB revealed that the temperature and the concentration of divalent cations, namely Ca++ or Mg++ affect both its assembly and ultrastructure (Esue et al., 2005). The remarkable thing about MreB is that it can produce straight (Vollmer, 2006), ring-like structures (Vats et al., 2009), or sheets (Popp et al., 2010b). It is plausible, on the basis of the suggestion of Esue et al. (2006) in their study of GTPase activity of MreB that the shape of the assembled MreB depends on the extent of ATP or GTP hydrolysis within the MreB polymer. An understanding of MreB assembly seems to be not an easy matter. Thus, Bean et al. (2008) have come across some significant findings when they tested the biochemical properties of Thermotoga maritima MreB. It turned out that the MreB protein can assemble across a wide range of temperatures. As for divalent ions, the authors assumed that Ca++ and Mg++ do not play a vital role at the nucleation stage of MreB polymerization. Mayer and Amann (2009) have succeeded in examining MreB from Bacillus subtilis and have understood its biochemical properties. By determining and evaluating the temperature range of polymerization, nucleotide and ion preferences, they showed the difference between the MreBs from Thermotoga maritima and Bacillus subtilis. Unlike these two representatives of quite different groups of bacteria, such as Thermotogae and Firmicutes, a member of the phylum Proteobacteria, Caulobacter, can produce long filamentous assemblies (Kim et al., 2006). However, the mechanism by which MreB structures are built has not been clarified. To explain uncommon MreB behavior, the authors proposed that bundling of short MreB filaments in a free global polarity manner causes formation of mysterious long structures. Even though MreB has received considerable attention with regard to its assembly (Graumann, 2009), the possibilities and limitations of the use of MreBs from various bacterial species in vitro have not been explored (discussed in chapter 9).
45 2.5.3.1.2. FtsA
Although there is no biochemical evidence that cytosolic cell division protein FtsA forms actin-like filaments, there are two highly conserved proteins (FtsA and tubulin orthologue FtsZ) involved in the cytokinesis of bacteria that are structurally related to components of the eukaryotic cytoskeleton and they have a central role in cytokinesis (Beuria et al., 2009). FtsA is recognized as the key cell division determinant, other than FtsZ, that lacks a clear membrane-spanning sequence (Pla et al., 1990). The crystal structure of FtsA did confirm that FtsA is a homologue of actin and the heat shock protein (Hsc70). However, unlike the members of actin family, the second subdomain of FtsA, namely 1C, is sitting on the opposite site of subdomain 1A from its location in actin (van den Ent and Lowe, 2000). It has been shown that the 1C domain of FtsA is engaged in the interplay between its own molecules and the other proteins that are recruited to the division ring assembly (Rico et al., 2004). The simple explanation for the unique structural features, such as insertions of entire domains, is that FtsA carries out specific cellular functions. Thus, the distinction between the position of the subdomain 2 homologue for FtsA and MreB, can only make MreB the true orthologue of actin (Egelman, 2003). Paradis-Bleau et al. (2005) have proved that of the two nucleotides, namely, ATP and GTP, the former is favored by FtsA. However, interaction between FtsA and ATP was found to be strongly dependent on the location of FtsA protein (Sanchez et al., 1994). Thus, only the cytoplasmic phosphorylated form of FtsA has the ability to bind ATP molecules. Studies on the contribution of FtsA to bacterial cell division have revealed that the conserved carboxy motif of FtsA operates as a membrane targeting sequence (MTS) by tying up the Z ring to the cell membrane (Pichoff and Lutkenhaus, 2005). The current understanding of the molecular mechanism of actin (described in subsection 2.5.2.) and/or MreB (described in the previous subsection) is that polymerization is not applicable to FtsA assembly. The work reviewed above helps us to understand why it was found that FtsA was unable to produce actin-like filaments that can be a reliable FtsZ anchor. Despite the lack of data on FtsA assembly
46 properties, some studies have shown that FtsA molecules can interact with each other (Lara et al., 2005). Strikingly, Lara and co-workers have provided ample evidence that the FtsA protein of cocci produces very stable corkscrew-shaped spirals. The authors have suggested that FtsA can form polymers in vivo due to its sufficient concentration, presence of a large number of ATP molecules and other proteins that can assist it. Feucht et al. (2001) have reported that both dimeric and multimeric FtsA forms exist in Bacillus subtilis. In their examination of the cytological and biochemical properties of FtsA, the authors indicated that FtsA predominantly exists as dimers. It is also interesting to note that the contributions of FtsA to cell function may vary depending on cell structure. For example, the number of FtsA molecules in gram- positive bacteria, namely, Bacillus subtilis, is 5-20 times higher than the number of the same molecules in gram-negative bacteria, for example, Escherichia coli. Feutch et al. (2001) have concluded that gram-positive bacteria need more ATP energy for cell division due to the broad peptidoglycan coat of the cell, greater intracellular osmotic pressure and/or lack of a ZipA protein. Indeed, Staphylococcus aureus has 40-nm thick cell wall, which is 7nm thicker than the periplasmic coat of Escherichia coli (Dubochet et al., 1983). Furthermore, osmotic pressure in gram-positive bacteria, such as Staphylococcus aureus, was estimated to reach 20-30 atmospheres, which was 6 times as high as in a gram-negative cell of Escherichia coli (Kuczynski-Halmann et al., 1958). However, it was observed that the ZipA homologues are missing not only in gram-positive but also in gram-negative bacteria, except for γ-Proteobacteria. Having established that Escherichia coli can get around the lack of ZipA, Geissler et al. (2003) proved that gram-negative bacteria can hire slightly modified FtsA for a ZipA role. In fact, all of these factors may trigger a higher level of expression of FtsA in gram-positive cells. So, questions related to FtsA assembly properties remain to be answered.
47 2.5.4. Evolution/Phylogeny of bacterial actin homologues
2.5.4.1. Overview
Over the last decade or so, there has been some interest in the relationship between actin and PARPs (Egelman, 2010, Lowe et al., 2010). In eukaryotic cells, actins are major players in the generation of various internal cytoskeletons. Acquisition of cytoskeletal motility is considered to be one of the major events in evolutionary history that enabled eukaryotic cells to perform variety of cellular functions (the contributions of MreB to the cytoskeletal organization is discussed in detail in chapter 2.5.6.). Except for some cell wall-less bacteria, such as Mycoplasma insons, which benefits from a harmless commensal interaction with green iguanas (Relich et al., 2009), all other prokaryotic and eukaryotic organisms have cytoskeletons. The classical characteristics commonly used to compare cytoskeleton counterparts, namely PARPs with actins, include atomic structure, biochemical properties, biophysical and DNA and/or amino acid composition.
2.5.4.2. Evolutionary/Phylogenetic comparison of MreB with FtsA
As has been demonstrated in previous subsections, actin, MreB and FtsA can use the energy from either ATP or GTP hydrolysis to produce mechanical motion. However, actin and FtsA prefer the former to the latter. Some subdomains of these proteins are predicted to adopt tertiary structures identical to ATP-ase subdomains of hexokinase (Bork et al., 1992). Comparison of MreB with FtsA subdomains (discussed in the previous subsections) reveals that MreB exhibits more similarity in atomic composition to actin than FtsA. In terms of polymerization properties, MreB shows actin-like behavior, except that it displays greater morphological variation and more efficient polymerization properties, such as a parsimonious critical concentration, time-saving nucleation (Esue et al., 2005), and flexibility in nucleotide choice (Esue et al., 2006). In contrast, the assembly behavior of FtsA is completely
48 different from its actin- or MreB-like behavior (discussed above in subsection 2.5.3.1.2.). A noteworthy feature of actin is that it belongs to a multigene family of evolutionary conserved proteins. For example, different members of the eukaryotic actin family, namely, plant and fungi show about 80-85 % identity to mammalian actin (Doolittle and York, 2002). The number of actin genes, however, can vary among species. Thus, the unicellular eukaryote Saccharomyces cerevisiae has only one isoactin (Gallwitz and Seidel, 1980); ascidians, invertebrate chordates, contain four actin genes encoding one alpha-muscle actin (Beach and Jeffery, 1992). In addition, the examination of amino-acid composition in vertebrate bovine tissues revealed a set of six actin isoforms (Vandekerckhove and Weber, 1978a), the set consists of two cytoplasmic (beta and gamma) (Vandekerckhove and Weber, 1978b) and four muscle ones, namely skeletal, cardiac, vascular, and nonvascular actins. A comparison of yeast actin with bovine beta-actin have showed that those proteins shared a high level (up to 90 %) of sequence similarity (Egelman, 2001). To account for the unique conservation, various possible explications have been proposed. One explanation for this phenomenon is that, in actin, a vitally important cellular player, the selection for subunit-subunit and subunit-filament contact sites, as well as ABP-sites, is sufficiently strong, so that the tertiary protein structure is maintained (Pollard, 1984). In order to estimate the extent of DNA conservation, Ponte et al. (1984) have examined human B- actin cDNA clones. The authors‘ suggestion that the 3‘-UT region of cDNA can drive the expression of actin genes was made on the basis of high homology between untranslated regions of cDNA of human and rat β-actins. It was thus concluded that the considerable high similarity may reflect strong evolutionary pressure on untranslated portions of cDNA. In fact, if eukaryotic actin was not well conserved, its structure could be easily affected by any of the ABPs (their influences on actin are discussed in subsections 2.5.2. and 2.5.6.2.). In addition, bacterial homologues of actin, MreB and FtsA have neither got large sets of accessory proteins (e.g., the only one is MreB‘s spiral-like configuration creator, RodZ (YfgA) so far), nor any introns. Moreover, despite their non ubiquitous nature, MreB as well as FtsA genes conserved throughout eubacterial lineage. Both
49 proteins display very low levels (e.g., MreB with 15 % similarity) of sequence identity to actin (Carballido-Lopez, 2006). Interestingly, the ftsA gene is much more conserved in bacteria than the other cell division genes and is almost always found in tandem with ftsZ (Dai and Lutkenhaus, 1992). Furthermore, the relationship between the ftsA and ftsZ gene products reflects the need to maintain a highly accurate expression ratio of these genes for a normal cell division. An interesting observation concerning the evolutionary relationships between eukaryotic cytoskeletal components and their bacterial counterparts has been made by Doolittle and York (2002). The authors revealed that the evolutionary scale positions of both MreB and actin drift away from the ubiquitous heat shock protein 70 (Hsp70) and glycolytic enzymes, such as enolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and triose- phosphate isomerase (TIM). Based on these results, they suggested that eukaryotes and prokaryotes shared a common ancestor long ago; the organism owned a protein with properties common to MreB. Since the majority of actin relatives cannot assemble filaments, Egelman (2003) has suggested that the ancestor had a monomeric structure. However, the idea of sharing a common monomeric ancestor by the members of the actin family ran contrary to Bork et al.‘s (1992) view that the ancestor is an ATP binding homodimer which carried on via a duplication event preceeded by structural divergence. Doolittle and York (2002) proposed that the ancestral protein was capable of self-assembling into plain filaments in an ATP-dependent manner. In order to understand MreB and FtsA phylogenies, one needs to take a close look at the phylogenies of bacteria that own them. Furthermore, the correlation between phylogenetic analyses based on MreB and/or FtsA and 16S rRNA sequences should be estimated to evaluate the possibility of using MreB and/or FtsA sequences as a chronometer to help unravel phylogenies of new and/or misplaced bacteria (see chapter 9).
2.5.4.3. Use of 16S rRNA as a molecular chronometer
With the recognition that ribosomal ribonucleic acid (rRNA) can be used as a molecular chronometer (Woese, 1987), the method of 16S rRNA sequencing became a
50 standard procedure for identification of any living organisms, including the producers of MreB and FtsA proteins. By using a molecular phylogenetic analysis method based on 16S rRNA sequences, Woese and Fox (1977) were able to present evolutionary relationships among different species. According to their three-domain model of the universal tree, prokaryotes include two subkingdoms (urkingdoms), namely, eubacteria and archaebacteria. Distribution of actins, MreB, FtsA, and Hsp70 proteins among the three domains of life has been investigated by Doolittle and York (2002). Notably, while actins are present in eukaryotes, they are lacking in prokaryotes. In contrast, MreBs are unevenly distributed across only two prokaryotic urkingdoms. The authors concluded that the striking structural similarity in the absence of sufficient sequence similarity of MreB and actin was due to early divergence. In addition, only Hsp70 was found in both prokaryotic and eukaryotic urkingdoms of life. As for FtsA, it was detected in the eubacterial domain of life. However, some researchers have doubts about the ability of 16S rRNA analysis to reflect a evolutionary history of life. Thus, Gupta et al. (1997) have proposed a molecular approach for estimating microbial phylogeny based on the presence or absence of conserved amino acid segments (called either indels or signatures). Using this method, the authors developed a model showing the reflection of hierarchical order of various bacterial groups. Furthermore, Gupta (2000a) has split bacteria into two major groups: monoderms (with single membranes) and diderms (with two membranes separated by periplasmic spaces). Four years later, Griffiths and Gupta (2004) provided evidence for placing Deinococcus into an intermediate group (its members possess not only two membranes but also a thick peptidoglycan). It should be emphasized that Gupta (2000b) has recognized the evolutionary value of members of the proteobacterial phylum. The author was able to demonstrate that not only mitochondria but also the nuclear cytosolic homologues of some eukaryotic genes originated from proteobacteria. Examination of protein sequences led to the finding of striking homology between MreB and the first half of the Hsp70 sequence (Gupta and Singh, 1992). Interestingly, in contrast to MreB, bovine actin did not display substantial similarity to Hsp70. This result might indicate that the MreB derived from a precursor of Hsp70. Although a ―protein signature‖ method can
51 provide valuable evolutionary insights, it cannot be employed for the analysis of noncoding sequences such as introns. Another limitation is the lack of reliable indels for all proteins. Apart from molecular genetic methods, transitional analysis together with the fossil record has been used for drafting a sequence of life-history events (Cavalier- Smith, 2006a). Cavalier-Smith (2006b) has offered an interesting explanation for the molecular events accountable for polarization of transitions. According to his model, the transformation of the MreB cytoskeleton into an actin one through gene duplication occurred with the participation of the Arp 2/3 complex (the product of MreB triplication in the pre-eukaryotic organism). It was noted that such a gene creation event was crucial for megaevolution (the term refers to quantum evolution). Though the approach is valuable in regard to gaining a deeper understanding of evolutionary relationships between phylogenetically-distant species, it is not suitable for microbial classification due to the lack of experimental evidence. It goes without saying that tremendous efforts have been devoted to the development of a precise approach for deducing prokaryotic phylogeny. However, none of the currently available methods can reflect the knottiness of microbial connections. Furthermore, the use of one criterion for figuring out the evolutionary history may lead to misinterpretation. Consequently, the choice of a sufficient number of reliable molecular chronometers may help us reach a deeper understanding of phylogeny (see chapter 9).
2.5.5. Classification of protein molecular motors
2.5.5.1. Overview
Motor proteins can be classified into two major groups such as linear and rotary according to the mode of operation. This subsection starts with structural comparison of different types of linear molecular motors and their in vitro performance (see also chapter 6). It continues with a comparison of eukaryotic and prokaryotic ATP synthases and their in vitro applications (see also chapter 8). While
52 linear molecular motors, namely eukaryotic actin (or its homologues MreB or FtsA proteins) can be used for building linear nanotracks (see chapters 6 and 9), prokaryotic producers of ATP may be incorporated into biosensors for supplying molecular motors with cheap ATP energy (see chapters 6 and 8).
2.5.5.2. Linear molecular motors
Despite principal differences in molecular properties (e.g., weight, dimension), the major linear motors, namely, myosins, kinesins, and dyneins, share some similarities. The three motors have two identical heads (or motor domains) that bind to the polymerized substrates (i.e., actin filaments for myosins, and microtubules for kinesins/dyneins) and that catalyse ATP hydrolysis in a cytoskeleton-dependent manner. It is important to note that these representatives of three superfamilies have one hallmark, the P-loop, designed by nature for ATP binding (Walker et al., 1982). Unlike myosin or kinesin, dynein has four consensus P-loops (Ogawa, 1991) and up to three heads (Gibbons et al., 1991) with numerous ATP binding sites (Gibbons et al., 1991). Furthermore, due to multisubunit architecture (Bruno et al., 1996), cytoplasmic dynein relies heavily on the presence of different accessory proteins (Burkhardt et al., 1997, Lam et al., 2010). In addition, it cannot walk with a uniform step size (Singh et al., 2005). Despite the possibility of the control of dynein performance at different levels, it is practical to employ less demanding and simple motors, such as myosin and kinesin, with their maximum capabilities to transport cargo in microdevices. As for myosin and kinesin, they are two-headed (except for the single-headed myosin I), molecular edifices that end with two-arm forks (Rayment, 1996), which are in charge of pushing. The common feature of kinesin and myosin is the structure of the motor domain. It has been shown that the kinesin‘s head domain is remarkably similar to the catalytic core of myosin (Kull et al., 1996). So, these proteins exhibit some structural similarity even though they share a low amino acid identity (Kull et al., 1996) and display distinct enzymatic properties (Johnson and Gilbert, 1995). Half-lying on the microtubule (Gibbons et al., 2001),
53 kinesin walks on two flexible legs with identical small feet (or motor domains) along the tubulin track (Kull et al., 1996) and takes a break when it meets an obstacle, such as another kinesin, in its way (Seitz and Surrey, 2006). Contrary to the assumption that kinesin remains permanently attached to the microtubule during the entire walk, Block et al. (1990) have demonstrated the inability of a small team of kinesins to continue their journey up to the final destination on the microtubule track. So the molecular motor team came off the track because it could not carry cargo over the expected distance. The authors suggested that kinesin spent a short time away from its substrate during any force-generating cycle. Further evidence confirmed kinesin‘s detachment. By using mutant and wild-type kinesins for creating either roadblocks or obstacles, Telley et al. (2009) have modulated crowded situations on a microtubule. Furthermore, the frequency and duration of time spent by kinesin in the waiting state was shown to depend upon the degree of molecular crowding on the microtubule track. Presumably, kinesin can go around an obstacle by changing its running path. However, it is not clear which road motor it is going to take when it faces a jammed section of a track. Consequently, kinesin movement strategies need to be better understood to permit an accurate qualitative as well as quantitative interpretation of its molecular behavior. In contrast to ―sticky‖ kinesin, which produces processive motion along microtubules (Seitz and Surrey, 2006), skeletal myosin generates ―rowing‖ movement along actin filaments (Leibler and Huse, 1993). Tawada and Sekimoto (1991) have presented the model in which muscle myosin works in collaboration with other myosin motors in two regimes: productive and nonproductive. According to this model, there are two major events that occur during the productive cycle: ATP splitting and force production. Subsequently, nonproductive connection/disconnection between myosin heads and actin results in the generation of friction drag and dissipation of heat. It is proven that reduction of the molecular friction may be achieved by minimizing connection/disconnection time (Lecarpentier et al., 2001). However, the problem of heat production is still not solved. As mentioned previously, the key part of the myosin molecule is a motor domain. To understand the contributions of different parts of this molecule, one should look closer at its structure.
54 The myosin is a product-inhibited ATPase consisting of three parts called a head, a neck and a tail with communicating functional units: the actin-binding site, the nucleotide-binding site (Korn, 2000), and the neck domain (or the ―lever arm‖), which magnifies the small transformations at the active site into the large ones needed to convey actin (Holmes, 1997). This nanomachine is strongly stimulated by binding to actin, which is a nucleotide exchange factor for myosin. With the hydrolyzed nucleotide the myosin binds to the actin filament. After recombining with actin, the cross-bridge goes through a conformational change allowing Pi and then ADP to be released, which also brings about the ―power stroke‖ (Holmes et al., 2003) producing movement along the actin filament. In order to utilize effectively the ability of myosin to interact with actin polymer, one should optimize the molecular environment of these biomolecules including the contact surfaces and fluid properties (as discussed in subsections 2.4.3. and 2.4.5.). However, this biomolecular couple cannot perform any nanotechnological tasks without an ATP energy supply.
2.5.5.3. Rotary molecular motors
Three protein motors have been described as rotary machines: the bacterial flagellar motor (BFM) and two portions (Fo and F1) of the ATP synthase (FoF1 ATPase). There are some other molecular motors believed to be driven by a rotary motion, including a dodecameric portal protein (a part of the genome packaging machine) (Simpson et al., 2000, Lander et al., 2009) and mini-chromosome maintenance (MCM) a protein complex that acts as the replicative DNA helicase (Brewster et al., 2010). Furthermore, we restrict our attention exclusively to three remarkable rotary machines. Of these, the bacterial flagellar motor, along with the Fo motor, is driven by the flow of ions across the cytoplasmic membrane – either + + hydrogen (H ) or sodium (Na ) ions depending on the organism, whereas the F1 motor is driven by ATP hydrolysis. Such rotary motor complexes play a major role in oxidative or photosynthetic phosphorylation, coupling the flow of protons down an electrochemical gradient to the synthesis of ATP (Mitchell, 1979). ATP synthesis diverges structurally depending on the source; it consists of eight distinctive subunits
55 in nonphotosynthetic eubacteria (Foster et al., 1980) and nine subunits in photosynthetic bacteria (Walker et al., 1990). BFM is discussed in detail in subsection 2.5.6.4. Both eukaryotes and prokaryotes have ATP synthases composed of two discrete sectors (F1 and F0) that are considered to be separate rotary motors working cooperatively (Muller and Gruber, 2003). The water-insoluble membrane portion (F0) and the water-soluble peripheral portion (F1) are joined together by a central shaft composed of γ (Omote et al., 1999) and ε (LaRoe and Vik, 1992) subunits. The direction of the shaft rotation affects the way the enzyme works (Diez et al., 2004). Whilst clockwise rotation is accompanied by ATP synthesis, anticlockwise rotation is energized by ATP hydrolysis. It is important to note that bacterial ATPase has the same rotation property, 120˚ step, as the eukaryotic one (Adachi et al., 2000). The spinning of the heterodimeric stalk is driven by the movement of the F0 rotor which is energized by a transmembrane ion gradient. There seems to be sufficient evidence from cross-linking studies that the F0 rotor is a ring oligomer of 12 c subunits (Jones and Fillingame, 1998). However, the ring stoichiometry may vary in the range between 10 (Jiang et al., 2001) and 15 c subunits (Pogoryelov et al., 2007) among living organisms. Additionally, there is data that suggests that the a1b2 subcomplex may fulfill the function of a stator (Fillingame, 1999) by preventing the shifting of the
α3β3 portion of the F1 domain during catalysis. Moreover, the b subunits (Perlin et al.,
1983) of the a1b2 trimer do not interact directly with the α3β3 spherical subcomplex.
The b dimer has been proven to be connected to an α subunit of the α3β3 hexamer via a bridging subunit delta (Wilkens et al., 1997). The importance of the b2δ structure has been indicated by Dunn (2000), showing that it is strongly bound to an α subunit. The authors believe that the b2δ stalk should be considered as a stator instead of an a1b2 subcomplex.
As for the stator part of the F1 motor, it is composed of an α3β3 hexameric assembly and a single δ subunit. Although all six homologous subunits of the hexamer are capable of binding nucleotides (Walker et al., 1982), only β subunits have their own catalytic sites (Boyer, 1993). It has been demonstrated that α and β subunits are coupled together to form the smallest functional protomers of enzyme (Hayashi et al.,
56 1989). From the analysis of the α subunit isoform association, Blanco et al. (1994) have concluded that the α subunits can oligomerize into stable structures. Furthermore, based on interaction specificity of the α subunits, the authors suggested that αβ protomerization contributes to stability and physiology of the entire enzyme. However, the exact contribution of the α subunit to enzymatic performance remains to be determined. The contribution of the rotor part, namely the γ and the ε subunits, of the F1 motor to the function of ATPase is discussed above. It is important to emphasize that integration of energy providers, such as rotary motors, into ATPase- powered devices is important for producing hybrid devices (Bachand and Montemagno, 2004).
Using F1-ATPase as the rotary motor, Noji et al. (1997) have assembled a hybrid structure resembling a propeller by connecting a central rotor of the motor to an actin filament. By incorporating the F1-ATPase motor into NEMS (nanoelectromechanical systems), Soon et al. (2000) have succeeded in constructing a rotory motor-supported nanodevice. They successfully replaced the actin filament with a nickel bar and mounted the motor protein on a nanometer-sized support structure.
However, the F1-F0 ATPase-powered hybrid device operated at only 50 % efficiency.
Omote et al. (1999) have found that bacterial F1-ATPase is capable of rotating actin filament in the flow cell with approximately 80 % efficiency. Seeing the potential applications of a natural ATP supplier, Montemagno and Bachand (1999) have tested the performance of modified bacterial F1-ATPase on different metal substrates to estimate its in vitro efficiency. The motor enzyme was shown to work at up to 100 % efficiency. The researchers concluded that their platform can be used for assembling devices that employ this kind of rotary motor as a main power source.
2.5.6. Native functions of molecular motors
2.5.6.1. Overview
Molecular motors, as discussed in the previous chapter, are produced in living organisms and serve vital functions for their owners. The contributions of these
57 natural nanomachines to the anatomy and physiology of cells count on their biochemical and mechanical aptitudes. Molecular motors play different key roles in such biological processes as cytoskeletal arrangement, metabolic reactions, flagella dynamics, bacteria pathogenesis and ATP generation. Although some motors appear to be sensitive to an in vitro environment, motor utilization seems to hold a great promise for helping to develop efficient and cheap microdevices. Potential utilization of motor proteins depends upon the ability to employ the proteins in order to benefit from their native properties.
2.5.6.2. Cytoskeleton
All living cells have an internal framework called a cytoskeleton (the example of an unusual organism has been given in chapter 2.5.4.). This big structure is composed of polarized actin filaments and various ABPs. The actin network allows the cytoskeleton to re-arrange rapidly, providing a supportive matrix that organizes the cytoplasm and holds the whole cell together, thereby regulating bacterial motility: (Mauriello et al., 2010), endocytosis (Suetsugu, 2010), cell division (Wong et al., 1997b), phagocytosis (Campos-Parra et al., 2010), segregation (Vats and Rothfield, 2007), polarity (Fanto and McNeill, 2004), chemotaxis (Swaney et al., 2010), adhesion (Hegge et al., 2010), cell migration (Gardel et al., 2010), organelle movement (Suetsugu et al., 2010), molecular and membrane trafficking (Okamoto and Forte, 2001, Molla-Herman et al., 2010). Furthermore, eukaryotic cells differ from prokaryotic cells by possessing the presence of a complex cytoskeleton consisting of an abundant array of proteins. The major ones are actin filaments, microtubules (MTs) and intermediate filaments (IFs). The filaments provide mechanical support to eukaryotic cells and serve as tracks for motor molecules to move along. These filament systems share one essential feature: they are composed of proteins that have the unique property of being able to self-assemble into linear polymers (Carballido- Lopez and Errington, 2003). Polymerization occurs at critical monomer concentration, where non-covalent reversible protein interactions mediate the assembly of cytoskeleton components into dynamic filaments (the actin assembly is discussed in
58 detail in subsection 2.5.2.). It should be noted that in vivo actin polymerization is not a fully independent intracellular act for building up a higher-order molecular structures. This process is controlled through interactions of actin with different members of the ABP family. Although the living cell was well recognized for its ability to produce actin/or an actin-like force generating system, the source of inspiration in this process came from the master of biomolecular mimicry, the model organism Listeria monocytogenes. The pathogen derives benefits from using the host‘s cytoskeleton. To stimulate actin assembly on the back surface of its own body for creating an elastic tail (Gerbal et al., 2000), the pathogen makes contact with the essential eukaryotic factor, Arp2/3 complex, and utilizes its properties (Boujemaa-Paterski et al., 2001). However, the contributions of this complex to the regulation of actin assembly and the network remained incompletely understood by scientists for a decade. Even though Welch et al. (1998) made quite interesting suggestions regarding the mechanism of Arp2/3 activation, they did not provide a solid explanation for Arp2/3-guided actin assembly. Later, Footer et al. (2008) shed more light on how actin nucleation can be activated by the ARP2/3 complex. In addition to the Arp2/3 complex, there is another important regulator, gelsolin (GLS) that is located both on the bacterial surface and in the ―comet tail‖ (Laine et al., 1998). The authors assume that the actin-uncapping capability of the pathogen is closely linked to its gelsolin-serving function. Interestingly, they heightened Listeria motility by elevating the intracellular concentration of GLS in the host cell. Despite the lack of experimental evidence for this process, the authors supposed that either an increase in recycling of monomeric actin or a decrease in the viscosity of the host cytoplasm was associated with accelerated bacterial movement. (The properties of this protein are discussed in subsection 2.5.2.). The dynamic structure, later called a cytoskeleton, was first extracted from erythrocytes by Yu and co-workers (1973). However, because of the lack of a reliable technique for non-brutal treatment of cells, the three dimensional arrangement of cytoskeletal counterparts was not clarified. To prepare muscle cytoskeleton for scanning electron microscopic (SEM) examination, Wallace and Fischman (1979) have developed the osmium-TCH method of producing evenly coated actin filaments.
59 The authors emphasized that cytoskeletal components could be regarded as protein assemblies as well as distinct organelles. For a long time, it was believed that the cytoskeleton was one of the key distinctive features of eukaryotes. That point of view was revolutionized in the 1990s by the analysis of FtsZ‘s contribution to the life of bacterium Escherichia coli (RayChaudhuri and Park, 1992). Moreover, structural and functional homologues of all three main eukaryotic cytoskeleton proteins: actin homologues, such as MreB (Takacs et al., 2010), FtsA (Shiomi and Margolin, 2007), ParM (Popp et al., 2010a), MamK (Katzmann et al., 2010); tubulin ones, such as FtsZ and BtubA/B (Sontag et al., 2009); and intermediate filament protein ones, such as CreS (Gitai et al., 2004), have been proven to make up cytoskeletal structures in bacterial cells. These prokaryotic homologues behave in many ways like eukaryotic cytoskeletal components. Thus, they are involved in a variety of essential cellular processes in bacteria (Michie and Lowe, 2006).
2.5.6.3. Cellular metabolism
A cell acquires and utilizes energy in order to move anything that a cell needs (Zimmerman and Walter, 1991) to assemble, develop and survive. A cell performs metabolic reactions, such as anabolic (Kwast and Hand, 1996) and catabolic ones (Reggiori et al., 2005), resulting in either the synthesis of complex molecules or decomposition of complex ones, respectively. The metabolism of substrate by a cell requires the participation of a transport system that allows for the transport of a certain organic or ionic molecule. There are two basic types of molecular transport, passive and active transport (Zeuthen, 1995). The principal means of passive transport is the diffusion that happens in all living cells spontaneously (Soh et al., 2010). It requires no energy to transfer a particle downwardly with respect to its concentration gradient. As an example, the passive transport of glucose (Bell et al., 1990) through the sarcolemma of the striated muscles (skeletal and cardiac) occurs by means of glucose- transporting proteins (GLUT1; GLUT4) (Santalucia et al., 1992). To study the insulin- stimulated transport of glucose in peripheral blood lymphocytes, Piatkiewicz et al. (2010) have applied a flow cytometry analysis. The authors proposed use of white
60 blood cells as a model due to the difficulty of performing the same experiment in living cells. Although these cells can be used for gaining a deeper understanding of insulin-related disorders, the huge difference between various types of cells must be taken into consideration. Meanwhile, there are substances that are too large to travel through a cell by means of diffusion. Furthermore, molecular motors need energy to move along polarized intracellular tracks. As an example, the active transport of membrane- enclosed organelles – such as Golgi stacks (Boevink et al., 1998), mitochondria (Tavi et al., 2010), lysosomes (Sontag et al., 1988, Demirel et al., 2010), peroxisomes (Li and Nebenfuhr, 2007) or secretory vesicles (Trifaro et al., 2008), as well as protein complexes – elements of the cytoskeleton, virus particles (Vaughan et al., 2009) – to their proper place in a living cell is mediated by motor proteins such as myosin and/or kinesins/dyneins. These proteins use the energy derived from repeated cycles of ATP hydrolysis. With respect to applications of molecular motors, myosin is useful for cargo transport. As discussed in subsection 2.5.5.2., the actin/myosin couple can be considered for participation in nanotechnological experiments. In addition, these proteins display essential molecular skills in vitro only in the presence of the source of energy, such as ATP. As discussed in subsection 2.5.5.3., ATP molecules in eukaryotic and prokaryotic cell membranes are synthesised by rotary motors (ATPases). Interestingly, some bacteria, which include pathogens (September et al., 2007) and marine bacteria (Ivanova et al., 2002b), use the same type of carbon metabolism, namely heterotrophic, as eukaryotic cells. Thus, heterotrophic species can obtain energy (ATP) through the fermentation or respiration of such organic compounds as carbohydrates, lipids, proteins and/or from decaying organic substrate. Furthermore, due to metabolic plasticity, some pathogens are capable of maintaining both parasitic and saprophytic lifestyles (Freitag et al., 2009) depending on their circumstances. It has been proven that the environment has an enormous effect on metabolic states of heterotrophic bacteria (Alexeeva et al., 2004b, Ivanova et al., 2003a). A recent study has demonstrated that short thermization has an impact on the metabolism of heterotrophic microorganisms (Samelis et al., 2009). In doing so, the attempt to
61 deactivate pathogenic bacteria in raw milk caused a significant change in its microbial community structure. Welch et al. (1995) have used arsenate, a toxic metalloid, that inhibits binding-protein-dependent transport systems such as PMF-dependent transport (Richarme, 1988) to check the symmetry of the flagellar motor. The authors applied two methods: a conventional one and an arsenate-incubation one. The comparison of these methods showed that motility was permanently lost in the former, while it was temporary affected in the latter. Although the results of these experiments indicated that the flagellar motor was asymmetrical, the underlying cause of the unidirectional rotation remained unclarified. As mentioned in subsection 2.5.5.3., unlike other motile cells, swimming bacteria power up flagellar rotary motors by proton motive force (PMF). It is important to note that metabolic processes, which use chemical energy to pump protons out of the cell allowing them to return, are the basis for PMF production.
2.5.6.4. Flagella-based motion
Motility is known to be generated by flagellated cells; however, due to the lack of understanding of the fine points of this mechanism, the full utilization of this cellular property for nanotechnological applications in microdevices has not been accomplished yet. It has been proven that both eukaryotic and prokaryotic motile cells can either swarm over surfaces or swim in a fluid environment. Although eukaryotic swarming techniques (Bonner, 2010, Gilbert, 1927), as well as bacterial ones (McCarter, 2010, Jones and Park, 1967), have been investigated for decades, they still remain to be studied in greater detail. For example, it has been demonstrated that of four main swarming strategies, namely, reversing, stalling, lateral or forward moving used by Escherichia coli swarmer cells to travel over an agar surface, the first one is a very specific one, which is not used by Escherichia coli swimmers (Turner et al., 2010). Nowadays, many questions have arisen concerning various aspects of bacterial swarming including its robustness and effectiveness (McCarter, 2010). Since the employment of microbial swimmers as energy generators (Zhang et al., 2010b) or use of their biomimetic actuator in a microfluidic environment may
62 become possible, in this subsection more attention is paid to swimmers. Thus, swimming microorganisms (Zonia and Bray, 2009) and some eukaryotic cells with flagellar motors (Woolley, 2010) can exhibit a variety of structures and movement patterns. These bacteria and eukaryotic cells manifest various locomotion styles, which are carried out by either rapid rotation or the beating motion of flagellar filaments that jut out from the swimmers. In animals, flagellated sperm cells propel themselves forward via symmetric sinusoidal-like and different asymmetric wavelike movements towards oocytes (Dillon et al., 2007, Gadelha et al., 2010). Many single- celled organisms related to protists (flagellated protozoa, some algae) use flagella (Lewin, 1953) or cilia (Manton, 1953, Zhang et al., 2010a) to move through their aquatic environment. It has been proven that ciliated epithelial cells (with short hair- like structures), which line body cavities like the respiratory tract (e.g., parts of the nasal cavities, trachea), need to beat in synchrony to push a mucous blanket and clean cellular debris off the epithelial surface (Sommer et al., 2010). As for bacteria, it has been demonstrated that the majority of them swim by rotating their flagella (Srigiriraju and Powers, 2006). In addition, different bacterial species have different numbers and arrangements of flagella on or around cell surfaces to inhibit not only swimming, and swarming but also such modes of locomotion as twitching (Hammond et al., 2010), and propulsion (Lin et al., 2010). For example, monotrichous bacteria (have one flagellum at a polar location) change the direction of motor rotation from forward to backward (Taylor and Koshland, 1974). Moreover, a flagellum can go back along the path it has swum along as well as move in a zigzag fashion (Goto et al., 2005). Interestingly, the swimming of lophotrichous bacteria (they have a bunch of polar flagella at the end of the cell) in a single direction is accompanied by sudden turns (Harwood et al., 1989). According to recent studies, a representative of amphitrichous bacteria (they have two flagella, one at each end of the cell) Helicobacter pylori, swims in a circular fashion (Celli et al., 2009). Peritrichous bacteria (with a high degree of flagellation) aggregate flagella into posterior bundles to propel themselves forward. (Darnton et al., 2007). Although eukaryotic and prokaryotic flagella look similar to each other, they have completely different structures (Engel et al., 2009). The eukaryotic one is a well-
63 preserved organelle (Hodges et al., 2010) in which an assembly of 9 doublet proteins is precisely arranged around two separate microtubules (Patel-King et al., 2004, Mitchell, 2007). It contains over 200 proteins that are arranged into sub-assemblies, such as dynein arms and radial spokes (Inaba, 2003), etc. It is important to note that dynein proteins can produce force as linear tension, compression (Lindemann, 2003) and torque on the doublets through ATP (Nevo and Rikmenspoel, 1970) and ADP hydrolysis (Lesich et al., 2008). Unlike the eukaryotic flagellum, the single bacterial one is composed of a hook-basal body (HBB) complex (Kubori et al., 1997, Wosten et al., 2010), and an extracellular flagellar filament (Hosogi et al., 2010, Macnab, 2003). A bacterial rotary motor has been proven to power up the corkscrew motion of a flagellar filament (Dreyfus et al., 2005). From the engineering point of view, the filament is a helical cylinder attached to the cell surface; it is constructed by means of self-assembly of 20,000 FliC monomers (Reid et al., 1999, Majander et al., 2005). As mentioned above, a bacterial motor can rotate filament in both clockwise and counterclockwise directions (Manson, 2010). The core of the bacterial nanomotor is a group of rings which rotates flagellar filaments (Sowa et al., 2005) and includes some 13 other proteins (Delalez et al., 2010). Even though significant efforts towards understanding the structure and functions of rotary motors have been made, the utilization of their force through recruitment of their owners still remains far from accomplished. In fact, such important motor characteristics as the conversion of energy (Nakamura et al., 2009) and its sustainability (Fukuoka et al., 2010) under various biochemical conditions (Thormann and Paulick, 2010), for example, change in temperature and/or pH, the effect of environmental homogeneity as well as shear stress, etc., need to be understood in greater detail. Furthermore, in order to recruit bacteria for nanotechnological work, one should find a microbial candidate that would be able to produce a sufficient amount of ATP in microfluidic conditions (see chapter 8).
64 2.5.6.5. Tactics of enteric pathogens
2.5.6.5.1. Overview
As discussed in subsections 2.4.5.4.4. and 2.5.2., the control of molecular self- assembly can be accomplished by means of different methods. A natural way for managing self-assembly is used by pathogenic bacteria. It is called the ―comet tail‖ technique. It has been proven that members of the Enterobacteriaceae family, such as pathogenic representatives of the genera Listeria and Shigella (Gouin et al., 1999, Lin et al., 2010, Adamovich et al., 2009), employ the method to propel themselves through host cells. To evaluate the benefits of utilization of pathogenic techniques for control of actin assembly, one should understand how enteric bacteria achieve high pathogenicity.
2.5.6.5.2. Common tactics of enteric pathogens
Pathogenic bacteria from the Enterobacteriaceae family can successfully inhabit intestines of humans and cause diseases such as bacillary dysentery (Fletcher, 1917), typhoid (Baker et al., 2010, Verma et al., 2010) and urinary tract infections (Yusha'u et al., 2010), etc., which result in three million annual deaths (Nel and Markotter, 2004). The pathogens can travel from the environment into the host cells through fecal-oral transmission. Accordingly, it predetermines the choice of therapy for the disease (Mai et al., 2010). It has been proven that enteric bacteria are not only physiologically but also metabolically adaptable to their surroundings (Freitag et al., 2009). Thus, they are able to withstand killing by acid or enzymes during the journey through the stomach (Barmpalia-Davis et al., 2008, Barmpalia-Davis et al., 2009). Moreover, pathogens can overcome intestinal peristalsis (which clears the gut), adhere to (Coconnier et al., 1993), and then invade epithelial cells (Sansonetti, 2002), in spite of the occupancy of competitors such as host specific faecal bacteria (Sekirov et al., 2010).
65 A pathogenic life cycle starts with host entry and colonization and is followed by the establishment of infection (Vazquez-Boland et al., 2001). After damaging host cells, enteric pathogens can continue cruising through the target organ or exit it. Although much research has been devoted to understanding pathogenic tactics, the mechanism of interactions of pathogenic Listeria with the surface of intestinal cells remains to be elucidated (Schuppler and Loessner, 2010). In fact, although several lines of research have indicated that enteropathogenic bacteria use virulence factors, such as the pore-forming toxin Listeriolysin O (LLO) (Yin et al., 2010, Meyer-Morse et al., 2010, Sashinami et al., 2010), ActA (Muller et al., 2010), internalins A and B (Pentecost et al., 2010), to besiege host defence mechanisms, it is not enough to convince all researchers. Thus, Conter et al. (2010) have shown that strains of Listeria monocytogenes differ in their ability to attack the HeLa (human epithelial carcinoma cell line) cells. Therefore, these researchers believe in the existence of other virulence factors that have not been discovered yet (Conter et al., 2010). Earlier work by Donnenberg (2000) has established that two major types of macromolecular structures, namely, an adhesion system and a secretion system, play a crucial role in enteropathogenic invasion. Pathogenic bacteria use adhesins (Beachey, 1981) to bind to the receptors of enterocyte surfaces. It has been demonstrated that adhesins can be located either at the surface of a gram-positive bacterium (Gilot et al., 1999, Reis et al., 2010) or at the end of its pili (De Greve et al., 2007). Remarkably, while some biomolecules, e.g., autolytic amidase (Ami) contribute to cell adhesion (Milohanic et al., 2001), other bacterial products such as internalins (Parida et al., 1998, Pentecost et al., 2010) and LapB (Reis et al., 2010) are responsible for both adhesion and invasion. Gram-negative pathogens are known to have about six secretion systems of different complexity. Moreover, all of them have specific functions, for example, the type I secretion system is necessary for transportation of biomolecules from the cytoplasm to the cell surface (Cescau et al., 2007). The type II secretion system is in charge of transport of biomolecules through the outer membrane (Francetic et al., 2007). This secretion system has been proven to be used by the human pathogen Vibrio cholera for the export of cholera toxin (Camberg and Sandkvist, 2005). Many enteropathogens, for example, Yersinia spp. (Brodsky et al., 2010), Salmonella spp.
66 (Van Engelenburg and Palmer, 2010), Shigella spp. (Newton et al., 2010), and enteropathogenic Escherichia coli (EPEC) use the type III secretion apparatus (Martinez et al., 2010) upon contacting target cells. This secretion system not only delivers biomolecules but also shoots them into the target cell (Kubori et al., 2000, Tamano et al., 2002, Galan and Wolf-Watz, 2006). Once transported into host cells, these effector biomolecules induce actin reorganization. Enteropathogenic bacteria have been proven to facilitate infection by exploiting host-cell actin (Lu and Walker, 2001). Of two enteropathogenic masters of harnessing actin dynamics for ―comet tail‖ formation, namely, Listeria monocytogenes and Shigella flexneri, the former has been chosen as a model organism for study of pathogenicity (Joyce and Gahan, 2010, Guillet et al., 2010).
2.5.6.5.3. Listeria as a regulator of actin assembly
The gram-positive bacillus Listeria monocytogenes is equipped with a set of surface proteins, such as internalins: A (InlA) (Van Stelten et al., 2010), B (InlB) (Auriemma et al., 2010) and J (InlJ) (Sabet et al., 2008, Bublitz et al., 2008); and the actin nucleator protein ActA (Conter et al., 2010). Remarkably, in order to succeed in intra/intercellular cruising, the facultative pathogen explores not only host cytoskeletal actin but also its Arp 2/3 complex (Sousa et al., 2007) and nucleation-promoting factors (NPFs) (Chong et al., 2009). As mentioned above, Listeria monocytogenes uses covalently linked internalins to invade the cells of intestinal epithelium. Travelling through the intestine, the pathogen is looking for the nearest multicellular junction (MCJ) located between partially separated enterocytes on the top of the villi in order to get access to hidden epithelial cadherin (E-cadherin) receptors. Though the study of host cell invasion has gained considerable attention during the last decade (Parida et al., 1998, Gao et al., 2009), it was not until recently that the contributions of InlB to this process were recognized. Thus, recent research has shown that InlB does not play a role in adhesion but triggers its cellular ligand c-Met, a receptor tyrosine kinase (RTK), to speed up endocytosis (Pentecost et al., 2010). Interestingly, although
67 InlB mimics the hepatocyte growth factor (HGF), it does not compete with HGF for a site on the cMet receptor (Shen et al., 2000). In contrast to InB, InlJ plays an important role in adhesion (Sabet et al., 2008). It should be emphasized that InlA participates in both the adhesion and invasion of pathogens (Schubert et al., 2002) in order that Listeria monocytogenes may breach host barriers (Lecuit, 2005) and subsequently multiply inside the host cell. In so doing, Listeria monocytogenes utilizes InlA to reach the actin cytoskeleton via the E-cadherin receptor; Listeria monocytogenes uses β-catenin as a link to the actin -connected anchor composed of α- catenin (Seveau et al., 2007). Furthermore, the intracellular protein ARHGAP10 has been proven to control the anchor (Sousa et al., 2005). It is important to note that there are two other key players, namely, myosin Vlla and its transmembrane receptor vezatin; they not only participate in the formation of direct contacts between cells at junctions but also contribute to InlA-initiated pathogen internalization (Sousa et al., 2004). Depending on the availability of a target cell, pathogens can invade either directly through enterocyte activity or indirectly via Peyer‘s patches (Jensen et al., 1998, Schuppler and Loessner, 2010, Marco et al., 1997). However, some researchers have doubts regarding the ability of Listeria monocytogenes to sneak indirectly through the host barrier (Pron et al., 1998). One of the key NPF, which is essential for invasion (Suarez et al., 2001) and motility (Chong et al., 2009, Portnoy et al., 2002), is ActA; it is produced by two pathogenic species of the genus Listeria only, namely, Listeria monocytogenes and Listeria ivanovii (Gouin et al., 1995). Even though ActA is not capable of making long polymers, it can homodimerize in vivo (Mourrain et al., 1997). The model has been proposed by the authors to explain the contributions of the ActA dimer to actin- mediated motility of Listeria monocytogenes. However, in contrast to dimerization, the closely spaced distribution of ActA on the bacterial surface has been recently proposed to explain its enhancing effect on actin polymerization (Footer et al., 2008). Strikingly, in order to initiate actin assembly at its own bacterial surface, the pathogen uses ActA for recruitment of essential host cell factors, such as the Arp 2/3 complex (Cossart, 2000) and representatives of the enabled homologue/vasodilator-stimulated phosphoprotein (Ena/VASP) family (Castellano et al., 2001, Lambrechts et al., 2008).
68 The Arp 2/3, evolutionary conserved multimeric protein complex, has been studied extensively due to its importance to nucleation of actin filaments and creation of the Y-shaped cross-linking actin network (May, 2001, Goley et al., 2010), which is crucial for actin-based motility (Zalevsky et al., 2001). It is important to note that the Arp 2/3 complex requires ATP energy to nucleate actin polymerization (LeClaire et al., 2008). Another key player in Listeria monocytogenes-induced ―comet tail‖ formation is VASP. It was not well understood until the beginning of the last decade (Loisel et al., 1999) how pathogens can benefit from hiring VASP. Experimental studies have revealed that Listeria monocytogenes uses VASP to employ profilin, a nucleotide exchange factor, which serves the barbed ends of actin filaments (Pasic et al., 2008) to cause hastening of actin growth at its own bacterial surface. In consequence, bacterium acquires an ―actin cloud‖ (Tilney et al., 1990) composed of young actin filaments (Lambrechts et al., 2008). By reorganizing the surrounding cloud at a pole enriched with ActA (Kocks et al., 1993) the pathogen creates a ―comet tail‖, which supports its directional movement through the host cell. It has been demonstrated that travelling pathogens display different behaviors. Thus, while gram- positive Listeria monocytogenes spins around, gram-negative Yersinia pseudotuberculosis and Escherichia coli do not (Robbins and Theriot, 2003). It has not been clarified whether this activity of Listeria monocytogenes is due to the different structure of cell envelopes or the distinctive properties of ActA and IcsA proteins. In addition, it has been noted that because of spinning, Listeria monocytogenes can produce various curvatures for its intracellular journey (Shenoy et al., 2007). Although the authors have shed some light on how Listeria monocytogenes cruises through the cell, a realistic model still remains to be developed. A remarkable study stating that ActA-covered beads can produce unidirectional motion in vitro has been reported (Cameron et al., 1999). The authors concluded that actin-based movement strongly depends on both the size of the bead and the number of ActA molecules sitting on it. It should be emphasized that some environmental factors, e.g., saturation of the cell extract with protein (Cameron et al., 2004) can affect the curvature of a bead route. It is important to note that key players, for example, actin and the Arp 2/3 complex require ATP energy for efficient operation.
69 2.5.6.6. Bacterial ATP generation
Owing to the importance of adenosine triphosphate (ATP) energy for various biological processes, including eukaryotic actin and its prokaryotic homologues, namely, MreB and FtsA, self-assembly; actin-myosin interaction and actin-based pathogenic motility, a great deal of research should be devoted to the study of ATP production by rotary molecular motors in bacterial cells and evaluation of employment of rotary motors as power suppliers in microdevices. Although eukaryotic organisms are capable of producing ATP, they utilize different metabolic pathways. Thus, the key organelles that eukaryotic cells use for energy production are either mitochondria in animal cells (Hogeboom et al., 1947, Wagner et al., 2010) or chloroplasts in plant cells (Arnon and Whatley, 1949, Krah et al., 2010). It is believed that eukaryotic cells acquired the ―power stations‖ for ATP production about 1.5 billion years ago when bacteria settled down as endosymbionts within eukaryotic cells (van der Giezen and Tovar, 2005). Those bacteria were free-living α-proteobacteria that developed endosymbiotic relationship with the ancestor of animals (Lang et al., 1999, Gabaldon and Huynen, 2007, Chang et al., 2010b) and/or fungal (Bullerwell and Lang, 2005) cells. Moreover, as a result of endosymbiosis between the ancestor of plants and cyanobacterium, it was a plant cell gained chloroplast that became the main energy- converting organelle of the cell (Raven and Allen, 2003, Ran et al., 2010). So, eukaryotic cells produce energy but they can only do it in vivo or under carefully optimized conditions. This means that for nanotechnological application they do not fit into microdevices. Nowadays, bacteria are considered to be more robust (Kitano, 2004, Kitano, 2010) for device-realistic conditions than eukaryotic cells. Thus, some members of marine bacteria have been shown to possess metabolic plasticity (Ivanova et al., 2000a, Bong et al., 2009), which is one of the features of biological robustness. Since both the biomolecular architecture and primary function of ATPase have been discussed in subsection 2.5.5.3., in this subsection (2.5.6.6.) more attention is given to the effects of various factors on ATP production by bacteria. It was not until the beginning of the 1960s that researchers shed some light on the energetic aspects of bacterial growth (Senez, 1962, Fukui and Hirata, 1968).
70 Interestingly, even though Senez did not have enough experimental evidence, he indicated that bacteria spend ATP as an energy source on biomolecular assembly, active transport and/or arrangement of cellular structures. Since the 1970s, energy- related studies have shown that apart from consumption of ATP for self-maintenance, bacteria can just simply spill it (Neijssel and Tempest, 1976, Neijssel et al., 1990, Russell, 2007). For example, Streptococcus bovis can waste a lot of energy on ineffectual transmembrane cycling of ions (Russell and Cook, 1995). In addition, it has been noted by Neijssel & Teixeira de Mattos (1994) that cultivation conditions can affect growth energetics. In fact, exposure of bacteria to laser radiation appears to cause a progressive decrease in ATP production (Nandakumar et al., 2003). The authors assume that irradiation of bacteria triggers destruction of cellular respiration. It is well known that both bacterial growth and metabolism can be controlled through temperature adjustment during cultivation. When the environmental temperature goes below the optimal level – which is related to bacterial physiology – bacterial membrane-embedded proteins become incapable of supporting essential molecular transport because of the altered flexibility of the membrane-associated lipids (Nedwell, 1999). For example, evidence has been presented that, while a psychrophilic bacterium produces ATP equally well at 30 °C and at 4 °C, a mesophilic bacterium struggles to do it at the latter (Theron et al., 1987). In addition, quantitative ATP analysis must consider bacterial lifestyle because it has a specific impact on the efficiency of ATP production. Thus, Hong and Brown (2009) have shown that planktonic bacteria produce less ATP than corresponding ones that go through an adhesion process. The authors have proposed the charge regulation effect to account for this observation. So, according to their hypothesis, negatively-charged surface groups have a positive effect on bacterial ATP production and adhesion, while positively-charged functional groups have an opposite influence on this kind of microbial behavior. Phenotypic change has been observed to happen when a bacterium changes its lifestyle and becomes one of the members of a biofilm community (Sauer and Camper, 2001). Interestingly, the authors suggest that at the early stage of adhesion (Dunne, 2002), bacteria may partially respond to their lifestyle change
71 through quorum sensing (QS), or communication by means of specific chemical language (Allison and Gilbert, 1995, Ren et al., 2010) It should be noted that bacteria can interact with living organisms at different levels: intraspecies, interspecies, intrakingdom and interkingdom. For example, a bacterium can contact members of the same species (Dawid et al., 2009), cross-talk to bacteria from other species (Kimura et al., 2009), communicate with the members of the same (Kendall et al., 2007, Weber et al., 2007) or different kingdoms (Kimura et al., 2009, Subramoni and Venturi, 2009). In order to employ a suitable ATP producer, one should understand that such cell phenotypes as bioluminescence (Miyamoto et al., 2000, Nelson et al., 2007), which is driven by ATP production, and biofilm creation (Ahmed et al., 2009), are dependent on QSs. Nowadays, the focus is on the evaluation of the possibility of the utilization of efficient, reliable and cheap biomaterials. For example, on the utilization of microbial-based therapy instead of an antibiotic one (Defoirdt et al., 2010). Since members of α-, β-, and γ-proteobacteria have been known to produce not only signal molecules but also enzymes, such as lactonases and acylases, they can participate in the QS-mediated communication as well as the inactivate QSs of pathogens (Uroz et al., 2009). So, proteobacteria have been proven to be promising candidates for ATP generation with regard to their application for construction of parts of microdevices (see chapter 8).
2.5.6.7. Use of MreB and FtsA proteins by bacteria
Actin homologues appear to play essential roles in the lives of different kinds of bacteria. It is generally accepted that MreB is in charge of maintaining a rod-shaped form, while FtsA is responsible for enhancing cell division. Although both proteins are capable of self-assembly, only MreB displays actin-like structure and polymerization behavior. Moreover, due to its unique ability to produce morphologically different structures, it can contribute to a broad range of bacterial cell affairs. Thus, MreB has been shown to be involved in cell growth (Robertson et al., 2007), shape morphogenesis (Takacs et al., 2010, Divakaruni et al., 2007, Margolin, 2009), viability maintenance (Burger et al., 2000, Carballido-Lopez, 2006, Kawai et al., 2009b),
72 polarization (Shih et al., 2005), protein positioning (Mauriello et al., 2010), organelle production (Cowles and Gitai, 2010), sporulation (Mazza et al., 2006), DNA replication (Defeu Soufo and Graumann, 2005, Munoz-Espin et al., 2009), chromosome segregation (Soufo and Graumann, 2003), etc. It should be emphasized that bacterial demand for such a multi-skilled MreB varies across species. The majority of rod-shaped bacteria lengthen their cells by means of peptidoglycan (PG) inclusion into the lateral wall, which is coordinated by the MreB helix (Kawai et al., 2009b, Varma and Young, 2009). Over the last decade, there has been increasing experimental evidence that the loss of MreB leads to the transition of rod-shaped cells which were rod-shaped from the beginning into rounded ones. Interestingly, a spherical shape is a typical characteristic of lack of MreB not only in gram-negative bacteria, such as Escherichia coli (Wachi et al., 1987), but also in gram-positive Bacillus subtilis (Soufo and Graumann, 2003). Furthermore, in the absence of MreB, the gram-negative bacterium Caulobacter crescentus has been observed to acquire a lemon-like look (Figge et al., 2004). It is also important to note that MreB plays a key role in handling stress and aging issues by bacteria. Having both MreB and CreS, which is the intermediate filament-like analogue (Ingerson-Mahar et al., 2010), Vibrio parahaemolyticus has been proven to use the former for adaptation to food deprivation and senescence (Chiu et al., 2008, Chen et al., 2009). Wang and co-workers (2010) have demonstrated that Escherichia coli may count on the MreB-based cytoskeleton to withstand changes in external or internal pressure. The researchers have proposed that Escherichia coli can accomplish it either through building up a thicker MreB cable-like biopolymer or by varying degrees of its cross-linking with the cell wall. In addition, results from a study using MreB from Thermotoga maritima have shown that MreB has sufficient rigidity so that it may manage to maintain its shape under high bending or compressive stress (Esue et al., 2006). Naturally, there are exceptions to the common view that MreB is a master of shape management. For example, Helicobacter pylori benefits from partnership between MreB and intracellular molecules in other ways. It has been recently demonstrated in Helicobacter pylori that its MreB protein participates in
73 chromosome segregation without being involved in any shape-related activity (Waidner et al., 2009). The role of MreB in coordinating localization of proteins has been recognized via testing dynamics of its whereabouts in Caulobacter crescentus (Gitai et al., 2004). The observation that MreB can reorganize itself has led to the assumption that such a dynamic behavior is important for cell polarity. The authors discussed the possibility of transformation of a molecular polarity into a cell one, though they did not reveal its mechanism. An investigation of the importance of MreB participation in pilus assembly in Pseudomonas aeruginosa (Robertson et al., 2007, Cowles and Gitai, 2010), and the gliding motility of Myxococcus xanthus (Patryn et al., 2010, Mauriello et al., 2010), has led to the conclusion that MreB is responsible for polar positioning of different proteins including virulence factors. As discussed above, MreB is essential for some pathogens. Apart from helping them get through environmental stress, it takes part in the development of predatory bacteria. For example, Bdellovibrio bacteriovorus uses it for transformation from vibrio-shaped to elongated cells (Fenton et al., 2010). It is noteworthy that some bacteria, such as Escherichia coli, have been found to use both MreB (Wachi and Matsuhashi, 1989, Madabhushi and Marians, 2009), and FtsA (Strahl and Hamoen, 2010) proteins for cell division. Although FtsA has only one specific aptitude for reproduction by binary fission (Ping, 2010), it can assemble into homo-, oligo- (Shiomi and Margolin, 2007) and polymeric (Lara et al., 2005) structures; (biochemical properties of FtsA are discussed in subsection 2.5.3.1.2.). In fact, FtsA is the first to support the assembly of the Z-ring, or the major cytokinetic structure (Adams and Errington, 2009). So, it is capable of making contact with FtsZ, a homologue of eukaryotic tubulin (Lutkenhaus and Addinall, 1997), and anchoring it to the cell membrane. Like MreB, it plays the role of leader for certain proteins (Schmidt et al., 2004, Karimova et al., 2009). However, due to a lack of data on the differences in the biochemical and the biophysical properties of MreB and FtsA proteins among different species of bacteria, it is hard to estimate fully the contributions of MreB and FtsA to bacterial lives. Studies have demonstrated that the inherent properties of FtsA may vary across species. For example, while FtsA is
74 dynamic in Escherichia coli (Karimova et al., 2005), it is stable in Streptococcus pneumoniae (Lara et al., 2005). In order to find the suitable actin homologue for application in microdevices, evaluation should be conducted of possible MreB and FtsA proteins belonging to different species of marine proteobacteria (see chapter 9).
75
CHAPTER 3
METHODOLOGY
76 3.1. Overview
This chapter consists of five subsections. It starts with a description of methods used to study protein-surface interactions including detection and quantification techniques (see subsection 3.2.) for the development of a novel approach to the design of surfaces for microdevices based on spatial immobilization of nonmotor proteins in micro/nano-channels fabricated via laser ablation (see chapters 4 and 5). Reproduced from references (Ivanova et al., 2006c) © 2006 With kind permission from IOP; (Ivanova et al., 2003b) © 2003 With kind permission from SPIE; and (Ivanova et al., 2004f) © 2004 With kind permission from SPIE. The following subsection 3.3. covers methods that allow controlled-self-assembly of actin filaments along microchannels in a continuous-flow system (see chapter 6). Reproduced from reference (Alexeeva et al., 2005) © 2005 With kind permission from Springer + Business Media. As environmental bacteria can be used as replacements for the energy sorce, and bacterial actin homologues as replacements for eukaryotic actin, the chapter continues with methods of bacterial taxonomy of valuable ATP, MreB and/or FtsA producers (see subsection 3.4. and chapter 7). This subsection starts with bacterial isolation (see subsection 3.4.1.), and is followed by bacterial characterization (see subsection 3.4.2.) including phenotypic (see subsection 3.4.2.1.), chemotaxonomic (see subsection 3.4.2.2.), genotypic (small subsection 3.4.2.3.) and phylogenetic (see subsection 3.4.2.4.) methods. Reproduced from references (Gorshkova et al., 2003) © 2003 With kind permission from IJSEM; (Ivanova et al., 2004e) © 2004 With kind permission from IJSEM; and (Ivanova et al., 2006b) © 2006 With kind permission from Microbiological journal. The chapter continues with a description of methods used to assess ATP production by bacteria. Reproduced from reference (Ivanova et al., 2006a) © 2006 With kind permission from International Microbiology. The chapter ends with subsection 3.5. devoted to methods used to assess MreB and FtsA proteins.
77 3.2. Methods used to study protein-surface interactions
3.2.1. Protein preparation for immobilization on polymeric surfaces
Prior to depositing onto polymeric surfaces, five proteins with different biochemical properties, namely human immunoglobulin G (HIgG), human serum albumin (HSA), lysozyme, myoglobin, and α-chymotrypsin (Sigma), were prepared as stock solutions (2mg ml-1), and at the later stage, after purification by column chromatography, were diluted with TBS to working solutions (100 μg ml-1). Alexa Fluor® 546, an orange-fluorescent phalloidin conjugate, was joined to the selected proteins (2mg ml-1) with the help of the Fluoro Tag Kit (Molecular Probes). The labelling procedure was performed strictly in accordance with the instructions provided by its manufacturer. After labelling, proteins were separated from unbound fluorescent dye by means of a Sephadex G-25 gel filtration. The concentrations of labelled proteins were determined by ultraviolet-visible (UV-Vis) absorption spectroscopy (Cary 50, Varian). Based on both protein adsorption value at 280 nm and the Alexa Fluor® 546 excitation maxima measurements, the fluorescent dye/protein molar ratio of the purified proteins was estimated. The measurements were taken in phosphate buffered saline (PBS), prepared by mixing 50 mM phosphate with 150 mM NaCl in filtered (0.2 µm) Nanopure water (18.2 MΏ/cm) and adjusting pH to 7.4 at RT (about 23 ºC).
3.2.2. Polymeric film preparation
Glass slides and cover slips (#1, 0.17 mm thick, 24 x 24 mm, Knittel) were sonicated in Nanopure water for 30 min and washed copiously with filtered (0.2 µm) Nanopure water (18.2 MΏ/cm), dried under a stream of high purity nitrogen, and then primed with hexamethyldisilazane (HDMS). A 4 wt% solution of poly(methyl methacrylate) (PMMA) and poly(tert-butyl methacrilate) P(tBuMA) in propylene glycol methyl ether acetate (PGMEA) 99 % (Sigma Aldrich Co.), polysterine-co- maleic acid (PSMA) (MW~225,000) (Aldrich) in tetrahydrofurane (THF) (99.9 %)
78 XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX were spin-coated at 3000 rpm for 40 s onto HMDS-primed #1 cover glass using a Specialty Coating System spin coater (Model P6708). The coated substrates were soft baked at 85 ºC for 1h, and stored in a desiccator prior to and after gold deposition. The cover slips covered with photosensitive polymeric substrates, namely, (P(tBuMA), PMMA) were subjected to the λ 254 nm ultraviolet (UV) light for 1 h. When exposed to the direct irradiation, the original polymer grows into complex phases of amorphous hydrogenated carbon (a-C:H). This allows the formation of uniform films of approximately 100-200 nm thick depending on the polymer nature as confirmed by ELM.
3.2.3. Preparation of microfabricated structures
A Specialty Coating Systems (SCS) spin-coater (Model P6708) was used to spin-cast a 4 wt % solution of PMMA in propylene glycol methyl ether acetate (PGMEA) 99 % (Sigma Aldrich Co.) at a high speed of 3000 rpm for 40 s. Once soft baked at 85 °C for 30 min, the substrates were placed in a desiccator and taken out only for gold deposition. With the SEM sputter coating unit E5100 (Polaron Equipment Ltd) depositing gold at 25 mA for 90 s at 0.1 Torr, the gold film coat of 50 nm was formed. To incubate with bovine serum albumin (BSA) required the gold- layered substrata immersion in a 1 % w/v BSA 10mM PBS solution (pH 7.4) at RT for 1h, followed by a rinse with PBS and subsequently with Nanopure water. A laser ablation of the gold and protein coats was carried out by means of a laser (Cell Robotics workstation, 337 nm, 20 pulses/s, 10 ns/pulses). The proteins were deposited either by sinking of the entire slides with created surfaces in protein solution or by spatially addressable deposition with a pipette mounted on an xy motorized table.
79 3.2.4. Protein adsorption on surfaces
3.2.4.1. Protein adsorption on flat surfaces
The proteins, either fluorescently labeled (15 μl of 100 μg ml−1) for the visualization and quantification of the protein attachment, or unlabeled (100 μg ml−1) for other experiments (e.g., the thickness estimation) were deposited onto polymeric surfaces. Adsorption of proteins began with their being incubated on the surfaces in humidity chambers at RT for 30 min. According to adsorption kinetics experiments reported elsewhere, the adsorption of relevant proteins was qualitatively the same after incubation periods of 30 min (Vasina and Dejardin, 2003, Tremsina et al., 1998). Here, 30 min incubation was considered sufficient to achieve formation of a saturated protein monolayer on the surface. With proteins adsorbed, slides were washed three times with 10 mM PBS (pH 7.4), and then two times with filtered Nanopure water (18.2 MΏ/cm) to take away non-adsorbed proteins.
3.2.4.2. Protein adsorption on micro/nano-fabricated structures
The proteins (20 μl of 70-330 μg/ml), either fluorescently labeled for the visualization and quantification of the protein attachment or unlabeled for the thickness estimation were deposited onto micropatterned ablated areas and on native PMMA polymeric surfaces in a ‗blanket‘ mode, flooding the whole surface of the micro-assay. For the ‗blanket‘ deposition, the slides were treated as described above.
3.2.5. Protein covalent binding onto surfaces
Protein immobilization via covalent linkage was accomplished by using 1-ethyl-3-d(3- imethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) crosslinking couple. The sulfo-NHS ester, a relatively more stable compound, was first produced by introducing 2 ml of a mixed aqueous solution of 75 mM EDC and 15 mM sulfo-NHS to the selected polymeric substrates for 2 h.
80 The surfaces modified by the reaction allowed protein binding. After a 30-minute reaction with proteins dissolved in 0.01 mM PBS buffer (pH 7.4), polymeric surfaces were washed three time with 10 mM PBS (pH 7.4), and then two times with filtered Nanopure water (18.2 MΏ/cm). The prepared samples were kept in an environmental chamber prior to analysis.
3.2.6. Detection and quantification techniques
3.2.6.1. Fluorescence spectroscopy of adsorbed proteins
The attachment of fluorescently labelled proteins on the ablated area was visualised and analysed using two different microscopic systems. One was the NIKON Microphot FX microscope with a UV light source (Nikon Mercury Lamp, HBO-100 W/2; Nikon C.SHG1 super high pressure mercury lamp power supply) at 100X objective. The images were captured and recorded by a Nikon camera (FX- 35WA). The second system was a Nikon inverted microscope (Nikon Eclipse TE-DH 100W, 12V) with an attached UV light source (Nikon TE-FM Epi-Fluorescence). The related images were taken using a Nikon Charged Coupling Device (CCD) camera. The fluorescence intensities were analysed using Gel-Pro Analyser software, version 4.0.
3.2.6.2. X-ray photoelectron spectroscopy
A Kratos Ultra Imaging X-ray Photoelectron Spectrometer (XPS) with monochromotised Al Kα (photon energy = 1486.6 eV) radiation at a source power of 150 W was used to perform elemental analysis of polymeric surfaces. The dimensions of areas analysed were nominally ~700x300 m2. The acquisitions of wide scan and region spectra were done using 160eV and 20eV pass energies, respectively. Electron binding energies were calibrated against the C1s emission at 285 eV.
81 3.2.6.3. Goniometry
Contact angle values were used to evaluate the hydrophobic properties of the films. The measurements were performed on sessile drops (2 l) of Nanopure water at RT in air employing using a contact angle goniometer. The measurement tool set included XY stage fitted with a (20 l) micro syringe, a 20 x magnification microscope (ISCO-OPTIC, Germany) and a fibre-optic illuminator. Six independent readings were taken to calculate an average value.
3.2.6.4. Ellipsometry
An elipsometry technique was used for measuring changes in light polarization reflected off polymeric films to determine the thickness of them as a function of 632.8 nm red Helium-Neon laser wavelength. A null-seeking type AutoE1-III ellipsometer (Rudolph Research, USA) was aligned with respect to the 70º angle of incidence, Φ (PHI). The data were analysed using elipsometric software, Version 3.9. The film polymeric/protein thickness was calculated according to De Feijter‘s equation (1978) that allows a film thickness and refractive index values to be transformed into an amount of adsorbed protein per unit area (Γ). The mathematical relationship between these parameters is represented by the following formula: 2 Γ (ng/mm ) = df[(nf-nm)/(dn/dc)], where df is the thickness of the adsorbed film (nm), nf is the refractive index of the adsorbed film, nm is the refractivs index of the ambient, and dn/dc is the refractivs index increment, a linear function of the protein concentration. Based on this formula, the same parameter for air/solid interface can be calculated: Γ (ng/mm2) = K·t, where K is the density of the protein ≈ 1.36 g/cm3 and t is the protein thickness (nm). Using the build-in software that evaluates film thickness and its refractive index concurrently, a polymer/protein coating thickness along with the corresponding values of the refractive indices were determined. The polymer-covered slides (24x24x5 mm) were incubated with 600 μl of sample containing 0.1 mg/ml protein in 10 mM
82 phosphate-buffered saline (PBS), pH 7.4, at RT for 1 h, followed by washing with PBS and Nanopure water.
3.2.6.5. Atomic force microscopy (AFM)
Atomic Force Microscopy (AFM) characterization was carried out on a TopoMetrix Explorer (Model No. 4400-11) in the non-contact mode using 2 m and 100 m scanners. The analyses were carried out under air-ambient conditions (temperature of 23 ºC and 45 % relative humidity). Silicon tips and cantilevers with a spring constant of 42 N/m and resonant frequency of 320 KHz were used. Scanning direction was perpendicular to the axis of the cantilever and the scanning rate was typically 4 Hz.
3.2.6.6. Calculation of protein-surface parameters
The distribution of surface-related molecular characteristics, e.g. surface charge, hydrophobicity at the protein surface was computed using the Protein Surface Properties Calculator program (Connolly, 1993); estimation of molecular properties was based on Connolly‘s algorithm. The algorithm was used beyond its original purpose for the calculation of the surface-related molecular properties (i.e. surface positive and negative charges; and surface hydrophobicity and hydrophilicity using Kyte-Doolittle scale of hydrophobicity/hydrophilicity) as well as the molecular surfaces related to these properties. The program calculated the surface properties using probing balls with different radius. The charges of individual amino acids have been calculated using a semi-empirical method (PM3 as implemented in HyperChem from HyperCube Inc.) for the structures relevant to a particular pH; then averaged according to acid-base equilibrium equations; then implemented in an input table read by the program. This procedure allowed the calculation of the charges on the protein surface as function of the pH of the solution, and therefore accounted for the modulation of the adsorption by the differences between the pH and the isoelectric point of the protein. The algorithm used by the program has been reported elsewhere
83 (Cao et al., 2002, Nicolau et al., 2003). The properties of the proteins have been calculated for a radius of the probing sphere between 1.4 Å and 10 Å. The Protein Data Bank (PDB) was searched to collect various protein structures (Bernstein et al., 1977).
3.3. Methods of actin/myosin preparation
3.3.1. Actin and heavy meromyosin (HMM) preparation
Rabbit skeletal muscle myosin and heavy meromyosin (HMM) were prepared as described by Margossian and Lowey (1982). Actin was prepared from acetone powder by the method of Pardee and Spudich (1982) and labeled with Rhodamine (Molecular Probes, R415) - or Alexa 488 (Molecular Probes, A12379)-labeled phalloidin (Faulstich et al., 1988). Gelsolin was purified from brevin (plasma gelsolin) according to the protocol described by Kurokawa et al. (1990). Concentrations of myosin, and HMM were determined from absorption at 280 nm (A280nm) using extinction coefficients of 0.56 mg-1mlcm-1, 0.65 mg-1mlcm-1, respectively, and the concentration of G-actin was determined from absorption at 290 nm (A290nm) using extinction coefficient of 0.62 mg-1mlcm-1. For the preparation of 2 µm-F-actin filaments (shown in Figure 19), the procedure adopted from Oda et al. (1998) was used. Briefly, G-actin was dissolved in buffer containing 0.2 mM CaCl2, 0.5 mM ATP, 1 mM DTT, 0.01 % NaN3, 5mM Tris- HCl (pH 8.0). Gelsolin segment 1 (GLS 1) was added into G-actin solution, at molar ratios of 1:800 and protein solution was kept for 30 min on ice. After addition of KCl up to 100 mM, GLS 1 treated G-actin was incubated at RT for 1 h. In order to remove large aggregates, the protein solution was centrifuged at 10,000 g for 20 min.
84 3.3.2. Preparation of the electrostatically condensed actin bundles
Bundles of actin filaments were formed by electrostatic condensation of labeled actin filaments (2 µm) with ions of Ba (108 µM) followed by method described by Angelini et al. (2003).
3.3.3. Preparation of the polymeric surfaces
Glass slides or cover slips (0.17 mm thick, 24 x 24 mm, Knittel) were sonicated in Nanopure water for 30 min and washed copiously with filtered (0.2 m) Nanopure water (18.2 M /cm), dried under a stream of high purity nitrogen, and then primed with hexamethyldisilazane (HMDS). The following polymers were used: poly(methyl methacrylate), 4 wt % solution of PMMA in propylene glycol methyl ether acetate PGMEA 99 % (purchased from Sigma Aldrich Co.) with an activator, 0.5 % of triphenylsulfonium triftalate (purchased from Sigma Aldrich Co.); poly(styrene-maleic acid), 2 wt % solution of PSMA in tetrahydrofurane (THF) 99.7 %; poly(tert-buthyl methacrylate), 4 wt % solution of P(tBuMA) in propylene glycol methyl ether acetate, PGMEA 99 %. The deposition of the polymers was done at 2000 rpm for 40 s using a Specialty Coating Systems spin coater (Model P6708). The coated substrates were then soft baked at 85 °C for 1 h. The photosensitive polymers [P(tBuMA), PMMA] were activated by irradiation with UV light of λ 254 nm for 1 h. The microstructures were initially fabricated by simple mechanical scratching of the polymeric surfaces using a stainless steel syringe needle (22 gauge, Sigma) and later on were readily accomplished by using commercially available microscope adaptations to ensure the reproducibility. The needle was held in a fix position vertically in relation to a polymeric surface and moved along the long axis of the glass slide, which was attached to a computer-controlled XYZ stage. The system (Cell Robotics, Inc.) was based on a Nikon Eclipse TE300 inverted microscope. All the operation procedures have been performed according to the Manual provided by the manufacturer.
85 3.3.4. Protein immobilization on the polymeric surfaces in the flow cell
The cell was constructed from a coverslip with fabricated microstructures on the polymeric surfaces. For covalent attachment, first the N-hydroxysulfosuccinimide, NHSS, (Pierce) ester was formed by mixing of 75 mM 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride, EDC, (Sigma) and 15 mM NHSS on selected polymeric surfaces for 1 h. The slides were rinsed 3 times with Nanopure water (18.2 M /cm), and then used for flow cell preparation. Two parallel strips of double sticky tape were placed symmetrically about 20 mm apart on one coverslip, another coverslip was placed on the top and pressed gently. Labeled G- actin (54 nM) was repeatedly (3 times) infused in the flow cell from one side while letting the solution freely pass through the cell from the other side. G-actin was either adsorbed physico-chemically or covalently bound (depending on the polymer) on polymeric surfaces and left for polymerization in the flow of buffer A containing 10 mM DTT, 10 mM ATP, pH 7.0 at 4 ˚C, during 1.5 h. The buffer flow rate of 0.06 ml per min was controlled by peristaltic pump (Ismatec RS 232). Nitrocellulose-coated coverslip/s was/re used as the reference substrate for the in vitro motility assay and/or actin immobilisation experiments. The nitrocellulose- coated glass was prepared as described elsewhere (Kron et al., 1991). The assay buffer solution contained 5 mM MgCl2, 20 mM KCl, 0.1 mM EGTA, 10 mM MOPS, 10 mM DTT, 2nM ATP, 10 mM MOPS (pH 7.2).
3.3.5. Beads functionalization
Monoclonal anti-skeletal myosin (MY32) (mouse IgG isotype, Sigma M4276) was covalently grafted via 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) (EDC) on the beads (1 μm) Dynabeads M450 (Dynal Biotech), according to Manufacturer‘s protocol. The Anti-HMM-coated Dynabeads were stored at 4 °C in storage buffer
(10 mM phosphate buffer, pH 7.4, 0.1 % BSA, 150 mM NaCl, 20 mM NaN2). Before the experiments the beads were incubated in solution of HMM 0.1 mg ml-1 for 60 min at 4 ˚C.
86 3.3.6. Fluorescence microscopy
Self-assembled actin filaments were observed at room temperature (22-24 ˚C) with an epifluorescence inverted microscope (Olympus IX71/IX51) and/or NIKON Microphot FX microscope system with a UV light source (Nikon Mercury Lamp, HBO-100 W/2; Nikon C.SHG1 super high pressure mercury lamp power supply) at 100 x NA 1.4 oil-immersion objective with Rhodamine or Alexa 488 specific filters. The images were recorded with an image-intensified CCD camera system (Coolview FDI) at eight frames per second, 1392 (h) x 1040 (v) pixels, 6.45 μm square. The recorded images were processed using ImagePro Plus (version 5.0 for Windows).The velocity of the beads was determined using ImagePro Plus software. Velocities are reported as the mean and standard deviation for at least six beads.
3.3.7. Scanning electron microscopy (SEM)
A conventional scanning electron microscopy (SEM) JEOL JSM840 was used for F-actin bundles examination. The cover slides (supporting the filaments) were mounted on pin-type aluminium SEM mounts with double-sided conducting carbon tape and then coated in a DYNAVAC CS300 coating unit with carbon and gold to achieve a conductivity of the specimen surface prior to the SEM examination. The thickness of the coating was not measured but would be in the order of a few nanometers. The excitation voltage (kV), the magnification, and working distance (WD) are given on the lower part of Figure 20. The working distance is the distance between final aperture and the specimen surface. The images are secondary electron images (SE). The sample was not tilted, j.e. the electron beam ―hits‖ the specimen surface at 90°.
3.3.8. X-ray photoelectron spectroscopy
Elemental analyses of polymeric surfaces were carried out on a Kratos Ultra Imaging X-Ray Photoelectron Spectrometer (XPS), using monochromatised Al Kα
87 (photon energy = 1486.6 eV) radiation at a source power of 150 W. The analysis areas were nominally ~ 700x300 m2. Wide scan and region scan spectra were acquired using 160eV and 20eV pass energies, respectively. Electron binding energies were calibrated against the C1s emission at 285 eV.
3.3.9. Rheological measurements
TA Instruments controlled stress rheometer AR 2000 with cone and plate measurement geometry (40 mm, 2°) was used to test liquid viscosity. Measurements were carried out at the controlled temperature 30 °C with accuracy 0.1 °C in shear rate sweep mode. Results of measurements were processed using ―Rheology Advantage‖ software package provided by company manufacturer.
3.4. Methods of bacterial taxonomy
3.4.1. Bacterial isolation
3.4.1.1. Isolation of gram-negative bacteria
3.4.1.1.1. Isolation of Marinobacter excellens
Bacteria of the genus Marinobacter were isolated from sediments collected in Chazhma Bay, Sea of Japan. This work was part of the taxonomic investigation of free-living marine bacteria dwelling in the Bay, sediments of which were contaminated by radionuclides (Ivanova et al., 2002d). Sediment samples were collected in 2001 from a depth of 0·5 m (salinity, 32 ‰; temperature, 12 °C) at Chazhma Bay, Sea of Japan. Bacteria were isolated by plating 0·1 ml of a suspension of 1 g sediment in 10 ml sterilized natural sea water onto marine 2216 agar plates (Difco) or plates with medium B, containing 0.2 % (w/v) Bacto Peptone (Difco, USA), 0.2 % (w/v) casein hydrolysate (Merck, USA), 0.2 % (w/v) Bacto Yeast
Extract (Difco, USA), 0.1 % (w/v) glucose, 0.002 % (w/v) KH2PO4, 0.005 % (w/v)
88 MgSO4 ·7H2O and 1.5 % (w/v) Bacto Agar (Difco, USA), 50 % (v/v) of natural seawater and 50 % (v/v) of distilled water at pH 7.5-7.8, as described elsewhere (Ivanova et al., 1996). Plates were incubated aerobically at RT for 5, 7 or 10 days. Strains were stored at -80 °C in marine 2216 broth (Difco) supplemented with 20 % (v/v) glycerol. In total, 145 viable bacterial strains have been recovered from sea water and sediment samples. During isolation studies, bacteria of different taxonomic groups, including Shewanella, Halomonas, Pseudoalteromonas and Kocuria, have been isolated (Ivanova et al., 2002d). From this collection, several bacterial strains with Marinobacter-like phenotypes were identified initially and studied further in detail.
3.4.1.1.2. Isolation of Sulfitobacter delicatus and Sulfitobacter dubius
This study extends our previous investigations into the biodiversity of marine proteobacteria from the Sea of Japan, the north-west Pacific Ocean and other geographical locations (Ivanova et al., 1996, Ivanova et al., 1998, Ivanova et al., 2000b, Sawabe et al., 2000). During isolation studies, bacteria of various taxonomic groups, including species of Shewanella, Marinobacter, Halomonas and Pseudoalteromonas, have been isolated (Kurilenko et al., 2001).. However, only two strains with Sulfitobacter -like phenotypes have been tentatively identified. The strains examined in this study were isolated from a starfish (Stellaster equestris) and sea grass (Zostera marina). The starfish was collected in October 1998 at a depth of 100 m (salinity 30 ‰, temperature 15 °C) in the South China Sea (26° 28·3' N 122° 29·0' E). The sea grass was collected in July 1998 at a depth of 5–8 m (salinity 33 ‰, temperature 12 °C) at the Pacific Institute of Bio-organic Chemistry Marine Experimental Station, Troitza Bay, Gulf of Peter the Great, Sea of Japan. The starfish and sea grass were pre-rinsed in sterilized sea water and pieces of tissue (about 3 g) were aseptically removed. Strains were isolated by plating samples of tissue homogenate (0·1 ml) onto marine agar 2216 (Difco) plates and medium B plates (the
89 composition of the medium is described in subsection 3.4.1.1.1.) and preserved in marine broth supplemented with 30 % glycerol at –80 °C.
3.4.1.2. Isolation of gram-positive bacteria
3.4.1.2.1. Isolation of Planococcus maritimus
Brown algae Fucus evanescens were collected by scuba divers in mid-summer (July 1999) at the Kraternaya Bight, Kuril Islands, N.W. Pacific Ocean, during the 23rd scientific expedition of the R/V ―Akademician Oparin‖. The enrichment experiments and bacterial isolation were peformed as described elsewhere (Ivanova et al., 2002a, Ivanova et al., 2002b) with the modification of adding a protein inhibitor for endo-(1->3)-beta-D-glucanases (Yermakova et al., 2002) to the enrichment culture. Cultures were maintained on Marine agar plates and medium B (the composition of the medium is described in subsection 3.4.1.1.1.) and in marine broth supplemented with 30 % of glycerol at –80 °C. All isolates were streaked on agar plates from broth cultures every six months to ensure purity and viability.
3.4.2. Bacterial characterization
3.4.2.1. Phenotypic analysis
Unless indicated otherwise, the phenotypic characteristics of gram-negative, namely, Marinobacter and Sulfitobacter species were studied using standard procedures (Baumann et al., 1972, Smibert and Krieg, 1994) as described previously (Ivanova et al., 1996, Ivanova et al., 1998). Phenotypic characteristics of gram- positive species, Planococcus maritimus, were assessed using standard procedures (Ivanova et al., 1996, Smibert and Krieg, 1994).
90 3.4.2.1.1. General phenotypic tests
The following physiological and biochemical properties were examined: oxidation/fermentation of glucose (Hugh and Leifson, 1953); Gram staining; nitrate and nitrite reduction; catalase (with 5 % H2O2) and oxidase (Kovacs, 1956) activities; gelatine liquefaction production of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, poly-β-hydroxybutyrate, and acetoin (Voges-Proskauer test); sodium requirement [0,1,3,6,8,10,12,15, 20) (w/v) NaCl]; indole and H2S production; the ability to hydrolyse starch, Tween 80, DNA, casein, chitin (1 %, w/v), alginate (0.1 %, w/v) and agar. The temperature range for bacterial growth was tested on marine agar plates incubated at 4, 10, 30, 35, 37, 42 and 45 ºC. To assess the effect of pH on bacterial growth, bacteria were cultivated in a pH range between 4.5 and 12.0; pH of medium was adjusted using HCl and NaOH. After 24 h-incubation of bacteria in marine broth, measurements of the optical density of the cultures at 660 nm were carried out. Cultures were incubated on a rotary shaker at 160 rpm for 24 h at 25 ºC. Haemolytic activity of the strains was detected on blood agar containing 40 g trypticase soy agar in 50 ml sheep blood and 950 ml water. Tests for utilization of various organic substrates as sole carbon sources (as described in subsection 3.4.2.1.1.2.) at a concentration of 0·1 % (w/v) were performed in 10 ml liquid BM medium (Baumann et al., 1972).
3.4.2.1.1.1. Microscopic examination
Cellular morphology and gram-stain were examined after 24 h incubation on medium B. The motility was determined by observing 18-h-old cultures under phase- contrast light microscope. To test for spreading growth and gliding motility, strains were grown on medium B with a reduced peptone content (0·2 g l–1). Motility was verified using phase-contrast microscopy (Nikon) of hanging drop preparations. Electron micrographs of negatively stained cells were prepared using a Zeiss EM 10 CA electronmicroscope (80 kV). A drop of particle-free (autoclaved and ultra-
91 centrifuged) distilled water was placed on the culture. The sample (30 μl) of resulting bacterial suspension was applied to carbon-and Formvar-coated 400-mesh copper grids, a drop of 1·25 % uranyl acetate was added and the bacteria were allowed to adhere for 1 min at RT. Superfluous liquid was gently removed using a piece of filter paper. Atomic force microscopy (AFM) was employed to characterize the morphology of the cells, by using a TopoMetrix Explorer (model no. 4400-11; ThermoMicroscopes) in the non-contact mode, with either a 2 µm liquid scanner (0·8 µm z-range; model no. 5270-00) or a 100 µm liquid scanner (10 µm z-range; model no. 5180-00). Silicon cantilevers with a spring constant of 42 N m-1 and resonant frequency of 320 kHz (model no. 1650.00) were used; all imaging was performed in ethanol. All samples were prepared on freshly cleaved mica.
3.4.2.1.1.2. Utilization of organic substrates
The tests for utilization of mono- and disaccharides, namely, D-arabinose, cellobiose, D-fructose, D-galactose, D-glucose, glycerol, lactose, maltose, mannitol, D-mannose, D-rhamnose, D-ribose, tagatose, L-fucose, sucrose, trehalose and D- xylose at a concentration of 0.1 % (wt/vol) were carried out in 10 ml per tube of liquid BM medium (Baumann et al., 1972). The ability to oxidize 95 carbon sources was tested using both test tubes and Biolog GN microplates (Rüger and Krambeck, 1994) as described elsewhere (Ivanova et al., 1998). The range of the substrates utilized according to Biolog profile is provided in the species descriptions.
3.4.2.1.1.3. Degradation of macromolecules
Degradation of macromolecules was tested using medium B. Chitin (1 %, w/v), elastin (0.1 %, w/v), and alginate (sodium salt; 0.1 %, w/v), hydrolysis was determined by development of clear zones around colonies. Cellulose hydrolysis was tested by using both cellulose overlay plates (1 % carbomethylcellulose), and
92 filter paper strips. The later strips were examined in liquid cell culture for dissolution (Smibert and Krieg, 1994). Starch, casein and gelatin hydrolysis was tested by the methods of Smibert and Krieg (1994).
3.4.2.1.1.4. Cytotoxic and antibacterial activities
Cytotoxic and antibacterial activities were assessed by the agar-diffusion assay, based on methods described elsewhere (Barry, 1980, Sasaki et al., 1985). Cultures (0·1 ml) of indicator test strains were spread on tryptic soy agar plates in which circular wells (diameter, 10 mm) had been cut. Areas of inhibited bacterial growth were measured after incubation for 48 h at 28 °C. Zones of inhibited growth of the indicator strains surrounding the wells were observed; mean diameters were measured and 10 mm was subtracted to represent the diameter of the well. Antimicrobial activities were tested against Staphylococcus aureus CIP 103594, Escherichia coli ATCC 25290, Proteus vulgaris NBRC 3851T, Enterococcus faecium CIP 104105, Bacillus subtilis ATCC 6051T and yeast Candida albicans KMM 455.
3.4.2.1.1.5. Susceptibility to antibiotics
Susceptibility to antibiotics was tested by the routine disc-diffusion plate method using medium B agar and disks (Oxoid) impregnated with following antibiotics: kanamycin (30 µg), ampicillin (10 µg), benzylpenicillin (10 µg), streptomycin (30 µg), gentamicin (30 µg), lincomycin (30 µg), neomycin (30 µg), polymyxin B (25 µg), and tetracycline (30 µg). Agar plates were seeded with light lawn of bacteria and incubated at 28 ºC for 24 h. A distinct inhibition zone indicated susceptibility to antibiotic.
3.4.2.1.2. Species-specific phenotypic tests
The ability of Sulfitobacter species to oxidize sulfite was tested as described by Pukall et al. (1999).
93 3.4.2.2. Chemotaxonomic methods
3.4.2.2.1. Polar lipid (PL) analysis
Lipids were extracted according to Bligh & Dyer (1959). Two-dimensional micro-TLC of polar lipids was carried out using the method of Svetashev &
Vaskovsky (1972), with chloroform/methanol/benzene/ 28 % NH4OH (65:30:10:6, by vol.) for the first dimension and chlorophorm/methanol/benzene/acetone/acetic acid/water (70:30:10:5:4:1, by vol.) for the second dimension (Vaskovsky and Terekhova, 1979). Non-specific detection of lipids on the TLC was performed with a
10 % solution of H2SO4 in methanol at 180 ºC (Kates, 1986). The following specific reagents were used: for phospholipids, see Vaskovsky et al. (1975); 2 % ninhydrin in acetone for amino-containing lipids; Dragendorff's reagent for choline lipids; and anthrone spray (0·5 % anthrone in benzene and 5 % H2SO4 in water) for glycolipids. Phosphorus analysis was carried out according to Vaskovsky et al. (1975).
3.4.2.2.2. Fatty acid (FA) analysis
Analyses of fatty acid methyl esters were carried out on a Shimadzu GC-14A GC with an FID using both a non-polar SPB-5 fused-silica column (30 m mmx0·25 i.d.) at 210 °C and a polar Supelcowax-10 fused-silica column (30 mx0·25 mm i.d.) at 200 °C. The FID was operated at 240 °C. Helium was used as the carrier gas (Carreau and Dubacq, 1978, Christie, 1988). Catalytic hydrogenation of fatty acid methyl esters was carried out as described by Appelquist (1972).
94 3.4.2.3. Genotypic analysis
3.4.2.3.1. DNA GC content determination
DNA was isolated from the strains by following the method of Marmur (1961). The G+C content of the DNA was determined by using the thermal denaturation method (Marmur and Doty, 1962).
3.4.2.3.2. DNA hybridization
Reference strains were routinely cultured on marine agar 2216 plates (Difco). DNA–DNA hybridization was performed spectrophotometrically and initial renaturation rates were recorded as described by De Ley et al. (1970). The method described by Christensen et al. (2000) was used to hybridize DNAs from Marinobacter and Sulfitobacter species. DNA–DNA hybridization experiments were performed by using covalent attachment of the DNA in micro-wells. Briefly, 300 ng DNA (400–700 bp fragments) diluted in ice-cold 1-methylimidazole (Sigma), pH 7·0 and 25 µl 40 mM 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide -1 (EDC; Sigma) dissolved in sterilized Nanopure H2O (18·2 M cm ) was added to each well of NucleoLink micro-well strips (Nalge Nunc International) to bind the DNA covalently to the NucleoLink surface. After incubation at 50 °C for 18 h (without shaking), unbound DNA was washed continuously. DNA labelling with photoactivated biotin, hybridization, detection and quantification were performed as described by Christensen et al. (2000). Marinobacter species were hybridized with the following type strains: Marinobacter hydrocarbonoclasticus (ATCC 49840T), Marinobacter aquaeolei (ATCC 700491T) and Marinobacter litoralis (KCCM 41591T). Sulfitobacter species were hybridized with Sulfitobacter pontiacus DSM 10014T, Sulfitobacter mediterraneus ATCC 700856T and Sulfitobacter brevis ATCC BAA-4T, obtained from the German Collection of Microorganisms and the American
95 Type Culture Collection and Staleya guttiformis DSM 11458T generously gifted by P. Hirsch (Institut für Allgemeine Mikrobiologie, Kiel, Germany).
3.4.2.4. Phylogenetic analysis
3.4.2.4.1. 16S rRNA gene analysis
DNAs from Sulfitobacter species for PCR were prepared using the Promega Wizard genomic DNA extraction kit according to the instruction manual. DNA templates (100 ng) were used for PCR amplification of small subunit rRNA genes as described previously (Sawabe et al., 1998a, Sawabe et al., 1998b). The PCR conditions were as follows: initial denaturation step at 94 °C for 180 s, annealing step at 55 °C for 60 s and extension step at 72 °C for 90 s. The thermal profile consisted of 30 cycles. The amplifcation primers used in this study gave a 1.5 kb PCR product. PCR products were purified using the Promega Wizard PCR preps DNA purification kit and were sequenced directly by using a Taq FS dye terminator sequencing kit (ABI) according to the protocol recommended by the manufacturer. DNA sequencing was performed with an Applied Biosystems model 373S automated sequencer. The 16S rDNA sequences of Sulfitobacter species were aligned automatically and then manually by reference to a database of previously aligned relevant bacterial 16S rDNA sequences. Phylogenetic trees were constructed according to three different methods (BIONJ, maximum-likelihood and maximum-parsimony). For the neighbour- joining (NJ) analysis, a distance matrix was calculated according to Kimura's two- parameter correction. Bootstraps were done using 500 replications, BIONJ and Kimura's two-parameter corrections. BIONJ was performed according to Gascuel (1997), and the maximum-likelihood (ML) and maximum-parsimony (MP) data were from PHYLIP (Phylogeny Inference Package, version 3.573c, distributed by J. Felsenstein, Department of Genetics, University of Washington, Seattle, WA, USA). Phylogenetic trees were drawn using NJPLOT (Perriere and Gouy, 1996) and CLARIS DRAW software for Apple Macintosh computers.
96 The 16S rRNA genes of Marinobacter and Planococcus species were amplified and sequenced by MIDI Laboratories (Newark, DE, USA). Briefly, primers used for amplification corresponded to Escherichia coli positions 5 and 1540. Amplification products were purified by using Microcon 100 (Millipore) molecular mass cut-off membranes and checked for quality and quantity on an agarose gel. Cycle sequencing of 16S rRNA gene amplification products was carried out by using AmpliTaq ES DNA polymerase and rhodamine dye terminators (Applied Biosystems). Samples were electrophoresed on an ABI Prism 377 DNA sequencer. Related sequences were selected according to previous phylogenetic analyses of a database of previously aligned bacterial 16S rRNA gene sequences and BLAST searches against the latest release of the EBI (European Bioinformatic Institute). In a preliminary analysis, relevant sequences were selected according to the result of a BLAST query. The construction of initial tree for each microbial species allowed closely related sequences to be selected from reference strains when available. When several sequences were available for a type species, the sequence with the fewest ambiguities was selected. Phylogenetic trees were constructed using three different methods: BIONJ, ML and MP. For the BIONJ analysis, distance matrices were calculated using Kimura's two-parameter correction. BIONJ analysis was performed according to Gascuel (1997). ML and MP were from PHYLIP (Felsenstein, 1985, Felsenstein, 1993). Phylogenetic trees were drawn using NJPLOT (Perriere and Gouy, 1996).
3.5. Methods used to assess ATP production by bacteria
3.5.1. Bacterial strains
The type strains and environmental (marine) bacterial isolates belonging to the 17 genera were used in this study (Table 1). Type strains were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), the Culture Collection of Pasteur Institute (CIP, Paris, France), the German Collection of Microorganisms (DSM, Braunschweig, Germany), the Institute of Molecular and Cellular Biosciences (IAM, Tokyo, Japan), and the National Collection of Industrial
97 and Marine Bacteria (NCIMB, UK). Other strains were from the Collection of Marine Microorganisms (KMM Vladivostok, Russia), and kindly provided by U. Simidu (University of Tokyo, Japan), M. Akagawa-Matsushita (University of Occupational and Environmental Health, Kitakyushu, Japan), P. Hirsch (Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel, Germany), J. Guinea, T. Sawabe (Hokkaido University Hakodate, Japan), A. Sánchez-Amat (University of Murcia, Spain), and C. Holmstrom (The University of New South Wales, Sydney, Australia). Strains used in this study were routinely cultured on Marine Agar 2216 (Difco, USA) and PYGV agar plates (Labrenz et al., 2000) and stored at -80 °C in marine broth 2216 (Difco) supplemented with 20 % (v/v) of glycerol.
98 Table 1. Strains and environmental (marine) bacterial isolates used in the study.
Genera/species Strain/ isolate Genera/species Strain/isolate Planomicrobium Marinobacter alkanoclasticum NCIMB 13489T hydrocarbonoclastis ATCC 49840T Planococcus antarcticus DSM 14505T Marinobacter litoralis KCCM 41591T Planomicrobium Marinobacter spp. 2-57, R9SW1 koreense YCM 10704T Marinobacterium Planomicrobium georgiensis ATCC 700074T mcmeekinii ATCC 700539T Microbulbifer T Planomicrobium hydrolyticus ATCC 700072 okeanokoites NCIMB 561T Cobetia marina F 6, F 15, F 57 T Planomicrobium Alteromonas macleodii ATCC 27126 psychrophylum DSM 14507T ‗Alteromonas infernus’ GY785 Planococcus citreus DSM 20549T Pseudoalteromonas T Planococcus kocurii DSM 20747T atlantica ATCC 19262 Planococcus Pseudoalteromonas maritimus KCCM 41587T carrageenovora ATCC 43555T KMM 3738, Pseudoalteromonas citrea ATCC 29719T KMM 3636, F 90 Pseudoalteromonas T Kocuria palustris CIP 105971 distincta ATCC 700518T T Kocuria polaris DSM 14382 Pseudoalteromonas Kocuria rhizophila CIP 105972T elyakovii ATCC 700519T Kocuria rosea CIP 71.15T, Pseudoalteromonas T KMM 3812 espejiana ATCC 29659 Kocuria varians CIP 8173T Pseudoalteromonas Bacillus algicola KMM 3737T haloplanktis ATCC 14393T Brevibacterium celere KMM 3637T, Pseudoalteromonas F 81, F 59 issachenkonii KMM 3549T Microbacterium sp. F 60 Pseudoalteromonas T Formosa algae KMM 3553 , maricaloris KMM 636T F 83 Pseudoalteromonas T Cytophaga lytica DSM 7489 marinaglutinosa NCIMB 1770T T Ruegeria algicola CIP 104267 Pseudoalteromonas Ruegeria spp. 1-30, R10SW5 nigrifaciens ATCC 19375T Erythrobacter vulgaris 022-2-9
99 Sulfitobacter brevis ATCC BAA-4T Pseudoalteromonas Sulfitobacter delicatus 2-77 ruthenica KMM 300T (=KMM 3584T) Pseudoalteromonas Sulfitobacter dubius Z-218 tetraodonis ATCC 51193T T (=KMM 3554 ) Pseudoalteromonas Sulfitobacter undina ATCC 29660T T mediterraneus ATCC 700856 Pseudoalteromonas spp. Z 2/2, SUT 3, T Sulfitobacter pontiacus DSM 10014 SUT 4, SUT 5, Sulfitobacter spp. Fg 1, Fg 36, SUT 11, SUT 12, Fg 116, Fg 117 SUT 13 Staleya guttiformis DSM 11458 T Shewanella affinis KMM 3821, Marinobacter aquaeolei ATCC 700491T KMM 3586, T Marinobacter excellens KMM 3809T, Fg 86 KMM 3587 T Shewanella colwelliana ATCC 35565 T Shewanella gelidimarina ACAM 456 T Shewanella japonica LMG 19691 T Shewanella pacifica KMM 3587 , R10SW14, R10SW16 T Shewanella pealeana ATCC 700345 T Shewanella woodyi ATCC 51908
3.5.2. Polymeric surface preparation
Poly(tert-butyl methacrylate) (Sigma-Aldrich, St. Louis, MO, USA) and mica (Ted Pella, Redding, CA, USA) were used as surfaces. The surfaces were prepared as described elsewhere (Ivanova et al., 2002e). Briefly, PtBMA (47 kDa, molecular weight/polydispersity, Mw/Mn = 2.33) dissolved in cyclohexanone (99.9 %) (Sigma- Aldrich) was spin-coated (substrates: #1 glass cover slips, 10-mm diameter), after previously being primed with hexamethyldisilasane (HMDS, Sigma-Aldrich). The substrates were sonicated in PriOH for 30 min, washed with copious amounts of filtered (0.2 mm) Nanopure water, and dried under a stream of high-purity nitrogen. The polymeric films were spin-coated on primed glass substrates by using tetrahydrofuran (THF) solution at concentrations of 2-5 mg/ml. The primer was spun 100 at 1000 rpm and polymers at 3000 rpm with a ramp acceleration of 1000 rpm using a spin coater (Model P6708, Specialty Coating Systems, Indianapolis, IN, USA). Finally, polymeric slides were baked at 95 ºC for 60 min. Muscovite mica sheets were freshly cleaved and used as received.
3.5.3. Contact angle measurements
Advancing contact angles were measured on sessile drops (2 ml) of Nanopure water at RT in air, using a contact-angle meter constructed from an XY stage fitted with a (20 ml) microsyringe, a 20× magnification microscope (Isco-Optic, Göttingen, Germany), and a fiber-optic illuminator. The images were captured using a digital camera (Aiptek, Tokyo, Japan) and analyzed using PaintShop Pro (Jasc Software). Observed values were averaged over six different readings. We defined the PtBMA polymeric surface as being hydrophobic with a measured water contact angle of 91º and mica as being hydrophilic with a much smaller contact angle of 5º.
3.5.4. Bacterial growth and sample preparation
For initial screening, bacterial suspensions of freshly grown cells (1.0-2.0 × 108 cells/ml, optical density, OD660 = 0.13-0.2) were used for inoculation of 0.5 l of Marine Broth 2216 (Difco). Bacteria were cultured for 18-24 h at RT without any growth-limiting factors and were harvested at the late exponential phase of growth. The growth phases were monitored spectrophotometrically. Bacterial strains were grown on Marine Agar 2216 plates at 28 °C for 48 h. Polymeric slides and freshly cleaved mica disks were placed in sterilized Nunc multidishes (12 wells). The polymer-lined wells were inoculated with exponential-phase cultures (3 ml). The cells were plated in duplicate for each polymeric surface and the experiment was repeated 12 times to monitor cell growth and ATP generation every 4 h over the course of the experiment. Cell density was adjusted to OD660 = 0.13 ± 0.05 by the addition of phosphate-buffered saline (PBS) containing 50 mM phosphate and 150 mM NaCl (pH 7.4). A 300-µl cell aliquot was added into 2700 µl of Marine Broth 2216. The same
101 suspension of each strain (3 ml, in triplicate) was added to an empty well and served as a control. Every 4 h, a correspondent aliquot (10 µl, in triplicate) of bacterial suspension was removed and the amount of extracellular ATP was measured. The optical density of bacterial cells in the wells was also monitored. The biofilms formed on the polymeric surfaces by statically grown bacteria were rinsed three times with PBS, and the attached cells were carefully scraped off and resuspended in 1 ml of PBS to determine the level of intracellular ATP.
3.5.5. Bioluminescence assay for ATP determination
Bioluminescence was monitored with a fluorimeter (FluorStar Galaxy, Offenburg, Germany) in white opaque 96-well microtiter plates (Nunc, Copenhagen, Denmark). The internal cellular ATP concentration and the external ATP concentration in the medium were analyzed separately. ATP generation was detected using the Enliten ATP Detection Kit (Promega). The homogeneous assay procedure involves adding a single reagent directly to bacterial cells cultured in medium and measuring ATP as an indicator of metabolically active cells. The procedure was carried out according to the manufacturer's protocol. Each well contained 10 µl of the bacterial suspension sample. Bioluminescence was recorded after the automatic injection of 90 µl rLuciferase/ Luciferin (rL/L) reagent. Light measurements were made in triplicate for each sample and for the negative control. ATP values are given as relative units, which define the amount of light emitted per unit of cell density. The levels of extracellular ATP were measured directly in bacterial suspension, and the levels of intracellular ATP in samples prepared via extraction of ATP by 1 % trichloroacetic acid (TCA, Sigma-Aldrich).
3.5.6. Cell-surface characterization by AFM
AFM characterization of the cell surfaces was carried out on a TopoMetrix Explorer (model no. 4400-11, Sebastopol, CA, USA) in both the non-contact and normal contact modes using 2-µm and 100-µm scanners. The analyses were done under air-
102 ambient conditions (23 °C, 45 % relative humidity). Pyramidal silicon-nitride tips attached to cantilevers with a spring constant of 0.032 N/m were used in the contact mode, whereas silicon tips and cantilevers with a spring constant of 42 N/m and a resonant frequency of 320 kHz were used in the non-contact mode. The scanning direction was perpendicular to the axis of the cantilever and the scanning rate was typically 4 Hz.…………………
3.6. Methods used to assess MreB and FtsA proteins
3.6.1. Analysis of mreB and ftsA genes
DNAs from 24 bacterial species belonging to the class γ-Proteobacteria, namely, Cobetia marina LMG 2217T, Marinobacter hydrocarbonoclasticus ATCC 49840T, Marinobacter aquaeolei, Pseudomonas fluorescens DSM 50030T, Pseudomonas extremorientalis KMM 3447T, Pseudoalteromonas issachenkonii KMM 3549 T, Pseudoalteromonas nigrifaciens ATCC 19375T, Pseudoalteromonas haloplanktis ATCC 14393T, Pseudoalteromonas atlantica ATCC 19262T, Alteromonas macleodii ATCC 27126T, Alteromonas addita R10SW13T, Oceanimonas doudoroffiin ATCC 27123T, Oceanimonas smirnovii 31 -1T, Marinomonas communis ATCC 27118T, Marinomonas vaga ATCC 27119T, Marinomonas pontica 46-16T, Aliivibrio fischeri DSM 507T, Idiomarina zobelii KMM 231T, Idiomarina loihiensis, Idiomarina baltica, Shewanella woodyi ATCC 51908T, Shewanella affinis KMM 3587T, Shewanella waksmanii KMM 3823T, Shewanella japonica KMM 3299T, 7 species belonging to the class α-Proteobacteria, namely, Sulfitobacter pontiakus DSM 10014T, Sulfitobacter mediterraneus ATCC 700856T, Sulfitobacter delicatus KMM 3584T, Sulfitobacter sp. Fg 107, Sulfitobacter sp. RIOSW6, Loktanella rosea Fg 1, Loktanella vestfoldensis and one Salegentibacter flavus Fg 69T relating to the CFB group were isolated following the method of Marmur (1961). MreB and FtsA genes were amplified, cloned and sequenced using specifically designed primers by AGRF Ltd (University of Queensland, Australia). Aligned and translated by AGRF sequences were utilized for phylogenetic analysis. Translated
103 sequences were compared with available MreB and FtsAs protein sequences of Escherichia coli K-12 substr. DH10B (γ-Proteobacteria); Bacillus subtilis, Listeria monocytogenes (Firmicutes); Thermotoga maritima MSB8 (class Thermotogae) and rabbit (Oryctolagus cuniculus) actin, alpha skeletal muscle. Also, FtsAs were compared with Streptococcus pneumoniae (Firmicutes) . Treecon software (Van de Peer and De Wachter, 1994) was used to build and draw NJ phylogenetic trees.
3.6.2. Computation of MreB and FtsA protein parameters
To estimate physico-chemical properties of bacterial MreB and FtsA proteins including their instability, ProtParam software (Gasteiger et al., 2005) was used.
104
CHAPTER 4
IMMOBILIZATION OF PROTEINS ON FLAT
SURFACES
105 4.1. Overview
This chapter presents a practical methodological approach to monitor the passive adsorption and covalent immobilization of two proteins, human immunoglobulin (HIgG) and lysozyme (LYZ), on modified poly(tert-butyl methacrylate) (PtBMA) surfaces. Reproduced from reference (Ivanova et al., 2006c) © 2006 With kind permission from IOP. As chemistry of surface affects the efficiency of protein immobilization, chapter starts with a characterization of PtBMA film. The chapter continues with an investigation of protein–surface interactions via physicochemical adsorption and covalent immobilization to clarify whether the protein immobilization behavior of these two attachment processes yields similar packing densities. Here, results of x-ray photoelectron spectroscopy, ellipsometry and AFM of two morphologically different proteins, i.e., a big β-sheet structured HIgG and a small α-helix/β-sheet structured LYZ, are presented and discussed. The chapter ends with a conclusion that covalent linkage of proteins to homologous polymeric surfaces can secure formation of reproducible protein layers. It was also concluded that the density of surface functional groups affected protein immobilization. The binding efficiency between the substrate and surface is crucial not only for the development of potential components of microfluidic devices described in the following chapters but also for the construction of microfluidic device presented in chapter 6.
4.2. Results and discussion
4.2.1. PtBMA film characterization
PtBMA has been frequently employed as a positive photoresist due to its excellent mechanical and optical properties, e.g. transparency (>90 % transmission), stiffness, low water absorption and high abrasion resistance. The PtBMA surface is typically hydrophobic (77◦ ± 3) due to the presence of the methyl groups on the polymer backbone and the tert-butyl ester. Following UV irradiation, and subsequent
106 heating (90 ◦C to catalyze the chemical amplification reaction), the modified PtBMA surface is found to be less hydrophobic (65◦ ± 2) due to the formation of surface carboxylic acid groups, as determined by water contact angle measurements consistent with previously reported values (Bahulekar et al., 1998). Exposure to UV (254 nm) radiation causes the PAG to release protons, which hydrolyze the ester linkages to produce carboxylic acid groups during the chemical amplification (heating) process.
∗ The tertiary carbon of the ester group (i.e., COO–C ), with binding energy of 286.6 eV, is absent from the spectrum indicating essentially a complete loss of the tert-butyl protecting group from the surface esters (Scheme 1).
Activated PtBMA
Scheme 1. Reaction scheme for the formation of sulfo-N-hydroxysuccinimide (sulfo- NHS) activated poly(tert-butyl methacrylate) (PtBMA).
107 The XPS analysis also confirms the presence of carboxylic groups on the irradiated PtBMA surface (Table 2, Figure 1). The root-mean-square (RMS) surface roughness values for the spin-coated polymers were determined from AFM topographical images using the TopoMetrix Explorer image processing software. Typical RMS roughness values ranged between 1.05 ± 0.5 nm for a field-of-view of 4.5 × 4.5 μm2 (data not shown).
Table 2. Atomic concentration ratios (determined by XPS) obtained for adsorbed and covalently immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers on activated P(tBMA) surfaces.
Surface Protein Integrated peak area ratios attachment (×103) N/C S/C P(tBuMA) (as received) - - - +Lysozyme Adsorbed 146.7 - +Human immunoglobulin Adsorbed 111.1 - P(tBuMA)(sulfo-NHS activated) - 20.2 5.05 +Lysozyme Covalent 87.3 4.31 +Human immunoglobulin Covalent 87.3 4.30
108
Figure 1. Representative surface topography of fluorescence images of human immunoglobulin (HIgG) adsorbed (top, left) and covalently immobilized (top, right) and lysozyme (LYZ) adsorbed (bottom, left) and covalently immobilized (bottom, right) on UV-irradiated PtBMA surfaces. Similar images were obtained in different regions of at least two different samples.
Overall, the surface modification of PtBMA provided moderately less hydrophobic surfaces than the commercially obtained polymers, which are suitable for both adsorption and covalent immobilization of biomolecules due to the presence of the surface carboxylic acid groups.
109 4.2.2. Adsorption and covalent binding of selected HIgG on PtBMA surface 4.2.2.1. X-ray photoelectron spectroscopy analyses
XPS analyses were carried out on the modified PtBMA polymer surfaces after incubation with either HIgG or LYZ. As mentioned earlier, the XPS analysis reveals conclusive evidence to support the presence of surface carboxylic acid groups on both modified PSMA and PtBMA substrates. The typical C 1s XPS spectrum of PtBMA + COOH surfaces is shown in Figure 2. To achieve chemically active surfaces suitable for protein immobilization, the standard water-soluble coupling reagents 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N- hydroxysulfosuccinimide (sulfo-NHS) were employed, resulting in the formation of an amide linkage on incubation with the exposed amines on certain proteins (Pan et al., 2005). XPS analysis revealed that after chemical treatment, as expected, both nitrogen and sulfur were present on the ‗activated‘ sample surfaces (i.e. EDC + sulfo-NHS only, see Tables 2 and 3). The chemical state of the surface after each derivatization reaction was monitored by XPS as shown in Figure 2, Table 2 and by the contact angle measurements summarized in Table 3.
Table 3. Elemental compositions (determined by XPS) obtained for adsorbed and covalently immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers on activated P(tBMA) surfaces.
Surface Contact Protein Relative elemental contribution (at%) angle (°) attachment N C O S P(tBuMA) 77 ± 3 - 0 19.5 80.5 0 (as received) a +(UV-irradiation) 65 ± 2 - 0 19.6 80.3 0 +Lysozyme Adsorbed 10.2 70.0 19.2 0.4 +Human Adsorbed 7.6 68.1 21.1 0.5 immunoglobulin P(tBuMA)(sulfo- - 1.2 59.6 31.5 0.3 NHS activated) +Lysozyme Covalent 3.9 73 20.7 0.3 +Human Covalent 6.1 69.9 20.9 0.3 immunoglobulin a Since the takeoff angle is 30◦, the data appeared similar.
110
Figure 2. XPS spectra of PtBMA+COOH surfaces: (a) typical C1s; (b) high- resolution N 1s spectra of samples ‗activated‘ by treatment with EDC and NHS; and (c) sample following covalent protein attachment; (d) high- resolution S 2p spectra of samples ‗activated‘ by treatment with EDC and NHS; and (e) of samples following covalent protein attachment.
111 Activation of the COOH-functional surface sites of the polymers by NHS/EDC mediation introduced a nitrogen 1s peak from NHS that is not present in the spectrum. The nitrogen peak persists in XPS spectra after NHS/EDC treatment: however, the N 1s peak position shifts from 402.2 to 401.1 eV. The N 1s signal centered at 402.2 eV is consistent with the electron-withdrawing nature of nitrogen in NHS, and the lower BE (401.1 eV) N 1s is also consistent with the nature of the nitrogen present in NHS/EDC. XPS-based elemental compositions and reaction yields, the latter calculated from N 1s/C 1s ratios, are summarized in Tables 2 and 3. The N 1s/C 1s ratio was used to estimate the reaction yield instead of the N/O or O/C ratios because (1) N is only present after the activation and amidation reactions and (2) most surface contaminants are of high oxygen content and their presence therefore leads to an overestimate of the oxygen atomic composition. The nitrogen level is much higher than that of sulfur. Given that the N:S ratio of sulfo-NHS is 1:1. As shown in Table 2, the attachment of HIgG and LYZ is evidenced by the increase in concentration of nitrogen compounds on the polymer surfaces. The level of sulfur on the samples following covalent immobilization of either protein suggests that most of the sulfo-NHS still remains on the surface. Moreover, given that EDC is displaced in favor of the more stable sulfo-NHS group, and that the N 1s contribution from sulfo-NHS would be minor, it is likely that most of the nitrogen signal derives from the attached protein molecules. It can be seen in Table 2 that the N/C values of samples following physicochemical adsorption are somewhat higher compared to those of their covalently-bound counterparts, suggesting that the physico-chemical adsorption was more effective than covalent immobilization in the current experimental regime, i.e. at pH 7.4 which is below the HIgG and LYZ isoelectric points, pI 7.8 and 11.1, respectively. This may occur due to the presence of deprotonated carboxylic groups on the modified polymer surfaces, which increases the electrostatic interactions between the oppositely charged polymer surface groups and the proteins molecules. Similar interactions of recombinant human growth hormone and lysozyme with different quartz surfaces containing either silyl groups, such as silanol, methylsilyl, or quaternary aminopropyldimethylsilyl surface
112 groups were observed by Buijs and Hlady (1997), who concluded that protein adsorption (in particular LYZ) was mostly affected by electrostatic interactions. High-resolution S 2p spectra, shown in Figures 2(b ) and (c), provide further insights into the nature of the bonding between the proteins and the polymer surfaces. The S 2p spectra for the ‗activated‘ samples (i.e., EDC + sulfo-NHS treatment only) exhibit a doublet at 168.0 eV. This doublet is due to oxidized sulfur and consistent with the presence of sulfo-NHS. Following the attachment of HIgG or LYZ, the S 2p spectra exhibited an additional peak at 163.8 eV, which is most likely due to sulfide species. Data obtained from curve fitting operation show that about 40 % of the surface sulfur is sulfite, the rest are oxidized species. High-resolution N 1s spectra of the ‗activated‘ samples exhibited two distinct peaks at 402.2 eV and 400.0 eV in a ratio of about 0.3, as shown in Figures 2(d) and (e). The peak at 400.0 eV is attributed to C–N or N–C=O species, whereas the peak at 402.2 eV is most likely due to NH 3 . It is interesting to note that following the covalent immobilization of the proteins, the
NH 3 peak disappears, and almost all the surface nitrogen is covalently bound. It is noted that the NH peak still remains on those samples with proteins attached physico-chemically. In a previous study, we employed EDC/sulfo-NHS coupling chemistry to activate the carboxylic acid groups on PSMA. Such ‗activated‘ surfaces were shown to react with the ε-amino groups of poly(L-lysine) to form amide bonds, with the concomitant release of sulfo-NHS (Ivanova et al., 2004g). Here, however, the XPS data indicated that much of the sulfo-NHS ester was still present after reaction with both proteins, as confirmed by the presence of a significant amount of sulfonyl groups (168.4 eV). Moreover, a high resolution N 1s analysis revealed that almost all of the detected nitrogen on the surface corresponds to covalently bound proteins, which suggests that the protein attachment mechanism described here may follow a different path to that described in the literature (Ivanova et al., 2004g). It is likely that the electrostatic attraction between the sulfonyl moiety on the sulfo-NHS ester backbone and protein amino groups is preferred to amide bond formation resulting from the subsequent displacement of the sulfo-NHS group.
113 4.2.2.2. Ellipsometry analysis
Ellipsometry yielded estimates of the amount of the proteins and the thickness of protein layers on the polymer surfaces similar to previous reports (Malmsten and Lassen, 1995). The amount of physically adsorbed HIgG on PtBMA was 23.0 ± 1.6 ng mm2, with corresponding protein layer thicknesses of 17.0 ± 1.2 nm, while those after covalent immobilization were 5.6–8 ng mm2 with a corresponding layer thickness of 5.9 ± 0.6 nm on PtBMA (Table 4).
Table 4. Ellipsometric measurements obtained for adsorbed and covalently immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers on activated P(tBuMA) surfaces.
Surface Protein Protein layer Amount attachment thickness (nm) of protein (ng mm2) P(tBuMA) (as received) - - - +Lysozyme Adsorbed 11.0 ± 3.2 15.0 ± 4.4 +Human immunoglobulin Adsorbed 17.0 ± 1.2 23.0 ± 1.6 P(tBuMA)(sulfo-NHS activated) - - - +Lysozyme Covalent 7.01 ± 0.6 7.8 ± 0.6 +Human immunoglobulin Covalent 5.9 ± 0.6 8.0 ± 0.8
HIgG is generally considered to adopt a typical Y- or T-shaped conformation having 3D dimensions of 10 × 15 × 13 nm3 and a thickness of 5 ± 0.5 nm (Day, 1990). According to these data, it can be inferred that the thickness of the adsorbed HIgG layers formed on modified polymer surfaces is consistent with the length of the HIgG arm (7 nm) and the base (6.5 nm), and that of covalently immobilized HIgG may correspond to proteins lying in a ‗side-on‘ configuration. The results obtained are consistent with that of Baszkin and Lyman (1980), who calculated theoretical values
114 for a monolayer of adsorbed HIgG molecules attached ‗end-on‘ (18.5 ng mm2) and ‗side-on‘ (2.7 ng mm2). The adsorption reaction between LYZ on PtBMA surfaces was similar to that observed for HIgG, where the amount of adsorbed protein was in the range 15 ng mm2 (Table 4), whilst the amount of protein after covalent immobilization was in the range of 8 ng mm2. The thickness of the adsorbed LYZ protein layers ranged at 11.0 ± 3.2 nm on PtBMA, suggesting the bulk of the compactly adsorbed protein occurred on the polymer surface (see also the AFM analysis section, Figure 4). It was also found that the thickness of the covalently immobilized LYZ layer of ~ 5–7 nm could be construed as a protein monolayer, since the LYZ dimensions [5 × 3.5 × 3.5 nm3] suggest that the maximum possible height of a single layer would be consistent with the largest dimension (i.e. 5 nm). Notably, in our previous study we observed that α- chymotrypsin exhibited a different attachment behavior whilst adsorbing to PMMA. We found that the amount of α-chymotrypsin adsorbed on PtBMA was typically 4 ng mm2, with a corresponding protein film thickness of ~ 3–6 nm (Ivanova et al., 2003b). These values agreed well with, for example, the theoretical value of 3 ng mm2 calculated for an α-chymotrypsin monolayer (based on the size of the protein as determined from its crystal structure (Aune and Tanford, 1969), while covalent immobilization resulted in similar or slightly greater amounts of immobilized protein on the polymer surfaces (Ivanova et al., 2003b). The most likely, on PtBMA HIgG and LYZ yielded greater adsorption values compared to that of α-chymotrypsin due to electrostatic interactions being positively charged in the buffer used in experiments (10 mM PBS, pH 7.4), in contrast to α-chymotrypsin, which is negatively charged under the same buffer conditions (Aune and Tanford, 1969). Our results correlate with those reported by Buijs and Hlady (Buijs and Hlady, 1997), who provided evidence to suggest that greater amounts of adsorbed LYZ are formed on polymeric surfaces when buffers of low ionic strength (10 mM PBS) are employed, pointing to the important role electrostatic interactions play on both hydrophobic and hydrophilic surfaces. It was also reported in previous studies that the build-up of the adsorbed proteins layers vary significantly for each protein, and LYZ in particular forms a more compact layer than that of other proteins (Malmsten and Lassen, 1995, Deere et al., 2004).
115 4.2.2.3. AFM analysis
AFM imaging was employed to analyze the protein layer formation at the molecular level following adsorption or covalent immobilization of HIgG and LYZ on PtBMA (Figures 3 and 4). A careful inspection of the AFM topographical images revealed that adsorbed/covalently-bound HIgG forms dense layers on PtBMA. A representative topographical image of surface-immobilized HIgG, together with the corresponding depth analysis, is presented in Figure 3.
Figure 3. Representative surface topography images and their corresponding line profile analyses of human immunoglobulin (HIgG) adsorbed (top) and covalently immobilized (bottom) on UV-irradiated PtBMA surfaces. Similar images were obtained in different regions of at least two different samples.
116 The adsorbed protein produced a dense surface coverage, with a protein layer thickness of approximately 15 ± 0.8 nm on PtBMA. The surface roughness of the covalently immobilized HIgG layer ranged from 4 to 6 ± 0.4nm (Figure 3). This finding is consistent with the ellipsometric measurements, which suggest that HIgG molecules physically adsorb in an ‗endon‘ configuration, while the covalently immobilized protein molecules adopt a ‗side-on‘ configuration. The physical adsorption of LYZ on the surface-modified PtBMA substrate produced a heterogeneous surface coverage, with a protein layer had a depth of about 20 ± 0.6 nm (Figure 4).
Figure 4. Representative surface topography images and their corresponding line profile analyses of lysozyme (LYZ) adsorbed (top) and covalently immobilized (bottom) on the UV-irradiated PtBMA surface.
117
Covalently immobilized LYZ on the other hand, formed more homogeneous layers on polymer surfaces, with depths ranging from 4 to 2 ± 0.3 nm (Figure 4). These depth values are comparable with those for a single layer of LYZ molecules adsorbed on mica, as previously imaged by Fritz et al. (1995). Interestingly, the same authors also noted that in some instances, the depth of the protein layer measured using AFM, was greater than that determined by x-ray diffraction (Fritz et al., 1995). The results obtained are consistent with each other, indicating that both the covalent immobilization and physico-chemical attachment used in the present study give rise to compatible levels of protein density on the modified surface of the polymers studied. Fluorescence images of labeled proteins confirmed the formation of continuous protein layers for both adsorbed and covalently immobilized proteins (data not shown). Overall, the ellipsometry, XPS and AFM analyses obtained for both adsorbed HIgG and LYZ on PtBMA, are in good agreement, implying that both proteins form protein layer(s). This observation is possibly a consequence of the non- specific nature of adhesion and/or the possible surface diffusion of the protein molecules (Tsukada and Blow, 1985, Tilton et al., 1990, Tarjus et al., 1990, HÖÖK et al., 1998), allowing the reorganization of the randomly adsorbed proteins into more tightly packed layers. One other possibility could result from the close co-existence of an initial, partially denatured layer of protein molecules, due to the mechanical stress exerted on the biomolecules during contact with the substrate surface, and a second (intact) protein layer on the surface (Malmsten and Lassen, 1995, Baszkin and Lyman, 1980, Aune and Tanford, 1969, Ivanova et al., 2003b, Deere et al., 2004, Fritz et al., 1995, Tsukada and Blow, 1985, Tilton et al., 1990, Tarjus et al., 1990, HÖÖK et al., 1998, Petrash et al., 1997, Norde et al., 1986, Garrison et al., 1992, Lenk et al., 1989, Castillo et al., 1984). In addition, the degree of conformational change for each particular protein can also depend on other factors such as pH and ionic stress, as well as on the hydrophobic effects mentioned earlier. It is noteworthy that denser, adsorbed and covalently immobilized HIgG and LYZ films were found to form on the surface-modified PtBMA substrate. At present, XXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
118 there is no clear explanation for this, however in the light of recent findings, where certain proteins (e.g. HSA) have been found to bind more tenaciously to surfaces modified by disordered alkyl-terminated self-assembled monolayers (Petrash et al., 2001), it is likely that a more flexible surface may conform better to the structure of the protein molecule (Petrash et al., 2001). Other workers have also noted that the orientation and packing of certain proteins on substrates is critically dependent on the surface/substratum characteristics and the immobilization strategy chosen (Vijayendran and Leckband, 2001, Chen et al., 2003, Zhou et al., 2003, Vikholm and Albers, 1998). For example, Denis et al. observed the formation of a 6 nm thick homogeneous collagen layer on smooth hydrophilic substrata, yet on a hydrophobic surface, this same protein formed a 20 nm thick layer exhibiting elongated, aggregated structures (Denis et al., 2002). Here, the fact that the modified PtBMA surface is hydrophobic and from XPS is deemed to consist of greater number of disordered carboxylic acid groups, could lead to a similar effect allowing a greater accommodation of protein.
4.3. Conclusion
From the protein thickness results, and given the known three-dimensional size of the proteins studied, in most cases one would be justified in treating the formed protein layers as monolayers. Notably, the covalent immobilization in the experiments performed was translated in good reproducibility in achieving protein monolayers on both PSMA and PtBMA surfaces. It is also worth noting that a protein concentration of 0.1 mg ml−1 was sufficient for both proteins to reach saturation point, and coat the surface completely, which is in good agreement with observations reported elsewhere (Castillo et al., 1984, Garrison et al., 1992). Here, the combination of analytical techniques, i.e. XPS, ellipsometry and AFM, was particularly revealing, providing comparable data during the investigation of HIgG and LYZ interactions with two surface-modified polymers. We have XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
119 demonstrated that covalent immobilization can secure the formation of reproducible protein layers on homologous polymer surfaces. Importantly, this approach can be effectively applied to proteins with different physical characteristics, e.g., isoelectric points. The density of substrate surface functional groups, i.e. carboxylic acids, however, will undoubtedly affect the efficiency of protein–surface interactions.
120
CHAPTER 5
ADVANTAGE OF IMMOBILIZATION OF PROTEINS
IN MICROCHANNELS
© 2004 NSTI http://nsti.org. Reprinted and revised, with permission, from ―Amplification of protein adsorption on micro/nanostructures for microarray applications‖, pp. 95-98, 2004, Boston, U.S.A.
121 5.1. Overview
This chapter describes the newly developed approach for surface design applicable for microdevices. The approach is based on spatial immobilization of proteins in micro/nano-channels fabricated via laser ablation. Reproduced from references (Ivanova et al., 2003b) © 2003 With kind permission from SPIE; and (Ivanova et al., 2004f) © 2004 With kind permission from SPIE. This work follows the study of protein immobilization on flat surfaces presented in chapter 4. The chapter begins with a brief characterization of utilized polymer, i.e., poly(methyl methacrylate), followed by a discussion of fabrication of microstructures in Au- deposited PMMA films. The chapter continuous with a comparative investigation of physico- chemical attachment of five morphologically different proteins, i.e., α- chymotrypsin, human serum albumin, human immunoglobulin, lysozyme and myoglobin, immobilized in laser ablated poly(methyl methacrylate)-based channels and on native poly(methyl methacrylate) films. Because the molecular properties of proteins and their interactions with surfaces affect protein adsorption, the following subsection 5.2.3. explains the impact of molecular descriptors on protein adsorption in microchannels. Subsection 5.2.4. discusses utilization of ellipsometry for evaluation of the thickness of polymeric films and attached proteins. Subsection 5.2.5. is devoted to the comparison of protein adsorption on flat surfaces with adsorption in microchannels, which was performed using fluorescent microscopy and quantified using Fluor reader. The chapter ends with a characterization of adsorption properties of selected proteins, and is followed by a conclusion that protein adsorption was greater in microchannels than that on flat surfaces. As protein immobilization was affected by microchannel, PSMA strips surrounded by channels were designed for control of self-assembly of actin filaments, as described in the following chapter.
122 5.2. Results and discussion
5.2.1. Characterization of poly(methyl methacrylate) polymeric films
Poly(methyl methacrylate) is a rather commonly used polymer due to its characteristics, e.g. transparency (>90 % transmission), stiffness with excellent UV stability, low water absorption and high abrasion resistance. The hydrophobicity of PMMA estimated by contact angle measurements was ranged from 68 θ to 72 θ indicating its moderate hydrophobic nature.
5.2.2. Fabrication of microstructures in Au-deposited PMMA films
The proposed technology produces surfaces that present to the proteins a large variation of the properties (in particular hydrophobicity and rugosity) of the surface concentrated in a small, micron-sized region. The fabrication consists in the ablation of the opaque thin metallic (e.g., Au) layer deposited on a thick transparent polymer (e.g., PMMA) layer. The ablation of the thin metallic layer (Figure 5) induces the pyrolysis and partial ―sculpturing‖ of the polymer, with more hydrophilic surfaces towards the edges of the channel and a hydrophobic hump in the middle.
123
Figure 5. Fabrication of micro/nano-structures for protein arrays using microablation and directed deposition.
The higher rugosity of the microstructures (Figure 6) translates in a 3 times more specific surface in than outside the channels.
124
Figure 6. AFM mapping of the ablated microchannels.
A more important feature than the increased rugosity in the microablated areas is the large variations in the relative hydrophobicity of different regions, as measured by AFM in lateral force mode. Figure 7 presents both the topography and the relative hydrophobicity of the surface of microablated lines. These structures have micron-size dimensions laterally but tens of nanometers in depth. The latter dimensions make the structures comparable with medium to large proteins (Figure 7).
125
Figure 7. AFM topographical (top left) and lateral force (top right) image of a channel fabricated via the ablation of a 30 nm Au layer on top of PMMA. The middle region (I) is the most hydrophobic, whereas comparative adsorption of the selected proteins in the channels of thin gold layer deposited on a poly(methyl methacrylate) film was visualised using fluorescence and atomic force microscopy and further quantified using a Fluor reader. Poly(methyl methacrylate) is a rather commonly used polymer due to its characteristics, e.g. transparency (>90 % transmission), stiffness with excellent UV stability, low water absorption and high abrasion resistance. The hydrophobicity of PMMA estimated by contact angle measurements was ranged from 68 θ to 72 θ indicating its moderate hydrophobic nature.
126 The AFM analysis of the microfabricated structures (Figure 7) showed the presence of a lateral variation of hydrophobicity with the edges of the channels being the most hydrophilic; and the centre being the most hydrophobic. This variation of surface chemistry can be attributed to the lateral distribution of the ablation energy, which translates in different energies delivered to the polymer, and subsequently different surface chemistries. The expected reactions, which translate to three spatial regions, would be, in order of increasing pyrolysis temperature, i.e. from the edges towards the centre, (i) the termination of the side ester groups at one of the C-O bonds, resulting in a more hydrophilic material; (ii) depolymerization of the main chain, preserving the same hydrophobicity; and if the pyrolysis process is quick enough (iii) the breaking of the side bonds, resulting in a more hydrophobic material. Although there are variations of the dimensions and distribution of hydrophobicity vs. laser power, the general structure of the microablated channels remains the same. Though the process can use different polymers and metals, we found that PMMA (and gold) are so far optimum choices. In particular PMMA can offer large possibilities to ‗combinatorialise‘ the chemistry of the surface upon thermolysis. Figure 8 depicts possible chemical pathways that would explain the AFM-measured variation in hydrophobicity.
127
Figure 8. Possible pyrolysis pathways of PMMA localized in micro-regions leading to the observed lateral distribution of hydrophobicities.
The spatial distribution of the surface chemistries/hydrophobicities reflects in the topography of the micro-fabricated structures, with an elevated (hydrophilic) region at the edges; a flat (medium hydrophobic) region between the edges and the centre; and a central region with a bump (hydrophobic). The micro/nano-topography of the microchannels, as well as the AFM lateral force mapping validated the mechanism proposed above. Finally, the rugosity of the surface is also distributed unevenly, with the region outside the channels and the plateaus (region II in Figure 7) being flatter than region I in Figure 7. The strategy behind this method was to allow different proteins, or different parts of the same large protein, to find the most appropriate in terms of adsorption and
128 preservation of bioactivity surface. Because the different micro/nano-surfaces are co- located in a small area (channel width around 10 μm or less) the florescence signal from the proteins would be perceived at the mm-range as being located in the same areas. Also it has been observed that the concentration of the proteins was apparently higher in the microstructures than on flat areas with similar material due to a higher specific surface and larger opportunities for attachment. The combinatorial character of the surface would in principle also allow the probing of several patches on the molecular surface of the proteins, or modulate their bioactivity. Figure 9 presents possible arrangements of IgG-like biomolecules on the combinatorial surfaces.
Figure 9. General concept of the probing of molecular surface of proteins.
The proposed technology for the fabrication of microarrays has the following potential advantages: (i) the combinatorial surfaces would improve the uniformity of biomolecule surface concentration, in particular for protein microarrays; (ii) the surface concentration of biomolecules (several very different proteins being tested) increases, and therefore the sensitivity increases accordingly, by 3-12 times, depending on the molecular properties of the biomolecules; (iii) it is possible -in principle- to probe different sides of the biomolecular surface and therefore the
129 bioactivity; and (iv) because the proposed method writes protein lines (that can encode information in a bar code manner) instead of dots, it can be used for the fabrication of informationally-addressable, as opposed to spatially addressable microarrays.
5.2.3. Impact of molecular descriptors on protein adsorption on microstructures
The molecular properties of proteins and their interactions with surfaces have an effect on protein adsorption, which is one of the first and most important events that occurs when a biological fluid contacts a surface. Interactions based on hydrophobicity (Tilton et al., 1991) and electrostatics (Lubarsky et al., 2005) have been found to be driving forces for protein adsorption. As the microstructures fabricated as described above comprise micro/nano-areas with very different chemistries, it is expected that both hydrophobicity and electrostatics would contribute to the adsorption on these ‗combinatorialized‘ micro/nano surface. Indeed an AFM analysis of the topography of the channels after the deposition of proteins showed that the initial topography of the channels (Figure 7) is partially smoothed, with IgG having a more pronounced effect. Essentially the adsorption of proteins is governed by (i) kinetic processes (i.e. diffusion of molecules to, and sometimes from the surface); and (ii) thermodynamic processes (i.e. electrostatic and hydrophobic interactions between the protein and the surface). These two types of processes are of course interconnected. In particular the electrostatic interactions which are long range interactions will influence in a larger degree the protein transport to the adsorbing surface. Also the electrostatic and hydrophobic interactions will be connected at the protein structure level (e.g. a protein which presents a hydrophobic molecular surface will, statistically speaking, have a less charged surface). However, when performing a sensitivity analysis of the ‗tug-of- war‘ between these three adsorption-relevant parameters, one should try to find the descriptors that are independent of each other. Three molecular descriptors have been found to both impact on protein adsorption (on flat and microstructured surfaces) and be largely independent of each other, namely: (i) total molecular surface (which modulates the transport of the protein on the surface and also has an effect on the
130 packing of the protein layer on the surface); (ii) ratio between the hydrophilic/hydrophobic specific density (the specific density is the total property – e.g. hydrophilicity index- per respective area –e.g. hydrophilic area-); and (iii) ration between positive and negative areas. The last two descriptors will account for the thermodynamic factors, i.e. hydrophobic and electrostatic interactions, respectively. It is worth noting that the hydrophobicity-related descriptors are much more statistically independent of the size of the molecule, i.e. molecular surface (i.e. R2 = 0.022 is a maximum), whereas the charge-related descriptors are less independent (i.e. R2 = 0.069 is a minimum). The transport of proteins to the surface is, to a large extent, diffusion- dependent as the convection will have a limited role at this scale. In the first approximation, the diffusion coefficient will be dependent -according to Stokes- Einstein theory- on the radius of a sphere, r, as D ~ r-1, or dependent on the surface, S (S ~ r2), D ~ S-0.5. Of course, these simple relationships will be altered by the shape of the object (apparent increase of the power applied to the size of the object and subsequently the surface) and environment in which the object diffuses (a confined environment will reflect in a decrease of power applied to the size of the object and subsequently the surface, down to -3/2 from -1/2, if an analogy with the Knudsen diffusion is applied). A sensitivity analysis of the impact of the total molecular surface and ratio of the hydrophilic/hydrophobic specific density (Figure 10) revealed a few interesting relationships.
131
Figure 10. Modulation of the amplification of protein adsorption in micro/nano- channels vs. the molecular surfaces of the respective protein.
First, the protein adsorption is amplified on microstructured rather than on flat surfaces (the larger coefficient in the fitted function, with a separate analysis to follow). Second, the adsorption on microstructured surfaces depends on the molecular surface with a power ~0.33, while the adsorption on flat surfaces is governed by an almost linear relationship (power ~1.2) versus molecular surface. Indeed on flat surfaces, for geometric reasons, a linearity of the adsorbed mass with the molecular surface would be expected, while in confined environments smaller molecules could capitalize better on newly created areas. Third, the specificity of adsorption on microstructured surfaces versus the hydrophobicity-related descriptor decreases dramatically (decrease two times of the respective power), possibly due to the
132 ‗combinatorialization‘ of the adsorbing surface in the channels (mechanism proposed in Figure 7). Fourth, as expected, a higher relative hydrophobicity (i.e. smaller ratio hydrophilic/hydrophobic specific density) induces a higher adsorption on both types of surfaces. Fifth, the quality of the fit decreases for microstructured surfaces, presumably because of the same ‗combinatorialization‘ that adds statistical noise to the data. A similar sensitivity analysis of the impact of the total molecular surface and ratio of the positive/negative areas revealed similar relationships. First, it is confirmed that the adsorption is much higher on microstructured than on flat surfaces. Second, it is confirmed that the adsorption is modulated by molecular surface with a power ~1 and ~0.33, for flat and microstructures, respectively. Third, the adsorption on microstructured surfaces depends less on the charges-related descriptor (decrease of the power from 0.6 to 0.25, for flat and microstructures, respectively), again possibly due to the ‗combinatorialization‘ of the adsorbing surface in the channels. Forth, interestingly, a higher positive charge (area) induces a higher adsorption, possibly due to the presence of carboxylic groups, created either parasitically or thermo-induced on flat surfaces and at the edges of the microstructures, respectively. Fifth, the same degradation of the quality of the fit is observed for the adsorption on flat and microstructured surfaces. It appeared that the small proteins can use the combinatorial surface better to amplify their adsorption, whereas larger proteins are less sensitive to the opportunities offered by various surfaces at least partially because they exhibit ‗combinatorial‘ molecular surfaces too. Finally, and for the concentrations used in this study, the adsorption on flat surfaces is proportional with d2-2.5 (where d is the average diameter of the protein) whereas on microstructured surfaces the adsorption is less sensitive to the diameter of the protein (proportional with d0.66-0.75). The role of rugosity on flat and micro-structured surfaces on protein adsorption can also be quantified. The shallow character of the channels (less than 50nm versus 5-10μm) does not suggest that the additional area created via ablation is large enough to explain alone the amplification of adsorption. These being said, it is clear that smaller protein can capitalize better on the newly created surfaces in the mid-valleys
133 in the channels (Figure 7) as well as on the rigidity of the surface than the larger proteins. Although the statistical relevance decreases for the analysis that considers only the molecular surface, this parameter is important enough to warrant a separate analysis. The molecular surface has been calculated using a probing sphere with 10Å radius, which offers the best statistical fit of the data, as well as being the most appropriate for the analysis of the adsorption on surfaces. The impact of the total molecular surface on the amplification of adsorption is presented in Figure 10. Whatever the circumstances, the amplification of adsorption (i.e., the ratio between the level of adsorption on micro/nano-structures and on flat surfaces) is important varying between 3- and 10-fold. The efficiency of the amplification of adsorption decreases with the molecular surface reaching a plateau at around 3-fold.
5.2.4. Characterization of thickness of polymeric films and attached proteins
Dependence of refractive index for PMMA on its thickness within nanoscale range between 67 and 259 nm displayed in Figure 11.
Figure 11. Correlation between nanothickness and refractive index of PMMA on glass surface treated with HMDS.
134 Strong correlation between refractive index and nanothickness for PMMA polymer films is observed. This relationship was approximated with function of
NPMMA=at-b, where N – refractive index of PMMA, t – nanothickness of PMMA polymer film, a=188.49 and b=0.88 are constant coefficients for given PMMA polymer films. This correlation makes sense only within the narrow nanoscale range.
Beyond the nanoscale range, when tPMMA → ∞, refractive index (NPMMA) should come nearer to 1.4846 that is value for PMMA as the bulk polymer (Wunderlich, 2000). This nanoscale limit for PMMA is reached starting approximately from ~250 nm. For thicker PMMA films, the refractive index should be constant (1.4846), and beyond the thickness of ~250 nm, the PMMA-films should be considered as the bulk PMMA- polymer. Further decreasing of thickness of the PMMA-nanofilms should lead to the refractive index of PMMA-monolayer. The refractive index of PMMA-films within nanoscale thicknesses increases in the factor of 3-4 in comparison with the refractive index for PMMA as the bulk polymer. Similar relationships for other polymer nanofilms on glass-surfaces treated with HMDS were obtained, namely for nitrocellulose (a=264.52, b=0.96), PtBMA (a=241.91, b=0.93), and PSMA (a=206.51, b=0.9). Values of the constants are defined with type of materials, which are used for nanofilms manufacturing, and can slightly vary, approaching to the true value, if number of statistical data increase. Such behaviour of refractive indexes within nanoscale thicknesses of protein films was observed as well. For example, correlation between refractive index and nanothicknesses for HSA is shown in Figure 12. One can see that the refractive index of HSA films within the nanoscale thicknesses is far from that for HSA as the bulk protein, i.e. Nbulk=1.465 (Benesch, -b 2001). Approximation of the relationship by function of NHSA=at gives the following constants a=200.1 and b=0.90. Similar relationships were obtained for myoglobin (b=1.04), α-chymotrypsin (b=0.99), and human IgG (b=1.1).
135
Figure 12. Correlation between nanothickness and refractive index of HSA in double nanolayered sandwich of HSA/PMMA on glass-surface treated with HMDS.
The thickness of the protein layers on native PMMA polymeric surfaces according to ellipsometric measurements significantly varied from ~61 nm for lysozyme and chicken IgG to ~122 nm for human IgG as shown in Table 5. The relationship between refractive index and nanothickness for both protein-nanolayers and polymer- naolayers demonstrates a rivers correlation, namely the thiner nanolayer the higher refractive index.
136 Table 5. Ellipsometric measurements of thicknesses of adsorbed proteins and correspondent PMMA polymeric films.
Proteins Protein Refractive PMMA Refractive
thickness (nm) index (Nproteins) thickness (nm) index (NPMMA) Myoglobin 70.6 ± 2.5 4.519 ± 0.167 170 ± 2.1 2.125 ± 0.020 Human IgG 121.6 ± 0.3 4.841 ± 0.012 78.4 ± 9.0 4.857 ± 0.603 Chymotrypsin 98.9 ± 15.6 4.703 ± 0.904 147.6 ± 3.6 2.362 ± 0.049 HSA 83.3 ± 6.9 4.158 ± 0.336 136.8 ± 2.5 2.518 ± 0.039 Lysozyme 61.2 ± 5.9 5.497 ± 0.504 170.6 ± 1.1 2.119 ± 0.011
5.2.5. Protein adsorption in PMMA-based channels and on native PMMA films
Comparative adsorption of the selected proteins in the channels of thin gold layer deposited on a poly(methyl methacrylate) film was visualised using fluorescence microscopy and further quantified using a Fluor reader. Fluorescent images of α- chymotrypsin, human serum albumin, human and chicken IgG, lysozyme, and myoglobin adsorbed in microchannels that were fabricated in a bar-code format are presented in Figure 13. In addition, in order to estimate the effectiveness of protein attachment in microfabricated structures we also studied the protein adsorption on native poly(methyl methacrylate) films with comparative quantification of fluorescently labelled proteins. The results obtained (Table 5, Figures 13-14) using three different methodologies are in a good correlation and indicated that in general the protein adsorption was dependent on protein and on initial concentration of protein solution. The protein adsorption in microfabricated channels was more effective comparing to that on native PMMA polymeric surface. Specifically, the adsorption of HSA, HIgG and myoglobin, in the microfabricated channels was as much as 2.5 times more than that on native PMMA surface (Figure 14) and the adsorption of lysozyme was comparable in both cases.
137
Figure 13. Protein adsorption in microstructured PMMA surface. The first row presents bright field images. The fluorescent images relate to different protein concentration in solution as follows: 0.014 mg/ml (second from the top); 0.07 mg/ml (third from the top); 0.14 mg/ml (bottom).
138
Figure 14. Protein adsorption in the channels of thin gold layer deposited on a poly(methyl methacrylate) film and on poly(methyl methacrylate) films.
5.2.6. Characterization of adsorption properties of selected proteins
Five proteins from the most common three classes, namely, myoglobin (the class of only-α-helices), IgGs, HSA, α-chymotrypsin (the class of almost exclusively β-sheets) and lysozyme (the class of α-helix and β-sheet tend to be segregated along the chain) were selected to compare their attachment behavior. Taking into account the isoelectric point of myoglobin (7.8) we assume that in our experiments it was mostly neutral. Our results also indicated that the adsorption yields of myoglobin on native PMMA polymeric surfaces were rather poor. However, its adsorption in microfabricated channels was greater. In an attempt to explain this phenomenon we compared its surface characteristics with those of other proteins studied (Table 6). The data in Table 6 shows that the solvent-accessible surface area of myoglobin is very small (7832.6 Å2) relative to its molecular weight.
139 Table 6. Characteristics of selected proteins.
Human IgG α-Chymo Human Myoglobin Lyso trypsin) serum zyme albumin Mol. weight, D 146000 24000 66500 66000 14000 Size (XxYxZ), Å 74x115x101 60x48x72 129x108x128 47x40x42 42x36x47 Isoelec. point, pI 7.36 4.6 4.7 7.8 10.7 Connolly surface 33677.9 23186 55270 7832.6 5793.5 area, Å2 Area with 7278.3 4016.9 12474.9 1659.9 1655.5 positive charge, Å2 Total positive 40.6 24.3 57.3 16.9 8.6 charge Area with 26398.8 19169.2 42792.8 6172.8 4137.9 negative charge, Å2 Total negative -255.4 -182.7 -389.2 -34.8 -25.2 charge Average surface -6.4 -6.8 -6.0 -2.3 -2.9 charge Hydrophilic area, 19670.5 12114.9 30699.6 4662.7 3848.0 Å2 Hydrophilicity 4,2 4.3 7.3 6.8 5.1 index Hydrophobic 14004.6 11070.8 24568 3169.9 1945.4 area, Å2 Hydrophobicity -4.7 -4.7 -4.5 -4.3 -5.1 index Total -66 -52.4 -111.3 -13.7 -9.9 hydrophobicity Total 82.3 52.1 225.3 31.8 19.6 hydrophilicity
140 Human serum albumin, for example, has molecular weight about 3.5 times greater than myoglobin but has a solvent-accessible area of 55270 Å2, or around 7 times more. A comparison of the dimensions of both molecules shows that myoglobin is far more compact than HSA, with a very smooth molecular surface, free of any atomic-size clefts. α-Chymotrypsin has a molecular weight of only 24000 D, but a surface area about 3 times larger than the area of myoglobin. The proteins from the second structural class were represented by three proteins, IgGs, HSA, α-chymotrypsin. The IgGs structure is highly asymmetric despite having two identical heavy and two identical light chains and can be considered a ―snapshot‖ of the broad range of conformations available in solution. The overall shape is between a Y and a T, with a 143° angle between the major axes of the two Fabs. The IgG spans 171 Å from the apex of one antigen-binding site to the other. According to our calculations presented in Table 6, the size of the protein was estimated as 74 x 115 x 101 Å. It should be noted that IgGs had the high adsorption yield on both native PMMA polymeric surfaces and in microfabricated channels. Interestingly that IgGs as well as myoglobin presumably were neutral, yet the attachment behavior was completely different. This fact might be one more evidence that proteins adsorption is controlled by a combination of factors. Another protein from the second structural class was human serum albumin, the most abundant protein in the blood. Because HSA acts as a fatty acid transporter, it has six binding sites for fatty acids. Petrash et al. (1997) have shown that specific binding of HSA is one of the main factors in binding tenacity. α-Chymotripsin contains intricate folding of the tertiary structure. This folding results in a hydrophobic core and an outer hydrophilic surface, thus allowing the protein to interact with other proteins in the cytoplasm. Chymotrypsin consists of three chains. Since the beta barrel is antiparallel, the interior is expected to be hydrophobic and exterior hydrophilic which favourably interact with water molecules (Tsukada and Blow, 1985). The adsorption capacity of the latter two proteins was quite similar (Figures 13-14) on plain surfaces although with greater yields in microfabricated channels especially for HSA. The isoeletric point of chymotrypsin and HSA is about 4.7, therefore we assumed that in our experiments these proteins were negatively charged.
141 Lysozyme belongs to the third structural class of the proteins. It has an alpha+beta fold, consisting of five to seven alpha helices and a three-stranded antiparallel beta sheet. The enzyme is approximately ellipsoidal in shape, with a large cleft in one side forming the active site (Tsukada and Blow, 1985). Since isoelectric point of lysozyme is 10.7, in our experiments the molecules were slightly positively charged. However, this protein was poorly attached to plain hydrophobic PMMA surfaces and in the channels in comparison to other proteins studied. In summary, there seems to be no single factor but the combination of a few/several that might control the adsorption of proteins on PMMA polymeric surfaces.
5.3. Conclusion
We propose a method for the fabrication of random ‗combinatorial‘ micro/nano-sized surfaces of 100 nm-range structures that allows a higher adsorption of proteins and possibly the immobilization of biomolecules on different sides of the molecular structure. The method, which is based on laser microablation of thin metal/blocking protein layers deposited on a polymer substrate, has proven to amplify the protein adsorption between 3 to 10 times depending on the molecular surface of the protein. It appears that smaller proteins can capitalize better on the newly created micro-level structure and nano-level rugosity. The fabrication of the microstructures, achieved by ablating a thin metallic layer deposited on a non-ablatable polymeric layer, induces the creation of ‗combinatorial‘ surfaces, with different surface chemistries. This surface ‗combinatorialization‘ makes the adsorption of proteins less dependent on the local molecular descriptors, i.e. hydrophobicity and charges. Consequently, molecularly different proteins will adsorb at increased levels with better chances for the preservation of bioactivity. The amplified and ‗combinatorialized‘ adsorption on micro/nano-structures has the potential of improving the detection of biomolecular recognition if used for muliplex analysis. Protein-binding assay with fluorescent detection and quantification enabled rapidly analyse the adsorption properties of different proteins belonged to three major structural classes. The physico-chemical adsorption of human serum albumin, human
142 immunoglobulin, α-chymotrypsin, lysozyme, and myoglobin in the microchannels fabricated via a localized laser ablation of a protein-blocked thin gold layer (50 nm) deposited on a poly(methyl methacrylate) films and native polymeric surfaces was 2.5-5 times greater than that on the plain PMMA polymeric surfaces. A surface mass density of adsorbed protein molecules on the latter defined with a protein-film thickness and a refractive index for the protein layer correlated with data obtained for fluorescently labeled proteins. So microchannel has an advantage over the flat surface.
143
CHAPTER 6
CONTROL OF SELF-ASSEMBLY OF ACTIN
FILAMENTS FOR DYNAMIC MICRODEVICES
144 6.1. Overview
This chapter presents a simple technique for actin-filament-bundle fabrication providing a convenient experimental system that is applicable for the development of a new device technology based on biomolecules. Reproduced from reference (Alexeeva et al., 2005) © 2005 With kind permission from Springer + Business Media. The chapter starts with a characterization of PtBuMA, PMMA and PSMA polymeric surfaces. In addition to adsorption and covalent binding of proteins on polymeric surfaces, as described in chapters 4 and 5, protein assembly was studied. Subsection 6.2.2. describes adsorption and self-assembly of G-actin on selected polymeric surfaces, and is followed by evaluation of covalent bonding of G-actin on the surfaces. Although the use of covalent binding resulted in more stable bonding and greater density of rhodamine phalloidin–labeled F-actin on all surfaces, polymerization under flow field did not cause actin alignment (see subsection 6.2.2.3. for details). As microchannel can affect protein-surface interactions (see chapter 5 for details), strips surrounded by microchannels for actin immobilization and alignment on the most suitable surface, namely PSMA (see subsection 6.2.3. for details), were constructed. The chapter continues with a description of the fabrication of electrostatically self-assembled actin filament bundles for the formation of tracks capable of supporting continuous bead movement, as described in subsection 6.2.4. The chapter ends with a conclusion that PSMA surface provided sufficient amount of binding sites for the covalent immobilization of actin. In addition, electrostatically condensed actin filament bundles can be assembled and aligned in a continuous-flow system. To provide molecular motor proteins with ATP energy, a search for bacterial ATP producers among 86 environmental (marine) bacteria belonging to 17 genera was performed, as described in chapter 8; some of the potential ATP producers are described in chapter 7.
145 6.2. Results and discussion
6.2.1. Polymeric surface characterization
PSMA is transparent in the visible region and non-fluorescent with appropriate thermomechanical characteristics, mildly hydrophilic with contact angles of 50°. There is a possibility that some of the carboxylic groups on the polymeric surface might have undergone reorientation toward the bulk of the polymer; however, this did not decrease polymer surface functionality (Ivanova et al., 2002c). The hydrophobic nature of PMMA is conferred by the methyl groups and the bonding arrangement around the oxygen (see subsection 5.2.1. for more details). The irradiation induces the evolution of the original polymer to complex phases of amorphous hydrogenated carbon (a-C:H). Following UV irradiation, the PMMA surface became less hydrophobic with a contact angle of 62°. The surface characteristics of P(tBuMA) are rather similar to those of PMMA, as these polymers only differ by the number of methyl groups. The non-irradiated surface was hydrophobic due to tert-butyl-termination (see chapter 4 for more details). Following UV irradiation, the polymer became hydrophilic due to COOH-termination.
In the presence of H2O P(tBuMA) will release (CH3)3COH. Loss of chemical species, along with possible bulk densification and surface reconstruction, could presumably account for the shrinkage of the polymer surface (Watson et al., 2002)
6.2.2. Effectiveness and stability of G-actin self-assembly 6.2.2.1. Adsorption and self-assembly of G-actin on selected polymeric surfaces
In the context of the surface characteristics (discussed above) we selected three polymeric surfaces PMMA, P(tBuMA), and PSMA, as appropriate substrates to investigate both adsorption and covalent bonding of G-actin followed by self- assembly of actin filaments. The results revealed that physicochemical adsorption of actin filaments in the continuous flow of the buffer for 0.5 h with a flow rate of 0.06 mL min–1 was most effective on PSMA (as monitored visually). The adsorption on
146 PSMA was comparable to that on nitrocellulose, which was used as the positive control. Actin filament attachment on PMMA and P(tBuMA) was less effective (Figure 15A–D). UV-irradiated surfaces appeared to have strong inherent fluorescence as observed in the microscope as bright regions as seen in Figure 15 (C–D), which probably was a result of an interaction of the buffer with irradiated surfaces. Dried irradiated surfaces had no or low fluorescence background. In addition, actin filaments might have been partly disintegrated and therefore washed in greater degree due to either (CH3)3COH release in water, oxygen release under exposure to light, or both from the surfaces of PMMA and P(tBuMA). The similar negative effect of PMMA on mictotubules was reported recently (Brunner et al., 2004).
Figure 15. Adsorption and polymerization of F-actin (23 mM) after 1.5 h in the continuous flow with the flow rate of 0.06 mL min–1 on polymeric surfaces: (A) NC, (B) PSMA, (C) PMMA (exposed), (D) P(tBuMA) (exposed). Scale bar, 10 μm.
147 Our efforts to mechanically induce the alignment of the adsorbed F-actin filaments using the flow field were not satisfactory. When 23 nM of F-actin was introduced into the flow cell, the initial alignment of actin filaments (up to 50 %) could be observed (data not shown), but over 1.5 h under the buffer flow, the alignment was not improved and most of the filaments were washed. At increased concentration, e.g., 23 mM of F-actin, a bulk of adsorbed filaments was observed. The adsorbing filaments were gradually and unevenly (depending on the polymeric surface) washing away over 1.5 h under the buffer flow. The desorption and washing of actin filaments occurred on P(tBuMA) and PMMA (shown in Figure 15) to a greater extent. Overall, 23 ± 3 %, 19 ± 5 %, 18 ± 3 % and 21 ± 3 % (n = 24) of actin filaments appeared aligned on nitrocellulose, PSMA, P(tBuMA), and PMMA, respectively. To determine the influence of fluid stress on the actin filaments in the flow cell, the shear stress at the wall in a flat-walled (actin-free) chamber (cell), υ, was found as a product of measured liquid viscosity (Figure 16), ω and shear rate, θ:
υ = ω · θ
Shear rate at the wall of the rectangular chamber is a function of the chamber width w = 22 mm and height h = 0.1 mm and applied flow rate Q = 0.06 mL min–1 (Han, 1998, Decave et al., 2003).
θ = 6Q/wh2 = 0.6 Pa
The attempts to force the flow-induced orientation of actin filaments by increasing the hydrodynamic forces in the flow cell have demonstrated that a shear rate greater than 0.6 Pa would lead to the breakage of filament, fast detachment, and washing from the surface. Notably, under similar experimental conditions, Fritzsche et al. (1998), Stracke et al. (2000), and Böhm et al. (2001) were able to align microtubules using mechanically induced flow fields. Indeed, microtubules can be more robust in such experiments due to their inherent rigidity and are able to stand rather strong hydrodynamic force. In contrast to actin, microtubules were able to
148 maintain its motility under 0.5–5 μm s–1 flow velocities applied in the cell (Stracke et al., 2000).
Figure 16. Estimation of the working buffer viscosity with and without BaSO4 (108 mM).
6.2.2.2. Evaluation of covalent bonding of G-actin on selected polymeric surfaces
To avoid the limitations of unstable physicochemical adsorption, we employed the cross-linking of G- and/or F-actin filaments with a water-soluble carbodiimide, EDC, by increasing the strength of the attachment. EDC is commonly used for the covalent attachment of proteins on surfaces as it catalyzes the formation of amide bonds between carboxylic groups of the polymeric surface and amine groups of proteins. The cross-linking reaction is favored by the presence of intermediate, N-
149 hydroxysulfo-succinimide (Grabarek and Gergely, 1990). In contrast to conventional agents, EDC does not remain as a part of that linkage but simply changes to water- soluble urea derivatives that have very low cytotoxicity (Tomihata and Ikada, 1997, Taguchi et al., 2002). The cross-linking reaction was confirmed by XPS analysis. Initially, XPS analysis of the surface of PSMA and P(tBuMA) + COOH showed peaks in the C 1s region at 285.0, 286.8 and 288.8 eV, which can be assigned to C–C/C–H, C–O and O–C = O species, respectively (Beamson and Briggs, 1992, Chastain, 1992).
6.2.2.3. Covalent bonding and self-assembly of G-actin on selected polymeric surfaces
The actin filament arrays were produced by polymerizing actin filaments covalently bound to the surface G-actin seeds under the constant flow. The use of covalent binding resulted in more stable bonding and greater density of rhodamine- phalloidin-labeled F-actin on all surfaces (Figure 17), which was further polymerized and aligned under the flow field (Figure 17).
Figure 17. Covalent bonding and polymerization of F-actin (23 mM) after 1.5 h in the continuous flow with the flow rate of 0.06 mL min-1 on polymeric surfaces: (A) PSMA, (B) PMMA (exposed), (C) P(tBuMA) (exposed). Scale bar, 10 μm.
150 It is noted that under the same experimental conditions over 1.5 h the amount of G-actin filaments was roughly three to four times greater on all polymeric surfaces tested; however, the degree of aligned F-actin filaments remained similar to that of adsorbed filaments, ranging from 21 ± 3 % to 17 ± 4 %. The degraded F-actin filaments were particularly noticeable on P(tBuMA), indicating again the poor biocompartibility of this type of polymeric surface. This observation is in agreement with the Bernheim-Groswasser et al. (2002) data, which showed that microtubules degraded rapidly in the presence of poly(dimethylsiloxane) (PDMS) and PMMA. We therefore excluded P(tBuMA) and PMMA from further experiments. The flow rate of 0.06 mL min–1 appeared to be optimal because the G-actin remained intact on the surface once it was covalently bound and was available for further polymerization.
6.2.3. Alignment of self-assembled actin filaments along fabricated microstructures
To concentrate F-actin on specific areas, we fabricated strips 10 μm wide and about 0.6 μm deep, where actin filaments were attached on the top of such strips (i.e., on the PSMA polymeric surface) rather than on the bottom (i.e., glass surface). The alignment of actin filaments was improved up to 80–90 % of the filaments. However, we could not achieve absolute selectivity of attachment as some filaments were stuck on the glass. According to our results (data not shown), the smaller than 5–10-μm- wide stripes were less effective, perhaps because when microstructures are smaller, filaments attach across the channels, break under the flow, and wash away. Dark field image of fabricated microstructures and fluorescence microscope images of rhodamine-phalloidin–labeled actin filaments along PSMA stipes and HMM-beads bound on actin filaments are shown in Figure 18.
151
Figure 18. Binding of self-assembled F-actin (23 nM) on functionalized PSMA polymeric surfaces. Dark field observation shows channels, 10 μm, fabricated on PSMA polymeric surfaces (A). Fluorescent observation shows self-assembled rhodamine-phalloidin-labeled F-actin filaments on the same field: (B) F-actin filaments aligned on the PSMA polymeric surfaces after 1.5 h in the continuous flow with the flow rate of 0.06 mL min-1; (C) the same as on (B) with antiHMM–HMM beads binding on F-actin. Scale bar, 10 μm.
6.2.4. Fabrication of electrostatically self-assembled actin filaments bundles
Even though we have demonstrated the feasibility of actin filaments self- assembly and alignment in the continous-flow system, the density of the filaments was not sufficient to form filament tracks capable of supporting continous bead movement. Besides, the directionality of motility is difficult to control as the motility remains random in such a system. In this regard we have designed the next experiment to achieve the fabrication of the tracks of actin bundles assembled from either F-actin or 2- μm-actin filaments with their barbed ends blocked by gelosin (Figures 19 A–F). We used gelsolin as an actin-modulating protein that binds to the plus (or barbed) end of actin monomers or filaments, preventing monomer exchange (end- blocking or capping). These F-actin fragments blocked by gelsolin (shown on Figure 19A) were used as ―seeds‖ for the self-assembly of actin filament bundles.
152 Gelsolin significantly affects the structure of actin polymerization and the rigidity of the filaments (Prochniewicz et al., 1996). The structural effect of gelsolin could be due to growth of the filaments on structurally altered nuclei: two monomers directly bound to gelsolin have been shown to be oriented in a different way than the monomers in spontaneously nucleated F-actin (Doi, 1992, Hesterkamp et al., 1993). A homogeneous increase of density in the bridge between two strands of the actin– gelsolin helix has also been shown (Prochniewicz et al., 1996).
153
Figure 19. Fluorescence images of (A) 2-μm actin filaments (23 nM) with their barbed ends blocked by gelsolin; and (B) their bundles condensed with Ba2+ (108 mM) during 45 min. Fluorescence images of electrostatically condensed and aligned actin- filament bundles assembled from 2-μm actin filaments (23 nM) (C) after 1.5 h; (D) after 3 h; and intact F-actin filaments: after 1.5 h (E) and 3 h (F) in the continuous flow system with the flow rate of 0.06 mL min-1. Scale bar, 10μm.
154 In order to assemble actin filament bundles, we adopted the methodology of like-charge attraction between polyelectrolytes induced by counterion charge density waves (Angelini et al., 2003, Tang et al., 1996, Butler et al., 2003). Angelini et al. (Angelini et al., 2003) have demonstrated that at high Ba2+ concentrations, F-actin condenses into closely packed bundles consisting of parallel arrays of three actin- filament units. This molecular mechanism is analogous to the formation of polarons in ionic solids (Wong et al., 1997a). In the case of F-actin, fluctuating counterions drag along soft helical distortions of the polyelectrolyte, and consequently freeze into static correlation, providing a transition between the extreme viewpoints of dynamic and static counterion correlations (Shklovskii, 1999). Electron microscopic images of electrostatically condensed F-actin/gelsolin bundles show the tightly packed parallel organization of F-actin filaments in the bundle (Figure 20). However, using SEM we were unable to visualise a helical structure and/or axial alignment of individual F-actin filaments (Tang et al., 1996).
155
Figure 20. SEM images of F-actin/gelsolin bundles formed from electrostatically condensed F-actin filaments. Accelerations voltage: 15 kV; magnification: 10,000x (top) and 20,000x (bottom).
156 In this study we went one step further using F-actin/gelsolin filaments to show that it can not only be successfully electrostatically condensed, but also form actin- filament bundles that were covalently bound on the surface. We were able to progressively form larger bundles aligned in the flow field that could be used in motility assays to support bead translocation. Intact F-actin filaments (Figure 19F) could be assembled into larger bundles compared to that assembled from 2-μm actin/gelsolin bundles (Figure 19D) over 3 h of condensation with Ba2+. We have also observed that over time, bundles of intact F-actin filaments become less regularly organized and form ―tree-like‖ tracks (Figure 19F), which, in fact, resulted in irregular-bead movement. The 2-μm actin/gelsolin fragment condensation after 45 min of incubation with 108 mM Ba2+ resulted in assembly of 15–20- μm-long bundles, which grew and aligned under the flow field over 3 h to produce elongated tracks. Morphologically, F-actin/gelsolin filaments after 3 h of electrostatic condensation appeared in more compact and organized bundles in contrast to F-actin filament bundles. This fact correlates with the earlier findings that binding of gelsolin to actin induces structural changes in the directly bound protomers, and these changes are subsequently propagated along the whole actin filament during its polymerization (Doi, 1992). Langford et al. (1994) reported that actin filaments assembled on the barbed end of acrosomal process of squid Limulus polyphmus maintained directionality toward the tip of actin filaments. Therefore, it is implied that the actin/gelsolin filaments in bundles also maintain a uniform polarity and therefore preserve more organized structure while condensing and aligning under the constant flow. However, the polar arrangement has not been discerned and unsubstantiated. Condensed into bundles, actin filaments preserved their functionality to support HMM-bead translocation. When the bead approached the bundle, it tended to accelerate along the bundle. The experiments with bead movement (illustrated in Figure 21) showed that the beads moved unidirectionally along the actin-filament bundles. The estimated average velocity of a 1 μm bead was 13.8 ± 5.1 μm s–1, which is faster than the average speed of rabbit actomyosin, 3–4.5 μm s–1 (Suzuki et al., 1997, Bunk et al., 2005, Sakamaki et al., 2003), and comparable with the sliding velocity of myosin-coated beads moving on actin filaments and/or bundles of different
157 origin (assembled on acrosomal process, on actin para-crystals, or algal actin bundles): 1.1–60 μm s–1.
Figure 21. Translocation of the antiHMM–HMM bead along the bundle formed from 2- μm-actin Alexa 488-phalloidin–labeled filaments (23 nM) and condensed with Ba2+ (108 mM). Scale bar, 10 μm.
These significant variations depended on samples and experimental setups (Chaen et al., 1995, Langford et al., 1994, Oiwa et al., 1990, Yamasaki and Nakayama, 1996, Suda and Ishikawa, 1997, Bernheim-Groswasser et al., 2002). The presence of Ba2+ ions did not affect the viscosity of the buffer (as shown in Figure 16) and hence could not slow down the bead velocity.
6.3. Conclusion
In summary, we have shown that among poly(styrene-maleic acid), poly(methyl methacrylate), and poly(t-butyl methacrylate) polymeric surfaces the former appeared to be more suitable for experiments as it lacked inherent fluorescence, had appropriate biocompatibility with actin–myosin in supramolecular manipulations, and provided sufficient amount of binding sites for the covalent immobilization of actin. The progressive formation of F-actin/gelsolin bundles by electrostatic condensation with Ba2+ and the alignment of such bundles can be easily performed 158 and controlled in the flow cell. Long-range cooperative transitions in actin induced by gelsolin represent a structural perturbation of the barbed end and presumably result in regularly organized bundles that supported directional bead movement. Our study also demonstrated that this simple technique for actin-filament-bundle fabrication provides a convenient experimental system that is applicable for the development of new device technology based on biomolecules.
159
CHAPTER 7
CHARACTERIZATION OF POTENTIAL
ATP, MreB AND FtsA PRODUCERS
160 7.1. Overview
In order to evaluate possibilities of both substituting eukaryotic linear molecular motor proteins with more robust bacterial homologues (see chapter 2.5 for more details) and providing molecular motors in microdevices with ATP energy (see chapter 6), a search for potential ATP and/or MreB/FtsA producers dwelling in marine environment was performed. Potential producers were isolated from both associated and free-living microbial communities. The former included bacteria, namely, Pseudoalteromonas issachenkonii, a producer of haemolysins, ectohydrolytic enzymes, biologically active compounds and unusual lipooligosaccharide (Ivanova et al., 2002b, Ivanova et al., 2002a, Alexeeva et al., 2002, Ivanova et al., 2003a, Kalinovskaya et al., 2004, Silipo et al., 2004, Alexeeva et al., 2004b), together with Formosa algae (Ivanova et al., 2004b), Brevibacterium celere (Ivanova et al., 2004c), Bacillus algicola (Ivanova et al., 2004a) and Planococcus maritimus isolated from the brown algae Fucus evanescens, Sulfitobacter delicatus from the starfish Stellaster equestris and Sulfitobacter dubius from the sea grass Zostera marina, while the latter included Marinobacter excellens isolated from radionuclide-contaminated sediments of Chazhma Bay, Sea of Japan. The chapter starts with a characterization of phenotypic and chemotaxonomic properties of potential marine ATP and/or MreB/FtsA producers, namely, Sulfitobacter (Reproduced from reference (Ivanova et al., 2004e) © 2004 With kind permission from IJSEM) and Marinobacter (Reproduced from reference (Gorshkova et al., 2003)) © 2003 With kind permission from IJSEM) belonging to the gram- negative group (see Table 10), and is followed by a characterization of the same properties of a member of the gram-positive group (see Table 10), namely, Planococcus (Reproduced from reference (Ivanova et al., 2006b) © 2006 With kind permission from Microbiological journal). The chapter continues with a description of genotypic and phylogenetic properties of these two groups. The chapter ends with a conclusion that all members of the gram-negative group were distinguished from described species by a number of phenotypic, chemotaxonomic, genotypic and phylogenetic traits. The following names and
161 numbers were assigned: Sulfitobacter delicatus KMM 3584T (=LMG 20554T = ATCC BAA-321T), Sulfitobacter dubius is KMM 3554T (=LMG 20555T = ATCC BAA-320T) and Marinobacter excellens KMM 3809T (=CIP 107686T). Remarkably, one member of the gram-negative group, namely, Marinobacter excellens KMM 3809T was found to have ―porous‖ features (dark spots in Figure 30) that may contain ATPases (discussed in subsection 2.5.5.3. and the following chapter). One member of the gram- positive group, namely, strain KMM 3738 was found to belong to Planococcus maritimus; it had unusual irregular coccoid shape of cells possessing a single flagellum. In contrast to Marinobacter excellens (Figures 30) , the surface of Planococcus spp. was found to be smooth with only 3 nm of cell-surface roughness (Figure 22). All these members of different microbial communities were screened for ATP and/or MreB/FtsA production (as described in the following chapters) to find the potential ATP producer to supply linear molecular motors in microdevices with ATP energy (see chapters 6 and 8) and/or substitute eukaryotic actin with more robust MreB or FtsA proteins (see chapters 2.5 and 9 for details).
7.2. Results and discussion
7.2.1. Phenotypic and chemotaxonomic classification
7.2.1.1. Gram-negative marine bacteria belonging to the genera Sulfitobacter and Marinobacter
7.2.1.1.1. Phenotypic and chemotaxonomic properties of Sulfitobacter delicatus
Sulfitobacter delicatus (de.li.ca'tus. L. masc. adj. delicatus beautiful).
Rod-shaped cells, single, about 0·7–0·9 μm in diameter. Gram-negative. Non- motile. Chemo-organotroph with respiratory metabolism. Colonies are uniformly round, 1–3 μm in diameter, regular, convex, smooth and slightly yellowish after
162 incubation for 48–74 h on marine agar. No diffusible pigment is produced in the medium. Does not form endospores. Accumulates poly-β-hydroxybutyrate as an intracellular reserve product. Oxidase- and catalase-positive. Required Na+ or sea water for growth (Table 7). Growth occurs in media containing 1–8 % NaCl. Mesophilic. Grows at 12–37 °C and pH 6·0–10·0; optimum growth is observed at 25 °C and pH 7·5–8·0. No growth is detected at 40 °C. Decomposes gelatin and alginate. Agar, starch, casein, laminarin, Tween 80 and DNA are not hydrolysed. From the 95 carbon sources tested, according to Biolog, α-cyclodextrin, glycogen, i-erythritol, psicose, D-raffinose, L-rhamnose, acetic acid, D-galactonic acid lactone, D-galacturonic acid, D-glucuronic acid, α-ketovaleric acid, glucuronamide, L-leucine, L-ornithine, D-serine, DL-carnitine, urocanic acid, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butandiol, DL-α-glycerolphosphate and glucose 1-phosphate are not utilized and L-fucose, D-galactose, gentiobiose, α-lactose, D- mannitol and hydroxyproline are only weakly utilized. Susceptible to ampicillin, benzylpenicillin, gentamicin, kanamycin, carbenicillin, neomycin, oleandomycin and streptomycin; not susceptible to polymyxin or tetracycline.
163 Table 7. Characteristics that differentiate Sulfitobacter delicatus KMM 3584T and Sulfitobacter dubius KMM 3554T from phylogenetically related species. Strains: 1, Sulfitobacter delicatus KMM 3584T; 2, Sulfitobacter dubius KMM 3554T; 3, Sulfitobacter pontiacus DSM 10014T; 4, Sulfitobacter mediterraneus ATCC 700856T; 5, Sulfitobacter brevis ATCC BAA-4T; 6, Staleya guttiformis DSM 11458T. None of the strains tested produced laminarinase or chitinase. +, Positive; -, negative; W, weak reaction. Data from this study and from Sorokin (1995), Pukall et al. (1999) and Labrenz et al. (2000).
Phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine are the major phospholipids. The main cellular fatty acid is cis-vaccenic acid (approx. 80 %).
164
7.2.1.1.2. Phenotypic and chemotaxonomic properties of Sulfitobacter dubius
Sulfitobacter dubius (du'bi.us. L. masc. adj. dubius doubtful).
Rod-shaped cells, single, about 0·6–0·8 μm in diameter and 1·2–1·5 μm long with a single subpolar flagellum. Gram-negative. Chemo-organotroph with respiratory metabolism. Colonies are uniformly round, 1–3 μm in diameter, regular, convex, smooth, slightly yellowish after incubation for 48 h on marine agar. No diffusible pigment is produced in the medium. Does not form endospores. Accumulates poly-β-hydroxybutyrate as an intracellular reserve product. Oxidase- and catalase- positive. Requires Na+ or sea water for growth. Growth occurs in media containing 1– 12 % NaCl (Table 7). Grows at 10–30 °C and pH 6·0–11·0; optimum growth is observed at 25 °C and pH 7·5–8·0. No growth is detected at 35 °C. Decomposes gelatin. Agar, starch, casein, laminarin, alginate, Tween 80 and DNA are not hydrolysed. From the 95 carbon sources tested, according to Biolog, i-erythritol, D- raffinose, thymidine, phenylethylamine, putrescine and 2-aminoethanol are not utilized and α-cyclodextrin, glycogen, L-fucose, D-galactose, L-rhamnose, D-sorbitol, D-galactonic acid lactone, D-galacturonic acid, glucuronamide, L-phenylalanine, L-pyroglutamic acid, D-serine and glucose 1-phosphate are only weakly utilized. Susceptible to ampicillin, benzylpenicillin, gentamicin, kanamycin, carbenicillin, neomycin, oleandomycin and streptomycin; not susceptible to polymyxin, tetracycline or lincomycin. Phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine are the major phospholipids. The main cellular fatty acid is cis-vaccenic acid (approx. 80 %).
165 7.2.1.1.3. Phenotypic and chemotaxonomic properties of Marinobacter excellens
Marinobacter excellens (ex'cell.ens. L. masc. adj. excellens remarkable, exceptional).
The majority of cells are rod-shaped, with lengths and widths that vary from 1 to 8 µm and from 0·6 to 1·4 µm, respectively. They are motile, polarly flagellated. Gram-negative strains that are strictly aerobic heterotrophs. Anaerobic growth occurs by fermentation of D-glucose by anaerobic respiration of nitrate. No endospores are formed. Colonies on marine 2216 agar are circular, smooth, and convex with an entire edge, transparent and 1–3 mm in diameter after 2 days of incubation at RT. Organic growth factors are not required. Growth occurs at 1–15 % NaCl. No growth at 20 % NaCl. Growth temperature ranges from 10 to 41 °C, with optimum growth at 20–25 °C. No growth is detected at 45 °C (Table 8). pH range for growth is 6·0–10·0, with optimum growth at pH 7·5. Oxidase-positive and weakly positive for catalase. Amylase and lipase are hydrolysed, whereas gelatin, casein, chitin, agar, alginate and laminaran are not. Bacteria are non-haemolytic on mouse blood agar, non-cytotoxic, do not exhibit antimicrobial activity, are susceptible to polymyxin and resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, carbenicillin, neomycin, tetracycline, lyncomycin, oleandomycin and streptomycin. Positive for lipase and amylase, but negative for agarase, chitinase, caseinase and gelatinase; able to utilize a limited range of carbohydrates. Of the 95 carbon sources in the Biolog system, strain KMM 3809T utilized Tween-85, N-acetyl-D-glucosamine, D-fructose, maltose, D-mannitol, L-rhamnose, D-sorbitol, methyl pyruvate, monomethyl succinate, cis-aconitic acid, D-galactonic acid lactone, α-hydroxybutyric acid, γ-hydroxybutyric acid, succinic acid, L-histidine, L-leucine, L-phenylalanine, L-proline, L-pyroglutamic acid, D-serine, DL-carnitine, urocanic acid and 2-aminoethanol. Major respiratory lipoquinone is Q9; PE, PG and DPG are major phospholipids.
166 Table 8. Characteristics that differentiate Marinobacter excellens from phylogenetically related species. Taxa: 1, Marinobacter excellens KMM 3809T; 2, Marinobacter hydrocarbonoclasticus; 3, Marinobacter aquaeolei. All strains are straight rod-shaped organisms, are oxidase-positive, exhibit lipase, grow in 15 % NaCl, do not hydrolyse gelatin, casein or chitin, are negative for haemolysis and are susceptible to polymyxin. W, Weakly positive. Data are from this study, Gauthier et al. (1992) and Nguyen et al. (1999).
*No. strains tested that are positive.
167 Three components, C16 : 0, C16 : 1ω9c and C18 : 1 ω 9c, accounted for >70 % of total fatty acids. Minor fatty acids included C12 : 0, C14 : 0, C15 : 0, C17 : 0, C18 : 0 and C17 : 1 ω 8c. In their main features, the fatty acid profiles were similar to those reported for Marinobacter species (Nguyen et al., 1999, Yoon et al., 2003a). These authors found a relatively high proportion of hydroxy fatty acids (up to 10 %), whilst in our experiments, C12 : 0 3-OH was detected in lower amounts (up to 0·7 %). Such variation in the proportion of fatty acids was observed previously for other Proteobacteria (Huys et al., 1994, Ivanova et al., 2000c) and can be explained by differing experimental conditions employed in different laboratories. In addition, a greater proportion of C16 : 1ω9c in the fatty acid profiles of the new isolates compared to those of other type strains and a number of differences in distribution of fatty acids present in minor amounts, i.e. accounting for <5–7 %, namely C15 : 0, C17 : 0 and C17 : 1ω8c, were also found. Notably, all bacteria of the genus Marinobacter exhibited an abundance of ω9c isomers of the fatty acids C16 : 1 and C18 : 1, which is in agreement with results reported previously for type strains grown under different cultivation conditions (Nguyen et al., 1999, Yoon et al., 2003a). We suggest that ω9c isomers of fatty acids C16 : 1 and C18 : 1 might be characteristic chemotaxonomic markers of the genus Marinobacter.
7.2.1.2. Gram-positive marine bacteria belonging to the genus Planococcus
7.2.1.2.1. Phenotypic and chemotaxonomic properties of Planococcus maritimus
Two bacteria were found to be oxidase- and catalase-positive, tolerant to 15 % NaCl levels, but not requiring Na+ ions for growth (Table 9). The effect of temperature on cell growth was monitored between 5-42 °C, with optimum growth found to occur at 25 °C, and weak growth around 45 °C. The pH range for growth was observed between 6.0-11.0, with optimum growth occurring at pH 8.5-9.0. Gelatin, casein, Tween 80, and alginate were all found to be hydrolyzed, while urea, starch, and agar were not. Both strains exhibited hemolytic and cytotoxic activities, but only
168 negative reactions towards Voges-Proskauer (acetoin production), indol, arginine dihydrolase, lysine decarboxylase, and ornithine decarboxylase tests. According to Biolog results, the following substrates were utilized: Tween 40, Tween 80, D-mannitol, methylpyruvate, D, L-Lactic acid, L-asparagine, D-serine, glycerol. In addition, strain KMM 3636 utilized glucose phosphate. The surface of Planococcus maritimus was found to be smooth with only 3 nm of cell-surface roughness (Figure 22).
169 Table 9. Differential phenotypic characteristics of Planococcus maritimus and other species of the genera Planococcus and Planomicrobium.
Characteristic 1 2 3 4 5 6 7 8 9 10
Colonies Orange Orange Orange Orange Yellow Orange Orange Orange Orange Yellow/ Orange
Cell co Irregular Cocci Rods Cocci Cocci Cocci Rods rods short Cocci/ Rods Rods/cocci
morphology
cci
Oxidase ------+ - W - Growth at 5-42 4-41 15- 0-30 4-37 4-37 0-30 4-38 20-37 0- temperature 41 37 (°C) NaCl no no yes no no no no no yes no requirement NaCl 15 17 12 12 10 3.3 12 6 15 7 tolerance, % Hydrolysis of: Starch - - + ------Casein + + ND ND ND ND ND + + + Gelatin + + + + + + + + + + Tween 80 + - - +/- - - + - - Nitrate v ------+ reduction Utilization of: D-Glucose - + - + d v - w - d D-Xylose - - - + - - + - + - Lactose ------+ - - D-Cellobiose ------+ - - Melibiose ------+ - - Glycerole + - - + - + + - - - G+C content 48 49 45 41.5 48- 40- 44.5 47 46 35 (mol%) 51 43
Taxa are identified as: 1 - Planococcus maritimus KMM 3738, KMM 3636; 2 - Planococcus maritimus KCCM 41587T; 3 – Planomicrobium alkanoclasticum NCIMB 13489T; 4 - Planococcus antarcticus DSM 14505T; 5 - Planococcus citreus DSM 20549T; Planococcus kocurii DSM 20747T; 7 – Planomicrobium psychrophilum DSM 14507T; 8 – Planomicrobium koreense JCM 10704T; 9 – Planomicrobium okeanokoites ATCC 700539T; 10 – Planococcus mcmeekinii NCIMB 561T; ―+‖ – positive; ―-‖ – negative, ―v(+)‖ – variable with most positive; d – different data in published articles; w – weak reaction; ND – no data available. Data from this study, Engelhardt et al. (2001), Yoon, et al. (2001, 2003b); Reddy et al. (2002). 170 The most relevant cellular fatty acids were branched chain saturated iso-methyl and anteiso branched acids, namely 14 : 0i, 15 : 0i, 15 : 0аi, and 16 : 0-i fatty acids the proportion of those has reached up to 60 %. More precisely, the amount of major fatty acids ranged as follows: 14 : 0-i - 15.1-16.2 %; 15 : 0-i – 12.9-13.2 %; 15 : 0-аi – 26.0- 25.6 %; 15 : 0 - 4.0-3.0 %; 16 : 0-i - 9.9-12.6 %; 17 : 0-аi – 3.0 - 3.4; 17 : 1ω8 – 3.7 – 2.6.
171 kind permission fromkind permission International Microbiology analys (non surface cell the 22 Figure
is (bottom) shows the roughness of the cell surface. cell the of roughness the shows (bottom) is . High - resolution AFM topographical images of images topographical AFM resolution - otc md, o) eeln dr sosprs Crepnet cross Correspondent spots/pores. dark revealing top) mode, contact .
Reproduced from reference from Reproduced Planococcus maritimus maritimus Planococcus F 90 cells and a close a and cells 90 F
( Ivanova et al., 2006a al., et Ivanova - section and line profiles profiles line and section - up of the area on on area the of up )
© 2006 With 2006 ©
172 7.2.2. Genotypic and phylogenetic characterization
7.2.2.1. Gram-negative marine bacteria belonging to the genera Sulfitobacter and Marinobacter
7.2.2.1.1. Genotypic and phylogenetic characterization of Sulfitobacter delicatus and Sulfitobacter dubius
The type strain of Sulfitobacter delicatus is KMM 3584T (= LMG 20554T = ATCC BAA-321T). The G + C content of the DNA of the type strain was 60·3 mol%. Isolated from a starfish (Stellaster equestris) collected from the South China Sea. The type strain of Sulfitobacter dubius is KMM 3554T (= LMG 20555T = ATCC BAA-320T). The G + C content of DNA of the type strain was 63·7 mol% (Table 7). Isolated from sea grass (Zostera marina) collected from the Sea of Japan. The level of DNA–DNA relatedness between the two strains studied was 33 % and they were therefore genotypically assigned to separate species. The genetic similarity of KMM 3584T to type strains of the genus Sulfitobacter, namely, Sulfitobacter pontiacus, Sulfitobacter brevis, Sulfitobacter mediterraneus and Staleya guttiformis was rather low (5–24 %); for KMM 3554T, the similarity was 10–41 %. Based on the generally accepted criterion of the definition of genomic species (Wayne et al., 1987), strains KMM 3584T and KMM 3554T are representatives of novel species. The most similar sequence to those of the novel isolates was that of ‗Oceanibulbus indoliflex‘ (99·5 %), followed by sequences from Sulfitobacter brevis, Sulfitobacter pontiacus, Sulfitobacter mediterraneus and Staleya guttiformis (≤97·8 %). An initial first analysis included the 79 most similar sequences as retrieved by BLAST on EMBL and EMBL new. Removal of sequences pertaining to species that were uncultured and not validly named led to a dataset of 14 sequences that were visually aligned and analysed by all three methods (Figure 23). The two KMM sequences grouped robustly with ‗Oceanibulbus indoliflex‘, a species that is yet to be described, but not with any species with validly published names, suggesting that each
173 of these sequences represents a novel bacterial species, as confirmed by DNA–DNA hybridization experiments. These two species clustered with the type species of the genus Sulfitobacter (Sulfitobacter pontiacus) according to NJ and ML, but not MP, and the degree of bootstrap replication was low (39 %), suggesting that the genus Sulfitobacter might be subject to revision in the future, probably to include only the type species of the genus. Such a revision may require phylogenetic analyses of more housekeeping genes; it is therefore suggested that the two novel species be assigned to the genus Sulfitobacter for the time being.
Figure 23. Phylogenetic position of Sulfitobacter delicatus KMM 3584T and Sulfitobacter dubius KMM 3554T according to 16S rRNA gene sequence analysis. The unrooted tree shown is the result of a neighbour-joining bootstrap analysis (1000 replications). Values shown are bootstrap percentages. Branches that were also retrieved by parsimony (three most parsimonious trees) and maximum-likelihood (ln=-3506) are respectively indicated by * and X (P<0·01).
174 7.2.2.1.2. Genotypic and phylogenetic characterization of Marinobacter excellens
The type strain is KMM 3809T(= CIP 107686T). Isolated from sediments collected in Chazhma Bay, Sea of Japan. DNA G + C content was 55·0–56·0 mol%. Domains used to construct the final phylogenetic trees (positions 88–1469 of KMM 3809T) were regions of the small-subunit rRNA gene sequences that were available for all sequences and excluded positions that were likely to show homoplasy or notoriously difficult to sequence, i.e. the 5' end of the sequences. 16S rRNA gene sequence analyses revealed that strain KMM 3809T is a member of the -Proteobacteria and, more precisely, that it is included in the clade formed by the genus Marinobacter (Figure 24). The topology of the phylogenetic tree shown in Figure 24 is that of the bootstrap analysis, as it has been demonstrated that this topology is often better than that of a simple NJ analysis (Gascuel, 1997). As a result, there is no distance bar in this tree; note also that the distance bar should be considered with caution in a tree, as it represents distances calculated after corrections (transversions being accounted for more than transitions) and branch-lengths do not represent the real number of differences between the sequences themselves. Bootstrap numbers are indicated only for branches that were also retrieved in the ML and MP trees (consensus tree). 16S rRNA gene sequence similarities with other available sequences were calculated by parsing the result of a BLAST analysis of KMM 3809T on the ‗Bacteria‘ division of GenBank (at 25 November 2002), with the options ‗no filter‘ and W = 7. The sequence of strain KMM 3809T had 97·3 % or less similarity to its nearest phylogenetic relatives, i.e. Marinobacter hydrocarbonoclasticus, Marinobacter aquaeolei and Marinobacter litoralis.
175
Figure 24. Phylogenetic position of Marinobacter excellens according to 16S rRNA gene sequence analysis. The topology shown was obtained by using the BIONJ algorithm and 1000 bootstrap replications with the Kimura two-parameter distance correction. Bootstrap values are indicated only for branches that were also retrieved by MP and ML (P<0·01); these branches should be considered as the only robust clusters identified by this analysis.
DNA–DNA hybridization data revealed a high level of DNA relatedness among KMM 3809T, KMM 3814, KMM 3817 and KMM 3818, ranging from 93 to 96 º%, which indicated that the strains belonged to the same species (Wayne et al., 1987). As the phenotypic and chemotaxonomic characteristics of KMM 3815 were identical to those of KMM 3814, the former was excluded from DNA–DNA hybridization experiments. Genetic similarity of KMM 3809T with type strains of the genus Marinobacter was 45–63 %. Based on the generally accepted criterion of the definition of genomic species (Wayne et al., 1987), strains KMM 3809T, KMM 3814, KMM 3817 and KMM 3818 are assigned to the novel species.
176 7.2.2.2. Gram-positive marine bacteria belonging to the genus Planococcus
7.2.2.2.1. Genotypic and phylogenetic characterization of Planococcus maritimus
The G + C content of the DNA ranged 48-49 mol%. The level of DNA hybridization between two novel strains isolated from algae was 98 %, suggesting these bacteria belong to a single genotypic species. The genetic similarity between the DNA of both KMM 3738 and KMM 3636 strains compared with that of the type strains of the genera Planomicrobium and Planococcus ranged between 12-15 % and 16-36 %, respectively, and with DNA from Planococcus maritimus 87 %. According to generally accepted criteria of the definition of the genomic species (Wayne et al., 1987), the strains isolated from brown algae Fucus evanescence can be assigned to Planococcus maritimus (Yoon et al., 2003b).
All phylogenetic analysis revealed that strain KMM 3738 was included in the clade formed the genera Planococcus and Planomicrobium (Figure 25).
177
Figure 25. Phylogenetic position of Planococcus maritimus KMM 3738 based on 16S rRNA gene sequence.
178 7.3. Conclusion
7.3.1. Classification of gram-negative marine isolates
On the basis of generally accepted criteria for the definition of genomic species (Wayne et al., 1987), gram-negative bacteria were assigned genotypically to different species. The following names and numbers were assigned: Sulfitobacter delicatus KMM 3584T (= LMG 20554T =ATCC BAA-321T), Sulfitobacter dubius KMM 3554T (= LMG 20555T = ATCC BAA-320T) and Marinobacter excellens KMM 3809T (= CIP 107686T). The two novel species can be distinguished from other Sulfitobacter species and Staleya guttiformis by phenotypic features (Table 7). For example, strain KMM 3584 T isolated from the starfish Stellaster equestris is able to hydrolyse gelatin and alginate and does not utilize melibiose, whereas strain KMM 3554T isolated from the sea grass Zostera marina is more halophilic, hydrolyses only gelatin and utilizes citrate and melibiose. Both novel species are unable to produce DNase or lipase. The most similar sequence to those of the novel isolates was that of ‗Oceanibulbus indoliflex‘ (99·5 %). The two KMM sequences grouped robustly with ‗Oceanibulbus indoliflex’, a species that was yet to be described, but not with any species with validly published names (Figure 23), suggesting that each of these sequences represented a novel bacterial species, as confirmed by DNA–DNA hybridization experiments. The genetic similarity of KMM 3584T to type strains of the genus Sulfitobacter was rather low (5–24 %); for KMM 3554 T, the similarity was 10–41 %. Based on the generally accepted criterion of the definition of genomic species (Wayne et al., 1987), strains KMM 3584T and KMM 3554 T are representatives of novel species. The names Sulfitobacter delicatus and Sulfitobacter dubius are proposed for KMM 3584T and KMM 3554T, respectively. Five strains of free-living marine bacteria (KMM 3809T, KMM 3814, KMM 3815, KMM 3817 and KMM 3818) isolated from radionuclide-contaminated sediments of Chazhma Bay, Sea of Japan, differed from other Marinobacter species by a number of phenotypic features (Table 8), for example, susceptibility to only one
179 antibiotic polymyxin. Remarkably, type strain KMM 3809T had ―porous‖ features (dark spots in Figure 30) that may contain ATPases (discussed in subsection 2.5.5.3. and the following chapter). Phylogenetic evidence, along with phenotypic and genotypic characteristics, showed that the bacteria constituted a novel species of the genus Marinobacter. Thus, phylogenetic 16S rRNA gene sequence-based analysis placed these bacteria in a clade within the genus Marinobacter- in the γ Proteobacteria. KMM 3809T showed highest 16S rRNA gene sequence similarity of 97·3 % to Marinobacter litoralis and 96·9 % to Marinobacter hydrocarbonoclasticus and Marinobacter aquaeolei (Figure 24). DNA–DNA hybridization between the five isolates was at the conspecific level (94–96 %) and that among the closest phylogenetic neighbours ranged from 45·0 to 62·5 %. The name Marinobacter excellens sp. nov. is proposed for this species, with the type strain KMM 3809T (= CIP 107686T).
7.3.2. Classification of gram-positive marine isolates
On the basis of generally accepted criteria for the definition of genomic species (Wayne et al., 1987), gram-positive bacteria were assigned genotypically to a single species. The following name and number were assigned: Planococcus maritimus KMM 3738. Two orange-pigmented bacteria (KMM 3738 and KMM 3636) isolated from enrichment culture during degradation of the thallus of the brown alga Fucus evanescens produced carotenoid pigments, were chemoorganotrophic, alkaliphilic and halo-tolerant growing well on nutrient media containing up to 15 % NaCl. Growth temperature ranged from 5 to 45 °C. The DNA base compositions were 48 mol% G + C and the level of DNA similarity of two strains was conspecific (98 %). A comparative phylogenetic analysis of 16S rRNA gene sequences (Figure 25) revealed that the strain KMM 3738 tightly clustered with Planococcus maritimus (99.9 % 16S rRNA gene sequence similarity). DNA-DNA hybridization experiments revealed that DNA from the KMM 3738 showed 12-15 % and 16-35 % of genetic relatedness with XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
180 the DNA of type strains of the genera Planomicrobium and Planococcus, respectively, and 87 % with DNA from Planococcus maritimus, indicating that new isolates belong to the later species. Interestingly, bacterium had unusual irregular coccoid shape of cells possessing a single flagellum. In contrast to Marinobacter excellens, the surface of Planococcus spp. was found to be smooth with only 3 nm of cell-surface roughness (Figure 22).
181
CHAPTER 8
CHARACTERIZATION OF ATPASES ACTIVITIES OF
MARINE BACTERIA
182 8.1. Overview
This chapter presents results of screening of several phylotypes of the domain Bacteria, comprising 86 environmental (marine and freshwater) bacteria of 17 genera. Reproduced from reference (Ivanova et al., 2006a) © 2006 With kind permission from International Microbiology. This work follows the study of self-assembly of actin filaments for microdevices presented in chapter 6. In order to evaluate the possibility of providing molecular motor proteins in microdevices with a cheap ATP energy, a search for bacterial ATP producers among strains belonging to different taxa was performed. The chapter starts with an estimate of the levels of extracellular ATP generated by bacteria, and is followed by a description of growth patterns of two selected ATP producers, gram-negative Sulfitobacter mediterraneus and gram- positive Planococcus maritimus (the latter is described in subsections 7.2.1.2. and 7.2.2.2.), on surfaces of different hydrophobicities: hydrophobic poly(tert-butyl methacrylate) (PtBMA) and hydrophilic (mica) surfaces (see subsection 8.2.2). The chapter continues with a discussion of effects of the polymeric surfaces on intracellular and extracellular ATP productions by two selected strains (see subsections 8.2.3. and 8.2.4.). As gram-negative bacteria belonging to the genera Sulfitobacter, Marinobacter and Staleya generated high amounts of extracellular ATP, while gram-positive strain belonging to the species Planococcus maritimus produced high amounts of intracellular ATP, their cell surfaces were examined. AFM (as described in subsection 3.5.6.) was used to reveal distinct features of ATP producers. The chapter ends with a description of particular features of potential ATP supplies. It was concluded that gram-negative extracellular ATP producers had porous cell surfaces (Figures 28, 30), while gram-positive intracellular ATP producer had a rough one (Figure 22). The former was assumed to have an effective membrane ultrastructure that facilitated the secretion of ATP.
183 8.2. Results and discussion
8.2.1. Levels of ATP detected in heterotrophic bacteria of different taxa
An estimate of the levels of ATP generated by 86 microorganisms of the 17 genera analyzed (Table 1) revealed that most of the frequently detected bacteria that secreted elevated amounts of ATP were members of the α-proteobacteria (e.g., Sulfitobacter spp. and related bacteria) and some γ-proteobacteria (in particular Marinobacter spp.), whereas members of certain gram-positive taxa, e.g., Kocuria spp. and Planococcus spp., secreted lesser amounts of ATP. In general, there were significant variations in the levels of secreted ATP, ranging from 190 μM ATP or 0.1 pM ATP per colony forming unit (cfu), as detected in Pseudoalteromonas spp., to 1.2–1.9 mM ATP or 6.0–9.8 pM ATP/cfu, as detected in Sulfitobacter spp., Staleya guttiformis, and Marinobacter spp. (Table 10). From the screening, two distantly related strains, gram-negative Sulfitobacter mediterraneus and gram-positive Planococcus maritimus, were selected in order to investigate the impact—if any—of surface hydrophobicity (in hydrophobic PtBMA and hydrophilic mica) on ATP production and secretion. The rationale of this selection was based on the notion that cellular membranes of gram-negative and gram-positive microorganisms differ significantly, and therefore it is of interest to understand whether the response of the attached cells reflects this difference. Both of the selected strains secreted the largest amounts of extracellular ATP in the culture fluid, in contrast to their counterparts of related and non-related phylotypes (Table 10).
184 Table 10. Levels of extracellular adenosine triphosphate (ATP) detected in heterotrophic bacteria of different taxa.
Taxon No. strains ATP; μM/ml ATP; pM/cfu*
*cfu: colony forming unit
A genus specific metabolic pattern could be also observed, even though there were some intra-species and intra-strain variations. Sulfitobacter spp., Staleya guttiformis, and Marinobacter spp. generated notable amounts of ATP (see Table 10). To our knowledge, this is the first report in which the ATP levels among diverse taxa have been estimated; therefore, no data are available that can be comprehensively compared with our experimental results. Nevertheless, our results are in agreement with previously reported data on the levels of ATP in microbial cells (Biteau et al., 2003, Di Tomaso et al., 2001, Fletcher, 1996) and comparable to the amounts detected in mammalian cells (Tornquist, 1991). For example, Di Tomaso et al. (2001) reported 8 that recombinant cells of phototrophic Rhodobacter capsulatus (OD660 = 0.5; 3 × 10 cells/ml) contained 1.35–2.64 mM ATP (0.6 pM ATP/cfu), and Biteau et al. (2003) found that Saccharomyces cerevisiae contained 1.78 mM ATP.
185 8.2.2. Pattern of bacterial growth on surfaces
The relative number of attached cells of Sulfitobacter mediterraneus increased slowly, and stabilized at 6 × 108 cfu/ml after about 32 h on the hydrophobic PtBMA surface. The number of attached cells on the hydrophilic mica remained low over the period studied (Figure 26).
Figure 26. Kinetics of adenosine triphosphate (ATP) production by Sulfitobacter mediterraneus ATCC 700856T during attachment on poly(tert-butyl methacrylate) (PtBMA) (top) and mica (bottom). ● number of cells in the culture medium, number of cells on the surface, production of • extra- and intracellular ATP.
186 In contrast, the number of attached cells of Planococcus maritimus increased rapidly within the first 20 h, up to 1 × 108 cfu/mlon PtBMA, and continued to increase over a period of 48 h. On the mica surface, the number of attached cells of this species reached 2 × 108 cfu/ml after 24 h and it stabilized at this level for the following 24 h (Figure 27). Notably, the growth pattern of planktonic cells of each strain in the correspondent wells with different surfaces was identical, although strain-specific features were retained all the time. Overall, it appeared that both strains showed a better propensity of attachment to hydrophobic surfaces than to hydrophilic ones.
Figure 27. Kinetics of ATP production by Planococcus maritimus F 90 during attachment on PtBMA (top) and mica (bottom). ● number of cells in the culture medium, number of cells on the surface, production of • extra- and intracellular
ATP.
187 Our study of the growth patterns of two bacterial strains on surfaces of different hydrophobicities and bacterial generation of intracellular and extracellular ATP revealed a few particular characteristics. Importantly, the generation of intracellular and extracellular ATP was followed by bacterial growth during attachment, which, in turn, was controlled by the type of surface. Both strains showed greater attachment to the hydrophobic PtBMA surface (other characteristics are discussed in subsections 8.2.3., 8.2.4. and 8.2.5.). This observation is in agreement with the well-known notion that most bacteria are more prone to attachment to hydrophobic than to hydrophilic surfaces. Yet, the driving mechanisms of this phenomenon remain unclear (Davey and O'Toole, 2000, Fletcher, 1996, Maechler et al., 1998, Pasmore and Costerton, 2003). While the physical environment provided by the PtBMA and mica surfaces no doubt exerts an effect on Planococcus maritimus and Sulfitobacter mediterraneus cells, remarkably, these bacteria responded differently, by producing increasing levels of either intracellular (Planococcus maritimus) or extracellular (Sulfitobacter mediterraneus) ATP.
8.2.3. Effect of polymeric surfaces on intracellular ATP generation
The levels of intracellular ATP in both species of bacteria were higher than those of extracellular ATP (Figures 26, 27). In addition, the levels of intracellular ATP varied during bacterial-cell attachment and biofilm formation over the 48-h experiments. For example, the level of ATP in Sulfitobacter mediterraneus increased significantly after 16 h of attachment on PtBMA and after 28 h on mica. Similar kinetics for intracellular ATP were observed in Planococcus maritimus albeit over a different time frame. A sharp increase of intracellular ATP production was detected in the early exponential phase of growth, after 8 h of attachment on PtBMA, and after 28 h on mica after a prolonged exponential phase of growth (Figures 26, 27). In general, the level of intracellular ATP correlated with the bacterial growth pattern, i.e., intracellular ATP levels reached maximum values when cells were at the exponential phase of growth, and they decreased when cells exited this phase and
188 entered the stationary growth phase. The average reduction in the amount of intracellular ATP produced by the two strains after 44 h was 70–90 %. It is not surprising that in both strains the levels of intracellular ATP were higher than those of extracellular ATP. It is logical that the generation of sufficient amounts of ATP by the cells, in particular during exponential growth, is essential for metabolic intracellular processes. During the attachment of either strain on hydrophilic mica, the highest increases in ATP production occurred after a prolonged lag period, while the same increase in ATP levels on hydrophobic PtBMA occurred earlier. Such changes in intracellular ATP may indicate that the chemical and/or physical properties of the surfaces affect cellular metabolism. The increase in the ATP level might be a reflection of the activities of several intensive metabolic processes as cells adapt, and then attach to the surface. Recently, it was shown that more than 200 bacterial genes are involved in the change from planktonic to biofilm life-style (Bassler, 2002, Kotra et al., 1999, Yan et al., 2003). Notably, the levels of intracellular ATP in the studied strains differed, in that the successful colonizer, Planococcus maritimus, contained up to five-fold more intracellular ATP than Sulfitobacter mediterraneus strains.
8.2.4. Variation in extracellular ATP generation
The levels of intracellular ATP of attached cells were in concert with both the extracellular ATP levels and the planktonic cell density in the same wells above the surfaces. The increase in the extracellular level of ATP of Sulfitobacter mediterraneus followed immediately the increase in its intracellular level of ATP. By contrast, some 4 h after intracellular ATP levels increased in Planococcus maritimus, its extracellular ATP levels increased. Similar patterns of intracellular and extracellular ATP production were observed on both surfaces. The levels and proportions of intracellular versus extracellular ATP significantly differed in the two strains (Figures 26 and 27). For example, the level of intracellular ATP in Sulfitobacter mediterraneus was 50-55 pM ATP/cfu on both polymeric surfaces, while Planococcus maritimus produced more intracellular ATP. In fact, Planococcus maritimus intracellular production was about 2.5–5 times higher and
189 ranged from 120 to 250 pM ATP/cfu depending on the surfaces, e.g., about two-fold more on mica. The amount of extracellular ATP generated by Planococcus maritimus planktonic cells was 6 pM ATP/cfu, and about the same for both PtBMA and mica, while the amount of extracellular ATP generated by Sulfitobacter mediterraneus ranged from 20 to 50 pM ATP/cfu, and was more than two-fold higher in the wells with mica (Figures 26 and 27). Interestingly, higher amounts of extracellular ATP were secreted by Sulfitobacter mediterraneus on mica, which appeared to be ‗difficult‘ for bacterial colonization, than on PtBMA. The cell response to this ‗unfriendly‘ physical environment might therefore have been an increase in the release of extracellular ATP. In contrast, during attachment on PtBMA, no dramatic changes in extracellular ATP levels were observed in either strain (Sulfitobacter mediterraneus secreted twice the amount found in our initial results). This observation can be partially explained by the fact that the bacterial densities of biofilms formed by both cultures on PtBMA polymeric surfaces did not reach the saturation level of 1012 cfu/cm3 (Bassler, 2002, Kuchma and O'Toole, 2000), so that the cell-density-dependent signaling system to control the production of cellular metabolites might not have been activated yet (Pasmore and Costerton, 2003). A biofilm-specific signaling system can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of cellular metabolites (Yan et al., 2003), so that an increase in ATP production would be also expected.
8.2.5. AFM investigation of bacterial surface ultrastructure
High-resolution AFM images of the cell surfaces at 0.5 μm (lateral dimension) of two representative strains that secreted high amounts of extracellular ATP, i.e., Staleya guttiformis and Marinobacter excellens, are shown in Figures 28 and 30. A few individual cells were selected and typical cell surfaces were imaged at closer range.
190
Figure 28. High-resolution atomic-force microscopy (AFM) topographical images of Staleya guttiformis DSM 11458T cells and a close-up of an area on the cell surface (non-contact mode, top) revealing dark spots/porous features. Correspondent cross- section and line profiles analysis (bottom) shows the tentative depth of the pores on the cell surface.
The surfaces of Staleya guttiformis and Marinobacter spp. cells appeared to be ―porous‖, with a surface roughness of about 11 nm. Although it was rather difficult to accurately estimate the depth of these surface features because of limitations of the AFM tip (Binnig et al., 1986, Dufrene, 2001, Dufrene, 2002, Dufrene, 2003, Grafstrom et al., 1993 ), there is no doubt about the presence of porous features on the surfaces of these bacteria. High-resolution cell surface images of bacteria that did not secrete pronounced amounts of extracellular ATP (see Table 10), e.g., Planococcus
191 maritimus and Formosa algae cells, were also obtained. In contrast to bacteria from the first group, the surface of Planococcus spp. was found to be smooth with only 3 nm of cell-surface roughness (Figure 22). Formosa algae produces extracellular polymeric material (most probably polysaccharides), as revealed by previous AFM analysis (Figure 29). As its surface appeared to be of amorphous ―gel-like‖ texture, it was not possible to obtain high- resolution images of those cells.
192 Reproduced reference from prof line and image Deflection (d) days. 2 after visualized and mica on deposited cell a of profile line and image Deflection (c) attachment. and/or gliding promote to la fine a revealed investigation Closer bacterium. the of edges the along noise of occurrence the Note cell. the of profiles transverse and longitudinal Deflectio 2 Figure n image and line profile of a cell freshly deposited on mica (30 min); the two curves correspond to the the to correspond curves two the min); (30 mica on deposited freshly cell a of profile line and image n 9 AM f el of cells of AFM . ( Ivanova et al., 2004b Ivanova yer of conditioning film (extracellular polymeric substances), secreted by the bacterium the by secreted substances), polymeric (extracellular film conditioning of yer oms algae Formosa
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T () elcin mg o cls eoie o mc. (b) mica. on deposited cells of image Deflection (a) .
193 So, the finding that Sulfitobacter spp., Staleya guttiformis, and Marinobacter spp. generated high amounts of ATP prompted further investigation into whether ATP generation and secretion might be reflected in distinct features of the cell surface. AFM imaging of the bacterial cell surface at high resolution revealed topographic peculiarities of those bacteria that secreted high amounts of extracellular ATP. These images showed ―porous‖ features on the surface of the studied strains (dark spots on Figures. 28, 30).
Figure 30. High-resolution AFM topographical images of Marinobacter excellens KMM 3809T cells and a close-up of the area on the cell surface (non-contact mode, top) revealing dark spots/porous features. Correspondent cross-section and line profiles analysis (bottom) shows the tentative depth of the pores on the cell surface.
194 There is no direct evidence yet that the ―porous‖ features found on the cell surface of Staleya guttiformis and Marinobacter excellens include ATP synthases that might facilitate ATP secretion. However, recently published research suggests that ATP synthesis is driven by a trans-membrane electrochemical gradient generated by light or oxidative reactions via the F0 part of ATP synthases incorporated into the cellular membrane (Fronzes et al., 2003, Hong and Pedersen, 2003, Müller et al., 2003). High-resolution AFM and transmission cryoelectron microscopy images of the ATPase from Ilyobacter tartaricus embedded into a lipid membrane (Lundin and Thore, 1975, Stahlberg et al., 2001) revealed the native structure and sizing of a single ATP synthase molecule. An average outer diameter of 5.4 ± 0.3 nm and a vertical roughness of about 3 nm were reported and are consistent with the sizes of the holes visualized on the surfaces of Staleya guttiformis and Marinobacter excellens. The dimensions of the protrusions (―bumps‖) on the cell surface of these strains were about 20–35 nm, with a vertical roughness of 4–11 nm. These measurements correlate well with the sizes of lipopolysaccharide (LPS) bundles reported recently (Kotra et al., 1999). While investigating the dynamics of LPS assembly on the surface of Escherichia coli, Kotra et al. (1999) obtained high-resolution images of the bacterial surface similar to those obtained in this study. The authors suggested that the spaces among these LPS bundles might be surface water-filled protein channels (Kotra et al., 1999). Within the outer membrane of gram-negative bacteria, particular proteins (antiporters, ABC transporters, symporters, porins, and other energy-transducing proteins) are incorporated in gated channels that facilitate entry of certain molecules into the cell (Beveridge, 1999, Biteau et al., 2003, Ferguson and Deisenhofer, 2004). This assumption does not exclude the possibility of the incorporation of ATPase into similar channels, which we observed on Staleya guttiformis and Marinobacter excellens cell surfaces. These bacteria may have an effective membrane ultrastructure that facilitates the secretion of ATP. Both α- and γ-proteobacteria represent abundant groups of marine prokaryotes (Buchan et al., 2000, Wagner-Dobler et al., 2003) that carry out several crucial ecological functions, including the reduction or oxidation of sulfur compounds (Pukall et al., 1999, Sorokin, 1995), the biodegradation of hydrocarbons and other compounds (Buchan et al., 2000, Doronina et al., 2000,
195 Gonzalez et al., 1997, Gonzalez et al., 2003), and the development of oxidant- dependent signal transduction systems (Allgaier et al., 2003, Shiba, 1991). These are thermodynamically unfavorable processes that are coupled to both an electrochemical proton gradient and the hydrolysis of ATP. In our experiments, the attachment of the bacteria onto hydrophilic mica might have imitated somewhat similar thermodynamically unfavorable/stressful processes, with a subsequent increase in the generation of ATP.
Conclusion
A survey of the extracellular ATP levels of 86 heterotrophic bacteria showed that gram-negative bacteria of the genera Sulfitobacter, Staleya, and Marinobacter secreted elevated amounts of extracellular ATP, ranging from 6.0 to 9.8 pM ATP/colony forming unit (cfu), and that gram-positive bacteria of the genera Kocuria and Planococcus secreted up to 4.1 pM ATP/cfu. The monitoring of variations in the levels of extra- and intracellular ATP- dependent luminescence in living cells of Sulfitobacter mediterraneus ATCC 700856T and Planococcus maritimus F 90 during 48 h of attachment on hydrophobic (PtBMA) and hydrophilic (mica) surfaces demonstrated that bacteria responded to different polymeric surfaces by producing either extra- or intracellular ATP. The level of intracellular ATP in Sulfitobacter mediterraneus ATCC 700856T attached to either surface was as high as 50–55 pM ATP/cfu, while in Planococcus maritimus F 90 it was 120 and 250 pM ATP/cfu on PtBMA and mica, respectively. Sulfitobacter mediterraneus ATCC 700856T generated about 20 and 50 pM of extracellular ATP/cfu on PtBMA and mica, respectively, while the amount generated by Planococcus maritimus F 90 was about the same for both surfaces, 6 pM ATP/cfu. The levels of extracellular ATP generated by Sulfitobacter mediterraneus during attachment on PtBMA and mica were two to five times higher than those detected during the initial screening. High-resolution atomic force microscopy imaging revealed a potentially interesting correlation between the porous cell-surface of certain α- and γ-proteobacteria and their ability to secrete high amounts of ATP.
196 Thus, our results have yielded useful insights in understanding the impact of hydrophilic and hydrophobic surfaces on bacterial attachment and ATP generation and further modeling of bacterial metabolism. So, gram-negative extracellular ATP producers belonging to the genera Sulfitobacter, Marinobacter and Staleya and/or gram-positive intracellular ATP producers belonging to the genera Planococcus and Kocuria can be considered as valuable candidates for nanotechnological application in microdevices.
197
CHAPTER 9 EVALUATION OF MreB AND FtsA PROTEINS
198 9.1. Overview
This chapter presents results of the screening of several phylotypes of the domain Bacteria, comprising 32 environmental (marine and freshwater) bacteria of 13 genera; 5 reference bacteria of 5 genera including 4 pathogenic and 1 thermophilic bacteria; and 1 eukaryotic actin for production of thermo- and inherently stable linear molecular motors with the longest half-lives. This work follows the study of self- assembly of actin filaments for dynamic microdevices presented in chapter 6 and study of ATP production by heterotrophic bacteria reported in chapter 8. In order to evaluate the possibility of substituting eukaryotic molecular motor proteins in microdevices with cheap prokaryotic homologues, a search for MreB and/or FtsA suppliers among strains belonging to different taxa was performed. The chapter starts with an estimate and comparison of the predicted stabilities of MreB proteins of selected bacterial taxa and actin, and is followed by evaluation of MreB parameters, namely, isoelectric point (pI) and overall hydrophobicity, that are important for immobilization of proteins in microdevices (see chapters 4, 5, 6 and subsection 9.2.1.2. for more details). Furthermore, determination of phylogenetic relationships based on MreB sequences was performed to evaluate the possibility of using PARP sequences as chronometers for rod-shaped bacteria (see subsections 2.5.4. and 9.2.1.3. for more details). The chapter continues with an estimate and a comparison of physicochemical properties of FtsA proteins and actin, and is followed by a discussion of phylogenetic positions of FtsA producers. The chapter ends with a conclusion that Pseudoalteromonas atlantica, Loktanella vestfoldensis and Thermotoga maritima can be used as MreB sources, whereas Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii and Salegentibacter flavus can be used as FtsA sources for substituting actin in microdevices, however, after preliminary evaluation of their biochemical properties in vitro. Moreover, it is concluded that FtsA gene may be used as a chronometer to help unravel phylogenies of new and/or misplaced bacteria.
199 9.2. Results and discussion
9.2.1. Comparison/Evaluation of predicted physicochemical properties of MreB proteins of selected bacterial taxa and actin
9.2.1.1. Stability of MreB proteins and actin
An estimate of stability of MreB proteins of 21 heterotrophic rod-shaped bacteria belonging to 12 genera and eukaryotic actin of rabbit skeletal muscle was based on the comparison of the following physicochemical properties: aliphatic index (AI), instability index (II) and estimated half-life. Calculation of AIs (Ikai, 1980), or measures of occupancy volumes of aliphatic side chains in proteins, revealed that all MreBs, including 3 reference MreBs of pathogens, namely, adherent invasive Escherichia coli (AIEC), Bacillus subtilis, Listeria monocytogenes and one reference MreB of thermophilic bacterium Thermotoga maritima, had higher AI values than eukaryotic actin (see Table 11 for details). The aliphatic indices of all MreBs ranged from 95.96 of Aliivibrio fischeri to 115.42 of Thermotoga maritima. Notably, five members of the γ-proteobacterial group, namely, Pseudomonas fluorescens, Pseudomonas extremorientalis, Pseudoalteromonas atlantica, Idiomarina loihiensis, Shewanella waksmanii, and two members of the α-proteobacterial group, namely, Sulfitobacter mediterraneus and Loktanella vestfoldensis appeared to have the highest aliphatic indices ranged between 100.89 and 103.58. Evaluation of thermostability of proteins by using AIs confirmed the dependence of thermostability of some MreBs and actin on thermostability of their origins. Thus, MreB protein with the highest AI was produced by the thermophilic bacterium Thermotoga maritima (Table 11). In fact, Thermotoga maritima owns the most thermostable MreB (see subsection 2.5.3.1.1. for details). Interestingly, all reference pathogens produced MreBs with similar thermostability ranged between 98.33 and 99.85. However, environmental bacteria produced very thermostable MreBs regardless of their own thermostabilities. For example, moderately thermophilic environmental bacteria, namely, Pseudomonas fluorescens, Pseudomonas
200 extremorientalis and Idiomarina loihiensis were able to produce MreBs of the similar thermostability as mesophilic ones, namely, Shewanella waksmanii, Loktanella vestfoldensis, Pseudoalteromonas atlantica and Sulfitobacter mediterraneus. These observations are in agreement with results of Ikai (1980). Assessment of MreB inherent stability based on dipeptide composition (Guruprasad et al., 1990) categorized 14 proteins including 7 MreBs of γ- proteobacteria belonging to Pseudoalteromonas atlantica, Alteromonas addita, Idiomarina zobellii, Idiomarina loihiensis, Pseudomonas fluorescens, Shewanella waksmanii, Pseudoalteromonas haloplanktis, 2 MreBs of α-proteobacteria belonging to Loktanella vestfoldensis and Loktanella rosea, 3 MreBs of pathogenic organisms, MreB of Thermotoga maritima and eukaryotic actin as stable proteins (see Table 11 for details). Once again, the most inherently stable MreB was produced by Thermotoga maritima. Interestingly, of 7 very thermostable MreBs belonging to environmental bacteria 5 were inherently stable (IIs < 40), 1 MreB of the α- proteobacterium Sulfitobacter mediterraneus was slightly unstable (II = 40.97) and 1 MreB of Pseudomonas extremorientalis was unstable (II = 47.49). As candidate protein for actin (AI = 81.78; II = 36.14) substitution in microdevices must be both very thermostable (AI > 100) and inherently stable (II < 36.14), only two out of 7 environmental bacteria, namely, Pseudoalteromonas atlantica and Loktanella vestfoldensis can be considered as the most valuable MreB producers. The results of evaluation of half-lives of proteins based on the N-end rule (Varshavsky, 1997) revealed only 3 very stable (up to 100 h in mammalian reticulocytes, in vitro) proteins: 1 MreB of the γ-proteobacterium Shewanella woodyi and 2 MreBs of the α-proteobacteria, namely, Sulfitobacter sp. RIOSW6 and Sulfitobacter sp. Fg 107 (see Table 11 for details). Interestingly, all three stable in model systems MreBs, but on the other hand, inherently unstable belonged to bacteria isolated from ecologically unusual environments. Thus, Shewanella woodyi was isolated from squid ink and intermediate seawater (Makemson et al., 1997); Sulfitobacter sp. RIOSW6 and Sulfitobacter sp. Fg 107 were isolated from sea water and sediments, respectively, of radionuclide-polluted area in Chazhma bay (Sea of Japan, Pacific Ocean). Besides, out of 7 very thermostable proteins, only MreBs of 3
201 γ-proteobacteria, namely, Pseudomonas fluorescens, Pseudoalteromonas atlantica, Idiomarina loihiensis, one α-proteobacterium Loktanella vestfoldensis and Thermotoga maritima were theoretically capable of living in mammalian reticulocytes 30 hours.
Table 11. Comparison of theoretical stability parameters (AI, II and half-life ) of MreB proteins of γ-Proteobacteria (1), α-Proteobacteria (2), Firmicutes (3), Thermotogae (4) and rabbit actin (5).
Bacte Strain Aliphatic Instabi Estimated half-life, h rial index lity group (AI) index, Mamma Yeast, Escheri (II) lian in vivo chia coli, reticulo in vivo cytes, in vitro
1 Pseudomonas 102.03 36.75 30 h > 20 h > 10 h fluorescens DSM 50030T (JF810206) Pseudomonas 100.90 47.49 1.1 h 3 min 10 h extremorientalis KMM 3447T (JF815021) Pseudoalteromonas 99.19 54.98 1.1 h 3 min > 10 h nigrifaciens ATCC 19375T (JF815022) Pseudoalteromonas 97.56 37.50 1.1 h 3 min > 10 h haloplanktis ATCC 14393T (JF815023) Pseudoalteromonas 100.89 33.70 30 h > 20 h > 10 h atlantica ATCC 19262T (JF815020) Alteromonas addita 97.98 37.21 > 20 h > 20 h ND R10SW13T (JF815024) Aliivibrio fischeri DSM 95.96 40.22 > 20 h > 20 h ND 507T (JF815027) Idiomarina zobellii 99.04 36.68 5.5 h 3 min 2 min KMM 231T (JF815025) Idiomarina loihiensis 102.31 39.55 30 h > 20 h > 10 h (YP_154773.1) Shewanella woodyi 97.50 45.79 100 h > 20 h > 10 h ATCC 51908T
202 Shewanella waksmanii 103.58 39.54 1.4 h 3 min > 10 h KMM 3823T (JF815026) Escherichia coli UM146 98.33 38.65 30 h > 20 h > 10 h (gb ADN69460.1) 2 Sulfitobacter 102.94 40.97 1 h 30 > 10 h mediterraneus min ATCC 700856T (JF825543) Sulfitobacter delicatus 96.06 47.73 1.1 h 3 min > 10 h KMM 3584T (JF825544) Sulfitobacter sp. 97.50 48.49 100 h > 20 h > 10 h Fg 107 (JF825545) Sulfitobacter sp. 97.50 45.79 100 h > 20 h > 10 h RIOSW6 (JF825546) Loktanella rosea Fg 1 97.05 37.79 1.1 h 3 min > 10 h (JF825547) Loktanella vestfoldensis 102.35 30.70 30 h > 20 h > 10 h (ZP_01001712.1) 3 Bacillus subtilis 99.85 28.58 30 h > 20 h > 10 h (gb AAA22397.1) Listeria monocytogenes 98.99 33.52 30 h > 20 h > 10 h (gb CAC99626.1) 4 Thermotoga maritima 115.42 27.75 30 h > 20 h > 10 h MSB8 (gb AAD35673.1) 5 Oryctolagus cuniculus, 81.78 36.14 30 h > 20 h > 10 h rabbit actin (P68135)
9.2.1.2. Isoelectric point (pI) and grand average of hydropathicity (GRAVY) of MreBs and actin
Since immobilization of proteins depends on both hydrophobicity of their amino acid residues and their molecular net charges (Ivanova et al., 2006c, Kyprianou et al., 2009, Muck et al., 2006), pI and grand average of hydropathicity (GRAVY) of MreBs and actin were calculated (see Table 12 for details). According to pI results, the majority of γ-proteobacterial MreBs were acidic, with the exception of MreBs of Pseudoalteromonas haloplanktis, Idiomarina zobellii and Shewanella waksmanii. Members of the α-proteobacterial group included 3 producers of acidic MreBs, namely, Sulfitobacter sp. Fg 107, Sulfitobacter sp. RIOSW6 and Loktanella vestfoldensis; and 3 producers of basic MreBs, namely,
203 Sulfitobacter mediterraneus, Sulfitobacter delicatus and Loktanella rosea. Notably, pI properties of MreBs turned out to be group (α/γ division) and genus independent. Moreover, while environmental bacteria produced MreBs with pIs ranged from 4.88 of Pseudoalteromonas nigrifaciens to 8.91 of Shewanella waksmanii, all pathogens, eubacterium and rabbit produced only acidic proteins, MreBs and actin, respectively. The GRAVY indices provided an evaluation of the overall hydrophobicities of MreBs and actin (see Table 12). All MreBs, with exception of actin (GRAVY = -0.232); two α-proteobacteria, Loktanella rosea (GRAVY = -0.049) and Sulfitobacter delicatus (GRAVY = -0.093); one γ-proteobacterium Pseudoalteromonas haloplanktis (GRAVY = -0.038) had hydrophobic features. Actin and Loktanella rosea had hydrophilic characteristics similar to those of two membrane-spanning proteins: Torpedo californica acetylcholine receptor (GRAVY = -0.22) and rabbit Ca2+-ATPase (GRAVY = -0.05) reported by Kyte (1982). As for hydrophobic proteins, all pathogens produced only slightly hydrophobic MreBs in the range between 0.04 - 0.061, while environmental bacteria produced MreBs in the range between 0.028 - 0.309. Notably, inherently unstable MreBs of Pseudomonas extremorientalis and Pseudoalteromonas nigrifaciens appeared to be the most hydrophobic ones. Besides, Thermotoga maritima produced moderately hydrophobic MreB (GRAVY = 0.193). To our knowledge, this is the first study on physicochemical properties of PARP reporting theoretical pIs and GRAVY indices; therefore, no data are available that can be comprehensively compared with these results. Nevertheless, our calculations correlate well with previously reported data on the average hydropathy of membrane-spanning proteins (Kyte and Doolittle, 1982). For example, membrane-spanning proteins such as human anion carrier, bovine rodopsin and human glucose carrier had 0.04, 0.28 and 0.37 GRAVY scores, respectively (Kyte and Doolittle, 1982). Kyte (1982) reported that GRAVY values for membrane-spanning proteins were higher than those for soluble proteins (-0.4). It is clear that GRAVY scores for membrane-spanning and/or membrane-associated proteins such as MreB (Defeu Soufo and Graumann, 2005) are higher than for soluble ones.
204 Table 12. Comparison of theoretical pI and GRAVY of MreB proteins of γ- Proteobacteria (1), α-Proteobacteria (2), Firmicutes, (3) Thermotogae (4) and rabbit actin (5).
Bacte Strain Theore Grand rial tical pI average of group hydropa thicity (GRAVY) 1 Pseudomonas fluorescens DSM 50030T (JF810206) 5.32 0.028 Pseudomonas extremorientalis KMM 3447T (JF815021) 5.61 0.309 Pseudoalteromonas nigrifaciens ATCC 19375T 4.88 0.300 (JF815022) Pseudoalteromonas haloplanktis ATCC 14393T 8.66 -0.038 (JF815023) Pseudoalteromonas atlantica ATCC 19262T (JF815020) 5.14 0.049 Alteromonas addita R10SW13T (JF815024) 8.28 0.033 Aliivibrio fischeri DSM 507T (DSM 507T) 6.38 0.035 Idiomarina zobellii KMM 231T (JF815025) 8.48 0.132 Idiomarina loihiensis (YP_154773.1) 5.04 0.106 Shewanella woodyi ATCC 51908T 5.27 0.085 Shewanella waksmanii KMM 3823T(JF815026) 8.91 0.198 Escherichia coli UM146 (gb ADN69460.1) 5.19 0.057 2 Sulfitobacter mediterraneus ATCC 700856T (JF825543) 8.00 0.171 Sulfitobacter delicatus KMM 3584T (JF825544) 8.68 -0.093 Sulfitobacter sp. Fg 107 (JF825545) 5.28 0.085 Sulfitobacter sp. RIOSW6 (JF825546) 5.27 0.085 Loktanella rosea Fg 1 (JF825547) 8.66 -0.049 Loktanella vestfoldensis (ZP_01001712.1) 6.03 0.050 3 Bacillus subtilis (gb AAA22397.1) 5.09 0.040 Listeria monocytogenes (gb CAC99626.1) 5.16 0.061 4 Thermotoga maritima MSB8 (gb AAD35673.1) 5.34 0.193 5 Oryctolagus cuniculus, actin, alpha skeletal muscle 5.23 -0.232 (P68135)
9.2.1.3. Phylogenetic relationships of MreB producers
Phylogenetic analysis of MreB sequences of 17 environmental bacteria, along with published MreB sequences from three pathogenic bacteria and one eubacterium Thermotoga maritima downloaded from GenBank, was carried out to examine the relationships among proteins (i.e., stable and unstable MreBs and/or actin). In general, inherently stable MreB proteins appeared to belong to evolutionary distant bacteria
205 such as the thermophilic eubacterium Thermotoga maritima ; γ-proteobacteria including seven environmental bacteria, namely, Pseudoalteromonas atlantica, Alteromonas addita, Idiomarina zobellii, Idiomarina loihiensis, Pseudomonas fluorescens, Shewanella waksmanii, Pseudoalteromonas haloplanktis and the pathogenic adherent invasive Escherichia coli (AIEC); the α-proteobacteria Loktanella vestfoldensis and Loktanella rosea; and pathogenic firmicutes. Thus, the neighbour-joining tree obtained in analysis of the MreB sequences and actin clearly demonstrated the presence of clades formed by distantly related bacteria such as Pseudoalteromonas haloplanktis (Ivanova et al., 2001) and Loktanella rosea (Ivanova et al., 2005). As discussed above, only MreBs of these two bacteria had negative GRAVY scores like actin. Since actin is distantly related to MreBs, its phylogenetic position was at the base of two GRAVY-negative sisters. Although some bacteria produced very thermo- and inherently stable MreBs, none of those MreBs was capable of living up to 100 hours in mammalian reticulocytes, with exception of MreBs belonging to three close phylogenetic relatives: one γ-proteobacterium Shewanella woodyi and two α-proteobacteria, namely Sulfitobacter sp. RIOSW6 and Sulfitobacter sp. Fg 107. It should be noted that there was a very strong support for the node uniting those three taxonomically distant bacteria as bacteria theoretically producing MreBs with the longest half-lives (up to 100 h in mammalian reticulocytes, in vitro). Furthemore, four very stable environmental bacteria were found to produce MreBs with 30-hour half-lives. Notably, one out of the four producers was the α- proteobacterium Loktanella vestfoldensis. This moderately thermophilic (grew at 45 ºC) resident of microbial mats in lake (Van Trappen et al., 2004) owned the most stable MreB with stability and half-life similar to Thermotoga maritima (see Table 11 for more details). This result correlates with the observation reported by Ikai (1980). The other very thermostable, but less inherently stable than Loktanella vestfoldensis’s MreBs, belonged to three γ-proteobacteria, namely, the freshwater inhabitant Pseudomonas fluorescens (does not grow at 42 ºC) (Lopez-Caballero et al., 2002), the seaweed-associated marine bacterium Pseudoalteromonas atlantica (does not grow at 40 ºC ) (Akagawa-Matsushita et al., 1992) and only one moderately thermophilic
206 hydrothermal vent bacterium Idiomarina loihiensis (grows at 46 ºC) (Donachie et al., 2003). Importantly, each of these three γ-proteobacteria formed clade with one of seven environmental γ-proteobacterial producers of thermo- and inherently stable MreBs. Interestingly, Loktanella vestfoldensis, a remarkable producer of MreB, had a phylogenetic brother, Sulfitobacter delicatus, and two distant relatives, namely, Pseudoalteromonas haloplanktis and Loktanella rosea. Although phylogenetic brother did not produce stable MreBs, two other members of the common clade of phylogenetic tree produced MreBs with actin-like overall hydrophobicity and inherent stability. It is important to note that two firmicutes, namely Bacillus subtilis and Listeria monocytogenes, produced MreBs with similar physicochemical properties. Phylogenetic positions of pathogens dictated by MreBs properties (see Figure 31) support the conclusion, reported in subsection 2.5.3.1.1., that MreBs of firmicutes differed from MreB of Thermotoga maritima. Moreover, MreB of the pathogenic γ-proteobacterium Escherichia coli appeared to have more similarity with MreB of the γ-proteobacterium Aliivibrio fischeri than with MreBs of pathogenic firmicutes.
207
Figure 31. Protein neighbor-joining phylogenetic tree shown is based on MreB sequences from heterotrophic bacteria using Thermotoga maritima as outgroup. Rabbit actin from Oryctolagus cuniculus is in red. Producers of in vivo stable proteins (instability indices < 40) are indicated by bold font; producers of in vitro stable proteins (half-lives of proteins in mammalian reticulocytes are 100 h) are indicated by underlined letters. Bootstrap values are shown as percentages of 1000 replicates. Bar, 0.05 amino acid substitutions per site.
208 9.2.2. Comparison/Evaluation of predicted physicochemical properties of FtsA proteins of selected bacterial taxa and actin
9.2.2.1. Stability of FtsA proteins and actin
The same stability parameters as for MreBs (see subsection 9.2.1.1 for details) were estimated for FtsA proteins of 30 heterotrophic bacteria belonging to 17 genera and eukaryotic actin of rabbit skeletal muscle. Calculation of AIs revealed that all FtsAs, including those of the thermophilic bacterium Thermotoga maritima and 3 reference FtsAs of rod-shaped pathogens and one FtsA of the pathogenic coccus Streptococcus pneumoniae, had higher AI values than eukaryotic actin (see Table 13 for details). The aliphatic indices of all FtsAs ranged from 88.40 of the radionuclide-sediment inhabitant Loktanella rosea Fg 1 (Ivanova et al., 2005) to 112.65 of the hydrocarbon-sediment inhabitant Marinobacter hydrocarbonoclasticus (Gauthier et al., 1992). Strikingly, nine members of the γ- proteobacterial group, namely, Cobetia marina, Marinobacter aquaeolei, Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii, Marinomonas communis, Marinomonas vaga, Marinomonas pontica, Idiomarina loihiensis and the pathogenic adherent invasive Escherichia coli (AIEC); one member of the CFB group, namely Salegentibacter flavus; one firmicute Streptococcus pneumoniae and the thermophilic bacterium Thermotoga maritima turned out to have the highest aliphatic indices climbing above 100. Again, similar to results reported in subsection 9.2.1.1, estimate of thermostability of proteins by means of AIs confirmed the dependence of thermostability of some FtsAs and actin on thermostability of their owners. Nevertheless, many questions concerning influence of protein origin on its stability remain unanswered. For example, not only FtsAs of moderately thermophilic Marinobacter aquaeolei, Idiomarina loihiensis and Marinobacter hydrocarbonoclasticus but also FtsAs of mesophilic Marinomonas pontica, Salegentibacter flavus, Marinomonas vaga and Oceanimonas doudoroffii appeared to be very thermostable. Moreover, FtsA with the highest AI was produced by a moderately thermophilic (grows at 45 ºC) Marinobacter hydrocarbonoclasticus
209 (Gauthier et al., 1992) even though Marinobacter hydrocarbonoclasticus is not as thermophilic as Thermotoga maritima. It is essential to note that MreB of the thermophilic eubacterium was more thermostable than FtsA (see subsection 9.2.1.1.). This evidence suggests that this bacterium can produce proteins of the same superfamily, for example actin superfamily, with different thermostabilities. In contrast to Thermotoga maritima, the γ-proteobacterium Escherichia coli had more thermostable FtsA than MreB. In case of protein production by firmicutes, there was no difference between thermostabilities of two PARPs of Bacillus subtilis and Listeria monocytogenes. From these results comes the conclusion that not only pathogenic lifestyle but also cell structure may contribute to thermostability of cell proteins. Assessment of FtsA inherent stability based on dipeptide composition (Guruprasad et al., 1990) categorized 13 proteins including 8 FtsAs of γ- proteobacteria belonging to Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii, Marinomonas communis, Marinomonas vaga, Aliivibrio fischeri, Shewanella woodyi, Shewanella japonica and a reference pathogen Escherichia coli; one representative of the Cytophaga–Flavo bacterium–Bacteroides (CFB) group Salegentibacter flavus; two FtsAs of pathogenic firmicutes Listeria monocytogenes and Streptococcus pneumoniae; one FtsA of Thermotoga maritima and eukaryotic actin as stable proteins (see Table 13 for details). Interestingly, none of α- proteobacteria appeared to produce inherently stable and/or very thermostable FtsAs. Strikingly, 2 out of 13 very thermostable FtsAs belonged to a model pathogen Listeria monocytogenes (see subsections 2.5.3.1.2. and 2.5.6.5.3. for details) and the thermophilic eubacterium Thermotoga maritima. Although 6 out of 13 inherently stable FtsAs (IIs < 40) were very thermostable, candidate protein for actin (AI = 81.78; II = 36.14) substitution in microdevices must be both very thermostable (AI > 100) and inherently stable (II < 36.14). So, only three out of 6 environmental bacteria, namely, Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii and Salegentibacter flavus can be considered the most valuable FtsA producers. An estimate of half-lives of proteins based on the N-end rule (Varshavsky, 1997) revealed 10 theoretically very stable (half-lives in mammalian reticulocytes up to 100 h) proteins: 8 FtsAs of the γ-proteobacteria, namely, Cobetia marina,
210 Marinobacter hydrocarbonoclasticus, Alteromonas macleodii, Oceanimonas doudoroffii, Oceanimonas smirnovii, Marinomonas vaga, Marinomonas pontica, Aliivibrio fischeri; one FtsA of the α-proteobacterium Loktanella rosea Fg 1 and one FtsA of the CFB bacterium Salegentibacter flavus (see Table 13 for details). Like MreBs of the eubacterium Thermotoga maritima and pathogens, FtsAs of the former and the latter had moderate half-lives (30 h in mammalian reticulocytes, in vitro). Remarkably, three FtsAs belonging to Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii and Salegentibacter flavus were not only very thermo- and inherently stable but also the longest living.
Table 13. Comparison of theoretical stability parameters (AI, II and half-li fe) of FtsA proteins of γ-Proteobacteria (1), α-Proteobacteria (2), CFB group (3), Firmicutes (4), Thermotogae (5) and rabbit actin (6).
Bacte Strain Alipha Insta Estimated half-life, h rial tic bility group index index Mamma Yeast, Escheri (AI) (II) lian in vivo chia reticulo coli, cytes, in vivo in vitro 1 Cobetia marina LMG 2217T 102.52 45.71 100 h > 20 h > 10 h (JF893438) Marinobacter aquaeolei 105.55 43.35 30 h > 20 h > 10 h (ABM19523.1) Marinobacter 112.65 32.02 100 h > 20 h > 10 h hydrocarbonoclasticus ATCC 49840T (JF893439) Pseudoalteromonas 97.45 44.14 30 h > 20 h > 10 h issachenkonii KMM 3549T (JF893436) Pseudoalteromonas 99.51 44.86 30 h > 20 h > 10 h nigrifaciens ATCC 19375T (JF893437) Pseudoalteromonas atlantica 95.55 41.66 30 h > 20 h > 10 h (ABG42017.1) Alteromonas macleodii 96.09 45.26 100 h > 20 h > 10 h ATCC 27126T Oceanimonas doudoroffii 110.13 32.02 100 h > 20 h > 10 h ATCC 27123T (JF893440)
211 Oceanimonas smirnovii 98.65 44.76 100 h > 20 h > 10 h 31-1T (JF893441) 1 Marinomonas communis 110.5 38.17 1.1 h 3 min > 10 h ATCC 27118T (JF893442) Marinomonas vaga 109.35 39.20 100 h > 20 h > 10 h ATCC 27119T (JF893443) Marinomonas pontica 46-16T 110.0 47.03 100 h > 20 h > 10 h (JF893444) Aliivibrio fischeri DSM 507T 98.65 39.32 100 h > 20 h > 10 h (JF893445) Idiomarina baltica 98.15 47.20 30 h > 20 h > 10 h (ZP_01043577.1) Idiomarina loihiensis 101.04 47.55 30 h > 20 h > 10 h (AAV81283.1) Shewanella woodyi 95.14 36.94 1.9 h > 20 h > 10 h ATCC 51908T Shewanella affinis 95.71 40.95 1.9 h > 20 h > 10 h KMM 3587T (JF893433) Shewanella waksmanii 94.60 42.91 > 20 h > 20 h ND KMM 3823T (JF893434) Shewanella japonica 92.47 36.88 1.9 h > 20 h > 10 h KMM 3299T (JF893435) Escherichia coli UM146 102.29 38.66 30 h > 20 h > 10 h (gb ADN73972.1) 2 Sulfitobacter pontiakus 91.51 47.73 0.8 h 10 min 10 h DSM 10014T (JF893447) Sulfitobacter delicatus 89.78 48.17 1 hour 2 min 2 min KMM 3584T (JF893448) Sulfitobacter sp. Fg 107 88.45 48.43 1 hour 2 min 2 min (JF893449) Sulfitobacter sp. RIOSW6 90.82 49.67 7.2 h > 20 h > 10 h (JF893450) Loktanella rosea Fg 1 88.40 47.53 100 h > 20 h > 10 h (JF893451) 3 Salegentibacter flavus 110.13 32.02 100 h > 20 h > 10 h Fg 69T (JF893446) 4 Bacillus subtilis 96.09 47.73 30 h > 20 h > 10 h (gb AAA22456.1) Listeria monocytogenes 98.59 29.19 30 h > 20 h > 10 h (gb CAD00111.1) Streptococcus pneumoniae 100.44 31.50 30 h > 20 h > 10 h DD39 (YP_816936.1) 5 Thermotoga maritima MSB8 106.10 29.21 30 h > 20 h > 10 h (NP_229082.1) 6 Oryctolagus cuniculus, actin, 81.78 36.14 30 h > 20 h > 10 h alpha skeletal muscle (P68135)
212 9.2.2.2. Isoelectric point (pI) and grand average of hydropathicity (GRAVY) of FtsAs and actin
An estimate of pI and grand average of hydropathicity (GRAVY) of FtsAs of 30 bacteria, including 19 environmental γ-proteobacteria and 1 pathogenic γ-proteobacterium Escherichia coli (AIEC), 5 environmental α-proteobacteria, 1 CFB group member, 3 pathogenic firmicutes, 1 thermophilic eubacterium, and eukaryotic actin of rabbit skeletal muscle revealed that all proteins were acidic (see Table 14 for details). The GRAVY indices provided an evaluation of the overall hydrophobicities of FtsAs and actin (see Table 14). Majority of FtsAs were slightly hydrophilic, with exception of Marinobacter aquaeolei (GRAVY = 0.125) , Marinobacter hydrocarbonoclasticus (GRAVY = 0.152) , Pseudoalteromonas nigrifaciens (GRAVY = 0.006 ), Oceanimonas doudoroffii (GRAVY = 0.145), Marinomonas communis (GRAVY = 0.125 ), Marinomonas vaga (GRAVY = 0.154), Marinomonas pontica (GRAVY = 0.096), Aliivibrio fischeri (GRAVY = 0.026), Idiomarina loihiensis (GRAVY = 0.036), one α-proteobacterium Loktanella rosea (GRAVY = 0.012) and a member of the CFB group, namely Salegentibacter flavus, (GRAVY = 0.135). So, the three thermo- and inherently stable FtsAs (see subsection 9.2.2.1. for details) were the most hydrophobic ones (see Table 14). To our knowledge, no data are available that can be comprehensively compared with these results except for data on the average hydropathy for membrane-spanning proteins (Kyte and Doolittle, 1982) discussed in subsection 9.2.1.2., which correlates well with calculations presented in this subsection.
213 Table 14. Comparison of theoretical pI and GRAVY of FtsA proteins of γ- Proteobacteria (1), α-Proteobacteria (2), CFB group (3), Firmicutes (4), Thermotogae (5) and rabbit actin (6).
Bacte Strain Theore Grand rial tical pI average group of hydropa thicity (GRAVY) 1 Cobetia marina LMG 2217T (JF893438) 4.7 -0.001 Marinobacter aquaeolei (ABM19523.1) 5.15 0.125 Marinobacter hydrocarbonoclasticus 4.82 0.152 ATCC 49840T (JF893439) Pseudoalteromonas issachenkonii KMM 3549T (JF893436) 4.61 -0.030 Pseudoalteromonas nigrifaciens ATCC 19375T (JF893437) 4.8 0.006 Pseudoalteromonas atlantica (ABG42017.1) 5.0 -0.017 Alteromonas macleodii ATCC 27126T 4.63 -0.096 Oceanimonas doudoroffii ATCC 27123T (JF893440) 4.82 0.145 Oceanimonas smirnovii 31-1T (JF893441) 4.79 -0.099 Marinomonas communis ATCC 27118T (JF893442) 4.4 0.125 Marinomonas vaga ATCC 27119T (JF893443) 4.73 0.154 Marinomonas pontica 46-16T (JF893444) 4.76 0.096 Aliivibrio fischeri DSM 507T (JF893445) 4.83 0.026 Idiomarina baltica (ZP_01043577.1) 4.93 -0.030 Idiomarina loihiensis (AAV81283.1) 4.88 0.036 Shewanella woodyi ATCC 51908T 4.73 -0.139 Shewanella affinis KMM 3587T (JF893433) 4.63 -0.139 Shewanella waksmanii KMM 3823T (JF893434) 4.63 -0.163 Shewanella japonica KMM 3299T (JF893435) 4.62 -0.178 Escherichia coli UM146 (gb ADN73972.1) 5.94 -0.040 2 Sulfitobacter pontiakus DSM 10014T (JF893447) 4.92 -0.024 Sulfitobacter delicatus KMM 3584T (JF893448) 5.71 -0.037 Sulfitobacter sp. Fg 107 (JF893449) 5.62 -0.100 Sulfitobacter sp. RIOSW6 (JF893450) 4.88 -0.018 Loktanella rosea Fg 1 (JF893451) 5.64 0.012 3 Salegentibacter flavus Fg 69T (JF893446) 4.82 0.135 4 Bacillus subtilis (gb AAA22456.1) 5.27 -0.249 Listeria monocytogenes (gb CAD00111.1) 4.63 -0.062 4 Streptococcus pneumoniae DD39 (YP_816936.1) 5.12 -0.063 5 Thermotoga maritima MSB8 (NP_229082.1) 5.21 -0.084 6 Oryctolagus cuniculus, actin, alpha skeletal muscle 5.23 -0.232 (P68135)
214
9.2.2.3. Phylogenetic relationships of FtsA producers
Phylogenetic analysis of FtsA sequences of 25 environmental bacteria, together with published FtsA sequences of 4 pathogenic bacteria and 1 eubacterium Thermotoga maritima downloaded from GenBank, was performed to examine the relationships among proteins (i.e., stable and unstable FtsAs and/or actin). In general, inherently stable FtsAs appeared to belong to evolutionary distant bacteria such as 8 γ- proteobacteria, 1 CFB group member, 2 firmicutes and 1 eubacterium. Thus, the neighbour-joining tree obtained in analysis of the FtsA sequences and actin clearly demonstrated that producers of inherently stable proteins branched with their closest phylogenetic relatives. For example, 4 members of the genus Shewanella, namely, 2 bacteria with inherently stable and 2 bacteria with slightly inherently unstable FtsAs (see Table 13) constituted one clade (see Figure 32). Furthermore, actin branched with α-proteobacteria with strong support by bootstrap analysis. Since the contribution of α-proteobacteria to cell evolution has been under discussion during the last decade (Vellai et al., 1998, Vellai and Vida, 1999, Vesteg and Krajcovic, 2008), this phylogenetic relationship may be taken into consideration; it can give us additional clue to unravel the mystery of cell origins. Moreover, phylogenetic positions of Aliivibrio fischeri, Escherichia coli and Oceanimonas doudoroffii shed some evolutionary light on their relationships by supporting the conclusion that they are phylogenetic neighbors (Ivanova et al., 2004h, Dorsch et al., 1992). Furthemore, three environmental bacteria, namely two γ-proteobacteria, Marinobacter hydrocarbonoclasticus and Oceanimonas doudoroffii, and the CFB bacterium Salegentibacter flavus, were found to produce FtsAs with 100-hour half- lives. Despite belonging to different groups, these three bacteria appeared to be three brothers producing FtsAs with remarkable physicochemical properties. Phylogenetic positions of pathogens dictated by FtsAs properties (see
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
215 Figure 32) support the conclusion that FtsAs of all firmicutes and Escherichia coli differed from FtsA of Thermotoga maritima. Moreover, MreB as well as FtsA protein of Escherichia coli appeared to have more similarity with MreB and FtsA of the
γ-proteobacterium Aliivibrio fischeri than with those of pathogenic firmicutes.
216
Figure 32. Protein neighbor-joining phylogenetic tree shown is based on FtsA sequences from heterotrophic bacteria using Thermotoga maritima as outgroup. Rabbit actin from Oryctolagus cuniculus is in red. Producers of in vivo stable proteins (instability indices < 40) are indicated by bold font; producers of in vitro stable proteins (half-lives of proteins in mammalian reticulocytes are 100 h) are indicated by underlined letters. Bootstrap values are shown as percentages of 1000 replicates. Bar, 0.05 amino acid substitutions per site.
217 9.3. Conclusion
The information gained from physicochemical and phylogenetic analyses of 21 MreB and 30 FtsA sequences of environmental, pathogenic, thermophilic bacteria and sequence of eukaryotic skeletal actin presented here provided some valuable insight into understanding the difference between eukaryotic actin and its prokaryotic homologues. Thus, some PARPs produced by environmental bacteria had the longest half-lives and/or were more thermo- and inherently stable than actin. The study of MreB sequences demonstrated that very thermo- and inherently stable MreB proteins with moderate half-lives (30 h in mammalian reticulocytes, in vitro) were produced by three phylogenetically distant bacteria, namely, Pseudoalteromonas atlantica, Loktanella vestfoldensis and Thermotoga maritima; these three MreBs can be used instead of actin for building linear molecular assemblies in microdevices, however, after preliminary in vitro evaluation of their biochemical properties. Although our results called attention to the finding that three members of the same phylogenetic clade, namely, Shewanella woodyi, Sulfitobacter sp. RIOSW6 and Sulfitobacter sp. Fg 107 (Figure 31), produced MreBs with 100-h half-lives (mammalian reticulocytes, in vitro), the MreB producers were not considered good candidates for nanothechnological application in microdevices due to moderate thermostabilities and inherent instabilities of their MreBs. Remarkably, 3 FtsAs belonging to Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii and Salegentibacter flavus were not only very thermo- and inherently stable but also the longest living (100 -h half-lives of proteins in mammalian reticulocytes, in vitro), therefore, if they can produce actin-like linear assemblies, they can be used as replacements for eukaryotic actin in microdevices. Moreover, data obtained pointed to a correlation between phylogenetic analyses based on FtsA and 16S rRNA sequences, therefore, FtsA gene may be used as a chronometer to help unravel phylogenies of new and/or misplaced bacteria.
218
CHAPTER 10 CONCLUSIONS AND FURTHER WORK
219 10.1. Conclusions
10.1.1. Overview
A novel approach for designing the surfaces of microdevices has been proposed. Our approach is based on ‗combinatorialized‘ micro/nano-channels that allow amplified protein immobilization in a highly controlled manner (Ivanova et al., 2004d, Ivanova et al., 2003b, Nicolau et al., 2010a, Ivanova et al., 2004f, Nicolau et al., 2010b). An innovative methodology allowing in vitro assembly of micron- and nano-scale tracks of protein (i.e., actin) which support unidirectional translocation of beads functionalized with motor proteins (i.e., myosin) was also developed. It was further suggested that in order to advance the stability and efficiency of microdevices based on molecular motor systems: i) substitution of commercial ATP with ATP produced by bacteria and b) substitution of eukaryotic actin with prokaryotic actin- related proteins, e.g., MreB or FtsA, may be considered. Employment of bacterial ATP, rather than a commercial ATP product, and real-time production of ATP are required to sustain the self-assembly of actin or its homologues and to make the molecular motor system as a whole more stable and long lasting. This can be achieved by incorporation of efficient bacterial ATP producers into the next generation of microdevices. The suitability of the employment of bacterial ATP producers and prokaryotic actin-related proteins such as MreB or FtsA protein as replacements for the energy source and eukaryotic actin (see subsection 2.5. and chapter 6 for more details), respectively, in the construction of the next generation of microdevices was also evaluated. From all bacteria isolated, bacteria of the genera Sulfitobacter, Marinobacter and Staleya and/or Planococcus and Kocuria were found to be the most promising producers of extracellular ATP.
10.1.2. Protein immobilization in ‘combinatorialized’ micro/nano-channels
A comparative study investigating the immobilization pattern of five proteins belonging to three major structural classes was carried out. The proteins were
220 immobilized along microchannels, fabricated by laser microablation of thin metal/blocking protein layers deposited on a polymeric substrate. It has been shown that the protein adsorption was amplified between 3 to 10 times depending on the molecular surface of the protein (see chapter 5). The results obtained demonstrated that physicochemical adsorption of HSA, HIgG, α-chymotrypsin, lysozyme, and myoglobin in the microchannels was at least 2.5 to 5 times greater than that on the plain PMMA polymeric surfaces (Ivanova et al., 2004d, Ivanova et al., 2003b, Nicolau et al., 2010a, Ivanova et al., 2004f, Nicolau et al., 2010b). A surface mass density of adsorbed protein molecules on the latter, defined by a protein-film thickness and a refractive index for the protein layer, correlated with the data obtained for fluorescently labeled proteins. Thus, different types of proteins were found to be immobilized at increased levels retaining their bioactivities. It was concluded that the amplified and ‗combinatorialized‘ adsorption on micro/nano-structures has the potential of improving detection of multiplex analytes if used for microdevices.
10.1.3. Controlled self-assembly of actin filaments along microchannels in a continuous-flow system
Although different methodologies have been applied to align actin and actin- based motility through a variety of techniques, e.g., myosin guiding (Butt et al., 2009), magnetic field (Kaur et al., 2010), electric field (Wigge et al., 2010), UV lithography (Yamamoto et al., 2008), they are not suitable for the fabrication of aligned actin tracks, which can support unidirectional bead translocation in vitro, due to the lack of precise control over them at the level of either individual or bundled linear assemblies. To solve this problem, we developed a methodology allowing assembly of F- actin filament tracks that can support the movement of cargo particles (Alexeeva et al., 2004a, Watson et al., 2004, Alexeeva et al., 2005). In this work PSMA polymeric surfaces were used for the immobilization of self-assembly of the actin filaments in vitro in a continuous-flow system. Gelsolin was used to induce cooperative transition in actin via a structural perturbation of the barbed end of monomeric actin resulting in formation of regularly organized actin/gelsolin bundles that supported directional bead
221 movement. The progressive formation of F-actin/gelsolin filaments by electrostatic condensation with Ba2+ and alignment of actin/gelsolin bundles was also demonstrated. This study established that the developed simple technique for actin- filament-bundle fabrication provides a convenient experimental system that may be applicable for the next generation of microdevices.
10.1.3.1. Search for bacterial ATP producers to be used as replacements for the energy source in microdevices
In order to evaluate the possibility of the employment of bacterial producers of ATP as replacements for the energy source in the construction of the next generation of microdevices, a search for ATP producers among 86 environmental strains belonging to several phylotypes of the domain Bacteria has been performed. A collection of environmental (marine and freshwater) bacteria comprising 17 genera is maintained at Swinburne University of Technology, Faculty of Life and Social Sciences. It was demonstrated that gram-negative bacteria of the genera Sulfitobacter, Staleya, and Marinobacter secreted elevated amounts of extracellular ATP while gram-positive bacteria of the genera Kocuria and Planococcus secreted high amounts of intracellular ATP. Variations in the levels of extracellular and intracellular ATP- dependent luminescence monitored in living cells of Sulfitobacter mediterraneus ATCC 700856T and Planococcus maritimus F 90 (the latter is described in subsections 7.2.1.2.1 and 7.2.2.2.1) during 48 h of attachment on different surfaces demonstrated that bacteria were capable of producing either extra- or intracellular ATP, depending on the experimental conditions (as described in subsections 8.2.2 – 8.2.4). It was found that the levels of extracellular ATP generated by Sulfitobacter mediterraneus during attachment on PtBMA and mica were two to five times higher than those detected during the initial screening. High-resolution AFM imaging revealed a potentially interesting correlation between the porous cell-surface of certain α- and γ- proteobacteria and their ability to secrete high amounts of ATP.
222 Thus, our results have provided important insights into understanding the impact of surface hydrophobicity on bacterial attachment, ATP generation, and further modeling of bacterial metabolism. It was concluded that gram-negative extracellular ATP producers belonging to the genera Sulfitobacter, Marinobacter and Staleya and/or gram-positive intracellular ATP producers belonging to the genera Planococcus and Kocuria can be considered as valuable candidates for the replacement of the energy source in the next generation of microdevices.
10.1.3.2. Evaluation of prokaryotic actin-related proteins, MreB and FtsA, as possible replacements for eukaryotic actin
Since the lifetime of microdevices depends primarily on the stability of their biological components, the possibility of replacement of eukaryotic actin with comparatively more stable prokaryotic homologue/s was evaluated. Several bacterial taxa were selected and tested as prospective candidates for MreB and/or FtsA production. The information gained from physicochemical and phylogenetic analyses of 21 MreB and 30 FtsA sequences of environmental, pathogenic, and thermophilic bacteria presented here provided some valuable insights into understanding the differences between eukaryotic actin and its prokaryotic homologues. After preliminary evaluation of their biochemical properties in vitro, analysis of available literature indicated that three phylogenetically distant bacteria, namely, Pseudoalteromonas atlantica, Loktanella vestfoldensis and Thermotoga maritima may be used as the sources of very thermo- and inherently-stable MreB proteins (Guruprasad et al., 1990, Ikai, 1980) with moderate half-lives (in mammalian reticulocytes in vitro up to 30 h) (Bachmair et al., 1986) to replace actin in microdevices. Similar analysis of FtsA sequences available in public databases indicated that Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii and Salegentibacter flavus may be used as the sources of thermo- and inherently-stable FtsA proteins with the longest half-lives (in mammalian reticulocytes in vitro up to 100 h).
223 10.2. Future work
10.2.1. Advancements of surface modification
It is anticipated that the newly developed approach for the fabrication of 100 nm-range microstructures with combinatorial surfaces based on laser microablation of thin metal/blocking protein layers deposited on a PMMA substrate can be used for production of surfaces suitable for other types of biomolecule immobilization. The direction of this research remains to be evaluated. Furthermore, to gain a better understanding of the degree of independence of molecular behavior on their descriptors on combinatorial surfaces, the distribution profile of structurally different molecules within channels should be estimated. Substitution of PMMA with an optically suitable substratum, e.g., PtBMA may be a useful alternative and should be tested to evaluate contributions of functional group interactions with the deposited biomolecules. Accordingly, the laser parameters (e.g., energy, intensity and fluency) should be adjusted for optimum ablation of other alternative fabrication materials.
10.2.2. Incorporation of ATP-producers into microdevices
The incorporation of bacterial producers in the next generation microdevices is another possible direction for future work. Bacterial producers can be immobilized on the suitable surfaces of separate microstructures which are connected directly to the channels with motor proteins. This is a challenging task as the establishment of a reliable network between ATP producing bacteria and motor proteins will be required.
10.2.3. Study of MreB and FtsA proteins in vitro
Optimization of prokaryotic actin-related protein extraction and comparative physicochemical analysis of eukaryotic actin and its prokaryotic homologues (MreB and FtsA) will be essential for realization of the employment of these proteins for design of new microdevices.
224
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