Microbial Bionanotechnology: Biological Self-Assembly Systems and Biopolymer-Based Nanostructures Chapter Abstracts

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Biopolyester (PHAs = polyhydroxyalkanoates) composed of hydroxy fatty acids represent a rather complex class of storage polymers synthesized by a various bacteria and archaea and are deposited as water-insoluble cytoplasmic nano-sized inclusions. These spherical particles are composed of a polyester core surrounded by phospholipids and proteins. The key enzymes of polyester biosynthesis and polyester particle formation are the polyester synthases, which catalyze the enantio-selective polymerization of (R)-hydroxyacyl-CoA thioesters to polyesters. Various metabolic routes have been identified and established in bacteria to provide substrate for polyester synthases. The role of the polyester synthases in morphogenesis and presumably self-assembly of these natural polyester particles will be described. Although not essential for particle formation, non-covalently attached proteins, the so-called phasins, can be found at the particle surface and are considered as structural proteins. A multiple alignment of 88 polyester synthases indicated an identity varying from 8% to 96% with eight strictly conserved amino acid residues. Protein engineering of polyester synthases and phasins was used to functionalize the polyester particle surface. The current knowledge enables the microbial and biocatalytic production of particles with controlled size, polyester core composition and surface functionality, which suggested numerous potential applications of these biocompatible and biodegradable nanostructures, particularly in the medical field.

Chapter 2
Bionanofabrication: a Tool for Creating Unique Structures Through In Vitro Polymer Synthesis
Soazig C. Delamarre, Nuttawee Niamsiri, and Carl A. Batt

Current "top-down" technologies for the fabrication of nanoscale structures are limited in terms of the minimum feature size that can be achieved. Natural macromolecules and the processes through which their highly controlled assembly is carried out have become a source of interest and inspiration for scientists. Using a "bottom up" process, which we have termed bionanofabrication, there is an opportunity to develop not only biomimetic polymers, but also wholly novel structures and functionalities beyond those of the biological world. This chapter describes two current examples of in vitro synthesized biological macromolecules and their use as innovative tools in nanotechnology. The first example is the use of specifically designed synthetic DNA molecules for constructing new nanoscale structures and their exploitations as "smart" materials with applications in nanoelectronics or nanodevice sensors. The other example is focused on a bacterial polymer, polyhydroxyalkanoate (PHA) that is both biodegradable and biocompatible and has properties that are similar to thermoplastics. The modification of solid surfaces though in vitro enzymatic polymerization of PHA onto various substrates was explored. Applications through the generation of complex, micropatterned surfaces as well as in the development of functional microfluidics are foreseen. The exploitation of nature in the field nanotechnology is just beginning, and is expected ultimately to lead to revolutionary materials at the nanometer scale.

Chapter 3
Polyhydroxyalkanoates in Nanobiotechnology: Application to ProteinProtein Interaction Studies
Sang Yup Lee and Jong Pil Park

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters that can be produced by fermentation from renewable resources. PHAs can be used as completely biodegradable plastics or elastomers. In this chapter, novel applications of PHAs in nanobiotechnology are described. A general platform technology was developed by using the substrate binding domain (SBD) of PHA depolymerase as a fusion partner to immobilize proteins of interest on PHA surface. It could be shown that the proteins fused to the SBD of PHA depolymerase could be specifically immobilized onto PHA film, PHA microbead, and microcontact printed PHA surface. By using enhanced green fluorescent protein, red fluorescent protein, single chain antibody against hepatitis B virus preS2 surface protein and severe acute respiratory syndrome virus surface antigen as model proteins, we demonstrate that specific immobilization of the proteins followed by proteinprotein interaction studies can be successfully performed. These results suggest that the specific interaction between the SBD and PHA can be employed for studying proteinprotein and possibly proteinbiomolecule interactions for various new nanobiotechnological applications.

Chapter 4
Cyanophycin Inclusions: Biosynthesis and Applications
Wolfgang Lockau and Karl Ziegler

Cyanophycin, also known as cyanophycin granule polypeptide or multi-l-arginyl-poly-l-aspartic acid, is a nitrogen rich substance found in most cyanobacteria and in some heterotrophic bacteria. The polymer consists of a poly(?-aspartic acid) backbone, with arginine residues linked to the ?-carboxyl groups of the aspartyl residues by isopeptide bonds. Cyanophycin is synthesized non-ribosomally by a single enzyme, denoted cyanophycin synthetase, in a reaction that requires a primer, the constituent amino acids aspartic acid and arginine, and ATP. Cyanophycin can be produced and extracted on a technical scale from recombinant bacteria and plants harboring the gene for cyanophycin synthetase. Technical applications for cyanophycin itself are not known. The compound is of biotechnological interest because it can be converted by chemical hydrolysis to polymers with a reduced arginine content, which may replace chemically synthesized polyaspartates in industrial processes.

Chapter 5
Biomineralization of Magnetosomes in Bacteria: Nanoparticles with Potential Applications
Claus Lang and Dirk Schüler

The ability of magnetotactic bacteria (MTB) to orient and migrate along magnetic field lines is caused by magnetosomes, which are membrane-enclosed intracellular crystals of a magnetic iron mineral. The biomineralization of magnetosomes is a process with genetic control over the accumulation of iron, the deposition of the magnetic crystal within a specific compartment, as well as the assembly, alignment and intracellular organization of particle chains. Magnetite crystals produced by MTB have uniform species-specific morphologies and sizes, which are mostly unknown from inorganic systems. The unusual characteristics of magnetosome particles have attracted a great interdisciplinary interest and inspired numerous ideas for their biotechnological application. In this chapter, we summarize the current knowledge of the physicochemical and molecular genetic basis of magnetosome biomineralization. In addition, we give an overview over current examples and the potential for future applications of magnetic nanoparticles produced by bacteria.

Chapter 6
Microbial Production of Alginates: Self-assembly and Applications
Mark Salzig and Bernd H. A. Rehm

Alginates are produced by brown algae and the two bacterial genera Pseudomonas and Azotobacter. Over the last two decades alginate biosynthesis has been intensively studied in the two bacterial genera providing insight into mainly the biosynthesis steps occurring in the cytosol and leading to the activated precursor GDP-mannuronic acid. Another focus was the analysis of alginate modifying enzymes, which were extensively studied. Genes involved in alginate biosynthesis had been identified and partially functionally assigned. Numerous orthologous alginate biosynthesis genes are present in recently sequenced genomes of bacteria belonging to the genera Pseudomonas and Azotobacter. The actual alginate polymerization and export is still not understood and requires in depth investigation of candidate proteins, e.g. Alg8, Alg44 and AlgE, presumably involved in these processes. However, the current knowledge of alginate biosynthesis and alginate modifying enzymes enables bioengineering approaches to produce tailor-made alginates with defined composition and material properties. The unique alginate properties implementing self-assembly processes were traditionally used in biotechnology for encapsulation processes and are increasingly considered for the production of tailor-made micro-/nanostructures suitable for medical applications. Alginates have been increasingly considered as biomaterials imposing a steadily growing need for defined and tailor-made alginates.

Chapter 7
Bacteriophages: Self-assembly and Applications
Jasna Rakonjac and James F. Conway

Bacteriophage biology ushered in the era of modern molecular and structural biology. Accumulated wealth of knowledge on phage assembly, structure and the life cycle permitted their utilization in broad range of applications, from basic molecular biology to nanotechnology and pharmaceutical industry. This chapter reviews current status of knowledge of bacteriophage assembly and structure represented by two morphologically different types, headed and filamentous bacteriophages. The principles of phage display are further presented, followed by a wide range of applications of this technology.

Chapter 8
Molecular Biomimetics: Linking Polypeptides to Inorganic Structures
Candan Tamerler and Mehmet Sarikaya

In developing novel materials, Mother Nature gave us enormous inspiration with its already existing highly organized structures varying from macro to nano- and molecular scales. Biological hard tissues are the examples of composite hybrid materials having both inorganic and organic phases that exhibit excellent physical properties, all based on their evolved architectural design. Biocomposites incorporate both structural macromolecules, such as proteins, lipids and polysaccharides and minerals, such as hydroxyapatite, silica, magnetite, and calcite. Among these, proteins are the most instrumental components for use in materials fabrication because of their molecular recognition, binding and self-assembly characteristics. Consequently, based on this premise, inorganic surface specific polypeptides could be a key in the molecular engineering of biomimetic materials. Peptides can now be selected by directed evolution, adapted from molecular biology, by using combinatorial peptide libraries, analogous to natural selection. Adapting genetic approaches further allow to redesign, modify or engineer the selected first generation peptides for their ultimate utilization in bionanotechnological applications as molecular erectors, couplers, growth modifiers and bracers.

Chapter 9
Bacterial Spores in Bionanotechnology
Simon M. Cutting, Ezio Ricca and Imrich Barák

Bacterial spores are well known for their ability to survive as dormant bioparticles in extreme environmental conditions. Dehydrated and extremely robust these unique organisms can survive for millions of years. These attributes make them attractive as "live" vehicles for bioengineering or more specifically, for nanoengineering. Forty years of effort has not only illuminated the biology and genetics of spore formation but also the potential of these heat stable organisms for drug and enzyme delivery, as vaccine vehicles and as biosensors. This chapter explores current studies in the field of nanobiotechnology and demonstrates what can be done with the current state of the art. Initially, the spore and how it is made are outlined. Then, the concept of using the spore surface for display of heterologous proteins is discussed. Finally, a summary of current studies using the spore as a vaccine vehicle and as a biosensor are reviewed.

Chapter 10
Supramolecular Assembly Using the Natural Specificies of Biological Macromolecules
Jarrod Clark and Steven S. Smith

Both directed evolution and rational design are rapidly producing a collection of supramolecular assemblies based on the biospecificty of nucleic acidnucleic acid interaction, proteinprotein interaction and proteinnucleic acid interaction. Open and closed geometries have been developed that provide proof-of-concept prototypes of addressable and self-assembling macro and supramolecular devices. Recent progress in this area is summarized.

Chapter 11
Bacterial Protein Complexes with Potential Applications in Nanotechnology
Shi-You Ding, Qi Xu, Edward A. Bayer, Xianghong Qian, Garry Rumbles, and Michael E. Himmel

Considerable effort has been recently devoted to developing novel strategies for organizing systems of nanometer length-scale objects. Electronics miniaturization, for example, provides a particularly strong motivation for this new technology, as does present-day lithography, which faces fundamental problems in achieving further reduction in feature sizes by orders of magnitude. One of the more striking challenges of nanotechnology is that at a length scale of approximately 30 nm, "normal" physics begin to fail and quantum mechanics begin to dominate. In the range of molecular scale and "supra" molecular scale structure and interaction; however, natural processes based in biology have long functioned. Many of these systems use an evolutionarily programmed template for organization of components that combine to effect complex and critical functions in nature. Although the ultimate basis for such templating resides in the DNA code, the proteins and enzymes created can form sophisticated assemblies of such detail that modern science will remain challenged for decades by the prospect of understanding the rules that govern these systems. In one example, the future use of quantum dots in novel photoconversion devices will require large arrays of known order and dimension in order to promote strong electronic interactions that facilitate charge and exciton transport. Assembling these arrays is non-trivial and requires knowledge of the forces governing interaction of inorganic and organic molecules at the nanometer scale. We will discuss new concepts for building arrays of quantum dots using the naturally occurring bacterial cellulosomes as templates used to capture ZnS-capped CdSe quantum dots and create new and useful assemblies.

Chapter 12
S-Layer Proteins: Potential Application in Nano(bio)technology
Margit Sára, Eva-Maria Egelseer, Carina Huber, Nicola Ilk, Magdalena Pleschberger, Dietmar Pum, and Uwe B. Sleytr

Crystalline bacterial cell surface layer (S-layer) proteins represent the outermost envelope component of many bacteria and archaea. Isolated S-layer proteins frequently recrystallize into monomolecular protein lattices on solid supports, Langmuir lipid films, or on liposomes. S-layer proteins from Bacillaceae specifically recognize a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. S-layer fusion proteins incorporating a foreign functional sequence, such as core-streptavidin, the hypervariable region of heavy chain camel antibodies, the Fc-binding Z-domain, the major birch pollen allergen, or enhanced green fluorescent protein, have been recrystallized on solid supports pre-coated with the S-layer-specific SCWP as biomimetic linker. Such monomolecular protein lattices are exploited as sensing layers for label free detection systems, as affinity matrix for binding of IgG, as anti-allergic vaccines, or in the case of liposomes, as targeting and delivery systems. Other developments are directed to the use of S-layers for non-life science applications. This includes their exploitation as templates for the formation of arrays of metal clusters or nanoparticles, as required in molecular electronics and non-linear optics. The fabrication of spatially well-defined S-layers on silicon wafers is achieved by optical lithography or micromolding in capillaries.

Chapter 13
Bacteriorhodopsin
Sergei P. Balashov and Janos K. Lanyi

The main features of light energy transformation by bacteriorhodopsin of purple membrane are reviewed. They include mechanisms involved in its cyclic light-induced reaction which underlies transmembrane proton transport, and the recently obtained high resolution structures of the photocycle intermediates which provide further mechanistic clues at atomic resolution. The inherent features of bacteriorhodopsin, such as reversible photochromic transitions and generation of photocurrents, are considered with respect to potential applications of the system for holography, real time information recording and processing, and optical memory.

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