Preparation and characterization of functional amyloids and amyloid-based biofilm in escherichia coli [electronic resource]
Keywords: Chemistry.

Amyloids are a special class of proteins possessing a common cross-beta fold. This cross-beta fold confers a number of unique properties including insolubility, resistance to thermal and chaotropic denaturation, protease resistance, and the ability to spontaneously self-template and auto-aggregate into fibers. Microorganisms have harnessed the power of the amyloid fold to produce a variety of functional amyloids. In microorganisms, amyloids are involved in many functions including: serving as non-chromosomal genetic elements in the form of prions; serving as surface modifiers in the form of hydrophobins and chaplins; and are used to form multifunctional extracellular fibrillar structures. Amyloid-based fimbriae appear to be prevalent in nature and play a huge number of roles from surface adhesive structures and virulence factors to structural components in biofilms. Curli fibers were the first bacterial functional amyloid fibers discovered. In Escherichia coli, curli are produced as extracellular surface adhesive structures mediating cell-cell interactions, binding to biotic and abiotic surfaces, and serving as major structural components in biofilm formation. The uropathogenic E. coli strain UTI89 is capable of forming a highly insoluble amyloid-mediated biofilm. Biofilms consist of bacteria plus their secreted extracellular matrix (ECM). The amyloid-mediated UTI89 ECM is known to contain curli and a polysaccharide believed to be cellulose; however, it is likely that other components are present. In this dissertation, two biological systems were developed, biochemically characterized, and analyzed by solid state NMR—(1) in vivo-assembled curli amyloid fibers and (2) the curli/cellulose-based ECM of E. coli strain UTI89. Solid state NMR was used for analysis due to its ability to provide data on both the structure and chemical composition of intact, insoluble biological assemblies. Examination of the intact biological assemblies required the use of the native bacterial expression and assembly systems. Thus high expression levels were achieved through manipulation of bacterial physiology via optimization of environmental conditions. Expression on both undefined, rich defined, and minimal defined media was optimized to enable maximum flexibility in the isotopic labeling of samples for detailed NMR studies. Minimally perturbative extractions were developed and optimized for curli and UTI89 ECM production. For both the curli and UTI89 ECM systems, > 100 mg yields are accessible. Most biochemical analyses were developed for soluble systems. Thus, the biochemical analysis of curli fibers and the UTI89 ECM required the development of new techniques and validation of pre-existing methods due to the insolubility of these systems. Additionally, new methods were developed to facilitate tracking and analysis of the curli and UTI89 ECM systems including a simple method for tracking the purification of extracellular structures using microbiological indicator dyes and an apparatus to simulate growth on agar using track-etched membranes and broth. Initial solid state NMR characterization of both the curli and UTI89 ECM systems were carried out. Isotopic labels were selectively incorporated into curli and the label incorporation was characterized. For the UTI89 ECM system, the ECM composition was examined and quantified by solid state NMR. It was found that the polysaccharide portion of the UTI89 ECM is a modified form of cellulose, and that the composition of our purified ECM is approximately 85% curli and 15% modified cellulose. Taken as a whole, the work presented in this thesis is an excellent beginning. The systems developed are naturally expressed, natively assembled, and biologically relevant yet are still robust and high yielding. Because these systems were optimized for solid state NMR, ample material is available for any other conceivable biochemical or biophysical assay. Basic characterization of these systems has been performed laying the foundation for more detailed future studies. Additionally, the indicator dye tracking and ECM purification tools have been found to be easily implementable and applicable to other biofilm systems thereby driving exploration and innovation.