@phdthesis{Irmscher2020,
  author    = {Irmscher, Tobias},
  title     = {Enzymatic remodelling of the exopolysaccharide stewartan network},
  doi       = {10.25932/publishup-47248},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-472486},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xiv, 170},
  year      = {2020},
  abstract  = {In nature, bacteria are found to reside in multicellular communities encased in self-produced extracellular matrices. Indeed, biofilms are the default lifestyle of the bacteria which cause persistent infections in humans. The biofilm assembly protects bacterial cells from desiccation and limits the effectiveness of antimicrobial treatments. A myriad of biomolecules in the extracellular matrix, including proteins, exopolysaccharides, lipids, extracellular DNA and other, form a dense and viscoelastic three dimensional network. Many studies emphasized that a destabilization of the mechanical integrity of biofilm architectures potentially eliminates the protective shield and renders bacteria more susceptible to the immune system and antibiotics. Pantoea stewartii is a plant pathogen which infects monocotyledons such as maize and sweet corn. These bacteria produce dense biofilms in the xylem of infected plants which cause wilting of plants and crops. Stewartan is an exopolysaccharide which is produced by Pantoea stewartii and secreted as the major component to the extracellular matrix. It consists of heptasaccharide repeating units with a high degree of polymerization (2-4 MDa). In this work, the physicochemical properties of stewartan were investigated to understand the contributions of this exopolysaccharide to the mechanical integrity and cohesiveness of Pantoea stewartii biofilms. Therefore, a coarse-grained model of stewartan was developed with computational techniques to obtain a model for its three dimensional structural features. Here, coarse-grained molecular dynamic simulations revealed that the exopolysaccharide forms a hydrogel in which the exopolysaccharide chains arrange into a three dimensional mesh-like network. Simulations at different concentrations were used to investigate the influence of the water content on the network formation. Stewartan was further purified from 72 h grown Pantoea stewartii biofilms and the diffusion of bacteriophage and differently-sized nanoparticles (which ranged from 1.1 to 193 nm diameter) was analyzed in reconstituted stewartan solutions. Fluorescence correlation spectroscopy and single-particle tracking revealed that the stewartan network impeded the mobility of a set of differently-sized fluorescent particles in a size-dependent manner. Diffusion of these particles became more anomalous, as characterized by fitting the diffusion data to an anomalous diffusion model, with increasing stewartan concentrations. Further bulk and microrheological experiments were used to analyze the transitions in stewartan fluid behavior and stewartan chain entanglements were described. Moreover, it was noticed, that a small fraction of bacteriophage particles was trapped in small-sized pores deviating from classical random walks which highlighted the structural heterogeneity of the stewartan network. Additionally, the mobility of fluorescent particles also depended on the charge of the stewartan exopolysaccharide and a model of a molecular sieve for the stewartan network was proposed. The here reported structural features of the stewartan polymers were used to provide a detailed description of the mechanical properties of typically glycan-based biofilms such as the one from Pantoea stewartii. In addition, the mechanical properties of the biofilm architecture are permanently sensed by the embedded bacteria and enzymatic modifications of the extracellular matrix take place to address environmental cues. Hence, in this work the influence of enzymatic degradation of the stewartan exopolysaccharides on the overall exopolysaccharide network structure was analyzed to describe relevant physiological processes in Pantoea stewartii biofilms. Here, the stewartan hydrolysis kinetics of the tailspike protein from the ΦEa1h bacteriophage, which is naturally found to infect Pantoea stewartii cells, was compared to WceF. The latter protein is expressed from the Pantoea stewartii stewartan biosynthesis gene cluster wce I-III. The degradation of stewartan by the ΦEa1h tailspike protein was shown to be much faster than the hydrolysis kinetics of WceF, although both enzymes cleaved the β D GalIII(1→3)-α-D-GalI glycosidic linkage from the stewartan backbone. Oligosaccharide fragments which were produced during the stewartan cleavage, were analyzed in size-exclusion chromatography and capillary electrophoresis. Bioinformatic studies and the analysis of a WceF crystal structure revealed a remarkably high structural similarity of both proteins thus unveiling WceF as a bacterial tailspike-like protein. As a consequence, WceF might play a role in stewartan chain length control in Pantoea stewartii biofilms.},
  language  = {en}
}