Abstract
Erwinia amylovora is the causative agent of the destructive fire blight disease affecting various Rosaceae plants. This Gram-negative bacterium's ability to infiltrate plants xylem and the lack of effective bactericides pose significant challenges for disease control, leading to reduced fruit production in susceptible apple and pear trees and possibly losses of entire orchards. Central to the pathogenesis of fire blight is the formation of biofilms, which offer protection and resistance to host immune responses and environmental stress, facilitate genetic exchange, and prevent water and nutrient loss. Biofilms rely on the production of the exopolysaccharide (EPS) amylovoran, whose biosynthesis involves multiple enzymatic steps within bacterial cells, followed by secretion into the extracellular environment. Despite its fundamental role, the molecular mechanisms governing amylovoran production have remained elusive, and the key enzyme structures involved in the process are uncharacterized. To address these knowledge gaps, this research thesis adopts a multidisciplinary approach, that encompasses bioinformatics analyses, molecular and structural biology techniques, and biophysical investigations. All direct towards the final aim of unravelling the molecular steps of amylovoran synthesis and paving the way for the development of novel strategies against the Erwinia pathogen. Firstly, a comprehensive examination of the exopolysaccharide operon within the Erwinia genus was conducted. Various bioinformatics analyses aided in the identification of pivotal genes and the exploration of their evolutionary relationships. Subsequently, a wide array of molecular and synthetic biology techniques were employed to produce and analyse each of the Ams proteins responsible for amylovoran subunit biosynthesis, with the primary aim of gaining an in-depth understanding of their three-dimensional structures. Different attempts were made to overexpress the six most important enzymes in Escherichia coli and purify them using chromatography techniques. In parallel, biophysical analyses were conducted on the purified proteins to solve folding and solubility issues that arise during their production. Hydrophobic patches within most of the proteins were found, and the formation of a complex and/or interactions with the membrane, to optimize the biosynthesis process or achieve successful amylovoran secretion, were hypothesised. Further study involved co-expression experiments, new constructs design, and multiple attempts to optimize the production of soluble proteins. Finally, a particular emphasis was placed on the membrane protein AmsG. Innovative techniques were utilized for its purification and crystallization, and a robust protocol for its purification was successfully established.