Escherichia coli is a Gram-negative bacterium that exhibits extensive diversity, ranging from a harmless commensal to a pathogen capable of causing serious intestinal and extra-intestinal infections. One feature that drives differences between commensal and pathogenic strains is the capacity to adhere to and colonise surfaces and form biofilms. In E. coli, these properties are often mediated by surface-located adhesins, including autotransporter (AT) proteins and chaperone-usher (CU) fimbriae. This work focussed on the characterisation of the YfaL AT and two CU fimbriae, Yad and S fimbriae.ATs represent a large outer membrane protein family and frequently associated with E. coli adhesion and biofilm formation. In Chapter 3, we determined the prevalence of genes encoding 49 different AT proteins from a curated set of 1378 completely sequenced E. coli genomes. The yfaL gene was highly prevalent and found in 98% of strains in the dataset. YfaL belongs to the AIDA-I subgroup of AT proteins frequently associated with aggregation and biofilm formation, yet its function has not been properly characterised. Here, we focused on YfaL, and employed an in-silico analysis to examine the YfaL phylogeny. We also selected three representative YfaL variants from this phylogeny and evaluated their functional properties. The gene encoding each YfaL variant was cloned and expressed, demonstrating cell surface localization of YfaL using a specific antibody in combination with fluorescence microscopy. To illuminate functional characteristics of each variant, recombinant cells expressing YfaL were tested for aggregation and biofilm formation under different growth conditions. While none of the YfaL variants mediated aggregation, all variants promoted biofilm formation following growth in low-nutrient one-tenth diluted LB broth. Surprisingly, this biofilm feature was growth media dependent, as biofilms were not formed in LB broth or M9 minimal media.Fimbriae are multi-subunit organelles produced by E. coli and many other bacteria and play a significant role in adhesion and biofilm formation. The assembly of fimbriae on the E. coli surface is largely driven by the CU assembly pathway, with 38 different CU fimbriae currently defined in the E. coli species. CU fimbriae are comprised of major and minor subunit proteins, as well as a dedicated assembly apparatus consisting of a periplasmic chaperone and an outer membrane usher. In Chapter 4, we investigated the function of S fimbriae, a type of CU fimbriae frequently associated with extra-intestinal E. coli strains that cause severe acute pyelonephritis and neonatal meningitis. Analysis of the curated 1378 completely sequenced E. coli genome dataset revealed that the genes encoding S fimbriae were present primarily in phylogroup B2 strains, with 95% of the S fimbriae-positive strains belonged to this group. To explore S fimbrial function, we focused on the fimbrial subunit proteins. S fimbriae have previously been shown to bind to sialic acid, with early studies showing this binding was largely mediated by the SfaA major subunit and SfaS minor subunit proteins. However, this is inconsistent with our current knowledge of CU fimbrial architecture and function, and thus we sought to determine if this binding property was mediated by the tip-located SfaH adhesin. The genes encoding SfaA, SfaS, SfaH and the SfaG subunit protein were cloned, and each protein was expressed and purified under non-denaturing conditions. Interestingly, and consistent with previous studies, the SfaA major subunit protein demonstrated the highest binding affinity to the sialic acid glycan N-Acetyl-D-glucosamine, with SfaS demonstrating low binding affinity and no binding observed by SfaG or SfaH. To elucidate the binding specificity of the SfaH adhesin, the lectin binding domain was purified, and its binding capacity was assessed using a glycan array. In total, 40 different glycans were bound by SfaH, including blood group antigens, Lewis antigen structures, structures with lactose, and GM2. Attempts were also made to elucidate the crystal structure of the SfaH lectin binding domain. However, only long thin needle-like crystals were observed with initial crystallization screenings, preventing high- resolution of its atomic structure.One type of CU fimbriae, Yad fimbriae, have been shown to mediate adherence to intestinal epithelial cells and contribute to colonisation of the mouse bladder, however detailed mechanistic insight into their function remains to be elucidated. Work in Chapter 5 we aimed to enhance knowledge of Yad fimbriae and determine the binding characteristics of the YadC tip adhesin. Firstly, the prevalence of Yad fimbriae was assessed in a dataset comprising 1378 completely sequenced E. coli genomes. Overall, the prevalence of yad fimbriae genes displayed a variable distribution based on E. coli phylogeny, with 86% of strains from the B2 phylogroup shown to possess yad fimbriae genes compared to strains from phylogroups A (62%), B1 (65%), C (43%), D (1%), E (0%) and F (19%). To further characterise the function of Yad fimbriae, four gene clusters representing allelic variation of the YadC adhesin were cloned and expressed in a recombinant E. coli strain. Despite repeated attempts, expression of the Yad fimbrial variants was inconsistent, with only the yad fimbrial cluster from the ST131 strain EC958 demonstrating detectable surface-expressed fimbriae production. Further work therefore focused on attempts to express, purify and characterise the YadC tip adhesin. Like other characterised fimbrial adhesins such as FimH and PapG, the YadC adhesin comprises two distinct domains, an N-terminal lectin-binding domain and a C-terminal pilin domain. As the lectin-binding domain was predicted to mediate interaction with specific glycan receptors and thus tissue tropism, the sequence corresponding to this region was amplified by PCR and cloned into a series of expression vectors. Using this strategy, the lectin domain of two variant YadC alleles was purified under non-denaturing conditions. Unfortunately, despite multiple attempts, we were unable to produce diffracting crystals that could enable high-resolution analysis of the YadC structure.Taken together, this work enhances our understanding of the prevalence of E. coli adhesins and uncovered the functions of the YfaL and S fimbriae. A new strategy was proposed to explore the function of the Yad fimbriae and additional work is required to investigate the contribution of Yad fimbriae to bacterial pathogenesis.