Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections (UTIs). UPEC possess a wide range of virulence factors, including adhesins that mediate attachment to the uroepithelium (e.g. fimbriae, curli and autotransporters), toxins that damage the uroepithelium (e.g. hemolysin) and surface polysaccharides that provide resistance against innate immune factors (e.g. capsule). Curli are amyloid fibres that contribute to UPEC adhesion and biofilm formation. Curli production is controlled by a complex regulatory network, which remains to be fully elucidated. In this study, we sequenced the genome of the curli-positive UPEC strain MS7163. In addition, we screened ~130,000 mini-Tn5 transposon mutants and employed transposon-directed insertion site sequencing (TraDIS) to identify genes involved in curli synthesis, which were then characterised using molecular and phenotypic analyses. Nineteen genes were identified to play a role in curli formation by MS7163, nine of which were known including the curli master regulator (mlrA) and the major curli subunit (csgA) genes. The remaining ten genes were novel and not previously linked to curli biosynthesis. They included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs regulatory system and a putative novel repressor. The involvement of these genes in curli production was confirmed through the construction of defined mutants and their complementation. All of the mutants did not express the curli major subunit CsgA based on western blot analysis, and failed to produce curli production based on their inability to bind Congo Red and their attenuated biofilm phenotype. Overall, our application of a high throughput forward genetic screen to study curli biogenesis revealed a role for the purine de novo biosynthesis pathway and two novel genes involved in curli production by UPEC, thus providing new insight into the regulation of this important adherence factor.