Strain CFT073 is a bona fide uropathogen, whereas strains 83972 and Nissle 1917 are harmless probiotic strains of urinary tract and faecal origin, respectively. Despite their different environmental origins and dispositions the three strains are very closely related and the ancestors of 83972 and Nissle 1917 must have been very similar to CFT073. Here, we report the first functional genome profiling of Nissle 1917 and the first biofilm profiling of a uropathogen. Transcriptomic profiling revealed that Nissle 1917 expressed many UPEC-associated genes and showed that the active genomic profiles of the three strains are closely related. The data demonstrate that the distance from a pathogen to a probiotic strain can be surprisingly short. We demonstrate that Nissle 1917, in spite of its intestinal niche origin, grows well in urine, and is a good biofilm former in this medium in which it also out-competes CFT073 during planktonic growth. The role in biofilm formation of three up-regulated genes, yhaK, yhcN and ybiJ, was confirmed by knockout mutants in Nissle 1917 and CFT073. Two of these mutants CFT073∆yhcN and CFT073∆ybiJ had significantly reduced motility compared with the parent strain, arguably accounting for the impaired biofilm formation. Although the three strains have very different strategies vis-à-vis the human host their functional gene profiles are surprisingly similar. It is also interesting to note that the only two Escherichia coli strains used as probiotics are in fact deconstructed pathogens.