A variety of bacterial cell surface structures and quorum signalling molecules play a role in biofilm development inEscherichia coli. However, here we show that an engineered reduced-genomeE. colimutant that lacks 17.6% of the parentalE. coligenome, including the genes involved in the synthesis of various cell surface structures, such as type 1 fimbriae, curli, exopolysaccharide polymers and the autoinducer-2 signalling molecule, is able to develop mature biofilms. Using temporal gene expression profiling, we investigated phenotypic changes in reduced-genome biofilms in relation with the genes encoding the synthesis of different amino acids that were differentially expressed during biofilm formation. We identified and characterizedentB,marR,dosC,mcbRandyahKgenes, as involved in biofilm formation by the reduced-genomeE. coli. Of these, for a first time, we demonstrated that overproduction ofentBandyahK, which encode an enterobactin for iron transport and a hypothetical oxidoreductase protein, respectively, promoted biofilm development and maturation. Our results indicate that specific types of genes contribute to phenotypic changes in reduced-genomeE. colibiofilms. In addition, this work demonstrates that the functions of biofilm-specific genes could be analysed through experiments using the reduced-genomeE. coli.