Biofilm formation increases antibiotic resistance among bacteria; therefore, a thorough understanding of the proteins and molecules affecting biofilm formation is necessary. PotD1, the substrate binding protein of the PotABCD1 spermidine transport system, negatively regulates biofilm formation in the intestinal pathogen Vibrio cholerae. This work characterized the binding properties of PotD1 to its ligands and its effect on biofilm formation through genetic characterization of the binding cleft and the ability of PotD1 to interact with various polyamines, which can alter biofilm levels in this bacterium. The results demonstrated that PotD1 is also responsible for the import of norspermidine. This is the first example of a norspermidine transporter reported. Ligand competition assays showed PotD1 has a higher binding affinity for spermidine over norspermidine. This work also showed the amino acids W252 and D254 play a large role in spermidine transport, and amino acids E168 and W32 play a less important role. Lastly, mutations in the amino acids that diminished spermidine import only had a partial effect on biofilm formation, suggesting spermidine uptake and biofilm formation are partially correlated. Thus, this work demonstrates that PotD1 alters biofilm formation by both its ability to uptake spermidine and through another yet unidentified mechanism
