Site-Specific Pilus Labeling Protocol

Pilus labeling was performed as previously described with minor modifications (23 (link), 50 (link)). Briefly, control strains or genetically modified bacteria with modified pilA alleles (encoding site-specific amino acid changes to knock-in a new cysteine residue) were pregrown overnight at 37°C, back diluted 1:100 in 2 ml LB (supplemented without or with l-arabinose, as indicated), and grown at 37°C until they reached an OD₆₀₀ of approximately 0.65. Alexa Fluor 488 C5 maleimide (AF-488-Mal; Thermo Fisher Scientific) was added to 100 μl of the bacterial culture at a final concentration of 25 μg/ml, gently mixed, and incubated for 15 min at room temperature in the dark. Cells were subsequently harvested by centrifugation (5,000 × g for 1 min.), washed once in 1× PBS, and resuspended in 30 μl of PBS before being imaged as described above.