Figure EV1. Characterization of the libraries and selection schemes.

• A–C
  Distribution of mutation counts per pilE read in each library assessed by NGS.
• D
  Number of single amino acid protein variants observed in each library relative to the theoretical maximum number of variants potentially obtained with single nucleotide mutations of pilE.
• E, F
  Piliation selection. Flow cytometry analysis of pili expression using the 20D9 monoclonal antibody. The Pili+ gate was used to separate piliated bacteria in the wild‐type SB strain (10.5%), pilD (0.1%), pilV (3%), and pilT (28%).
• G, H
  Aggregation selection. (G) Ratio between the colony forming units of pilE _SB and pilD after passage of a 1:1 mixture through a 5‐μm pore filters. Mean ratio ± SEM is indicated. N ≥ 3 independent experiments. Paired t‐test. P < 0.01 (**). (H) Brightfield images of aggregates after different rounds of selection.
• I
  Ratio between the colony‐forming units of pilE _SB over pilF or pilV mutants after passage of a 1:1 mixture on HUVEC cells for 4 h of infection (MOI 100). Mean ratio ± SEM is indicated. N ≥ 3 independent experiments. Paired t‐test. P < 0.001 (***).