Figure 2. Effect of mutants in regulators of type IV pili on twitching, biofilm formation and swarming. Various regulators showed alterations in some but not all of these pilus-related phenotypes. (A) Twitching motility along the agar-plastic interface of LB plates containing 1% agar was reduced in pilG, pilH and pilR regulator mutants but not in pilS. The non-piliated pilB was used as a control. (B) Pilus-related regulatory mutants demonstrated alterations in their abilities to produce static biofilm. Overnight cultures were diluted into LB and grown for 24 hours in polystyrene 96-well plates. The cultures were then removed, the wells washed, and the remaining biofilm stained with crystal violet. The amount of crystal violet bound corresponded directly to the amount of biofilm formed. (C) Swarming motility across the surface of semi-solid 0.5% agar. Only the non-piliated pilB mutant demonstrated a reduction in swarming. Student's t-test, mean average with standard error. *p < 0.05, **p < 0.01, NS not significant.