FIG 2.

The HPT domain of SypF is necessary and sufficient for biofilm formation in TBS containing pABA and Ca²⁺. (A) Colony biofilm formation was evaluated following growth on tTBS with pABA and calcium (TPC) of the following strains: WT ES114, the ΔsypG mutant (KV1787), the sypG complement (KV6475), the ΔsypF mutant (KV5367), the sypF complement (KV6659), and the sypF mutant complemented with only the SypF HPT domain (KV7226). Pictures were taken using the Zeiss Stemi 2000-c microscope with ×6.5 magnification at 72 h before and after disruption using a toothpick. Pictures are representative of three separate experiments. Arrows indicate where “pulling,” indicating cohesion, was observed. Dotted arrows represent where “pulling” was observed but to a lesser extent, with portions of the colony breaking off during disruption. (B) Biofilm cohesion was quantified from images as described in Materials and Methods with scores of 1 assigned to a null phenotype and 4 to the most biofilm formation/the strongest phenotypes (most/strongest). Statistics for panel B were performed via a one-way ANOVA using Tukey’s multiple-comparison test, where biofilm strength was the dependent variable. *, P = 0.0210; **, P = 0.0017; ****, P < 0.0001. (C) sypA promoter activity (Miller units) was measured using a PsypA-lacZ reporter present in the parent (KV8079) and ΔsypG and ΔsypF derivatives (KV10307 and KV10317, respectively) following subculture for 22 h in tTBS (T), tTBS+calcium (TC), tTBS+pABA (TP), and tTBS+pABA/calcium (TPC). Statistics for panel C were performed via a two-way ANOVA using Tukey’s multiple-comparison test, where Miller units was the dependent variable. ****, P < 0.0001.