FIGURE 5.

ActS interacts with AmiC and binds PG. A, AmiC is pulled down by His‐ActS. Coomassie blue‐stained SDS–PAGE gel showing the pull‐down of proteins to Ni²⁺‐NTA beads. (a) applied sample; E, eluted sample. (b) Fluorescent (NT‐647)‐labeled ActS (FL‐ActS) interacts with AmiC, but no AmiA or AmiB, in microscale thermophoresis (MST). FL‐ActS (100 nM) was titrated against two‐fold serial dilutions of unlabeled AmiA, AmiB, or AmiC, and the thermophoresis (movement along a temperature gradient) of FL‐ActS was followed and quantified as normalized fluorescence (Fnorm). A change in Fnorm indicates ligand‐binding. (c) ActS is pulled down by PG. Coomassie blue‐stained SDS–PAGE gel showing the pull‐down of ActS to PG from strain BW25113Δ6LDT. ActS was present in the pellet fraction (lane P) in samples with PG but not without PG. S, supernatant; W, wash [Colour figure can be viewed at wileyonlinelibrary.com]