Depletion or overproduction of FzlA impacts FtsW movement. (A) Histogram comparing single-molecule speeds of FtsW in cells (EG3523) producing FzlA (+FzlA, 0.001% xylose, n = 230 total from three biological replicates) or depleted of FzlA for 7 h (−FzlA [7 h], 0.001% glucose, n = 117 total from three biological replicates). (B) Plot comparing the move time of single FtsW molecules moving at <20 nm/s in cells producing FzlA (+FzlA, 0.001% xylose, n = 230 total from three biological replicates) or depleted of FzlA for 7 h (−FzlA [7 h], 0.001% glucose, n = 117 total from three biological replicates). Error bars are SEM. (C i and ii) Histogram comparing speeds of FtsW or FtsW** in cells with an EV control or with an fzlA overexpression (OE) construct in either (i) a halo-ftsW (EV; EG3537, n = 472 total from three biological replicates. fzlA OE; EG3519, n = 300 total from three biological replicates) or (ii) halo-ftsW**; ftsI* (EV; EG3538, n = 160 total from four biological replicates. fzlA OE; EG3525, n = 205 total from three biological replicates) background. (D i and ii) Plot comparing the move time of single FtsW and FtsW** molecules moving at speeds <20 nm/s with and without FzlA overproduction in a (i) halo-ftsW or (ii) halo-ftsW**; ftsI* background. Error bars are SEM. (E) Fraction of the population that is moving overall (% moving), slow moving, or fast moving for the indicated strains and conditions.