Fig. 4: PBP2 is responsible for unidirectional PG synthesis at the midcell in A. excentricus.

a Schematic depicting pulse-chase experiments in A. excentricus ZapA-sfGFP cells with (right) or without (left) mecillinam treatment. Whole-cell PG was labeled with TADA (magenta) over two generations, followed by washing and growth with or without mecillinam (50 µg ml⁻¹) over one generation before imaging. Representative phase, fluorescence (TADA and TADA overlaid with ZapA-sfGFP), and merged images with WGA fluorescence are shown (n  =  3 biological replicates). See Supplementary Fig. 5c for population-level demographs. Scale bar: 2 µm. b ShapePlots of A. excentricus cells after an FDAA pulse-chase experiment, with mecillinam. Top: Shape plots showing bulge localization. Bottom: ShapePlots show ZapA-sfGFP and FDAA signal loss. Each ShapePlot is divided longitudinally (black line) with FDAA signal loss on the left and overlaid with ZapA-sfGFP on the right. ShapePlots show four categories of cells binned by cell length (left) and the entire population of 160 cells (right) oriented using WGA-labeled holdfast (old pole at the bottom). Horizontal white lines represent the midcell. c Top: Schematic of a dual short-pulse experiment with or without mecillinam (50 µg ml⁻¹). A. excentricus cells were grown over 120 min with or without mecillinam, then labeled with TADA (magenta), washed, allowed to grow, and labeled with BADA (green). Cells were washed again and imaged with microscopy. Middle: Representative phase and merged images from both treatment conditions (n  =  3 biological replicates). Merged images show phase contrast overlaid with fluorescence signals from the two FDAAs and WGA-labeled holdfast (cyan). Scale bar: 2 µm. Look-up tables (LUTs) were adjusted for each condition to have a visible FDAA signal. Bottom: Population-level demographs showing the fluorescence intensities of both FDAA signals, with and without mecillinam. Cells were arranged by length, with the old pole to the left. 50% of the maximum fluorescence intensities are shown. d Dot plots graphs showing quantification of fluorescence intensities for each FDAA pulse, with or without mecillinam (n = 686 cells control and n = 514 mecillinam treated-cells). **** P < 0.0001, unpaired two-tailed t-test with Welch’s correction. Error bars show the SEM. Source data are provided as a Source Data file.