Extended Data Fig. 4: FacZ structural predictions and functionality of the FacZ-mCherry fusion.
a) Predicted membrane topology of FacZ (Protter)1. FacZ is predicted to have one transmembrane helix with a short extracellular N-terminal region. Most of the protein is predicted to extend into the cytoplasm, with a coiled-coil region (blue box) and a disordered C-terminal extension (green box). b) AlphaFold2 model of full-length FacZ, colored by pLDDT confidence score. Predicted domains and orientation in the plasma membrane are labeled. c) AlphaFold2-multimer predictions of FacZ assemblies from one copy to six copies. d) A ∆facZ strain harboring facZ-mCherry under control of a Ptet promoter [aTB373] was grown into mid-log phase with 0 or 50 ng/µl aTC, pulse-labeled with HADA as described, and imaged on PBS pads containing 2% agarose. Cells depleted of or overexpressing FacZ-mCherry (red) had aberrant sites of PG synthesis (yellow arrowheads), while aTC (50 ng/mL) induction of facZ-mCherry restored normal morphology and PG incorporation. Insets, bottom right: When FacZ-mCherry is expressed at levels that restore normal cell morphology to ∆facZ cells, the fluorescent fusion is enriched at periseptal regions, flanking actively-growing zones of septal PG synthesis (yellow arrowheads) (scale bar = 2 µm). e) Diagram showing two potential models for FacZ-mCherry localization at the periseptum. FacZ may be enriched at the highly-curved periseptal rim, or may be equally enriched along the curved peripheral surface and extend slightly into the flat septal membrane. f) Schematic showing regions of cell surface used to measure periseptal:peripheral fluorescence ratios. Periseptal regions were defined as the six pixels centred around the septum (the peak of HADA labeling in dividing cells). The peripheral regions were defined as the six pixels half the distance between the periseptum and the ends of hemispherical arcs along which fluorescence intensity was measured. These intermediate regions were chosen because the extreme ends of the hemispherical arcs could display increased membrane abundance due to previously-formed septa in cells with immature division planes, or nascent orthogonal septa in cells with completed division planes. Periseptal enrichment was measured by taking the ratio of periseptal fluorescence intensity to peripheral fluorescence intensity for FacZ-mCherry (2.10-fold periseptal enrichment) or Nile Red (1.79-fold periseptal enrichment), a non-specific membrane label (see Fig. 4). FacZ-mCherry periseptal enrichment was 1.18 times greater than Nile Red periseptal enrichment (Student’s t-test, p = 2.8 × 10-4). g) WT [aTB003], ∆facZ [aTB251], and ∆facZ cells expressing untagged facZ [aTB372] or facZ-mCherry [aTB373] under the control of the Ptet promoter were normalized to OD600 = 1.0, serially diluted, and spotted onto TSA agar plates with aTC (50 ng/mL) and with or without PC190723 (100 ng/mL) as indicated.
