Extended Data Fig. 6: ∆gpsB corrects the morphological defects of ∆facZ mutants.
a) Representative images of WT (aTB003), ∆gpsB (aTB525), FacZ- (aTB372, ∆facZ Ptet -facZ), and FacZ- ∆gpsB (aTB540, ∆facZ Ptet -facZ ∆gpsB) grown into mid-log phase without induction of facZ, and pulse-labeled with sBADA to label active PG insertion and Nile Red to label cell membranes (see Fig. 5e). Inactivation of gpsB has minimal impact on cell morphology or localization of envelope probes. Depletion of FacZ causes characteristic envelope and morphological defects (yellow arrowheads), which are rescued by inactivation of gpsB (bar = 2 µm). b) Spot titers of the same strains imaged in panel A confirms that depletion of FacZ and/or inactivation of gpsB has negligible impact on cell viability. c) Growth curves of WT (aTB003), ∆facZ [aTB251], ∆gpsB [aTB492], and ∆facZ ∆gpsB [aTB497], grown at 30 C. Values are means of six biological replicates (error bars = 95% CI). Inactivation of FacZ or GpsB causes minor growth defects, while the double mutant is largely restored to WT growth rates. d) Median cell size and coefficient of variance of cell size (CoeV) of the indicated strains from replicated experiments (see pooled data in Fig. 5f). Graphs display mean values from four biological replicates, with individual replicate values included as circles, and bars showing standard error. Inactivation of gpsB alone has no significant impact on size or variability, but rescues defects caused by depletion of FacZ (Student’s t-test, p = 0.01). e–g) Cells were imaged to measure the localization of FacZ and GpsB in different stages of cell division (see Fig. 6a, b). E) Graphs showing the fluorescence intensity of FacZ-mCherry (light and dark red) and GpsB-mNeon (light and dark green) along lines parallel to the septum. Cells were separated into two groups based on the GpsB-mNeon signal: cells with discontinuous GpsB foci were considered early-division cells (top left) whereas cells with a continuous GpsB-mNeon band at the septum were considered late-division cells (top right). (n ≥ 50 cells for each group). F) Graph showing the linear regression of the inter-peak distance of FacZ (red) and GpsB (green) in dividing cells (error bars = 95% CI). Cells were sorted by decreasing inter-peak distance of GpsB, so that measurements are pairwise for individual cells. FacZ and GpsB overlap in early division, but FacZ is left behind at the periseptum as GpsB departs with the closing divisome. G) Violin plots of raw interpeak distance of FacZ in early and late division show no significant change in FacZ localization between early and late division (n = 50 cells). H) Spot titers testing the synthetic interactions of facZ, gpsB, and ezrA. FacZ [aTB378] and GpsB [aTB663] were inactivated separately and together [aTB679] in an EzrA depletion strain [aTB264], and spot titers prepared with or without aTC as in Fig. 4b. Inactivation of facZ and ezrA is synthetically lethal, while inactivation of gpsB and ezrA is not. Inactivation of gpsB somewhat, but not completely, restores growth in a ∆facZ EzrA- strain. I) A plasmid containing Ptet-gpsB was integrated into WT [aTB632] or ∆facZ S. aureus [aTB639], and cell viability was assayed by spot titer alongside their parental strains lacking Ptet-gpsB [aTB003 & aTB251] as described (see Methods, Fig. 4b).
