Extended Data Fig. 3: Inactivation of FacZ impairs cell division and morphogenesis.
a) Representative images showing membrane labeling and cytoplasmic fluorescence of WT [aTB523] and ∆facZ [aTB527] cells used to quantify morphological and membrane defects in Fig. 3b, c. Cells were grown overnight in the presence of Tmp (5 µg/ml) to maintain the RFP-bearing plasmid, then subcultured into medium free of antibiotics, and grown into mid-log phase. These cells were then labeled with TMA-DPH, imaged on M9 pads (2% agarose), and segmented on cytoplasmic fluorescence signal. Cell size measurements for violin plots (Fig. 3b) were automated using MicrobeJ; red arrowheads point to unusually large cells. For each cell identified by MicrobeJ, the number of aberrant membrane foci (blue arrowheads) was recorded manually (Fig. 3c). b) Z−stack of 3D-SIM reconstruction of ΔfacZ cells [aTB251] stained identically to Fig. 3 shows aberrant features. Red arrowheads follow membrane features (Nile Red, top) through Z−planes (left to right); features are continuous throughout the cell in the Z−dimension, and do not emerge in the middle of the cytoplasm, consistent with these membrane features being continuous invaginations of the cell membrane, rather than completely internalized structures. Analogous continuous features are apparent when imaging labeled cell wall (green arrowheads, sBADA, middle), and correspond to local exclusion of the nucleoid (blue arrowheads, DAPI, bottom), both of which also extend throughout all Z−slices. c) FtsZ-GFP was induced at low levels in exponentially-growing S. aureus to determine which cells were dividing and to determine the orientation of the division plane. In WT S. aureus [aTB219], most cells exhibited a single-ring, consistent with normal cell division. In cells depleted of FacZ [aTB390], aberrant FtsZ structures (defined as multiple Z-structures, drastically off-centre Z-structures, or diffuse cytoplasmic FtsZ-GFP signal) were apparent. A similar phenotype was observed in cells depleted for the division protein EzrA [aTB391]. d) Stacked bar graphs showing the frequency of aberrant FtsZ structures (black) and normal FtsZ structures (gray) from a representative experiment, with horizontal bars indicating significant differences (p-value < 0.001). Left: The division defect associated with inactivation of FacZ was largely rescued in the presence of aTC (25 ng/mL) to induce facZ expression (∆facZ Ptet-facZ vs. ∆ezrA Ptet-ezrA: p = 1.5 × 10−5, chi-squared test); WT: n = 129 cells, ∆ezrA Ptet-ezrA: n = 100 cells; ∆facZ Ptet-facZ: n = 114 cells. Right: Depletion of EzrA or FacZ causes division defects (WT vs. ∆ezrA Ptet-ezrA: p = 2.7 × 10−25; WT vs. ∆facZ Ptet-facZ: p = 1.2 × 10−27; ∆facZ Ptet-facZ vs. ∆ezrA Ptet-ezrA: p = 5.3 × 10−6, chi-squared test); WT: n = 105 cells, ∆ezrA Ptet-ezrA: n = 143 cells; ∆facZ Ptet-facZ: n = 100 cells. e) Micrographs showing localization of divisome PG synthases FtsW-GFP and GFP-Pbp1 expressed from a multicopy pLOW plasmid in WT and ∆facZ cells as described (see Methods). Left: FtsW-GFP localizes to the divisome in WT cells [aTB666] but mislocalizes to the envelope foci characteristic of ∆facZ mutants [aTB675]. Right: similar localization patterns are observed with GFP-Pbp1 in WT [aTB665] and ∆facZ cells [aTB673]. f) WT S. aureus with [aTB341] and without [aTB003] an integrated Ptet-facZ expression construct were imaged alongside ∆facZ cells with [aTB372] and without [aTB251] the same integrated facZ expression construct. Cells were grown to mid-log phase, treated with aTC (25 ng/mL) to induce FacZ expression from the Ptet promoter, and exposed to HADA to label active zones of PG insertion. Only ∆facZ cells lacking the complementing allele exhibited morphological defects (yellow arrowheads). g) Representative full fields of view corresponding to electron micrograph conditions displayed in Fig. 3a insets. (All scale bars = 2 µm).
