Fig. 2: StlP localizes to hyphal tips and affects cell wall synthesis.

A Localization of StlP-mCherry (red) in the wild-type strain carrying plasmid pXZ16. White arrowheads indicate the location of StlP-mCherry at hyphal tips (bright spots). BF brightfield. B Localization of DivIVA-mCherry (red) in the wild-type strain and the stlP mutant, both of which carry plasmid pXZ17. Strains were grown in L-phase broth (LPB) medium for 16 h prior to imaging. The arrowheads indicate the location of DivIVA-mCherry (bright spots). BF brightfield. C Visualization of nascent peptidoglycan in S. coelicolor strains using fluorescent vancomycin (Van^FL; green) staining. White arrows indicate peptidoglycan (PG) synthesis sites (bright spots). BF brightfield. D Deletion of stlP increases hyphal diameter. Quantification was done by measuring the hyphal diameter after staining with FM5-95. Box plots display the distribution of hyphal diameter measurements. The center line represents the median (50th percentile). The box spans from the first quartile (25th percentile) to the third quartile (75th percentile), representing the interquartile range (IQR). Whiskers extend to the smallest and largest values within 1.5 times the IQR. Data points beyond this range are considered outliers and are shown as individual dots. ***p < 0.001; ns, not significant (p = 0.37); unpaired two-tailed Student’s t test without corrections. E The absence of StlP affects proper deposition of the cellulose-like glycan. Foci are either splitting at hyphal tips (bright spots) or diffused (bright spots indicated by white arrowheads) along the filament in the stlP mutant. Strains were grown in LPB medium and fluorescent images were taken after 16 h of growth. Bars represent 20 μm. F Quantification of the cellulose-like glycan at hyphal tips using calcofluor white staining. For each tip, the total fluorescence in a square (1.5 μm by 1.5 μm) at the hyphal tip was measured using ImageJ software. For each strain, fluorescence at 10 tips was measured, and a square (1.5 μm by 1.5 μm) without fluorescence near the hyphal tip was used as the blank. Box plots display the distribution of apical fluorescence measurements. The center line represents the median (50th percentile). The box spans from the first quartile (25th percentile) to the third quartile (75th percentile), representing the interquartile range (IQR). Whiskers extend to the smallest and largest values within 1.5 times the IQR. Data points beyond this range are considered outliers and are shown as individual dots. ***p < 0.001; ns, not significant (p = 0.91); unpaired two-tailed Student’s t test. G The absence of the cellulose-like glycan at hyphal tips increases lysozyme sensitivity of the stlP mutant. For each strain, ~1000 spores were plated on nutrient agar plates, and colony numbers were counted after 3 days. The percentage (number of colonies from plates with 0.25 mg ml⁻¹ lysozyme divided by the number of colonies from plates without lysozyme) was used to evaluate the sensitivity of strains for lysozyme. Colony counts for plates with lysozyme were: wild-type strain (M145), 160/161/160; stlP mutant, 3/5/3; complemented mutant, 145/142/113. Colony counts for plates without lysozyme were: wild-type strain (M145), 929/822/708; stlP mutant, 861/814/773; complemented mutant, 935/1003/1179. n = three biological replicates; ****p < 0.0001; ns, not significant (p = 0.10); Mann–Whitney test. Error bars represent the standard error of the mean. H Stills from Supplementary Movie 3 and 4 show cryo-ET images of sacculi from the wild-type (top left panel) and the stlP mutant strain (top right panel). The bottom panel depicted the corresponding straightened cell wall at the apical region of each strain. I Cell wall thickness measurements of the wild-type and stlP mutant strain. Our observation here could not be statistically analyzed due to generating large dataset from Cryo-ET is challenging. n = three technical replicates; ****p < 0.0001; ***p < 0.001; unpaired two-tailed Student’s t test. Error bars represent the standard error of the mean. Bars represent 20 μm (A), 5 μm (B), 1 μm (C), 10 μm (E), 200 nm (H, top panel), and 50 nm (H, bottom panel), respectively.