Fig. 6: Enhanced robustness of K. viridifaciens growth under hyperosmotic stress conditions through constitutive StlP expression.
A Visualization of fluid membrane regions at hyphal tips of a K. viridifaciens derivative constitutively expressing StlP (K. viridifaciens + pXZ15) compared to the parental wild-type. Images were obtained after DilC12 staining (yellow) of 16 h-old cultures followed by growth for 3 h in LPB liquid medium. White arrows indicate fluid regions (bright spots) at hyphal tips. BF brightfield. B, C Quantitively analysis of membrane fluidity using Laurdan staining. Mycelia from 16 h-old cultures were collected and labeled with 1 mM Laurdan (B). BF brightfield. The values in the graph (C) indicate the generalized polarization (GP), which ranges from −1 (more fluid) to +1 (less fluid). Red and blue pixels represent high and low fluidity, respectively. The number of tips measured was 37 (K. viridifaciens) and 45 (K. viridifaciens + pXZ15). 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; unpaired two-tailed Student’s t test. D Constitutive expression of stlP from the gapAp promoter increases the average colony size of K. viridifaciens. Colonies of the wild-type strain and a derivative constitutively expressing StlP (K. viridifaciens + pXZ15) are shown after 7 days of growth. E Quantitative assessment of the average colony diameter (n = 88 for K. viridifaciens and n = 84 for K. viridifaciens + pXZ15). Box plots display the distribution of colony 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 < 0.0001; unpaired two-tailed Student’s t test. F Constitutive expression of stlP from the gapAp promoter prevents hyperbranching. Individual hyphae were imaged after 16 h of growth on cellophane membranes overlaying solid L-phase medium agar (LPMA) plates. G Histograms indicating the distribution of tip-to-branch distances of K. viridifaciens with or without pXZ15. The number of filaments measured for each strain were 219 (K. viridifaciens) and 189 (K. viridifaciens + pXZ15). H Constitutive expression of StlP (from plasmid pXZ15) blocks the extrusion of wall-deficient cells in K. viridifaciens. 16 h-old mycelium was labeled with SYTO-9 (stains nucleic acids; green) and FM5-95 (lipid stain; red). BF, brightfield. I Quantification of cell wall-deficient cells was assessed after growing strains in LPB medium for 40 h (n = three biological replicates). Scale bars represent 5 μm (A, F), 10 μm (B, main images), 0.5 μm (B, insets), 5 mm (D) and 20 μm (H), respectively.
