Reorganization of cell wall biosynthesis in the absence of phosphate.

(A) Major growth zones of phosphate-starved wild-type cells. Cells were cultivated in M2G-P medium for 6 h or 24 h and exposed to a short (2 min) pulse of HADA. The subcellular distribution of the fluorescence signals was quantified by demographic analysis of a random subpopulation of cells (n = 200). To generate the graphs, single-cell fluorescence profiles were sorted according to cell length and stacked on top of each other (scale bars: 3 μm). (B) Changes in stalk and cell body lengths during phosphate starvation. Wild-type cells were incubated in M2G-P medium for 8 h, 18 h, 28 h, and 40 h prior to imaging. The data are shown as box plots, with the horizontal line indicating the median, the box the interquartile range, and the whiskers the 2th and 98th percentile (0 h: n = 108, 8 h: n = 100, 18 h: n = 100, 28 h: n = 105, 40 h: n = 102) (*** p −6; t-test). See S1 File for the raw data. (C and D) Slow turnover of PG in the stalk compartment. Cells producing StpB-mCherry as a proxy for crossbands (SW30) were cultivated in M2G-P medium for 18 h and exposed to HADA for an extended period of time (1.5 h). Subsequently, they were washed, transferred into fresh in M2G-P medium and grown for 2 h, 4 h, and 6 h in the absence of the label (scale bars: 3 μm). To quantify the changes in HADA fluorescence overtime, fluorescence profiles were obtained from random subpopulations of cells (n = 200 per time point). The lengths of the profiles in each quintile of the cell length distribution were normalized to the maximum cell length in the respective quintile. Subsequently, the fluorescence intensities were averaged and used to generate violin plots. Shown is a representative part of the data depicting the fluorescence distributions in the fourth quintile at each of the time points (D). The full analysis is presented in S2C Fig.