Extended Data Fig. 8: Removal of the S protein specifically decreases the frequency of circumferentially moving PBP1a.
From: Pneumococcal S protein coordinates cell wall modification and repair to resist host antimicrobials
n = the total number of molecules analyzed from two biological replicates and ns (not significant), P > 0.05. (A) Deleting ess does not affect the velocity or duration of circumferentially moving iHT-PBP1a. All strains express a functional fusion of the HaloTag (iHT) domain fused to PBP1a. (left) Velocities and (right) durations of circumferentially moving HT-labeled single molecules of PBP1a are shown. Dots represent individual measurements, black horizontal line shows median, error bars denote interquartile range. Mean, standard deviation (±SD) and n = circumferential molecules. (B-C) Deleting ess decreases the frequency of circumferentially moving iHT-aPBP1a molecules, but does not affect their velocity. Both strains are merodiploids that express a functional fusion of the HaloTag (iHT) domain fused to PBP1a from the native locus of pbp1a as well as from an ectopic site under the control of a zinc-inducible promoter. (B) Deleting ess decreases the frequency of circumferentially moving iHT-aPBP1a in unencapsulated D39W. Merodiploid strains expressed a functional fusion of the HaloTag (iHT) domain to PBP1a from both the native locus of pbp1a as well as from an ectopic site under the control of a zinc-inducible promoter. Movement of >400 iHT-aPBP1a single molecules were recorded and analyzed as described for Fig. 5a. Similar frequency distributions were obtained in independent experiments using comparable single-copy strains (see Fig. 5a). See Methods (Single-molecule dynamics) for details of statistical tests. * P ≤ 0.0254; ** P ≤ 0.0098; all other comparisons (not shown) were not significant, P > 0.05. (C) (left) Velocities and (right) durations of circumferentially moving HT-labeled single molecules of PBP1a are shown and plotted as described above. (D-E) Deleting ess does not affect the frequency or velocity of circumferentially moving molecules of iHT-PBP2b, but reduces their duration. Merodiploid strains of iHT-bPBP2b were constructed similarly to the iHT-aPBP1a merodiploids described in panel B. (D) Movement of >220 iHT-bPBP2b single molecules were recorded and analyzed as described for Fig. 5a (E) Velocities and durations of circumferentially moving HT-labeled single molecules of PBP2b are shown. Mean, standard deviation (±SD) and n = circumferential molecules; ****, P = 0.0001. See Methods (Single-molecule dynamics) for details of statistical tests.
