Fig. 6: Identification and characterization of CIS^Sc effectors (Sco4256–4258).

a, Schematic showing the domain organization of Sco4256, Sco4257 and Sco4258. All three proteins include one predicted transmembrane domain. Sco4257/4258 contain a ricin-like domain on the C terminus. b, Survival of E. coli expressing Sco4256, Sco4257, Sco4258 or YFP-GypA (used as a negative control of toxicity). Images are representatives of three independent experiments. c, CryoET slice of a hypha from the S. coelicolor effector-deficient mutant (∆sco4256–4258), showing empty CIS^Sc but otherwise WT-like particles (arrowhead). Tomograms (90) were collected from 3 independent datasets. Scale bars, 75 nm. d, Fractions of empty CIS^Sc particles are increased in the effector mutant compared with the WT (ScoWT, n_total = 91; ∆sco4256–4258, n_total = 73). e, Representative images of filled and empty CIS^Sc particles. Tomograms (60) were collected from 2 independent datasets. Scale bars, 50 nm. f, fLM (shown are representative images) was used to determine the ratio between live cells (cytoplasmic sfGFP) and total cells (membrane dye FM5-95) after growth in the absence of stress or in the presence of nisin stress for S. coelicolor ∆sco4256–58/sfGFP. Scale bars, 10 µm. g, The quantification of the experiments in f showed no notable difference between the ∆sco4256–58/sfGFP strain grown without stress and with nisin. Superplots show the area ratio of live to total hyphae. The three different colours (green, grey and pink) indicate the three biological repeats. Black line indicates the mean ratio derived from biological triplicate experiments (n = 100 images for each experiment). Significance was calculated using one-way ANOVA and Tukey’s post-test. h, Representative brightfield images of surface imprints of plate-grown colonies of WT S. coelicolor (from Fig. 5b) and the CIS^Sc effector mutant strain ∆sco4256–4258. Images were taken at the indicated timepoints. Insets show magnified regions of the colony surface containing spores and spore chains. Note that strains with functional CIS sporulate later. Scale bars, 50 µm. i, Quantification of spore production (c.f.u.) in the same strains as above, revealing much higher c.f.u.s (spores) at 72 h in the effector-deficient CIS mutant. Data show mean ± s.d. obtained from biological triplicate experiments.