Contraction of CISSc in situ is dependent on CisA.

a. Schematic of the mode of action of CISSc in Streptomyces coelicolor (18,19). CISSc are assembled as free-floating particles in the cytoplasm of vegetative hyphae. In response to cellular stress and/or an unknown cellular signal, CISSc particles contract, which results in regulated cell death (mediated by released effectors) and impacts cellular development.

b/c. Negative-stain electron micrographs of purified CISSc particles from S. coelicolor wildtype (WT) (b) and the ΔcisA mutant (c), show that all CISSc particles are contracted upon purification. These experiments were performed three independent times. Bars, 100 nm.

d-f. Shown are representative images of cryo-electron tomogram slices (thickness 11 nm) of vegetative hyphae (top: intact cells; bottom: ghost cells) of S. coelicolor WT (d), ΔcisA mutant (e), and the complemented ΔcisA/cisA+ mutant (f). CISSc particles remain almost exclusively in an extended state (white arrowheads) in the ΔcisA mutant, whereas in ghost cells derived from the WT and the complemented mutant, CISSc particles (black arrowheads) are mostly contracted. See also Supplementary Fig. 1. PG, peptidoglycan; CM, cytoplasmic membrane; Bars, 50 nm.

CryoEM structure of the extended CISSc assembly reveals its composition.

a. Schematic illustrating the CISSc gene cluster (adapted from (18)). Asterisks indicate gene products that were detected by mass spectrometry analyzing crude preparations of CISSc particles (red). See also Supplementary Table 1.

b/c. Single-particle cryoEM structure of an extended CISSc particle obtained from purified particles from a non-contractile CISSc mutant. Shown is the composite atomic model in surface (left) and ribbon (right) rendering (b). Subunits are color-coded according to (a) and their copy numbers in the assembly are indicated in (c).

d. Perpendicular views of the ribbon representation of the CISSc model of the cap and baseplate modules.

e. Top view (left) and side view (right) of ribbon diagrams showing the cap module of CISSc, revealing that the cap is composed of a Cis16 hexamer.

f. Ribbon diagrams showing a single baseplate wedge subunit, which is composed of two copies of Cis11 and one copy of Cis12.

CisA is a single-pass membrane protein.

a. Schematic and Alphafold2 model of CisA. CisA is predicted to contain a largely unstructured N-terminal domain (grey), a transmembrane domain (TM, purple) and a C-terminal immunoglobulin-like domain (green).

b/c. Representative micrographs (left panels: phase contrast, Ph3; right panel: mCherry) of strains of S. coelicolor vegetative hyphae either constitutively expressing CisA-mCherry (ΔcisA/cisA-mcherry) in trans (b) or WT cells carrying an empty vector (e.v.) to control for background fluorescence (c). Box shows a magnified region of hyphae with CisA-mCherry accumulation in the cellular periphery. Bars, 5 µm.

d. Western blot of the cellular fractionation of samples from the S. coelicolor WT or the ΔcisA mutant constitutively expressing a CisA-3xFLAG fusion in trans. Lysate and soluble and membrane fractions were probed for the presence of CisA-3xFLAG with an α-FLAG antibody (top). Fractionation efficiency was assessed using an α-WhiA antibody to detect the soluble transcription factor WhiA (bottom). CisA-3xFLAG was detected in the lysate and the membrane fraction. The experiment was performed in biological duplicates and shown is a representative image.

e/f. Experimental determination of the CisA membrane topology using the dual phoA-lacZα reporter in E. coli. The schematic diagram (e) depicts the four CisA constructs used in the assay and indicates the relevant amino acid (aa) deleted in the three CisA mutant derivates. The analysis of colony coloration (f); pink coloration for cytosolic proteins and blue coloration for periplasmic proteins, indicates that the CisA N-terminus is localized in the cytoplasm and the CisA C-terminus localized to the periplasm and this topology is dependent on the transmembrane domain (TM). E. coli strains carrying an empty reporter plasmid or reporter plasmids with the Streptomyces genes for SepH (cytoplasmic) and RsbN (periplasmic) were used as controls. Note that expression of full-length CisA in E. coli is toxic, leading to reduced growth and lighter blue coloration.

CisA is required for regulated cell death and cellular function of CISSc.

a-d. Light microscopy-based viability assay showing that CISSc do not mediate cell death in CisA-deficient hyphae in response to exogenous stress. Shown are representative micrographs of the WT/sfGFP, the ΔcisA/sfGFP and the complemented ΔcisA/cisA+/sfGFP mutant expressing cytosolic sfGFP (indicator for live cells). Strains were grown for 48 h and then either incubated without (a) or with (b) the membrane-disrupting antibiotic nisin (1 µg/ml nisin for 90 min). Samples were subsequently stained with the fluorescent membrane dye FM5-95 to visualize the cytoplasmic membrane. Bars, 10 µm. Z-stacks were taken of the samples and the relative fluorescence ratio from sfGFP to FM5-95 was calculated in c/d. WT and ΔcisA/cisA+ complemented cells showed a significantly higher rate of cell death in response to nisin treatment than ΔcisA cells. There is no significant difference in the viability of the tested strains in the absence of nisin. Experiments were performed in three biological replicates (green, grey and pink data points). Black lines indicate the mean ratio derived from biological triplicate experiments (n=100 images per replicate). ns (not significant) and **** (p < 0.0001) were determined using a one-way ANOVA and Tukey’s post-test.

e. Visualization of spore production in WT, ΔcisA mutant and ΔcisA/cisA+mutant reveal accelerated cellular development of the ΔcisA mutant. Shown are representative brightfield images of surface impressions of plate-grown colonies of each strain taken at the indicated time points. Only sporulating hyphae will attach to the hydrophobic cover glass surface. Insets show magnified regions of the colony surface containing spores and spore chains. Bars, 50 µm.

f. Quantification of sporulation shown in (e) via counts of colony-forming units (c.f.u.). The graph shows a 6-fold higher c.f.u. count at 72 h in the ΔcisA mutant. Strains were grown on R2YE agar and spores were harvested after 48 h, 72 h, and 96 h of incubation. Data show mean ± s.d. obtained from biological triplicate experiments.

Cytoplasmic CISSc require the membrane protein CisA for contraction and function.

Proposed model illustrating a possible role of CisA. In response to exogenous stress or an unknown cellular signal, membrane-bound CisA either directly or indirectly mediates the association of free-floating CISSc particles with the cytoplasmic membrane, leading to CISSc firing and regulated cell death, which impacts the Streptomyces developmental life cycle.

CisA is conserved among CIS-positive Streptomyces and Actinomycete species.

Protein alignment of CisA homologs identified via reciprocal BLAST search in genomes of Streptomyces and Actinomycete species previously reported to encode an eCIS class IId region. Protein sequences (n=75) were aligned with Clustal Omega and visualized using JalView. CisA domain structure is indicated on top. Genome accession numbers and BLAST results are listed in Supplementary Data 1 and 2.

Sheath contraction in situ is linked to the presence of CisA.

a-c. Shown are additional cryo-tomographic slices (thickness 11 nm) of S. coelicolor ΔcisA vegetative hyphae, which were observed as ‘Intact hyphae’ (a), ‘Partially lysed hyphae’ (b) and ‘Ghost cells’ (c). Note that all visible CISSc particles (white arrowheads) are in the extended conformation. PG, peptidoglycan; CM, cytoplasmic membrane/membranes. Bars, 50 nm.

d. Shown is a quantification of CISSc assemblies per tomogram and their conformations as observed in different classes of ΔcisA hyphae. Almost all CISSc particles were seen in the extended conformation, indicating that sheath contraction in situ correlates with the presence of CisA. Data shows mean values and standard deviations obtained from biological triplicate experiments, with n=30 tomograms for each class of hyphae.

Workflow for the cryoEM structural determination of the extended CISSc.

Flowchart for cryoEM reconstruction of the extended CISSc particle. See METHODS and Supplementary Table 1 for details.

Map qualities of different CISSc modules.

a-f. Local resolution maps of the CISSc baseplate with C6 symmetry (a), CISSc baseplate with C3 symmetry (c), and the CISSc cap with C6 symmetry (e). (d-f) shows regions of the cryoEM density map (mesh) that were superimposed with the atomic models (ribbons and sticks), demonstrating the agreement between the observed and modeled amino acid side chains for one beta-sheet (left) and one alpha helix (right). Shown are examples of the baseplate with C6 symmetry (b), the baseplate with C3 symmetry (d), and the cap with C6 symmetry (f).

Structures of the cap modules of other CISs.

a-d. Top views (left) and side views (right) of ribbon representations of the structures of CIS cap modules of Pyocin R2 (a, PDB 6U5B), PVC (b, PDB 6J0N), AFP (c, PDB 6RAP) and AlgoCIS (d, PDB 7ADZ).

Structures of the baseplate modules of other CISs.

a-d. Ribbon representations of the full structures (left) and the individual wedge subunit (right) of the CIS baseplate components (Cis11/12) of Pyocin R2 (a, PDB 6U5F), PVC (b, PDB 6J0F), AFP (c, PDB 6RAO) and AlgoCIS (d, PDB 7AEB).

Control experiment showing that CISSc particles are comparable in the strains used in the viability assay.

Representative negative-stain electron micrographs of purified CISSc particles from the WT/sfGFP, the ΔcisA/sfGFP mutant and the ΔcisA/ΔcisA+/sfGFP complemented mutant exposed to no stress (upper row) or 1 µg/ml nisin (lower row). No differences were observed between the overall structure of CISSc particles. These experiments were performed three independent times. Bars, 100 nm.

CisA impacts secondary metabolite production.

a. Comparison of cultures of WT, ΔcisA mutant and ΔcisA/ΔcisA+mutant in R2YE liquid medium. The coloration of the culture is indicative of actinorhodin (purple) and undecylprodigiosin (red) production (33). Images of each culture flask were taken at the indicated time points.

b. Quantification of total actinorhodin production by the strains shown in (a), revealing reduced production in the ΔcisA mutant. Samples were taken at the indicated time points. Actinorhodin was extracted and quantified by measuring the optical density OD640 of the culture supernatant (65) and normalized to the wet pellet weight. Analysis was performed in biological triplicate experiments. The mean values and standard error are shown. ns (not significant) and **** (p < 0.0001) were calculated using one-way ANOVA and Tukey’s post-test.

In silico protein-protein interaction analyses predict that CisA binds a CISSc baseplate component.

a. An AlphaFold2-Multimer-based protein-protein interaction screen between monomeric CisA and the 19 gene products of the CISSc gene cluster suggests an interaction between CisA and the baseplate component Cis11. The likelihood of a protein-protein interaction was ranked based on the interface pTM (ipTM) confidence score, with an ipTM greater than 0.7 indicating a potential protein-protein interaction.

b. Zoomed-in view of the SPA-cryoEM structure of the CISSc baseplate complex (Fig. 2b/d), illustrating the peripheral and surface-exposed position of Cis11.

c/d Predicted CisA-Cis11 complex. (c) The Alphafold3 model of the CisA-Cis11 complex. The “IgG” domain at the C-terminal end of CisA is located in the periplasm. (d) The Alphafold3 predicted aligned error (%) heatmap plot of the concatenated Cis11 and CisA input sequence.

The cytosolic part of CisA is predicted to interact with Cis11.

a. Schematic showing the CisA domain organization. Relevant amino acid (aa) positions are shown above. TM, transmembrane domain.

b. AlphaFold2-Multimer based protein-protein interaction screen between monomers of CisA and truncated versions of CisA and Cis11. The likelihood of a protein-protein interaction was ranked based on the interface pTM (ipTM) confidence score.

c/d. Predicted CisA-Cis11 complexes using truncated versions of CisA (amino acids 1-285 and 1-310) show that the largely unstructured cytosolic portion of CisA is required to interact with Cis11. (c) The Alphafold3 predicted aligned error (%) heatmap plot of the concatenated Cis11 and truncated CisA 1-285 and CisA 1-310 input sequences. (d) The Alphafold3 model of the CisA 1-285-Cis11 (left) and CisA 1-310-Cis11 (right) complexes.

Western blot confirming similar levels of cytosolic sfGFP in strains used for viability assays.

Control experiments to verify similar protein levels of sfGFP which was expressed either from the ϕC31 or the ϕBT1 phage attachment site in the S. coelicolor genome. Expression of sfgfp was driven from the constitutive ermE* promoter (PermE*). Equal amounts of total protein were loaded and sfGFP levels were detected with an anti-GFP antibody. As a control, the same cell lysates were also probed with an anti-WhiA antibody. Shown are representative results of duplicate experiments.