Cannibalism enhances biofilm development in Bacillus subtilis

Abstract

Cannibalism is a mechanism to delay sporulation in Bacillus subtilis. Cannibal cells express the skf and sdp toxin systems to lyse a fraction of their sensitive siblings. The lysed cells release nutrients that serve to feed the community, effectively delaying spore formation. Here we provide evidence that the subpopulation of cells that differentiates into cannibals is the same subpopulation that produces the extracellular matrix that holds cells together in biofilms. Cannibalism and matrix formation are both triggered in response to the signalling molecule surfactin. Nutrients released by the cannibalized cells are preferentially used by matrix-producing cells, as they are the only cells expressing resistance to the Skf and Sdp toxins. As a result this subpopulation increases in number and matrix production is enhanced when cannibalism toxins are produced. The cannibal/matrix-producing subpopulation is also generated in response to antimicrobials produced by other microorganisms and may thus constitute a defense mechanism to protect B. subtilis from the action of antibiotics in natural settings.

Fig. 1

Activation of cannibalism by the quorum-sensing molecule surfactin. Flow cytometry monitoring the expression of the reporter P_skfA-yfp in LB cultures treated with surfactin after 8 h of incubation. The addition of surfactin induced higher expression of the reporter in a subpopuation of cells, as evidenced by the shoulder observed in the expression profile of treated cultures. This subpopulation was not observed when surfactin was added in the absence of the membrane kinase KinC. Fluorescence is shown in arbitrary units.

Fig. 2

Subpopulations of cannibals and matrix producers are the same cells. Flow cytometry monitoring the subpopulations of cannibals (P_skfA-yfp) and matrix producers (P_yqxM-cfp). Fluorescence intensity for the YFP channel is presented on the X-axis and for the CFP channel is presented on the Y-axis. Fluorescence is measured in arbitrary units. (A) Wild-type strain expressing no fluorescence protein was used as the negative control. (B) Strain harboring only the P_skfA-yfp (cannibalism) reporter. (C) Strain harboring only the P_yqxM-cfp (matrix) reporter. (D) Double-labeled strain P_skfA-yfp, P_yqxM-cfp.

Fig. 3

The Δabh Δdlt mutant over-expresses cannibalism genes. (A)β-galactosidase assay of the transcriptional gene expression of the cannibalism operons (skf and sdp) in the absence of their repressor Abh. Assays were performed on extracts of cells grown in MSgg medium for 72 h. (B) Antibiogram of the supernatant produced by wild type or the Δabh mutant in a disc over a lawn of wild type or the Δdlt mutant strain. Lawns and extracts were obtained from cells grown in MSgg medium. Assay was performed on MSgg medium for 24 h prior to imaging.

Fig. 4

The hypercannibal ΔdltΔabh mutant overproduced extracellular matrix and sporulating-cells are absent. (A) Colony pictures of the strains grown on MSgg medium for 72 h. Scale bar is 1 cm. (B) Magnified images detailing the occurrence of aerial structures where spores localize on the surface of the biofilm. Scale bar is 1 mm. (C) Microscopy pictures used to determine the presence of spores in each biofilm. Representative spores and vegetative cells are denoted using arrows. Scale bar is 3 μm.

Fig. 5

(A) Sporulation is delayed in the hypercannibal ΔdltΔabh mutant. Quantification of vegetative CFU after heat treatment over time in different mutants. Samples were grown as colonies on MSgg and incubated at 30°C for the indicated time prior to analysis. Percent of spores is compared to the number of wild-type spores at day six of the assay. (B) Hypercannibalism increased the subpopulation of matrix-producing cells. Flow cytometry monitoring matrix producers using the reporter P_yqxM-cfp. The ΔdltΔabh mutant showed a two-fold increase in cells differentiated as matrix producers compared to the wild type.

Fig. 6

The signaling molecule surfactin and the histidine kinase KinC are essential for the hypercannibalism phenotype. Effect of the Δsrf and the ΔkinC mutations on wild-type and hypercannibalism ΔdltΔabh mutant biofilms. Colonies were grown on MSgg at 30°C and photographed after 72 h.

Fig. 7

Other peptide antimicrobials mimic the effect of cannibalism. (A) Disc impregnated with nisin (0.6 μM) set near the growth area of B. subtilis. Image was taken after 72 h of incubation on a MSgg plate at 30°C. (B) Microscopy images detailing the occurrence of spores in the region affected and non-affected with nisin. Scale bar is 3μm. Representative spores and vegetative cells are denoted with arrows.