Escherichia coli self-organizes developmental rosettes

Abstract

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.

Keywords: bacteria; development; dynamics; multicellular.

Fig. 1.

Sister-cell folding produces rosettes and follows an angular random walk. (A) Micrographs from a representative movie (2-s resolution) of E. coli sister-cell folding and parallel alignment. Time and scale indicated on all micrographs. New (blue) and old (red) poles are indicated. Inset: Illustration of cell folding documented in movies. (B) Later micrographs from the same video. (C) Micrographs from a representative movie (30-s resolution) of rosette formation in cells constitutively expressing GFP. New (blue) and old (red) poles are indicated. (D) Dynamics of sister-cell angle (θ) during cellular folding (2-s resolution; mean ± SD, n = 5). Folding (red) and prefolding (blue) are indicated. Inset: Illustration of θ calculation. (E) Representative example of sister cell θ (blue) and Ф (red). Inset: Illustration of Ф calculation. (F) Trajectory of sister-cell angles from representative example of folding. Start and end positions and times are indicated. (G) Representative example of 3D trajectory of cell pole during folding. (H) Dynamics of angular strokes (movement > 0.8 µm) for five representative sister-cell pairs. Inset: Illustration of distance calculation. (I) Distribution of angle change between strokes (δ) (59 recorded strokes across five independent sister-cell pairs). Inset: Illustration of δ calculation.

Fig. 2.

Flagella propulsion generates sister-cell folding. (A) Diagram of the flagellum (not to scale). Micrographs from representative movies of (B, C) ΔfliC and (D, E) ΔmotA cells. Time and scale indicated on all micrographs. (F) Dynamics of ΔfliC (blue) and ΔmotA (red) sister-cell angle (30-s resolution; mean ± SD, n = 3). An example wild-type trajectory is included for comparison. (G) Dynamics of angular speed for ΔfliC and ΔmotA cells (30-s resolution; mean ± SD, n = 3). Inset: Average maximum speed. (H) Length dynamics of ΔfliC and ΔmotA communities (mean ± SD; n = 3). An example wild-type trajectory is included for comparison. Inset: Illustration of halving length during folding. (I) Dynamics of angular movement for representative ΔcheY (blue) and ΔcheZ (red) sister-cell pairs (2-s resolution). Inset: Representative wild-type example. (J) Trajectory of sister-cell angles from representative ΔcheY example. (K) Trajectory of sister-cell angles from representative ΔcheZ example.

Fig. 3.

Rosette formation requires de novo flagella biosynthesis. (A) Representative micrographs of AW405 cells grown (A) with glucose and (B) without glucose and stained with Alexa Fluor 488 (green). Scale is indicated on micrographs. Micrographs from representative movies of (C) AW405 and (D) AW405 ΔfliC cells grown without glucose. (E) Micrographs from a representative movie of AW405 transitioned from glucose (+) to glucose (−) media. New (blue) and old (red) poles are indicated. (F) Diagram of the observed angular cell motion of strains and conditions used throughout this study and correspondence to community structure. ΔcheY and ΔcheZ cells produce heterogeneous outcomes and thus overlap depicted regions. (G) Cellular fluorescence (arbitrary units) from fliE-GFP promoter reporter during chain morphogenesis (red) in AW405 on transition from glucose (+) to (−) (5-min resolution; mean ± SD, n = 3). The gray zone indicates fliE-GFP fluorescence in nonflagellated cells in glucose (+) media (SI Appendix, Fig. S37). The blue zone indicates fliE-GFP cellular fluorescence in flagellated cells (1 to 4 flagella per cell) in glucose (−) media (SI Appendix, Fig. S37). The approximate times of rosette formation and dissociation of single swimming cells are indicated by arrows.

Fig. 4.

Developmental consequences and generality of rosette formation. Micrographs at 5 h from representative movies of (A) wild-type and (B) ΔfliC community formation (scale is indicated). (C) Cellular fluorescence (arbitrary units) from rpoS-GFP promoter reporter during chain morphogenesis in wild type (blue) and ΔfliC (red) (20-min resolution; mean ± SD, n = 3). The approximate time of chain attachment to surfaces is indicated. Micrographs of hydrostatic biofilms formed by (D) wild-type, (E) ΔfliC, and (F) ΔmotA cells. Cells contained either mScarlet (red) or GFP (cyan) at a 10:1 ratio (scale, as well as air and liquid sides of biofilms, are indicated). Example micrographs of (G) wild-type, (H) ΔfliC, and (I) Δflu cells after ~2 h on motility agar (2-layer 0.25% agar; scale is indicated; see also SI Appendix, Fig. S40).

Fig. 5.

Postulated stages of E. coli multicellular morphogenesis in hydrostatic environments. The data presented here and in our previous study (8), together with past characterization of adhesins, support the following working model. 2-cell: Self-recognizing Ag43 enables adhesion of sister cells after the first division event. Cells induce de novo flagella biosynthesis, and the propulsion of the growing flagella tail causes sister cells to perform cell folding by an angular random walk to align side by side. 4-cell: Sister cells adhere after the second division event and again perform cell folding to align in parallel and produce rosettes with quatrefoil configuration. Rosettes transition into chains, whose square cross-sectional arrangement is approximately maintained. ~8-cell stage: Radial-projecting extracellular polymer type-1 fimbriae (indicated in red) control chain stability. ~16-cell stage: Extracellularly polymerizing curli (indicated in blue) maintain cellular positioning. ~200-cell stage: Transcriptional master regulator σ³⁸ (encoded by rpoS) is induced (indicated by cells turning blue). ~1,000-cell stage: Extracellular polysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PGA, indicated in orange) is produced and attaches multicellular communities to surfaces. The representations of adhesins are not to scale and have yet to be confirmed by electron microscopy.