Crowning: a novel Escherichia coli colonizing behaviour generating a self-organized corona

Abstract

Background: Encased in a matrix of extracellular polymeric substances (EPS) composed of flagella, adhesins, amyloid fibers (curli), and exopolysaccharides (cellulose, β-1,6-N-acetyl-D-glucosamine polymer-PGA-, colanic acid), the bacteria Escherichia coli is able to attach to and colonize different types of biotic and abiotic surfaces forming biofilms and colonies of intricate morphological architectures. Many of the biological aspects that underlie the generation and development of these E. coli's formations are largely poorly understood.

Results: Here, we report the characterization of a novel E. coli sessile behaviour termed "crowning" due to the bacterial generation of a new 3-D architectural pattern: a corona. This bacterial pattern is formed by joining bush-like multilayered "coronal flares or spikes" arranged in a ring, which self-organize through the growth, self-clumping and massive self-aggregation of cells tightly interacting inside semisolid agar on plastic surfaces. Remarkably, the corona's formation is developed independently of the adhesiveness of the major components of E. coli's EPS matrix, the function of chemotaxis sensory system, type 1 pili and the biofilm master regulator CsgD, but its formation is suppressed by flagella-driven motility and glucose. Intriguingly, this glucose effect on the corona development is not mediated by the classical catabolic repression system, the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Thus, corona formation departs from the canonical regulatory transcriptional core that controls biofilm formation in E. coli.

Conclusions: With this novel "crowning" activity, E. coli expands its repertoire of colonizing collective behaviours to explore, invade and exploit environments whose critical viscosities impede flagella driven-motility.

Figure 1

Old macrocolony biofilms are producers of E. coli’s corona. (A) Typical volcano-like appearance (morphotype) of a 14-old-day macrocolony biofilm of E. coli K-12 MG1655 strain developed over a semisolid 0.6% ABE agar surface visualized by reflected light. (B) Schematic representation of characteristic structures observed in this macrocolony. EC, external colony corresponds to the superficial aerial, visible part of macrocolony developed on the semisolid agar surface. IB, represent the internal biofilm, an agar-entrapped formation, composed of "root" (R) and "corona" (C) developed inside semisolid agar. The root is the structure generated by bacterial "fingers" that penetrate inside the agar along the toothpick-punctured zone, while the corona (C) is the biofilm structure that develops in contact with the plastic surface (PS) of the base of the Petri dish. (C) Typical 14-old-day corona of E. coli K-12 MG1655 strain. (D-E) Close-up of the corona and a coronal spike. (F-J) Zooming view of a coronal spike removed from a corona and placed under a microscope at different magnifications. (F) Part of a corona viewed to × 40, the box indicates the coronal spike removed and observed at different magnifications: (G) × 100 (H) × 400 (I-J) ×1000. (K) Typical 14-old-day E. coli K-12 BW25113 strain volcano-like colony and their corona (L-M), (L) × 40 (M) × 100 (N-O). Typical 14-old-day corona of E. coli K-12 W3110 strain. (N) × 40 (O) × 100 magnifications. Each box (right) represents the enlargement region in the following image (left). Scale bars: (A, K) 0.5 cm (C) 0.1 cm (D) 500 μm (E, M, O) 100 μm (F) 350 μm (G) 150 μm (L, N) 200 μm (H) 40 μm (I-J) 20 μm.

Figure 2

Environmental and biological factors that promote the E. coli K-12 corona formation or else preclude this formation. (A) The colony (A-1) produced the E. coli’s corona when it was inoculated with a toothpick that punctured the 0.6% ABE semisolid agar and contacted the Petri dish’s plastic surface. The colony (A-2) by contrast did not generate a corona when the bacterial inoculation was carried out with a drop containing 5 μl of a stationary culture grown in LB medium of MG1655 strain deposited carefully on semisolid agar surface (B-C) The flagella-driven swimming interstitial/internal motility abolished the corona formation. Typical 14-old-day macrocolony swimming colony of E. coli K-12 MG1655 strain (B) View from the top (C) Reverse view (D) Corona developed on a plastic surface, but not on a glass surface (E). (F) A typical 14-old-day macrocolony of E.coli K-12 W3110 strain (G) Appearance of the same colony when the external colony was removed (H-I)E. coli’s corona developed inside semisolid 1.0% ABE agar concentration. (H) View from the top (I) Reverse view. (J-K) The enlarged box region in (H-I) showing the corona at × 40 (J) and ×100 (K) magnifications. The closed white arrowheads indicate the situation of corona. The open white arrowheads indicated the coronal root. Scale bars: (A-G), 0.5 cm; (H-I) 0.25 cm; (J) 300 μm; (K) 100 μm.

Figure 3

(A-G) Glucose represses corona formation independently of the regulatory activity of the cAMP-CRP complex. (A) A typical 14-old-day biofilm macrocolony of E.coli K-12 strain grown over 0.6% ABE semisolid agar with Luria Bertani medium supplemented with 0.5% of D-(+)-glucose does not produce a corona (B-C) Circular cellular formation surrounding the inoculation point does not produce coronal flares or spikes (B) × 40 (C) ×100 (D) × 400 magnifications (E) Individual elongated cells 5–10 μm long, typical "swarm" cells [24,25], removed from macrocolony and observed under optical microscope at × 1000 augmentation (F-G) Corona generated by a E.coli K-12 Δcrp mutant strain (GS0549) lacking CRP protein. (G) Enlargement of the boxed region in F. Scale bars: (A) 0.25 cm (B) 400 μm (C) 200 μm (D) 40 μm (E) 20 μm (F) 350 μm (G) 150 μm. (H-I) Corona formation in relation to the canonical "core" transcriptional network that controls switching between motility and biofilm formation. In E. coli K-12, the transition from a planktonic/foraging lifestyle to biofilming behaviour is regulated by two inversely controlled transcriptional feedforward cascades, the FlhDC + σ⁷⁰/σ^F "flagellar" cascade and the σ^S/MlrA/CsgD cascade (adapted from references [17,22,28]). Remarkably, while CRP in conjunction with the small RNA McsA has a dual role in the control of both cascades (forming a coherent feedforward loop (FFL) in order to regulate the expression of flhDC and an incoherent FFL to control csgD expression, [33]) apparently it plays no role in E. coli’s corona formation at all. Arrowheads indicate positive regulation; perpendicular lines indicate negative regulation. Scale bar: (I) 800 μm.