Vibrio cholerae filamentation promotes chitin surface attachment at the expense of competition in biofilms

Abstract

Collective behavior in spatially structured groups, or biofilms, is the norm among microbes in their natural environments. Though biofilm formation has been studied for decades, tracing the mechanistic and ecological links between individual cell morphologies and the emergent features of cell groups is still in its infancy. Here we use single-cell-resolution confocal microscopy to explore biofilms of the human pathogen Vibrio cholerae in conditions mimicking its marine habitat. Prior reports have noted the occurrence of cellular filamentation in V. cholerae, with variable propensity to filament among both toxigenic and nontoxigenic strains. Using a filamenting strain of V. cholerae O139, we show that cells with this morphotype gain a profound competitive advantage in colonizing and spreading on particles of chitin, the material many marine Vibrio species depend on for growth in seawater. Furthermore, filamentous cells can produce biofilms that are independent of primary secreted components of the V. cholerae biofilm matrix; instead, filamentous biofilm architectural strength appears to derive at least in part from the entangled mesh of cells themselves. The advantage gained by filamentous cells in early chitin colonization and growth is countered in long-term competition experiments with matrix-secreting V. cholerae variants, whose densely packed biofilm structures displace competitors from surfaces. Overall, our results reveal an alternative mode of biofilm architecture that is dependent on filamentous cell morphology and advantageous in environments with rapid chitin particle turnover. This insight provides an environmentally relevant example of how cell morphology can impact bacterial fitness.

Keywords: Vibrio cholerae; biofilm; cell shape; chitin; extracellular matrix.

Fig. 1.

Cell morphology and biofilm structures of V. cholerae N16961 and CVD112. (A) Cell-cluster biofilms of O1 El Tor strain N16961 [3D render is 80 × 80 × 15 μm length (L) × width (W) × depth (D), planktonic cells, Inset]. (B) Filamented biofilms of O139 strain CVD112 [3D render is 80 × 80 × 50 μm (L × W × D), planktonic cells, Inset]. Biofilm images in A and B were captured in glass-bottom chambers containing M9 minimal media with 0.5% glucose. (C and D) Growth kinetics of CVD112 and N16961 in LB, artificial seawater with 0.5% GlcNAc, and M9 minimal media with 0.5% glucose, as measured by (C) colony-forming unit count and (D) optical density at 600 nm (for each growth curve and condition, n = 3 biological replicates, each with three technical replicates). Error bars denote the SEM.

Fig. 2.

Filamentous V. cholerae CVD112 has an increased chitin colonization rate and produces VPS- and RbmA-independent biofilms on chitin in seawater. (A) The rates of CVD112 (red data) and N16961 (yellow data) accumulation onto fresh chitin particles in artificial seawater (n = 6 biological replicates). (B) As in A, but here N16961 was pretreated for 60 min with cefalexin, which causes it to filament in a manner similar to CVD112 without reducing cell viability (n = 4 biological replicates). NS, not significant. (C) CVD112 cells (red) and N16961 (yellow) bound to pieces of chitin (blue) in artificial seawater [3D render is 85 × 85 × 60 μm (L × W × D)]. (D) Mean biomass production (black bars, left vertical axis) and matrix normalized to biomass (purple bar, right vertical axis) for wild type and matrix-deficient ΔvpsL derivatives of CVD112 and N16961 (n = 5 biological replicates). Error bars denote the SEM. (E–H) Wild-type N16961 (E) (yellow), N16961 ΔvpsL (F) (yellow), CVD112 (G) (red), and CVD112 ΔvpsL (H) (red) on chitin (blue) in seawater. Matrix stain (Cy3-conjugated antibody to RbmA-FLAG) is shown in purple [3D renders in E–H are 175 × 175 × 40 μm (L × W × D)].

Fig. 3.

Competition between short-cell N16961 (yellow) and filamenting CVD112 (red) on chitin (blue) in seawater. The two strains were grown together with different disturbance/recolonization regimes for 12 d. A shows the frequency (fraction of total biomass) of CVD112, while B shows the total biomass in the chambers for each disturbance condition. Chambers were either undisturbed (red traces in A and B; images in C), disrupted and reinoculated into new chitin chambers once per 72 h (black traces in A and B; images in D), or disrupted and reinoculated into chambers every 24 h (blue traces in A and B; images in E). Above each image series the treatment regime is shown with imaging times marked in green (representative images noted with black dots) and disturbance/recolonization events shown in magenta. All 3D renders in C–E are 385 × 385 × 32 μm (L × W × D).