Extracellular-matrix-mediated osmotic pressure drives Vibrio cholerae biofilm expansion and cheater exclusion

Abstract

Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmotic pressure difference between the biofilm and the external environment. This pressure difference promotes biofilm expansion on nutritious surfaces by physically swelling the colony, which enhances nutrient uptake, and enables matrix-producing cells to outcompete non-matrix-producing cheaters via physical exclusion. Osmotic pressure together with crosslinking of the matrix also controls the growth of submerged biofilms and their susceptibility to invasion by planktonic cells. As the basic physicochemical principles of matrix crosslinking and osmotic swelling are universal, our findings may have implications for other biofilm-forming bacterial species.

Interspecies interactions in bacterial colonies are determined by physiological traits and the environment

“…For K. pneumoniae, lowering the agar concentration allowed the wild-type to colonize 445 more area, which was similar to how a rugose strain of V. cholerae that hypersecretes ECM 446 formed larger colonies on lower concentrations of agar [25]. In V. cholerae, it was 447 demonstrated that matrix secretion generates an osmotic pressure gradient between the agar 448 and the biofilm, allowing the biofilm to expand by physical swelling and increased nutrient 449 uptake.…”

“…When the two were co-cultured together, the NMV 257 covered less area, ~4 mm 2 , compared to when grown alone, ~12 mm 2 , at 24 h, but there was 258 no change at 48 h. The area covered by the NMV was also decreased when paired with P. 259 protegens, however, neither of these differences persisted at 48 h. In contrast, the area 260 covered by P. aeruginosa was decreased when grown in the presence of the K. pneumoniae 261 NMV at 24 h but not 48 h. When paired with P. aeruginosa, the area covered by the NMV 262 was not significantly increased as was observed for the parental K. pneumoniae strain when 263 paired with P. aerguinosa. The percentage of agar influences motility, as lower percentages enable swarming 290 and/or swimming motility and affect the colony growth of highly mucoid strains [25]. 291…”

“…[51] and V. cholerae [52]. 422 affects the ability of biofilms to extract nutrients as it determines the osmotic pressure of an 425 environment [25]. Here we showed that lowering the agar concentration from 1.5% to 0.6% 426 allowed the TFP motility deficient SCV of P. aeruginosa to colonize as much area as the 427 wild-type and was not encircled by the parental wild-type when they were co-cultured (Table 428 3).…”

“…55Colony biofilms, where bacteria are grown on a nutrient surface, are an efficient 56 system for manipulating and visualizing the spatial distribution of different bacteria, but have 57 mostly been used for studying eco-evolutionary dynamics [20]. Colony biofilms have been 58 used to characterize various spatiotemporal aspects of microbial interactions including the co-59 localization of mutually auxotrophic strains of Saccharomyces cerevisiae [21, 22], the 60 vertical separation of differently sized cells of Escherichia coli [23], the sequential range 61 expansion of nitrate reducing Pseudomonas stutzeri [24], the spatial separation patterns of 62 strains of Vibrio cholerae [25] or Bacillus subtilis [26] that differ in the quantity of 63 extracellular matrix produced, and the proportion of different antibiotic resistant/sensitive overlapping excitation and emission spectra. When multiple combinations were possible (e.g.…”

Dynamics of spreading microbial swarms and films

“…In both cases, an interplay between mechanical constraints and biological organization sets limits on the overall colony morphology and expansion dynamics [9]. The forces driving colony expansion are generated by non-homogeneous patterns of biological activity, originating from spatial localizations in cell growth and division [10], extracellular polymer matrix production [11][12][13], osmolyte secretion [14] and active stresses [15,16]. Conversely, the formation of localized biologically active zones is tightly coupled to the heterogeneity of the environment, including the diffusion and transport of nutrients [17], accumulation of metabolic by-products [18,19] and presence of quorum sensing and signaling agents that regulate cell-differentiation and development.…”