Morphogenesis and cell ordering in confined bacterial biofilms

Abstract

Biofilms are aggregates of bacterial cells surrounded by an extracellular matrix. Much progress has been made in studying biofilm growth on solid substrates; however, little is known about the biophysical mechanisms underlying biofilm development in three-dimensional confined environments in which the biofilm-dwelling cells must push against and even damage the surrounding environment to proliferate. Here, combining single-cell imaging, mutagenesis, and rheological measurement, we reveal the key morphogenesis steps of Vibrio cholerae biofilms embedded in hydrogels as they grow by four orders of magnitude from their initial size. We show that the morphodynamics and cell ordering in embedded biofilms are fundamentally different from those of biofilms on flat surfaces. Treating embedded biofilms as inclusions growing in an elastic medium, we quantitatively show that the stiffness contrast between the biofilm and its environment determines biofilm morphology and internal architecture, selecting between spherical biofilms with no cell ordering and oblate ellipsoidal biofilms with high cell ordering. When embedded in stiff gels, cells self-organize into a bipolar structure that resembles the molecular ordering in nematic liquid crystal droplets. In vitro biomechanical analysis shows that cell ordering arises from stress transmission across the biofilm–environment interface, mediated by specific matrix components. Our imaging technique and theoretical approach are generalizable to other biofilm-forming species and potentially to biofilms embedded in mucus or host tissues as during infection. Our results open an avenue to understand how confined cell communities grow by means of a compromise between their inherent developmental program and the mechanical constraints imposed by the environment.

Roughening instability of growing 3D bacterial colonies

“…This growth-induced roughening is markedly different from that observed in Ref. [72], where much smaller bacterial colonies growing as inclusions in stiff, cross-linked bulk gels retained smooth surfaces. Moreover, while highly-branched shapes have been previously observed for colonies growing on planar 2D surfaces, they are thought to only arise when the underlying surface is depleted in nutrients, thereby generating stochasticity in the ability of cells to access nutrient [14–20, 24, 25, 37]; in our case, however, the surrounding matrix is nutrient-replete.…”

“…For instance, external fluid flows are now known to trigger the formation of streamers that stem from an initially surface-attached colony [11, 71]. Under quiescent conditions, small (≲ 10s of cells across) biofilm colonies constrained in strongly-cross-linked bulk gels adopt internally-ordered structures as they grow and push outward [72], mediated by elastic stresses arising at the interface between the colony and its stiff environment. However, the behavior of larger bacterial colonies growing freely in quiescent 3D environments remains underexplored, despite the fact that they represent a fundamental building block of more complex natural colonies.…”

Chemotactic smoothing of collective migration

“…Indeed, while studies of chemotaxis typically focus on the role of the external nutrient gradient in driving cellular migration, our work highlights the distinct and pivotal role played by the cellular chemotactic response function in regulating migration and large-scale population morphology more broadly. Our work therefore contributes a new factor to be considered in descriptions of morphogenesis, which thus far have focused on the role of other factors—such as differential forcing by signaling gradients, differential proliferation, intercellular mechanics, substrate interactions, and osmotic stresses ( McLennan et al, 2012 ; Fujikawa and Matsushita, 1989 ; Bonachela et al, 2011 ; Nadell et al, 2010 ; Farrell et al, 2013 ; Trinschek et al, 2018 ; Allen and Waclaw, 2019 ; Beroz et al, 2018 ; Fei et al, 2020 ; Yan et al, 2019 ; Yan et al, 2017 ; Copenhagen et al, 2020 ; Smith et al, 2017 ; Ghosh et al, 2015 ; Zhang et al, 2021 )—in regulating the overall morphology of cellular communities and active matter in general.…”

Microbial Biofilms: Structural Plasticity and Emerging Properties

“…From these observations, they better predicted developmental dynamics and 3D structure emergence of biofilm through a better deciphering of cellular interactions. Complementarily, by integrating rheological measurements, single-cell imaging by spinning disc confocal microscopy, and modeling, Zhang et al [33] built a comprehensive view of V. cholerae biofilm development in confined environments using an agarose gel entrapment model. Thereby, they demonstrated that mechanical stress governs morphogenetic development and cell ordering in V. cholerae confined biofilms.…”