Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities1-7. Biofilm-forming bacteria such as the opportunistic pathogen7-10 Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors impacting surface adhesion and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as a ‘molecular glue’11-15. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here, we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface via a search-engine based approach16. By combining these techniques with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generate positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Zipf's Law17 indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system18, a ‘rich-get-richer’ mechanism of Psl accumulation that results in a small number of ‘elite’ cells extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl levels determine post-division cell fates and that high local Psl levels ultimately allow ‘elite’ cells to serve as the founding population for initial microcolony development.

Biofilms are surface-attached multicellular communities. Using single-cell tracking microscopy, we showed that a pilY1 mutant of Pseudomonas aeruginosa is defective in early biofilm formation. We leveraged the observation that PilY1 protein levels increase on a surface to perform a genetic screen to identify mutants altered in surface-grown expression of this protein. Based on our genetic studies, we found that soon after initiating surface growth, cyclic AMP (cAMP) levels increase, dependent on PilJ, a chemoreceptor-like protein of the Pil-Chp complex, and the type IV pilus (TFP). cAMP and its receptor protein Vfr, together with the FimS-AlgR two-component system (TCS), upregulate the expression of PilY1 upon surface growth. FimS and PilJ interact, suggesting a mechanism by which Pil-Chp can regulate FimS function. The subsequent secretion of PilY1 is dependent on the TFP assembly system; thus, PilY1 is not deployed until the pilus is assembled, allowing an ordered signaling cascade. Cell surface-associated PilY1 in turn signals through the TFP alignment complex PilMNOP and the diguanylate cyclase SadC to activate downstream cyclic di-GMP (c-di-GMP) production, thereby repressing swarming motility. Overall, our data support a model whereby P. aeruginosa senses the surface through the Pil-Chp chemotaxis-like complex, TFP, and PilY1 to regulate cAMP and c-di-GMP production, thereby employing a hierarchical regulatory cascade of second messengers to coordinate its program of surface behaviors.

When a monolayer of hard microscale square platelets, produced lithographically, is osmotically concentrated in a flat plane to raise the particle area fraction ϕ A , an order-order transition occurs between a hexagonal rotator crystal and a rhombic crystal. Strikingly, phases having fourfold symmetry are not observed at any ϕ A . The rhombic lattice angle α increases continuously with ϕ A , as the system maximizes its total rotational and translational entropy. A cage model, based on packing rotationally swept squares, or "squaroids," reasonably predicts the measured αðϕ A Þ, indicating that rotational entropy and the square particle shape combine to produce the rhombic unit cell.colloid | phase behavior | structure | two dimensions | thermal fluctuations T hermodynamic structures of liquid crystalline mesophases depend in an important way on the geometrical shapes of their constituent molecules. This shape dependence is particularly clear for hard-core repulsive interactions, because the state having minimum free energy is determined by maximizing the entropy through the available free volume per molecule. For solutions of long hard rods, Onsager first showed that the onset of nematic liquid crystalline order depends on molecular geometry through the excluded volume (1). Molecules in two dimensions (2D) are particularly interesting because their shapes and symmetries can determine whether or not a first-order freezing phase transition is replaced by an intervening mesophase, which exhibits a spatial power-law decay in orientational order but only short-range translational order. For hard disks, a sixfold hexatic phase can interpolate between the isotropic liquid and hexagonal 2D crystalline phases (2-9). For hard squares, Monte Carlo (MC) simulations (10) predict that a high-density crystal phase having square symmetry will melt into a tetratic mesophase having a fourfold orientational order at lower densities: The fourfold symmetries of the square crystal and the tetratic mesophase reflect the underlying symmetry of the constituent square particle. However, no experiments have yet been made on systems of Brownian squares that test these predictions.Lithographic methods have facilitated the synthesis of uniform particulate dispersions containing shape-designed colloids that can serve as model systems for molecular liquid crystals (11-16). When properly controlled, these customized dispersions can be used to study interesting and fundamental problems of statistical mechanics of dense many-particle systems. Although the 2D phase behavior and jammed states of a thermal system of hard pentagons, which cannot fully tile a plane, have been examined (17), a very different set of phases and phase transitions could be observed by investigating a system of Brownian squares which can fully tile a plane.To investigate this, we have explored the phase behavior of a model aqueous dispersion of lithographic square platelets (i.e., "squares") that diffuse in a plane. We form a monolayer of microscale squares near the bottom s...

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay<30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.