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1. Hierarchical Transitions and Fractal Wrinkling Drive Bacterial Pellicle Morphogenesis

   Qin, B. ; Fei, C. ; Wang, B. ; Stone, H.A. ; Wingreen, N.S. ; Bassler, B.L. ( April 2021 , Proceedings of the National Academy of Sciences of the United States of America)

   null (Ed.)

   Bacterial cells can self-organize into structured communities at fluid-fluid interfaces. These soft, living materials composed of cells and extracellular matrix are called pellicles. Cells residing in pellicles garner group-level survival advantages such as increased antibiotic resistance. The dynamics of pellicle formation and, more generally, how complex morphologies arise from active biomaterials confined at interfaces are not well understood. Here, using Vibrio cholerae as our model organism, a custom-built adaptive stereo microscope, fluorescence imaging, mechanical theory, and simulations, we report a fractal wrinkling morphogenesis program that differs radically from the well-known coalescence of wrinkles into folds that occurs in passive thin films at fluid-fluid interfaces. Four stages occur: growth of founding colonies, onset of primary wrinkles, development of secondary curved ridge instabilities, and finally the emergence of a cascade of finer structures with fractal-like scaling in wavelength. The time evolution of pellicle formation depends on the initial heterogeneity of the film microstructure. Changing the starting bacterial seeding density produces three variations in the sequence of morphogenic stages, which we term the bypass, crystalline, and incomplete modes. Despite these global architectural transitions, individual microcolonies remain spatially segregated, and thus the community maintains spatial and genetic heterogeneity. Our results suggest that the memory of the original microstructure is critical in setting the morphogenic dynamics of a pellicle as an active biomaterial. 

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2. Quorum-sensing control of matrix protein production drives fractal wrinkling and interfacial localization of Vibrio cholerae pellicles

   https://doi.org/10.1038/s41467-022-33816-6  

   Qin, Boyang ; Bassler, Bonnie L. ( October 2022 , Nature Communications)

   Bacterial cells at fluid interfaces can self-assemble into collective communities with stunning macroscopic morphologies. Within these soft, living materials, called pellicles, constituent cells gain group-level survival advantages including increased antibiotic resistance. However, the regulatory and structural components that drive pellicle self-patterning are not well defined. Here, usingVibrio choleraeas our model system, we report that two sets of matrix proteins and a key quorum-sensing regulator jointly orchestrate the sequential mechanical instabilities underlying pellicle morphogenesis, culminating in fractal patterning. A pair of matrix proteins, RbmC and Bap1, maintain pellicle localization at the interface and prevent self-peeling. A single matrix protein, RbmA, drives a morphogenesis program marked by a cascade of ever finer wrinkles with fractal scaling in wavelength. Artificial expression ofrbmArestores fractal wrinkling to a ΔrbmAmutant and enables precise tuning of fractal dimensions. The quorum-sensing regulatory small RNAs Qrr1-4 first activate matrix synthesis to launch pellicle primary wrinkling and ridge instabilities. Subsequently, via a distinct mechanism, Qrr1-4 suppress fractal wrinkling to promote fine modulation of pellicle morphology. Our results connect cell-cell signaling and architectural components to morphogenic patterning and suggest that manipulation of quorum-sensing regulators or synthetic control ofrbmAexpression could underpin strategies to engineer soft biomaterial morphologies on demand.

    

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3. Metabolic Remodeling during Biofilm Development of Bacillus subtilis

   https://doi.org/10.1128/mBio.00623-19  

   Pisithkul, Tippapha ; Schroeder, Jeremy W. ; Trujillo, Edna A. ; Yeesin, Ponlkrit ; Stevenson, David M. ; Chaiamarit, Tai ; Coon, Joshua J. ; Wang, Jue D. ; Amador-Noguez, Daniel ; Ghigo, Jean-Marc ; et al ( June 2019 , mBio)

   ABSTRACT Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology. IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis , very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism. 

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4. High-Reynolds-number fractal signature of nascent turbulence during transition

   https://doi.org/10.1073/pnas.1916636117  

   Wu, Zhao ; Zaki, Tamer A. ; Meneveau, Charles ( February 2020 , Proceedings of the National Academy of Sciences)

   Transition from laminar to turbulent flow occurring over a smooth surface is a particularly important route to chaos in fluid dynamics. It often occurs via sporadic inception of spatially localized patches (spots) of turbulence that grow and merge downstream to become the fully turbulent boundary layer. A long-standing question has been whether these incipient spots already contain properties of high-Reynolds-number, developed turbulence. In this study, the question is posed for geometric scaling properties of the interface separating turbulence within the spots from the outer flow. For high-Reynolds-number turbulence, such interfaces are known to display fractal scaling laws with a dimension$D\approx 7/3$, where the 1/3 excess exponent above 2 (smooth surfaces) follows from Kolmogorov scaling of velocity fluctuations. The data used in this study are from a direct numerical simulation, and the spot boundaries (interfaces) are determined by using an unsupervised machine-learning method that can identify such interfaces without setting arbitrary thresholds. Wide separation between small and large scales during transition is provided by the large range of spot volumes, enabling accurate measurements of the volume–area fractal scaling exponent. Measurements show a dimension of$D=2.36\pm 0.03$over almost 5 decades of spot volume, i.e., trends fully consistent with high-Reynolds-number turbulence. Additional observations pertaining to the dependence on height above the surface are also presented. Results provide evidence that turbulent spots exhibit high-Reynolds-number fractal-scaling properties already during early transitional and nonisotropic stages of the flow evolution.

    

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5. Bacterial scattering in microfluidic crystal flows reveals giant active Taylor–Aris dispersion

   https://doi.org/10.1073/pnas.1819613116  

   Dehkharghani, Amin ; Waisbord, Nicolas ; Dunkel, Jörn ; Guasto, Jeffrey S. ( June 2019 , Proceedings of the National Academy of Sciences)

   The natural habitats of planktonic and swimming microorganisms, from algae in the oceans to bacteria living in soil or intestines, are characterized by highly heterogeneous fluid flows. The complex interplay of flow-field topology, self-propulsion, and porous microstructure is essential to a wide range of biophysical and ecological processes, including marine oxygen production, remineralization of organic matter, and biofilm formation. Although much progress has been made in the understanding of microbial hydrodynamics and surface interactions over the last decade, the dispersion of active suspensions in complex flow environments still poses unsolved fundamental questions that preclude predictive models for microbial transport and spreading under realistic conditions. Here, we combine experiments and simulations to identify the key physical mechanisms and scaling laws governing the dispersal of swimming bacteria in idealized porous media flows. By tracing the scattering dynamics of swimming bacteria in microfluidic crystal lattices, we show that hydrodynamic gradients hinder transverse bacterial dispersion, thereby enhancing stream-wise dispersion ∼ 100 -fold beyond canonical Taylor–Aris dispersion of passive Brownian particles. Our analysis further reveals that hydrodynamic cell reorientation and Lagrangian flow structure induce filamentous density patterns that depend upon the incident angle of the flow and disorder of the medium, in striking analogy to classical light-scattering experiments. 

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