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1. Analysis of gene expression within individual cells reveals spatiotemporal patterns underlying Vibrio cholerae biofilm development

   https://doi.org/10.1371/journal.pbio.3003187  

   Johnson, Grace E; Fei, Chenyi; Wingreen, Ned S; Bassler, Bonnie L (May 2025, PLOS Biology)

   Brown, Sam Paul (Ed.)

   Bacteria commonly exist in multicellular, surface-attached communities called biofilms. Biofilms are central to ecology, medicine, and industry. TheVibrio choleraepathogen forms biofilms from single founder cells that, via cell division, mature into three-dimensional structures with distinct, yet reproducible, regional architectures. To define mechanisms underlying biofilm developmental transitions, we establish a single-molecule fluorescence in situ hybridization (smFISH) approach that enables accurate quantitation of spatiotemporal gene-expression patterns in biofilms at cell-scale resolution. smFISH analyses ofV. choleraebiofilm regulatory and structural genes demonstrate that, as biofilms mature, overall matrix gene expression decreases, and simultaneously, a pattern emerges in which matrix gene expression becomes largely confined to peripheral biofilm cells. Both quorum sensing and c-di-GMP-signaling are required to generate the proper temporal pattern of matrix gene expression. Quorum sensing signaling is uniform across the biofilm, and thus, c-di-GMP-signaling alone sets the regional matrix gene expression pattern. The smFISH strategy provides insight into mechanisms conferring particular fates to individual biofilm cells. 

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2. Breakdown of clonal cooperative architecture in multispecies biofilms and the spatial ecology of predation

   https://doi.org/10.1073/pnas.2212650120  

   Wucher, Benjamin R.; Winans, James B.; Elsayed, Mennat; Kadouri, Daniel E.; Nadell, Carey D. (February 2023, Proceedings of the National Academy of Sciences)

   Biofilm formation, including adherence to surfaces and secretion of extracellular matrix, is common in the microbial world, but we often do not know how interaction at the cellular spatial scale translates to higher-order biofilm community ecology. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacteriumBdellovibrio bacteriovorus. This predator can kill and consume many different Gram-negative bacteria, includingVibrio choleraeandEscherichia coli.V. choleraecan protect itself from predation within densely packed biofilm structures that it creates, whereasE. colibiofilms are highly susceptible toB. bacteriovorus. We explore how predator–prey dynamics change whenV. choleraeandE. coliare growing in biofilms together. We find that in dual-species prey biofilms,E. colisurvival underB. bacteriovoruspredation increases, whereasV. choleraesurvival decreases.E. colibenefits from predator protection when it becomes embedded within expanding groups of highly packedV. cholerae. But we also find that the ordered, highly packed, and clonal biofilm structure ofV. choleraecan be disrupted ifV. choleraecells are directly adjacent toE. colicells at the start of biofilm growth. When this occurs, the two species become intermixed, and the resulting disordered cell groups do not block predator entry. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the surface colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure toB. bacteriovorus. 

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3. Bacterial Biofilm Material Properties Enable Removal and Transfer by Capillary Peeling

   https://doi.org/10.1002/adma.201804153  

   Yan, Jing; Moreau, Alexis; Khodaparast, Sepideh; Perazzo, Antonio; Feng, Jie; Fei, Chenyi; Mao, Sheng; Mukherjee, Sampriti; Košmrlj, Andrej; Wingreen, Ned_S; et al (October 2018, Advanced Materials)

   Abstract Biofilms, surface‐attached communities of bacterial cells, are a concern in health and in industrial operations because of persistent infections, clogging of flows, and surface fouling. Extracellular matrices provide mechanical protection to biofilm‐dwelling cells as well as protection from chemical insults, including antibiotics. Understanding how biofilm material properties arise from constituent matrix components and how these properties change in different environments is crucial for designing biofilm removal strategies. Here, using rheological characterization and surface analyses ofVibrio choleraebiofilms, it is discovered how extracellular polysaccharides, proteins, and cells function together to define biofilm mechanical and interfacial properties. Using insight gained from our measurements, a facile capillary peeling technology is developed to remove biofilms from surfaces or to transfer intact biofilms from one surface to another. It is shown that the findings are applicable to other biofilm‐forming bacterial species and to multiple surfaces. Thus, the technology and the understanding that have been developed could potentially be employed to characterize and/or treat biofilm‐related infections and industrial biofouling problems. 

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4. Multispecies biofilm architecture determines bacterial exposure to phages

   https://doi.org/10.1371/journal.pbio.3001913  

   Winans, James B.; Wucher, Benjamin R.; Nadell, Carey D. (December 2022, PLOS Biology)

   Barr, Jeremy J. (Ed.)

   Numerous ecological interactions among microbes—for example, competition for space and resources, or interaction among phages and their bacterial hosts—are likely to occur simultaneously in multispecies biofilm communities. While biofilms formed by just a single species occur, multispecies biofilms are thought to be more typical of microbial communities in the natural environment. Previous work has shown that multispecies biofilms can increase, decrease, or have no measurable impact on phage exposure of a host bacterium living alongside another species that the phages cannot target. The reasons underlying this variability are not well understood, and how phage–host encounters change within multispecies biofilms remains mostly unexplored at the cellular spatial scale. Here, we study how the cellular scale architecture of model 2-species biofilms impacts cell–cell and cell–phage interactions controlling larger scale population and community dynamics. Our system consists of dual culture biofilms ofEscherichia coliandVibrio choleraeunder exposure to T7 phages, which we study using microfluidic culture, high-resolution confocal microscopy imaging, and detailed image analysis. As shown previously, sufficiently mature biofilms ofE.colican protect themselves from phage exposure via their curli matrix. Before this stage of biofilm structural maturity,E.coliis highly susceptible to phages; however, we show that these bacteria can gain lasting protection against phage exposure if they have become embedded in the bottom layers of highly packed groups ofV.choleraein co-culture. This protection, in turn, is dependent on the cell packing architecture controlled byV.choleraebiofilm matrix secretion. In this manner,E.colicells that are otherwise susceptible to phage-mediated killing can survive phage exposure in the absence of de novo resistance evolution. While co-culture biofilm formation withV.choleraecan confer phage protection toE.coli, it comes at the cost of competing withV.choleraeand a disruption of normal curli-mediated protection forE.colieven in dual species biofilms grown over long time scales. This work highlights the critical importance of studying multispecies biofilm architecture and its influence on the community dynamics of bacteria and phages. 

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5. Morphogenesis and cell ordering in confined bacterial biofilms

   https://doi.org/10.1073/pnas.2107107118  

   Zhang, Qiuting; Li, Jian; Nijjer, Japinder; Lu, Haoran; Kothari, Mrityunjay; Alert, Ricard; Cohen, Tal; Yan, Jing (July 2021, Proceedings of the National Academy of Sciences)

   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 ofVibrio choleraebiofilms 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. 

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