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1. Vibrio cholerae RbmB is an α-1,4-polysaccharide lyase with biofilm-disrupting activity against Vibrio polysaccharide (VPS)

   https://doi.org/10.1371/journal.ppat.1012750  

   Weerasekera, Ranjuna; Moreau, Alexis; Huang, Xin; Nam, Kee-Myoung; Hinbest, Alexander J; Huynh, Yun; Liu, Xinyu; Ashwood, Christopher; Pepi, Lauren E; Paulson, Eric; et al (December 2024, PLOS Pathogens)

   Paczkowski, Jon (Ed.)

   Many pathogenic bacteria form biofilms as a protective measure against environmental and host hazards. The underlying structure of the biofilm matrix consists of secreted macromolecules, often including exopolysaccharides. To escape the biofilm, bacteria may produce a number of matrix-degrading enzymes, including glycosidic enzymes that digest exopolysaccharide scaffolds. The human pathogenVibrio choleraeassembles and secretes an exopolysaccharide called VPS (Vibriopolysaccharide) which is essential in most cases for the formation of biofilms and consists of a repeating tetrasaccharide unit. Previous studies have indicated that a secreted glycosidase called RbmB is involved inV.choleraebiofilm dispersal, although the mechanism by which this occurs is not understood. To approach the question of RbmB function, we recombinantly expressed and purified RbmB and tested its activity against purified VPS. Using a fluorescence-based biochemical assay, we show that RbmB specifically cleaves VPSin vitrounder physiological conditions. Analysis of the cleavage process using mass spectrometry, solid-state NMR, and solution NMR indicates that RbmB cleaves VPS at a specific site (at the α-1,4 linkage between D-galactose and a modified L-gulose) into a mixture of tetramers and octamers. We demonstrate that the product of the cleavage contains a double bond in the modified guluronic acid ring, strongly suggesting that RbmB is cleaving using a glycoside lyase mechanism. Finally, we show that recombinant RbmB fromV.choleraeand the related aquatic speciesVibrio coralliilyticusare both able to disrupt livingV.choleraebiofilms. Our results support the role of RbmB as a polysaccharide lyase involved in biofilm dispersal, as well as an additional glycolytic enzyme to add to the toolbox of potential therapeutic antibacterial enzymes. 

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2. 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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3. Non-disruptive matrix turnover is a conserved feature of biofilm aggregate growth in paradigm pathogenic species

   https://doi.org/10.1128/mbio.03935-24  

   Reichhardt, Courtney; Matwichuk, Michael L; Lewerke, Lincoln T; Jacobs, Holly M; Yan, Jing; Parsek, Matthew R (February 2025, mBio)

   Whiteley, Marvin (Ed.)

   ABSTRACT Bacteria form multicellular aggregates called biofilms. A crucial component of these aggregates is a protective matrix that holds the community together. Biofilm matrix composition varies depending upon bacterial species but typically includes exopolysaccharides (EPS), proteins, and extracellular DNA.Pseudomonas aeruginosais a model organism for the study of biofilms, and in non-mucoid biofilms, it uses the structurally distinct EPS Psl and Pel, the EPS-binding protein CdrA, and eDNA as key matrix components. An interesting phenomenon that we and others have observed is that the periphery of a biofilm aggregate can be EPS-rich and contain very few cells. In this study, we investigated two possible models of assembly and dynamics of this EPS-rich peripheral region: (i) newly synthesized EPS is inserted and incorporated into the existing EPS-rich region at the periphery during biofilm aggregate growth or (ii) EPS is continuously turned over and newly synthesized EPS is deposited at the outermost edge of the aggregate. Our results support the latter model. Specifically, we observed that new EPS is continually deposited at the aggregate periphery, which is necessary for continued aggregate growth but not aggregate stability. We made similar observations in another paradigm biofilm-forming species,Vibrio cholerae. This pattern of deposition raises the question of how EPS is retained. Specifically, forP. aeruginosabiofilms, the matrix adhesin CdrA is thought to retain EPS. However, current thinking is that cell-associated CdrA is responsible for this retention, and it is not clear how CdrA might function in the relatively cell-free aggregate periphery. We observed that CdrA is enzymatically degraded during aggregate growth without negatively impacting biofilm stability and that cell-free CdrA can partially maintain aggregation and Psl retention. Overall, this study shows that the matrix ofP. aeruginosabiofilms undergoes both continuous synthesis of matrix material and matrix turnover to accommodate biofilm aggregate growth and that cell-free matrix can at least partially maintain biofilm aggregation and EPS localization. Furthermore, our similar observations forV. choleraebiofilms suggest that our findings may represent basic principles of aggregate assembly in bacteria. IMPORTANCEHere, we show that, to accommodate growing cellular biomass, newly produced Psl is deposited over existing Psl at the periphery of biofilm aggregates. We demonstrated thatV. choleraeemploys a similar mechanism with its biofilm matrix EPS, VPS. In addition, we found that the protease LasB is present in the biofilm matrix, resulting in degradation of CdrA to lower molecular weight cell-free forms. We then show that the released forms of CdrA are retained in the matrix and remain functional. Together, our findings support that theP. aeruginosabiofilm matrix is dynamic during the course of aggregate growth and that other species may employ similar mechanisms to remodel their matrix. 

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4. 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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5. 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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