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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. Impacts of hydrodynamic conditions and microscale surface roughness on the critical shear stress to develop and thickness of early‐stage Pseudomonas putida biofilms

   https://doi.org/10.1002/bit.28409  

   Wei, Guanju; Yang, Judy Q (July 2023, Biotechnology and Bioengineering)

   Abstract Biofilms can increase pathogenic contamination of drinking water, cause biofilm‐related diseases, alter the sediment erosion rate, and degrade contaminants in wastewater. Compared with mature biofilms, biofilms in the early‐stage have been shown to be more susceptible to antimicrobials and easier to remove. Mechanistic understanding of physical factors controlling early‐stage biofilm growth is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and microscale surface roughness on the development of early‐stagePseudomonas putidabiofilm through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that early‐stage biofilm growth is suppressed under high flow conditions and that the local velocity for early‐stageP. putidabiofilms (growth time < 14 h) to develop is about 50 μm/s, which is similar toP. putida's swimming speed. We further illustrate that microscale surface roughness promotes the growth of early‐stage biofilms by increasing the area of the low‐flow region. Furthermore, we show that the critical average shear stress, above which early‐stage biofilms cease to form, is 0.9 Pa for rough surfaces, three times as large as the value for flat or smooth surfaces (0.3 Pa). The important control of flow conditions and microscale surface roughness on early‐stage biofilm development, characterized in this study, will facilitate future predictions and managements of early‐stageP. putidabiofilm development on the surfaces of drinking water pipelines, bioreactors, and sediments in aquatic environments. 

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3. Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses

   https://doi.org/10.1371/journal.ppat.1010742  

   Nicastro, Lauren K.; de Anda, Jaime; Jain, Neha; Grando, Kaitlyn C.; Miller, Amanda L.; Bessho, Shingo; Gallucci, Stefania; Wong, Gerard C.; Tükel, Çagla (August 2022, PLOS Pathogens)

   Baumler, Andreas J. (Ed.)

   Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro . The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity. 

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4. Antibiotic Susceptibility of Escherichia coli Cells during Early-Stage Biofilm Formation

   https://doi.org/10.1128/JB.00034-19  

   Gu, Huan; Lee, Sang Won; Carnicelli, Joseph; Jiang, Zhaowei; Ren, Dacheng; O'Toole, George (September 2019, Journal of Bacteriology)

   ABSTRACT Bacteria form complex multicellular structures on solid surfaces known as biofilms, which allow them to survive in harsh environments. A hallmark characteristic of mature biofilms is the high-level antibiotic tolerance (up to 1,000 times) compared with that of planktonic cells. Here, we report our new findings that biofilm cells are not always more tolerant to antibiotics than planktonic cells in the same culture. Specifically, Escherichia coli RP437 exhibited a dynamic change in antibiotic susceptibility during its early-stage biofilm formation. This phenomenon was not strain specific. Upon initial attachment, surface-associated cells became more sensitive to antibiotics than planktonic cells. By controlling the cell adhesion and cluster size using patterned E. coli biofilms, cells involved in the interaction between cell clusters during microcolony formation were found to be more susceptible to ampicillin than cells within clusters, suggesting a role of cell-cell interactions in biofilm-associated antibiotic tolerance. After this stage, biofilm cells became less susceptible to ampicillin and ofloxacin than planktonic cells. However, when the cells were detached by sonication, both antibiotics were more effective in killing the detached biofilm cells than the planktonic cells. Collectively, these results indicate that biofilm formation involves active cellular activities in adaption to the attached life form and interactions between cell clusters to build the complex structure of a biofilm, which can render these cells more susceptible to antibiotics. These findings shed new light on bacterial antibiotic susceptibility during biofilm formation and can guide the design of better antifouling surfaces, e.g., those with micron-scale topographic structures to interrupt cell-cell interactions. IMPORTANCE Mature biofilms are known for their high-level tolerance to antibiotics; however, antibiotic susceptibility of sessile cells during early-stage biofilm formation is not well understood. In this study, we aim to fill this knowledge gap by following bacterial antibiotic susceptibility during early-stage biofilm formation. We found that the attached cells have a dynamic change in antibiotic susceptibility, and during certain phases, they can be more sensitive to antibiotics than planktonic counterparts in the same culture. Using surface chemistry-controlled patterned biofilm formation, cell-surface and cell-cell interactions were found to affect the antibiotic susceptibility of attached cells. Collectively, these findings provide new insights into biofilm physiology and reveal how adaptation to the attached life form may influence antibiotic susceptibility of bacterial cells. 

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5. Cyclic di-GMP Is Integrated Into a Hierarchal Quorum Sensing Network Regulating Antimicrobial Production and Biofilm Formation in Roseobacter Clade Member Rhodobacterales Strain Y4I

   https://doi.org/10.3389/fmars.2021.681551  

   Armes, April C.; Buchan, Alison (July 2021, Frontiers in Marine Science)

   null (Ed.)

   Microbial biofilms associated with marine particulate organic matter carry out transformations that influence local and regional biogeochemical cycles. Early microbial colonizers are often hypothesized to “set the stage” for biofilm structure, dynamics, and function via N -acyl homoserine lactone (AHL)-mediated quorum sensing (QS). Production of AHLs, as well as antimicrobials, contributes to the colonization success of members of the Roseobacter clade. One member of this group of abundant marine bacteria, Rhodobacterales sp. Y4I, possesses two QS systems, phaRI (QS1) and pgaRI (QS2). Here, we characterize mutants in both QS systems to provide genetic evidence that the two systems work in hierarchical fashion to coordinate production of the antimicrobial indigoidine as well as biofilm formation. A mutation in pgaR (QS2) results in decreased expression of genes encoding both QS systems as well as those governing the biosynthesis of indigoidine. In contrast, mutations in QS1 did not significantly influence gene expression of QS2. Addition of exogenous AHLs to QS1 and QS2 mutants led to partial restoration of indigoidine production (45–60% of WT) for QS1 but not QS2. Mutational disruptions of QS1 had a more pronounced effect on biofilm development than those in QS2. Finally, we demonstrate that c-di-GMP levels are altered in QS and indigoidine biosynthesis Y4I mutants. Together, these results indicate that pgaRI (QS2) is at the top of a regulatory hierarchy governing indigoidine biosynthesis and that the global regulatory metabolite, c-di-GMP, is likely integrated into the QS circuitry of this strain. These findings provide mechanistic understanding of physiological processes that are important in elucidating factors driving competitiveness of Roseobacters in nature. 

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