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1. Quorum Sensing and Antimicrobial Production Orchestrate Biofilm Dynamics in Multispecies Bacterial Communities

   https://doi.org/10.1128/spectrum.02615-22  

   Armes, April C.; Walton, Jillian L.; Buchan, Alison (October 2022, Microbiology Spectrum)

   DeAngelis, Kristen M. (Ed.)

   ABSTRACT Microbial interactions are often mediated by diffusible small molecules, including secondary metabolites, that play roles in cell-to-cell signaling and inhibition of competitors. Biofilms are often “hot spots” for high concentrations of bacteria and their secondary metabolites, which make them ideal systems for the study of small-molecule contributions to microbial interactions. Here, we use a five-member synthetic community consisting of Roseobacteraceae representatives to investigate the role of secondary metabolites on microbial biofilm dynamics. One synthetic community member, Rhodobacterales strain Y4I, possesses two acylated homoserine lactone (AHL)-based cell-to-cell signaling systems ( pgaRI and phaRI ) as well as a nonribosomal peptide synthase gene ( igi ) cluster that encodes the antimicrobial indigoidine. Through serial substitution of Y4I with mutants deficient in single signaling molecule pathways, the contribution of these small-molecule systems could be assessed. As secondary metabolite production is dependent upon central metabolites, the influence of growth substrate (i.e., complex medium versus defined medium with a single carbon substrate) on these dynamics was also considered. Depending on the Y4I mutant genotype included, community dynamics ranged from competitive to cooperative. The observed interactions were mostly competitive in nature. However, the community harboring a Y4I variant that was both impaired in quorum sensing (QS) pathways and unable to produce indigoidine ( pgaR variant) shifted toward more cooperative interactions over time. These cooperative interactions were enhanced in the defined growth medium. The results presented provide a framework for deciphering complex, small-molecule-mediated interactions that have broad application to microbial biology. IMPORTANCE Microbial biofilms play critical roles in marine ecosystems and are hot spots for microbial interactions that play a role in the development and function of these communities. Roseobacteraceae are an abundant and active family of marine heterotrophic bacteria forming close associations with phytoplankton and carrying out key transformations in biogeochemical cycles. Group members are aggressive primary colonizers of surfaces, where they set the stage for the development of multispecies biofilm communities. Few studies have examined the impact of secondary metabolites, such as cell-to-cell signaling and antimicrobial production, on marine microbial biofilm community structure. Here, we assessed the impact of secondary metabolites on microbial interactions using a synthetic, five-member Roseobacteraceae community by measuring species composition and biomass production during biofilm growth. We present evidence that secondary metabolites influence social behaviors within these multispecies microbial biofilms, thereby improving understanding of bacterial secondary metabolite production influence on social behaviors within marine microbial biofilm communities. 

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2. Active pH regulation facilitates Bacillus subtilis biofilm development in a minimally buffered environment

   https://doi.org/10.1128/mbio.03387-23  

   Tran, Peter; Lander, Stephen M; Prindle, Arthur (March 2024, mBio)

   Kline, Kimberly A (Ed.)

   ABSTRACT Biofilms provide individual bacteria with many advantages, yet dense cellular proliferation can also create intrinsic metabolic challenges including excessive acidification. Because such pH stress can be masked in buffered laboratory media—such as MSgg commonly used to studyBacillus subtilisbiofilms—it is not always clear how such biofilms cope with minimally buffered natural environments. Here, we report howB. subtilisbiofilms overcome this intrinsic metabolic challenge through an active pH regulation mechanism. Specifically, we find that these biofilms can modulate their extracellular pH to the preferred neutrophile range, even when starting from acidic and alkaline initial conditions, while planktonic cells cannot. We associate this behavior with dynamic interplay between acetate and acetoin biosynthesis and show that this mechanism is required to buffer against biofilm acidification. Furthermore, we find that buffering-deficient biofilms exhibit dysregulated biofilm development when grown in minimally buffered conditions. Our findings reveal an active pH regulation mechanism inB. subtilisbiofilms that could lead to new targets to control unwanted biofilm growth.IMPORTANCEpH is known to influence microbial growth and community dynamics in multiple bacterial species and environmental contexts. Furthermore, in many bacterial species, rapid cellular proliferation demands the use of overflow metabolism, which can often result in excessive acidification. However, in the case of bacterial communities known as biofilms, these acidification challenges can be masked when buffered laboratory media are employed to stabilize the pH environment for optimal growth. Our study reveals thatB. subtilisbiofilms use an active pH regulation mechanism to mitigate both growth-associated acidification and external pH challenges. This discovery provides new opportunities for understanding microbial communities and could lead to new methods for controlling biofilm growth outside of buffered laboratory conditions. 

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3. Biofabrication and characterization of multispecies electroactive biofilms in stratified paper-based scaffolds

   https://doi.org/10.1039/d2an01059c  

   Elhadad, Anwar; Choi, Seokheun (September 2022, The Analyst)

   Bioelectrochemical technologies have attracted significant scientific interest because the effective bacterial electron exchange with external electrodes can provide a sustainable solution that joins environmental remediation and energy recovery. Multispecies electroactive bacterial biofilms are catalysts that will drive the operation of bioelectrochemical devices. Unfortunately, there is a lack of understanding of key mechanisms determining their electron-generating capabilities and syntrophic relations within microbial communities in biofilms. This is because there are no universally standardized models for simple, rapid, reliable, and cost-effective fabrication and characterization of electroactive multispecies biofilms. The heterogeneous and long-term nature of biofilm formation has hampered the development of those models. This work develops novel biofabrication and analysis platforms by creating innovative, paper-based 3-D systems that accurately recapitulate the structure, function, and physiology of living multispecies biofilms. Multiple layers of paper containing bacterial cells were stacked to simulate different layered 3-D biofilm models with defined cellular compositions and microenvironments. Overall bacterial electrogenic capabilities through the biofilm structures were characterized by thoroughly monitoring collective electron flows through different external resistors. Changes in the type of species and order of stacking created biofilm modeling which allowed for the study of their electrogenic performance via variation in electron flow rate output. Furthermore, multi-laminate structures allowed for straightforward de-stacking and layer-by-layer separation for analyses of pH distribution and cellular viability. Our multi-laminate structures provide a new strategy for (i) controlling the biofilm geometry of 3-D bacterial cultures, (ii) monitoring the microbial electoral properties, and (iii) constructing an artificial biofilm layer by layer. 

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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. Mechanical instability and interfacial energy drive biofilm morphogenesis

   https://doi.org/10.7554/eLife.43920  

   Yan, Jing; Fei, Chenyi; Mao, Sheng; Moreau, Alexis; Wingreen, Ned S; Košmrlj, Andrej; Stone, Howard A; Bassler, Bonnie L (March 2019, eLife)

   Engineers have long studied how mechanical instabilities cause patterns to form in inanimate materials, and recently more attention has been given to how such forces affect biological systems. For example, stresses can build up within a tissue if one layer grows faster than an adjacent layer. The tissue can release this stress by wrinkling, folding or creasing. Though ancient and single-celled, bacteria can also develop spectacular patterns when they exist in the lifestyle known as a biofilm: a community of cells adhered to a surface. But do mechanical instabilities drive the patterns seen in biofilms? To investigate, Yan, Fei, Mao et al. grew biofilms of the bacterium called Vibrio cholerae – which causes the disease cholera – on solid, non-growing ‘substrates’. This work revealed that as the biofilms grow, their expansion is constrained by the substrate, and this situation generates mechanical stresses. To release the stresses, the biofilm initially folds to form wrinkles. Later, as the biofilm expands further, small parts of it detach from the substrate to form blisters. The same forces that keep water droplets spherical (known as interfacial forces) dictate how the blisters evolve, interact, and eventually shape the expanding biofilm. Using these principles, Yan et al. could engineer the biofilm into desired shapes. Collectively, the results presented by Yan et al. connect the shape of the biofilm surface with its material properties, in particular its stiffness. Understanding this relationship could help researchers to develop new ways to remove harmful biofilms, such as those that cause disease or that damage underwater structures. The stiffness of biofilms is already known to affect how well bacteria can resist antibiotics. Future studies could look for new genes or compounds that change the material properties of a biofilm, thereby altering the biofilm surface. 

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