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1. 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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2. 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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3. Omics-Inferred Partitioning and Expression of Diverse Biogeochemical Functions in a Low-O ₂ Cyanobacterial Mat Community

   https://doi.org/10.1128/mSystems.01042-21  

   Grim, Sharon L.; Voorhies, Alexander A.; Biddanda, Bopaiah A.; Jain, Sunit; Nold, Stephen C.; Green, Russ; Dick, Gregory J. (December 2021, mSystems)

   Bernstein, Hans C. (Ed.)

   ABSTRACT Cyanobacterial mats profoundly influenced Earth’s biological and geochemical evolution and still play important ecological roles in the modern world. However, the biogeochemical functioning of cyanobacterial mats under persistent low-O 2 conditions, which dominated their evolutionary history, is not well understood. To investigate how different metabolic and biogeochemical functions are partitioned among community members, we conducted metagenomics and metatranscriptomics on cyanobacterial mats in the low-O 2 , sulfidic Middle Island sinkhole (MIS) in Lake Huron. Metagenomic assembly and binning yielded 144 draft metagenome assembled genomes, including 61 of medium quality or better, and the dominant cyanobacteria and numerous Proteobacteria involved in sulfur cycling. Strains of a Phormidium autumnale -like cyanobacterium dominated the metagenome and metatranscriptome. Transcripts for the photosynthetic reaction core genes psaA and psbA were abundant in both day and night. Multiple types of psbA genes were expressed from each cyanobacterium, and the dominant psbA transcripts were from an atypical microaerobic type of D1 protein from Phormidium . Further, cyanobacterial transcripts for photosystem I genes were more abundant than those for photosystem II, and two types of Phormidium sulfide quinone reductase were recovered, consistent with anoxygenic photosynthesis via photosystem I in the presence of sulfide. Transcripts indicate active sulfur oxidation and reduction within the cyanobacterial mat, predominately by Gammaproteobacteria and Deltaproteobacteria , respectively. Overall, these genomic and transcriptomic results link specific microbial groups to metabolic processes that underpin primary production and biogeochemical cycling in a low-O 2 cyanobacterial mat and suggest mechanisms for tightly coupled cycling of oxygen and sulfur compounds in the mat ecosystem. IMPORTANCE Cyanobacterial mats are dense communities of microorganisms that contain photosynthetic cyanobacteria along with a host of other bacterial species that play important yet still poorly understood roles in this ecosystem. Although such cyanobacterial mats were critical agents of Earth’s biological and chemical evolution through geological time, little is known about how they function under the low-oxygen conditions that characterized most of their natural history. Here, we performed sequencing of the DNA and RNA of modern cyanobacterial mat communities under low-oxygen and sulfur-rich conditions from the Middle Island sinkhole in Lake Huron. The results reveal the organisms and metabolic pathways that are responsible for both oxygen-producing and non-oxygen-producing photosynthesis as well as interconversions of sulfur that likely shape how much O 2 is produced in such ecosystems. These findings indicate tight metabolic reactions between community members that help to explain the limited the amount of O 2 produced in cyanobacterial mat ecosystems. 

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4. Host-Associated Biofilms: Vibrio fischeri and Other Symbiotic Bacteria Within the Vibrionaceae

   https://doi.org/10.3390/microorganisms13061223  

   Lucero, Joaquin; Nishiguchi, Michele K (May 2025, Microorganisms)

   Biofilm formation is important for microbial survival, adaptation, and persistence within mutualistic and pathogenic systems in the Vibironaceae. Biofilms offer protection against environmental stressors, immune responses, and antimicrobial treatments by increasing host colonization and resilience. This review examines the mechanisms of biofilm formation in Vibrio species, focusing on quorum sensing, cyclic-di-GMP signaling, and host-specific adaptations that influence biofilm structure and function. We discuss how biofilms differ between mutualistic and pathogenic species based on environmental and host signals. Recent advances in omics technologies such as transcriptomics and metabolomics have enhanced research in biofilm regulation under different conditions. Horizontal gene transfer and phase variation promote the greater fitness of bacterial biofilms due to the diversity of environmental isolates that utilize biofilms to colonize host species. Despite progress, questions remain regarding the long-term effects of biofilm formation and persistence on host physiology and biofilm community dynamics. Research integrating multidisciplinary approaches will help advance our understanding of biofilms and their implications for influencing microbial adaptation, symbiosis, and disease. These findings have applications in biotechnology and medicine, where the genetic manipulation of biofilm regulation can enhance or disrupt microbiome stability and pathogen resistance, eventually leading to targeted therapeutic strategies. 

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5. A Heterogeneously Expressed Gene Family Modulates the Biofilm Architecture and Hypoxic Growth of Aspergillus fumigatus

   https://doi.org/10.1128/mBio.03579-20  

   Kowalski, Caitlin H.; Morelli, Kaesi A.; Stajich, Jason E.; Nadell, Carey D.; Cramer, Robert A. (February 2021, mBio)

   Doering, Tamara L. (Ed.)

   ABSTRACT The genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger . In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. However, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A ( bafA ), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus . Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatus bafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together, these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings. IMPORTANCE The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here, we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. fumigatus . Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in aspergilli. 

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