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The international workshop "Interdisciplinary life of microbes: from single cells to multicellular aggregates," following a virtual preassembly in November 2021, was held in person in Dresden, from 9 to 13 November 2022. It attracted not only prominent experts in biofilm research but also researchers from broadly neighboring disciplines, such as medicine, chemistry, and theoretical and experimental biophysics, both eukaryotic and prokaryotic. Focused brainstorming sessions were the special feature of the event and are at the heart of this commentary., Competing Interests: The authors declare no conflict of interest.

Bacillus subtilis is a soil-dwelling bacterium that can form biofilms, or communities of cells surrounded by a self-produced extracellular matrix. In biofilms, genetically identical cells often exhibit heterogeneous transcriptional phenotypes, so that subpopulations of cells carry out essential yet costly cellular processes that allow the entire population to thrive. Surprisingly, the extent of phenotypic heterogeneity and the relationships between subpopulations of cells within biofilms of even in well-studied bacterial systems like B. subtilis remains largely unknown. To determine relationships between these subpopulations of cells, we created 182 strains containing pairwise combinations of fluorescent transcriptional reporters for the expression state of 14 different genes associated with potential cellular subpopulations. We determined the spatial organization of the expression of these genes within biofilms using confocal microscopy, which revealed that many reporters localized to distinct areas of the biofilm, some of which were co-localized. We used flow cytometry to quantify reporter co-expression, which revealed that many cells "multi-task," simultaneously expressing two reporters. These data indicate that prior models describing B. subtilis cells as differentiating into specific cell types, each with a specific task or function, were oversimplified. Only a few subpopulations of cells, including surfactin and plipastatin producers, as well as sporulating and competent cells, appear to have distinct roles based on the set of genes examined here. These data will provide us with a framework with which to further study and make predictions about the roles of diverse cellular phenotypes in B. subtilis biofilms. IMPORTANCE Many microbes differentiate, expressing diverse phenotypes to ensure their survival in various environments. However, studies on phenotypic differentiation have typically examined only a few phenotypes at one time, thus limiting our knowledge about the extent of differentiation and phenotypic overlap in the population. We investigated the spatial organization and gene expression relationships for genes important in B. subtilis biofilms. In doing so, we mapped spatial gene expression patterns and expanded the number of cell populations described in the B. subtilis literature. It is likely that other bacteria also display complex differentiation patterns within their biofilms. Studying the extent of cellular differentiation in other microbes may be important when designing therapies for disease-causing bacteria, where studying only a single phenotype may be masking underlying phenotypic differentiation relevant to infection outcomes., Competing Interests: The authors declare no conflict of interest.

Many microbes coexist within biofilms, or multispecies communities of cells encased in an extracellular matrix. However, little is known about the microbe-microbe interactions relevant for creating these structures. In this study, we explored a striking dual-species biofilm between Bacillus subtilis and Pantoea agglomerans that exhibited characteristics that were not predictable from previous work examining monoculture biofilms. Coculture wrinkle formation required a P. agglomerans exopolysaccharide as well as the B. subtilis amyloid-like protein TasA. Unexpectedly, other B. subtilis matrix components essential for monoculture biofilm formation were not necessary for coculture wrinkling (e.g., the exopolysaccharide EPS, the hydrophobin BslA, and cell chaining). In addition, B. subtilis cell chaining prevented coculture wrinkling, even though chaining was previously associated with more robust monoculture biofilms. We also observed that increasing the relative proportion of P. agglomerans (which forms completely featureless monoculture colonies) increased coculture wrinkling. Using microscopy and rheology, we observed that these two bacteria assemble into an organized layered structure that reflects the physical properties of both monocultures. This partitioning into distinct regions negatively affected the survival of P. agglomerans while also serving as a protective mechanism in the presence of antibiotic stress. Taken together, these data indicate that studying cocultures is a productive avenue to identify novel mechanisms that drive the formation of structured microbial communities. IMPORTANCE In the environment, many microbes form biofilms. However, the interspecies interactions underlying bacterial coexistence within these biofilms remain understudied. Here, we mimic environmentally relevant biofilms by studying a dual-species biofilm formed between Bacillus subtilis and Pantoea agglomerans and subjecting the coculture to chemical and physical stressors that it may experience in the natural world. We determined that both bacteria contribute structural elements to the coculture, which is reflected in its overall viscoelastic behavior. Existence within the coculture can be either beneficial or detrimental depending on the context. Many of the features and determinants of the coculture biofilm appear distinct from those identified in monoculture biofilm studies, highlighting the importance of characterizing multispecies consortia to understand naturally occurring bacterial interactions., (Copyright © 2019 American Society for Microbiology.)

There is a growing appreciation for the impact that bacteria have on higher organisms. Plant roots often harbor beneficial microbes, such as the Gram-positive rhizobacterium Bacillus subtilis , that influence their growth and susceptibility to disease. The ability to form surface-attached microbial communities called biofilms is crucial for the ability of B. subtilis to adhere to and protect plant roots. In this study, strains harboring deletions of the B. subtilis genes known to synthesize and degrade the second messenger cyclic di-adenylate monophosphate (c-di-AMP) were examined for their involvement in biofilm formation and plant attachment. We found that intracellular production of c-di-AMP impacts colony biofilm architecture, biofilm gene expression, and plant attachment in B. subtilis We also show that B. subtilis secretes c-di-AMP and that putative c-di-AMP transporters impact biofilm formation and plant root colonization. Taken together, our data describe a new role for c-di-AMP as a chemical signal that affects important cellular processes in the environmentally and agriculturally important soil bacterium B. subtilis These results suggest that the "intracellular" signaling molecule c-di-AMP may also play a previously unappreciated role in interbacterial cell-cell communication within plant microbiomes. IMPORTANCE Plants harbor bacterial communities on their roots that can significantly impact their growth and pathogen resistance. In most cases, however, the signals that mediate host-microbe and microbe-microbe interactions within these communities are unknown. A detailed understanding of these interaction mechanisms could facilitate the manipulation of these communities for agricultural or environmental purposes. Bacillus subtilis is a plant-growth-promoting bacterium that adheres to roots by forming biofilms. We therefore began by exploring signals that might impact its biofilm formation. We found that B. subtilis secretes c-di-AMP and that the ability to produce, degrade, or transport cyclic di-adenylate monophosphate (c-di-AMP; a common bacterial second messenger) affects B. subtilis biofilm gene expression and plant attachment. To our knowledge, this is the first demonstration of c-di-AMP impacting a mutualist host-microbe association and suggests that c-di-AMP may function as a previously unappreciated extracellular signal able to mediate interactions within plant microbiomes., (Copyright © 2018 Townsley et al.)

Cell-cell communication enables bacteria to coordinate their behavior through the production, recognition, and response to chemical signals produced by their microbial neighbors. An important example of coordinated behavior in bacteria is biofilm formation, where individual cells organize into highly complex, matrix-encased communities that differentiate into distinct cell types and divide labor among individual cells. Bacteria rely on environmental cues to influence biofilm development, including chemical cues produced by other microbes. A multitude of recent studies have demonstrated that natural-product antibiotics at subinhibitory concentrations can impact biofilm formation in neighboring microbes, supporting the hypothesis that these compounds may have evolved as signaling molecules that mediate cell-cell interactions. In this review we discuss the role of antibiotics in modulating biofilm formation and interspecies communication in bacteria., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bacteria have evolved the ability to produce a wide range of structurally complex natural products historically called "secondary" metabolites. Although some of these compounds have been identified as bacterial communication cues, more frequently natural products are scrutinized for antibiotic activities that are relevant to human health. However, there has been little regard for how these compounds might otherwise impact the physiology of neighboring microbes present in complex communities. Bacillus cereus secretes molecules that activate expression of biofilm genes in Bacillus subtilis. Here, we use imaging mass spectrometry to identify the thiocillins, a group of thiazolyl peptide antibiotics, as biofilm matrix-inducing compounds produced by B. cereus. We found that thiocillin increased the population of matrix-producing B. subtilis cells and that this activity could be abolished by multiple structural alterations. Importantly, a mutation that eliminated thiocillin's antibiotic activity did not affect its ability to induce biofilm gene expression in B. subtilis. We go on to show that biofilm induction appears to be a general phenomenon of multiple structurally diverse thiazolyl peptides and use this activity to confirm the presence of thiazolyl peptide gene clusters in other bacterial species. Our results indicate that the roles of secondary metabolites initially identified as antibiotics may have more complex effects--acting not only as killing agents, but also as specific modulators of microbial cellular phenotypes.

Many different systems of bacterial interactions have been described. However, relatively few studies have explored how interactions between different microorganisms might influence bacterial development. To explore such interspecies interactions, we focused on Bacillus subtilis, which characteristically develops into matrix-producing cannibals before entering sporulation. We investigated whether organisms from the natural environment of B. subtilis--the soil--were able to alter the development of B. subtilis. To test this possibility, we developed a coculture microcolony screen in which we used fluorescent reporters to identify soil bacteria able to induce matrix production in B. subtilis. Most of the bacteria that influence matrix production in B. subtilis are members of the genus Bacillus, suggesting that such interactions may be predominantly with close relatives. The interactions we observed were mediated via two different mechanisms. One resulted in increased expression of matrix genes via the activation of a sensor histidine kinase, KinD. The second was kinase independent and conceivably functions by altering the relative subpopulations of B. subtilis cell types by preferentially killing noncannibals. These two mechanisms were grouped according to the inducing strain's relatedness to B. subtilis. Our results suggest that bacteria preferentially alter their development in response to secreted molecules from closely related bacteria and do so using mechanisms that depend on the phylogenetic relatedness of the interacting bacteria.

Interspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacterium Bacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identified Pseudomonas putida and Pseudomonas protegens as bacteria that secrete compounds that inhibit biofilm gene expression in B. subtilis. The active compound produced by P. protegens was identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies of B. subtilis grown adjacent to a DAPG-producing P. protegens strain had altered colony morphologies relative to B. subtilis colonies grown next to a DAPG-null P. protegens strain (phlD strain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced in B. subtilis liquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria. [ABSTRACT FROM AUTHOR]