Where bacteria and eukaryotes meet.

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.

Thiocillin contributes to the ecological fitness of Bacillus cereus ATCC 14579 during interspecies interactions with Myxococcus xanthus.

The soil-dwelling delta-proteobacterium Myxococcus xanthus is a model organism to study predation and competition. M. xanthus preys on a broad range of bacteria mediated by lytic enzymes, exopolysaccharides, Type-IV pilus-based motility, and specialized metabolites. Competition between M. xanthus and prey bacterial strains with various specialized metabolite profiles indicates a range of fitness, suggesting that specialized metabolites contribute to prey survival. To expand our understanding of how specialized metabolites affect predator-prey dynamics, we assessed interspecies interactions between M. xanthus and two strains of Bacillus cereus. While strain ATCC 14579 resisted predation, strain T was found to be highly sensitive to M. xanthus predation. The interaction between B. cereus ATCC 14579 and M. xanthus appears to be competitive, resulting in population loss for both predator and prey. Genome analysis revealed that ATCC 14579 belongs to a clade that possesses the biosynthetic gene cluster for production of thiocillins, whereas B. cereus strain T lacks those genes. Further, purified thiocillin protects B. cereus strains unable to produce this specialized metabolite, strengthening the finding that thiocillin protects against predation and contributes to the ecological fitness of B. cereus ATCC 14579. Lastly, strains that produce thiocillin appear to confer some level of protection to their own antibiotic by encoding an additional copy of the L11 ribosomal protein, a known target for thiopeptides. This work highlights the importance of specialized metabolites affecting predator-prey dynamics in soil microenvironments.

Extensive cellular multi-tasking within Bacillus subtilis biofilms.

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.

Microclimate is a strong predictor of the native and invasive plant-associated soil microbiome on San Cristóbal Island, Galápagos archipelago.

Understanding the drivers that affect soil bacterial and fungal communities is essential to understanding and mitigating the impacts of human activity on vulnerable ecosystems like those on the Galápagos Islands. The volcanic slopes of these Islands lead to steep elevation gradients that generate distinct microclimates across small spatial scales. Although much is known about the impacts of invasive plant species on the above-ground biodiversity of the Galápagos Islands, little is known about their resident soil microbial communities and the factors shaping them. Here, we investigate the bacterial and fungal soil communities associated with invasive and native plant species across three distinct microclimates on San Cristóbal Island (arid, transition zone and humid). At each site, we collected soil at three depths (rhizosphere, 5 cm and 15 cm) from multiple plants. Sampling location was the strongest driver of both bacterial and fungal communities, explaining 73% and 43% of variation in the bacterial and fungal community structure, respectively, with additional minor but significant impacts from soil depth and plant type (invasive vs. native). This study highlights the continued need to explore microbial communities across diverse environments and demonstrates how both abiotic and biotic factors impact soil microbial communities in the Galápagos archipelago.

Imaging and Direct Sampling Capabilities of Nanospray Desorption Electrospray Ionization with Absorption-Mode 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Nanospray desorption electrospray ionization mass spectrometry, a powerful ambient sampling and imaging technique, is herein coupled as an isolated source with 21 Tesla (21T) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Absorption-mode data, enabled by an external data acquisition system, is applied for improved mass resolution, accuracy, and dynamic range without compromising spectral acquisition rates. Isotopic fine structure (IFS) information is obtained from the ambient sampling of living Bacillus and Fusarium species, allowing for high confidence in molecular annotations with a resolution >830 k (at m/z 825). Tandem mass spectrometry experiments for biological samples are shown to retain the IFS in addition to gained fragmentation information, providing a further degree of annotation confidence from ambient analyses. Rat brain was imaged by nanospray desorption electrospray ionization (nano-DESI) 21T FTICR MS in ∼5 h using 768 ms transients, producing over 800 molecular annotations using the METASPACE platform and low-parts-per-billion mass accuracy at a spatial resolution of ∼25 × 180 µm. Finally, nano-DESI 21T FTICR MS imaging is demonstrated to reveal images corresponding to the IFS, as well as hundreds of additional molecular features (including demonstrated differences as low as 8.96 mDa) that are otherwise undetected by a more conventional imaging methodology.

Direct Visualization of Chemical Cues and Cellular Phenotypes throughout Bacillus subtilis Biofilms.

Bacillus subtilis is a soil bacterium that can form biofilms, which are communities of cells encased by an extracellular matrix. In these complex communities, cells perform numerous metabolic processes and undergo differentiation into functionally distinct phenotypes as a survival strategy. Because biofilms are often studied in bulk, it remains unclear how metabolite production spatially correlates with B. subtilis phenotypes within biofilm structures. In many cases, we still do not know where these biological processes are occurring in the biofilm. Here, we developed a method to analyze the localization of molecules within sagittal thin sections of B. subtilis biofilms using high-resolution mass spectrometry imaging. We correlated the organization of specific molecules to the localization of well-studied B. subtilis phenotypic reporters determined by confocal laser scanning fluorescence microscopy within analogous biofilm thin sections. The correlations between these two data sets suggest the role of surfactin as a signal for extracellular matrix gene expression in the biofilm periphery and the role of bacillibactin as an iron-scavenging molecule. Taken together, this method will help us generate hypotheses to discover relationships between metabolites and phenotypic cell states in B. subtilis and other biofilm-forming bacteria. IMPORTANCE Bacterial biofilms are complex and heterogeneous structures. Cells within biofilms carry out numerous metabolic processes in a nuanced and organized manner, details of which are still being discovered. Here, we used multimodal imaging to analyze B. subtilis biofilm processes at the metabolic and gene expression levels in biofilm sagittal thin sections. Often, imaging techniques analyze only the top of the surface of the biofilm and miss the multifaceted interactions that occur deep within the biofilm. Our analysis of the sagittal planes of B. subtilis biofilms revealed the distributions of metabolic processes throughout the depths of these structures and allowed us to draw correlations between metabolites and phenotypically important subpopulations of B. subtilis cells. This technique provides a platform to generate hypotheses about the role of specific molecules and their relationships to B. subtilis subpopulations of cells.

Selective Bacterial Community Enrichment between the Pitcher Plants Sarracenia minor and Sarracenia flava.

The interconnected and overlapping habitats present in natural ecosystems remain a challenge in determining the forces driving microbial community composition. The cuplike leaf structures of some carnivorous plants, including those of the family Sarraceniaceae, are self-contained ecological habitats that represent systems for exploring such microbial ecology questions. We investigated whether Sarracenia minor and Sarracenia flava cultivate distinct bacterial communities when sampled at the same geographic location and time. This sampling strategy eliminates many abiotic environmental variables present in other studies that compare samples harvested over time, and it could reveal biotic factors driving the selection of microbes. DNA extracted from the decomposing detritus trapped in each Sarracenia leaf pitcher was profiled using 16S rRNA amplicon sequencing. We identified a surprising amount of bacterial diversity within each pitcher, but we also discovered bacteria whose abundance was specifically enriched in one of the two Sarracenia species. These differences in bacterial community representation suggest some biotic influence of the Sarracenia plant on the bacterial composition of their pitchers. Overall, our results suggest that bacterial selection due to factors other than geographic location, weather, or prey availability is occurring within the pitchers of these two closely related plant species. This indicates that specific characteristics of S. minor and S. flava may play a role in fostering distinct bacterial communities. These confined, naturally occurring microbial ecosystems within Sarracenia pitchers may provide model systems to answer important questions about the drivers of microbial community composition, succession, and response to environmental perturbations. IMPORTANCE This study uses amplicon sequencing to compare the bacterial communities of environmental samples from the detritus of the leaf cavities of Sarracenia minor and Sarracenia flava pitcher plants. We sampled the detritus at the same time and in the same geographic location, eliminating many environmental variables present in other comparative studies. This study revealed that different species of Sarracenia contain distinct bacterial members within their pitchers, suggesting that these communities are not randomly established based on environmental factors and the prey pool but are potentially enriched for by the plants' chemical or physical environment. This study of these naturally occurring, confined microbial ecosystems will help further establish carnivorous pitcher plants as a model system for answering important questions about the development and succession of microbial communities.

Rhizobacteria Impact Colonization of Listeria monocytogenes on Arabidopsis thaliana Roots.

In spite of its relevance as a foodborne pathogen, we have limited knowledge about Listeria monocytogenes in the environment. L. monocytogenes outbreaks have been linked to fruits and vegetables; thus, a better understanding of the factors influencing its ability to colonize plants is important. We tested how environmental factors and other soil- and plant-associated bacteria influenced L. monocytogenes' ability to colonize plant roots using Arabidopsis thaliana seedlings in a hydroponic growth system. We determined that the successful root colonization of L. monocytogenes 10403S was modestly but significantly enhanced by the bacterium being pregrown at higher temperatures, and this effect was independent of the biofilm and virulence regulator PrfA. We tested 14 rhizosphere-derived bacteria for their impact on L. monocytogenes 10403S, identifying one that enhanced and 10 that inhibited the association of 10403S with plant roots. We also characterized the outcomes of these interactions under both coinoculation and invasion conditions. We characterized the physical requirements of five of these rhizobacteria to impact the association of L. monocytogenes 10403S with roots, visualizing one of these interactions by microscopy. Furthermore, we determined that two rhizobacteria (one an inhibitor, the other an enhancer of 10403S root association) were able to similarly impact 10 different L. monocytogenes strains, indicating that the effects of these rhizobacteria on L. monocytogenes are not strain specific. Taken together, our results advance our understanding of the parameters that affect L. monocytogenes plant root colonization, knowledge that may enable us to deter its association with and, thus, downstream contamination of, food crops. IMPORTANCE Listeria monocytogenes is ubiquitous in the environment, being found in or on soil, water, plants, and wildlife. However, little is known about the requirements for L. monocytogenes' existence in these settings. Recent L. monocytogenes outbreaks have been associated with contaminated produce; thus, we used a plant colonization model to investigate factors that alter L. monocytogenes' ability to colonize plant roots. We show that L. monocytogenes colonization of roots was enhanced when grown at higher temperatures prior to inoculation but did not require a known regulator of virulence and biofilm formation. Additionally, we identified several rhizobacteria that altered the ability of 11 different strains of L. monocytogenes to colonize plant roots. Understanding the factors that impact L. monocytogenes physiology and growth will be crucial for finding mechanisms (whether chemical or microbial) that enable its removal from plant surfaces to reduce L. monocytogenes contamination of produce and eliminate foodborne illness.

Defining the Expression, Production, and Signaling Roles of Specialized Metabolites during Bacillus subtilis Differentiation.

Bacterial specialized (or secondary) metabolites are structurally diverse molecules that mediate intra- and interspecies interactions by altering growth and cellular physiology and differentiation. Bacillus subtilis, a Gram-positive model bacterium commonly used to study biofilm formation and sporulation, has the capacity to produce more than 10 specialized metabolites. Some of these B. subtilis specialized metabolites have been investigated for their role in facilitating cellular differentiation, but only rarely has the behavior of multiple metabolites been simultaneously investigated. In this study, we explored the interconnectivity of differentiation (biofilm and sporulation) and specialized metabolites in B. subtilis. Specifically, we interrogated how development influences specialized metabolites and vice versa. Using the sporulation-inducing medium DSM, we found that the majority of the specialized metabolites examined are expressed and produced during biofilm formation and sporulation. Additionally, we found that six of these metabolites (surfactin, ComX, bacillibactin, bacilysin, subtilosin A, and plipastatin) are necessary signaling molecules for proper progression of B. subtilis differentiation. This study further supports the growing body of work demonstrating that specialized metabolites have essential physiological functions as cell-cell communication signals in bacteria. IMPORTANCE Bacterially produced specialized metabolites are frequently studied for their potential use as antibiotics and antifungals. However, a growing body of work has suggested that the antagonistic potential of specialized metabolites is not their only function. Here, using Bacillus subtilis as our model bacterium, we demonstrated that developmental processes such as biofilm formation and sporulation are tightly linked to specialized metabolite gene expression and production. Additionally, under our differentiation-inducing conditions, six out of the nine specialized metabolites investigated behave as intraspecific signals that impact B. subtilis physiology and influence biofilm formation and sporulation. Our work supports the viewpoint that specialized metabolites have a clear role as cell-cell signaling molecules within differentiated populations of bacteria.

Expanding Molecular Coverage in Mass Spectrometry Imaging of Microbial Systems Using Metal-Assisted Laser Desorption/Ionization.

Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (∼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.

Transparent soil microcosms for live-cell imaging and non-destructive stable isotope probing of soil microorganisms.

Microscale processes are critically important to soil ecology and biogeochemistry yet are difficult to study due to soil's opacity and complexity. To advance the study of soil processes, we constructed transparent soil microcosms that enable the visualization of microbes via fluorescence microscopy and the non-destructive measurement of microbial activity and carbon uptake in situ via Raman microspectroscopy. We assessed the polymer Nafion and the crystal cryolite as optically transparent soil substrates. We demonstrated that both substrates enable the growth, maintenance, and visualization of microbial cells in three dimensions over time, and are compatible with stable isotope probing using Raman. We applied this system to ascertain that after a dry-down/rewetting cycle, bacteria on and near dead fungal hyphae were more metabolically active than those far from hyphae. These data underscore the impact fungi have facilitating bacterial survival in fluctuating conditions and how these microcosms can yield insights into microscale microbial activities.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system's reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

A Dual-Species Biofilm with Emergent Mechanical and Protective Properties.

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.

Inter-Modular Linkers play a crucial role in governing the biosynthesis of non-ribosomal peptides.

MOTIVATION: Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic machines that catalyze the ribosome-independent production of structurally complex small peptides, many of which have important clinical applications as antibiotics, antifungals and anti-cancer agents. Several groups have tried to expand natural product diversity by intermixing different NRPS modules to create synthetic peptides. This approach has not been as successful as anticipated, suggesting that these modules are not fully interchangeable. RESULTS: We explored whether Inter-Modular Linkers (IMLs) impact the ability of NRPS modules to communicate during the synthesis of NRPs. We developed a parser to extract 39 804 IMLs from both well annotated and putative NRPS biosynthetic gene clusters from 39 232 bacterial genomes and established the first IMLs database. We analyzed these IMLs and identified a striking relationship between IMLs and the amino acid substrates of their adjacent modules. More than 92% of the identified IMLs connect modules that activate a particular pair of substrates, suggesting that significant specificity is embedded within these sequences. We therefore propose that incorporating the correct IML is critical when attempting combinatorial biosynthesis of novel NRPS. AVAILABILITY AND IMPLEMENTATION: The IMLs database as well as the NRPS-Parser have been made available on the web at https://nrps-linker.unc.edu. The entire source code of the project is hosted in GitHub repository (https://github.com/SWFarag/nrps-linker). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Considering the Lives of Microbes in Microbial Communities.

Over the last decades, sequencing technologies have transformed our ability to investigate the composition and functional capacity of microbial communities. Even so, critical questions remain about these complex systems that cannot be addressed by the bulk, community-averaged data typically provided by sequencing methods. In this Perspective, I propose that future advances in microbiome research will emerge from considering "the lives of microbes": we need to create methods to explicitly interrogate how microbes exist and interact in native-setting-like microenvironments. This approach includes developing approaches that expose the phenotypic heterogeneity of microbes; exploring the effects of coculture cues on cellular differentiation and metabolite production; and designing visualization systems that capture features of native microbial environments while permitting the nondestructive observation of microbial interactions over space and time with single-cell resolution.

Cyclic di-AMP Acts as an Extracellular Signal That Impacts Bacillus subtilis Biofilm Formation and Plant Attachment.

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.

Design of synthetic bacterial communities for predictable plant phenotypes.

Specific members of complex microbiota can influence host phenotypes, depending on both the abiotic environment and the presence of other microorganisms. Therefore, it is challenging to define bacterial combinations that have predictable host phenotypic outputs. We demonstrate that plant-bacterium binary-association assays inform the design of small synthetic communities with predictable phenotypes in the host. Specifically, we constructed synthetic communities that modified phosphate accumulation in the shoot and induced phosphate starvation-responsive genes in a predictable fashion. We found that bacterial colonization of the plant is not a predictor of the plant phenotypes we analyzed. Finally, we demonstrated that characterizing a subset of all possible bacterial synthetic communities is sufficient to predict the outcome of untested bacterial consortia. Our results demonstrate that it is possible to infer causal relationships between microbiota membership and host phenotypes and to use these inferences to rationally design novel communities.

Large-Scale Bioinformatics Analysis of Bacillus Genomes Uncovers Conserved Roles of Natural Products in Bacterial Physiology.

Bacteria possess an amazing capacity to synthesize a diverse range of structurally complex, bioactive natural products known as specialized (or secondary) metabolites. Many of these specialized metabolites are used as clinical therapeutics, while others have important ecological roles in microbial communities. The biosynthetic gene clusters (BGCs) that generate these metabolites can be identified in bacterial genome sequences using their highly conserved genetic features. We analyzed an unprecedented 1,566 bacterial genomes from Bacillus species and identified nearly 20,000 BGCs. By comparing these BGCs to one another as well as a curated set of known specialized metabolite BGCs, we discovered that the majority of Bacillus natural products are comprised of a small set of highly conserved, well-distributed, known natural product compounds. Most of these metabolites have important roles influencing the physiology and development of Bacillus species. We identified, in addition to these characterized compounds, many unique, weakly conserved BGCs scattered across the genus that are predicted to encode unknown natural products. Many of these "singleton" BGCs appear to have been acquired via horizontal gene transfer. Based on this large-scale characterization of metabolite production in the Bacilli, we go on to connect the alkylpyrones, natural products that are highly conserved but previously biologically uncharacterized, to a role in Bacillus physiology: inhibiting spore development. IMPORTANCEBacilli are capable of producing a diverse array of specialized metabolites, many of which have gained attention for their roles as signals that affect bacterial physiology and development. Up to this point, however, the Bacillus genus's metabolic capacity has been underexplored. We undertook a deep genomic analysis of 1,566 Bacillus genomes to understand the full spectrum of metabolites that this bacterial group can make. We discovered that the majority of the specialized metabolites produced by Bacillus species are highly conserved, known compounds with important signaling roles in the physiology and development of this bacterium. Additionally, there is significant unique biosynthetic machinery distributed across the genus that might lead to new, unknown metabolites with diverse biological functions. Inspired by the findings of our genomic analysis, we speculate that the highly conserved alkylpyrones might have an important biological activity within this genus. We go on to validate this prediction by demonstrating that these natural products are developmental signals in Bacillus and act by inhibiting sporulation.

Natural-Product Antibiotics: Cues for Modulating Bacterial Biofilm Formation.

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.