Metabolic interactions in microbial communities

The Shank laboratory studies the chemical and physical interactions of microbes with each other and their hosts. Microbes live everywhere, and their activities can have profound impacts on their hosts as well as on ecosystem‐level processes. How microbes interact within these communities, however, remains largely unknown. We are fascinated by the idea that microbes are able to generate and secrete chemical cues (known as specialized or secondary metabolites) that can act as interspecies signals to influence the physiology and metabolism of their microbial neighbors, and thus contribute to the stability and functioning of complex microbial communities. Our research dissects these microbial interactions using traditional microbiology, fluorescent co-culture, bioinformatics, mass spectrometry imaging, and native-like microcosms. We aim to define the molecular basis of how microbial specialized metabolites impact bacterial cellular differentiation, discover chemical tools to kill and modulate pathogens, and dynamically visualize microbial interactions at the single-cell level. In doing so, we are gaining insights into microbial ecology. We are also identifying novel bioactive compounds as potential therapeutics and chemical tools to achieve our long-term goal of manipulating microbial communities to improve host health and the environment. Presented virtually during the New Faculty Talks session at the 25th Annual University of Massachusetts Medical School Research Retreat 2020 on October 27, 2020

Profiling the metabolic signals involved in chemical communication between microbes using imaging mass spectrometry

The ability of microbes to secrete bioactive chemical signals into their environment has been known for over a century. However, it is only in the last decade that imaging mass spectrometry has provided us with the ability to directly visualize the spatial distributions of these microbial metabolites. This technology involves collecting mass spectra from multiple discrete locations across a biological sample, yielding chemical ‘maps’ that simultaneously reveal the distributions of hundreds of metabolites in two dimensions. Advances in microbial imaging mass spectrometry summarized here have included the identification of novel strain- or coculture-specific compounds, the visualization of biotransformation events (where one metabolite is converted into another by a neighboring microbe), and the implementation of a method to reconstruct the 3D subsurface distributions of metabolites, among others. Here we review the recent literature and discuss how imaging mass spectrometry has spurred novel insights regarding the chemical consequences of microbial interactions

Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens

ABSTRACT 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. IMPORTANCE Biofilms are communities of bacteria adhered to surfaces by an extracellular matrix; such biofilms can have important effects in both clinical and agricultural settings. To identify chemical compounds that inhibited biofilm formation, we used a fluorescent reporter to screen for bacteria that inhibited biofilm gene expression in Bacillus subtilis . We identified Pseudomonas protegens as one such bacterium and found that the biofilm-inhibiting compound it produces was the antibiotic 2,4-diacetylphloroglucinol (DAPG). We showed that even at subinhibitory concentrations, DAPG inhibits biofilm formation and sporulation in B. subtilis . These findings have potential implications for understanding the interactions between these two microbes in the natural world and support the idea that many compounds considered antibiotics can impact bacterial development at subinhibitory concentrations

Pirated Siderophores Promote Sporulation in Bacillus subtilis

ABSTRACT In microbial communities, bacteria chemically and physically interact with one another. Some of these interactions are mediated by secreted specialized metabolites that act as either intraspecies or interspecies signals to alter gene expression and to change cell physiology. Bacillus subtilis is a well-characterized soil microbe that can differentiate into multiple cell types, including metabolically dormant endospores. We were interested in identifying microbial interactions that affected sporulation in B. subtilis . Using a fluorescent transcriptional reporter, we observed that coculturing B. subtilis with Escherichia coli promoted sporulation gene expression via a secreted metabolite. To identify the active compound, we screened the E. coli Keio Collection and identified the sporulation-accelerating cue as the siderophore enterobactin. B. subtilis has multiple iron acquisition systems that are used to take up the B. subtilis- produced siderophore bacillibactin, as well as to pirate exogenous siderophores such as enterobactin. While B. subtilis uses a single substrate binding protein (FeuA) to take up both bacillibactin and enterobactin, we discovered that it requires two distinct genes to sporulate in response to these siderophores (the esterase gene besA for bacillibactin and a putative esterase gene, ybbA , for enterobactin). In addition, we found that siderophores from a variety of other microbial species also promote sporulation in B. subtilis . Our results thus demonstrate that siderophores can act not only as bacterial iron acquisition systems but also as interspecies cues that alter cellular development and accelerate sporulation in B. subtilis . IMPORTANCE While much is known about the genetic regulation of Bacillus subtilis sporulation, little is understood about how other bacteria influence this process. This work describes an interaction between Escherichia coli and B. subtilis that accelerates sporulation in B. subtilis . The interaction is mediated by the E. coli siderophore enterobactin; we show that other species' siderophores also promote sporulation gene expression in B. subtilis . These results suggest that siderophores not only may supply bacteria with the mineral nutrient iron but also may play a role in bacterial interspecies signaling, providing a cue for sporulation. Siderophores are produced by many bacterial species and thus potentially play important roles in altering bacterial cell physiology in diverse environments

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\u27 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\u27 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\u27 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

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\u27s 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

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 ( approximately 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

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis

Thiazolyl peptides are known antibiotics produced by diverse bacterial taxa. It has been believed that antibiotics are deployed by bacteria as weapons, providing them with an evolutionary advantage over other microbes. We show here that these weapons can also act as chemical tools that elicit biofilm production in the model bacterium Bacillus subtilis. Importantly, the biofilm-inducing (and therefore signaling) properties of these compounds are independent of their killing activity. We go on to use this biofilm-inducing activity to identify and confirm the presence of thiazolyl peptide gene clusters in other bacteria. These results indicate that thiazolyl peptides, and potentially other antibiotics, have the ability to alter bacterial behavior in ways important both to the environment and to human health

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

ABSTRACT 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. IMPORTANCE Bacilli 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

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