Environmental purines decrease Pseudomonas aeruginosa biofilm formation by disrupting c-di-GMP metabolism.

Cyclic di-guanosine monophosphate (c-di-GMP) is a bacterial second messenger that governs the lifestyle switch between planktonic and biofilm states. While substantial investigation has focused on the proteins that produce and degrade c-di-GMP, less attention has been paid to the potential for metabolic control of c-di-GMP signaling. Here, we show that micromolar levels of specific environmental purines unexpectedly decrease c-di-GMP and biofilm formation in Pseudomonas aeruginosa. Using a fluorescent genetic reporter, we show that adenosine and inosine decrease c-di-GMP even when competing purines are present. We confirm genetically that purine salvage is required for c-di-GMP decrease. Furthermore, we find that (p)ppGpp prevents xanthosine and guanosine from producing an opposing c-di-GMP increase, reinforcing a salvage hierarchy that favors c-di-GMP decrease even at the expense of growth. We propose that purines can act as a cue for bacteria to shift their lifestyle away from the recalcitrant biofilm state via upstream metabolic control of c-di-GMP signaling.

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

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 study Bacillus subtilis biofilms-it is not always clear how such biofilms cope with minimally buffered natural environments. Here, we report how B. subtilis biofilms 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 in B. subtilis biofilms 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 that B. subtilis biofilms 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.

Engineered calprotectin-sensing probiotics for IBD surveillance in humans.

Inflammatory bowel disease (IBD) is a spectrum of autoimmune diseases affecting the gastrointestinal tract characterized by a relapsing and remitting course of gut mucosal inflammation. Disease flares can be difficult to predict, and the current practice of IBD disease activity surveillance through endoscopy is invasive and requires medical expertise. Recent advancements in synthetic biology raise the possibility that symbiotic microbes can be engineered to selectively detect disease biomarkers used in current clinical practice. Here, we introduce an engineered probiotic capable of detecting the clinical gold standard IBD biomarker, calprotectin, with sensitivity and specificity in IBD patients. Specifically, we identified a bacterial promoter in the probiotic strain Escherichia coli Nissle 1917 (EcN) which exhibits a specific expression increase in the presence of calprotectin. Using murine models of colitis, we show that the reporter signal is activated in vivo during transit of the GI tract following oral delivery. Furthermore, our engineered probiotic can successfully discriminate human patients with active IBD from those in remission and without IBD using patient stool samples, where the intensity of reporter signal quantitatively tracks with clinical laboratory-measured levels of calprotectin. Our pilot study sets the stage for probiotics that can be engineered to detect fecal calprotectin for precise noninvasive disease activity monitoring in IBD patients.

Toward manipulating serotonin signaling via the microbiota-gut-brain axis.

It is now well established in humans that there is a bidirectional pathway of communication between the central and enteric nervous systems in which members of the microbiome participate. This microbiota-gut-brain axis (MGBA) is crucial for normal development and physiology, and its dysregulation has been implicated in a range of neurological and intestinal disorders. Investigations into the mechanistic underpinnings of the MGBA have identified serotonin as a molecule of particular interest. In this review, we highlight recent advances toward understanding the role of endogenous serotonin in microbial communities, how microbial communities bidirectionally interact with host serotonin, and potential future engineering opportunities to leverage these novel mechanisms for biomedical applications.

Vesicle-Based Sensors for Extracellular Potassium Detection.

INTRODUCTION: The design of sensors that can detect biological ions in situ remains challenging. While many fluorescent indicators exist that can provide a fast, easy readout, they are often nonspecific, particularly to ions with similar charge states. To address this issue, we developed a vesicle-based sensor that harnesses membrane channels to gate access of potassium (K+) ions to an encapsulated fluorescent indicator. METHODS: We assembled phospholipid vesicles that incorporated valinomycin, a K+ specific membrane transporter, and that encapsulated benzofuran isophthalate (PBFI), a K+ sensitive dye that nonspecifically fluoresces in the presence of other ions, like sodium (Na+). The specificity, kinetics, and reversibility of encapsulated PBFI fluorescence was determined in a plate reader and fluorimeter. The sensors were then added to E. coli bacterial cultures to evaluate K+ levels in media as a function of cell density. RESULTS: Vesicle sensors significantly improved specificity of K+ detection in the presence of a competing monovalent ion, sodium (Na+), and a divalent cation, calcium (Ca2+), relative to controls where the dye was free in solution. The sensor was able to report both increases and decreases in K+ concentration. Finally, we observed our vesicle sensors could detect changes in K+ concentration in bacterial cultures. CONCLUSION: Our data present a new platform for extracellular ion detection that harnesses ion-specific membrane transporters to improve the specificity of ion detection. By changing the membrane transporter and encapsulated sensor, our approach should be broadly useful for designing biological sensors that detect an array of biological analytes in traditionally hard-to-monitor environments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00688-7.

Potential Roles for Gamma-Aminobutyric Acid Signaling in Bacterial Communities.

It is now established that the gut microbiome influences human neurology and behavior, and vice versa. Distinct mechanisms underlying this bidirectional communication pathway, termed the gut-brain axis, are becoming increasingly uncovered. This review summarizes recent interkingdom signaling research focused on gamma-aminobutyric acid (GABA), a human neurotransmitter and ubiquitous signaling molecule found in bacteria, fungi, plants, invertebrates, and mammals. We detail how GABAergic signaling has been shown to be a crucial component of the gut-brain axis. We further describe how GABA is also being found to mediate interkingdom signaling between algae and invertebrates, plants and invertebrates, and plants and bacteria. Based on these emerging results, we argue that obtaining a complete understanding of GABA-mediated communication in the gut-brain axis will involve deciphering the role of GABA signaling and metabolism within bacterial communities themselves.

A Two-Dimensional Model of Potassium Signaling and Oscillatory Growth in a Biofilm.

Biofilms are complex communities of bacteria that exhibit a variety of collective behaviors. These behaviors improve their ability to survive in many different environments. One of these collective behaviors seen in Bacillus subtilis is the ability for starving cells to stop the growth of other cells using potassium signaling and voltage changes. This signaling produces an oscillatory growth pattern so that during periods of low growth the nutrients diffuse deeper into the biofilm and reach the nutrient-starved, interior regions of the biomass. In this paper, we develop a mathematical model to describe this oscillatory behavior, and we use this model to develop a two-dimensional simulation that reproduces many of the important features seen in the experimental data. This simulation allows us to examine the spatial patterning of the oscillatory behavior to better understand the relationships between the various regions of the biofilm. Studying the spatial components of the metabolic and voltage oscillations could allow for the development of new control techniques for biofilms with complex shapes.

Synthetic biology in biofilms: Tools, challenges, and opportunities.

The field of synthetic biology seeks to program living cells to perform novel functions with applications ranging from environmental biosensing to smart cell-based therapeutics. Bacteria are an especially attractive chassis organism due to their rapid growth, ease of genetic manipulation, and ability to persist across many environmental niches. Despite significant progress in bacterial synthetic biology, programming bacteria to perform novel functions outside the well-controlled laboratory context remains challenging. In contrast to planktonic laboratory growth, bacteria in nature predominately reside in the context of densely packed communities known as biofilms. While biofilms have historically been considered environmental and biomedical hazards, their physiology and emergent behaviors could be leveraged for synthetic biology to engineer more capable and robust bacteria. Specifically, bacteria within biofilms participate in complex emergent behaviors such as collective organization, cell-to-cell signaling, and division of labor. Understanding and utilizing these properties can enable the effective deployment of engineered bacteria into natural target environments. Toward this goal, this review summarizes the current state of synthetic biology in biofilms by highlighting new molecular tools and remaining biological challenges. Looking to future opportunities, advancing synthetic biology in biofilms will enable the next generation of smart cell-based technologies for use in medicine, biomanufacturing, and environmental remediation.

Bioelectrical understanding and engineering of cell biology.

The last five decades of molecular and systems biology research have provided unprecedented insights into the molecular and genetic basis of many cellular processes. Despite these insights, however, it is arguable that there is still only limited predictive understanding of cell behaviours. In particular, the basis of heterogeneity in single-cell behaviour and the initiation of many different metabolic, transcriptional or mechanical responses to environmental stimuli remain largely unexplained. To go beyond the status quo, the understanding of cell behaviours emerging from molecular genetics must be complemented with physical and physiological ones, focusing on the intracellular and extracellular conditions within and around cells. Here, we argue that such a combination of genetics, physics and physiology can be grounded on a bioelectrical conceptualization of cells. We motivate the reasoning behind such a proposal and describe examples where a bioelectrical view has been shown to, or can, provide predictive biological understanding. In addition, we discuss how this view opens up novel ways to control cell behaviours by electrical and electrochemical means, setting the stage for the emergence of bioelectrical engineering.

Encoding Membrane-Potential-Based Memory within a Microbial Community.

Cellular membrane potential plays a key role in the formation and retrieval of memories in the metazoan brain, but it remains unclear whether such memory can also be encoded in simpler organisms like bacteria. Here, we show that single-cell-level memory patterns can be imprinted in bacterial biofilms by light-induced changes in the membrane potential. We demonstrate that transient optical perturbations generate a persistent and robust potassium-channel-mediated change in the membrane potential of bacteria within the biofilm. The light-exposed cells respond in an anti-phase manner, relative to unexposed cells, to both natural and induced oscillations in extracellular ion concentrations. This anti-phase response, which persists for hours following the transient optical stimulus, enables a direct single-cell resolution visualization of spatial memory patterns within the biofilm. The ability to encode robust and persistent membrane-potential-based memory patterns could enable computations within prokaryotic communities and suggests a parallel between neurons and bacteria.

Metabolic basis of brain-like electrical signalling in bacterial communities.

Information processing in the mammalian brain relies on a careful regulation of the membrane potential dynamics of its constituent neurons, which propagates across the neuronal tissue via electrical signalling. We recently reported the existence of electrical signalling in a much simpler organism, the bacterium Bacillus subtilis. In dense bacterial communities known as biofilms, nutrient-deprived B. subtilis cells in the interior of the colony use electrical communication to transmit stress signals to the periphery, which interfere with the growth of peripheral cells and reduce nutrient consumption, thereby relieving stress from the interior. Here, we explicitly address the interplay between metabolism and electrophysiology in bacterial biofilms, by introducing a spatially extended mathematical model that combines the metabolic and electrical components of the phenomenon in a discretized reaction-diffusion scheme. The model is experimentally validated by environmental and genetic perturbations, and confirms that metabolic stress is transmitted through the bacterial population via a potassium wave. Interestingly, this behaviour is reminiscent of cortical spreading depression in the brain, characterized by a wave of electrical activity mediated by potassium diffusion that has been linked to various neurological disorders, calling for future studies on the evolutionary link between the two phenomena. This article is part of the theme issue 'Liquid brains, solid brains: How distributed cognitive architectures process information'.

Signal Percolation within a Bacterial Community.

Signal transmission among cells enables long-range coordination in biological systems. However, the scarcity of quantitative measurements hinders the development of theories that relate signal propagation to cellular heterogeneity and spatial organization. We address this problem in a bacterial community that employs electrochemical cell-to-cell communication. We developed a model based on percolation theory, which describes how signals propagate through a heterogeneous medium. Our model predicts that signal transmission becomes possible when the community is organized near a critical phase transition between a disconnected and a fully connected conduit of signaling cells. By measuring population-level signal transmission with single-cell resolution in wild-type and genetically modified communities, we confirm that the spatial distribution of signaling cells is organized at the predicted phase transition. Our findings suggest that at this critical point, the population-level benefit of signal transmission outweighs the single-cell level cost. The bacterial community thus appears to be organized according to a theoretically predicted spatial heterogeneity that promotes efficient signal transmission.

SnapShot: Electrochemical Communication in Biofilms.

The role of electricity in biological systems was first appreciated through electrical stimulation experiments performed by Luigi Galvani in the 18th century. These pioneering experiments demonstrated that the behavior of living tissues is governed by the flow of electrochemical species-an insight that gave rise to the modern field of electrophysiology. Since then, electrophysiology has largely remained a bastion of neuroscience. However, exciting recent developments have demonstrated that even simple bacteria residing in communities use electrochemical communication to coordinate population-level behaviors. These recent works are defining the emerging field of bacterial biofilm electrophysiology. To view this SnapShot, open or download the PDF.

Coupling between distant biofilms and emergence of nutrient time-sharing.

Bacteria within communities can interact to organize their behavior. It has been unclear whether such interactions can extend beyond a single community to coordinate the behavior of distant populations. We discovered that two Bacillus subtilis biofilm communities undergoing metabolic oscillations can become coupled through electrical signaling and synchronize their growth dynamics. Coupling increases competition by also synchronizing demand for limited nutrients. As predicted by mathematical modeling, we confirm that biofilms resolve this conflict by switching from in-phase to antiphase oscillations. This results in time-sharing behavior, where each community takes turns consuming nutrients. Time-sharing enables biofilms to counterintuitively increase growth under reduced nutrient supply. Distant biofilms can thus coordinate their behavior to resolve nutrient competition through time-sharing, a strategy used in engineered systems to allocate limited resources.

Species-Independent Attraction to Biofilms through Electrical Signaling.

Bacteria residing within biofilm communities can coordinate their behavior through cell-to-cell signaling. However, it remains unclear if these signals can also influence the behavior of distant cells that are not part of the community. Using a microfluidic approach, we find that potassium ion channel-mediated electrical signaling generated by a Bacillus subtilis biofilm can attract distant cells. Integration of experiments and mathematical modeling indicates that extracellular potassium emitted from the biofilm alters the membrane potential of distant cells, thereby directing their motility. This electrically mediated attraction appears to be a generic mechanism that enables cross-species interactions, as Pseudomonas aeruginosa cells also become attracted to the electrical signal released by the B. subtilis biofilm. Cells within a biofilm community can thus not only coordinate their own behavior but also influence the behavior of diverse bacteria at a distance through long-range electrical signaling. PAPERCLIP.