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2.
Science ; 378(6615): 43-49, 2022 10 07.
Article in English | MEDLINE | ID: mdl-36201591

ABSTRACT

The dormant state of bacterial spores is generally thought to be devoid of biological activity. We show that despite continued dormancy, spores can integrate environmental signals over time through a preexisting electrochemical potential. Specifically, we studied thousands of individual Bacillus subtilis spores that remain dormant when exposed to transient nutrient pulses. Guided by a mathematical model of bacterial electrophysiology, we modulated the decision to exit dormancy by genetically and chemically targeting potassium ion flux. We confirmed that short nutrient pulses result in step-like changes in the electrochemical potential of persistent spores. During dormancy, spores thus gradually release their stored electrochemical potential to integrate extracellular information over time. These findings reveal a decision-making mechanism that operates in physiologically inactive cells.


Subject(s)
Bacillus subtilis , Potassium-Hydrogen Antiporters , Spores, Bacterial , Bacillus subtilis/physiology , Electrophysiological Phenomena , Models, Biological , Potassium/physiology , Potassium-Hydrogen Antiporters/physiology , Spores, Bacterial/physiology
3.
Cell Syst ; 13(6): 488-498.e4, 2022 06 15.
Article in English | MEDLINE | ID: mdl-35512710

ABSTRACT

Biological systems ranging from bacteria to mammals utilize electrochemical signaling. Although artificial electrochemical signals have been utilized to characterize neural tissue responses, the effects of such stimuli on non-neural systems remain unclear. To pursue this question, we developed an experimental platform that combines a microfluidic chip with a multielectrode array (MiCMA) to enable localized electrochemical stimulation of bacterial biofilms. The device also allows for the simultaneous measurement of the physiological response within the biofilm with single-cell resolution. We find that the stimulation of an electrode locally changes the ratio of the two major cell types comprising Bacillus subtilis biofilms, namely motile and extracellular-matrix-producing cells. Specifically, stimulation promotes the proliferation of motile cells but not matrix cells, even though these two cell types are genetically identical and reside in the same microenvironment. Our work thus reveals that an electronic interface can selectively target bacterial cell types, enabling the control of biofilm composition and development.


Subject(s)
Bacillus subtilis , Biofilms , Bacillus subtilis/metabolism , Cell Proliferation , Electric Stimulation , Extracellular Matrix/metabolism
4.
Cell ; 185(1): 145-157.e13, 2022 01 06.
Article in English | MEDLINE | ID: mdl-34995513

ABSTRACT

Contrary to multicellular organisms that display segmentation during development, communities of unicellular organisms are believed to be devoid of such sophisticated patterning. Unexpectedly, we find that the gene expression underlying the nitrogen stress response of a developing Bacillus subtilis biofilm becomes organized into a ring-like pattern. Mathematical modeling and genetic probing of the underlying circuit indicate that this patterning is generated by a clock and wavefront mechanism, similar to that driving vertebrate somitogenesis. We experimentally validated this hypothesis by showing that predicted nutrient conditions can even lead to multiple concentric rings, resembling segments. We additionally confirmed that this patterning mechanism is driven by cell-autonomous oscillations. Importantly, we show that the clock and wavefront process also spatially patterns sporulation within the biofilm. Together, these findings reveal a biofilm segmentation clock that organizes cellular differentiation in space and time, thereby challenging the paradigm that such patterning mechanisms are exclusive to plant and animal development.


Subject(s)
Bacillus subtilis/growth & development , Bacillus subtilis/genetics , Biofilms/growth & development , Body Patterning/genetics , Bacillus subtilis/metabolism , Gene Expression , Gene Expression Regulation, Developmental , Kinetics , Models, Biological , Nitrogen/metabolism , Signal Transduction/genetics , Somites/growth & development , Spores, Bacterial/growth & development , Stress, Physiological/genetics , Time Factors
5.
Cell Syst ; 12(6): 497-508, 2021 06 16.
Article in English | MEDLINE | ID: mdl-34139162

ABSTRACT

Metal ions are essential for life and represent the second most abundant constituent (after water) of any living cell. While the biological importance of inorganic ions has been appreciated for over a century, we are far from a comprehensive understanding of the functional roles that ions play in cells and organisms. In particular, recent advances are challenging the traditional view that cells maintain constant levels of ion concentrations (ion homeostasis). In fact, the ionic composition (metallome) of cells appears to be purposefully dynamic. The scientific journey that started over 60 years ago with the seminal work by Hodgkin and Huxley on action potentials in neurons is far from reaching its end. New evidence is uncovering how changes in ionic composition regulate unexpected cellular functions and physiology, especially in bacteria, thereby hinting at the evolutionary origins of the dynamic metallome. It is an exciting time for this field of biology, which we discuss and refer to here as IonoBiology.


Subject(s)
Metals , Neurons , Bacteria , Homeostasis , Ions
6.
Cell Syst ; 10(5): 417-423.e3, 2020 05 20.
Article in English | MEDLINE | ID: mdl-32343961

ABSTRACT

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.


Subject(s)
Membrane Potentials/physiology , Memory/physiology , Microbiota/genetics , Bacteria/metabolism , Biofilms , Membrane Potentials/genetics , Microbiota/physiology , Models, Theoretical , Optical Phenomena , Potassium Channels/physiology , Voltage-Sensitive Dye Imaging/methods
7.
Biophys J ; 118(7): 1721-1732, 2020 04 07.
Article in English | MEDLINE | ID: mdl-32105650

ABSTRACT

Many multicellular communities propagate signals in a directed manner via excitable waves. Cell-to-cell heterogeneity is a ubiquitous feature of multicellular communities, but the effects of heterogeneity on wave propagation are still unclear. Here, we use a minimal FitzHugh-Nagumo-type model to investigate excitable wave propagation in a two-dimensional heterogeneous community. The model shows three dynamic regimes in which waves either propagate directionally, die out, or spiral indefinitely, and we characterize how these regimes depend on the heterogeneity parameters. We find that in some parameter regimes, spatial correlations in the heterogeneity enhance directional propagation and suppress spiraling. However, in other regimes, spatial correlations promote spiraling, a surprising feature that we explain by demonstrating that these spirals form by a second, distinct mechanism. Finally, we characterize the dynamics using techniques from percolation theory. Despite the fact that percolation theory does not completely describe the dynamics quantitatively because it neglects the details of the excitable propagation, we find that it accounts for the transitions between the dynamic regimes and the general dependency of the spiral period on the heterogeneity and thus provides important insights. Our results reveal that the spatial structure of cell-to-cell heterogeneity can have important consequences for signal propagation in cellular communities.

8.
PLoS Comput Biol ; 15(12): e1007508, 2019 12.
Article in English | MEDLINE | ID: mdl-31790383

ABSTRACT

Signal propagation over long distances is a ubiquitous feature of multicellular communities, but cell-to-cell variability can cause propagation to be highly heterogeneous. Simple models of signal propagation in heterogenous media, such as percolation theory, can potentially provide a quantitative understanding of these processes, but it is unclear whether these simple models properly capture the complexities of multicellular systems. We recently discovered that in biofilms of the bacterium Bacillus subtilis, the propagation of an electrical signal is statistically consistent with percolation theory, and yet it is reasonable to suspect that key features of this system go beyond the simple assumptions of basic percolation theory. Indeed, we find here that the probability for a cell to signal is not independent from other cells as assumed in percolation theory, but instead is correlated with its nearby neighbors. We develop a mechanistic model, in which correlated signaling emerges from cell division, phenotypic inheritance, and cell displacement, that reproduces the experimentally observed correlations. We find that the correlations do not significantly affect the spatial statistics, which we rationalize using a renormalization argument. Moreover, the fraction of signaling cells is not constant in space, as assumed in percolation theory, but instead varies within and across biofilms. We find that this feature lowers the fraction of signaling cells at which one observes the characteristic power-law statistics of cluster sizes, consistent with our experimental results. We validate the model using a mutant biofilm whose signaling probability decays along the propagation direction. Our results reveal key statistical features of a correlated signaling process in a multicellular community. More broadly, our results identify extensions to percolation theory that do or do not alter its predictions and may be more appropriate for biological systems.


Subject(s)
Microbiota/physiology , Models, Biological , Bacillus subtilis/genetics , Bacillus subtilis/physiology , Biofilms , Computational Biology , Electrophysiological Phenomena , Lab-On-A-Chip Devices , Microbial Interactions/physiology , Mutation , Potassium/metabolism , Signal Transduction/physiology
9.
Philos Trans R Soc Lond B Biol Sci ; 374(1774): 20180382, 2019 06 10.
Article in English | MEDLINE | ID: mdl-31006362

ABSTRACT

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'.


Subject(s)
Bacillus subtilis/physiology , Biofilms , Bacillus subtilis/metabolism , Brain/physiology , Diffusion , Electrophysiological Phenomena , Models, Biological
10.
Cell ; 177(2): 352-360.e13, 2019 04 04.
Article in English | MEDLINE | ID: mdl-30853217

ABSTRACT

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.


Subject(s)
Bacterial Outer Membrane/metabolism , Magnesium/metabolism , Ribosomes/metabolism , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Proteomics , Ribosomal Proteins/metabolism
11.
Proc Natl Acad Sci U S A ; 115(36): E8333-E8340, 2018 09 04.
Article in English | MEDLINE | ID: mdl-30127028

ABSTRACT

Biofilm communities of Bacillus subtilis bacteria have recently been shown to exhibit collective growth-rate oscillations mediated by electrochemical signaling to cope with nutrient starvation. These oscillations emerge once the colony reaches a large enough number of cells. However, it remains unclear whether the amplitude of the oscillations, and thus their effectiveness, builds up over time gradually or if they can emerge instantly with a nonzero amplitude. Here we address this question by combining microfluidics-based time-lapse microscopy experiments with a minimal theoretical description of the system in the form of a delay-differential equation model. Analytical and numerical methods reveal that oscillations arise through a subcritical Hopf bifurcation, which enables instant high-amplitude oscillations. Consequently, the model predicts a bistable regime where an oscillating and a nonoscillating attractor coexist in phase space. We experimentally validate this prediction by showing that oscillations can be triggered by perturbing the media conditions, provided the biofilm size lies within an appropriate range. The model also predicts that the minimum size at which oscillations start decreases with stress, a fact that we also verify experimentally. Taken together, our results show that collective oscillations in cell populations can emerge suddenly with nonzero amplitude via a discontinuous transition.


Subject(s)
Bacillus subtilis/physiology , Biofilms/growth & development , Biological Clocks/physiology , Models, Biological
12.
Cell Syst ; 7(2): 137-145.e3, 2018 08 22.
Article in English | MEDLINE | ID: mdl-30056004

ABSTRACT

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.


Subject(s)
Bacteria/metabolism , Microbial Interactions , Single-Cell Analysis/methods , Bacteria/cytology , Biofilms , Electrochemistry , Microbiota , Models, Biological , Phase Transition
13.
Cell ; 170(1): 214-214.e1, 2017 Jun 29.
Article in English | MEDLINE | ID: mdl-28666120

ABSTRACT

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.


Subject(s)
Biofilms , Bacteria/classification , Bacteria/metabolism , Bacterial Physiological Phenomena , Electrophysiological Phenomena
14.
Science ; 356(6338): 638-642, 2017 05 12.
Article in English | MEDLINE | ID: mdl-28386026

ABSTRACT

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.


Subject(s)
Bacillus subtilis/classification , Bacillus subtilis/physiology , Biofilms , Microbial Interactions , Bacillus subtilis/growth & development , Electrophysiological Phenomena , Models, Biological , Signal Transduction
15.
Cell ; 168(1-2): 200-209.e12, 2017 Jan 12.
Article in English | MEDLINE | ID: mdl-28086091

ABSTRACT

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.


Subject(s)
Bacillus subtilis/physiology , Biofilms , Electrophysiological Phenomena , Pseudomonas aeruginosa/physiology , Biofilms/classification , Membrane Potentials , Microfluidic Analytical Techniques , Models, Biological , Potassium/metabolism
16.
Mol Syst Biol ; 12(5): 871, 2016 05 23.
Article in English | MEDLINE | ID: mdl-27216630

ABSTRACT

How can changes in growth rate affect the regulatory networks behavior and the outcomes of cellular differentiation? We address this question by focusing on starvation response in sporulating Bacillus subtilis We show that the activity of sporulation master regulator Spo0A increases with decreasing cellular growth rate. Using a mathematical model of the phosphorelay-the network controlling Spo0A-we predict that this increase in Spo0A activity can be explained by the phosphorelay protein accumulation and lengthening of the period between chromosomal replication events caused by growth slowdown. As a result, only cells growing slower than a certain rate reach threshold Spo0A activity necessary for sporulation. This growth threshold model accurately predicts cell fates and explains the distribution of sporulation deferral times. We confirm our predictions experimentally and show that the concentration rather than activity of phosphorelay proteins is affected by the growth slowdown. We conclude that sensing the growth rates enables cells to indirectly detect starvation without the need for evaluating specific stress signals.


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Spores, Bacterial/growth & development , Transcription Factors/metabolism , Chromosomes, Bacterial/genetics , Culture Media/chemistry , Gene Expression Regulation, Bacterial , Models, Theoretical
17.
PLoS Comput Biol ; 12(3): e1004793, 2016 Mar.
Article in English | MEDLINE | ID: mdl-27003682

ABSTRACT

Gene regulatory circuits must contend with intrinsic noise that arises due to finite numbers of proteins. While some circuits act to reduce this noise, others appear to exploit it. A striking example is the competence circuit in Bacillus subtilis, which exhibits much larger noise in the duration of its competence events than a synthetically constructed analog that performs the same function. Here, using stochastic modeling and fluorescence microscopy, we show that this larger noise allows cells to exit terminal phenotypic states, which expands the range of stress levels to which cells are responsive and leads to phenotypic heterogeneity at the population level. This is an important example of how noise confers a functional benefit in a genetic decision-making circuit.


Subject(s)
Adaptation, Physiological/genetics , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Gene Regulatory Networks/genetics , Genetic Fitness/genetics , Models, Genetic , Computer Simulation , Models, Statistical , Signal-To-Noise Ratio , Stress, Physiological/genetics
18.
Nature ; 527(7576): 59-63, 2015 Nov 05.
Article in English | MEDLINE | ID: mdl-26503040

ABSTRACT

The study of bacterial ion channels has provided fundamental insights into the structural basis of neuronal signalling; however, the native role of ion channels in bacteria has remained elusive. Here we show that ion channels conduct long-range electrical signals within bacterial biofilm communities through spatially propagating waves of potassium. These waves result from a positive feedback loop, in which a metabolic trigger induces release of intracellular potassium, which in turn depolarizes neighbouring cells. Propagating through the biofilm, this wave of depolarization coordinates metabolic states among cells in the interior and periphery of the biofilm. Deletion of the potassium channel abolishes this response. As predicted by a mathematical model, we further show that spatial propagation can be hindered by specific genetic perturbations to potassium channel gating. Together, these results demonstrate a function for ion channels in bacterial biofilms, and provide a prokaryotic paradigm for active, long-range electrical signalling in cellular communities.


Subject(s)
Bacillus subtilis/cytology , Bacillus subtilis/metabolism , Biofilms/growth & development , Electric Conductivity , Potassium Channels/metabolism , Bacillus subtilis/growth & development , Feedback, Physiological , Ion Channel Gating , Membrane Potentials , Models, Biological , Potassium/metabolism , Signal Transduction
19.
Cell ; 162(2): 328-337, 2015 Jul 16.
Article in English | MEDLINE | ID: mdl-26165942

ABSTRACT

Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here, we demonstrate a functional role for the arrangement of Bacillus subtilis sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin and the other close to the terminus, leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A∼P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell cycle spent in starvation. The simplicity of this coordination mechanism suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings. VIDEO ABSTRACT.


Subject(s)
Bacillus subtilis/physiology , Spores, Bacterial/physiology , Bacillus subtilis/genetics , Bacterial Proteins/metabolism , Chromosomes, Bacterial , DNA Replication , Feedback , Gene Dosage , Phosphorylation , Transcription Factors/metabolism
20.
PLoS One ; 10(7): e0132948, 2015.
Article in English | MEDLINE | ID: mdl-26196509

ABSTRACT

Bacteria predominantly reside in microbial communities known as biofilms, where cells are encapsulated and protected by the extracellular matrix (ECM). While all biofilm cells benefit from the ECM, only a subgroup of cells carries the burden of producing this public good. This dilemma provokes the question of how these cells balance the cost of ECM production. Here we show that ECM producing cells have a higher gene expression response to quorum sensing (QS) signals, which can lead to a private benefit. Specifically, we constructed a synthetic quorum-sensing system with designated "Sender" and "Receiver" cells in Bacillus subtilis. This synthetic QS system allowed us to uncouple and independently investigate ECM production and QS in both biofilms and single cells. Results revealed that ECM production directly enhances the response to QS signals, which may offset the cost of ECM production.


Subject(s)
Bacillus subtilis/physiology , Biofilms , Quorum Sensing/physiology , Bacterial Proteins/metabolism , Chromosomes, Bacterial/genetics , Cysteine/chemistry , Esters/chemistry , Extracellular Matrix/metabolism , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Protein Structure, Tertiary , Signal Transduction , Staphylococcus epidermidis/physiology , Sulfhydryl Compounds/chemistry
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