Enforced specificity of an animal symbiosis.

Insect diversification has been catalyzed by widespread specialization on novel hosts - a process underlying exceptional radiations of phytophagous beetles, lepidopterans, parasitoid wasps, and inordinate lineages of symbionts, predators and other trophic specialists. The strict fidelity of many such interspecies associations is posited to hinge on sensory tuning to host-derived cues, a model supported by studies of neural function in host-specific model species. Here, we investigated the sensory basis of symbiotic interactions between a myrmecophile rove beetle and its single, natural host ant species. We show that host cues trigger analogous behaviors in both ant and symbiont. Cuticular hydrocarbons - the ant's nestmate recognition pheromones - elicit partner recognition by the beetle and execution of ant grooming behavior, integrating the beetle into the colony via chemical mimicry. The beetle also follows host trail pheromones, permitting inter-colony dispersal. Remarkably, the rove beetle also performs its symbiotic behaviors with ant species separated by ~95 million years, and shows minimal preference for its natural host over non-host ants. Experimentally validated agent-based modeling supports a scenario in which specificity is enforced by physiological constraints on symbiont dispersal, and negative fitness interactions with alternative hosts, rather than via sensory tuning. Enforced specificity may be a pervasive mechanism of host range restriction of specialists embedded within host niches. Chance realization of latent compatibilities with alternative hosts may facilitate host switching, enabling deep-time persistence of obligately symbiotic lineages.

Phenotypic memory in quorum sensing.

Quorum sensing (QS) is a regulatory mechanism used by bacteria to coordinate group behavior in response to high cell densities. During QS, cells monitor the concentration of external signals, known as autoinducers, as a proxy for cell density. QS often involves positive feedback loops, leading to the upregulation of genes associated with QS signal production and detection. This results in distinct steady-state concentrations of QS-related molecules in QS-ON and QS-OFF states. Due to the slow decay rates of biomolecules such as proteins, even after removal of the initial stimuli, cells can retain elevated levels of QS-associated biomolecules for extended periods of time. This persistence of biomolecules after the removal of the initial stimuli has the potential to impact the response to future stimuli, indicating a memory of past exposure. This phenomenon, which is a consequence of the carry-over of biomolecules rather than genetic inheritance, is known as "phenotypic" memory. This theoretical study aims to investigate the presence of phenotypic memory in QS and the conditions that influence this memory. Numerical simulations based on ordinary differential equations and analytical modeling were used to study gene expression in response to sudden changes in cell density and extracellular signal concentrations. The model examined the effect of various cellular parameters on the strength of QS memory and the impact on gene regulatory dynamics. The findings revealed that QS memory has a transient effect on the expression of QS-responsive genes. These consequences of QS memory depend strongly on how cell density was perturbed, as well as various cellular parameters, including the Fold Change in the expression of QS-regulated genes, the autoinducer synthesis rate, the autoinducer threshold required for activation, and the cell growth rate.

Red-Light-Induced Genetic System for Control of Extracellular Electron Transfer.

Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.

Utilizing a divalent metal ion transporter to control biogenic nanoparticle synthesis.

Biogenic synthesis of inorganic nanomaterials has been demonstrated for both wild and engineered bacterial strains. In many systems the nucleation and growth of nanomaterials is poorly controlled and requires concentrations of heavy metals toxic to living cells. Here, we utilized the tools of synthetic biology to engineer a strain of Escherichia coli capable of synthesizing cadmium sulfide nanoparticles from low concentrations of reactants with control over the location of synthesis. Informed by simulations of bacterially-assisted nanoparticle synthesis, we created a strain of E. coli expressing a broad-spectrum divalent metal transporter, ZupT, and a synthetic CdS nucleating peptide. Expression of ZupT in the outer membrane and placement of the nucleating peptide in the periplasm focused synthesis within the periplasmic space and enabled sufficient nucleation and growth of nanoparticles at sub-toxic levels of the reactants. This strain synthesized internal CdS quantum dot nanoparticles with spherical morphology and an average diameter of approximately 3.3 nm. ONE-SENTENCE SUMMARY: Expression of a metal ion transporter regulates synthesis of cadmium sulfide nanoparticles in bacteria.

Stochastic effects in bacterial communication mediated by extracellular vesicles.

Quorum sensing (QS) allows bacterial cells to sense changes in local cell density and, hence, to regulate multicellular processes, including biofilm formation, regulation of virulence, and horizontal gene transfer. While, traditionally, QS was thought to involve the exchange of extracellular signal molecules free in solution, recent experiments have shown that for some bacterial systems a substantial fraction of signal molecules are packaged and delivered in extracellular vesicles. How the packaging of signal molecules in extracellular vesicles influences the ability of cells to communicate and coordinate multicellular behaviors remains largely unknown. We present here a stochastic reaction-diffusion model of QS that accounts for the exchange of both freely diffusing and vesicle-associated signal molecules. We find that the delivery of signal molecules via extracellular vesicles amplifies local fluctuations in the signal concentration, which can strongly affect the dynamics and spatial range of bacterial communication. For systems with multiple bacterial colonies, extracellular vesicles provide an alternate pathway for signal transport between colonies, and may be crucial for long-distance signal exchange in environments with strong degradation of free signal molecules.

Membrane-Binding Biomolecules Influence the Rate of Vesicle Exchange between Bacteria.

The exchange of bacterial extracellular vesicles facilitates molecular exchange between cells, including the horizontal transfer of genetic material. Given the implications of such transfer events on cell physiology and adaptation, some bacterial cells have likely evolved mechanisms to regulate vesicle exchange. Past work has identified mechanisms that influence the formation of extracellular vesicles, including the production of small molecules that modulate membrane structure; however, whether these mechanisms also modulate vesicle uptake and have an overall impact on the rate of vesicle exchange is unknown. Here, we show that membrane-binding molecules produced by microbes influence both the formation and uptake of extracellular vesicles and have the overall impact of increasing the vesicle exchange rate within a bacterial coculture. In effect, production of compounds that increase vesicle exchange rates encourage gene exchange between neighboring cells. The ability of several membrane-binding compounds to increase vesicle exchange was demonstrated. Three of these compounds, nisin, colistin, and polymyxin B, are antimicrobial peptides added at sub-inhibitory concentrations. These results suggest that a potential function of exogenous compounds that bind to membranes may be the regulation of vesicle exchange between cells. IMPORTANCE The exchange of bacterial extracellular vesicles is one route of gene transfer between bacteria, although it was unclear if bacteria developed strategies to modulate the rate of gene transfer within vesicles. In eukaryotes, there are many examples of specialized molecules that have evolved to facilitate the production, loading, and uptake of vesicles. Recent work with bacteria has shown that some small molecules influence membrane curvature and induce vesicle formation. Here, we show that similar compounds facilitate vesicle uptake, thereby increasing the overall rate of vesicle exchange within bacterial populations. The addition of membrane-binding compounds, several of them antibiotics at subinhibitory concentrations, to a bacterial coculture increased the rate of horizontal gene transfer via vesicle exchange.

Light-Induced Patterning of Electroactive Bacterial Biofilms.

Electroactive bacterial biofilms can function as living biomaterials that merge the functionality of living cells with electronic components. However, the development of such advanced living electronics has been challenged by the inability to control the geometry of electroactive biofilms relative to solid-state electrodes. Here, we developed a lithographic strategy to pattern conductive biofilms of Shewanella oneidensis by controlling aggregation protein CdrAB expression with a blue light-induced genetic circuit. This controlled deposition enabled S. oneidensis biofilm patterning on transparent electrode surfaces, and electrochemical measurements allowed us to both demonstrate tunable conduction dependent on pattern size and quantify the intrinsic conductivity of the living biofilms. The intrinsic biofilm conductivity measurements enabled us to experimentally confirm predictions based on simulations of a recently proposed collision-exchange electron transport mechanism. Overall, we developed a facile technique for controlling electroactive biofilm formation on electrodes, with implications for both studying and harnessing bioelectronics.

Intertemporal trade-off between population growth rate and carrying capacity during public good production.

Public goods are biomolecules that benefit cellular populations, such as by providing access to previously unutilized resources. Public good production is energetically costly. To reduce this cost, populations control public good biosynthesis, for example using density-dependent regulation accomplished by quorum sensing. Fitness costs and benefits of public good production must be balanced, similar to optimal investment decisions used in economics. We explore the regulation of a public good that increases the carrying capacity, through experimental measurements of growth in Escherichia coli and analysis using a modified logistic growth model. The timing of public good production showed a sharply peaked optimum in population fitness. The cell density associated with maximum public good benefits was determined by the trade-off between the cost of public good production, in terms of reduced growth rate, and benefits received from public goods, in the form of increased carrying capacity.

Formation, collective motion, and merging of macroscopic bacterial aggregates.

Chemotactic bacteria form emergent spatial patterns of variable cell density within cultures that are initially spatially uniform. These patterns are the result of chemical gradients that are created from the directed movement and metabolic activity of billions of cells. A recent study on pattern formation in wild bacterial isolates has revealed unique collective behaviors of the bacteria Enterobacter cloacae. As in other bacterial species, Enterobacter cloacae form macroscopic aggregates. Once formed, these bacterial clusters can migrate several millimeters, sometimes resulting in the merging of two or more clusters. To better understand these phenomena, we examine the formation and dynamics of thousands of bacterial clusters that form within a 22 cm square culture dish filled with soft agar over two days. At the macroscale, the aggregates display spatial order at short length scales, and the migration of cell clusters is superdiffusive, with a merging acceleration that is correlated with aggregate size. At the microscale, aggregates are composed of immotile cells surrounded by low density regions of motile cells. The collective movement of the aggregates is the result of an asymmetric flux of bacteria at the boundary. An agent-based model is developed to examine how these phenomena are the result of both chemotactic movement and a change in motility at high cell density. These results identify and characterize a new mechanism for collective bacterial motility driven by a transient, density-dependent change in motility.

Simulations to Aid in the Design of Microbes for Synthesis of Metallic Nanomaterials.

Microbes are champions of nanomaterial synthesis. By virtue of their incredible native range─from thermal vents to radioactive soil─microbes evolved tools to thrive on inorganic material, and, in their normal course of living, forge nanomaterials. In recent decades, synthetic biologists have engineered a vast array of functional nanomaterials using genetic tools that control the natural ability of bacteria to perform complex redox chemistry, maintain steep chemical gradients, and express biomolecular scaffolds. Leveraging microbial biology can lead to intricate nanomaterial architectures whose design and assembly exists beyond the ken of inorganic methods. Theories enumerating microbial nanomaterial synthesis are spare, however, despite the advantage they could offer. Here, we describe a theoretical approach to simulating biogenic nanomaterial synthesis that incorporates key features and parameters of Gram-negative bacteria. By adapting previously verified inorganic theories of nanoparticle synthesis, we recapitulate past biogenic experiments, such as the ability to localize nanoparticle synthesis or regulate nucleation of specific nanomaterials. Moreover, the simulation offers direction in the design of future experiments. Our results demonstrate the promise of marrying experimental and theoretical approaches to microbial nanomaterial synthesis.

Engineering Biological Electron Transfer and Redox Pathways for Nanoparticle Synthesis.

Many species of bacteria are naturally capable of types of electron transport not observed in eukaryotic cells. Some species live in environments containing heavy metals not typically encountered by cells of multicellular organisms, such as arsenic, cadmium, and mercury, leading to the evolution of enzymes to deal with these environmental toxins. Bacteria also inhabit a variety of extreme environments, and are capable of respiration even in the absence of oxygen as a terminal electron acceptor. Over the years, several of these exotic redox and electron transport pathways have been discovered and characterized in molecular-level detail, and more recently synthetic biology has begun to utilize these pathways to engineer cells capable of detecting and processing a variety of metals and semimetals. One such application is the biologically controlled synthesis of nanoparticles. This review will introduce the basic concepts of bacterial metal reduction, summarize recent work in engineering bacteria for nanoparticle production, and highlight the most cutting-edge work in the characterization and application of bacterial electron transport pathways.

Computation in bacterial communities.

Bacteria across many scales are involved in a dynamic process of information exchange to coordinate activity and community structure within large and diverse populations. The molecular components bacteria use to communicate have been discovered and characterized, and recent efforts have begun to understand the potential for bacterial signal exchange to gather information from the environment and coordinate collective behaviors. Such computations made by bacteria to coordinate the action of a population of cells in response to information gathered by a multitude of inputs is a form of collective intelligence. These computations must be robust to fluctuations in both biological, chemical, and physical parameters as well as to operate with energetic efficiency. Given these constraints, what are the limits of computation by bacterial populations and what strategies have evolved to ensure bacterial communities efficiently work together? Here the current understanding of information exchange and collective decision making that occur in microbial populations will be reviewed. Looking toward the future, we consider how a deeper understanding of bacterial computation will inform future direction in microbiology, biotechnology, and biophysics.

A neural network model predicts community-level signaling states in a diverse microbial community.

Signal crosstalk within biological communication networks is common, and such crosstalk can have unexpected consequences for decision making in heterogeneous communities of cells. Here we examined crosstalk within a bacterial community composed of five strains of Bacillus subtilis, with each strain producing a variant of the quorum sensing peptide ComX. In isolation, each strain produced one variant of the ComX signal to induce expression of genes associated with bacterial competence. When strains were combined, a mixture of ComX variants was produced resulting in variable levels of gene expression. To examine gene regulation in mixed communities, we implemented a neural network model. Experimental quantification of asymmetric crosstalk between pairs of strains parametrized the model, enabling the accurate prediction of activity within the full five-strain network. Unlike the single strain system in which quorum sensing activated upon exceeding a threshold concentration of the signal, crosstalk within the five-strain community resulted in multiple community-level quorum sensing states, each with a unique combination of quorum sensing activation among the five strains. Quorum sensing activity of the strains within the community was influenced by the combination and ratio of strains as well as community dynamics. The community-level signaling state was altered through an external signal perturbation, and the output state depended on the timing of the perturbation. Given the ubiquity of signal crosstalk in diverse microbial communities, the application of such neural network models will increase accuracy of predicting activity within microbial consortia and enable new strategies for control and design of bacterial signaling networks.

Disruption of microbial communication yields a two-dimensional percolation transition.

Bacteria communicate with each other to coordinate macroscale behaviors including pathogenesis, biofilm formation, and antibiotic production. Empirical evidence suggests that bacteria are capable of communicating at length scales far exceeding the size of individual cells. Several mechanisms of signal interference have been observed in nature, and how interference influences macroscale activity within microbial populations is unclear. Here we examined the exchange of quorum sensing signals to coordinate microbial activity over long distances in the presence of a variable amount of interference through a neighboring signal-degrading strain. As the level of interference increased, communication over large distances was disrupted and at a critical amount of interference, large-scale communication was suppressed. We explored this transition in experiments and reaction-diffusion models, and confirmed that this transition is a two-dimensional percolation transition. These results demonstrate the utility of applying physical models to emergence in complex biological networks to probe robustness and universal quantitative features.

Connecting single-cell properties to collective behavior in multiple wild isolates of the Enterobacter cloacae complex.

Some strains of motile bacteria self-organize to form spatial patterns of high and low cell density over length scales that can be observed by eye. One such collective behavior is the formation in semisolid agar media of a high cell density swarm band. We isolated 7 wild strains of the Enterobacter cloacae complex capable of forming this band and found its propagation speed can vary 2.5 fold across strains. To connect such variability in collective motility to strain properties, each strain's single-cell motility and exponential growth rates were measured. The band speed did not significantly correlate with any individual strain property; however, a multilinear analysis revealed that the band speed was set by a combination of the run speed and tumbling frequency. Comparison of variability in closely-related wild isolates has the potential to reveal how changes in single-cell properties influence the collective behavior of populations.

Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles.

Horizontal gene transfer is responsible for the exchange of many types of genetic elements, including plasmids. Properties of the exchanged genetic element are known to influence the efficiency of transfer via the mechanisms of conjugation, transduction, and transformation. Recently, an alternative general pathway of horizontal gene transfer has been identified, namely, gene exchange by extracellular vesicles. Although extracellular vesicles have been shown to facilitate the exchange of several types of plasmids, the influence of plasmid characteristics on genetic exchange within vesicles is unclear. Here, a set of different plasmids was constructed to systematically test the impact of plasmid properties, specifically, plasmid copy number, size, and origin of replication, on gene transfer in vesicles. The influence of each property on the production, packaging, and uptake of vesicles containing bacterial plasmids was quantified, revealing how plasmid properties modulate vesicle-mediated horizontal gene transfer. The loading of plasmids into vesicles correlates with the plasmid copy number and is influenced by characteristics that help set the number of plasmids within a cell, including size and origin of replication. Plasmid origin also has a separate impact on both vesicle loading and uptake, demonstrating that the origin of replication is a major determinant of the propensity of specific plasmids to transfer within extracellular vesicles.IMPORTANCE Extracellular vesicle formation and exchange are common within bacterial populations. Vesicles package multiple types of biomolecules, including genetic material. The exchange of extracellular vesicles containing genetic material facilitates interspecies DNA transfer and may be a promiscuous mechanism of horizontal gene transfer. Unlike other mechanisms of horizontal gene transfer, it is unclear whether characteristics of the exchanged DNA impact the likelihood of transfer in vesicles. Here, we systematically examine the influence of plasmid copy number, size, and origin of replication on the loading of DNA into vesicles and the uptake of DNA containing vesicles by recipient cells. These results reveal how each plasmid characteristic impacts gene transfer in vesicles and contribute to a greater understanding of the importance of vesicle-mediated gene exchange in the landscape of horizontal gene transfer.

Single-cell variability of growth interactions within a two-species bacterial community.

Cell-cell interaction networks have been examined in many high diversity microbial communities using macroscale approaches. Microscale studies of multispecies communities are lacking and it remains unclear how macroscale trends scale down to small groups of cells. Experimental approaches using microfluidic devices have revealed heterogeneity in the behavior of single cells, however, this analysis has not been extended towards the variability of cell-cell interactions. Using a microwell device, we analyzed cell growth within hundreds of replicate microbial communities consisting of two species and small population sizes. The wells of the devices were inoculated with a coculture of Escherichia coli and Enterobacter cloacae. Each species expressed a unique fluorescent protein enabling simultaneously tracking of cell number for each species over time. Growth dynamics within the device were consistent with bulk measurements. The device enabled monitoring of replicate, isolated coculture populations at high magnification, revealing both the growth interaction between the two species and the variability of such cell-cell interactions within small groups of cells. The device enables new experimental measurements of the heterogeneity of interactions within small, multispecies populations of bacteria.

Engineering bacteria for biogenic synthesis of chalcogenide nanomaterials.

Microbes naturally build nanoscale structures, including structures assembled from inorganic materials. Here, we combine the natural capabilities of microbes with engineered genetic control circuits to demonstrate the ability to control biological synthesis of chalcogenide nanomaterials in a heterologous host. We transferred reductase genes from both Shewanella sp. ANA-3 and Salmonella enterica serovar Typhimurium into a heterologous host (Escherichia coli) and examined the mechanisms that regulate the properties of biogenic nanomaterials. Expression of arsenate reductase genes and thiosulfate reductase genes in E. coli resulted in the synthesis of arsenic sulfide nanomaterials. In addition to processing the starting materials via redox enzymes, cellular components also nucleated the formation of arsenic sulfide nanomaterials. The shape of the nanomaterial was influenced by the bacterial culture, with the synthetic E. coli strain producing nanospheres and conditioned media or cultures of wild-type Shewanella sp. producing nanofibres. The diameter of these nanofibres also depended on the biological context of synthesis. These results demonstrate the potential for biogenic synthesis of nanomaterials with controlled properties by combining the natural capabilities of wild microbes with the tools from synthetic biology.

Modeling Multispecies Gene Flow Dynamics Reveals the Unique Roles of Different Horizontal Gene Transfer Mechanisms.

Horizontal gene transfer within diverse bacterial populations occurs through multiple mechanisms of exchange. The most established routes of gene transfer, transduction, transformation, and conjugation, have been characterized in detail, revealing the advantages and limitations of each mechanism. More recently, interspecies gene exchange via extracellular vesicles has been reported and characterized, making vesicle-mediated exchange a fourth, general mechanism of gene transfer. Despite an understanding of each individual pathway, how all of these mechanisms act in concert has not been explored. Here we develop a model of gene exchange in a multispecies bacterial community that takes into account the rates and limitations of all four gene transfer mechanisms. Our results reveal unique roles for each gene exchange mechanism, and highlight how multiple pathways working together are required for widespread gene exchange within diverse bacterial populations.

Quantifying the strength of quorum sensing crosstalk within microbial communities.

In multispecies microbial communities, the exchange of signals such as acyl-homoserine lactones (AHL) enables communication within and between species of Gram-negative bacteria. This process, commonly known as quorum sensing, aids in the regulation of genes crucial for the survival of species within heterogeneous populations of microbes. Although signal exchange was studied extensively in well-mixed environments, less is known about the consequences of crosstalk in spatially distributed mixtures of species. Here, signaling dynamics were measured in a spatially distributed system containing multiple strains utilizing homologous signaling systems. Crosstalk between strains containing the lux, las and rhl AHL-receptor circuits was quantified. In a distributed population of microbes, the impact of community composition on spatio-temporal dynamics was characterized and compared to simulation results using a modified reaction-diffusion model. After introducing a single term to account for crosstalk between each pair of signals, the model was able to reproduce the activation patterns observed in experiments. We quantified the robustness of signal propagation in the presence of interacting signals, finding that signaling dynamics are largely robust to interference. The ability of several wild isolates to participate in AHL-mediated signaling was investigated, revealing distinct signatures of crosstalk for each species. Our results present a route to characterize crosstalk between species and predict systems-level signaling dynamics in multispecies communities.