Spatial biology of Ising-like synthetic genetic networks.

BACKGROUND: Understanding how spatial patterns of gene expression emerge from the interaction of individual gene networks is a fundamental challenge in biology. Developing a synthetic experimental system with a common theoretical framework that captures the emergence of short- and long-range spatial correlations (and anti-correlations) from interacting gene networks could serve to uncover generic scaling properties of these ubiquitous phenomena. RESULTS: Here, we combine synthetic biology, statistical mechanics models, and computational simulations to study the spatial behavior of synthetic gene networks (SGNs) in Escherichia coli quasi-2D colonies growing on hard agar surfaces. Guided by the combined mechanisms of the contact process lattice simulation and two-dimensional Ising model (CPIM), we describe the spatial behavior of bi-stable and chemically coupled SGNs that self-organize into patterns of long-range correlations with power-law scaling or short-range anti-correlations. These patterns, resembling ferromagnetic and anti-ferromagnetic configurations of the Ising model near critical points, maintain their scaling properties upon changes in growth rate and cell shape. CONCLUSIONS: Our findings shed light on the spatial biology of coupled and bistable gene networks in growing cell populations. This emergent spatial behavior could provide insights into the study and engineering of self-organizing gene patterns in eukaryotic tissues and bacterial consortia.

Ecological succession and the competition-colonization trade-off in microbial communities.

BACKGROUND: During range expansion in spatially distributed habitats, organisms differ from one another in terms of their patterns of localization versus propagation. To exploit locations or explore the landscape? This is the competition-colonization trade-off, a dichotomy at the core of ecological succession. In bacterial communities, this trade-off is a fundamental mechanism towards understanding spatio-temporal fluxes in microbiome composition. RESULTS: Using microfluidics devices as structured bacterial habitats, we show that, in a synthetic two-species community of motile strains, Escherichia coli is a fugitive species, whereas Pseudomonas aeruginosa is a slower colonizer but superior competitor. We provide evidence highlighting the role of succession and the relevance of this trade-off in the community assembly of bacteria in spatially distributed patchy landscapes. Furthermore, aggregation-dependent priority effects enhance coexistence which is not possible in well-mixed environments. CONCLUSIONS: Our findings underscore the interplay between micron-scale landscape structure and dispersal in shaping biodiversity patterns in microbial ecosystems. Understanding this interplay is key to unleash the technological revolution of microbiome applications.

Variance in Landscape Connectivity Shifts Microbial Population Scaling.

Understanding mechanisms shaping distributions and interactions of soil microbes is essential for determining their impact on large scale ecosystem services, such as carbon sequestration, climate regulation, waste decomposition, and nutrient cycling. As the functional unit of soil ecosystems, we focus our attention on the spatial structure of soil macroaggregates. Emulating this complex physico-chemical environment as a patchy habitat landscape we investigate on-chip the effect of changing the connectivity features of this landscape as Escherichia coli forms a metapopulation. We analyze the distributions of E. coli occupancy using Taylor's law, an empirical law in ecology which asserts that the fluctuations in populations is a power law function of the mean. We provide experimental evidence that bacterial metapopulations in patchy habitat landscapes on microchips follow this law. Furthermore, we find that increased variance of patch-corridor connectivity leads to a qualitative transition in the fluctuation scaling. We discuss these results in the context of the spatial ecology of microbes in soil.

Emergence of Resistant Escherichia coli Mutants in Microfluidic On-Chip Antibiotic Gradients.

Spatiotemporal structures and heterogeneities are common in natural habitats, yet their role in the evolution of antibiotic resistance is still to be uncovered. We applied a microfluidic gradient generator device to study the emergence of resistant bacteria in spatial ciprofloxacin gradients. We observed biofilm formation in regions with sub-inhibitory concentrations of antibiotics, which quickly expanded into the high antibiotic regions. In the absence of an explicit structure of the habitat, this multicellular formation led to a spatial structure of the population with local competition and limited migration. Therefore, such structures can function as amplifiers of selection and aid the spread of beneficial mutations. We found that the physical environment itself induces stress-related mutations that later prove beneficial when cells are exposed to antibiotics. This shift in function suggests that exaptation occurs in such experimental scenarios. The above two processes pave the way for the subsequent emergence of highly resistant specific mutations.

Do microenvironmental changes disrupt multicellular organisation with ageing, enacting and favouring the cancer cell phenotype?

Cancer is a singular cellular state, the emergence of which destabilises the homeostasis reached through the evolution to multicellularity. We present the idea that the onset of the cellular disobedience to the metazoan functional and structural architecture, known as the cancer phenotype, is triggered by changes in the cell's external environment that occur with ageing: what ensues is a breach of the social contract of multicellular life characteristic of metazoans. By integrating old ideas with new evidence, we propose that with ageing the environmental information that maintains a multicellular organisation is eroded, rewiring internal processes of the cell, and resulting in an internal shift towards an ancestral condition resulting in the pseudo-multicellular cancer phenotype. Once that phenotype emerges, a new local social contract is built, different from the homeostatic one, leading to tumour formation and the foundation of a novel local ecosystem.

Expansion, Exploitation and Extinction: Niche Construction in Ephemeral Landscapes.

We aim to understand general consequences of niche construction on metapopulation dynamics in ephemeral landscapes. To this effect, a contact process-like stochastic spatial model is introduced where local populations colonize and go extinct on a dynamic landscape of habitable and destroyed patches. In contrast to previous models, where the extinction threshold is a consequence of available niche rendered by global rates of patch destruction/renewal, here we investigate how the metapopulation persists when they are the sole generators of their own niche. Niche construction is full-filled by localized populations through the transformation of destroyed patches in their neighborhood to viable habitat for future colonization. With this theoretical framework we are able to address the dual nature of niche construction by investigating the ephemerality of the landscape (destruction rate) and the continuum of population level strategies, where construction comes at a cost to colonization. Using mean field theory and Monte Carlo simulations of the model, we are able to quantify optimal population level strategies in a wide range of ephemeral landscapes. Interestingly, we observe qualitative differences at the extinction threshold between analytic and numeric results. Investigating this discrepancy further, we find that increasing niche construction neighborhood in the spatial model leads to two interrelated effects i) an increased rate in range expansion ii) a loss in resiliency and return of the discontinuous transition at the extinction threshold. Furthermore, in the discontinuous regime of the model, spatial clustering prior to a critical transition disappears. This is a significant finding as spatial clustering has been considered to be an early warning signal before ecosystems reach their 'tipping point'. In addition to maintaining stability, we find local niche construction strategies have an advantage when in scramble competition with an exploiter strategy because of their ability to monopolize the constructed niche due to spatial adjacency. As the niche construction neighborhood expands this advantage disappears and the exploiter strategy out-competes the niche constructor. In some cases the exploiter pushes the niche constructor to extinction, thus a tragedy of the commons ensues leading to 'ecological suicide' and a collapse of the niche.

Application of Microfluidics in Experimental Ecology: The Importance of Being Spatial.

Microfluidics is an emerging technology that is used more and more in biology experiments. Its capabilities of creating precisely controlled conditions in cellular dimensions make it ideal to explore cell-cell and cell-environment interactions. Thus, a wide spectrum of problems in microbial ecology can be studied using engineered microbial habitats. Moreover, artificial microfluidic ecosystems can serve as model systems to test ecology theories and principles that apply on a higher level in the hierarchy of biological organization. In this mini review we aim to demonstrate the versatility of microfluidics and the diversity of its applications that help the advance of microbiology, and in more general, experimental ecology.

Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering.

The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential use in a high school environment to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for education, scientific research and bioengineering. All the resources developed here are available under open source licenses.

What Does Not Kill You Makes You Stronger.

Colicin production is an extreme form of labor division; cells lyse after making the toxin! Stochastic phenotype switching allows producers to outcompete sensitive strains since colicin release frees up vacancy. If patch dynamics does not kill you, it stimulates adaptation to a dynamic habitat landscape which selects for rapid dispersal.

Density-dependent adaptive resistance allows swimming bacteria to colonize an antibiotic gradient.

During antibiotic treatment, antibiotic concentration gradients develop. Little is know regarding the effects of antibiotic gradients on populations of nonresistant bacteria. Using a microfluidic device, we show that high-density motile Escherichia coli populations composed of nonresistant bacteria can, unexpectedly, colonize environments where a lethal concentration of the antibiotic kanamycin is present. Colonizing bacteria establish an adaptively resistant population, which remains viable for over 24 h while exposed to the antibiotic. Quantitative analysis of multiple colonization events shows that collectively swimming bacteria need to exceed a critical population density in order to successfully colonize the antibiotic landscape. After colonization, bacteria are not dormant but show both growth and swimming motility under antibiotic stress. Our results highlight the importance of motility and population density in facilitating adaptive resistance, and indicate that adaptive resistance may be a first step to the emergence of genetically encoded resistance in landscapes of antibiotic gradients.

Multi-color imaging of the bacterial nucleoid and division proteins with blue, orange, and near-infrared fluorescent proteins.

Studies of the spatiotemporal protein dynamics within live bacterial cells impose a strong demand for multi-color imaging. Despite the increasingly large collection of fluorescent protein (FP) variants engineered to date, only a few of these were successfully applied in bacteria. Here, we explore the performance of recently engineered variants with the blue (TagBFP), orange (TagRFP-T, mKO2), and far-red (mKate2) spectral colors by tagging HU, LacI, MinD, and FtsZ for visualizing the nucleoid and the cell division process. We find that, these FPs outperformed previous versions in terms of brightness and photostability at their respective spectral range, both when expressed as cytosolic label and when fused to native proteins. As this indicates that their folding is sufficiently fast, these proteins thus successfully expand the applicable spectra for multi-color imaging in bacteria. A near-infrared protein (eqFP670) is found to be the most red-shifted protein applicable to bacteria so far, with brightness and photostability that are advantageous for cell-body imaging, such as in microfluidic devices. Despite the multiple advantages, we also report the alarming observation that TagBFP directly interacts with TagRFP-T, causing interference of localization patterns between their fusion proteins. Our application of diverse FPs for endogenous tagging provides guidelines for future engineering of fluorescent fusions in bacteria, specifically: (1) The performance of newly developed FPs should be quantified in vivo for their introduction into bacteria; (2) spectral crosstalk and inter-variant interactions between FPs should be carefully examined for multi-color imaging; and (3) successful genomic fusion to the 5(')-end of a gene strongly depends on the translational read-through of the inserted coding sequence.

Symmetry and scale orient Min protein patterns in shaped bacterial sculptures.

The boundary of a cell defines the shape and scale of its subcellular organization. However, the effects of the cell's spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be 'sculpted' into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shaped Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2 × 1 × 1 to 11 × 6 × 1 µm(3), Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale with the cell size within a characteristic length range of 3-6 µm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling and an adaptive range, when and only when facilitated by the three-dimensional confinement of the cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood.

The idiosyncrasy of spatial structure in bacterial competition.

BACKGROUND: The spatial structure of a habitat can have a strong impact on community dynamics. Different experimental approaches exist to explore the effect of spatial structure on bacterial communities. To investigate the effect of 'space', a single implementation of spatial structure is often contrasted to bacterial community dynamics in well-mixed cultures. While such comparisons are useful, it is likely that the observed dynamics will be particular to the specific experimental implementation of spatial structure. In order to address this question, we track the community dynamics of a two-strain Escherichia coli community in various spatial habitats and relate the observed dynamics to the structure of a habitat. RESULTS: By tracking the community dynamics of rpoS wild-type and mutant E. coli in radially expanding colonies on solid and semi-solid agar plates, we find that the mutant strain outcompetes the wild-type on semi-solid agar plates, whereas the two strains coexist on solid agar. We compare these results to previous studies in which the same two strains were shown to coexist in habitats spatially structured by microfabrication, while the mutant outcompeted the wild-type in well-mixed batch cultures. Together, these observations show that different implementations of space may result in qualitatively different community dynamics. Furthermore, we argue that the same competitive outcome (e.g. coexistence) may arise from distinct underlying dynamics in different experimental implementations of spatial structure. CONCLUSIONS: Our observations demonstrate that different experimental implementations of spatial structure may not only lead to quantitatively different communities (changes in the relative abundance of types) but can also lead to qualitatively different outcomes of long-term community dynamics (coexistence versus extinction and loss of biodiversity).

Nutrient-responsive regulation determines biodiversity in a colicin-mediated bacterial community.

BACKGROUND: Antagonistic interactions mediated by antibiotics are strong drivers of bacterial community dynamics which shape biodiversity. Colicin production by Escherichia coli is such an interaction that governs intraspecific competition and is involved in promoting biodiversity. It is unknown how environmental cues affect regulation of the colicin operon and thus influence antibiotic-mediated community dynamics. RESULTS: Here, we investigate the community dynamics of colicin-producing, -sensitive, and -resistant/non-producer E. coli strains that colonize a microfabricated spatially-structured habitat. Nutrients are found to strongly influence community dynamics: when growing on amino acids and peptides, colicin-mediated competition is intense and the three strains do not coexist unless spatially separated at large scales (millimeters). Surprisingly, when growing on sugars, colicin-mediated competition is minimal and the three strains coexist at the micrometer scale. Carbon storage regulator A (CsrA) is found to play a key role in translating the type of nutrients into the observed community dynamics by controlling colicin release. We demonstrate that by mitigating lysis, CsrA shapes the community dynamics and determines whether the three strains coexist. Indeed, a mutant producer that is unable to suppress colicin release, causes the collapse of biodiversity in media that would otherwise support co-localized growth of the three strains. CONCLUSIONS: Our results show how the environmental regulation of an antagonistic trait shapes community dynamics. We demonstrate that nutrient-responsive regulation of colicin release by CsrA, determines whether colicin producer, resistant non-producer, and sensitive strains coexist at small spatial scales, or whether the sensitive strain is eradicated. This study highlights how molecular-level regulatory mechanisms that govern interference competition give rise to community-level biodiversity patterns.

The effects of chemical interactions and culture history on the colonization of structured habitats by competing bacterial populations.

BACKGROUND: Bacterial habitats, such as soil and the gut, are structured at the micrometer scale. Important aspects of microbial life in such spatial ecosystems are migration and colonization. Here we explore the colonization of a structured ecosystem by two neutrally labeled strains of Escherichia coli. Using time-lapse microscopy we studied the colonization of one-dimensional arrays of habitat patches linked by connectors, which were invaded by the two E. coli strains from opposite sides. RESULTS: The two strains colonize a habitat from opposite sides by a series of traveling waves followed by an expansion front. When population waves collide, they branch into a continuing traveling wave, a reflected wave and a stationary population. When the two strains invade the landscape from opposite sides, they remain segregated in space and often one population will displace the other from most of the habitat. However, when the strains are co-cultured before entering the habitats, they colonize the habitat together and do not separate spatially. Using physically separated, but diffusionally coupled, habitats we show that colonization waves and expansion fronts interact trough diffusible molecules, and not by direct competition for space. Furthermore, we found that colonization outcome is influenced by a culture's history, as the culture with the longest doubling time in bulk conditions tends to take over the largest fraction of the habitat. Finally, we observed that population distributions in parallel habitats located on the same device and inoculated with cells from the same overnight culture are significantly more similar to each other than to patterns in identical habitats located on different devices inoculated with cells from different overnight cultures, even tough all cultures were started from the same -80°C frozen stock. CONCLUSIONS: We found that the colonization of spatially structure habitats by two interacting populations can lead to the formation of complex, but reproducible, spatiotemporal patterns. Furthermore, we showed that chemical interactions between two populations cause them to remain spatially segregated while they compete for habitat space. Finally, we observed that growth properties in bulk conditions correlate with the outcome of habitat colonization. Together, our data show the crucial roles of chemical interactions between populations and a culture's history in determining the outcome of habitat colonization.

Nanoscale probing the kinetics of oriented bacterial cell growth using atomic force microscopy.

Probing oriented bacterial cell growth on the nanoscale: A novel open-top micro-channel is developed to facilitate the AFM imaging of physically trapped but freely growing bacteria. The growth curves of individual Escherichia coli cells with nanometer resolution and their kinetic nano-mechanical properties are quantitatively measured.

Spatial structure facilitates cooperation in a social dilemma: empirical evidence from a bacterial community.

Cooperative organisms are ubiquitous in nature, despite their vulnerability to exploitation by cheaters. Although numerous theoretical studies suggest that spatial structure is critical for cooperation to persist, the spatial ecology of microbial cooperation remains largely unexplored experimentally. By tracking the community dynamics of cooperating (rpoS wild-type) and cheating (rpoS mutant) Escherichia coli in well-mixed flasks and microfabricated habitats, we demonstrate that spatial structure stabilizes coexistence between wild-type and mutant and thus facilitates cooperator maintenance. We develop a method to interpret our experimental results in the context of game theory, and show that the game wild-type and mutant bacteria play in an unstructured environment changes markedly over time, and eventually obeys a prisoner's dilemma leading to cheater dominance. In contrast, when wild-type and mutant E. coli co-inhabit a spatially-structured habitat, cooperators and cheaters coexist at intermediate frequencies. Our findings show that even in microhabitats lacking patchiness or spatial heterogeneities in resource availability, surface growth allows cells to form multi-cellular aggregates, yielding a self-structured community in which cooperators persist.

Robustness and accuracy of cell division in Escherichia coli in diverse cell shapes.

Cell division in typical rod-shaped bacteria such as Escherichia coli shows a remarkable plasticity in being able to adapt to a variety of irregular cell shapes. Here, we investigate the roles of the Min system and the nucleoid-occlusion factor SlmA in supporting this adaptation. We study "squeezed" E. coli in narrow nanofabricated channels where these bacteria exhibit highly irregular shapes and large volumes. Despite the severely anomalous morphologies we find that most of these bacteria maintain their ability to divide into two equally sized daughters with an accuracy comparable to that of normal rod-shaped cells (about 4%). Deletion of either slmA or minC shows that the molecular systems associated with these genes are largely dispensable for accurate cell division in these irregular cell shapes. Using fluorescence time-lapse microscopy, we determine that the functionality of the Min system is affected by the cell shape, whereas the localization of a nucleoid relative to the cell division proteins (the divisome) remains unperturbed in a broad spectrum of morphologies, consistent with nucleoid occlusion. The observed positioning of the nucleoid relative to the divisome appears not to be affected by the nucleoid-occlusion factor SlmA. The current study underscores the importance of nucleoid occlusion in positioning the divisome and shows that it is robust against shape irregularities.

Bacterial growth and motility in sub-micron constrictions.

In many naturally occurring habitats, bacteria live in micrometer-size confined spaces. Although bacterial growth and motility in such constrictions is of great interest to fields as varied as soil microbiology, water purification, and biomedical research, quantitative studies of the effects of confinement on bacteria have been limited. Here, we establish how Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria can grow, move, and penetrate very narrow constrictions with a size comparable to or even smaller than their diameter. We show that peritrichously flagellated E. coli and B. subtilis are still motile in microfabricated channels where the width of the channel exceeds their diameters only marginally (approximately 30%). For smaller widths, the motility vanishes but bacteria can still pass through these channels by growth and division. We observe E. coli, but not B. subtilis, to penetrate channels with a width that is smaller than their diameter by a factor of approximately 2. Within these channels, bacteria are considerably squeezed but they still grow and divide. After exiting the channels, E. coli bacteria obtain a variety of anomalous cell shapes. Our results reveal that sub-micron size pores and cavities are unexpectedly prolific bacterial habitats where bacteria exhibit morphological adaptations.

Computation of mutual fitness by competing bacteria.

Competing populations in shared spaces with nonrenewable resources do not necessarily wage a battle for dominance at the cost of extinction of the less-fit strain if there are fitness advantages to the presence of the other strain. We report on the use of nanofabricated habitat landscapes to study the population dynamics of competing wild type and a growth advantage in stationary phase (GASP) mutant strains of Escherichia coli in a sealed and heterogeneous nutrient environment. Although GASP mutants are competitors with wild-type bacteria, we find that the 2 strains cooperate to maximize fitness (long-term total productivity) via spatial segregation: despite their very close genomic kinship, wild-type populations associate with wild-type populations and GASP populations with GASP populations. Thus, wild-type and GASP strains avoid each other locally, yet fitness is enhanced for both strains globally. This computation of fitness enhancement emerges from the local interaction among cells but maximizes global densities. At present we do not understand how fluctuations in both spatial and temporal dimensions lead to the emergent computation and how multilevel aggregates produce this collective adaptation.