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Habitat spatial structure has a profound influence on bacterial life, yet there currently are no low-cost equipment-free laboratory techniques to reproduce the intricate structure of natural bacterial habitats. Here, we demonstrate the use of paper scaffolds to create landscapes spatially structured at the scales relevant to bacterial ecology. In paper scaffolds, planktonic bacteria migrate through liquid-filled pores, while the paper’s cellulose fibres serve as anchor points for sessile colonies (biofilms). Using this novel approach, we explore bacterial colonisation dynamics in different landscape topographies and characterise the community composition of Escherichia coli strains undergoing centimetre-scale range expansions in habitats structured at the micrometre scale. The bacteria-in-paper platform enables quantitative assessment of bacterial community dynamics in complex environments using everyday materials., BN/Cees Dekker Lab

Habitat spatial structure has a profound influence on bacterial life, yet there currently are no low-cost equipment-free laboratory techniques to reproduce the intricate structure of natural bacterial habitats. Here, we demonstrate the use of paper scaffolds to create landscapes spatially structured at the scales relevant to bacterial ecology. In paper scaffolds, planktonic bacteria migrate through liquid-filled pores, while the paper’s cellulose fibres serve as anchor points for sessile colonies (biofilms). Using this novel approach, we explore bacterial colonisation dynamics in different landscape topographies and characterise the community composition of Escherichia coli strains undergoing centimetre-scale range expansions in habitats structured at the micrometre scale. The bacteria-in-paper platform enables quantitative assessment of bacterial community dynamics in complex environments using everyday materials., BN/Cees Dekker Lab

Soil is a microenvironment with a fragmented (patchy) spatial structure in which many bacterial species interact. Here, we explore the interaction between the predatory bacterium Bdellovibrio bacteriovorus and its prey Escherichia coli in microfabricated landscapes. We ask how fragmentation influences the prey dynamics at the microscale and compare two landscape geometries: a patchy landscape and a continuous landscape. By following the dynamics of prey populations with high spatial and temporal resolution for many generations, we found that the variation in predation rates was twice as large in the patchy landscape and the dynamics was correlated over shorter length scales.We also found that while the prey population in the continuous landscapewas almost entirely driven to extinction, a significant part of the prey population in the fragmented landscape persisted over time. We observed significant surface-associated growth, especially in the fragmented landscape and we surmise that this sub-population is more resistant to predation. Our results thus show that microscale fragmentation can significantly influence bacterial interactions., Accepted Author Manuscript, BN/Cees Dekker Lab

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., BN/Cees Dekker Lab, BN/Bertus Beaumont Lab, BN/Juan Keymer Lab

Soil is a microenvironment with a fragmented (patchy) spatial structure in which many bacterial species interact. Here, we explore the interaction between the predatory bacterium Bdellovibrio bacteriovorus and its prey Escherichia coli in microfabricated landscapes. We ask how fragmentation influences the prey dynamics at the microscale and compare two landscape geometries: a patchy landscape and a continuous landscape. By following the dynamics of prey populations with high spatial and temporal resolution for many generations, we found that the variation in predation rates was twice as large in the patchy landscape and the dynamics was correlated over shorter length scales.We also found that while the prey population in the continuous landscapewas almost entirely driven to extinction, a significant part of the prey population in the fragmented landscape persisted over time. We observed significant surface-associated growth, especially in the fragmented landscape and we surmise that this sub-population is more resistant to predation. Our results thus show that microscale fragmentation can significantly influence bacterial interactions., Accepted Author Manuscript, BN/Cees Dekker Lab

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., BN/Cees Dekker Lab, BN/Bertus Beaumont Lab, BN/Juan Keymer Lab

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)., BN/Bionanoscience, Applied Sciences

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)., BN/Bionanoscience, Applied Sciences

In this thesis, I describe experiments aimed at understanding bacterial population dynamics in ecosystems that are spatially structured at the micro-scale. We combine microfabrication and microfluidics to create synthetic ecosystems that have a complex yet well-defined geometry and chemical composition. Bacteria that inhabit such ecosystems can be observed at high spatiotemporal resolution using fluorescence microscopy. Using this experimental approach we have gained deeper insight into diverse aspects of bacterial life., Bionanoscience, Applied Sciences

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, BN/Bionanoscience, Applied Sciences

In this thesis, I describe experiments aimed at understanding bacterial population dynamics in ecosystems that are spatially structured at the micro-scale. We combine microfabrication and microfluidics to create synthetic ecosystems that have a complex yet well-defined geometry and chemical composition. Bacteria that inhabit such ecosystems can be observed at high spatiotemporal resolution using fluorescence microscopy. Using this experimental approach we have gained deeper insight into diverse aspects of bacterial life., Bionanoscience, Applied Sciences

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, BN/Bionanoscience, Applied Sciences

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., BN/Bionanoscience, Applied Sciences

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., Bionanoscience, Applied Sciences

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., Bionanoscience, Applied Sciences

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., BN/Bionanoscience, Applied Sciences

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., BN/Bionanoscience, Applied Sciences