From microbiome composition to functional engineering, one step at a time.

SUMMARYCommunities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N+1/N-1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N-1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N-1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies.

The engineering of spatially linked microbial consortia - potential and perspectives.

Traditional biotechnological applications of microorganisms employ mono-cultivation or co-cultivation in well-mixed vessels disregarding the potential of spatially organized cultures. Metabolic specialization and guided species interactions facilitated through spatial isolation would enable consortia of microbes to accomplish more complex functions than currently possible, for bioproduction as well as biodegradation processes. Here, we review concepts of spatially linked microbial consortia in which spatial arrangement is optimized to increase control and facilitate new species combinations. We highlight that genome-scale metabolic network models can inform the design and tuning of synthetic microbial consortia and suggest that a standardized assembly of such systems allows the combination of 'incompatibles', potentially leading to countless novel applications.

Life on a leaf.

Microscopic water films allow bacteria to survive the seemingly dry surface of plant leaves.

Rapid Shifts in Bacterial Community Assembly under Static and Dynamic Hydration Conditions in Porous Media.

The complexity of natural soils presents a challenge to the systematic identification and disentanglement of governing processes that shape natural bacterial communities. Studies have highlighted the critical role of the soil aqueous phase in shaping interactions among soil bacterial communities. To quantify and improve the attributability of soil aqueous-phase effects, we introduced a synthetic and traceable bacterial community to simple porous microcosms and subjected the community to constant or dynamic hydration conditions. The results were expressed in terms of absolute abundance and show species-specific responses to hydration and nutrient conditions. Hydration dynamics exerted a significant influence on the fraction of less-abundant species, especially after extended incubation periods. Phylogenetic relationships did not explain the group of most abundant species. The ability to quantify species-level dynamics in a bacterial community offers an important step toward deciphering the links between community composition and functions in dynamic terrestrial environments.IMPORTANCE The composition and activity of soil bacteria are central to various ecosystem services and soil biogeochemical cycles. A key factor for soil bacterial activity is soil hydration, which is in a constant state of change due to rainfall, drainage, plant water uptake, and evaporation. These dynamic changes in soil hydration state affect the structure and function of soil bacterial communities in complex ways often unobservable in natural soil. We designed an experimental system that retains the salient features of hydrated soil yet enables systematic evaluation of changes in a representative bacterial community in response to cycles of wetting and drying. The study shows that hydration cycles affect community abundance, yet most changes in composition occur with the less-abundant species (while the successful ones remain dominant). This research offers a new path for an improved understanding of bacterial community assembly in natural environments, including bacterial growth, maintenance, and death, with a special focus on the role of hydrological factors.

Bridging the Holistic-Reductionist Divide in Microbial Ecology.

Microbial communities are inherently complex systems. To address this complexity, microbial ecologists are developing new, more elaborate laboratory models at an ever-increasing pace. These model microbial communities and habitats have opened up the exploration of new territories that lie between the simplicity and controllability of "synthetic" systems and the convolution and complexity of natural environments. Here, we discuss this classic methodological divide, we propose a conceptual perspective that integrates new research developments, and we sketch a 3-point possible roadmap to cross the divide between controllability and complexity in microbial ecology.

Cell-to-cell bacterial interactions promoted by drier conditions on soil surfaces.

Bacterial cell-to-cell interactions are in the core of evolutionary and ecological processes in soil and other environments. Under most conditions, natural soils are unsaturated where the fragmented aqueous habitats and thin liquid films confine bacterial cells within small volumes and close proximity for prolonged periods. We report effects of a range of hydration conditions on bacterial cell-level interactions that are marked by plasmid transfer between donor and recipient cells within populations of the soil bacterium Pseudomonas putida Using hydration-controlled sand microcosms, we demonstrate that the frequency of cell-to-cell contacts under prescribed hydration increases with lowering water potential values (i.e., under drier conditions where the aqueous phase shrinks and fragments). These observations were supported using a mechanistic individual-based model for linking macroscopic soil water potential to microscopic distribution of liquid phase and explicit bacterial cell interactions in a simplified porous medium. Model results are in good agreement with observations and inspire confidence in the underlying mechanisms. The study highlights important physical factors that control short-range bacterial cell interactions in soil and on surfaces, specifically, the central role of the aqueous phase in mediating bacterial interactions and conditions that promote genetic information transfer in support of soil microbial diversity.

Spatial organization of bacterial populations in response to oxygen and carbon counter-gradients in pore networks.

Microbial activity in soil is spatially heterogeneous often forming spatial hotspots that contribute disproportionally to biogeochemical processes. Evidence suggests that bacterial spatial organization contributes to the persistence of anoxic hotspots even in unsaturated soils. Such processes are difficult to observe in situ at the microscale, hence mechanisms and time scales relevant for bacterial spatial organization remain largely qualitative. Here we develop an experimental platform based on glass-etched micrometric pore networks that mimics resource gradients postulated in soil aggregates to observe spatial organization of fluorescently tagged aerobic and facultative anaerobic bacteria. Two initially intermixed bacterial species, Pseudomonas putida and Pseudomonas veronii, segregate into preferential regions promoted by opposing gradients of carbon and oxygen (such persistent coexistence is not possible in well-mixed cultures). The study provides quantitative visualization and modeling of bacterial spatial organization within aggregate-like hotspots, a key step towards developing a mechanistic representation of bacterial community organization in soil pores.

Resolving Species Level Changes in a Representative Soil Bacterial Community Using Microfluidic Quantitative PCR.

Rapid advances in genome sequencing technologies enable determination of relative bacterial abundances and community composition, yet, changes at the species level remain difficult to detect despite importance for certain ecological inferences. We present a method for extraction and direct quantification of species composition of a predefined multispecies bacterial community using microfluidic-based quantitative real-time PCR (qPCR). We employ a nested PCR approach based on universal 16S rRNA gene pre-amplification followed by detection and quantification of absolute abundance of bacterial species using microfluidic array of parallel singleplex qPCR reactions. Present microfluidic qPCR supports 2,304 simultaneous reactions on a single chip, while automatic distribution of samples and reactants minimizes pipetting errors and technical variations. The utility of the method is illustrated using a synthetic soil bacterial community grown in two contrasting environments - sand microcosms and batch cultures. The protocol entails extraction of total nucleic acid, preparation of genomic DNA, and steps for qPCR assessment of bacterial community composition. This method provides specific and sensitive quantification of bacterial species requiring only 2 ng of community DNA. Optimized extraction protocol and preamplification step allow for rapid, quantitative, and simultaneous detection of candidate species with high throughput. The proposed method offers a simple and accurate alternative to present sequencing methods especially when absolute values of species abundance are required. Quantification of changes at the species level contributes to the mechanistic understanding of the roles of particular species in a bacterial community functioning.

Biophysical processes supporting the diversity of microbial life in soil.

Soil, the living terrestrial skin of the Earth, plays a central role in supporting life and is home to an unimaginable diversity of microorganisms. This review explores key drivers for microbial life in soils under different climates and land-use practices at scales ranging from soil pores to landscapes. We delineate special features of soil as a microbial habitat (focusing on bacteria) and the consequences for microbial communities. This review covers recent modeling advances that link soil physical processes with microbial life (termed biophysical processes). Readers are introduced to concepts governing water organization in soil pores and associated transport properties and microbial dispersion ranges often determined by the spatial organization of a highly dynamic soil aqueous phase. The narrow hydrological windows of wetting and aqueous phase connectedness are crucial for resource distribution and longer range transport of microorganisms. Feedbacks between microbial activity and their immediate environment are responsible for emergence and stabilization of soil structure-the scaffolding for soil ecological functioning. We synthesize insights from historical and contemporary studies to provide an outlook for the challenges and opportunities for developing a quantitative ecological framework to delineate and predict the microbial component of soil functioning.

Cooperation in carbon source degradation shapes spatial self-organization of microbial consortia on hydrated surfaces.

Mounting evidence suggests that natural microbial communities exhibit a high level of spatial organization at the micrometric scale that facilitate ecological interactions and support biogeochemical cycles. Microbial patterns are difficult to study definitively in natural environments due to complex biodiversity, observability and variable physicochemical factors. Here, we examine how trophic dependencies give rise to self-organized spatial patterns of a well-defined bacterial consortium grown on hydrated surfaces. The model consortium consisted of two Pseudomonas putida mutant strains that can fully degrade the aromatic hydrocarbon toluene. We demonstrated that obligate cooperation in toluene degradation (cooperative mutualism) favored convergence of 1:1 partner ratio and strong intermixing at the microscale (10-100 µm). In contrast, competition for benzoate, a compound degraded independently by both strains, led to distinct segregation patterns. Emergence of a persistent spatial pattern has been predicted for surface attached microbial activity in liquid films that mediate diffusive exchanges while permitting limited cell movement (colony expansion). This study of a simple microbial consortium offers mechanistic glimpses into the rules governing the assembly and functioning of complex sessile communities, and points to general principles of spatial organization with potential applications for natural and engineered microbial systems.

Symplasmata are a clonal, conditional, and reversible type of bacterial multicellularity.

Microorganisms are capable of remarkable social behaviours, such as forming transient multicellular assemblages with properties and adaptive abilities exceeding those of individual cells. Here, we report on the formation and structure of genets known as symplasmata produced by Pantoea eucalypti bacteria. Each symplasmatum develops clonally and stochastically from a single bacterium into a membrane-delimited, capsule-embedded cluster of progeny cells and with a frequency that depends on temperature, pH, and nutrient availability. Transposon mutagenesis identified several gene products required for symplasmata formation, including master regulator LrhA, replication inhibitor CspD, polysaccharide transporter RfbX3, and autoinducer synthase PhzI. We also show that bacteria inside symplasmata are shaped irregularly with punctuated cell-to-cell contacts, metabolically responsive to environmental stimuli, dispersal-ready, and transcriptionally reprogrammed to anticipate multiple alternative futures in terms of carbon source availability. The structured and conditionable nature of symplasmata offers exciting prospects towards a mechanistic understanding of multicellular behaviours and their ecological significance.

Bacterial flagellar motility on hydrated rough surfaces controlled by aqueous film thickness and connectedness.

Recent studies have shown that rates of bacterial dispersion in soils are controlled by hydration conditions that define size and connectivity of the retained aqueous phase. Despite the ecological implications of such constraints, microscale observations of this phenomenon remain scarce. Here, we quantified aqueous film characteristics and bacterial flagellated motility in response to systematic variations in microhydrological conditions on porous ceramic surfaces that mimic unsaturated soils. We directly measured aqueous film thickness and documented its microscale heterogeneity. Flagellar motility was controlled by surface hydration conditions, as cell velocity decreased and dispersion practically ceased at water potentials exceeding -2 kPa (resulting in thinner and disconnected liquid films). The fragmentation of aquatic habitats was delineated indirectly through bacterial dispersal distances within connected aqueous clusters. We documented bacterial dispersal radii ranging from 100 to 10 µm as the water potential varied from 0 to -7 kPa, respectively. The observed decrease of flagellated velocity and dispersal ranges at lower matric potentials were in good agreement with mechanistic model predictions. Hydration-restricted habitats thus play significant role in bacterial motility and dispersal, which has potentially important impact on soil microbial ecology and diversity.

Explaining bacterial dispersion on leaf surfaces with an individual-based model (PHYLLOSIM).

We developed the individual-based model PHYLLOSIM to explain observed variation in the size of bacterial clusters on plant leaf surfaces (the phyllosphere). Specifically, we tested how different 'waterscapes' impacted the diffusion of nutrients from the leaf interior to the surface and the growth of individual bacteria on these nutrients. In the 'null' model or more complex 'patchy' models, the surface was covered with a continuous water film or with water drops of equal or different volumes, respectively. While these models predicted the growth of individual bacterial immigrants into clusters of variable sizes, they were unable to reproduce experimentally derived, previously published patterns of dispersion which were characterized by a much larger variation in cluster sizes and a disproportionate occurrence of clusters consisting of only one or two bacteria. The fit of model predictions to experimental data was about equally poor (<5%) regardless of whether the water films were continuous or patchy. Only by allowing individual bacteria to detach from developing clusters and re-attach elsewhere to start a new cluster, did PHYLLOSIM come much closer to reproducing experimental observations. The goodness of fit including detachment increased to about 70-80% for all waterscapes. Predictions of this 'detachment' model were further supported by the visualization and quantification of bacterial detachment and attachment events at an agarose-water interface. Thus, both model and experiment suggest that detachment of bacterial cells from clusters is an important mechanism underlying bacterial exploration of the phyllosphere.

Impact of mycelia on the accessibility of fluorene to PAH-degrading bacteria.

Mycelia have been recently shown to actively transport polycyclic aromatic hydrocarbons (PAH) in water-unsaturated soil over the range of centimeters, thereby efficiently mobilizing hydrophobic PAH beyond their purely diffusive transport in air and water. However, the question if mycelia-based PAH transport has an effect on PAH biodegradation was so far unsolved. To address this, we developed a laboratory model microcosm mimicking air-water interfaces in soil. Chemical analyses demonstrated transport of the PAH fluorene (FLU) by the mycelial oomycete Pythium ultimum that was grown along the air-water interfaces. Furthermore, degradation of mycelia-transported FLU by the bacterium Burkholderia sartisoli RP037-mChe was indicated. Since this organism expresses eGFP in response to a FLU flux to the cell, it was also as a bacterial reporter of FLU bioavailability in the vicinity of mycelia. Confocal laser scanning microscopy (CLSM) and image analyses revealed a significant increase of eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU. Hence, we could show that physically separated FLU becomes bioavailable to bacteria after transport by mycelia. Experiments with silicon coated glass fibers capturing mycelia-transported FLU guided us to propose a three-step mechanism of passive uptake, active transport and diffusion-driven release. These experiments were also used to evaluate the contributions of these individual steps to the overall mycelial FLU transport rate.

Draft Genome Sequence of the Phyllosphere Model Bacterium Pantoea agglomerans 299R.

Bacteria belonging to the genus Pantoea are common colonizers of plant leaf surfaces. Here, we present the draft genome sequence of Pantoea agglomerans 299R, a phyllosphere isolate that has become a model strain for studying the ecology of plant leaf-associated bacterial commensals.

The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology.

Bacteria that colonize the leaves of terrestrial plants often occur in clusters whose size varies from a few to thousands of cells. For the formation of such bacterial clusters, two non-mutually exclusive but very different mechanisms may be proposed: aggregation of multiple cells or clonal reproduction of a single cell. Here we assessed the contribution of both mechanisms on the leaves of bean plants that were colonized by the bacterium Pantoea agglomerans. In one approach, we used a mixture of green and red fluorescent P. agglomerans cells to populate bean leaves. We observed that this resulted in clusters made up of only one colour as well as two-colour clusters, thus providing evidence for both mechanisms. Another P. agglomerans bioreporter, designed to quantify the reproductive success of bacterial colonizers by proxy to the rate at which green fluorescent protein is diluted from dividing cells, revealed that during the first hours on the leaf surface, many bacteria were dividing, but not staying together and forming clusters, which is suggestive of bacterial relocation. Together, these findings support a dynamic model of leaf surface colonization, where both aggregative and reproductive mechanisms take place. The bioreporter-based approach we employed here should be broadly applicable towards a more quantitative and mechanistic understanding of bacterial colonization of surfaces in general.

Variation in local carrying capacity and the individual fate of bacterial colonizers in the phyllosphere.

Using a phyllosphere model system, we demonstrated that the term 'carrying capacity', as it is commonly used in microbial ecology, needs to be understood as the sum of many 'local carrying capacities' in order to better explain and predict the course and outcome of bacterial colonization of an environment. Using a green fluorescent protein-based bioreporter system for the quantification of reproductive success (RS) in individual Erwinia herbicola cells, we were able to reconstruct the contribution of individual immigrants to bacterial population sizes on leaves. Our analysis revealed that plant foliage represents to bacteria an environment where individual fate is determined by the local carrying capacity of the site where an immigrant cell lands. With increasing inoculation densities, the RS of most immigrants declined, suggesting that local carrying capacity under the tested conditions was linked to local nutrient availability. Fitting the observed experimental data to an adapted model of phyllosphere colonization indicated that there might exist three types of sites on leaves, which differ in their frequency of occurrence and local carrying capacity. Specifically, our data were consistent with a leaf environment that is characterized by few sites where individual immigrants can produce high numbers of offspring, whereas the remainder of the leaf offered an equal number of sites with low and medium RS. Our findings contribute to a bottom-up understanding of bacterial colonization of leaf surfaces, which includes a quantifiable role of chance in the experience at the individual level and in the outcome at the population level.

Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2.

Long-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect long-chain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspension assays but very slightly only to tetradecane, in line with what is expected from its low water solubility. In contrast, long-term assays (up to 5 days) with A. borkumensis bioreporters showed strong induction with tetradecane and crude oil. Gel-immobilized A. borkumensis reporter cells were used to demonstrate tetradecane and crude oil bioavailability at a distance from a source. Alcanivorax borkumensis bioreporters induced fivefold more rapid and more strongly when allowed physical contact with the oil phase in standing flask assays, suggesting a major contribution of adhered cells to the overall reporter signal. Using the flask assays we further demonstrated the effect of oleophilic nutrients and biosurfactants on oil availability and degradation by A. borkumensis. The fluorescence signal from flask assays could easily be captured with a normal digital camera, making such tests feasible to be carried out on, e.g. marine oil responder vessels in case of oil accidents.

Effect of two types of biosurfactants on phenanthrene availability to the bacterial bioreporter Burkholderia sartisoli strain RP037.

Biosurfactants are tensio-active agents that have often been proposed as a means to enhance the aqueous solubility of hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). Biosurfactant-producing bacteria such as those belonging to the genus Pseudomonas might therefore enhance PAH availability to PAH-degrading bacteria. We tested the effects of two types of biosurfactants produced by Pseudomonas sp., cyclic lipopeptides and rhamnolipids, on phenanthrene bioavailability. Bioavailability was judged from growth rates on phenanthrene and from specific induction of a phenanthrene-responsive GFP-reporter in Burkholderia sartisoli strain RP037. Co-culturing of strain RP037 with the lipopeptide-producing bacterium Pseudomonas putida strain PCL1445 enhanced GFP expression compared to a single culture, but this effect was not significantly different when strain RP037 was co-cultivated with a non-lipopeptide-producing mutant of P. putida. The addition of partially purified supernatant extracts from the P. putida lipopeptide producer equally did not unequivocally enhance phenanthrene bioavailability to strain RP037 compared to controls. In contrast, a 0.1% rhamnolipid solution strongly augmented RP037 growth rates on phenanthrene and led to a significantly larger proportion of cells in culture with high GFP expression. Our data therefore suggest that biosurfactant effects may be strongly dependent on the strain and type of biosurfactant.

Development of a multistrain bacterial bioreporter platform for the monitoring of hydrocarbon contaminants in marine environments.

Petroleum hydrocarbons are common contaminants in marine and freshwater aquatic habitats, often occurring as a result of oil spillage. Rapid and reliable on-site tools for measuring the bioavailable hydrocarbon fractions, i.e., those that are most likely to cause toxic effects or are available for biodegradation, would assist in assessing potential ecological damage and following the progress of cleanup operations. Here we examined the suitability of a set of different rapid bioassays (2-3 h) using bacteria expressing the LuxAB luciferase to measure the presence of short-chain linear alkanes, monoaromatic and polyaromatic compounds, biphenyls, and DNA-damaging agents in seawater after a laboratory-scale oil spill. Five independent spills of 20 mL of NSO-1 crude oil with 2 L of seawater (North Sea or Mediterranean Sea) were carried out in 5 L glass flasks for periods of up to 10 days. Bioassays readily detected ephemeral concentrations of short-chain alkanes and BTEX (i.e., benzene, toluene, ethylbenzene, and xylenes) in the seawater within minutes to hours after the spill, increasing to a maximum of up to 80 muM within 6-24 h, after which they decreased to low or undetectable levels. The strong decrease in short-chain alkanes and BTEX may have been due to their volatilization or biodegradation, which was supported by changes in the microbial community composition. Two- and three-ring PAHs appeared in the seawater phase after 24 h with a concentration up to 1 muM naphthalene equivalents and remained above 0.5 muM for the duration of the experiment. DNA-damage-sensitive bioreporters did not produce any signal with the oil-spilled aqueous-phase samples, whereas bioassays for (hydroxy)biphenyls showed occasional responses. Chemical analysis for alkanes and PAHs in contaminated seawater samples supported the bioassay data, but did not show the typical ephemeral peaks observed with the bioassays. We conclude that bacterium-based bioassays can be a suitable alternative for rapid on-site quantitative measurement of hydrocarbons in seawater.