The genetic basis for adaptation of model-designed syntrophic co-cultures.

Understanding the fundamental characteristics of microbial communities could have far reaching implications for human health and applied biotechnology. Despite this, much is still unknown regarding the genetic basis and evolutionary strategies underlying the formation of viable synthetic communities. By pairing auxotrophic mutants in co-culture, it has been demonstrated that viable nascent E. coli communities can be established where the mutant strains are metabolically coupled. A novel algorithm, OptAux, was constructed to design 61 unique multi-knockout E. coli auxotrophic strains that require significant metabolite uptake to grow. These predicted knockouts included a diverse set of novel non-specific auxotrophs that result from inhibition of major biosynthetic subsystems. Three OptAux predicted non-specific auxotrophic strains-with diverse metabolic deficiencies-were co-cultured with an L-histidine auxotroph and optimized via adaptive laboratory evolution (ALE). Time-course sequencing revealed the genetic changes employed by each strain to achieve higher community growth rates and provided insight into mechanisms for adapting to the syntrophic niche. A community model of metabolism and gene expression was utilized to predict the relative community composition and fundamental characteristics of the evolved communities. This work presents new insight into the genetic strategies underlying viable nascent community formation and a cutting-edge computational method to elucidate metabolic changes that empower the creation of cooperative communities

Functional responses of methanogenic archaea to syntrophic growth.

Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism

Increasing sulfate levels show a differential impact on synthetic communities comprising different methanogens and a sulfate reducer

Methane producing microbial communities are of ecological and biotechnological interest. Syntrophic interactions among sulphate reducers and aceto/hydrogenotrophic and obligate hydrogenotrophic methanogens form a key component of these communities, yet, the impact of these different syntrophic routes on methane production and their stability against sulphate availability are not well understood. Here, we construct model synthetic communities using a sulphate reducer and two types of methanogens representing different methanogenesis routes. We find that tri-cultures with both routes increase methane production by almost two-fold compared to co-cultures, and are stable in the absence of sulphate. With increasing sulphate, system stability and productivity decreases, and does so faster in communities with aceto/hydrogenotrophic methanogens despite the continued presence of acetate. We show that this is due to a shift in these methanogens’ metabolism towards co-utilisation of hydrogen with acetate. These findings indicate the important role of hydrogen dynamics in the stability and productivity of syntrophic communities

Controlling microbial community dynamics through engineered metabolic dependencies

Metabolic cross-feeding is an important process that can broadly shape microbial communities. Comparative genomic analysis of >6000 sequenced bacteria from diverse environments provides evidence to suggesting that amino acid biosynthesis has been broadly optimized to reduce individual metabolic burden in favor of enhanced cross-feeding to support synergistic growth across the biosphere. Still, little is known about specific cross-feeding principles that drive the formation and maintenance of individuals within a mixed population. Here, we devised a series of synthetic syntrophic communities to probe the complex interactions underlying metabolic exchange of amino acids. We experimentally analyzed multi-member, multi-dimensional communities of Escherichia coli of increasing sophistication to assess the outcomes of synergistic cross-feeding. We find that biosynthetically costly amino acids including methionine, lysine, isoleucine, arginine and aromatics, tend to promote stronger cooperative interactions than amino acids that are cheaper to produce. Furthermore, cells that share common intermediates along branching pathways yielded more synergistic growth, but exhibited many instances of both positive and negative epistasis when these interactions scaled to higher-dimensions. This system enabled the identification of synergistic pairings and optimal expression levels of amino acid exporters of arginine, threonine and aromatics towards drastic improvements of ecosystem productivity. Tradeoffs identified in these mutualistic systems between secretion, relative abundance and absolute community productivity have implication in the evolution of cooperative behaviors. Long-term evolution of these synthetic communities highlight transporter over-expression, amino acid pool redistribution, and perturbations to nitrogen regulation as strategies to circumvent imposed metabolic dependencies. To address this potentially problematic genomic plasticity, a genetically reassigned organism is leveraged to investigate synthetic metabolic dependencies showing improved biocontainment and potential for microbial consortia control. These results improve our basic understanding of microbial syntrophy while also highlighting the utility and limitations of current approaches to modeling and controlling the dynamic complexities of microbial ecosystems. This work sets a foundation for future endeavors in microbial ecology and evolution, and presents a platform to develop better and more robust engineered synthetic communities for industrial biotechnology

Mechanism for microbial population collapse in a fluctuating resource environment.

Managing trade-offs through gene regulation is believed to confer resilience to a microbial community in a fluctuating resource environment. To investigate this hypothesis, we imposed a fluctuating environment that required the sulfate-reduce

Thermodynamic modelling of synthetic communities predicts minimum free energy requirements for sulfate reduction and methanogenesis

Microbial communities are complex dynamical systems harbouring many species interacting together to implement higher-level functions. Among these higher-level functions, conversion of organic matter into simpler building blocks by microbial communities underpins biogeochemical cycles and animal and plant nutrition, and is exploited in biotechnology. A prerequisite to predicting the dynamics and stability of community-mediated metabolic conversions is the development and calibration of appropriate mathematical models. Here, we present a generic, extendable thermodynamic model for community dynamics and calibrate a key parameter of this thermodynamic model, the minimum energy requirement associated with growth-supporting metabolic pathways, using experimental population dynamics data from synthetic communities composed of a sulfate reducer and two methanogens. Our findings show that accounting for thermodynamics is necessary in capturing the experimental population dynamics of these synthetic communities that feature relevant species using low energy growth pathways. Furthermore, they provide the first estimates for minimum energy requirements of methanogenesis (in the range of −30 kJ mol−1) and elaborate on previous estimates of lactate fermentation by sulfate reducers (in the range of −30 to −17 kJ mol−1 depending on the culture conditions). The open-source nature of the developed model and demonstration of its use for estimating a key thermodynamic parameter should facilitate further thermodynamic modelling of microbial communities

Stability of Cross-Feeding Polymorphisms in Microbial Communities

This is the final version of the article. Available from Public Library of Science via the DOI in this record.Cross-feeding, a relationship wherein one organism consumes metabolites excreted by another, is a ubiquitous feature of natural and clinically-relevant microbial communities and could be a key factor promoting diversity in extreme and/or nutrient-poor environments. However, it remains unclear how readily cross-feeding interactions form, and therefore our ability to predict their emergence is limited. In this paper we developed a mathematical model parameterized using data from the biochemistry and ecology of an E. coli cross-feeding laboratory system. The model accurately captures short-term dynamics of the two competitors that have been observed empirically and we use it to systematically explore the stability of cross-feeding interactions for a range of environmental conditions. We find that our simple system can display complex dynamics including multi-stable behavior separated by a critical point. Therefore whether cross-feeding interactions form depends on the complex interplay between density and frequency of the competitors as well as on the concentration of resources in the environment. Moreover, we find that subtly different environmental conditions can lead to dramatically different results regarding the establishment of cross-feeding, which could explain the apparently unpredictable between-population differences in experimental outcomes. We argue that mathematical models are essential tools for disentangling the complexities of cross-feeding interactions.IG was supported by a Natural Environment Research Council (NERC) Advanced Fellowship NE/E013007/3 and a European Research Council (ERC) Consolidator grant MathModExp 647292, MK was funded by a National Aeronautics and Space Administration (NASA) NPP Fellowship and NASA NNX12AD87G, IG and PR were funded by a Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/J010340/1, KS was supported by National Human Genome Research Institute (NHGRI) 2R01HG003328 - 07A1 and FR was supported by NASA NNX12AD87G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Syntrophic entanglements for propionate and acetate oxidation under thermophilic and high-ammonia conditions

Propionate is a key intermediate in anaerobic digestion processes and often accumulates in association with perturbations, such as elevated levels of ammonia. Under such conditions, syntrophic ammonia-tolerant microorganisms play a key role in propionate degradation. Despite their importance, little is known about these syntrophic microorganisms and their cross-species interactions. Here, we present metagenomes and metatranscriptomic data for novel thermophilic and ammonia-tolerant syntrophic bacteria and the partner methanogens enriched in propionate-fed reactors. A metagenome for a novel bacterium for which we propose the provisional name 'Candidatus Thermosyntrophopropionicum ammoniitolerans' was recovered, together with mapping of its highly expressed methylmalonyl-CoA pathway for syntrophic propionate degradation. Acetate was degraded by a novel thermophilic syntrophic acetate-oxidising candidate bacterium. Electron removal associated with syntrophic propionate and acetate oxidation was mediated by the hydrogen/formate-utilising methanogens Methanoculleus sp. and Methanothermobacter sp., with the latter observed to be critical for efficient propionate degradation. Similar dependence on Methanothermobacter was not seen for acetate degradation. Expression-based analyses indicated use of both H2 and formate for electron transfer, including cross-species reciprocation with sulphuric compounds and microbial nanotube-mediated interspecies interactions. Batch cultivation demonstrated degradation rates of up to 0.16 g propionate L-1 day-1 at hydrogen partial pressure 4-30 Pa and available energy was around -20 mol-1 propionate. These observations outline the multiple syntrophic interactions required for propionate oxidation and represent a first step in increasing knowledge of acid accumulation in high-ammonia biogas production systems

Common principles and best practices for engineering microbiomes

Despite broad scientific interest in harnessing the power of Earth's microbiomes, knowledge gaps hinder their efficient use for addressing urgent societal and environmental challenges. We argue hat structuring research and technology developments around a design-build-test-learn (DBTL) cycle will advance microbiome engineering and spur new discoveries on the basic scientific principles governing microbiome function. In this Review, we present key elements of an iterative DBTL cycle for microbiome engineering, focusing on generalizable approaches, including top-down and bottom-up design processes, synthetic and self-assembled construction methods, and emerging tools to analyze microbiome function. These approaches can be used to harness microbiomes for broad applications related to medicine, agriculture, energy, and the environment. We also discuss key challenges and opportunities of each approach and synthesize them into best practice guidelines for engineering microbiomes. We anticipate that adoption of a DBTL framework will rapidly advance microbiome-based biotechnologies aimed at improving human and animal health, agriculture, and enabling the bioeconomy

METHANOGEN METABOLIC FLEXIBILITY

Methanogens are obligately anaerobic archaea which produce methane as a byproduct of their respiration. They are found across a wide diversity of environments and play an important role in cycling carbon in anaerobic spaces and the removal of harmful fermentation byproducts which would otherwise inhibit other organisms. Methanogens subsist on low-energy substrates which requires them to utilize a highly efficient central metabolism which greatly favors respiratory byproducts over biomass. This metabolic strategy creates high substrate:product conversion ratios which is industrially relevant for the production of biomethane, but may also allow for the production of value-added commodities. Particularly of interest are terpene compounds, as methanogen membranes are composed of isoprenoid lipids resulting in a higher flux through isoprenoid biosynthetic pathways compared to Eukarya and Bacteria. To assess the metabolic plasticity of methanogens, our laboratory has engineered the methanogen Methanosarcina acetivorans to produce the hemiterpene isoprene. We hypothesized that isoprene producing strains would result in a decreased growth phenotype corresponding to a depletion of metabolic precursors needed for isoprenoid membrane production. We found that the engineered methanogens responded well to the modification, directing up to 4% of total towards isoprene production and increasing overall biomass despite the additional metabolic burden. Using flux balance analysis and RNA sequencing we investigated how the engineered strains respond to isoprene production and how production can be enhanced. Advisor: Nicole R. Bua