The microbiome actively influences antimicrobial resistance (AMR) dynamics by shaping both ecological and evolutionary processes. However, the extent of its role in resistance emergence, transmission and persistence remains unclear. Traditional AMR research has mainly focused on genetic mechanisms and pathogen-level dynamics. In contrast, the intersection of AMR and the microbiome, including resistance-gene reservoirs, microbial competition and community-mediated selection, remains poorly represented, especially in a modelling context. Here we present a structured framework for incorporating microbiome-AMR interactions into predictive models. We identify key microbiome-mediated processes shaping AMR across different levels of complexity, describe how these can be quantitatively integrated into models, and identify critical data gaps that limit current approaches. By bridging microbiome ecology, AMR biology and mathematical modelling, we set out research priorities and strategies to improve resistance prediction and guide microbiome-targeted interventions.

Crohn’s disease (CD) and ulcerative colitis (UC) are chronic relapsing inflammatory bowel disorders (IBD), the pathogenesis of which is uncertain but includes genetic susceptibility factors, immune-mediated tissue injury and environmental influences, most of which appear to act via the gut microbiome. We hypothesized that host-microbe alterations could be used to prognostically stratify patients experiencing relapses up to four years after endoscopy. We therefore examined multiple omics data, including published and new datasets, generated from paired inflamed and non-inflamed mucosal biopsies from 142 patients with IBD (54 CD; 88 UC) and from 34 control (non-diseased) biopsies. The relapse-predictive potential of 16S rRNA gene and transcript amplicons (standing and active microbiota) were investigated along with host transcriptomics, epigenomics and genetics. While standard single-omics analysis could not distinguish between patients who relapsed and those that remained in remission within four years of colonoscopy, we did find an association between the number of flares and a patient’s succinotype. Our multi-omics machine learning approach was also able to predict relapse when combining features from the microbiome and human host. Therefore multi-omics, rather than single omics, better predicts relapse within 4 years of colonoscopy, while a patient’s succinotype is associated with a higher frequency of relapses.

Background: Bilberries are effective in inducing clinical, endoscopic, and biochemical improvement in ulcerative colitis (UC) patients. The aim of this study was to investigate the efficacy of anthocyanin-rich extract (ACRE), the bioactive ingredient of bilberries, in a controlled clinical trial in moderate-to-severe UC. Methods: A multi-center, randomized, placebo-controlled, double-blind study with a parallel group was conducted. Initially, the study was planned for 100 patients; nevertheless, it prematurely ended due to COVID-19. Patients had moderate-to-severe active UC at screening (a Mayo score of 6–12, an endoscopic sub-score ≥ 2) and were randomized at baseline. The primary endpoint was a clinical response (week 8, a total Mayo score reduction ≥ 3 points). Fecal calprotectin (FC) and a centrally read endoscopic response were among the secondary endpoints. Results: Out of 48 patients (6 Swiss centers), 34 were randomized. Eighteen ACRE and eight placebo patients could be analyzed (per protocol set). Half (9/18) of ACRE patients and 3/8 of placebo patients responded clinically (p = 0.278). An improvement in the Mayo score was observed in the ACRE arm (77.8% vs. 62.5% placebo). FC dropped from 1049 ± 1139 to 557 ± 756 μg/g for ACRE but not for the placebo group (947 ± 1039 to 1040 ± 1179; p = 0.035). Serious adverse events were rare. Conclusions: ACRE treatment did not yield significant superiority to the placebo. Furthermore, the placebo response was unusually high. Moreover, there was a significant calprotectin decrease at the end of treatment, indicative of ACRE efficacy in UC.

Diversity-generating retroelements (DGRs) are used by bacteria, archaea, and viruses as a targeted mutagenesis tool. Through error-prone reverse transcription, DGRs introduce random mutations at specific genomic loci, enabling rapid evolution of these targeted genes. However, the function and benefits of DGR-diversified proteins in cellular hosts remain elusive. We find that 82% of DGRs from one of the major monophyletic lineages of DGR reverse transcriptases are encoded by multicellular bacteria, which often have two or more DGR loci in their genomes. Using the multicellular purple sulfur bacterium Thiohalocapsa sp. PB-PSB1 as an example, we characterized nine distinct DGR loci capable of generating 10282 different combinations of target proteins. With environmental metagenomes from individual Thiohalocapsa aggregates, we show that most of PB-PSB1's DGR target genes are diversified across its biogeographic range, with spatial heterogeneity in the diversity of each locus. In Thiohalocapsa PB-PSB1 and other bacteria hosting this lineage of cellular DGRs, the diversified target genes are associated with NACHT-domain anti-phage defenses and putative ternary conflict systems previously shown to be enriched in multicellular bacteria. We propose that these DGR-diversified targets act as antigen sensors that confer a form of adaptive immunity to their multicellular consortia, though this remains to be experimentally tested. These findings could have implications for understanding the evolution of multicellularity, as the NACHT-domain anti-phage systems and ternary systems share both domain homology and conceptual similarities with the innate immune and programmed cell death pathways of plants and metazoans.

The human gut microbiome produces and consumes a variety of compounds that interact with the host and impact health. Succinate is of particular interest as it intersects with both host and microbiome metabolism. However, which gut bacteria are most responsible for the consumption of intestinal succinate is poorly understood. Here, we build upon an enrichment-based whole fecal sample culturing approach and identify two main bacterial taxa that are responsible for succinate consumption in the human intestinal microbiome,PhascolarctobacteriumandDialister. These two taxa have the hallmark of a functional guild and are strongly mutual exclusive across over 20,000 fecal samples in nearly 100 cohorts and can thus be used to assign a robust ‘succinotype’ to an individual. We show that they differ with respect to their rate of succinate consumptionin vitroand that this is associated with higher concentrations of fecal succinate. Finally, individuals suffering from inflammatory bowel disease (IBD) are more likely to have theDialistersuccinotype compared to healthy subjects. The functionally meaningful classification of human intestinal microbiota based on ‘succinotype’ thus builds a bridge between microbiome function and IBD pathophysiology related to succinate.

AbstractObjectiveAnthocyanins (AC) have been shown to elicit anti-inflammatory and antioxidant effects in several animal models of colitis. Furthermore, AC lowered biochemical disease activity in our double-blind randomized trial in patients with ulcerative colitis. Here, we report on the changes in the faecal microbiome composition in the patient upon AC exposure in this trial.MethodsUlcerative colitis patients received a 3g daily dose of an AC-rich bilberry extract (ACRE) for eight weeks. We determined the microbiome composition in longitudinal stool samples from 24 patients and quantified the degree of change over time. We also correlated the relative abundances of individual microbial taxa at different time points to faecal concentration measurements of calprotectin.ResultsMicrobiome compositions did not change over time as a result of the intervention, both in terms of alpha and beta diversity. Before the intervention, Haemophilus parainfluenzae was positively correlated with faecal calprotectin concentrations, and this correlation persisted in placebo-treated subjects throughout the study. In contrast, the correlation between H. parainfluenzae and the concentration of faecal calprotectin vanished in ACRE-treated subjects, while the relative abundance of H. parainfluenzaae did not change. Conclusion: Our results suggest that ACRE treatment mitigates the contribution of H. parainfluenzae to inflammation. Further research is warranted to better comprehend the role of microbial composition in response to medical therapy including AC-rich extract in ulcerative colitis patients.Key messages1. What is already known on this topicAnthocyanins are a promising therapeutic alternative for ulcerative colitis patients given their effect on faecal calprotectin, excellent safety profile, and low costs.2. What this study addsThe administration of anthocyanin-rich extract to ulcerative colitis patients in a Phase 2a study reduced faecal calprotectin concentrations by mitigating the contribution from the pathobiont genus Haemophilus but did not affect the overall microbiome composition.3. How this study might affect research, practice or policyOur results could aid in targeted treatment of ulcerative colitis patients with anthocyanins, according to individual microbial composition.

Anthocyanins (ACs) have been shown to elicit anti-inflammatory and antioxidant effects in animal models of ulcerative colitis (UC). Furthermore, we previously observed in a double-blind randomized trial in UC patients that biochemical disease activity tended to be lower in patients that were exposed to AC. Here, we report on the changes in the fecal microbiome composition in these patients upon AC exposure. UC patients received a 3 g daily dose of an AC-rich bilberry extract (ACRE) for eight weeks. We determined the microbiome composition in longitudinal stool samples from 24 patients and quantified the degree of change over time. We also correlated the relative abundances of individual microbial taxa at different timepoints to fecal concentrations of calprotectin, a proxy for inflammation. Microbiome composition did not change over time as a result of the intervention, in terms of both alpha and beta diversity. However, before the intervention, the abundance of Haemophilus parainfluenzae was positively correlated with fecal calprotectin concentrations, and this correlation persisted in placebo-treated subjects throughout the study. In contrast, the correlation between H. parainfluenzae and calprotectin vanished in ACRE-treated subjects, while the relative abundance of H. parainfluenzae did not change. Our results suggest that ACRE treatment mitigates the contribution of H. parainfluenzae to inflammation. Further research is warranted to better comprehend the role of microbial composition in response to medical therapy including AC-rich extract in UC patients.

The anaerobic cultivation of fecal microbiota is a promising approach to investigating how gut microbial communities respond to specific intestinal conditions and perturbations. Here, we describe a flexible protocol using 96-deepwell plates to cultivate stool-derived gut microbiota. Our protocol aims to address gaps in high-throughput culturing in an anaerobic chamber. We characterized the influence of the gas phase on the medium chemistry and microbial physiology and introduced a modular medium preparation process to enable the testing of several conditions simultaneously. Furthermore, we identified a medium formulation that maximized the compositional similarity of ex vivo cultures and donor microbiota while limiting the bloom of Enterobacteriaceae. Lastly, we validated the protocol by demonstrating that cultivated fecal microbiota responded similarly to dietary fibers (resistant dextrin, soluble starch) and drugs (ciprofloxacin, 5-fluorouracil) as reported in vivo. This high-throughput cultivation protocol has the potential to facilitate culture-dependent studies, accelerate the discovery of gut microbiota-diet-drug-host interactions, and pave the way to personalized microbiota-centered interventions.

AbstractBackgroundIn an open label pilot study dried bilberries were effective in inducing clinical, endoscopic and biochemical improvement in ulcerative colitis (UC) patients. Aim was the investigation of efficacy of anthocyanin rich extract (ACRE), the presumptive active ingredient of bilberries, in a controlled clinical trial in moderate-severe UC.MethodsWe performed a multicenter randomized, placebo-controlled, double-blind study (planned initially for 100 patients; premature termination due to COVID-19 pandemic). Patients had moderate-severe active UC at screening (Mayo-score 6-12, endoscopic sub-score at least 2) and were randomized at baseline (verum: placebo, 2:1). Continuation of all UC-directed stable medical therapy was allowed. Primary endpoint was clinical response at week 8 (reduction of total Mayo-score at least 3 points). Biochemical (fecal calprotectin) and centrally-read endoscopic response were amongst the secondary endpoints.ResultsOut of 48 patients screened in six Swiss trial centers, 34 were randomized. Eighteen ACRE and eight placebo patients could be analyzed in the Per-Protocol-Set. Half (9/18) of ACRE patients and 3/8 of placebo patients revealed clinical response at week 8 (CI 0.399-6.963; p=0.278). An improvement of the Mayo-score was observed in 77.8% of ACRE treated patients (62.5% of placebo). Fecal calprotectin dropped from 1049+/-1139 to 557+/-756μg/g feces in the ACRE but not in the placebo group (947+/-1039 to 1040+/-1179; p=0.035). Adverse events were rare.ConclusionsACRE therapy was not significantly superior to placebo at inducing a clinical response. However, placebo response was unusual high. Moreover, there was a significant calprotectin decrease at end of treatment, indicative of ACRE biochemical efficacy in UC.Study HighlightsWhat is knownDried bilberries have been reported to ameliorate active ulcerative colitis (UC) in an uncontrolled pilot trialAnthocyanins (flavonoids) are regarded to be the active anti-inflammatory compound of bilberriesAn anthocyanin rich extract (ACRE) of bilberries was reported to ameliorate colitis in mouse modelsWhat is new hereIn a multi-center randomized, double-blind, placebo controlled, parallel group study in patients with moderate to severe active UC, ACRE did not reach the statistical endpoint of clinical responseAn unusually high placebo response was observedACRE induced significant biochemical response with significant decrease in calprotectin levels

Microbiomes are an essential contributor to the metabolic activity in the human gastrointestinal tract. The fermentation of otherwise indigestible nutritional components like dietary fibers relies on a complex interplay of metabolic pathways that are distributed across the individual bacteria. Yet, which of the bacteria are responsible for which parts of the distributed metabolism and how they should be grouped together is insufficiently understood. Here, we present the NicheMap™, an approach to map the different bacterial taxa that make up the gut microbiome onto the different functional niches of microbial carbohydrate fermentation. Our approach uses in vitro measurements of bacterial growth and metabolic activity to identify which bacterial taxa are responsible for which metabolic function in the relevant complex context of whole human fecal microbiomes. We identified ‘characteristic taxa’ selected for by a panel growth substrates representative of dietary components that are resistant to digestion by host enzymes. These characteristic taxa offer predictions of which bacteria are stimulated by the various components of human diet. We validated these predictions using microbiome data from a human nutritional supplementation study. We suggest a template of how bacterial taxonomic diversity is organized along the trophic cascade of intestinal carbohydrate fermentation. We anticipate that our results and our approach will provide a key contribution towards building a structure-function map for gut microbiomes. Having such a map on hand is an important step in moving the microbiome from a descriptive science to an interventional one.

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.

The success of fecal microbiota transplants (FMT) has provided the necessary proof-of-concept for microbiome therapeutics. Yet, feces-based therapies have many associated risks and uncertainties, and hence defined microbial consortia that modify the microbiome in a targeted manner have emerged as a promising safer alternative to FMT. The development of such live biotherapeutic products has important challenges, including the selection of appropriate strains and the controlled production of the consortia at scale. Here, we report on an ecology- and biotechnology-based approach to microbial consortium construction that overcomes these issues. We selected nine strains that form a consortium to emulate the central metabolic pathways of carbohydrate fermentation in the healthy human gut microbiota. Continuous co-culturing of the bacteria produces a stable and reproducible consortium whose growth and metabolic activity are distinct from an equivalent mix of individually cultured strains. Further, we showed that our function-based consortium is as effective as FMT in counteracting dysbiosis in a dextran sodium sulfate mouse model of acute colitis, while an equivalent mix of strains failed to match FMT. Finally, we showed robustness and general applicability of our approach by designing and producing additional stable consortia of controlled composition. We propose that combining a bottom-up functional design with continuous co-cultivation is a powerful strategy to produce robust functionally designed synthetic consortia for therapeutic use.

Sometimes I imagine being able to observe metabolism at its multiple scales and quickly zoom out from the level of a single electron all the way to the outskirts of our atmosphere, where I see solar energy set in motion cycles of energy and matter. At these two extremes, everything seems to be governed by the principles of physics, from quantum mechanics to thermodynamics. Yet somewhere in between these two levels, as chemistry arises and microbial cells appear, the rules of the game seem completely different: genetic regulatory processes determine the short-term dynamics of microbes, and natural selection determines their long-term evolution. And as I continue zooming out and watch thousands of species in complex ecosystems, the rules change yet again in a way that is quite mysterious: a dynamical dance of taxa, determined by the molecular composition of the environment and by inter-microbial interactions mediated by signals and metabolites. The traditional biological view fades, and we start wondering about new ecological principles and the role of microbes in biogeochemical cycles. Perhaps at some scale we may expect the emergence of some form of metabolic determinism—even if the metabolism of an individual species is unstable and unpredictable, that of a community may be stable and predictable based on its environment. So, microbial community metabolism is at the same time the bottom-up emergent property of a collection of microbes with a myriad of pathways and strategies and the top-down property of a planet subject to an energy flow. Reconciling these two perspectives is to me one of the most fascinating questions about microbial communities. It is also a question with ramifications in other fundamental research areas, including the ancient history of life on our planet and the possibility of engineering communities whose collective metabolism could help mitigate climate change.

The gut microbiome lies at the intersection between the environment and the host, with the ability to modify host responses to disease-relevant exposures and stimuli. This is evident in how enteric microbes interact with the immune system, e.g., supporting immune maturation in early life, affecting drug efficacy via modulation of immune responses, or influencing development of immune cell populations and their mediators. Many factors modulate gut ecosystem dynamics during daily life and we are just beginning to realise the therapeutic and prophylactic potential of microbiome-based interventions. These approaches vary in application, goal, and mechanisms of action. Some modify the entire community, such as nutritional approaches or faecal microbiota transplantation, while others, such as phage therapy, probiotics, and prebiotics, target specific taxa or strains. In this review, we assessed the experimental evidence for microbiome-based interventions, with a particular focus on their clinical relevance, ecological effects, and modulation of the immune system.

Genomic data has revealed that genotypic variants of the same species, that is, strains, coexist and are abundant in natural microbial communities. However, it is not clear if strains are ecologically equivalent, and at what characteristic genetic distance they might exhibit distinct interactions and dynamics. Here, we address this problem by tracking 10 taxonomically diverse microbial communities from the pitcher plant Sarracenia purpurea in the laboratory for more than 300 generations. Using metagenomic sequencing, we reconstruct their dynamics over time and across scales, from distant phyla to closely related genotypes. We find that most strains are not ecologically equivalent and exhibit distinct dynamical patterns, often being significantly more correlated with strains from another species than their own. Although even a single mutation can affect laboratory strains, on average, natural strains typically decouple in their dynamics beyond a genetic distance of 100 base pairs. Using mathematical consumer-resource models, we show that these taxonomic patterns emerge naturally from ecological interactions between community members, but only if the interactions are coarse-grained at the level of strains, not species. Finally, by analyzing genomic differences between strains, we identify major functional hubs such as transporters, regulators, and carbohydrate-catabolizing enzymes, which might be the basis for strain-specific interactions. Our work suggests that fine-scale genetic differences in natural communities could be created and stabilized via the rapid diversification of ecological interactions between strains.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) recently emerged as a promising cancer immunotherapy target. We set out to investigate the functional role of PTPN2 in the pathogenesis of human colorectal carcinoma (CRC), as its role in immune-silent solid tumors is poorly understood. We demonstrate that in human CRC, increased PTPN2 expression and activity correlated with disease progression and decreased immune responses in tumor tissues. In particular, stage II and III tumors displayed enhanced PTPN2 protein expression in tumor-infiltrating T cells, and increased PTPN2 levels negatively correlated with expression of PD-1, CTLA4, STAT1, and granzyme A. In vivo, T cell- and DC-specific PTPN2 deletion reduced tumor burden in several CRC models by promoting CD44+ effector/memory T cells, as well as CD8+ T cell infiltration and cytotoxicity in the tumor. In direct relevance to CRC treatment, T cell-specific PTPN2 deletion potentiated anti-PD-1 efficacy and induced antitumor memory formation upon tumor rechallenge in vivo. Our data suggest a role for PTPN2 in suppressing antitumor immunity and promoting tumor development in patients with CRC. Our in vivo results identify PTPN2 as a key player in controlling the immunogenicity of CRC, with the strong potential to be exploited for cancer immunotherapy.

Despite overall success, T cell checkpoint inhibitors for cancer treatment are still only efficient in aminority of patients. Recently, intestinal microbiota was found to critically modulate anti-cancer immunity and therapy response. Here, we identify Clostridiales members of the gut microbiota associated with a lower tumor burden in mouse models of colorectal cancer (CRC). Interestingly, these commensal species are also significantly reduced in CRC patients compared with healthy controls. Oral application of a mix of four Clostridiales strains (CC4) in mice prevented and even successfully treated CRC as stand-alone therapy. This effect depended on intratumoral infiltration and activation of CD8(+) T cells. Single application of Roseburia intestinalis or Anaerostipes caccae was even more effective than CC4. In a direct comparison, the CC4 mix supplementation outperformed anti-PD-1 therapy in mouse models of CRC and melanoma. Our findings provide a strong preclinical foundation for exploring gut bacteria as novel stand-alone therapy against solid tumors.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Previously, we found that in glucose-limited Saccharomyces cerevisiae colonies, metabolic constraints drive cells into groups exhibiting gluconeogenic or glycolytic states. In that study, threshold amounts of trehalose - a limiting, produced carbon-resource, controls the emergence and self-organization of cells exhibiting the glycolytic state, serving as a carbon source that fuels glycolysis (Varahan et al., 2019). We now discover that the plasticity of use of a non-limiting resource, aspartate, controls both resource production and the emergence of heterogeneous cell states, based on differential metabolic budgeting. In gluconeogenic cells, aspartate is a carbon source for trehalose production, while in glycolytic cells using trehalose for carbon, aspartate is predominantly a nitrogen source for nucleotide synthesis. This metabolic plasticity of aspartate enables carbon-nitrogen budgeting, thereby driving the biochemical self-organization of distinct cell states. Through this organization, cells in each state exhibit true division of labor, providing growth/survival advantages for the whole community. Introduction During the development of microbial communities, groups of cells come together and exhibit heterogeneity within spatial organization (Ackermann, 2015). As the community develops, cells can present specialization of function, which allows the community as a whole to perform various tasks including the acquisition of food, defense against competing microorganisms, or more efficient growth (Newman, 2016; Niklas, 2014; West and Cooper, 2016). This division of labor allows breakdown of complex biological processes into simpler steps, eliminating the need for individual cells to perform several tasks simultaneously, thereby enhancing the overall efficiency with which cells in the community function (Giri et al., 2019; Johnson et al., 2012; Rueffler et al., 2012; van Gestel et al., 2015). Due to these advantages, division of labor is widely prevalent across diverse microbial communities and can be found at different levels of biological organization (Gordon, 2016; Kirk, 2003; Tarnita et al., 2013). However, the underlying rules that enable division of labor within cell populations remain to be deciphered. In particular, microbial community development is commonly triggered by nutrient limitation (Ackermann, 2015; Hoehler and Jørgensen, 2013; Johnson et al., 2012). Clearly, an optimal allocation of resources is critical for maximizing overall fitness within a microbial community, especially when the availability of nutrients is limiting (Litchman et al., 2015; Wessely et al., 2011). One strategy by which the community can manage the requirement of different resources is by sharing metabolic products, and this is employed by many microbial communities (D'Souza et al., 2018; Liu et al., 2015). Since resources can often be insufficient, the sharing of such resources might incur a cost to the cell. Hence, different cells of the community exhibit metabolic interdependencies, presumably to balance out trade-offs arising from resource sharing. While this concept has been demonstrated for example, in synthetically engineered systems, where required metabolic dependencies are created between non-isogenic cells (Campbell et al., 2016; Campbell et al., 2015), this has been exceptionally challenging to demonstrate within a clonal community of cells. We recently discovered that metabolic constraints are sufficient to enable the emergence and maintenance of cells in specialized biochemical states within a clonal S. cerevisiae community (Varahan et al., 2019). Remarkably, this occurs through a simple, self-organized biochemical system. In yeast growing in low glucose, cells are predominantly gluconeogenic. As the colony matures, groups of cells exhibiting glycolytic metabolism emerge with spatial organization. Strikingly, this occurs through the production (via gluconeogenesis) and accumulation of a limiting metabolic resource, trehalose. As this resource builds up, some cells spontaneously switch to utilizing trehalose for carbon, which then drives a glycolytic state. This also depletes the resource, and therefore a self-organized system of trehalose producers and utilizers establish themselves, enabling structured phenotypic heterogeneity (Varahan et al., 2019). This observation raises a deeper question, of how such groups of heterogeneous cells can sustain themselves in this self-organized biochemical system. In particular, is it sufficient to only have the build-up of this limiting, controlling resource? How are carbon and nitrogen requirements balanced within the cells in the heterogeneous states? In this study, we uncover how a non-limiting resource with plasticity in function can control the organization of this entire system. We find that the amino acid aspartate, through distinct use of its carbon or nitrogen backbone, enables the emergence and organization of heterogeneous cells. In gluconeogenic cells, aspartate is utilized in order to produce the limiting carbon resource, trehalose, which in turn is utilized by other cells that switch to and stabilize in a glycolytic state. Combining biochemical, computational modeling and analytical approaches, we find that aspartate is differentially utilized by the oppositely specialized cells of the community as a carbon or a nitrogen source to sustain different metabolism. This carbon/nitrogen budgeting of aspartate is crucial for the emergence of distinct cell states in this isogenic community. Through this, cell groups show complete division of labor, and each specialized state provides distinct proliferation and survival advantages to the colony. Collectively, we show how the carbon/nitrogen economy of a cell community enables a self-organizing system based on non-limiting and limiting resources, and this allows organized phenotypic heterogeneity in cells. Results Amino acid driven gluconeogenesis is critical for emergence of metabolic heterogeneity In a previous study (Varahan et al., 2019), we discovered that trehalose controls the emergence of spatially organized, metabolically heterogeneous groups of cells within a S. cerevisiae colony growing in low glucose. Within this colony were cells with high gluconeogenic activity, and other cells showing high glycolytic/pentose phosphate pathway (PPP) activity (Figure 1A). The high glycolytic/PPP activity cells could be distinguished as ‘light’ cells, and the highly gluconeogenic cells as ‘dark’, based purely on optical density as observed by brightfield microscopy, as shown in Figure 1A (Varahan et al., 2019). In this system, cells start in a gluconeogenic state, and these cells (dark) produce trehalose. When a threshold concentration of external trehalose is reached, a subpopulation of cells switch to trehalose consumption that drives a glycolytic state, and these cells continue to proliferate as light cells (Figure 1A). Trehalose is a limiting resource since it is not freely available in the glucose limited external environment, and must be synthesized via gluconeogenesis (François et al., 1991). We therefore first asked how the loss of gluconeogenesis affects the emergence of metabolically specialized light cells. For this, we genetically generated mutants that lack two key gluconeogenic enzymes (PCK1 and FBP1). These gluconeogenic mutants (Δpck1 and Δfbp1) expectedly formed smooth colonies completely lacking structured morphology (which correlates with the absence of metabolic heterogeneity; Figure 1B and Figure 1—figure supplement 1A). Further, these mutants had essentially undetectable cells with high PPP activity (light cells), based on the fluorescence-signal of a PPP reporter, as compared to a wild-type colony, although the total number of viable cells in all the colonies were comparable (Figure 1C and Figure 1—figure supplement 1B). This confirms that gluconeogenesis is critical for the emergence and maintenance of metabolic heterogeneity in the colony. Figure 1 with 1 supplement see all Download asset Open asset Amino acid driven gluconeogenesis is critical for the emergence of phenotypic heterogeneity. (A) External trehalose controls the emergence of light cells. Trehalose synthesized by the dark cells fuels glycolysis and pentose phosphate pathway in light cells. (B) Gluconeogenesis is required for development of structural morphology in the colonies. The panel shows the morphology of mature wild-type and gluconeogenic mutant (∆pck1 and ∆fbp1) yeast colonies in rich medium, with supplemented glucose as the sole variable. Scale bar: 2 mm. Also see Figure 1—figure supplement 1A for more information. (C) Spatial distribution of mCherry fluorescence across a colony, indicating the activity of a reporter for pentose phosphate pathway (TKL1) activity in wild-type and gluconeogenesis defective mutants (∆pck1 and ∆fbp1). (D) Amino acids and in particular aspartate is required for the development of structural morphologies in the colonies in a gluconeogenesis dependent manner. The panel shows the morphology of mature wild-type and gluconeogenesis-defective (∆pck1) yeast colonies in minimal medium (low glucose), with and without amino acid supplementation, or with only aspartate supplementation. (E) Spatial distribution of mCherry fluorescence across a colony, indicating the activity of a reporter for pentose phosphate pathway (TKL1) activity in wild-type colonies grown either in minimal media or minimal media supplemented with all amino acids or just aspartate. Trehalose, the produced resource controlling the switch to the light state (Varahan et al., 2019), is a disaccharide made up of two molecules of glucose and is produced via gluconeogenesis. This two-state community of cells requires a continuous supply of trehalose to sustain itself. Therefore, in order to address how dark cells maintained threshold concentrations of trehalose, we asked how this resource itself is produced. Notably, the media conditions under which these colonies develop essentially have non-limiting amounts of amino acid resources (2% yeast extract and 2% peptone). We therefore hypothesized that amino acids (available in non-limiting levels) could act as carbon sources (via possible anaplerotic processes) to fuel trehalose production in dark cells. We tested this by growing wild-type cells in media devoid of free amino acids, but with sufficient ammonium sulfate (Minimal medium). Wild-type colonies failed to develop structured colonies (which correlates with the lack of metabolic heterogeneity) in the absence of free amino acids, and this could be rescued by adding back amino acids to this media (Figure 1D). Expectedly, this amino acid dependent rescue of colony morphology depended on gluconeogenesis, since a Δpck1 strain failed to develop morphology even after the addition of amino acids to the medium (Figure 1D). This shows that non-limiting amino acids promote the development of structured colonies exhibiting metabolic heterogeneity, in a gluconeogenesis dependent manner. Interestingly this amino acid dependent effect is specific. In add-back experiments in minimal medium, amongst all amino acids tested, aspartate supplementation strongly promoted the development of structured colonies exhibiting metabolic heterogeneity (Figure 1D). This ‘rescue’ by aspartate was stronger than that seen with the addition of any other amino acids individually or in combination (Figure 1D and Figure 1—figure supplement 1C). This was further validated via experiments wherein wild-type colonies that developed in minimal medium, supplemented either with all amino acids, or only aspartate alone, exhibited spatially restricted metabolic heterogeneity comparable to the wild-type colonies grown in rich media. The light cell population was estimated using the fluorescent PPP reporter, which serves as an excellent proxy for light cells (Varahan et al., 2019; Figure 1E and Figure 1—figure supplement 1B). Collectively, these results reveal that aspartate is sufficient for the development of metabolically specialized colonies in a gluconeogenesis-dependent manner. The addition of other amino acids (particularly glutamine/glutamate) only show a delayed, weaker emergence of light cells. This would be consistent with their eventual, steady conversion to aspartate, which will not result in a build-up of excess amounts of this amino acid. Aspartate promotes light cell emergence by directly fueling trehalose synthesis In contrast to their canonical roles as nitrogen sources, amino acids can also act as carbon donors for several metabolic processes (Boyle, 2005). While many amino acids can enter the tricarboxylic acid (TCA) cycle via anaplerosis, and TCA intermediates in turn can enter gluconeogenesis, aspartate is unique. It is the only amino acid that can directly enter gluconeogenesis, without entering into the TCA cycle. This is via the conversion of aspartate into oxaloacetate directly in the cytosol through the activity of aspartate transaminase. In this cytosolic process, aspartate and 2-oxoglutarate combine to give one molecule each of oxaloacetate and glutamate. All the other amino acids have to be first transported to the mitochondria and enter the TCA cycle, and these TCA intermediates must then be transported back to the cytosol to enter gluconeogenesis (Brunengraber and Roe, 2006). Since the addition of aspartate alone to minimal media was sufficient for light cells to emerge, we tested if aspartate is a direct carbon source required for trehalose production within the colony, since trehalose is a pre-requisite for light cell emergence. Wild-type colonies were grown in minimal media supplemented with all amino acids, or aspartate alone, or all amino acids without aspartate (aspartate dropout) and total trehalose levels in the 7 day old colonies were measured. As controls, trehalose levels in the Δpck1 colonies (gluconeogenesis defective) and Δtps1 colonies (trehalose synthesis defective) were measured. Compared to colonies grown in minimal medium, colonies grown in minimal medium supplemented with all amino acids, or aspartate alone, had significantly higher amounts of trehalose (Figure 2A). Notably, the level of trehalose in wild-type colonies grown in aspartate dropout minimal medium was significantly lower compared to colonies grown in minimal media supplemented with all amino acids or just aspartate, demonstrating that aspartate can be the primary carbon contributor towards trehalose synthesis (Figure 2A). As expected, Δpck1 colonies (gluconeogenesis defective) and Δtps1 (trehalose synthesis defective) had background levels of trehalose (Figure 2A). Furthermore, colonies grown on aspartate dropout medium had fewer light cells (quantified using the PPP reporter activity) compared to colonies grown in minimal media supplemented with all amino acids or just aspartate (Figure 2B and Figure 2—figure supplement 1). This shows that aspartate enables trehalose production, which in turn controls the emergence of metabolic heterogeneity in these clonal colonies (Figure 2A and B). To demonstrate that aspartate directly provides the carbon backbone of trehalose, we grew colonies in minimal medium (low glucose) supplemented with 13C-labeled aspartate, and measured intracellular levels of 13C–labeled gluconeogenic intermediates or end-products directly by targeted mass spectrometric methods described earlier (Vengayil et al., 2019; Figure 2C). Cells in wild-type colonies accumulated 13C-labeled 3-phosphoglycerate (3 PG) and 13C-labeled trehalose, while these labeled metabolites were undetectable in a gluconeogenic mutant (Δpck1) (Figure 2D). Collectively, these data show that aspartate provides the carbon skeleton for trehalose production via gluconeogenesis, and this turn is essential for the emergence of spatially restricted metabolic heterogeneity. Figure 2 with 1 supplement see all Download asset Open asset Aspartate enables light cell emergence by fueling trehalose synthesis. (A) Comparative steady-state amounts of trehalose measured in wild-type, ∆pck1 (gluconeogenesis defective) and ∆tps1 (trehalose synthesis defective) colonies grown in minimal medium, or minimal medium supplemented with either all amino acids, or aspartate alone, or all amino acids without aspartate (aspartate dropout) (n = 3). Colony insets represent wild-type colony morphology in different media conditions. Statistical significance was calculated using unpaired t test (*** indicates p<0.001) and error bars represent standard deviation. (B) Aspartate significantly contributes to colony development and emergence of light cells. Spatial distribution of mCherry fluorescence across a colony, indicating the activity of a reporter for pentose phosphate pathway (TKL1) activity in wild-type yeast colonies grown in minimal medium (low glucose), supplemented with either all amino acids, or aspartate alone, or all amino acids without aspartate (aspartate dropout). Also see Figure 2—figure supplement 1A for more information. (C and D) Metabolic-flux based analysis comparing relative 13C incorporation from 13C-labeled aspartate into newly synthesized gluconeogenic intermediates (3-phosphoglycerate and glucose-6-phosphate) and trehalose, in wild-type and ∆pck1 colonies. An agent-based model suggests how differential aspartate utilization drives the emergence of self-organized, metabolically heterogeneous states We had previously noted that the light cells had higher rates of nucleotide synthesis (Varahan et al., 2019). Synthesis of the nucleotide backbone requires an assimilation of carbon (typically from glucose derived metabolites, notably pentose sugars from the PPP), as well as nitrogen that comes from amino acids (primarily glutamine and aspartate) (Boyle, 2005). Indeed, this donation of nitrogen by aspartate towards nucleotide synthesis is considered a primary role of this amino acid. Interestingly, within the dark cells of the colony, aspartate is also being used as a carbon source for the synthesis of trehalose (Figure 2D). This raises the central idea of molecular budgeting: how is the utilization of aspartate as a carbon/nitrogen source managed in different types of cells? To theoretically address this question, we refined our originally coarse-grained mathematical model from Varahan et al., 2019. In the original model that simulates the development of the colony with dark and light cells, the resource driving the emergence of light cells was featureless and could only be used to drive hypothetically opposite metabolism (Varahan et al., 2019). In our new model, we now build-in molecule specificity. Based on experimental data, we incorporate aspartate utilization for the emergence of metabolic subpopulations, and self-organization within the colony. The processes now included in the model are explained below (See Materials and methods for a detailed description): Both dark and light cells utilize externally available resources to synthesize and accumulate the metabolites needed for growth. We can now assign two specific categories for these accumulating metabolites: carbon (C) and nitrogen (N). The dark cells utilize a single resource, aspartate, to serve both C and N requirements. Aspartate itself is a molecule that is in excess in the environment (non-limiting). We propose that the dark cells budget the aspartate flux for both these requirements, and some of the accumulated C (as trehalose) becomes available in the extracellular environment. From our earlier findings (Varahan et al., 2019), we know that the extracellular trehalose controls when some dark cells switch to being light cells. The light cells utilize the available trehalose for their C needs (driving glycolysis and the PPP). However, aspartate remains readily available for their N requirements, which includes nucleotide synthesis (this is illustrated in the model schematic and sample colony in Figure 3A). We now implement this revised model as an agent-based simulation, and monitor colony growth with these new assumptions of aspartate utilization. The specific modifications from the original model and the new parameters are introduced below: Figure 3 with 5 supplements see all Download asset Open asset An agent-based model for carbon-nitrogen budgeting reveals principles of metabolic heterogeneity via self-organization. (A) A model schematic based on an experimental understanding of aspartate utilization by the two cell types in the system. Dark and light cells are colored accordingly. Dark cells take in aspartate, budget it for nitrogen (N) and carbon (C) needs. Some of the accumulating C is released into the extracellular environment as trehalose, triggering the switching into light cells and also acting as the primary C source for light cells as it diffuses in the 2D space. On the right, we have a representative simulated colony generated from a default parameter set. Parameters are indicated in the parentheses. (B) Varying the fraction of aspartate flux allocated towards nitrogen (N) in order to observe the simulated colony over the same length of time. When the majority of the flux is used for carbon (C) needs, the simulated colonies resemble experimental colonies. If less aspartate is allocated for C rather than N, the developed colonies no longer resemble the experimental colonies. (C) Varying the relative rate of aspartate uptake compared to trehalose uptake by the light cells in order to observe simulated colonies over the same length of time. If the rate is the same, as shown in the first simulated colony where AspU = 1.0, the colony is underdeveloped. A middle value of AspU = 4.0 generates colonies similar to experimental colonies, while for a large value of AspU on the right, the dark cell blocks and light cell blocks have similar division times and the final colony is larger. Both light and dark [cell blocks] take up aspartate from the external environment at the same rate. Light cells can also take up trehalose from the surroundings. If the maximum amount of trehalose taken up per time step is Cmax, the rate of aspartate uptake is AspU*Cmax. Dark cells budget the aspartate utilization for different ends. A fraction, ‘f’ is utilized for nitrogen (N) needs, the remainder, (1 f), is utilized for carbon (C) needs. The aspartate to C conversion requires a yield coefficient, Y. Since aspartate is used as a carbon source (gluconeogenesis and nucleotide synthesis), and/or nitrogen source (nucleotide synthesis and other functions), and for protein synthesis, we must assume this parameter is less than 1. A fraction, Pf, of this accumulated C inside dark cell blocks is secreted into the extracellular environment as trehalose. Thus, we can couple the trehalose production by dark cells to their aspartate consumption and utilization. Additionally, there will be an imposed upper limit to this secreted amount, but for our simulation this extra constraint is not limiting to cells (see Figure 3—figure supplement 1). In the new model, the two cell types (dark and light) accumulate both C and N to a minimum amount before division. We assume that dark and light cell [blocks] need the same minimum amount of C, normalized to a value of 1.0 units. However, experimental observations show that light cells have a higher rate of nucleotide synthesis, corresponding with faster growth rates and nitrogen consumption compared to dark cells (as observed in Varahan et al., 2019). Hence, while the dark cell blocks have a minimum N requirement normalized to 1.0, the light cells need ExN*1.0. Once cells accumulate the minimum amount, the probability of division for both cell types are the same. See Table 2 for values of these parameters and see Figure 3—figure supplement 2 for a comparison of the division rate of the dark and light cells. Our first aim here is simply to show that adding a simple carbon-nitrogen budgeting to our previous model (Varahan et al., 2019) does not destroy the spatial patterns. Figure 3A shows that this model is sufficient to produce spatial patterns of dark and light cells that are similar to what we observe experimentally. Having established the sufficiency of the model, a second aim was to understand, within this framework, how the carbon-nitrogen budgeting could be set up to be consistent with the patterns. In essence, we ask what constraints from the carbon-nitrogen budgeting are necessary to produce the spatial patterns observed. By varying the two main parameters in this study, the model makes the following predictions: More of the aspartate taken up by dark cells is allocated for carbon metabolism and trehalose synthesis We vary the fraction of the aspartate flux allocated to nitrogen, f, from 0.0 to 1.0 (0%–100%) only in the dark cells. The colonies formed from some selected values are shown in Figure 3B to show the general trend. Low values of ‘f’ generate virtual colonies which are similar to experimental ones. As the value of f increases, enough resources cannot be allocated to fulfil carbon requirements for light cells to divide. Aspartate uptake rate by both types of cells is higher than the rate of uptake of trehalose by light cells The parameter AspU dictates the relative rate of aspartate uptake compared to trehalose uptake rate by light cells. Dark cells take up aspartate at the same rate as light cells. However, in dark cells, aspartate is responsible for carbon metabolism and trehalose generated in the system. Varying this parameter as shown in Figure 3C, we see that if the rate of uptake for aspartate is the same as the uptake rate for trehalose (AspU = 1.0), the colonies cannot grow like the wild-type colony (Figure 3B). This can be attributed to the fact that 4 molecules of aspartate are required for the synthesis of 1 molecule of trehalose. Hence it will be impossible for cells to synthesize sufficient amounts of trehalose required for the emergence of light cells, if the uptake rate of aspartate by dark cells is equal to the uptake of trehalose by light cells. Therefore, in simple simulations, a higher value of AspU (=4.0) provides enough carbon for the dark cells despite budgeting, to synthesize trehalose (C for light cells in the model) that is required for the proliferation of light cells. Since aspartate drives the growth of both dark and light cells in a direct and indirect manner, larger values of AspU give larger colonies and vice versa, as shown in Figure 3C. Also see Figure 3—figure supplements 3, 4 and 5 and Videos 1, 2, 3, 4. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Development of a simulated wild-type colony. A simulation movie of the WT colony over 750 time-steps (~6 days in real time). The colony starts with 95–99% dark cells, which go through switching and growth phases as observed. This colony is generated using default parameter values in the model. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Aspartate is allocated equally for Carbon and Nitrogen by dark cells. A simulation movie where the budgeting fraction ‘f’ is 50%, i.e., 50% of the aspartate flux is allocated towards nitrogen reserves. The dark cell blocks cannot allocate sufficient carbon for themselves, leading to almost no divisions by the dark cell blocks while the light cells at the edge keep proliferating. For comparison, the default value of ‘f’ is 12.5%. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Aspartate uptake rate by both types of cells is equal to trehalose uptake rate by light cells. A simulation movie where the relative rate of aspartate uptake is equal to the trehalose uptake rate. (i.e. AspU = 1.0). In this case, the aspartate uptake by dark cells is much slower, which also leads to slower trehalose production, resulting in smaller colonies consisting of predominantly dark cell blocks. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Aspartate uptake rate by both types of cells is much higher than trehalose uptake rate by light cells. A simulation movie where the relative rate of aspartate uptake is high. (i.e. AspU = 8.0). In this case, the dark cells allocate adequate aspartate for both nitrogen and carbon requirements rapidly enough. This leads to both dark and light cells having nearly the same division rate. Aspartate allows differential carbon/nitrogen budgeting in light and dark cells of the colony in vivo Our agent-based model suggested that the emergence of light cells and the spatial patterns similar to those observed experimentally arise when aspartate is used predominantly as a carbon source in dark cells. We therefore hypothesized that distinct cells in the colony might differentially utilize aspartate predominantly as either a carbon or a nitrogen source. We previously showed that light cells exhibit high PPP activity and nucleotide biosynthesis, using carbon precursors derived from the trehalose, provided by the dark cells (Var

Niche construction through interspecific interactions can condition future community states on past ones. However, the extent to which such history dependency can steer communities towards functionally different states remains a subject of active debate. Using bacterial communities collected from wild pitchers of the carnivorous pitcher plant, Sarracenia purpurea, we test the effects of history on composition and function across communities assembled in synthetic pitcher plant microcosms. We find that the diversity of assembled communities is determined by the diversity of the system at early, pre-assembly stages. Species composition is also contingent on early community states, not only because of differences in the species pool, but also because the same species have different dynamics in different community contexts. Importantly, compositional differences are proportional to differences in function, as profiles of resource use are strongly correlated with composition, despite convergence in respiration rates. Early differences in community structure can thus propagate to mature communities, conditioning their functional repertoire.