Cross-feeding Of Metabolites Research Articles

Activation of denitrification potential within anammox consortia to exceed nitrogen removal thresholds via Fe-Mn functionalized biochar: Enhanced interspecies electron transfer and metabolite cross-feeding

Anammox exerts a crucial role in nitrogenous effluents remediation; nevertheless, nitrogen removal efficiency thresholds triggered by additional nitrate production poses a critical obstacle for anammox performance. Herein, Fe-Mn functionalized biochar (FeMn#BC) was synthesized and incorporated into anammox consortia to elicit the exogenous organics-independent metabolic potential of denitrifying bacteria, which decreased nitrate accumulation and achieved up to 90.69 ± 0.48 % total nitrogen removal efficiency. Enzyme content analysis indicated that FeMn#BC bolstered biometabolic vigour and electron transport system activity by stimulating the synthesis of heme c and NADH. Microbial community succession results demonstrated that FeMn#BC facilitated the proliferation of anammox bacteria (AnAOB) and nitrate-reducing functional bacteria. Metagenomic analysis uncovered that FeMn#BC increased the relative abundance of genes related to energy metabolism, nutrient-substrate ingestion, cofactor synthesis, and iron-manganese transport pathways. Candidatus Brocadia sp013360945 interacted nitrogen metabolism substrates and electrons with dominated denitrifying species PR03 sp. (dependent on endogenous organics and FeMn#BC-released Fe(II)), while interacted cofactors with other mutualistic bacteria possessing nitrate-reducing potential (e.g. ATM1 sp003577165). FeMn#BC-released Mn(II) served as an additional electron donor, augmenting electron availability for non-dominant anammox species like Candidatus Jettenia caeni and Candidatus. Kuenenia stuttgartiensis. This research highlights the critical mechanism of Fe-Mn synergism for overcoming the bottleneck of anammox nitrogen removal efficiency, and demonstrates the substantial potential of Fe-Mn functionalized materials to revolutionize the existing wastewater treatment technologies towards a carbon-free approach.

Fungal-Bacterial Mutualism: Species and Strain-Dependent Simultaneous Modulation of Branched-Chain Esters and Indole Derivatives in Fermented Sausages through Metabolite Cross-Feeding.

The precise impact of species and strain diversity on fungal-bacterial interactions and the overall community functioning has remained unclear. First, our study revealed how Debaryomyces hansenii influences diverse bacteria to accumulate key metabolites in a simulated fermented food system. For flavor, D. hansenii promoted the accumulation of branched-chain esters in Staphylococcus xylosus by promoting growth and facilitating the precursor branched-chain acids transformations but hindered the accumulation of Staphylococcus equorum. Furthermore, fungal-bacterial interactions displayed diversity among S. equorum strains. For bioactive compounds, species and strain diversity of lactic acid bacteria (LAB) also influences the production of indole derivatives. Then, we investigated specific metabolic exchanges under reciprocal interaction. Amino acids, rather than vitamins, were identified as the primary drivers of the bacterial growth promotion. Moreover, precursor transformations by D. hansenii played a significant role in branched-chain esters production. Finally, a synthetic community capable of producing high concentrations of branched-chain esters and indole derivatives was successfully constructed. These results provide valuable insights into understanding and designing synthetic communities for fermented sausages.

Citrate cross-feeding by Pseudomonas aeruginosa supports lasR mutant fitness.

Cross-feeding of metabolites between subpopulations can affect cell phenotypes and population-level behaviors. In chronic Pseudomonas aeruginosa lung infections, subpopulations with loss-of-function (LOF) mutations in the lasR gene are common. LasR, a transcription factor often described for its role in virulence factor expression, also impacts metabolism, which, in turn, affects interactions between LasR+ and LasR- genotypes. Prior transcriptomic analyses suggested that citrate, a metabolite secreted by many cell types, induces virulence factor production when both genotypes are together. An unbiased analysis of the intracellular metabolome revealed broad differences including higher levels of citrate in lasR LOF mutants. Citrate consumption by LasR- strains required the CbrAB two-component system, which relieves carbon catabolite repression and is elevated in lasR LOF mutants. Within mixed communities, the citrate-responsive two-component system TctED and its gene targets OpdH (porin) and TctABC (citrate transporter) that are predicted to be under catabolite repression control were induced and required for enhanced RhlR/I-dependent signaling, pyocyanin production, and fitness of LasR- strains. Citrate uptake by LasR- strains markedly increased pyocyanin production in co-culture with Staphylococcus aureus, which also secretes citrate and frequently co-infects with P. aeruginosa. This citrate-induced restoration of virulence factor production by LasR- strains in communities with diverse species or genotypes may offer an explanation for the contrast observed between the markedly deficient virulence factor production of LasR- strains in monocultures and their association with the most severe forms of cystic fibrosis lung infections. These studies highlight the impact of secreted metabolites in mixed microbial communities.IMPORTANCECross-feeding of metabolites can change community composition, structure, and function. Here, we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes in chronic Pseudomonas aeruginosa lung infections. We illustrate an example of how clonally derived diversity in a microbial communication system enables intra- and inter-species cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa and Staphylococcus aureus, was differentially consumed between genotypes. Since these two pathogens frequently co-occur in the most severe cystic fibrosis lung infections, the cross-feeding-induced virulence factor expression and fitness described here between diverse genotypes exemplify how co-occurrence can facilitate the development of worse disease outcomes.

Inferring bacterial interspecific interactions from microcolony growth expansion.

Bacterial species interactions significantly shape growth and behavior in communities, determining the emergence of community functions. Typically, these interactions are studied through bulk population measurements, overlooking the role of cell-to-cell variability and spatial context. This study uses real-time surface growth measurements of thousands of sparsely positioned microcolonies to investigate interactions and kinetic variations in monocultures and cocultures of Pseudomonas putida and P. veronii under substrate competition (succinate) or substrate independence (d-mannitol and putrescine). In monoculture, microcolonies exhibited expected substrate-dependent expansion rates, but individual colony sizes were affected by founder cell density, spatial positioning, growth rates, and lag times. In coculture, substrate competition favored P. putida, but unexpectedly, reduced the maximum growth rates of both species. In contrast, 10% of P. veronii microcolonies under competition grew larger than expected, likely due to founder cell phenotypic variation and stochastic spatial positioning. These effects were alleviated under substrate independence. A linear relationship between founder cell ratios and final colony area ratios in local neighborhoods (6.5-65µm radius) was observed in coculture, with its slope reflecting interaction type and strength. Measured slopes in the P. putida to P. veronii biomass ratio under competition were one-third reduced compared to kinetic predictions using a cell-agent growth model, which exometabolite analysis and simulations suggested may be due to metabolite cross-feeding or inhibitory compound production. This indicates additional factors beyond inherent monoculture growth kinetics driving spatial interactions. Overall, the study demonstrates how microcolony growth experiments offer valuable insights into bacterial interactions, from local to community-level dynamics.

Metabolite cross-feeding enables concomitant catabolism of chlorinated methanes and chlorinated ethenes in synthetic microbial assemblies.

Isolate studies have been a cornerstone for unraveling metabolic pathways and phenotypical (functional) features. Biogeochemical processes in natural and engineered ecosystems are generally performed by more than a single microbe and often rely on mutualistic interactions. We demonstrate the rational bottom-up design of synthetic, interdependent co-cultures to achieve concomitant utilization of chlorinated methanes as electron donors and organohalogens as electron acceptors. Specialized anaerobes conserve energy from the catabolic conversion of chloromethane or dichloromethane to formate, H2, and acetate, compounds that the organohalide-respiring bacterium Dehalogenimonas etheniformans strain GP requires to utilize cis-1,2-dichloroethenene and vinyl chloride as electron acceptors. Organism-specific qPCR enumeration matched the growth of individual dechlorinators to the respective functional (i.e. dechlorination) traits. The metabolite cross-feeding in the synthetic (co-)cultures enables concomitant utilization of chlorinated methanes (i.e. chloromethane and dichloromethane) and chlorinated ethenes (i.e. cis-1,2-dichloroethenene and vinyl chloride) without the addition of an external electron donor (i.e. formate and H2). The findings illustrate that naturally occurring chlorinated C1 compounds can sustain anaerobic food webs, an observation with implications for the development of interdependent, mutualistic communities, the sustenance of microbial life in oligotrophic and energy-deprived environments, and the fate of chloromethane/dichloromethane and chlorinated electron acceptors (e.g. chlorinated ethenes) in pristine environments and commingled contaminant plumes.

Why does increased microbial fermentation in the human colon shift toward butyrate?

The microbial community of the human large intestine mainly ferments dietary fiber to short chain fatty acids (SCFAs), which are efficiently absorbed by the host. The three major SCFAs (acetate, propionate, and butyrate) have different fates within the body and different effects on health. A recent analysis of 10 human volunteer studies established that the proportions of these SCFA in fecal samples significantly shifted towards butyrate as the overall concentration of SCFA increased. Butyrate plays a key role in gut health and is preferentially utilized as an energy source by the colonic epithelium. Here we discuss possible mechanisms that underlie this 'butyrate shift'; these include the selection for butyrate-producing bacteria within the microbiota by certain types of fiber, and the possibility of additional butyrate formation from lactate and acetate by metabolite cross-feeding. However, a crucial factor appears to be the pH in the proximal colon, which decreases as the SCFA concentrations increase. A mildly acidic pH has been shown to have an important impact on microbial competition and on the stoichiometry of butyrate production. Understanding these complex interactions has been greatly aided by the refinement of theoretical models of the colonic microbiota that assume a small number (10) of microbial functional groups (MFGs).

Regulated bacterial interaction networks: A mathematical framework to describe competitive growth under inclusion of metabolite cross-feeding.

When bacterial species with the same resource preferences share the same growth environment, it is commonly believed that direct competition will arise. A large variety of competition and more general 'interaction' models have been formulated, but what is currently lacking are models that link monoculture growth kinetics and community growth under inclusion of emerging biological interactions, such as metabolite cross-feeding. In order to understand and mathematically describe the nature of potential cross-feeding interactions, we design experiments where two bacterial species Pseudomonas putida and Pseudomonas veronii grow in liquid medium either in mono- or as co-culture in a resource-limited environment. We measure population growth under single substrate competition or with double species-specific substrates (substrate 'indifference'), and starting from varying cell ratios of either species. Using experimental data as input, we first consider a mean-field model of resource-based competition, which captures well the empirically observed growth rates for monocultures, but fails to correctly predict growth rates in co-culture mixtures, in particular for skewed starting species ratios. Based on this, we extend the model by cross-feeding interactions where the consumption of substrate by one consumer produces metabolites that in turn are resources for the other consumer, thus leading to positive feedback in the species system. Two different cross-feeding options were considered, which either lead to constant metabolite cross-feeding, or to a regulated form, where metabolite utilization is activated with rates according to either a threshold or a Hill function, dependent on metabolite concentration. Both mathematical proof and experimental data indicate regulated cross-feeding to be the preferred model to constant metabolite utilization, with best co-culture growth predictions in case of high Hill coefficients, close to binary (on/off) activation states. This suggests that species use the appearing metabolite concentrations only when they are becoming high enough; possibly as a consequence of their lower energetic content than the primary substrate. Metabolite sharing was particularly relevant at unbalanced starting cell ratios, causing the minority partner to proliferate more than expected from the competitive substrate because of metabolite release from the majority partner. This effect thus likely quells immediate substrate competition and may be important in natural communities with typical very skewed relative taxa abundances and slower-growing taxa. In conclusion, the regulated bacterial interaction network correctly describes species substrate growth reactions in mixtures with few kinetic parameters that can be obtained from monoculture growth experiments.

Discovery of novel amino acid production traits by evolution of synthetic co-cultures

BackgroundAmino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities.ResultsHere we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either l-leucine or l-arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The l-leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na+/H+-antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to l-leucine transport. The l-arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only l-arginine uptake system of C. glutamicum with an affinity of KD = 30 nM. Finally, deletion of argTUV in an l-arginine producer strain resulted in a faster and 24% higher l-arginine production in comparison to the parental strain.ConclusionOur work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.

Metabolite Cross-Feeding Promoting NADH Production and Electron Transfer during Efficient SMX Biodegradation by a Denitrifier and S. oneidensis MR-1 in the Presence of Nitrate.

Antibiotics often coexist with other pollutants (e.g., nitrate) in an aquatic environment, and their simultaneous biological removal has attracted widespread interest. We have found that sulfamethoxazole (SMX) and nitrate can be efficiently removed by the coculture of a model denitrifier (Paracoccus denitrificans, Pd) and Shewanella oneidensis MR-1 (So), and SMX degradation is affected by NADH production and electron transfer. In this paper, the mechanism of a coculture promoting NADH production and electron transfer was investigated by proteomic analysis and intermediate experiments. The results showed that glutamine and lactate produced by Pd were captured by So to synthesize thiamine and heme, and the released thiamine was taken up by Pd as a cofactor of pyruvate and ketoglutarate dehydrogenase, which were related to NADH generation. Additionally, Pd acquired heme, which facilitated electron transfer as heme, was the important composition of complex III and cytochrome c and the iron source of iron sulfur clusters, the key component of complex I in the electron transfer chain. Further investigation revealed that lactate and glutamine generated by Pd prompted So chemotactic moving toward Pd, which helped the two bacteria effectively obtain their required substances. Obviously, metabolite cross-feeding promoted NADH production and electron transfer, resulting in efficient SMX biodegradation by Pd and So in the presence of nitrate. Its feasibility was finally verified by the coculture of an activated sludge denitrifier and So.

GUT-BIOME MODULATION WITH HUMAN MILK OLIGOSACCHARIDE (HMO) BASED SYNBIOTIC FOR A COMPLETE AND DEEP REMISSION IN CROHN’S DISEASE: A CASE STUDY

Abstract BACKGROUND Gut microbiome plays a crucial role in modulation of inflammatory and immune pathways. Diverse bacterial species from gut microbiota feed on specific prebiotics (non-digestible dietary fibers) producing metabolites, such as short chain fatty acids (SCFA). These metabolites beneficially affect the intestinal mucosa with their role in metabolic and immunologic homeostasis, and gut barrier integrity. Recently gut-biome modulation and use of metabolites gained interest as potential targets for therapeutic development, for instance in Inflammatory Bowel Disease (IBD) which includes Crohn’s disease (CD) and ulcerative colitis (UC). HYPOTHESIS Nourishing the gut-biome thru synbiotic formulation can enhance the interspecies synergy with production of metabolites and improved gut barrier integrity more naturally. This is a superior and holistic approach to reduce gut-inflammation. METHODS (CLINICAL CASE) An 11 year old male with refractory iron deficiency anemia, fecal calprotectin at 1375 µg/g, on video capsule endoscopy showed discrete linear and serpiginous ulcers with surrounding erythema in duodenum and proximal jejunum. Biopsy of colon showed multiple granulomas in terminal ileum and entire colon suggesting CD (Inflammatory phenotype). Exclusive Enteral Nutrition (EEN) with polymeric formula proved unsustainable. To nourish the gut-biome and its ecosystem a synbiotic formulation (Gastrosyn) that has a blend of specific prebiotics with HMO and complimentary probiotic blend with strains of Lactobacilli and Bifidobacterium was administered and followed for 20 weeks. Formulation also contained powerful antioxidant for synergistic effect in reducing inflammation. RESULTS After 4 weeks on synbiotic, fecal calprotectin dropped to 665ug/g which further dropped to 110 ug/g by week 12. Complete and deep remission was observed by 15 weeks as analyzed by endoscopy/colonoscopy (images), histology and lab tests results repeated several times (Calprotectin 65ug/g by week 15). Anemia also improved significantly, and patient continued to tolerate solid foods with no relapse even after 1year. CONCLUSIONS Synbiotics utilizing prebiotics that are specific to probiotic strains used is crucial for successful engraftment and biome modulation as established in recent literature. HMO based Synbiotic formulation has proven to nurture the ecosystem, and foster interspecies interactions, cross feeding, and generation of metabolites (SCFAs), providing a holistic and superior approach for remission in IBD.

Dual species biofilms are enhanced by metabolite cross-feeding.

Enterococcus faecalis and Staphylococcus aureus are frequently co-isolated from biofilm-associated infections. A new study by Ch'ng et al. revealed that S. aureus-released heme feeds E. faecalis respiration, augmenting E. faecalis growth and overall biofilm biomass. Their finding further supports the theory that metabolite cross-feeding is a critical aspect shaping polymicrobial biofilm interactions.

Non-obligate pairwise metabolite cross-feeding suggests ammensalic interactions between Bacillus amyloliquefaciens and Aspergillus oryzae

Bacterial-fungal metabolite trade-offs determine their ecological interactions. We designed a non-obligate pairwise metabolite cross-feeding (MCF) between Bacillus amyloliquefaciens and Aspergillus oryzae. Cross-feeding Aspergillus metabolites (MCF-1) affected higher growth and biofilm formation in Bacillus. LC-MS/MS-based multivariate analyses (MVA) showed variations in the endogenous metabolite profiles between the cross-fed and control Bacillus. We observed and validated that Aspergillus-derived oxylipins were rapidly depleted in Bacillus cultures concomitant with lowered secretion of cyclic lipopeptides (CLPs). Conversely, Bacillus extracts cross-fed to Aspergillus (MCF-2) diminished its mycelial growth and conidiation. Fungistatic effects of Bacillus-derived cyclic surfactins were temporally reduced following their hydrolytic linearization. MVA highlighted disparity between the cross-fed (MCF-2) and control Aspergillus cultures with marked variations in the oxylipin levels. We conclude that the pairwise MCF selectively benefitted Bacillus while suppressing Aspergillus, which suggests their ammensalic interaction. Widening this experimental pipeline across tailored communities may help model and simulate BFIs in more complex microbiomes.

Sulfide alters microbial functional potential in a methane and nitrogen cycling biofilm reactor.

Cross-feeding of metabolites between coexisting cells leads to complex and interconnected elemental cycling and microbial interactions. These relationships influence overall community function and can be altered by changes in substrate availability. Here, we used isotopic rate measurements and metagenomic sequencing to study how cross-feeding relationships changed in response to stepwise increases of sulfide concentrations in a membrane-aerated biofilm reactor that was fed with methane and ammonium. Results showed that sulfide: (i) decreased nitrite oxidation rates but increased ammonia oxidation rates; (ii) changed the denitrifying community and increased nitrous oxide production; and (iii) induced dissimilatory nitrite reduction to ammonium (DNRA). We infer that inhibition of nitrite oxidation resulted in higher nitrite availability for DNRA, anammox, and nitrite-dependent anaerobic methane oxidation. In other words, sulfide likely disrupted microbial cross-feeding between AOB and NOB and induced cross-feeding between AOB and nitrite reducing organisms. Furthermore, these cross-feeding relationships were spatially distributed between biofilm and planktonic phases of the reactor. These results indicate that using sulfide as an electron donor will promote N2 O and ammonium production, which is generally not desirable in engineered systems.

Syntrophic splitting of central carbon metabolism in host cells bearing functionally different symbiotic bacteria

Insects feeding on the nutrient-poor diet of xylem plant sap generally bear two microbial symbionts that are localized to different organs (bacteriomes) and provide complementary sets of essential amino acids (EAAs). Here, we investigate the metabolic basis for the apparent paradox that xylem-feeding insects are under intense selection for metabolic efficiency but incur the cost of maintaining two symbionts for functions mediated by one symbiont in other associations. Using stable isotope analysis of central carbon metabolism and metabolic modeling, we provide evidence that the bacteriomes of the spittlebug Clastoptera proteus display high rates of aerobic glycolysis, with syntrophic splitting of glucose oxidation. Specifically, our data suggest that one bacteriome (containing the bacterium Sulcia, which synthesizes seven EAAs) predominantly processes glucose glycolytically, producing pyruvate and lactate, and the exported pyruvate and lactate is assimilated by the second bacteriome (containing the bacterium Zinderia, which synthesizes three energetically costly EAAs) and channeled through the TCA cycle for energy generation by oxidative phosphorylation. We, furthermore, calculate that this metabolic arrangement supports the high ATP demand in Zinderia bacteriomes for Zinderia-mediated synthesis of energy-intensive EAAs. We predict that metabolite cross-feeding among host cells may be widespread in animal-microbe symbioses utilizing low-nutrient diets.

Uncoupling of invasive bacterial mucosal immunogenicity from pathogenicity

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection.

The microbial exometabolome: ecological resource and architect of microbial communities.

All microorganisms release many metabolites, collectively known as the exometabolome. The resultant multi-way cross-feeding of metabolites among microorganisms distributes resources, thereby increasing total biomass of the microbial community, and promotes the recruitment and persistence of phylogenetically and functionally diverse taxa in microbial communities. Metabolite transfer can also select for evolutionary diversification, yielding multiple closely related but functionally distinct strains. Depending on starting conditions, the evolved strains may be auxotrophs requiring metabolic outputs from producer cells or, alternatively, display loss of complementary reactions in metabolic pathways, with increased metabolic efficiency. Metabolite cross-feeding is widespread in many microbial communities associated with animals and plants, including the animal gut microbiome, and these metabolic interactions can yield products valuable to the host. However, metabolite exchange between pairs of intracellular microbial taxa that share the same host cell or organ can be very limited compared to pairs of free-living microorganisms, perhaps as a consequence of host controls over the metabolic function of intracellular microorganisms. Priorities for future research include the development of tools for improved quantification of metabolite exchange in complex communities and greater integration of the roles of metabolic cross-feeding and other ecological processes, including priority effects and antagonistic interactions, in shaping microbial communities. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Abstract P1-06-03: Breast cancers exhibit diverse metabolic cross-feeding behaviors associated with differing long-term survival

Abstract Introduction Intrinsic breast cancer subtypes (e.g., Normal-like, Luminal A/B, HER2-enriched, Triple Negative) vary in their responsiveness to neoadjuvant chemotherapy (NACT) and are associated with differing long-term prognosis. Expression dis-regulation of critical regulatory pathways partially accounts for variance in cancer phenotype responsiveness which has allowed development of targeted therapies to improve prognosis for some subtypes. Expression dis-regulation also plays a role in regulating cancer metabolism—the Warburg Effect being the most notable example. Metabolic dis-regulation opens avenues for cancer cells to exploit environmental niches arising in the tumor microenvironment (TME) (e.g., lactate cross-feeding). Focusing on the role TME plays in defining cancer behavior, we undertook a theoretical investigation to uncover how metabolic cross-feeding affects subtype behavior. Methods An in silico analysis of cross-feeding variability arising from metabolic differences in Luminal A/B (LA, LB), HER2-enriched (HER2+) and Triple Negative (TN) breast cancers was performed. Community models of the TME were created by coupling metabolic models of adipocytes, glandular, and cancer tissues derived from a comprehensive reconstruction of human metabolism (Pornputtapong, Nookaew, Jensen, Database, 2015). A parsimonious flux balance analysis technique was used to elucidate the consumption, production, and cross-feeding of metabolites by the three tissues (Lewis et al. Mol. Syst. Biol. 2010). Constraints were applied to the model to limit the uptake of 701 metabolites to levels measured in the blood (Wishart et al., Nucleic Acids Res. 2018). In total, 1222 community models were generated by personalizing cancer models using expression levels of metabolic enzyme transcripts for breast cancer patients in the TCGA BRCA study (The Cancer Genome Atlas Network, Nature, 2019). Results Previously characterized gene expression features were recapitulated via metabolic consumption/production in the models: TNBC/HER2+ had higher asparagine, glutamine (Glu), tryptophan, phenylalanine, consumption than LA/LB (van Geldermolsen et al. Oncogone 2016), Glu was cross-fed from cancer to fat in TNBC (Cao et al. BMC Cancer, 2014), TNBC produced methionine and proline (Kanaan et al. Can. Gen. Prot. 2014), and TNBC/HER2+ cancers consumed high density lipoprotein produced by cancer (Balabum et al. Cancer Met. 2017). The community models also revealed novel findings. TNBC/HER2+ relied on glucose as the primary energy source, while LA/LB relied on amino acids alanine, glycine and Glu. TNBC/HER2+ produced high levels of lactate which was consumed by adipose tissues. Uniquely, glycine produced by cancer was consumed by fat in TNBC. Several environmental niches were induced in the healthy tissue by the presence of the cancer; for example, ornithine was predicted to be cross-fed from fatty to glandular tissues in TNBC/HER2+ cancers, and glutamate and inosine from glandular to fatty tissues in HER2+ cancers. To identify metabolic differences that could explain variability in survival, hierarchical clustering was performed resulting in 13 distinct metabolic categories. As one illustrative example, two clusters enriched in LA/LB cancers had different 10-year survival rates. The poorer prognosis cluster lacked valine, leucine, isoleucine biosynthesis, and arginine, proline and pyruvate metabolism, suggesting resource starvation. Since many NACT therapies act by killing proliferating cells and pathological complete response is correlated to better prognosis, we posit that such starving tumors will be less susceptible to treatment. Conclusion While preliminary, these results suggest that metabolic pathway usage can lead to difference in growth, cross-feeding and potentially drug efficacy. Citation Format: Joseph R Peterson, John A Cole Jr., Tyler M. Earnest, Michael J. Hallock, Tushar Pandey, Katherine Ritchie, Eduardo Braun. Breast cancers exhibit diverse metabolic cross-feeding behaviors associated with differing long-term survival [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-06-03.

Environmental stimuli drive a transition from cooperation to competition in synthetic phototrophic communities.

Phototrophic communities of photosynthetic algae or cyanobacteria and heterotrophic bacteria or fungi are pervasive throughout the environment1. How interactions between members contribute to the resilience and affect the fitness of phototrophic communities is not fully understood2,3. Here, we integrated metatranscriptomics, metabolomics and phenotyping with computational modelling to reveal condition-dependent secretion and cross-feeding of metabolites in a synthetic community. We discovered that interactions between members are highly dynamic and are driven by the availability of organic and inorganic nutrients. Environmental factors, such as ammonia concentration, influenced community stability by shifting members from collaborating to competing. Furthermore, overall fitness was dependent on genotype and streamlined genomes improved growth of the entire community. Our mechanistic framework provides insights into the physiology and metabolic response to environmental and genetic perturbation of these ubiquitous microbial associations.

Metabolite Cross-Feeding between Rhodococcus ruber YYL and Bacillus cereus MLY1 in the Biodegradation of Tetrahydrofuran under pH Stress.

Bacterial consortia are among the most basic units in the biodegradation of environmental pollutants. Pollutant-degrading strains frequently encounter different types of environmental stresses and must be able to survive with other bacteria present in the polluted environments. In this study, we proposed a noncontact interaction mode between a tetrahydrofuran (THF)-degrading strain, Rhodococcus ruber YYL, and a non-THF-degrading strain, Bacillus cereus MLY1. The metabolic interaction mechanism between strains YYL and MLY1 was explored through physiological and molecular studies and was further supported by the metabolic response profile of strain YYL, both monocultured and cocultured with strain MLY1 at the optimal pH (pH 8.3) and under pH stress (pH 7.0), through a liquid chromatography-mass spectrometry-based metabolomic analysis. The results suggested that the coculture system resists pH stress in three ways: (i) strain MLY1 utilized acid metabolites and impacted the proportion of glutamine, resulting in an elevated intracellular pH of the system; (ii) strain MLY1 had the ability to degrade intermediates, thus alleviating the product inhibition of strain YYL; and (iii) strain MLY1 produced some essential micronutrients for strain YYL to aid the growth of this strain under pH stress, while strain YYL produced THF degradation intermediates for strain MLY1 as major nutrients. In addition, a metabolite cross-feeding interaction with respect to pollutant biodegradation is discussed.IMPORTANCERhodococcus species have been discovered in diverse environmental niches and can degrade numerous recalcitrant toxic pollutants. However, the pollutant degradation efficiency of these strains is severely reduced due to the complexity of environmental conditions and limitations in the growth of the pollutant-degrading microorganism. In our study, Bacillus cereus strain MLY1 exhibited strong stress resistance to adapt to various environments and improved the THF degradation efficiency of Rhodococcus ruber YYL by a metabolic cross-feeding interaction style to relieve the pH stress. These findings suggest that metabolite cross-feeding occurred in a complementary manner, allowing a pollutant-degrading strain to collaborate with a nondegrading strain in the biodegradation of various recalcitrant compounds. The study of metabolic exchanges is crucial to elucidate mechanisms by which degrading and symbiotic bacteria interact to survive environmental stress.

A Mathematical Model to Facilitate Study of Hydrogen Cross-feeding by the Human Colonic Microbiota (P13-036-19)

ObjectivesThe role of hydrogen cross-feeding microbes in digestive function is unclear, but several such organisms have been implicated in functional gastrointestinal disorders. In order to study the dynamics of hydrogen cross-feeders, we require a computational model that provides realistic predictions of food metabolism and metabolite cross-feeding by the human intestinal microbiota. The goal is to produce a model that captures the relationships between the concentrations of all major metabolites in the colon and the microbial population. MethodsWe adapted the existing model microPop [Kettle et al., Methods in Ecology and Evolution, 9, 399–409, (2017)] to the human colonic environment. The model divides the microbiota into functional groups, determined by the metabolites that they feed upon and produce. We introduced alterations to the bacterial functional groups in the original model, including the addition of sulphate-reducing bacteria (SRB), which have an important role in hydrogen cross-feeding. Further adaptions included running the model through three sequentially connected compartments representing the proximal, transverse and distal colon. To enhance the applicability of the model to the colon, the production of sulphated colonic mucins by the host was included. ResultsThe model predicts comparable conditions to those found in experimental work. The sulphated mucins were degraded by saccharolytic members of the microbiota to smaller molecules, including hydrogen, short-chain sugars and free sulphate. These metabolites formed a food source for hydrogen cross-feeders, including SRB, as has been seen in rodent models. Cross-feeding for sulphate released from mucins may be more significant in the metabolism of SRB than dietary sulphate. ConclusionsThe model may be used to make predictions about the consequences of certain diets on the production of microbial-derived metabolites and the composition of the microbiota. It also provides predictions about the availability of nutrients in the colon to the host. Finally, the model allows us to perform theoretical studies on the role of hydrogen cross-feeders and the metabolites they secrete in digestive function. Funding SourcesThis work was funded by the Riddet Institute, a New Zealand Centre of Research Excellence.