Genome-scale metabolic reconstructions of Bifidobacterium adolescentis L2-32 and Faecalibacterium prausnitzii A2-165 and their interaction
© El-Semman et al.; licensee BioMed Central Ltd. 2014
Received: 17 November 2013
Accepted: 21 March 2014
Published: 3 April 2014
The gut microbiota plays an important role in human health and disease by acting as a metabolic organ. Metagenomic sequencing has shown how dysbiosis in the gut microbiota is associated with human metabolic diseases such as obesity and diabetes. Modeling may assist to gain insight into the metabolic implication of an altered microbiota. Fast and accurate reconstruction of metabolic models for members of the gut microbiota, as well as methods to simulate a community of microorganisms, are therefore needed. The Integrated Microbial Genomes (IMG) database contains functional annotation for nearly 4,650 bacterial genomes. This tremendous new genomic information adds new opportunities for systems biology to reconstruct accurate genome scale metabolic models (GEMs).
Here we assembled a reaction data set containing 2,340 reactions obtained from existing genome-scale metabolic models, where each reaction is assigned with KEGG Orthology. The reaction data set was then used to reconstruct two genome scale metabolic models for gut microorganisms available in the IMG database Bifidobacterium adolescentis L2-32, which produces acetate during fermentation, and Faecalibacterium prausnitzii A2-165, which consumes acetate and produces butyrate. F. prausnitzii is less abundant in patients with Crohn’s disease and has been suggested to play an anti-inflammatory role in the gut ecosystem. The B. adolescentis model, iBif452, comprises 699 reactions and 611 unique metabolites. The F. prausnitzii model, iFap484, comprises 713 reactions and 621 unique metabolites. Each model was validated with in vivo data. We used OptCom and Flux Balance Analysis to simulate how both organisms interact.
The consortium of iBif452 and iFap484 was applied to predict F. prausnitzii’s demand for acetate and production of butyrate which plays an essential role in colonic homeostasis and cancer prevention. The assembled reaction set is a useful tool to generate bacterial draft models from KEGG Orthology.
Metagenomic sequencing facilitates the study of a large number of microorganisms in environmental samples . This technique has been used to study the composition of gut microbiota , its role in human metabolism [3, 4] and its relation to diseases such as atherosclerosis , obesity [6, 7] and Crohn’s disease . In functional metagenomic studies, it is common to use KEGG Orthology (KO)  to annotate gene functions . KO can be used to predict the composition ratio of microbial gene families and pathways from the human microbiome project . The functional annotation for a large number of sequenced bacteria, nearly 4,650 bacterial genomes, is stored in the Integrated Microbial Genomes (IMG) database, and the genomes are mapped to KEGG pathway images . This tremendous new genomic information adds a new opportunity for systems biology, as it enables use of information about genome content for prediction of metabolic phenotypes of species in the gut , or to develop community systems  or supra-model organisms . Therefore, it is relevant to reconstruct accurate genome scale metabolic models (GEMs) from KO annotated by metagenomic analysis.
Several methods have been developed to reconstruct genome scale models from GEMs of closely related organisms , KEGG [16–18], and the Model SEED . The RAVEN toolbox  has been used to generate GEMs for the eukaryotic microorganisms Pichia stipitis and Pichia pastoris using iIN800, a GEM of Saccharomyces cerevisiae. However, this method requires a GEM of a closely related organism. The RAVEN toolbox has another function to solve this problem by assigning gene to KO using MUSCLE  and HMMER . Then it generates the draft model by mapping KO to KEGG reactions. The web-based methods, FAME  and MicrobesFlux , are able to produce draft models for ~750 and ~1,200 KEGG genomes, respectively. The disadvantage of both FAME and MicrobesFlux is that they are limited to organisms already annotated in KEGG. The Model SEED can generate a draft model for a desired organism based on RAST annotation of genes . Even though some of these methods have computational gap filling methods, there is still a need for manual curation to obtain a functional model. Manual curation is generally cumbersome and time-consuming. The lack of visualization, such as organization and readability of reactions and genes names into the model Excel file and KEGG maps, can hamper manual curation of generated draft models.
To facilitate generation of a draft model and manual gap filling, we assembled an organized reference reaction data set consisting of common microbial reactions, where every reaction is assigned with KOs. The reactions were collected from high quality GEMs and from Rhea, a manually annotated database of chemical reactions , but not from KEGG reactions. In spite of the accuracy of KEGG reactions, some reactions need to substantially manual curation for substrate and co-factor usage, and the reactions in reconstructed GEMs generally have to be well annotated in terms of substrate and co-factor usage and elemental balancing. Our reaction data set can, in principle, be used to generate draft models for all 4,650 bacteria in the IMG database, KEGG organisms, or other user-defined organisms annotated by KO.
Here, we used this reaction data set to generate high quality GEMs for two bacterial genomes from the IMG database: B. adolescentis L2-32 and F. prausnitzii A2-165. Bifidobacterium is a dominating genus in the phyla Actinobacteria present in the human gut microbiota and Faecalibacterium is the most abundant genus among the Firmicutes. Firmicutes, Bacteroidetes and Actinobacteria are the most highly abundant phyla in the human gut microbiota . Both Bifidobacterium and Faecalibacterium interact with Bacteroidetes[25, 26]. Moreover, Bifidobacterium produces acetate to protect the host from infection , and Faecalibacterium has a relation with Crohn disease . Furthermore, the production of butyrate by Faecalibacterium, among others, has been associated with a healthy state [5, 29, 30].
Step 1 (Collecting reference reaction data set)
We assembled a reference reaction data set containing reactions assigned with KO for each KEGG map (see Additional file 1: Figure S1). For organisms Escherichia coli K-12 MG1655, Staphylococcus aureus N315 and Saccharomyces cerevisiae, we mapped the genes from each KEGG map to the corresponding reactions contained in the respective GEMs iAF1260 , iSB619  and iTO977 . The GEM reactions, together with the associated KOs, were then added to the reference reaction data set. In the case the organism had no genes for the enzyme or all GEMs lacked the reaction, we downloaded it from Rhea by the Matlab function get_reaction_from_Rhea. This function downloads Rhea reactions as XML format and prints them. To avoid the mismatched metabolite names between Rhea reactions and the reference reaction set, we evaluated the metabolite names in the Rhea reaction using its corresponding KEGG reaction. We retained Rhea metabolite names if they did not exist in the reference reaction data set, and the Rhea reaction and its corresponding KO were subsequently inserted into the reference reaction data set. iAF1260 reactions not present in any of the KEGG maps, especially transporters and reactions occurring in the cell wall, were inserted into the reference reaction data set with the corresponding KO as the corresponding gene. Moreover, we added the capsular polysaccharide and teichoic acid biosynthesis reactions, which are required for cell wall biosynthesis, from the GEM of Lactobacillus plantarum WCFS , because cell wall could be a significant fraction of gram dry weight of Gram-positive bacteria. Also we added the methane metabolism from the GEM of Methanosarcina barkeri and added the siderophore group biosynthesis from the GEM of Mycobacterium tuberculosis. Finally, the reactions were organized, ordered and made readable to facilitate the manual gap filling process.
Step2 (Generating draft models)
We downloaded the gene annotation for each studied organism from the IMG database . We extracted the set GK = (gene,KO) for each organism from the downloaded IMG file using the function get_gene_ko_from_img. For organisms available in KEGG, the set GK can be obtained directly using the function get_gene_ko_from_kegg_org_id, otherwise the users can build the set GK themselves. The set GK was passed to the function buildDraftModel to extract reactions using KO identifiers from the reaction data set. The draft model was exported to an Excel file by the function saveDraftModel. Finally, we removed the exchange reaction for metabolites that were not participating in any cytosolic reaction. All the described functions are provided in Additional file 2 and can be used in the RAVEN toolbox.
Step 3 (Gap filling)
The gaps in each model were filled manually by mapping the model to KEGG maps and inserting the required reactions to ensure full connectivity in the model. To find genes for the filled reactions or metabolic genes, we extended the search to other available gene annotation in the IMG database. Both studied organisms have genes annotated by Pfam , TIGRFAMs , TC families  and METACYC . Moreover, B. adolescentis L2-32 has gene annotations by SEED. Annotation with TC families has no specific gene assignment, so we ran a bidirectional blast between the TC protein sequence and each organism sequence. Also, we searched for the residual filled reactions by their enzyme name or EC number in the Pfam database to get the corresponding Pfam identifiers, and the corresponding genes were searched in IMG file. Additional file 3: Table S1 contains the results of this analysis.
Flux balance analysis
Both B. adolescentis L2-32 and F. prausnitzii A2-165 models grow anaerobically in rich media. We assumed that each model consumed ammonia as a source of nitrogen, phosphate, H2S or cysteine as a source of sulfur, nicotinate and all amino acids having transporter reactions. In addition, The F. prausnitzii A2-165 model consumed folic acid from the medium. Xanthine, uracil and urea transporters were closed.
The compositions of protein, RNA and DNA in the biomass were estimated from Neidhardt et al. . The compositions of peptidoglycan and capsular polysaccharide are the same as in the GEM of Lactobacillus plantarum WCFS .
We used flux variability analysis  to evaluate the predicted fluxes by the COBRA toolbox function fluxVariability (see Additional file 4: Table S2). To determine if a studied organism is able to grow on different carbon sources such as galactose, xylose or fructose, the transporter flux was fixed to 1 mmol/gDW/h and the biomass formation was optimized.
Description of community using XML
We described a community structure without details of each model as XML format (see Additional file 5), because LibSBML fails to read an SBML containing a user defined attribute or XML tag for community features . Both iBif452 and iFap484 competed for glucose while iFap484 consumed acetate produced by iBif452. The functions generateOptComModel and generateComModel used XML files to generate OptCom and FBA models.
Results and discussion
Reconstruction of reference reaction data set
GEMs elucidate how organisms consume nutrients, carbon source, ammonia, phosphate and autotrophic metabolites to build their biomass precursors and produce chemical byproducts . The biochemical reactions included in a GEM are based on experimental or predicted function of enzymes contained by the studied organism . Reconstructed GEMs share many components: exchange flux, transport, central metabolism, nucleotide, amino acids, cofactor biosynthesis, cell wall and lipid. Most GEMs use KEGG maps and literature to illustrate the content of each component, where it contains one or more KEGG maps. Additional file 1: Figure S1 (adapted from ) shows these components and the KEGG map name for each component and how flux distributes to each component and builds the necessary biomass precursors.
To cover the reactions in Additional file 1: Figure S1, we built a reference reaction data set from published GEMs and a manually curated reaction database Rhea. This reaction data set contained reactions for the central carbon metabolism (glycolysis, PP pathway, TCA, pyruvate), amino acids and nucleotide biosynthesis, cell wall (peptidoglycan, capsular polysaccharide and teichoic acid biosynthesis) and cofactors (folate, CoA and NAD+). We adopted the fatty acids biosynthesis and glycerophospholipd metabolism in iAF1260. Then we included reactions that connect other carbon resources, such as galactose and maltose to the main network.
The reactions were organized to facilitate manual revision and editing of newly reconstructed GEMs. Each reaction was assigned with KOs obtained from KEGG maps. The reference reaction data set comprises 2,340 reactions out of which 214 came from Rhea, 1256 unique metabolite and 2146 KOs (see Additional file 6: Table S3). The reference reaction data set was used to generate a draft model with an input file containing a gene and its KO. Additional file 7: Figure S2 shows how the reaction set covers KEGG maps.
Description of GEMS: iBif452 and iFap484 of B. adolescentis L2-32 and F. prausnitzii A2-165 in comparison with draft model generating by Model SEED
B. adolescentis L2-32
F. prausnitzii A2-165
Reactions without genes
Genes from other annotations
Both iBif452 and iFap484 have reactions for the central carbon metabolism and can utilize other sole carbon sources than glucose, as reported in in vivo studies. iBif452 can utilize galactose, fructose and maltose, which is consistent with in vivo studies . The iFap484 can also utilize galactose and maltose but cannot utilize xylose, which is also consistent with in vivo studies . iBif452 features the bifid shunt pathway or the F6PPK pathway representing a special Bifidobacteria pathway converting glucose to pyruvate (see Additional file 9: Figure S3) [57–59]. The F6PPK pathway includes fructose-6-phosphate phosphoketolase converting D-Fructose 6-phosphate to Acetyl phosphate and D-Erythrose 4-phosphate, compared to the common part of glycolysis with 6-phosphofructokinase and fructose-1,6-bisphosphate aldolase. iFap484 has a Faecalibacterium prausnitzii butyrate producing pathway (see Additional file 10: Figure S4) . Neither iBif452 nor iFap484 produces anything when the glucose uptake rate is 0 mmol/gDW/h and the objective function is biomass or ATP non-growth association maintenance, so the models did not generate energy or matter from nothing. In spite of the two models were validated with FBA in the following two sections, the comprehensive validation of the GEMs needs extensive experiments. Since these bacteria are not yet well-studied, we think the two models based on recently sequenced, may assist a lot to overcome some important questions, computing phenotypic states and describing the genotype-phenotype relationships.
Bifidobacterium adolescentis adolescentis L2-32 validation
Bifidobacterium has predicted genes for biosynthesis of all 20 amino acids, purines and pyrimidines . However, Bifidobacterium only grows in complex media, probably because some of the genes in the biosynthetic pathway for amino acids are non-functional . We assumed that iBif452 grows in media containing 12 amino acids for which it has transporter reactions.
The iBif452 model did not produce lactate when biomass or ATP production was optimized, while Bifidobacterium produces acetate, lactate, formate and ethanol in vivo. Under glucose limitation, Bifidobacterium does not produce lactate because it tries to maximize energy production by cleaving pyruvate to acetyl phosphate and formate . Furthermore, the specific rate of sugar consumption affects the amount of lactate production. For example, the organism produces a large amount of lactate when it has a rapid sugar consumption, but produces a small amount of lactate when it consumes a less preferred sugar like oligofructose .
To study the ability of the model iBif452 to produce lactate, we maximized ATP production for non-growth association maintenance, i.e., the reaction ATP + H2O = > ADP + Phosphate + H. The model produced 3 mmol of ATP per 1 mmol of glucose and produced only acetate, formate and ethanol. When the model was constrained to produce 1 mmol of lactate per 1 mmol of glucose, it produced 2.5 mmol of ATP and 1.5 mmol of acetate per 1 mmol of glucose.
The last results showed that the model aimed to generate ATP by converting pyruvate to acetate through acetyl-CoA and acetyl-phosphate and it therefore has to regenerate NAD + by forming ethanol, as this is only way this co-factor can be balanced when there is formation of acetate (See Additional file 9: Figure S3). Although the model predicts a flux distribution for the theoretical ratio between acetate and lactate in Bifidobacterium, it fails to predict the amount of lactate just like previous GEMs of lactic acid bacteria . To overcome this problem, Oliveira et al. constrained the pyruvate formate lyase reaction to an interval to deal with lactate production in a GEM of L. Lactic. Bas Teusink et al. fixed the measured flux in the GEM of L. plantarum WCFS1 . Milan et al. added new enzyme turnover parameter to avoid metabolism overflow in GEM of L.lactis based on flux balance analysis with molecular crowding . Finally, Bas Teusink et al. showed that L. plantarum optimizes its yield when it grows with glycerol to support the prediction of GEM in lactic acid bacteria .
Comparison between in-silco prediction of short chain fatty acids of iBif452 and iFap484 with experimental data
Faecalibacterium prausnitzii A2-165 validation
To study the effect of external acetate on butyrate production in Faecalibacterium prausnitzii A2-165, biomass production was used as an objective function. The model produces butyrate with a yield of 1.62 (mmol butyrate/mmol glucose) and co-consumes 1.39 mmol of acetate per mmol of glucose. The ratio of acetate uptake to butyrate production was 85.8%, which is close to the 85-90% observed in in vivo studies of F. prausnitzii. Flux variability analysis shows that the acetate, butyrate, and formate have a difference of 0.0005, 0.014, and 0.014 mmol/gDW/h between maximum and minimum fluxes, respectively. Table 2 shows the comparison between these values with in vivo studies , where F. prausnitzii consumed 10 mM of glucose and 9.55 ± 1.2 mM of acetate to produce 10.45 ± 1.53 mM of butyrate.
Both OptCom and FBA methods were applied to iBif452 and iFap484 to simulate how B. adolescentis L2-32 and F. prausnitzii A2-165 co-culture together. Both organisms compete for 1 mmol/gDW/h of glucose to maximize their growth. The model iBif452 generates acetate and iFap484 consumes acetate to produce butyrate, which plays a critical role in colonic homeostasis and cancer prevention [71–73].
We assembled a reaction set from published GEMs, where each reaction is assigned with KO. This reaction set was used to generate draft GEMs for each non-KEGG organisms. It represented a simple method to generate bacterial draft models from KEGG KO, instead of generating it from KGML . The description of a community as a XML format can be used together with the two community simulation methods Optcom and FBA. This saves time and effort when performing community modeling.
Community simulations of an acetate producer B. adolescentis adolescentis L2-32 and an acetate consumer F. prausnitzii A2-165 provided insights into metabolic cross talk between these two members of the gut microbiota. It shows the importance of acetate supply to butyrate production, since the growth and production of Faecalibacterium prausnitzii is severely hampered by limited acetate supply. This is an initial attempt to approach the very complex ecosystem and metabolic organ that the gut microbiota constitutes.
We thank Ali Zomorrodi and Prof. Costas Maranas for providing us with OptCom implementation. Ibrahim E. El-Semman appreciates the Egyptian Cultural Affairs and Missions Sector for their financial support. We acknowledge funding from the Knut and Alice Wallenberg Foundation. We thank Chalmers Library for funding the open access charge.
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