Genome-Scale Model of Streptococcus thermophilus LMG18311 for Metabolic Comparison of Lactic Acid Bacteria

Construction of the genome-scale model.Genome-scale models are based on annotated genome sequences and experimental data and have become available for an increasing number of organisms, including various LAB (20, 30). A useful tool for the construction of these in silico models is the SimPheny software package (Genomatica Inc., San Diego, CA). The in silico models are based on a thorough metabolic reconstruction of well-annotated genome sequences (29). The reconstruction of the network of S. thermophilus LMG18311 (2, 11) was initiated by an automatic first reconstruction using the autograph method (automatic transfer by orthology of gene reaction associations for pathway heuristics) as described in much detail elsewhere (18). The automatic output of the autograph method was subsequently curated extensively to accommodate the available annotation and literature on metabolic pathways and enzymes, a process described in detail elsewhere (8). Also part of the curation was the comparison of the gene reaction associations with the available annotations in KEGG (http://www.genome.jp/kegg/ ) and in the ERGO bioinformatics suite (http://ergo.integratedgenomics.com/ERGO/ ) (26).

Bacterial strains, media, and growth conditions.The strains used in this study were S. thermophilus LMG18311 (2), L. lactis MG1363 (35), and L. plantarum WCFS1 (14). Cells were grown anaerobically in chemically defined medium (CDM) (15, 21, 23), containing the amino acids (see Table S5 in the supplemental material), at 42°C, 30°C, and 37°C, respectively.

Amino acid omissions.Cells of S. thermophilus were grown overnight in CDM (15) containing all 20 amino acids in the concentrations shown in Table S5 in the supplemental material. The overnight cultures were washed twice at 4°C in a Megafuge 1.0R (Heraeus Instruments, Germany) in phosphate-buffered saline.

CDM without amino acids was freshly prepared for each experiment. Different combinations of amino acids were added to this medium. The amino acids were added in the same concentrations as those used in complete CDM. We started with single omissions of amino acids followed by multiple omissions until we found the most minimal combination. The concentrations of the different amino acids supplied are listed for the different experiments (see Table 2). The different minimal defined media were inoculated 0.5% in triplicate with the washed overnight culture, and growth was followed by measuring the optical density at 600 nm (OD₆₀₀).

Growth on defined medium (chemostat).Fermentations were performed in duplicate as described by Teusink et al. (30). S. thermophilus LMG18311 was grown at 42°C in CDM in a 50-ml tube and used as the inoculum of 500 ml of pH-controlled (pH 6.5) CDM, while the medium was 1% inoculated. Fermentations were performed in a 2-liter fermentor (Applikon Biotechnology BV, The Netherlands). The fermentations were controlled by a Bio Controller ADI 1010 and by a Bio Console ADI 1025 (Applikon Biotechnology BV, The Netherlands). The headspace was flushed with nitrogen (10 ml min⁻¹) at a stirring speed of 100 rpm. At an OD₆₀₀ of ∼0.5, the medium pump was switched on to reach a dilution rate of 0.4 h⁻¹. Steady-state conditions were achieved within five volume changes (30). The dilution rate was changed three times, so a total of four dilution rates was achieved (0.1 h⁻¹, 0.2 h⁻¹, 0.3 h⁻¹, and 0.4 h⁻¹). At each steady state, four 50-ml samples were taken and spun down at 4°C in a UniCen MR centrifuge (Herolab, The Netherlands). Supernatant was used for high-performance liquid chromatography (HPLC) analysis of organic compounds (28).

GC analyses.For the identification of volatile components in the samples, purge-and-trap thermal desorption cold-trap gas chromatography (GC) was used as described previously (7, 27). The headspace samples were concentrated on a Fisons MFA 815 cold trap (CE Instruments, Milan, Italy), followed by separation on a GC-8000 top gas chromatograph (CE Instruments) equipped with a CP-Sil 5 CB low-bleed column (Chrompack, Middelburg, The Netherlands) and detection by a flame ionization detector. The GC data were processed in MetAlign, a tool (developed by Plant Research International, The Netherlands) to align spectra and to identify significant differences between the spectra (6, 16).

HPLC analyses.Extracellular metabolites present in the supernatant of fermentation samples were measured using reversed-phase HPLC analysis with a C₁₈ column as described elsewhere (28).

Genome-scale model development.A genome-scale metabolic model for S. thermophilus has been developed based on the annotated genome of strain LMG18311 (2, 11). The available models of L. plantarum (30) and L. lactis, which were constructed using the autograph method (18), were used for the construction and development of the S. thermophilus model. Based on these models, many gene-protein relationships and non-gene-associated reactions could be incorporated into our model, resulting in a metabolic map of S. thermophilus (Fig. 1).

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FIG. 1.

Primary metabolism of Streptococcus thermophilus. Part of the total genome-scale metabolic model developed for S. thermophilus. Large bold capital italic letters indicate the enzymes, and smaller lowercase roman letters indicate the metabolites. For the complete model, see Fig. S1 in the supplemental material.

Different features of every gene, such as correct annotation, function, and corresponding EC number, were checked manually before they were included in (or excluded from) the model. Examples of excluded genes are truncated, hypothetical, and nonmetabolic. Excluded genes are not deleted and can be included again later when the function of such a gene has been identified. Genes coding for metabolic enzymes have been included and associated with the corresponding reactions (30). Also, non-gene-associated reactions, based on biochemical and experimental evidence (fermentations, amino acid omissions), were added to close gaps in the biochemical network. These included the following: (i) vitamin transport systems such as nicotinic acid uptake, (ii) specific S. thermophilus protein synthesis based on experimental data, and (iii) different uptake systems such as oxygen diffusion, a proton symporter for lactate. The current model consists of 429 genes (23% of the total number of genes) and 522 model reactions, 79 (15%) of which are non-gene associated. Moreover, the biomass composition of this strain was determined in this study and compared with two other LAB (Table 1). The closely related strains L. lactis and S. thermophilus have comparable amounts of protein. Organic compounds in fermentation samples were measured by HPLC, on the basis of which fluxes were calculated (30). Both biomass data and fluxes were used for in silico simulations. The model of S. thermophilus is now at a stage where in silico growth can be simulated under different conditions.

Amino acid omissions.Experiments with single amino acid omissions in S. thermophilus have shown that the number and type of essential amino acids is strain dependent (9, 15, 17). In general, S. thermophilus has a much lower degree of auxotrophy for amino acids than other LAB (4), showing no growth only in the absence of histidine and clearly reduced growth in the absence of cysteine (see Table S1 in the supplemental material).

Multiple omissions of amino acids, performed in our laboratory, showed that S. thermophilus LMG18311 needs only histidine and one of the sulfur-containing amino acids (cysteine or methionine) in the presence of citrate for (minimal) growth (Table 2). We have performed the growth experiments on a minimal defined medium with histidine, cysteine, and glutamic acid, since the addition of glutamic acid improved the growth rate significantly and growth experiments showed that cysteine is preferred over methionine.

In silico predictions of the amino acid biosynthesis pathways of S. thermophilus LMG18311 were performed (11), and this strain indeed seems to contain all the genes coding for the enzymes required for the biosynthesis of all amino acids except histidine. This analysis also showed that yhcE is truncated by a conserved stop codon. The product of yhcE shows similarity to the vitamin B₁₂-independent 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase. Its orthologue in L. lactis is involved in the synthesis of cysteine from methionine. This gene inactivation may explain the auxotrophy for one of the two sulfur amino acids. Even though the genome of LMG18311 lacks a glutamate synthase gene, the strain shows (minimal) growth in the presence of citrate when both glutamate and glutamine were depleted from the medium. However, S. thermophilus possesses a pathway for the synthesis of glutamate from citrate via 2-oxoglutarate involving glutamate dehydrogenase and glutamine synthetase for interconversion between glutamic acid and glutamine.

Different LAB have different absolute requirements for amino acids; S. thermophilus only needs 2 amino acids, as described above, whereas L. lactis and L. plantarum need 6 and 11 amino acids for minimal growth, respectively (12, 30) (Table 3).

GC analyses.In order to get an overview of flavor formation by the three different LAB, we compared fermentation samples using GC. The headspaces of steady-state samples of S. thermophilus LMG18311, L. lactis MG1363, and L. plantarum WCFS1 grown on CDM (containing all amino acids) were compared. The metabolic activities of the fermenting microbes (22) were investigated through flavor profiles in the fermentation fluids corrected for the medium components at the start of the experiments. An overview of the volatile metabolic products is shown in Fig. 2, 3, and 4, and they show multiple differences in the volatile profiles of different strains. Many volatiles or flavors are produced during amino acid metabolism. When the results of the GC analyses of the three LAB are compared, they show that S. thermophilus is able to produce a broad variety of flavors. In combination with the low requirements of amino acids (only two), this reflects a relatively complete set of amino acid biosynthetic and amino acid-converting pathways. When S. thermophilus grows on CDM, all amino acids are consumed in small amounts (data not shown). L. lactis and L. plantarum need more amino acids (6 and 11, respectively) for minimal growth, and especially L. plantarum produces less flavors.

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FIG. 2.

Major volatiles formed during growth by L. lactis on CDM. Relative peak areas are expressed as arbitrary units, and the areas of three peaks are indicated since they are beyond the scale. Some important peaks are indicated. For all the identified metabolites for L. lactis, see Table S2 in the supplemental material.

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FIG. 3.

Major volatiles formed during growth by L. plantarum on CDM. Relative peak areas are expressed as arbitrary units. Some important peaks are indicated. For all the identified metabolites for L. plantarum, see Table S3 in the supplemental material.

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FIG. 4.

Major volatiles formed during growth by S. thermophilus on CDM. Relative peak areas are expressed as arbitrary units, and the areas of three peaks are indicated since they are beyond the scale. Some important peaks are indicated. For all the identified metabolites for S. thermophilus, see Table S4 in the supplemental material.

One of the identified compounds produced by all three LAB is acetaldehyde. As described previously (5), S. thermophilus can convert threonine into acetaldehyde and glycine by threonine aldolase activity. L. lactis and L. plantarum, among others, can produce acetaldehyde during lactose metabolism by pyruvate decarboxylation (3). This difference in pathways leading to the same compound can also be visualized in the SimPheny models, as was shown in our previous paper (22).

Homofermentative metabolism. S. thermophilus was grown under chemostat conditions on CDM containing all amino acids. Steady-state fermentation samples (dilution rate, 0.1 to 0.4 h⁻¹) of S. thermophilus were used for different analyses. The supernatant of these samples was analyzed by using HPLC and was compared with the composition of the growth medium to determine which compounds are produced and consumed during growth (Table 4).

The HPLC analysis shows that S. thermophilus consumes all the glucose and some of the citric acid. S. thermophilus produces mainly lactate, and only small amounts of pyruvate, succinate, and formate are formed. The model strongly suggests that homofermentative lactic acid production is the only primary metabolism operating in S. thermophilus, and this is confirmed by our fermentation data and also by others (11). The mixed acid fermentation (acetate, formate, and ethanol) is metabolically the most efficient route for LAB, whereas the homolactic route is catalytically more efficient (10). Both L. lactis and L. plantarum can grow via homolactic (high dilution rates) or mixed-acid (low dilution rates) fermentation (10, 30). Because S. thermophilus has pseudogenes in the primary metabolism that prevent the formation of ethanol, acetate formation will cause a redox problem, and hence, the only possible route is the homolactic fermentation at both high and low dilution rates.

Flux balance analysis (FBA) was carried out within the SimPheny software (30). FBA is an optimization technique that can be used as a tool to predict the metabolic possibilities given mass balance and capacity constraints (24). FBA correctly predicted homolactic fermentation in S. thermophilus, in contrast to what was found for L. plantarum (30) and L. lactis (20). Based on the sequenced genome of strain LMG18311 that was visualized on the model, it is known that this strain does not have the oxidative part of the pentose phosphate pathway (PPP). The absence of a complete PPP may have important consequences for the redox balance and thereby may potentially influence primary metabolism.