Within this approach, the authors identify the metabolic cores of these organisms. These metabolic cores represent connected sets of reactions that are used in all tested environments. When cross-correlating the predicted cores with experimental data, the authors find that the cores display a significantly higher fraction, both of essential and evolutionary conserved enzymes, than their noncore counterparts. Additionally, the extended mRNA half-lives of the core enzymes give further support to the notion that core reactions represent main integrators of metabolic activity.

The two types of response mechanisms described by flux and structural plasticity imply the possible existence of a group of reactions that are not subject to structural plasticity, being active under all environmental conditions. Indeed, some metabolic reactions were found to carry non-zero fluxes in S. cerevisiae under nine different growth conditions [6]. In itself, however, this finding is compatible with a scenario in which the active reactions are randomly distributed: if typically a q fraction of the metabolic reactions are active under a specific growth condition, for n distinct conditions one can predict a non-zero overlap encompassing at least qⁿ fraction of the reactions. Indeed, we find that as we increase the number of inspected conditions, n, the number of reactions carrying non-zero flux in each condition decreases rapidly. However, it eventually saturates at a constant value (Figure 1A–C), identifying a group of reactions that have non-zero flux under all 30,000 simulated growth conditions. Specifically, we find that in 138 of 381 H. pylori (36.2%), 90 of 758 E. coli (11.9%), and 33 of 1,172 S. cerevisiae (2.8%), metabolic reactions are always active (Figure 1D and and1E).1E). While these reactions respond to environmental changes only through flux-based plasticity, the rest of the reactions are only conditionally active, being turned on only in specific growth conditions and thus being subject to both structural- and flux-based plasticity.

Figure 2 displays those metabolic reactions of E. coli that remain active in all 30,000 simulated growth conditions. The striking feature of this diagram is the fact that these reactions form a single connected cluster, encompassing each of the 90 reactions. This is not a unique feature of E. coli; in H. pylori, all 138 reactions form a single cluster, and in S. cerevisiae all 33 reactions are connected. Given the relatively low number of reactions that are always active, the likelihood that they form a single large cluster by chance alone is negligible, with p < 1e⁻⁶ for H. pylori and E. coli, and p ~ 2e⁻⁶ for S. cerevisiae. Given the compact and clustered nature of the group of reactions that are always active, we will refer to them collectively as the metabolic core.

The metabolic core contains two types of reactions: The first type consists of those that are essential for biomass formation under all environmental conditions (81 out of 90 reactions in E. coli), while the second type of reaction is required only to assure optimal metabolic performance (Table S1). In case of the inactivation of the second type, alternative sub-optimal pathways can be used to ensure cellular survival. Interestingly, in the compact core of S. cerevisiae, all 33 reactions were predicted to be indispensable for biomass formation under all growth conditions. Moreover, when assuming a 10% reduction in the growth rate (compared to optimal growth), the size and identity of the E. coli metabolic core remains largely unchanged, retaining 83 of the original 90 reactions, of which two are nonessential. This indicates that the concept of the metabolic core is valid under both optimal and suboptimal growth, although with some difference in the identity of individual reactions. Of note, the metabolic core represents a subset of the minimal reaction sets [10,11] and the overlap of alternative (i.e., degenerate) minimal reaction sets [4,5]. The minimal reaction set of [11] contains the metabolic core in addition, however, to reactions necessary for the sustained growth on any chosen substrate, whereas the minimal reaction set of [5] consists of the 201 reactions that are always active in E. coli for all 136 aerobic and anaerobic single-carbon-source minimal environments capable of sustaining optimal growth. The latter finding is in excellent agreement with Figure 1B (upper curve), and demonstrates that the analysis of a low number of growth conditions will significantly overestimate the set of reactions that are always active.

To identify some of the factors that determine the size of the metabolic core, we note that the number of core metabolic reactions systematically decreases as we move from H. pylori to S. cerevisiae (Figures 3A and and1E),1E), the relative size of the core decreasing from 36.2% of all reactions in H. pylori to 11.9% in E. coli and 2.8% in S. cerevisiae. This trend can be explained as a collective network effect: the relatively small size of the H. pylori metabolic network leaves little flexibility for biomass production, requiring the continuous activity of a high fraction of the available metabolic reactions. Indeed, we find that on average approximately 61% of the H. pylori reactions are active in a given environmental condition. The larger number of metabolic reactions present in E. coli offers a higher degree of metabolic flexibility, allowing for a significant fraction of the biomass to be produced by alternative pathways. Indeed, the average utilization of the E. coli metabolic network in a given growth condition is only 35.3%. For S. cerevisiae, whose metabolic network size significantly exceeds that of both H. pylori and E. coli, there is an even higher metabolic flexibility, and the activity of only 19.7% of the reactions are required in a typical environment.

The fact that the core reactions are active under all investigated environmental conditions suggests that they must play a key role in maintaining the metabolism's overall functional integrity. Therefore, the absence of individual reactions that are part of the metabolic core may lead to significant metabolic disruptions. Indeed, using genome-scale deletion-phenotype data obtained in rich growth media [12,13], we find that 74.7% of those E. coli enzymes that catalyze core metabolic reactions (i.e., core enzymes) are essential, compared with a 19.6% lethality fraction characterizing the noncore enzymes. The gap between the core and noncore enzymes is also significant for S. cerevisiae, for which essential enzymes catalyze 84% of the core reactions, whereas the average essentiality of the conditionally active enzymes is only 15.6% (Figure 3B). Note that the likelihood of a random concentration of so many essential enzymes in the core is extremely small, with p-values of 3.3e⁻²³ and 9.0e⁻¹³ for E. coli and yeast, respectively, even if we presume the presence of errors in the deletion-phenotype data (Protocol S1). Taken together, these results indicate that an organism's ability to adapt to changing environmental conditions rests, to a large extent, on the continuous activity of the metabolic core, regardless of the environmental conditions.

Intuitively, one could assume that the core represents a subset of high-flux reactions characterizing the activity of metabolic networks [3]. However, our measurements indicate that, on average, the fluxes of the core and noncore reactions are highly comparable (Figure 3C). Alternatively, we could also assume that the main biological role of the metabolic core is to ensure the continuous production of biomass under all growth conditions. In contrast, we find that 20 out of the 51 metabolites (39%) considered necessary for biomass production in E. coli [8] are not produced by any of the core reactions; instead—in a growth-condition-dependent fashion—they are produced by various alternative metabolic pathways. The core, however, contains a large number of reactions for selected anabolic pathways, including those of membrane lipid-, cell envelope-, and peptidoglycan-biosynthesis pathways (Table S1). These core reactions represent network bottlenecks, being the only paths for the synthesis of certain biomass components. Therefore, it appears that the composition of the metabolic core is determined by two factors. First, those metabolic reactions that directly contribute to biomass production tend to be part of the metabolic core of the organism. Second, however, this tendency is offset by network-induced redundancy: reactions or pathways whose end-products can be synthesized by at least two alternative pathways show environmental redundancy and structural plasticity, and are thus eliminated from the core. Therefore, the more reactions a metabolic network possesses (see Figure 1D), the stronger is the network-induced redundancy, and the smaller is the core (Figure 1E).

Given the important functional role played by the metabolic core in a given organism, one would expect significant parts of the core to be conserved in different organisms. Indeed, the E. coli and H. pylori cores have 63 reactions in common, and 18 of the 33 core reactions in S. cerevisiae are present in both the E. coli and the H. pylori metabolic cores (see Figure 3A). Also, when considering enzyme orthologs among 32 divergent bacteria [13], we find that the metabolic core enzymes of E. coli display a high degree of evolutionary conservation, the average core enzyme having orthologs in 71.7% of the reference bacteria (p < 1e⁻⁶). In contrast, the conditionally active noncore enzymes have an evolutionary retention of only 47.7% [13]. Given the observed correlation between evolutionary retention and the essentiality of gene products [13], this difference may be a simple consequence of the high-lethality fraction of the core enzymes. However, further analysis indicates that this is not the case: random selection of 90 enzymes with a 74.7% lethality ratio has an average evolutionary retention of only 63.4%. Taken together, these results indicate that major portions of the metabolic core have been conserved, displaying a higher evolutionary retention than the individual essentiality of its participating enzymes would indicate, suggesting that maintaining the core's integrity is a collective need of the organism.

The requirement for the continuous activity of the core reactions may well also impact the regulation of its catalytic enzymes. We would expect that the activity of core reactions—for which only the flux magnitude needs to be modulated—should display a higher degree of stability and a different regulatory control than the reactions outside of the core. Evidence for such regulatory effects is provided by two measurements. First, we inspected the experimentally determined mRNA half-lives of the E. coli metabolic enzymes when cells were grown in Luria-Bertani medium [14]. We found that the distribution of mRNA half-life times for the core enzymes displays a shift to higher values, with the average half-life time of their mRNAs being 14.0 min compared with 10.5 min for the noncore mRNAs (p = 0.016). Furthermore, only 2% of the core enzymes have corresponding mRNA half-life times shorter than 5 min, compared to 23% for the ones catalyzing conditionally active noncore reactions (see Materials and Methods). Therefore, the continuous metabolic need for these reactions has apparently affected the mechanisms responsible for mRNA decay of their catalytic enzymes, providing a higher dynamic inertia under environmental changes.

Additionally, for each enzyme-encoding operon, we counted the number of activating and repressive regulatory links in the E. coli transcriptional regulatory network [15,16]. As regulatory interactions are currently known for only 13 of the core enzyme-encoding operons, we considered the extended core, representing the set of 234 reactions that are active in more than 90% of the 30,000 simulated growth conditions. The results indicate that the fraction of repressive regulatory links in the extended core is 52.3%, while the fraction of activating interactions is only 35.7%, and the remaining 12% represents regulatory links that can either activate or suppress the enzyme's mRNA synthesis rate. In contrast, for noncore enzyme-encoding operons, there is no difference between the fraction of activating and repressing links, both representing 45% of the regulatory interactions. These results offer evidence for the co-evolution of the core metabolic network and its corresponding regulatory network: given the requirement for the continuously active nonredundant core, regulatory mechanisms have shifted both the mRNA decay rate and the nature of the regulatory interactions, offering a higher regulatory stability for the core enzymes.

The finding that the core reactions form a single cluster would suggest that the activity of the participating reactions is highly synchronized, with changes in the flux of one reaction affecting the flux of other reactions as well. Thus, we should be able to discover the core by inspecting the correlations between the activities of all metabolic reactions. To test this hypothesis, we calculated the flux correlation coefficient, C_ij, for each reaction pair in the E. coli metabolism [17] by inspecting the flux values of all reactions under each of the 30,000 simulated growth conditions. Using the C_ij matrix as the metric of a hierarchical clustering algorithm [18], we observed the emergence of a group of reactions whose fluxes change simultaneously under environmental shifts. Interestingly, these highly correlated reactions significantly overlap with the metabolic core (Figure 4A), indicating that when the environmental conditions are altered, the fluxes of the core reactions are increased or decreased in a synchronized fashion.

The identification of the metabolic core also has important practical implications: given the continuous activity and high degree of essentiality of the core enzymes, they represent potential targets for antimicrobial intervention. Specifically, while many bacterial and yeast gene products are essential, a high fraction of them are essential only in specific environments. For example, recent measurements indicate that 76% of the S. cerevisiae genes that are inactive in nutrient-rich conditions are in fact not only active, but are also essential in some other growth conditions [6]. However, an effective antimicrobial drug needs to be able to kill its target organism under all physiological conditions in which it can exist. Thus pharmacological interventions targeting a specific pathway will not be effective in environments where the pathway is not needed and its corresponding enzyme is turned off. Instead, the most effective antimicrobials must target the activity of the core reactions, as their disruption will have an impact on the microorganism's ability to function under all environmental conditions. Indeed, among the currently used antibiotics, fosfomycin and cycloserine act by inhibiting cell-wall peptidoglycan, while sulfonamides and trimethoprim inhibit tetrahydrofolate biosynthesis, both pathways being present in the H. pylori as well as the E. coli core (see Figure 2). In addition, core pathways involved in the synthesis of flavin adenine dinucleotide, 2-dehydro-3-deoxy-d-octonate, and lipopolysaccharide are among potential antibiotic targets [21].