Energy-spilling reactions of Streptococcus bovis and resistance of its membrane to proton conductance.

Glucose-excess cultures of Streptococcus bovis consumed glucose faster than the amount that could be explained by growth or maintenance, and nongrowing chloramphenicol-treated cells had a rate of glucose consumption that was 10-fold greater than the maintenance rate. Because N,N-dicyclohexylcarbodiimide, an inhibitor of the membrane-bound F1F0 ATPase, eliminated the nongrowth energy dissipation (energy spilling) without a decrease in ATP and the rate of energy spilling could be increased by the protonophore 3,3',4',5-tetrachlorosalicylanilide, it appeared that a futile cycle of protons through the cell membrane was responsible for most of the energy spilling. When the rate of energy spilling was decreased gradually with iodoacetate, there was only a small decrease in the phosphorylation potential (delta G'p) and the theoretical estimate of H+ per ATP decreased from 4.2 to 3.6. On the bases of this ratio of H+ to ATP and the rate of ATP production, the flux of protons (amperage) across the cell membrane was directly proportional to the rate of energy spilling. Amperage values estimated from delta G'p were, however, nearly twice as great as values which were estimated from the heat production (delta H) of the cells [amperage = (0.38 x wattage)/delta p]. The last comparison indicated that only a fraction of the delta G of ATP hydrolysis was harvested by the F1F0 ATPase to pump protons. Both estimates of amperage indicated that the resistance of the cell membrane to proton conductance was inversely proportional to the log of the energy-spilling rate.     

Carbonyl Stress in Bacteria: Causes and Consequences

Pathways of synthesis of the α-reactive carbonyl compound methylglyoxal (MG) in prokaryotes are described in this review. Accumulation of MG leads to development of carbonyl stress. Some pathways of MG formation are similar for both pro- and eukaryotes, but there are reactions specific for prokaryotes, e.g. the methylglyoxal synthase reaction. This reaction and the glyoxalase system constitute an alternative pathway of glucose catabolism–the MG shunt not associated with the synthesis of ATP. In violation of the regulation of metabolism, the cell uses MG shunt as well as other glycolysis shunting pathways and futile cycles enabling stabilization of its energetic status. MG was first examined as a biologically active metabolic factor participating in the formation of phenotypic polymorphism and hyperpersistent potential of bacterial populations. The study of carbonyl stress is interesting for evolutionary biology and can be useful for constructing highly effective producer strains.     

Constraint-Based Model of Shewanella oneidensis MR-1 Metabolism: A Tool for Data Analysis and Hypothesis Generation

Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems level.     

Cell energy metabolism: An update

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD⁺ molecules play a key role. Cell energy metabolism hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metabolism: glycolysis/fermentation and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodynamic and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metabolism will be reviewed using examples such as the Crabtree and the Warburg effect.     

A Role for Fructose 1,6-Diphosphate in the ATPase-Mediated Energy-Spilling Reaction of Streptococcus bovis

The amount of ATP produced by Streptococcus bovis was larger than the amount that could be attributed to growth and maintenance, and even glucose-limited continuous cultures used ATP inefficiently (spilled ATP). Rapid-dilution-rate cultures always spilled more ATP than those growing at slow dilution rates, but rates of ATP spilling could also be enhanced by amino acid deprivation (with only ammonia as a nitrogen source). Energy spilling and intracellular ATP were not correlated, but energy spilling was always greatest when the rate of lactate production was high. The relationship between lactate production and energy spilling was supported by the observation that amino acid deprivation increased lactate production and ATP spilling. The lactate production rate of nongrowing (energy-spilling) S. bovis cells was fructose 1,6-diphosphate (FDP) dependent, and previous work showed that the lactate dehydrogenase of S. bovis was activated by FDP (M. J. Wolin, Science 146:775-777, 1964). The role of FDP in energy spilling was supported by the observation that the membrane-bound ATPase of S. bovis could be stimulated by FDP. FDP decreased the K(infm) for ATP by as much as fivefold. Other glycolytic intermediates could not stimulate the ATPase of washed membrane preparations, and FDP had no effect on soluble ATPase activity.     

Detecting and investigating substrate cycles in a genome-scale human metabolic network

Substrate cycles, also known as futile cycles, are cyclic metabolic routes that dissipate energy by hydrolysing cofactors such as ATP. They were first described to occur in the muscles of bumblebees and brown adipose tissue in the 1970s. A popular example is the conversion of fructose?6-phosphate to fructose?1,6-bisphosphate and back. In the present study, we analyze a large number of substrate cycles in human metabolism that consume ATP and discuss their statistics. For this purpose, we use two recently published methods (i.e. EFMEvolver and the K-shortest EFM method) to calculate samples of 100?000 and 15?000 substrate cycles, respectively. We find an unexpectedly high number of substrate cycles in human metabolism, with up to 100 reactions per cycle, utilizing reactions from up to six different compartments. An analysis of tissue-specific models of liver and brain metabolism shows that there is selective pressure that acts against the uncontrolled dissipation of energy by avoiding the coexpression of enzymes belonging to the same substrate cycle. This selective force is particularly strong against futile cycles that have a high flux as a result of thermodynamic principles.     

On the analysis of substrate cycles in large metabolic systems

The simultaneous operation of paired, opposing reactions (substrate cycles) or parallel reactions (dual pathways) with seeming wastage of ATP is widespread in cellular metabolism. Analysis of such “futile” pathways has hitherto been limited to loci with only two or three interconnecting fluxes. We introduce here a method that allows straightforward analysis of more complex systems. The method involves the linear superposition of “fundamental” modes, one or more of which may be energetically wasteful. Decomposition of a flux pattern into such modes allows computation of the amount of free energy “wasted” at any locus. Appropriate normalizations of energy wastage yield a number of indices useful for assessing the energetic impact of futile pathways on the cell and for comparing the degree of regulation of substrate cycles or dual pathways under different metabolic conditions. This approach is applied to steady-state flux data obtained in the protozoanTetrahymena pyriformis and in isolated rat hepatocytes under a variety of conditions.     

The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria

In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.     

The Energy Costs of Insulators in Biochemical Networks

Complex networks of biochemical reactions, such as intracellular protein signaling pathways and genetic networks, are often conceptualized in terms of modules—semiindependent collections of components that perform a well-defined function and which may be incorporated in multiple pathways. However, due to sequestration of molecular messengers during interactions and other effects, collectively referred to as retroactivity, real biochemical systems do not exhibit perfect modularity. Biochemical signaling pathways can be insulated from impedance and competition effects, which inhibit modularity, through enzymatic futile cycles that consume energy, typically in the form of ATP. We hypothesize that better insulation necessarily requires higher energy consumption. We test this hypothesis through a combined theoretical and computational analysis of a simplified physical model of covalent cycles, using two innovative measures of insulation, as well as a possible new way to characterize optimal insulation through the balancing of these two measures in a Pareto sense. Our results indicate that indeed better insulation requires more energy. While insulation may facilitate evolution by enabling a modular plug-and-play interconnection architecture, allowing for the creation of new behaviors by adding targets to existing pathways, our work suggests that this potential benefit must be balanced against the metabolic costs of insulation necessarily incurred in not affecting the behavior of existing processes.     

HEPNet: A Knowledge Base Model of Human Energy Pool Network for Predicting the Energy Availability Status of an Individual

HEPNet is an electronic representation of metabolic reactions occurring within human cellular organization focusing on inflow and outflow of the energy currency ATP, GTP and other energy associated moieties. The backbone of HEPNet consists of primary bio-molecules such as carbohydrates, proteins and fats which ultimately constitute the chief source for the synthesis and obliteration of energy currencies in a cell. A series of biochemical pathways and reactions constituting the catabolism and anabolism of various metabolites are portrayed through cellular compartmentalization. The depicted pathways function synchronously toward an overarching goal of producing ATP and other energy associated moieties to bring into play a variety of cellular functions. HEPNet is manually curated with raw data from experiments and is also connected to KEGG and Reactome databases. This model has been validated by simulating it with physiological states like fasting, starvation, exercise and disease conditions like glycaemia, uremia and dihydrolipoamide dehydrogenase deficiency (DLDD). The results clearly indicate that ATP is the master regulator under different metabolic conditions and physiological states. The results also highlight that energy currencies play a minor role. However, the moiety creatine phosphate has a unique character, since it is a ready-made source of phosphoryl groups for the rapid synthesis of ATP from ADP. HEPNet provides a framework for further expanding the network diverse age groups of both the sexes, followed by the understanding of energetics in more complex metabolic pathways that are related to human disorders.     

Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model

A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.     

Metabolic futile cycles and their functions: a systems analysis of energy and control

It has long been hypothesised that futile cycles in cellular metabolism are involved in the regulation of biochemical pathways. Following the work of Newsholme and Crabtree, a quantitative theory was developed for this idea based on open-system thermodynamics and metabolic control analysis. It is shown that the stoichiometric sensitivity of an intermediary metabolite concentration with respect to changes in steady-state flux is governed by the effective equilibrium constant of the intermediate formation, and the equilibrium can be regulated by a futile cycle. The direction of the shift in the effective equilibrium constant depends on the direction of operation of the futile cycle. High stoichiometric sensitivity corresponds to ultrasensitivity of an intermediate concentration to net flow through a pathway; low stoichiometric sensitivity corresponds to super-robustness of concentration with respect to changes in flux. Both cases potentially play important roles in metabolic regulation. Futile cycles actively shift the effective equilibrium by expending energy; the magnitude of changes in effective equilibria and sensitivities is a function of the amount of energy used by a futile cycle. This proposed mechanism for control by futile cycles works remarkably similar to kinetic proofreading in biosynthesis. The sensitivity of the system is also intimately related to the rate of concentration fluctuations of intermediate metabolites. The possibility of different roles for the two major mechanisms within cellular biochemical regulation, namely reversible chemical modifications via futile cycles and shifting equilibrium by macromolecular binding, are discussed.     

Model of dissolved organic carbon distribution for substrate-sufficient continuous culture.

The growth yields (Yobs) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism. It appears that the excess substrate could determine metabolic pathways of microorganisms, which further control dissolved organic carbon (DOC) distribution under substrate-sufficient conditions. However, how to quantitatively describe the DOC distribution remains unclear in substrate-sufficient continuous culture. Based on a balanced DOC reaction, a DOC distribution model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. Results showed that a considerable portion of the DOC consumed was directly oxidized to carbon dioxide through energy spilling under substrate-sufficient conditions. The proposed model for the first time quantified the DOC distribution between nongrowth-associated and growth-associated metabolisms of cells. The proposed model was verified with literature data very well.     

Carbon and energy balances in cell-recycle cultures of Schizosaccharomyces pombe

A strain of the fission yeast Schizosaccharomyces pombe was aerobically grown in a cell-recycle fermentor under various operating conditions, i.e., different bleeding rates and various separate feed rates of glucose and basal medium. Carbon and energy balances were analyzed during steady-state culture regimes, allowing growth yields and maintenance coefficients to be determined under glucose-limited and glucose-excess environments. Special attention was given to the metabolic shift from purely oxidative to respirofermentative glucose catabolism resulting from a change in the growth-limiting factor. No maintenance requirements for the carbon source and for energy were observed during glucose-limited culture regimes and oxidative catabolism. Under glucose excess and respirofermentative metabolism, the m(G) coefficient was shown to be growth-linked, whereas the enhancement of the apparent m(e) coefficient observed for increased residual glucose concentrations could be assigned to a decline in the ATP yield. (c) 1993 John Wiley & Sons, Inc.     

Metabolism of arginine and its positive effect on growth and revival of Oenococcus oeni

Oenococcus oeni is the main lactic acid bacteria species which induces malolactic fermentation during wine-making. It is able to break down arginine via the arginine deiminase pathway, a potential source of energy already considered for many bacteria. The production of ATP by starved cells from arginine was quantified with a bioluminescence assay, and efficient coupling of amino acid catabolism and cell growth was monitored. Therefore, molecular growth yield was determined after glucose exhaustion. With colony plate counting and a direct epifluorescence technique, it was shown that addition of arginine to viable but non-culturable cells obtained after nutrient starvation restored their ability to grow during its degradation. Therefore, arginine produced more than maintenance energy. It is concluded that strains which are able to metabolize arginine might take advantage of this additional energy source for growth.     

Why is metabolic labour divided in nitrification?

Winogradsky discovered in 1890 that nitrification is carried out in two consecutive steps by two distinct groups of bacteria: ammonia-oxidizing bacteria and nitrite-oxidizing bacteria. An explanation for this division of labour is offered based on the kinetic theory of optimal design of metabolic pathways, which postulates the existence of an optimal length for a pathway that maximizes the rate of ATP production. Shortening long pathways could, therefore, increase growth rate. However, this would reduce growth yield if the shorter pathway has fewer ATP-generating steps. High yields would be advantageous when bacteria grow in clonal clusters, as is typical for biofilms. It is postulated that bacteria that completely oxidize ammonia to nitrate exist in such environments.