Flux Balance Analysis of Cyanobacterial Metabolism: The Metabolic Network of Synechocystis sp. PCC 6803

Cyanobacteria are versatile unicellular phototrophic microorganisms that are highly abundant in many environments. Owing to their capability to utilize solar energy and atmospheric carbon dioxide for growth, cyanobacteria are increasingly recognized as a prolific resource for the synthesis of valuable chemicals and various biofuels. To fully harness the metabolic capabilities of cyanobacteria necessitates an in-depth understanding of the metabolic interconversions taking place during phototrophic growth, as provided by genome-scale reconstructions of microbial organisms. Here we present an extended reconstruction and analysis of the metabolic network of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Building upon several recent reconstructions of cyanobacterial metabolism, unclear reaction steps are experimentally validated and the functional consequences of unknown or dissenting pathway topologies are discussed. The updated model integrates novel results with respect to the cyanobacterial TCA cycle, an alleged glyoxylate shunt, and the role of photorespiration in cellular growth. Going beyond conventional flux-balance analysis, we extend the computational analysis to diurnal light/dark cycles of cyanobacterial metabolism.     

Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.     

The Ability of Analysts' Recommendations to Predict Optimistic and Pessimistic Forecasts

Previous researches show that buy (growth) companies conduct income increasing earnings management in order to meet forecasts and generate positive forecast Errors (FEs). This behavior however, is not inherent in sell (non-growth) companies. Using the aforementioned background, this research hypothesizes that since sell companies are pressured to avoid income increasing earnings management, they are capable, and in fact more inclined, to pursue income decreasing Forecast Management (FM) with the purpose of generating positive FEs. Using a sample of 6553 firm-years of companies that are listed in the NYSE between the years 2005–2010, the study determines that sell companies conduct income decreasing FM to generate positive FEs. However, the frequency of positive FEs of sell companies does not exceed that of buy companies. Using the efficiency perspective, the study suggests that even though buy and sell companies have immense motivation in avoiding negative FEs, they exploit different but efficient strategies, respectively, in order to meet forecasts. Furthermore, the findings illuminated the complexities behind informative and opportunistic forecasts that falls under the efficiency versus opportunistic theories in literature.     

Elementary Flux Mode Analysis Revealed Cyclization Pathway as a Powerful Way for NADPH Regeneration of Central Carbon Metabolism

NADPH regeneration capacity is attracting growing research attention due to its important role in resisting oxidative stress. Besides, NADPH availability has been regarded as a limiting factor in production of industrially valuable compounds. The central carbon metabolism carries the carbon skeleton flux supporting the operation of NADPH-regenerating enzyme and offers flexibility in coping with NADPH demand for varied intracellular environment. To acquire an insightful understanding of its NADPH regeneration capacity, the elementary mode method was employed to compute all elementary flux modes (EFMs) of a network representative of central carbon metabolism. Based on the metabolic flux distributions of these modes, a cluster analysis of EFMs with high NADPH regeneration rate was conducted using the self-organizing map clustering algorithm. The clustering results were used to study the relationship between the flux of total NADPH regeneration and the flux in each NADPH producing enzyme. The results identified several reaction combinations supporting high NADPH regeneration, which are proven to be feasible in cells via thermodynamic analysis and coincident with a great deal of previous experimental report. Meanwhile, the reaction combinations showed some common characteristics: there were one or two decarboxylation oxidation reactions in the combinations that produced NADPH and the combination constitution included certain gluconeogenesis pathways. These findings suggested cyclization pathways as a powerful way for NADPH regeneration capacity of bacterial central carbon metabolism.     

有鳞目动物鳞片表面超微结构的适应性进化

爬行动物鳞片的微结构是对环境的一种适应。本研究运用扫描电子显微镜观察了北草蜥(Takydromus septentrionalis)、脆蛇蜥(Dopasia harti)和王锦蛇(Elaphe carinata)头部、背部和腹部鳞片的微皮纹结构及感受器特征。结果表明,3个物种的微皮纹和感受器存在种间差异。北草蜥和王锦蛇背部及腹部微皮纹均为狭长带状,脆蛇蜥为不规则多边形。北草蜥和王锦蛇颔片上有感受器,北草蜥无。脆蛇蜥腹部微皮纹上无小齿状凸起,北草蜥和王锦蛇有,与北草蜥相比王锦蛇的小齿状凸起更宽更长。王锦蛇的眼部微皮纹为向上竖起的脊,而其他部位的鳞片为具有小齿状凸起的狭长带状结构。本研究共收集整理17科99种的背鳞微皮纹数据和8科25种的感受器数据,对微皮纹特征和感受器形态进行祖先重建发现,狭长带状背鳞微皮纹主要存在于蜥蜴科(Lacertidae)、游蛇科(Colubridae)和石龙子科(Scincidae)中,而鬛蜥科(Agamidae)、蛇蜥科(Anguidae)、蟒蛇科(Boidae)以及蝰蛇科(Viperidae)的大多为多边形;较原始的感受器形态为无感觉毛的透镜状,这一结构在有...     

Growth Rate-Dependent Modulation of Carbon Flux through Central Metabolism and the Kinetic Consequences for Glucose-Limited Chemostat Cultures of Corynebacterium glutamicum

The physiological behavior of Corynebacterium glutamicum in glucose-limited chemostat cultures was examined from both growth kinetics and enzymatic viewpoints. Metabolic fluxes within the central metabolism were calculated from growth kinetics and analyzed in relation to specific enzyme activities. At high growth rates, incomplete glucose removal was observed, and this was attributed to rate-limiting capacity of the phosphotransferase system transporter and the probable contribution of a low-affinity permease uptake mechanism. The improved biomass yield observed at high growth rates was related to a shift in the profile of anaplerotic carboxylation reactions, with pyruvate carboxylase replacing malic enzyme. Phosphoenolpyruvate carboxylase, an activity often assumed to be the major anaplerotic reaction during growth of C. glutamicum on glucose, was present at only low levels and is unlikely to contribute significantly to tricarboxylic acid cycle fuelling other than at low growth rates.     

Using Knowledge of Protein Structural Constraints to Predict the Evolution of HIV-1

The high levels of sequence diversity and rapid rates of evolution of HIV-1 represent the main challenges for developing effective therapies. However, there are constraints imposed by the three-dimensional protein structure that affect the sequence space accessible to the evolution of HIV-1. Here, we present a strategy for predicting the set of possible amino acid replacements in HIV. Our approach is based on the identification of likely amino acid changes in the context of these structural constraints using environment-specific substitution matrices as well as considering the physical constraints imposed by local structure. Assessment of the power of various published algorithms in predicting the evolution of HIV-1 Gag P17 shows that it is possible to use these methods to make accurate predictions of the sequence diversity. Our own method, SubFit, uses knowledge of local structural constraints; it achieves similar prediction success with the best-performing methods. We also show that erroneous predictions are largely due to infrequently occurring amino acids that will probably have severe fitness costs for the protein. Future improvements; for example, incorporating covariation and immunological constraints will permit more reliable prediction of viral evolution.     

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

The Ability of Virulence Factor Expression by Pseudomonas aeruginosa to Predict Clinical Disease in Hospitalized Patients

Background

Pseudomonas aeruginosa is an opportunistic pathogen that frequently causes hospital acquired colonization and infection. Accurate identification of host and bacterial factors associated with infection could aid treatment decisions for patients with P. aeruginosa cultured from clinical sites.

Methods

We identified a prospective cohort of 248 hospitalized patients with positive P. aeruginosa cultures. Clinical data were analyzed to determine whether an individual met predefined criteria for infection versus colonization. P. aeruginosa isolates were tested for the expression of multiple phenotypes previously associated with virulence in animal models and humans. Logistic regression models were constructed to determine the degree of association between host and bacterial factors with P. aeruginosa infection of the bloodstream, lung, soft tissue and urinary tract.

Results

One host factor (i.e. diabetes mellitus), and one bacterial factor, a Type 3 secretion system positive phenotype, were significantly associated with P. aeruginosa infection in our cohort. Subgroup analysis of patients with P. aeruginosa isolated from the urinary tract revealed that the presence of a urinary tract catheter or stent was an additional factor for P. aeruginosa infection.

Conclusions

Among hospitalized patients with culture-documented P. aeruginosa, infection is more likely to be present in those with diabetes mellitus and those harboring a Type 3 secretion positive bacterial strain.     

Focus on Metabolism: A Method of Accounting for Enzyme Costs in Flux Balance Analysis Reveals Alternative Pathways and Metabolite Stores in an Illuminated Arabidopsis Leaf

Flux balance analysis of plant metabolism is an established method for predicting metabolic flux phenotypes and for exploring the way in which the plant metabolic network delivers specific outcomes in different cell types, tissues, and temporal phases. A recurring theme is the need to explore the flexibility of the network in meeting its objectives and, in particular, to establish the extent to which alternative pathways can contribute to achieving specific outcomes. Unfortunately, predictions from conventional flux balance analysis minimize the simultaneous operation of alternative pathways, but by introducing flux-weighting factors to allow for the variable intrinsic cost of supporting each flux, it is possible to activate different pathways in individual simulations and, thus, to explore alternative pathways by averaging thousands of simulations. This new method has been applied to a diel genome-scale model of Arabidopsis (Arabidopsis thaliana) leaf metabolism to explore the flexibility of the network in meeting the metabolic requirements of the leaf in the light. This identified alternative flux modes in the Calvin-Benson cycle revealed the potential for alternative transitory carbon stores in leaves and led to predictions about the light-dependent contribution of alternative electron flow pathways and futile cycles in energy rebalancing. Notable features of the analysis include the light-dependent tradeoff between the use of carbohydrates and four-carbon organic acids as transitory storage forms and the way in which multiple pathways for the consumption of ATP and NADPH can contribute to the balancing of the requirements of photosynthetic metabolism with the energy available from photon capture.Computational modeling of metabolism is increasingly used to analyze the complexity of plant metabolic networks and to understand system-level properties such as carbon use efficiency (Sweetlove and Ratcliffe, 2011; Nägele and Weckwerth, 2012; de Oliveira Dal’Molin and Nielsen, 2013; Kruger and Ratcliffe, 2015). Flux balance analysis (FBA), which is a method for predicting steady-state flux distributions using a stoichiometric model of the network, is particularly well suited to this task, because it can be applied to large-scale metabolic networks (Lewis et al., 2012). It is also computationally efficient, meaning that models of different cell types (de Oliveira Dal’Molin et al., 2010), different temporal phases (Cheung et al., 2014), and different tissues (Borisjuk et al., 2013; Grafahrend-Belau et al., 2013) can be combined.FBA can generate accurate predictions of plant metabolic fluxes (Williams et al., 2010; Hay and Schwender, 2011; Cheung et al., 2013), but the analysis is complicated by the presence of alternative pathways that share the same function within the network. For example, mitochondria and chloroplasts have several potential mechanisms for maintaining energetic homeostasis, including alternative electron flow pathways, metabolite shuttles for the transfer of reducing power or ATP, and uncoupling mechanisms (Millar et al., 2011; Taniguchi and Miyake, 2012). More generally, the distributed robustness of metabolic networks means that they have the inherent property of being able to achieve cellular objectives in different ways (Wagner, 2005). However, FBA does not automatically identify these alternative flux distributions, because the immediate output of the analysis is a single flux distribution that satisfies the constraints and objectives applied to the model. This has the effect of masking the potential contribution of alternative pathways, and to avoid this, it is necessary to extend the analysis in a way that will reveal them.The most commonly used approach to this problem is flux variability analysis (FVA), which defines the permissible flux ranges for each reaction in the optimal flux space (Mahadevan and Schilling, 2003). Another possibility is random sampling of the optimal flux space, using a uniform sampling algorithm that was originally introduced to characterize the entire feasible flux solution space (Price et al., 2004). While both approaches are useful for exploring the capability of the metabolic system in achieving the cellular objectives, they do not give any indication of which alternative optimal flux solutions are more likely to be found in vivo, and they do not generate flux distributions that represent the biological reality in which alternative pathways may be operating simultaneously.Here, we develop a methodology that permits alternative pathways to be explored efficiently and that allows the consequences of the simultaneous operation of alternative pathways on the rest of the metabolic network to be examined. Our approach emerged from a reconsideration of the use of flux minimization as the objective function. Minimization of the sum of the absolute flux values supported by all the reactions in the network is often used as an objective function in FBA on the principle that cells have evolved to minimize the costs for the synthesis of the enzymes and membrane transporters that support growth and cell maintenance (Holzhütter, 2004). However, no weighting is applied when calculating the sum of fluxes, so there is an implicit assumption that the machinery cost per unit of flux is the same for all reactions. This assumption is invalid in vivo, as enzymes vary in terms of their size, number of subunits, and catalytic capacity. Ideally, each reaction should be weighted by its enzyme machinery costs per unit of flux, but such information is not available for the majority of the reactions in large-/genome-scale metabolic models. Here, we develop a modeling method, cost-weighted FBA, that avoids the invalid assumption of equal costs and that allows the evaluation of alternative metabolic routes in a complex network. The method was used to demonstrate the flexibility of leaf metabolism in meeting the metabolic requirements of an Arabidopsis (Arabidopsis thaliana) leaf in the light. A genome-scale diel FBA model, in which the light and dark phases of the diel cycle were solved as a single optimization problem, was used for the analysis because this approach currently provides the most realistic constraints-based framework for modeling leaf metabolism (Cheung et al., 2014; de Oliveira Dal’Molin et al., 2015).     

Semi-automated Curation of Metabolic Models via Flux Balance Analysis: A Case Study with Mycoplasma gallisepticum

Primarily used for metabolic engineering and synthetic biology, genome-scale metabolic modeling shows tremendous potential as a tool for fundamental research and curation of metabolism. Through a novel integration of flux balance analysis and genetic algorithms, a strategy to curate metabolic networks and facilitate identification of metabolic pathways that may not be directly inferable solely from genome annotation was developed. Specifically, metabolites involved in unknown reactions can be determined, and potentially erroneous pathways can be identified. The procedure developed allows for new fundamental insight into metabolism, as well as acting as a semi-automated curation methodology for genome-scale metabolic modeling. To validate the methodology, a genome-scale metabolic model for the bacterium Mycoplasma gallisepticum was created. Several reactions not predicted by the genome annotation were postulated and validated via the literature. The model predicted an average growth rate of 0.358±0.12, closely matching the experimentally determined growth rate of M. gallisepticum of 0.244±0.03. This work presents a powerful algorithm for facilitating the identification and curation of previously known and new metabolic pathways, as well as presenting the first genome-scale reconstruction of M. gallisepticum.     

Experimental Approaches to Studying the Initial Evolution of Conspicuous Aposematic Signalling

Aposematism, where prey species conspicuously advertise their unprofitability to predators, is a widespread defensive strategy. One feature of an aposematic anti-predatory strategy that is especially puzzling is conspicuousness. While conspicuousness aids associative learning in predators, it involves being more visible, which probably increases predation risk. Although aposematism is an old evolutionary question, experimental studies to its evolution have been scarce. Only 11 experiments address the potential benefits of conspicuousness, which have successfully manipulated conspicuousness. This is probably because it is difficult to separate conspicuousness from other characters of aposematic prey, e.g. colour. Furthermore since predators and prey species have coexisted for a long time, and there might be special adaptations other than conspicuous signalling, our experimental results might be confounded with, e.g. predatory biases. In this review, I will examine the problems of studying the costs and benefits of conspicuousness as well as the initial evolution of conspicuousness and the recent progress in the study of aposematism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Computational Modeling to Predict the Temporal Regulation of Chondrocyte Metabolism in Response to Various Dynamic Compression Regimens

Based on previously published experimental work, computational models were developed to simulate the effect of different dynamic compression regimens on the activity of chondrocytes seeded in agarose constructs. In particular, the balance between proliferation and matrix synthesis can be adjusted by applying different intervals of continuous or intermittent mechanical compression. A phenomenological compartment based-modeling approach was used as first model. A more mechanistic cell cycle model was used as the second model. The compartment-based modeling approach was found to be useful in representing a balance between proliferation and proteoglycan synthesis, when the effect of a certain stimulation protocol is known. In order to predict the response to different intervals of mechanical stimulation, however, a more mechanistic cell cycle-based approach is required. The cell cycle model supports an important role of the onset of loading. In addition, an inhibitory effect of further loading is required, which is more likely to be related to cell cycle progression velocity than to a decreased probability of commitment to the cell cycle. The mechanisms behind this inhibitory effect and the computational implementation, however, require further investigation.     

Metabolic Changes in Klebsiella oxytoca in Response to Low Oxidoreduction Potential,as Revealed by Comparative Proteomic Profiling Integrated with Flux Balance Analysis

Oxidoreduction potential (ORP) is an important physiological parameter for biochemical production in anaerobic or microaerobic processes. However, the effect of ORP on cellular physiology remains largely unknown, which hampers the design of engineering strategies targeting proteins associated with ORP response. Here we characterized the effect of altering ORP in a 1,3-propanediol producer, Klebsiella oxytoca, by comparative proteomic profiling combined with flux balance analysis. Decreasing the extracellular ORP from −150 to −240 mV retarded cell growth and enhanced 1,3-propanediol production. Comparative proteomic analysis identified 61 differentially expressed proteins, mainly involved in carbohydrate catabolism, cellular constituent biosynthesis, and reductive stress response. A hypothetical oxidoreductase (HOR) that catalyzes 1,3-propanediol production was markedly upregulated, while proteins involved in biomass precursor synthesis were downregulated. As revealed by subsequent flux balance analysis, low ORP induced a metabolic shift from glycerol oxidation to reduction and rebalancing of redox and energy metabolism. From the integrated protein expression profiles and flux distributions, we can construct a rational analytic framework that elucidates how (facultative) anaerobes respond to extracellular ORP changes.     

The Effects of Drought on the Water Balance and Nitrogen Metabolism in Sand-fixing Plants

The changes in water balance and nitrogen metabolism of sandfixing plants under soil drought condition in desert area were studied. During sustained drought, the free water content was decreased and the bound water content was increased in all of the experimental plants. Nitrogen metabolism in the plants was converted by drought. The protein synthesis was inhibited, in the meantime the non-protein nitrogen was accumulated. It was found that a notable difference exists in various plants as far as accumulated non-protein nitrogen is concerned, amide nitrogen was accumulated mainly by xerophyte Hedysarium scoparium and nitrate nitrogen by mesophyte Robinia psudoacacia. The water balance of plants and the role of bound water in drought resistance are also discussed.