Predicting synthetic rescues in metabolic networks

An important goal of medical research is to develop methods to recover the loss of cellular function due to mutations and other defects. Many approaches based on gene therapy aim to repair the defective gene or to insert genes with compensatory function. Here, we propose an alternative, network-based strategy that aims to restore biological function by forcing the cell to either bypass the functions affected by the defective gene, or to compensate for the lost function. Focusing on the metabolism of single-cell organisms, we computationally study mutants that lack an essential enzyme, and thus are unable to grow or have a significantly reduced growth rate. We show that several of these mutants can be turned into viable organisms through additional gene deletions that restore their growth rate. In a rather counterintuitive fashion, this is achieved via additional damage to the metabolic network. Using flux balance-based approaches, we identify a number of synthetically viable gene pairs, in which the removal of one enzyme-encoding gene results in a nonviable phenotype, while the deletion of a second enzyme-encoding gene rescues the organism. The systematic network-based identification of compensatory rescue effects may open new avenues for genetic interventions.Comment: Supplementary Information is available at the Molecular Systems Biology website: http://www.nature.com/msb/journal/v4/n1/full/msb20081.htm

Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection'—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/)

Fitness of Escherichia coli during Urinary Tract Infection Requires Gluconeogenesis and the TCA Cycle

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium's ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes

Mapping an atlas of tissue-specific drosophila melanogaster metabolomes by high resolution mass spectrometry

Metabolomics can provide exciting insights into organismal function, but most work on simple models has focussed on the whole organism metabolome, so missing the contributions of individual tissues. Comprehensive metabolite profiles for ten tissues from adult Drosophila melanogaster were obtained here by two chromatographic methods, a hydrophilic interaction (HILIC) method for polar metabolites and a lipid profiling method also based on HILIC, in combination with an Orbitrap Exactive instrument. Two hundred and forty two polar metabolites were putatively identified in the various tissues, and 251 lipids were observed in positive ion mode and 61 in negative ion mode. Although many metabolites were detected in all tissues, every tissue showed characteristically abundant metabolites which could be rationalised against specific tissue functions. For example, the cuticle contained high levels of glutathione, reflecting a role in oxidative defence; the alimentary canal (like vertebrate gut) had high levels of acylcarnitines for fatty acid metabolism, and the head contained high levels of ether lipids. The male accessory gland uniquely contained decarboxylated S-adenosylmethionine. These data thus both provide valuable insights into tissue function, and a reference baseline, compatible with the FlyAtlas.org transcriptomic resource, for further metabolomic analysis of this important model organism, for example in the modelling of human inborn errors of metabolism, aging or metabolic imbalances such as diabetes

BMP Signaling Modulates Hepcidin Expression in Zebrafish Embryos Independent of Hemojuvelin

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv

Production of phi mesons at mid-rapidity in sqrt(s_NN) = 200 GeV Au+Au collisions at RHIC

We present the first results of meson production in the K^+K^- decay channel from Au+Au collisions at sqrt(s_NN) = 200 GeV as measured at mid-rapidity by the PHENIX detector at RHIC. Precision resonance centroid and width values are extracted as a function of collision centrality. No significant variation from the PDG accepted values is observed. The transverse mass spectra are fitted with a linear exponential function for which the derived inverse slope parameter is seen to be constant as a function of centrality. These data are also fitted by a hydrodynamic model with the result that the freeze-out temperature and the expansion velocity values are consistent with the values previously derived from fitting single hadron inclusive data. As a function of transverse momentum the collisions scaled peripheral.to.central yield ratio RCP for the is comparable to that of pions rather than that of protons. This result lends support to theoretical models which distinguish between baryons and mesons instead of particle mass for explaining the anomalous proton yield.Comment: 326 authors, 24 pages text, 23 figures, 6 tables, RevTeX 4. To be submitted to Physical Review C as a regular article. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

From bit to it: How a complex metabolic network transforms information into living matter

Organisms live and die by the amount of information they acquire about their environment. The systems analysis of complex metabolic networks allows us to ask how such information translates into fitness. A metabolic network transforms nutrients into biomass. The better it uses information on available nutrient availability, the faster it will allow a cell to divide. I here use metabolic flux balance analysis to show that the accuracy I (in bits) with which a yeast cell can sense a limiting nutrient's availability relates logarithmically to fitness as indicated by biomass yield and cell division rate. For microbes like yeast, natural selection can resolve fitness differences of genetic variants smaller than 10-6, meaning that cells would need to estimate nutrient concentrations to very high accuracy (greater than 22 bits) to ensure optimal growth. I argue that such accuracies are not achievable in practice. Natural selection may thus face fundamental limitations in maximizing the information processing capacity of cells. The analysis of metabolic networks opens a door to understanding cellular biology from a quantitative, information-theoretic perspective

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of 13C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-13C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-13C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides