Enrichment of sequencing targets from the human genome by solution hybridization

Background

Genome sequences, now available for most pathogens, hold promise for the rational design of new therapies. However, biological resources for genome-scale identification of gene function (notably genes involved in pathogenesis) and/or genes essential for cell viability, which are necessary to achieve this goal, are often sorely lacking. This holds true for Neisseria meningitidis, one of the most feared human bacterial pathogens that causes meningitis and septicemia.

Results

By determining and manually annotating the complete genome sequence of a serogroup C clinical isolate of N. meningitidis (strain 8013) and assembling a library of defined mutants in up to 60% of its non-essential genes, we have created NeMeSys, a biological resource for Neisseria meningitidis systematic functional analysis. To further enhance the versatility of this toolbox, we have manually (re)annotated eight publicly available Neisseria genome sequences and stored all these data in a publicly accessible online database. The potential of NeMeSys for narrowing the gap between sequence and function is illustrated in several ways, notably by performing a functional genomics analysis of the biogenesis of type IV pili, one of the most widespread virulence factors in bacteria, and by identifying through comparative genomics a complete biochemical pathway (for sulfur metabolism) that may potentially be important for nasopharyngeal colonization.

Conclusions

By improving our capacity to understand gene function in an important human pathogen, NeMeSys is expected to contribute to the ongoing efforts aimed at understanding a prokaryotic cell comprehensively and eventually to the design of new therapies.     

Integrative network analysis reveals active microRNAs and their functions in gastric cancer

Background

Ralstonia eutropha H16, found in both soil and water, is a Gram-negative lithoautotrophic bacterium that can utillize CO₂ and H₂ as its sources of carbon and energy in the absence of organic substrates. R. eutropha H16 can reach high cell densities either under lithoautotrophic or heterotrophic conditions, which makes it suitable for a number of biotechnological applications. It is the best known and most promising producer of polyhydroxyalkanoates (PHAs) from various carbon substrates and is an environmentally important bacterium that can degrade aromatic compounds. In order to make R. eutropha H16 a more efficient and robust biofactory, system-wide metabolic engineering to improve its metabolic performance is essential. Thus, it is necessary to analyze its metabolic characteristics systematically and optimize the entire metabolic network at systems level.

Results

We present the lithoautotrophic genome-scale metabolic model of R. eutropha H16 based on the annotated genome with biochemical and physiological information. The stoichiometic model, RehMBEL1391, is composed of 1391 reactions including 229 transport reactions and 1171 metabolites. Constraints-based flux analyses were performed to refine and validate the genome-scale metabolic model under environmental and genetic perturbations. First, the lithoautotrophic growth characteristics of R. eutropha H16 were investigated under varying feeding ratios of gas mixture. Second, the genome-scale metabolic model was used to design the strategies for the production of poly[R-(-)-3hydroxybutyrate] (PHB) under different pH values and carbon/nitrogen source uptake ratios. It was also used to analyze the metabolic characteristics of R. eutropha when the phosphofructokinase gene was expressed. Finally, in silico gene knockout simulations were performed to identify targets for metabolic engineering essential for the production of 2-methylcitric acid in R. eutropha H16.

Conclusion

The genome-scale metabolic model, RehMBEL1391, successfully represented metabolic characteristics of R. eutropha H16 at systems level. The reconstructed genome-scale metabolic model can be employed as an useful tool for understanding its metabolic capabilities, predicting its physiological consequences in response to various environmental and genetic changes, and developing strategies for systems metabolic engineering to improve its metabolic performance.     

Further developments towards a genome-scale metabolic model of yeast

Background

To date, several genome-scale network reconstructions have been used to describe the metabolism of the yeast Saccharomyces cerevisiae, each differing in scope and content. The recent community-driven reconstruction, while rigorously evidenced and well annotated, under-represented metabolite transport, lipid metabolism and other pathways, and was not amenable to constraint-based analyses because of lack of pathway connectivity.

Results

We have expanded the yeast network reconstruction to incorporate many new reactions from the literature and represented these in a well-annotated and standards-compliant manner. The new reconstruction comprises 1102 unique metabolic reactions involving 924 unique metabolites - significantly larger in scope than any previous reconstruction. The representation of lipid metabolism in particular has improved, with 234 out of 268 enzymes linked to lipid metabolism now present in at least one reaction. Connectivity is emphatically improved, with more than 90% of metabolites now reachable from the growth medium constituents. The present updates allow constraint-based analyses to be performed; viability predictions of single knockouts are comparable to results from in vivo experiments and to those of previous reconstructions.

Conclusions

We report the development of the most complete reconstruction of yeast metabolism to date that is based upon reliable literature evidence and richly annotated according to MIRIAM standards. The reconstruction is available in the Systems Biology Markup Language (SBML) and via a publicly accessible database http://www.comp-sys-bio.org/yeastnet/.     

NeMeSys: a biological resource for narrowing the gap between sequence and function in the human pathogen Neisseria meningitidis

Background

Genome sequences, now available for most pathogens, hold promise for the rational design of new therapies. However, biological resources for genome-scale identification of gene function (notably genes involved in pathogenesis) and/or genes essential for cell viability, which are necessary to achieve this goal, are often sorely lacking. This holds true for Neisseria meningitidis, one of the most feared human bacterial pathogens that causes meningitis and septicemia.

Results

By determining and manually annotating the complete genome sequence of a serogroup C clinical isolate of N. meningitidis (strain 8013) and assembling a library of defined mutants in up to 60% of its non-essential genes, we have created NeMeSys, a biological resource for Neisseria meningitidis systematic functional analysis. To further enhance the versatility of this toolbox, we have manually (re)annotated eight publicly available Neisseria genome sequences and stored all these data in a publicly accessible online database. The potential of NeMeSys for narrowing the gap between sequence and function is illustrated in several ways, notably by performing a functional genomics analysis of the biogenesis of type IV pili, one of the most widespread virulence factors in bacteria, and by identifying through comparative genomics a complete biochemical pathway (for sulfur metabolism) that may potentially be important for nasopharyngeal colonization.

Conclusions

By improving our capacity to understand gene function in an important human pathogen, NeMeSys is expected to contribute to the ongoing efforts aimed at understanding a prokaryotic cell comprehensively and eventually to the design of new therapies.     

General metabolism of Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes and pathways of the general metabolism of L. hongkongensis and correlated them with its phenotypic characteristics.

Results

The L. hongkongensis genome possesses the pentose phosphate and gluconeogenesis pathways and tricarboxylic acid and glyoxylate cycles, but incomplete Embden-Meyerhof-Parnas and Entner-Doudoroff pathways, in agreement with its asaccharolytic phenotype. It contains enzymes for biosynthesis and β-oxidation of saturated fatty acids, biosynthesis of all 20 universal amino acids and selenocysteine, the latter not observed in Neisseria gonorrhoeae, Neisseria meningitidis and Chromobacterium violaceum. The genome contains a variety of dehydrogenases, enabling it to utilize different substrates as electron donors. It encodes three terminal cytochrome oxidases for respiration using oxygen as the electron acceptor under aerobic and microaerophilic conditions and four reductases for respiration with alternative electron acceptors under anaerobic conditions. The presence of complete tetrathionate reductase operon may confer survival advantage in mammalian host in association with diarrhea. The genome contains CDSs for incorporating sulfur and nitrogen by sulfate assimilation, ammonia assimilation and nitrate reduction. The existence of both glutamate dehydrogenase and glutamine synthetase/glutamate synthase pathways suggests an importance of ammonia metabolism in the living environments that it may encounter.

Conclusions

The L. hongkongensis genome possesses a variety of genes and pathways for carbohydrate, amino acid and lipid metabolism, respiratory chain and sulfur and nitrogen metabolism. These allow the bacterium to utilize various substrates for energy production and survive in different environmental niches.     

A genome-scale metabolic model of potato late blight suggests a photosynthesis suppression mechanism

Background

Phytophthora infestans is a plant pathogen that causes an important plant disease known as late blight in potato plants (Solanum tuberosum) and several other solanaceous hosts. This disease is the main factor affecting potato crop production worldwide. In spite of the importance of the disease, the molecular mechanisms underlying the compatibility between the pathogen and its hosts are still unknown.

Results

To explain the metabolic response of late blight, specifically photosynthesis inhibition in infected plants, we reconstructed a genome-scale metabolic network of the S. tuberosum leaf, PstM1. This metabolic network simulates the effect of this disease in the leaf metabolism. PstM1 accounts for 2751 genes, 1113 metabolic functions, 1773 gene-protein-reaction associations and 1938 metabolites involved in 2072 reactions. The optimization of the model for biomass synthesis maximization in three infection time points suggested a suppression of the photosynthetic capacity related to the decrease of metabolic flux in light reactions and carbon fixation reactions. In addition, a variation pattern in the flux of carboxylation to oxygenation reactions catalyzed by RuBisCO was also identified, likely to be associated to a defense response in the compatible interaction between P. infestans and S. tuberosum.

Conclusions

In this work, we introduced simultaneously the first metabolic network of S. tuberosum and the first genome-scale metabolic model of the compatible interaction of a plant with P. infestans.

     

Detecting positive selection from genome scans of linkage disequilibrium

Background

During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.

Results

Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.

Conclusions

Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.     

Pattern of antioxidant enzyme activities and hydrogen peroxide content during developmental stages of rhizogenesis from hypocotyl explants of Mesembryanthemum crystallinum L.

Key message

H ₂ O ₂ is necessary to elicit rhizogenic action of auxin. Activities of specific catalase and manganese superoxide dismutase forms mark roots development.

Abstract

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots on medium containing 2,4-dichlorophenoxyacetic acid. Explants became competent to respond to the rhizogenic action of auxin on day 3 of culture, when hydrogen peroxide content in cultured tissue was the highest. l-Ascorbic acid added to the medium at 5 μM lowered the H₂O₂ level, inhibited rhizogenesis and induced non-regenerative callus, suggesting that certain level of H₂O₂ is required to promote root initiation. Coincident with the onset of rhizogenic determination, meristemoids formed at the periphery of the hypocotyl stele and the activity of the manganese form of superoxide dismutase, MnSOD-2 was induced. Once induced, MnSOD-2 activity was maintained through the post-determination phase of rooting, involving root growth. MnSOD-2 activity was not found in non-rhizogenic explants maintained in the presence of AA. Analyses of the maximum photochemical efficiency of photosystem II and the oxygen uptake rate revealed that the explants were metabolically arrested during the predetermination stage of rhizogenesis. Respiratory and photosynthetic rates were high during root elongation and maturation. Changes in catalase and peroxidase activities correlated with fluctuations of endogenous H₂O₂ content throughout rhizogenic culture. Expression of a specific CAT-2 form accompanied the post-determination stage of rooting and a high rate of carbohydrate metabolism during root growth. On the other hand, the occurrence of MnSOD-2 activity did not depend on the metabolic status of explants. The expression of MnSOD-2 activity throughout root development seems to relate it specifically to root metabolism and indicates it as a molecular marker of rhizogenesis in M. crystallinum.     

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

Background

FeFe-hydrogenases are the most active class of H₂-producing enzymes known in nature and may have important applications in clean H₂ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O₂.

Results

We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O₂ levels eliminate growth. This pathway forms the basis for a genetic selection for O₂ tolerance. Genetically selected hydrogenases did not show improved stability in O₂ and in many cases had lost H₂ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.

Conclusions

Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H₂ metabolism. Our results also indicate a H₂-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H₂-activating catalysts.     

Genome-wide patterns of genetic variation in sweet and grain sorghum (Sorghum bicolor)

Background

Sorghum (Sorghum bicolor) is globally produced as a source of food, feed, fiber and fuel. Grain and sweet sorghums differ in a number of important traits, including stem sugar and juice accumulation, plant height as well as grain and biomass production. The first whole genome sequence of a grain sorghum is available, but additional genome sequences are required to study genome-wide and intraspecific variation for dissecting the genetic basis of these important traits and for tailor-designed breeding of this important C₄ crop.

Results

We resequenced two sweet and one grain sorghum inbred lines, and identified a set of nearly 1,500 genes differentiating sweet and grain sorghum. These genes fall into ten major metabolic pathways involved in sugar and starch metabolisms, lignin and coumarin biosynthesis, nucleic acid metabolism, stress responses and DNA damage repair. In addition, we uncovered 1,057,018 SNPs, 99,948 indels of 1 to 10 bp in length and 16,487 presence/absence variations as well as 17,111 copy number variations. The majority of the large-effect SNPs, indels and presence/absence variations resided in the genes containing leucine rich repeats, PPR repeats and disease resistance R genes possessing diverse biological functions or under diversifying selection, but were absent in genes that are essential for life.

Conclusions

This is a first report of the identification of genome-wide patterns of genetic variation in sorghum. High-density SNP and indel markers reported here will be a valuable resource for future gene-phenotype studies and the molecular breeding of this important crop and related species.     

Filling the gap - COI barcode resolution in eastern Palearctic birds

Background

Many fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.

Results

Hierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.

Conclusion

Early responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.     

A systems biology approach to investigate the antimicrobial activity of oleuropein

Oleuropein and its hydrolysis products are olive phenolic compounds that have antimicrobial effects on a variety of pathogens, with the potential to be utilized in food and pharmaceutical products. While the existing research is mainly focused on individual genes or enzymes that are regulated by oleuropein for antimicrobial activities, little work has been done to integrate intracellular genes, enzymes and metabolic reactions for a systematic investigation of antimicrobial mechanism of oleuropein. In this study, the first genome-scale modeling method was developed to predict the system-level changes of intracellular metabolism triggered by oleuropein in Staphylococcus aureus, a common food-borne pathogen. To simulate the antimicrobial effect, an existing S. aureus genome-scale metabolic model was extended by adding the missing nitric oxide reactions, and exchange rates of potassium, phosphate and glutamate were adjusted in the model as suggested by previous research to mimic the stress imposed by oleuropein on S. aureus. The developed modeling approach was able to match S. aureus growth rates with experimental data for five oleuropein concentrations. The reactions with large flux change were identified and the enzymes of fifteen of these reactions were validated by existing research for their important roles in oleuropein metabolism. When compared with experimental data, the up/down gene regulations of 80% of these enzymes were correctly predicted by our modeling approach. This study indicates that the genome-scale modeling approach provides a promising avenue for revealing the intracellular metabolism of oleuropein antimicrobial properties.     

Mesothelioma mortality in Europe: impact of asbestos consumption and simian virus 40

Background

Hypophosphatasia (HP) is an inborn error of bone metabolism characterized by a genetic defect in the gene encoding the tissue-nonspecific alkaline phosphatase (TNSALP). There is a lack of knowledge as to how the variability and clinical severity of the HP phenotype (especially pain and walking impairment) are related to metabolic disturbances or impairments, subsequent to the molecular defect.

Methods

We analyzed the changes in clinical symptoms and the prostaglandin (PG) metabolism in response to treatment with non-steroidal anti-inflammatory drugs (NSAIDs) in six children affected by childhood HP. In addition, by exposing HP fibroblasts to pyridoxal phosphate and/or calcium pyrophosphate in vitro, we analyzed whether the alterations in PG levels are sequelae related to the metabolic defect.

Results

Childhood HP patients, who often complain about pain in the lower limbs without evident fractures, have systemic hyperprostaglandinism. Symptomatic anti-inflammatory treatment with NSAIDs significantly improved pain-associated physical impairment. Calcium pyrophosphate, but not pyridoxal phosphate, induced cyclooxygenase-2 (COX-2) gene expression and PG production in HP and normal fibroblasts in vitro.

Conclusion

Clinical features of childhood HP related to pain in the lower legs may be, at least in part, sequelae related to elevated PG levels, secondary to the primary metabolic defect. Consequently, NSAID treatment does improve the clinical features of childhood HP.