Publications

Abstract (Expand)

Atlantic salmon migrates from rivers to sea to feed, grow and develop gonads before returning to spawn in freshwater. The transition to marine habitats is associated with dramatic changes in the environment, including water salinity, exposure to pathogens, and shift in dietary lipid availability. Many changes in physiology and metabolism occur across this life-stage transition, but little is known about the molecular nature of these changes. Here we use a long term feeding experiment to study transcriptional regulation of lipid metabolism in Atlantic salmon gut and liver in both fresh- and saltwater. We find that lipid metabolism becomes significantly less plastic to differences in dietary lipid composition when salmon transitions to saltwater and experiences increased dietary lipid availability. Expression of genes in liver relating to lipogenesis and lipid transport decrease overall and become less responsive to diet, while genes for lipid uptake in gut become more highly expressed. Finally, analyses of evolutionary consequences of the salmonid specific whole-genome duplication on lipid metabolism reveals several pathways with significantly different (p<0.05) duplicate retention or duplicate regulatory conservation. We also find a limited number of cases where the whole genome duplication has resulted in an increased gene dosage. In conclusion, we find variable and pathway-specific effects of the salmonid genome duplication on lipid metabolism genes. A clear life-stage associated shift in lipid metabolism regulation is evident, and we hypothesize this to be, at least partly, driven by non-dietary factors such as the preparatory remodeling of gene regulation and physiology prior to sea migration. This article is protected by copyright. All rights reserved.

Authors: Gareth Gillard, Thomas Harvey, Arne Gjuvsland, Yang Jin, Magny Sidsel Thomassen, S. Lien, M. Leaver, Jacob Seilø Torgersen, Torgeir R. Hvidsten, Jon Olav Vik, Sandve Simen

Date Published: No date defined

Journal: Mol Ecol

Abstract (Expand)

Predicting a multicellular organism's phenotype quantitatively from its genotype is challenging, as genetic effects must propagate up time and length scales. Circadian clocks are intracellular regulators that control temporal gene expression patterns and hence metabolism, physiology and behaviour, from sleep/wake cycles in mammals to flowering in plants1-3. Clock genes are rarely essential but appropriate alleles can confer a competitive advantage4,5 and have been repeatedly selected during crop domestication3,6. Here we quantitatively explain and predict canonical phenotypes of circadian timing in a multicellular, model organism. We used metabolic and physiological data to combine and extend mathematical models of rhythmic gene expression, photoperiod-dependent flowering, elongation growth and starch metabolism within a Framework Model for growth of Arabidopsis thaliana7-9. The model predicted the effect of altered circadian timing upon each particular phenotype in clock-mutant plants. Altered night-time metabolism of stored starch accounted for most but not all of the decrease in whole-plant growth rate. Altered mobilisation of a secondary store of organic acids quantitatively explained the remaining defect. Our results link genotype through specific processes to higher-level phenotypes, formalising our understanding of a subtle, pleiotropic syndrome at the whole-organism level, and validating the systems approach to understand complex traits starting from intracellular circuits.

Authors: Yin Hoon Chew, Daniel Seaton, Virginie Mengin, Anna Flis, Sam T. Mugford, Alison M. Smith, Mark Stitt, Andrew Millar

Date Published: No date defined

Journal: Not specified

Abstract (Expand)

Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Authors: Z. Xu, Y. Wang, K. F. Chater, H. Y. Ou, H. H. Xu, Z. Deng, M. Tao

Date Published: 8th Jan 2017

Journal: Appl Environ Microbiol

Abstract (Expand)

BACKGROUND: Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders. RESULTS: First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients' metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach. CONCLUSION: We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

Authors: Karen Van Eunen, C. M. Volker-Touw, A. Gerding, A. Bleeker, J. C. Wolters, W. J. van Rijt, A. M. Martines, K. E. Niezen-Koning, R. M. Heiner, H. Permentier, A. K. Groen, D. J. Reijngoud, Terry G.J. Derks, Barbara Bakker

Date Published: 7th Dec 2016

Journal: BMC Biol

Abstract (Expand)

MOTIVATION: A major goal of drug development is to selectively target certain cell types. Cellular decisions influenced by drugs are often dependent on the dynamic processing of information. Selective responses can be achieved by differences between the involved cell types at levels of receptor, signaling, gene regulation or further downstream. Therefore, a systematic approach to detect and quantify cell type-specific parameters in dynamical systems becomes necessary. RESULTS: Here, we demonstrate that a combination of nonlinear modeling with L1 regularization is capable of detecting cell type-specific parameters. To adapt the least-squares numerical optimization routine to L1 regularization, sub-gradient strategies as well as truncation of proposed optimization steps were implemented. Likelihood-ratio tests were used to determine the optimal regularization strength resulting in a sparse solution in terms of a minimal number of cell type-specific parameters that is in agreement with the data. By applying our implementation to a realistic dynamical benchmark model of the DREAM6 challenge we were able to recover parameter differences with an accuracy of 78%. Within the subset of detected differences, 91% were in agreement with their true value. Furthermore, we found that the results could be improved using the profile likelihood. In conclusion, the approach constitutes a general method to infer an overarching model with a minimum number of individual parameters for the particular models. AVAILABILITY AND IMPLEMENTATION: A MATLAB implementation is provided within the freely available, open-source modeling environment Data2Dynamics. Source code for all examples is provided online at http://www.data2dynamics.org/ CONTACT: bernhard.steiert@fdm.uni-freiburg.de.

Authors: Bernhard Steiert, Jens Timmer, C. Kreutz

Date Published: 3rd Sep 2016

Journal: Bioinformatics

Abstract (Expand)

Clock-regulated pathways coordinate the response of many developmental processes to changes in photoperiod and temperature. We model two of the best-understood clock output pathways in Arabidopsis, which control key regulators of flowering and elongation growth. In flowering, the model predicted regulatory links from the clock to cycling DOF factor 1 (CDF1) and flavin-binding, KELCH repeat, F-box 1 (FKF1) transcription. Physical interaction data support these links, which create threefold feed-forward motifs from two clock components to the floral regulator FT. In hypocotyl growth, the model described clock-regulated transcription of phytochrome-interacting factor 4 and 5 (PIF4, PIF5), interacting with post-translational regulation of PIF proteins by phytochrome B (phyB) and other light-activated pathways. The model predicted bimodal and end-of-day PIF activity profiles that are observed across hundreds of PIF-regulated target genes. In the response to temperature, warmth-enhanced PIF4 activity explained the observed hypocotyl growth dynamics but additional, temperature-dependent regulators were implicated in the flowering response. Integrating these two pathways with the clock model highlights the molecular mechanisms that coordinate plant development across changing conditions.

Authors: Daniel Seaton, R. W. Smith, Y. H. Song, D. R. MacGregor, K. Stewart, G. Steel, J. Foreman, S. Penfield, T. Imaizumi, Andrew Millar, K. J. Halliday

Date Published: 21st Jan 2015

Journal: Mol Syst Biol

Abstract (Expand)

BACKGROUND: Polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) with harmful effects in animals and humans. Although PCB 153 is one of the most abundant among PCBs detected in animal tissues, its mechanism of toxicity is not well understood. Only few studies have been conducted to explore genes and pathways affected by PCB 153 by using high throughput transcriptomics approaches. To obtain better insights into toxicity mechanisms, we treated juvenile Atlantic cod (Gadus morhua) with PCB 153 (0.5, 2 and 8 mg/kg body weight) for 2 weeks and performed gene expression analysis in the liver using oligonucleotide arrays. RESULTS: Whole-genome gene expression analysis detected about 160 differentially regulated genes. Functional enrichment, interactome, network and gene set enrichment analysis of the differentially regulated genes suggested that pathways associated with cell cycle, lipid metabolism, immune response, apoptosis and stress response were among the top significantly enriched. Particularly, genes coding for proteins in DNA replication/cell cycle pathways and enzymes of lipid biosynthesis were up-regulated suggesting increased cell proliferation and lipogenesis, respectively. CONCLUSIONS: PCB 153 appears to activate cell proliferation and lipogenic genes in cod liver. Transcriptional up-regulation of marker genes for lipid biosynthesis resembles lipogenic effects previously reported for persistent organic pollutants (POPs) and other environmental chemicals. Our results provide new insights into mechanisms of PCB 153 induced toxicity.

Authors: F. Yadetie, O. A. Karlsen, M. Eide, C. Hogstrand, A. Goksoyr

Date Published: 19th Jun 2014

Journal: BMC Genomics

Abstract (Expand)

Systems metabolomics, the identification and quantification of cellular metabolites and their integration with genomics and proteomics data, promises valuable functional insights into cellular biology. However, technical constraints, sample complexity issues and the lack of suitable complementary quantitative data sets prevented accomplishing such studies in the past. Here, we present an integrative metabolomics study of the genome-reduced bacterium Mycoplasma pneumoniae. We experimentally analysed its metabolome using a cross-platform approach. We explain intracellular metabolite homeostasis by quantitatively integrating our results with the cellular inventory of proteins, DNA and other macromolecules, as well as with available building blocks from the growth medium. We calculated in vivo catalytic parameters of glycolytic enzymes, making use of measured reaction velocities, as well as enzyme and metabolite pool sizes. A quantitative, inter-species comparison of absolute and relative metabolite abundances indicated that metabolic pathways are regulated as functional units, thereby simplifying adaptive responses. Our analysis demonstrates the potential for new scientific insight by integrating different types of large-scale experimental data from a single biological source.

Authors: T. Maier, J. Marcos, J. A. Wodke, B. Paetzold, M. Liebeke, R. Gutierrez-Gallego, Luis Serrano

Date Published: 20th Apr 2013

Journal: Mol Biosyst

Abstract (Expand)

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see (1,2,3)). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy). Here, we provide a detailed description of a microscopy method used in several recent studies (4, 5, 6, 7), which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Authors: Imke De Jong, Katrin Beilharz, Oscar Kuipers, Jan-Willem Veening

Date Published: 16th Aug 2011

Journal: J Vis Exp

Abstract (Expand)

Abstract A new YNB medium containing very low concentrations of alkali metal cations has been developed to carry out experiments to study potassium homoeostasis. Physiological characterization of Saccharomyces cerevisiae BY4741 strain and the corresponding mutant lacking the main potassium uptake systems (trk1 trk2) under potassium nonlimiting and limiting concentrations was performed, and novel important differences between both strains were found. At nonlimiting concentrations of KCl, the two strains had a comparable cell size and potassium content. Nevertheless, mutants were hyperpolarized, had lower pH and extruded fewer protons compared with the BY4741 strain. Upon transfer to K(+)-limiting conditions, cells of both strains became hyperpolarized and their cell volume and K(+) content diminished; however, the decrease was more relevant in BY4741. In low potassium, trk1 trk2 cells were not able to accomplish the cell cycle to the same extent as in BY4741. Moreover, K(+) limitation triggered a high-affinity K(+)/Rb(+) uptake process only in BY4741, with the highest affinity being reached as soon as 30 min after transfer to potassium-limiting conditions. By establishing basic cellular parameters under standard growth conditions, this work aims to establish a basis for the investigation of potassium homoeostasis at the system level.

Authors: Clara Navarrete, Silvia Petrezselyova, Lina Patricia Barreto Parra, José L Martínez, Jaromir Zahrádka, Joaquin Ariño, Hana Sychrova, Jose Ramos

Date Published: 25th May 2010

Journal: Not specified

Abstract (Expand)

Protein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.

Authors: Janine Kirstein, Henrik Strahl, Noël Molière, Leendert Hamoen, Kürşad Turgay

Date Published: 10th Sep 2008

Journal: Mol. Microbiol.

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