Anika Erxleben

Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens

The degradation of aromatic compounds follows different biochemical principles in aerobic and anaerobic microorganisms. While aerobes dearomatize and cleave the aromatic ring by oxygenases, facultative anaerobes utilize an ATP‐dependent ring reductase for the dearomatization of the activated key intermediate benzoyl‐coenzyme A (CoA). In this work, the aromatic metabolism was studied in the obligately anaerobic model organism Geobacter metallireducens. The gene coding for a putative carboxylic acid‐CoA ligase was heterologously overexpressed and the gene product was characterized as a highly specific benzoate‐CoA ligase catalysing the initial step of benzoate metabolism. However, no evidence for the presence of an ATP‐dependent benzoyl‐CoA reductase as observed in facultative anaerobes was obtained. In a proteomic approach benzoate‐induced proteins were identified; the corresponding genes are organized in two clusters comprising 44 genes. Induction of representative genes during growth on benzoate was confirmed by reverse transcription polymerase chain reaction. The results obtained suggest that benzoate is activated to benzoyl‐CoA, which is then reductively dearomatized to cyclohexa‐1,5‐diene‐1‐carbonyl‐CoA, followed by β‐oxidation reactions to acetyl‐CoA units, as in facultatively anaerobic bacteria. However, in G. metallireducens the process of reductive benzene ring dearomatization appears to be catalysed by a set of completely different protein components comprising putative molybdenum and selenocysteine containing enzymes.

StreptomeDB: a resource for natural compounds isolated from Streptomyces species

Bacteria from the genus Streptomyces are very important for the production of natural bioactive compounds such as antibiotic, antitumour or immunosuppressant drugs. Around two-thirds of all known natural antibiotics are produced by these bacteria. An enormous quantity of crucial data related to this genus has been generated and published, but so far no freely available and comprehensive database exists. Here, we present StreptomeDB (http://www.pharmaceutical-bioinformatics.de/streptomedb/). To the best of our knowledge, this is the largest database of natural products isolated from Streptomyces. It contains >2400 unique and diverse compounds from >1900 different Streptomyces strains and substrains. In addition to names and molecular structures of the compounds, information about source organisms, references, biological role, activities and synthesis routes (e.g. polyketide synthase derived and non-ribosomal peptides derived) is included. Data can be accessed through queries on compound names, chemical structures or organisms. Extraction from the literature was performed through automatic text mining of thousands of articles from PubMed, followed by manual curation. All annotated compound structures can be downloaded from the website and applied for in silico screenings for identifying new active molecules with undiscovered properties.

Metabolite profiling of the moss Physcomitrella patens reveals evolutionary conservation of osmoprotective substances

The moss Physcomitrella patens is suitable for systems biology studies, as it can be grown axenically under standardised conditions in plain mineral medium and comprises only few cell types. We report on metabolite profiling of two major P. patens tissues, filamentous protonema and leafy gametophores, from different culture conditions. A total of 96 compounds were detected, 21 of them as yet unknown in public databases. Protonema and gametophores had distinct metabolic profiles, especially with regard to saccharides, sugar derivates, amino acids, lignin precursors and nitrogen-rich storage compounds. A hydroponic culture was established for P. patens, and was used to apply drought stress under physiological conditions. This treatment led to accumulation of osmoprotectants, such as altrose, maltitol, ascorbic acid and proline. Thus, these osmoprotectants are not unique to seed plants but have evolved at an early phase of the colonization of land by plants.

StreptomeDB 2.0—an extended resource of natural products produced by streptomycetes

Over the last decades, the genus Streptomyces has stirred huge interest in the scientific community as a source of bioactive compounds. The majority of all known antibiotics is isolated from these bacterial strains, as well as a variety of other drugs such as antitumor agents, immunosuppressants and antifungals. To the best of our knowledge, StreptomeDB was the first database focusing on compounds produced by streptomycetes. The new version presented herein represents a major step forward: its content has been increased to over 4000 compounds and more than 2500 host organisms. In addition, we have extended the background information and included hundreds of new manually curated references to literature. The latest update features a unique scaffold-based navigation system, which enables the exploration of the chemical diversity of StreptomeDB on a structural basis. We have included a phylogenetic tree, based on 16S rRNA sequences, which comprises more than two-thirds of the included host organisms. It enables visualizing the frequency, appearance, and persistence of compounds and scaffolds in an evolutionary context. Additionally, we have included predicted MS- and NMR-spectra of thousands of compounds for assignment of experimental data. The database is freely accessible via http://www.pharmaceutical-bioinformatics.org/streptomedb.

Genome Sequence of the Fungus Glarea lozoyensis: the First Genome Sequence of a Species from the Helotiaceae Family

ABSTRACT The anamorphic fungus Glarea lozoyensis mutant strain 74030 is an overproducer of pneumocandin B0, which is chemically converted into Cancidas, a potent antibiotic against clinically important fungal pathogens. Pneumocandins are acylated, cyclic hexapeptides with unusual hydroxylated amino acids. With the Glarea lozoyensis genome, the first species from the large polyphyletic family Helotiaceae has been sequenced.

The RNA workbench: best practices for RNA and high-throughput sequencing bioinformatics in Galaxy

Abstract RNA-based regulation has become a major research topic in molecular biology. The analysis of epigenetic and expression data is therefore incomplete if RNA-based regulation is not taken into account. Thus, it is increasingly important but not yet standard to combine RNA-centric data and analysis tools with other types of experimental data such as RNA-seq or ChIP-seq. Here, we present the RNA workbench, a comprehensive set of analysis tools and consolidated workflows that enable the researcher to combine these two worlds. Based on the Galaxy framework the workbench guarantees simple access, easy extension, flexible adaption to personal and security needs, and sophisticated analyses that are independent of command-line knowledge. Currently, it includes more than 50 bioinformatics tools that are dedicated to different research areas of RNA biology including RNA structure analysis, RNA alignment, RNA annotation, RNA-protein interaction, ribosome profiling, RNA-seq analysis and RNA target prediction. The workbench is developed and maintained by experts in RNA bioinformatics and the Galaxy framework. Together with the growing community evolving around this workbench, we are committed to keep the workbench up-to-date for future standards and needs, providing researchers with a reliable and robust framework for RNA data analysis. Availability: The RNA workbench is available at https://github.com/bgruening/galaxy-rna-workbench.

Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.

High throughput metabolic screen of Physcomitrella transformants

Abstract A large-scale metabolic screen was performed for 51,180 targeted knockout mutants of the haploid moss Physcomitrella patens (Hedw.) Bruch & Schimp. The growth ability of each mutant was compared to the wild type. Plants were cultured on a minimal medium which contained only macroelements, as well as on a supplemented medium, additionally containing microelements, glucose, vitamins, ammonium tartrate, adenine, peptone and Na-palmitic acid. The screen resulted in the identification of 20 (0.04 %) auxotrophs. Medium supplementation tests were performed for five auxotrophs, which showed no growth on minimal medium, but were rescued on supplemented medium. One vitamin-deficient mutant was identified as p-aminobenzoic acid auxotroph, three plants were nitrate assimilation deficient mutants and one transformant showed undefined growth requirements. The screen resulted further in the identification of two physiological mutants, exhibiting an albino phenotype on minimal medium but a green wild type phenotype on the supplemented medium. The culture of both albinos under low light intensities could not prevent bleaching, revealing that the missing production of chlorophyll was not caused by light sensitivity. Astonishingly, the supplementation of the minimal medium with selected compounds of the supplemented medium did also not prevent bleaching. Moreover, both mutants produced green protonemata, even if single substances of the supplemented medium were lacking. It was concluded that a complex network of interactions related to photosynthesis in Physcomitrella was disturbed. This is the first detailed study of auxotrophic and albino Physcomitrella mutants produced by transformation-mediated gene disruption. The described physiological mutants provide valuable resources for the identification of essential gene functions of plant metabolism.

Carbimazole is an inhibitor of protein synthesis and protects from neuronal hypoxic damage in vitro

Oxygen deprivation during ischemic or hemorrhagic stroke results in ATP depletion, loss of ion homeostasis, membrane depolarization, and excitotoxicity. Pharmacologic restoration of cellular energy supply may offer a promising concept to reduce hypoxic cell injury. In this study, we investigated whether carbimazole, a thionamide used to treat hyperthyroidism, reduces neuronal cell damage in oxygen-deprived human SK-N-SH cells or primary cortical neurons. Our results revealed that carbimazole induces an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) that was associated with a marked inhibition of global protein synthesis. Translational inhibition resulted in significant bioenergetic savings, preserving intracellular ATP content in oxygen-deprived neuronal cells and diminishing hypoxic cellular damage. Phosphorylation of eEF2 was mediated by AMP-activated protein kinase and eEF2 kinase. Carbimazole also induced a moderate calcium influx and a transient cAMP increase. To test whether translational inhibition generally diminishes hypoxic cell damage when ATP availability is limiting, the translational repressors cycloheximide and anisomycin were used. Cycloheximide and anisomycin also preserved ATP content in hypoxic SK-N-SH cells and significantly reduced hypoxic neuronal cell damage. Taken together, these data support a causal relation between the pharmacologic inhibition of global protein synthesis and efficient protection of neurons from ischemic damage by preservation of high-energy metabolites in oxygen-deprived cells. Furthermore, our results indicate that carbimazole or other translational inhibitors may be interesting candidates for the development of new organ-protective compounds. Their chemical structure may be used for computer-assisted drug design or screening of compounds to find new agents with the potential to diminish neuronal damage under ATP-limited conditions.

Genome Sequence of Streptomyces sp. Strain Tü6071

Streptomyces sp. Tü6071 is a soil-dwelling bacterium which has a highly active isoprenoid biosynthesis. Isoprenoids are important precursors for biopharmaceutical molecules such as antibiotics or anticancer agents, e.g., landomycin. Streptomyces sp. Tü6071 produces the industrially important terpene glycosides phenalinolactones, which have antibacterial activity against several Gram-positive bacteria. The availability of the genome sequence of Streptomyces sp. Tü6071 allows for understanding the biosynthesis of these pharmaceutical molecules and will facilitate rational genome modification to improve industrial use.