UV-Licht induzierte Proteine aus Nidula niveo-tomentosa

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Coexisting/Coexpressing Genomic Libraries (CoGeL) identify interactions among distantly located genetic loci for developing complex microbial phenotypes

In engineering novel microbial strains for biotechnological applications, beyond a priori identifiable pathways to be engineered, it is becoming increasingly important to develop complex, ill-defined cellular phenotypes. One approach is to screen genomic or metagenomic libraries to identify genes imparting desirable phenotypes, such as tolerance to stressors or novel catabolic programs. Such libraries are limited by their inability to identify interactions among distant genetic loci. To solve this problem, we constructed plasmid- and fosmid-based Escherichia coli Coexisting/Coexpressing Genomic Libraries (CoGeLs). As a proof of principle, four sets of two genes of the l-lysine biosynthesis pathway distantly located on the E. coli chromosome were knocked out. Upon transformation of these auxotrophs with CoGeLs, cells growing without supplementation were found to harbor library inserts containing the knocked-out genes demonstrating the interaction between the two libraries. CoGeLs were also screened to identify genetic loci that work synergistically to create the considerably more complex acid-tolerance phenotype. CoGeL screening identified combination of genes known to enhance acid tolerance (gadBC operon and adiC), but also identified the novel combination of arcZ and recA that greatly enhanced acid tolerance by 9000-fold. arcZ is a small RNA that we show increases pH tolerance alone and together with recA

A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments

BACKGROUND: Alkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome. RESULTS: The gene encoding a novel AP was isolated from a metagenomic library constructed with ocean-tidal flat sediments from the west coast of Korea. The metagenome-derived AP (mAP) gene composed of 1,824 nucleotides encodes a polypeptide with a calculated molecular mass of 64 kDa. The deduced amino acid sequence of mAP showed a high degree of similarity to other members of the AP family. Phylogenetic analysis revealed that the mAP is shown to be a member of a recently identified family of PhoX that is distinct from the well-studied classical PhoA family. When the open reading frame encoding mAP was cloned and expressed in recombinant Escherichia coli, the mature mAP was secreted to the periplasm and lacks an 81-amino-acid N-terminal Tat signal peptide. Mature mAP was purified to homogeneity as a monomeric enzyme with a molecular mass of 56 kDa. The purified mAP displayed typical features of a psychrophilic enzyme: high catalytic activity at low temperature and a remarkable thermal instability. The optimal temperature for the enzymatic activity of mAP was 37°C and complete thermal inactivation of the enzyme was observed at 65°C within 15 min. mAP was activated by Ca(2+) and exhibited maximal activity at pH 9.0. Except for phytic acid and glucose 1-phosphate, mAP showed phosphatase activity against various phosphorylated substrates indicating that it had low substrate specificity. In addition, the mAP was able to remove terminal phosphates from cohesive and blunt ends of linearized plasmid DNA, exhibiting comparable efficiency to commercially available APs that have been used in molecular biology. CONCLUSIONS: The presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

Biotransformation of N-alkyl- and N,N-dialkylarylamines by Bacillus megaterium

In der vorliegenden Arbeit wurden Studien zur bakteriellen Biotransformation von N Alkyl- sowie N,N Dialkylarylaminen mit dem Ziel der arylischen Hydroxy-lierung und N-Dealkylierung durchgeführt. Bodenproben wurden einem Screening-Verfahren unterworfen, um selektiv nach Mikroorganismen zu suchen, die zu den genannten Biotransformationen fähig waren. In einem Screeningverfahren wurden unter Verwendung von N-Ethyl-N methyl-anilin als Standardsubstrat aus Boden-proben Mikroorganismen isoliert und auf ihre Fähigkeit zur Biotransformation überprüft. Einer der isolierten Stämme setzte das zugeführte Substrat effizient und reproduzierbar zu drei Hauptprodukten um. Deren Strukturaufklärung erfolgte mittels Gaschromatographie-Massenspektrometrie (GC-MS) sowie anhand ein-dimensionaler NMR-Experimente (1H-NMR und 13C-NMR). Identifiziert wurden zwei hydroxylierte Produkte, N Ethyl-N-methyl-2-aminophenol und N-Ethyl-N-methyl-4-aminophenol, ferner wurde N-Ethylanilin, das N Demethylierungsprodukt gebildet. Die Identitäten wurden durch synthetisierte Referenzsubstanzen bestätigt. Die phänotypische und genotypische Charakterisierung dieses Bodenisolates als Bacillus megaterium erfolgte mittels mikroskopischer und färbetechnischer Methoden sowie der 16S rDNA-Sequenzierung. Zur eindeutigen Zuordnung wurde eine DNA/DNA-Hybridisierung gegenüber dem Bacillus megaterium Type-strain (DSM 32, ATCC 14581t) durchgeführt. In einem erweiterten Substratscreening wurden die strukturellen Voraussetzungen zur Biotransformation von N-Alkyl- und N,N Dialkylarylaminen durch Bacillus megaterium ermittelt. Ausführlich sind Sub-stituenteneffekte in Bezug auf Hydroxylierung und N-Dealkylierung untersucht worden. Sperrige und räumlich große Substrate wie N,N,N´,N´-Tetramethyl-p,p´ benzidin als auch N,N,N´,N´-Tetramethyl-p-benzidin wurden nicht hydro-xyliert; allerdings fand bei beiden Substrate eine N-Demethylierung statt, wobei das Erstere stärker N demethyliert wurde. Der Effekt des Austausches von Stickstoff gegen Phosphor in einigen Substraten wurde ebenfalls untersucht. So kamen Dimethylphenylphosphin und Diethylphenylphosphin mit Bacillus megaterium zur Anwendung. Phosphor-haltige Substrate wurden von B. megaterium nicht umgesetzt. Durch Versuche mit verschiedenen Induktoren und Repressoren wurde die Cytochrom-P-450-Aktvität des isolierten Bacillus megaterium bei der Bio-transformation von N,N-Diethylanilin untersucht. Hierbei wurde gezeigt, dass durch bestimmte Barbiturate die arylische Hydroxylierung als auch die N Deethylierung gesteigert wurden. Repression der Hydroxylierungs- und N Dealkylierungsaktivität zeigte sich durch Metyrapon, n-Octylamin, Pyridin und Imidazol. Die von Bacillus megaterium durchgeführte N-Demethylierung von N,N Dimethylanilin sowie N-Ethyl-N-methylanilin wurde im Hinblick auf Formaldehydbildung untersucht. Dies erfolgte im Inkubationsmedium direkt in-situ, mittels Cysteamin-Zugabe in das Medium, mit Umsetzung zu Thiazolidin. Anhand von Inkubationsversuchen mit den stabil-isotopenmarkierten Substraten N,N-Di-(trideuteromethyl)-anilin und N-Ethyl-N (trideuteromethyl)-anilin sowie N,N-Di-[methyl-13C]-anilin und N-Ethyl-N [methyl-13C]-anilin wurde anhand der Massenspektren der gebildeten Thiazolidine eindeutig festgestellt, dass die Methylgruppe als Formaldehyd abgespalten wird. Das Potential von Bacillus megaterium zur N-Dealkylierung wurde zur mikrobiellen N Demethylierung von natürlichem N-Methyl-methyl-anthranilat genutzt. Dadurch wurde der natürliche Aromastoff Methylanthranilat gewonnen. Zur Optimierung der bakteriellen Biotransformation von N-Methyl-methyl-anthranilat zu Methylanthranilat wurden in Bezug auf Wachstumsmedium, Inkubationstemperatur, pH-Wert des Inkubationsmediums sowie Substrat-konzentration verschiedene Variationen durchgeführt. Die antimikrobiellen Eigenschaften von N-Methyl-methylanthranilat und Methylanthranilat gegenüber Bacillus megaterium wurden durch Hemmhofbildung sowie der Bestimmung der Inhibierungskonzentration im Flüssigmedium nachgewiesen. Sowohl Substrat als auch Produkt erwiesen sich in den unterschiedlichen Tests als antibakteriell. Im Anschluß an Versuche zur Immobilisierung, die zu geringeren Ausbeuten führten, erfolgten schließlich Umsetzungen im Bioreaktor. Hierbei ließen sich die bei den Schüttelkulturen erhaltenen Ergebnisse direkt proportional auf den Fermenter übertragen.The goal of this study was to conduct the microbial biotransformation of N-alkyl- and N,N dialkylarylamines. Main objective was the arylic hydroxylation and N-dealkylation of this class of substrates. By this, microorganisms were isolated from topsoil and a selective screening process was started to search for microorganisms capable of con-ducting the biotransformations mentioned above. In a screening process and by the use of N-ethyl-N methylaniline as substrate, microorganisms of soil samples were isolated and proven of their ability to biotransformate N-ethyl-N-methylaniline. One isolate showed three biotransformation products. Structure determination was done by gaschromatography-masspectrometry (GC-MS) and nuclear magnetic resonance experiments (1H-NMR and 13C-NMR). N-ethyl-N-methylaniline was biohydroxylated to N-ethyl-N-methylaniline-2-aminophenol and N ethyl-N-methyl-4-aminophenol, N demethylation of N-ethyl-N-methyl-aniline afforded N-ethylaniline. The soil isolate was characterized and identified phenotypical and genotypical by colour reactions and the standard 16S rDNA method being Bacillus megaterium. To complete the assignment of the isolate to the species Bacillus megaterium, DNA/DNA-hybridization analysis against the Bacillus megaterium type strain (DSM 32, ATCC 14581t) was conducted. In an extended substrate screening, structural properties for a successful bio-transformation of N-alkyl- and N,N-dialkylarylamines by Bacillus megaterium were investigated. Substituent influences were proven in a further substrate screening regarding hydroxylation as well as N-dealkylation. Bulky and large substrates like N,N,N´,N´-tetramethyl-p,p´-benzidine and N,N,N´,N´-tetramethyl-p-benzidine were not hydroxylated by the microorganisms. But, N-demethylation was observed whereas the first one was more accepted. Exchange of nitrogen against phosphorus by using dimethylphenylphosphine and diethylphenylphosphine as substrates finally stopped all biotransformation activity of Bacillus megaterium. Neither arylic hydroxylation nor P dealkylation occurred. Trials to influence the bacterial activity towards the substrate N,N-diethylaniline, elicitors and inhibitors were used to affect cytochrome-P-450. Certain barbiturates clearly increased arylic hydroxylation as well as N dealkylation. Repression of these metabolites was shown by metyrapone, pyridine, n-octylamine and imidazole. N-demethylation reaction of Bacillus megaterium was further investigated for a detection of formaldehyde. Formaldehyde, built after N-demethylation of N,N dimethylaniline or N ethyl-N-methylaniline, was caught by added cysteamine and derivatized to thiazolidine. Incubation of the stable isotope labelled substrates N,N-di-(trideuteromethyl)-aniline and N-ethyl-N-(trideuteromethyl)-aniline as well as N,N-di[methyl 13C]-aniline and N-ethyl-N-[methyl-13C]-aniline and the resulting mass spectral fragmentations of the build labelled thiazolidines clearly stated that formaldehyde comes from methylgroups. Observed N-dealkylation reactions of Bacillus megaterium were used to produce the food flavour methyl anthranilate by microbial N-demethylation of N methyl methyl anthranilate. Several variations of the standard conditions were proven for the optimization of the bacterial biotransformation from N-methyl methyl anthranilate to methyl anthranilate: incubation temperature, pH-value, substrate concentration and media. Antimicrobial activities of N-methyl methyl anthranilate and methyl anthranilate against Bacillus megaterium were tested by using the paper disc diffusion assay and determination of the minimum inhibitory concentration in liquid medium. Substrate as well as product exhibited antimicrobial activity in both tests. To improve the stability of Bacillus megaterium, bacterial cells were immobilized in sodium-alginate-globules resulting in very low yields of methyl anthranilate. Finally, as a summarization of all obtained methyl anthranilate results, an upscaling was done in the bioreactor in a direct proportional way

Harbour seal counts in the Elbe Estuary, Germany, between Wedel and Cuxhaven in 2018/2019

Aerial surveys were conducted in the Elbe Estuary, Germany, to count harbour seals on their haul-out sites. Haul-out sites were located within the intertidal zone, usually close to the waterway or tidal creeks. The north-western border of the study area was located at river-km 728 (city of Cuxhaven) and the south-eastern border was located at river-km 632 (Mühlenberger Loch). Flights were performed monthly from August 2018 until July 2019 (12 flights in total) at low tide. Within each month, a flight date in the middle of the month was chosen, depending on the tide and weather conditions. Flights were performed with a twin-engined aircraft at a height of approximately 600 feet and at an average speed of 100 knots. The flight routes were tracked via GPS. Sighted harbour seals were photographed on their haul-out sites and the corresponding geographic position was recorded. In the office, the photos were used to count the animals. In the beginning of the pupping season in June, adult and juvenile individuals were counted separately. The sizes of individual seals were used to distinguish between both age classes. Later in the year, juvenile seals could no longer visually be distinguished from adult individuals due to the pups' rapid growth. Consequently, count data of juvenile harbour seals are only available for June 2019

Dehydrierung des Cholesterins im aktiviert adsorbierten Zustand unter Normalbedingungen, ein Beitrag zur Frage der Endogenese carcinogener, polycyclischer Kohlenwasserstoffe

Die Ergebnisse einer Selendehydrierung des Cholesterins bei 350°C werden mit Produkten verglichen, die sich aus dem Steroid im aktiviert absorbierten Zustand auf Kieselgel durch Oxydation mit Joddampf bei Raumtemperatur bilden. Alkylsubstituierte Cyclopentenophenanthrene konnten hierbei mit Sicherheit gefaßt werden. Da die äußeren Versuchsbedingungen, im Gegensatz zu allen bisherigen Dehydrierungsversuchen an Steroiden, am ehesten mit physiologischen verglichen werden können (20°C, ein dem freien Sauerstoff entsprechendes Oxydationspotential von ca. +0,4 V und eine aktivierende Grenzfläche), werfen die Ergebnisse ein neues Licht auf die alte Hypothese der endogenen Bildung carcinogener Kohlenwasserstoffe im Organismus. Der erleichterte Reaktionsverlauf im Adsorpt wird durch Erhöhung der Adsorptionswärme im Zuge der Aromatisierung erklärt