Sources and pathways of methane formed in oxidative environments

Methane is the most abundant reduced organic compound in the atmosphere. As the strongest known long-lived greenhouse gas after water vapour and carbon dioxide methane perturbs the radiation balance of Earth’s atmosphere. The abiotic formation of methane requires ultraviolet irradiation of organic matter or takes place in locations with high temperature and/or pressure, e.g. during biomass burning or serpentinisation of olivine, under hydrothermal conditions in the oceans deep or below tectonic plates. The biotic methane formation was traditionally thought to be formed only by methanogens under strictly anaerobic conditions, such as in wetland soils, rice paddies and agricultural waste. rnIn this dissertation several chemical pathways are described which lead to the formation of methane under aerobic and ambient conditions. Organic precursor compounds such as ascorbic acid and methionine were shown to release methane in a chemical system including ferrihydrite and hydrogen peroxide in aquatic solution. Moreover, it was shown by using stable carbon isotope labelling experiments that the thio-methyl group of methionine was the carbon precursor for the methane produced. Methionine, a compound that plays an important role in transmethylation processes in plants was also applied to living plants. Stable carbon isotope labelling experiments clearly verified that methionine acts as a precursor compound for the methane from plants. Further experiments in which the electron transport chain was inhibited suggest that the methane generation is located in the mitochondria of the plants. The abiotic formation of methane was shown for several soil samples. Important environmental parameter such as temperature, UV irradiation and moisture were identified to control methane formation. The organic content of the sample as well as water and hydrogen peroxide might also play a major role in the formation of methane from soils. Based on these results a novel scheme was developed that includes both biotic and chemical sources of methane in the pedosphere.rn

Na +-coupled betaine symporters involved in osmotic stress response in prokaryotes and eukaryotes

The betaine/GABA transporter BGT1 is one of the most important osmolyte transporters in the kidney. BGT1 is a member of the neurotransmitter sodium symporter (NSS) family, facilitates Na+/Cl--coupled betaine uptake to cope with hyperosmotic stress. Betaine transport in kidney cells is upregulated under hypertonic conditions by a yet unknown mechanism when increasing amounts of intracellular BGT1 are inserted into the plasma membrane. Re-establishing isotonicity results in ensuing depletion of BGT1 from the membrane. BGT1 phosphorylation on serines and threonines might be a regulation mechanism. In the present study, four potential PKC phosphorylation sites were mutated to alanines and the responses to PKC activators, phorbol 12-myristate acetate (PMA) and dioctanoyl-sn-glycerol (DOG) were determined. GABA-sensitive currents were diminished after 30 min preincubation with these PKC activators. Staurosporine blocked the response to DOG. Three mutants evoked normal GABA-sensitive currents but currents in oocytes expressing the mutant T40A were greatly diminished. [3H]GABA uptake was also determined in HEK-293 cells expressing EGFP-tagged BGT1 with the same mutations. Three mutants showed normal upregulation of GABA uptake after hypertonic stress, and downregulation by PMA was normal compared to EGFP-BGT1. In contrast, GABA uptake by the T40A mutant showed no response to hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells, grown on glass or filters, revealed that T40A was present in the cytoplasm after 24 h hypertonic stress while the other mutants and EGFP-BGT1 were predominantely present in the plasma membrane. All four mutants co-migrated with EGFP-BGT1 on Western blots suggesting they are full-length proteins. In conclusion, T235, S428, and S564 are not involved in downregulation of BGT1 due to phosphorylation by PKC. However, T40 near the N-terminus may be part of a hot spot important for normal trafficking or insertion of BGT1 into the plasma membrane. Additionally, a link between substrate transport regulation, insertion of BGT1 into the plasma membrane and N-glycosylation in the extracellular loop 2 (EL2) could be revealed. The functional importance of two predicted N-glycosylation sites, which are conserved in EL2 within the NSS family were investigated for trafficking, transport and regulated plasma membrane insertion by immunogold-labelling, electron microscopy, mutagenesis, two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radioactive-labelled substrate into MDCK cells. Trafficking and plasma membrane insertion of BGT1 was clearly promoted by proper N-glycosylation in both, oocytes and MDCK cells. De-glycosylation with PNGase F or tunicamycin led to a decrease in substrate affinity and transport rate. Mutagenesis studies revealed that in BGT1 N183 is the major N-glycosylation site responsible for full protein activity. Replacement of N183 with aspartate resulted in a mutant, which was not able to bind N-glycans suggesting that N171 is a non-glycosylated site in BGT1. N183D exhibited close to WT transport properties in oocytes. Surprisingly, in MDCK cells plasma membrane insertion of the N183D mutant was no longer regulated by osmotic stress indicating unambiguously that association with N-glycans at this position is linked to osmotic stress-induced transport regulation in BGT1. The molecular transport mechanism of BGT1 remains largely unknown in the absence of a crystal structure. Therefore investigating the structure-function relationship of BGT1 by a combination of structural biology (2D and 3D crystallization) and membrane protein biochemistry (cell culture, substrate transport by radioactive labeled GABA uptake into cells and proteoliposomes) was the aim of this work. While the functional assays are well established, structure determination of eukaryotic membrane transporters is still a challenge. Therefore, a suitable heterologous expression system could be defined, starting with cloning and overexpression of an optimized gene. The achieved expression levels in P. pastoris were high enough to proceed with isolation of BGT1. Furthermore, purification protocols could be established and resulted in pure protein, which could even be reconstituted in an active form. The quality and homogeneity of the protein allowed already 2D and 3D crystallization, in which initial crystals could be obtained. Interestingly, the striking structural similarity of BGT1 to the bacterial betaine transporter BetP, which became a paradigm for osmoregulated betaine transport, provided information on substrate coordination in BGT1. The structure of a BetP mutant that showed activity for GABA was solved to 3.2Å in complex with GABA in an inward facing open state. This structure shed some light into the molecular transport mechanisms in BGT1 and might help in future to design conformationally locked BGT1 to enforce the on-going structure determination.

Surfactants and their aerobic degradation products

Die Ziele der vorliegenden Arbeit waren 1) die Entwicklung und Validierung von sensitiven und substanz-spezifischen Methoden für die quantitative Bestimmung von anionischen, nichtionischen und amphoteren Tensiden und deren Metaboliten in wässrigen Umweltproben unter Einsatz leistungsfähiger, massenspektrometrischer Analysengeräte,2) die Gewinnung von aeroben, polaren Abbauprodukten aus Tensiden in einem die realen Umweltbedingungen simulierenden Labor-Festbettbioreaktor (FBBR), dessen Biozönose oberflächenwasserbürtig war,3) zur Aufklärung des Abbaumechanismus von Tensiden neue, in 2) gewonnene Metabolite zu identifizieren und massenspektrometrisch zu charakterisieren ebenso wie den Primärabbau und den weiteren Abbau zu verfolgen,4) durch quantitative Untersuchungen von Tensiden und deren Abbauprodukten in Abwasser und Oberflächenwasser Informationen zu ihrem Eintrag und Verhalten bei unterschiedlichen hydrologischen und klimatischen Bedingungen zu erhalten,5) das Verhalten von persistenten Tensidmetaboliten in Wasserwerken, die belastetes Oberflächenwasser aufbereiten, zu untersuchen und deren Vorkommen im Trinkwasser zu bestimmen,6) mögliche Schadwirkungen von neu entdeckten Metabolite mittels ökotoxikologischer Biotests abzuschätzen,7) durch Vergleich der Felddaten mit den Ergebnissen der Laborversuche die Umweltrelevanz der Abbaustudien zu belegen. Die Auswahl der untersuchten Verbindungen erfolgte unter Berücksichtigung ihres Produktionsvolumens und der Neuheit auf dem Tensidmarkt. Sie umfasste die Waschmittelinhaltsstoffe lineare Alkylbenzol-sulfonate (LAS), welches das Tensid mit der höchsten Produktionsmenge darstellte, die beiden nichtionischen Tenside Alkylglucamide (AG) und Alkylpolyglucoside (APG), ebenso wie das amphotere Tensid Cocamidopropylbetain (CAPB). Außerdem wurde der polymere Farbübertragungsinhibitor Polyvinylpyrrolidon (PVP) untersucht.

Investigations on DNA methylation by Dnmt2 and impact of tRNA modifications on TLR7 stimulation

The incorporation of modified nucleotides into ribonucleic acids (RNAs) is important for their structure and proper function. These modifications are inserted by distinct catalytic macromolecules one of them being Dnmt2. It methylates the Cytidine (C) at position 38 in tRNA to 5-methylcytidine (m5C). Dnmt2 has been a paradigm in this respect, because all of its nearest neighbors in evolution are DNA-cytosine C5-methyltransferases and methylate DNA, while its (own) DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. This work determines whether the biochemical potential for DNA methylation is present in the enzyme. It was discovered that DNA fragments, when presented as covalent RNA:DNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Additional minor deviations from a native tRNA structure that were seen to be tolerated by Dnmt2 were used for a stepwise development of a composite system of guide RNAs that enable the enzyme to perform cytidine methylation on single stranded DNA in vitro. Furthermore, a proof-of-principle is presented for utilizing the S-adenosyl methionine-analog cofactor SeAdoYn with Dnmt2 to search for new possible substrates in a SELEX-like approach.rnIn innate immunity, nucleic acids can function as pathogen associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs). The modification pattern of RNA is the discriminating factor for toll-like receptor 7 (TLR7) to distinguish between self and non-self RNA of invading pathogens. It was found that a 2'-O-methylated guanosine (Gm) at position18, naturally occurring at this position in some tRNAs, antagonizes recognition by TLR7. In the second part of this work it is pointed out, that recognition extends to the next downstream nucleotide and the effectively recognized molecular detail is actually a methylated dinucleotide. The immune silencing effect of the ribose methylation is most pronounced if the dinucleotide motif is composed of purin nucleobases whereas pyrimidines diminish the effect. Similar results were obtained when the Gm modification was transposed into other tRNA domains. Point mutations abolishing base pairings important for a proper tertiary structure had no effect on the immune stimulatory potential of a Gm modified tRNA. Taken together these results suggest a processive type of RNA inspection by TLR7.rn