Regulation des anaeroben Abbaus aromatischer Kohlenwasserstoffe in Aromatoleum aromaticum EbN1

Das genomsequenzierte β-Proteobakterium Aromatoleum aromaticum Stamm EbN1 und einige verwandte Arten weisen die ungewöhnliche Stoffwechselaktivität des anaeroben Abbaus von Kohlenwasserstoffen sowie phenolischen Verbindungen auf. Hierfür besitzt A. aromaticum mehrere sehr substratspezifische Stoffwe...

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Bibliographic Details
Main Author: Muhr, Enrico
Contributors: Heider, Johann (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2016
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Table of Contents: The genome-sequenced Aromatoleum aromaticum strain EbN1 and several related β-proteobacterial genera have the unusual metabolic capability of degrading aromatic hydrocarbons as well as phenolic compounds under anaerobic conditions. A. aromaticum possesses numerous highly substrate-specific metabolic pathways which are activated only in the presence of the respective substrate and not by chemically analogous compounds that leads to the induction of highly specific and interesting catabolic enzymes. Focus of this PhD Thesis was the investigation of the mechanism of specific sensing of hydrocarbons and phenolic substrates as well as the metabolic regulation of induction of the respective Degradation pathways. By the combination of newly developed and already established biochemical and molecular biological methods, it was possible to show that, despite having a very similar chemical structure, ethylbenzene and p-ethylphenol were metabolized by completely separate pathways with specifically induced enzymes. Therefore, cultures of strain EbN1 were grown under denitrifying conditions on ethylbenzene, p-ethylphenol and acetophenone as important intermediate in the anaerobic ethylbenzene metabolism. The results of all determined Enzyme activities, heme-staining as well as immuno- and biotin-blotting analyses allowed the identification and differentiation of the key enzymes involved in both anaerobic Degradation pathways. Additionally, the proposed p-ethylphenol degradation pathway was substantiated by newly identified enzymes including a novel biotin-dependent p-hydroxyacetophenone carboxylase. The aromatic ketone acetophenone, an intermediate of anaerobic ethylbenzene degradation, is metabolized by A. aromaticum either anaerobically or aerobically by an ATP-dependent acetophenone carboxylase (ApcABCDE). A mCherry reporter strain was constructed to study the conditions under which the genes encoding the carboxylase were expressed. This Reporter strain provides a basis for the development of a specific acetophenone bioreporter system. For this purpose, a gene fusion of the mCherry gene and the first gene of the apc-bal operon (apcA)was created and integrated into the chromosome of A. aromaticum. It could be shown that the induction of the fusion protein and its fluorescence activity responded consistently with the expression pattern of the acetophenone-metabolic enzymes. Furthermore, the recorded amounts of ApcA-mCherry production were proportional to the applied acetophenone concentrations within a range of 25 to 250 μM. With the exception of both 1-phenylethanol enantiomers which are easily converted to acetophenone by A. aromaticum, all further tested analogous compounds showed a significantly weaker signal response or none at all. Furthermore, this thesis investigates the adiRS operon encoding a two-component regulatory system which was proposed to regulate the acetophenone-dependent induction of the apc-bal operon in A. aromaticum. Therefore, a chloramphenicol resistant deletion mutant lacking the adiRS operon was constructed by homologous recombination. In contrast to the expectations, the adiRS gene deletion strain still grew on acetophenone and ethylbenzene. However, reduced acetophenone carboxylase activities were detectable in extracts of these cultures. Surprisingly, the mutant strain showed uncommon induction of EbDH and two paralogous biotin-containing carrier proteins (XccB and XccB2) which are not induced in the wildtype strain grown on acetophenone or ethylbenzene, respectively. Restoring the adiRS genes in trans only resulted in a partial reversal of the observed deviations. Moreover, a mCherry reporter strain lacking the adiRS operon was constructed as described above. The results indicated a probable timedelayed and decreased expression of the apcA-mCherry gene fusion in the reporter strain lacking the genes encoding AdiRS. The obtained data led to the assumption, that acetophenonedependent induction of the apc-bal operon is possibly mediated by a second two-component regulatory system (XdiRS) closely related to AdiRS. Finally, this study presents a tentative model of the potential role of the AdiRS and XdiRS regulatory systems in substrate recognition and discrimination in A. aromaticum strain EbN1. Finally, it was possible to map putative transcription start sites of the catabolic operons involved in the anaerobic ethylbenzene and acetophenone degradation by using primer extension Analysis for the first time experimentally. Upstream of all determined starting points putative -35 and -10 boxes could be identified by comparison with consensus sequences of σ70-dependent promoters. In case of the apc-bal operon an extensive 5’-untranslated region and the predicted secondary structure with a calculated minimum free energy of -61.4 kcal mol-1 could indicate that this RNA structure has an influence on transcriptional or translational regulation.